volume 1 number 01 - Prevention and Research

Transcript

Freely available online
ISSN:2240-2594
International Open Access Journal of Prevention and Research in Medicine
Director Prof. Francesco Tomei
VOLUME 1 NUMBER 01
OCT-DEC 2011
IN THIS NUMBER
Jean-Claude Andrè
For the development of a common understanding for innovation in lifenanosciences through
interdisciplinarity
pag. 01-07
D. A. Giorgi, S. Palmieri, G. Renzi, F. Massoni, S. Ricci
Rischio biologico Legionella presso strutture sanitarie di Roma e provincia
pag. 08-15
C. Angioni, G. Montanari Vergallo, I. Catarinozzi, L. Iovenitti, P. Frati
Il valore giuridico e medico-legale delle linee guida
pag.16-21
G. Palaia, R. Lo Giudice, C. Cavallini, G. Gaimari, G. Tenore
Papilloma virus e lesioni della mucosa orale. Infezione e aumento del potenziale di
degenerazione maligna
G. Tenore, F. Carpenteri, R. Lo Giudice, C. Cavallini, G. Gaimari, G. Palaia
Biopsia nelle lesioni del cavo orale: mezzo per la certezza della diagnosi
G. Tomei, A. Sancini
La Devianza come problema sociale: le risposte della Psicologia
R. Romano, S. Gramolelli, F. Tabacchi, G. Russo, S. Verzaro, F. Marinucci, G.M.
Paganotti, A. Gaeta, M. Coluzzi
Human herpes virus 8 (HHV-8): salivary shedding in mothers and children from Uganda: risk
factors and clues about transmission
pag.22-28
pag.29-35
pag.36-43
pag.44-52
P. Andreozzi, G. Viscogliosi, E. Cipriani, A. Servello, B. Marigliano, E. Ettorre, V.
Marigliano
Predictive medicine in Cardiovascular Diseases. What next?
pag.53-59
A. Sancini, S. De Sio, M. Ciarrocca, M. Fioravanti, G. Andreozzi, O. Sarlo, R. D’Amelio, A.
Anselmi, E. Mascia, G. De Lorenzo, E. Ferrante, F. Gaudioso, A. Rauccio, V. Zelano, F.
Tomei , G. Tomei
Estimated risk assessment of the exposed to asbestos
pag.60-71
M. Pieri, N. Miraglia, G. Genovese, R. Guadagni, A. Acampora, N. Sannolo
New perspectives in hemoglobin adducts analysis: selective digestion with Calpain I
pag.72-86
Sancini A, Caciari T, Di Pastena C, Sinibaldi F, Scala B, Fiaschetti M, De Sio S,
Maurizi D, Nardone N, Scimitto L, Miracco P, Tomei F, Tomei G, Ciarrocca M
Meta-analysis: cardiovascular effects in workers occupationally exposed to urban pollution
pag.87-100
P&R Scientific
FOR THE DEVELOPMENT OF A COMMON UNDERSTANDING FOR
INNOVATION IN LIFENANOSCIENCES THROUGH
INTERDISCIPLINARITY
Jean-Claude Andrè
1
1
CNRS SCIENTIFIC ADVISER PARIS-FRANCE
Citation: Andrè JC. For the development of a common understanding for innovation in lifenanosciences through
interdisciplinarity. Prevent Res 2011; 1 (1): 01-07
Key words: nano-medicine, innovation, risks and precautionary principle
Abstract
Background: Research is more and more divided up into scientific disciplines leading to difficulties to associate different
specialized knowledge, inside projects of public usefulness.
Objectives: The object of this essay is to explore this topic by applying it to a domain supported by decision-makers,
that of nanotechnologies and particularly their development as nano-drugs for medical applications. The choice of this
selected topic corresponds to an ambivalent situation: rejection of new risks induced by the development of industrial
production of nano-systems and need of new medical treatments leading to the improvement of the citizen health,
including nano-medicine.
The presentation is meant to be interrogative with open questions.
Methods: The author, consequently, wish to receive your opinions and some comments, you active readers who act in
the joint domains of research and prevention of medical risks. Results: Your answers will be the object of a synthesis which will be later published in “Prevention and Research” in a
few months. The reader vision is of course of importance in a media system presently stimulated by active associations in
which the scientists have a too little space of expression.
Conclusions: No final conclusion is proposed in this essay, only a Provisory closure called: For an operational proposal.
I-Background
Scientific Disciplines exist because the Man in his wish to understand the real cannot embrace complexity by a unique
question which would cover the multiplicity of the possible disciplinary approaches. Research is subjected to numerous
paradoxical injunctions linked to modes of social and financial regulations, which would probably need to be “re – visited”
in order to take into account new “needs” of the Society (1, 2). The inter-disciplinarity or the cross-disciplinarity which
are never well and deeply defined are nevertheless the object of a visible support on behalf of tutelage, but with a
supervision still hard controlled by disciplines and the peers evaluation system (3, 4, 5, 6).
II-Objectives
The goal of this essay is to explore the missions of the scientific research in a society in profound mutation, and so to
explore, without taboo, means to satisfy them “at the best way“by means of science. It is a question of finding ways of
progress supporting responsible innovation as a motor of the dynamics of an existent research of the academic world to
the advantage of the Society (7). In this document, the author asks questions which seem to him necessary to hire a
useful debate which should decrease the gap between decision makers, stakeholders, researchers and the citizens. From
your answers to some of these questions, a synthesis will be accomplished constituting a form of collective expertise
leading to recommendations linked to points of view concerning means to be implemented to reach targets of social
usefulness, chosen in the joint domain of life sciences and nanotechnologies chosen as an example. All these comments
and proposals should be the object of a synthesis serving to pro-active readers of “Prevention and Research” in the
responsible development for inter-disciplinarity as a normal way of action for a high quality research associating social
progress and mastering of risks (risk assessment and risk management).
II-1- Why such proposal?
The necessary cutting up of knowledge, its distribution in a disciplinary silo, is a thing which cannot be any more called
into question (8). For complex systems, a unique knowledge doesn’t exists, but several options of knowledge have to be
www.preventionandresearch.com
1
Oct-Dec 2011|P&R Scientific|Volume 1|N°1
P&R Scientific
taken into consideration, a particular understanding of question through each cultural and knowledge spectra leading to
specific answers. Conceptual walls were born separating disciplinary knowledge in more restrained units, or silos, allowing
the deepening of researches and knowledge. Every new research unit develops its own methods, its own procedures,
refining them to answer questions very different from the historian to the biologist, by way of the musician and by the
chemist or the engineer: First group of specialists search in texts recreating a past bit by bit, second dissects, experiment,
makes out a will to understand the details of the mechanics of the living being, when in the third domain, it whets its
practice by a methodology which would not know how to be essential in the two others, finally the chemist plays with a
“partition” imposed by Mendeleyev...At the same time, as this specialization of tools and the Men who create knowledge,
knowledge increases faster and faster, requiring new specialized paradigms, new disciplinary silos and new specific
languages. It is then the hatching of a new series of smaller entities, favoring the development of particular and more
specific knowledge, with a vernacular specialized language in which will bloom other persons become specialists of the
first specialty. This mechanism is particularly visible, for instance, in the scientific domains, where it is not rare to see
researchers working on the single gene, on a single equation, leaving care others to attach their researches to more
global concerns, integrating an ensemble of separated knowledge (8). This obvious situation has for consequence a real
segregation of the different areas (and, in association, a certain linked implicit hierarchy, some being more equals than
others…), transforming sciences into “a true Babel Tower” where each, in its own domain, pose and treats its tiny
problems without caring too much about signification or consequences which these can have in other domains.
The
paradox is then put down. Disciplines are the main actors there, conservative shelters were created to shelter our divided
and specialized up knowledge.To keep one of the above-mentioned examples, musical training or the music theory such
as they teach them, reveals the same symptoms as grammar. What are the purposes followed by the practice of the
reading of notes and rhythmic reading? Allow the reading of a code, which is at the root of the majority of musical
practices in a context of western learned music! However the reading of note as such, is reserved for a pedagogic usage,
there is not together reading, which would constitute a “musical reference practice”. We can draw similar conclusions if we
wonder about dictations. This result means then that it is necessary to know how to use, to manipulate specialized
knowledge, confined by the scientific disciplines with the aim of a specific use, and it is at this moment that
interdisciplinary research intervenes, by the need, result of a specific intellectual constructs, a divergent way of thinking,
finally disruptive of a system organized by the scientists themselves.Numerous constraints exist and restrict the
development of innovations for a progress induced by Science, and supported by the Society. For instance, numerous
researchers suffer particularly from the “syndrome of the Kwai river” because formatted and fascinated by their own
production (and the “h” factor for evaluation of the researchers which goes with! (9), they may lose the context view in
which the research has to be used as well as the social world in which and for which they act. It therefore required there
to remember the ethical missions of science, particularly those of support of progress for the Society (and for its
sustainable survival) (10,11,12,13). In this context, the development of interdisciplinary research can be not only useful
in a society in doubt (exhaustion of mineral reservations, warming and climate change, pollution, epidemics, new risks,
improvement of health and well-being, etc) but also to act as “laboratory” to optimize links between disciplines and for the
accomplishment of social usefulness. This consideration leads to work on the critical interfaces of interdisciplinary actions
in research which correspond to organizational and cultural “knots” of coordination of researchers issued from different
scientific disciplines, key instants of incorporation of research, within which near relations, of friendship, of deep
motivation for the opening can be decisive for the orientation and sequence of interdisciplinary process. The scientific
mutation which they observe in numerous developed countries can be considered to be the result of the junction of
“organized research” and societal needs, rather close to the socioeconomic needs or demands… In these conditions, the
selection of the best “heads” in research is more and more performed on axes considered by scientific organizations as
hard priorities because strongly interesting the decision-makers; amazingly, the present situation is translated, for a very
broad part, by an accumulative and apparent random progress of knowledge without re-visit of the researcher
responsibility, hardly protected (Galileo's syndrome) and practically innocent of everything… Besides, the search of
immediacy leads to a pressure of optimization on the main principal variables of an innovation, those considered as
secondary being abandoned (remember that for basic researchers that risks are present in the second category, may be
in a third one…).
And, for a decision-maker distant from any inventive process, specialization (or the legibility of a
research unit) is translated in principle by:
1.
A better clarity of modes of production of knowledge;
2.
An optimize use of the specialized equipment and facilities;
2
P&R Scientific
3.
An amplified productivity (linked often with disciplinary scientific papers);
4.
A quicker training associated with the internal “culture” of specialized laboratory;
5.
A “mass production” of scientific papers (allowing confrontation/quantitative competition through figures between
States, etc.) with a risk of a research disconnected from the “true “needs;
6.
Finally, a separation between creativity, comprehension and production of knowledge.
These elements are close to what was searched by Taylor and applied by Ford in the “chain” working activities (14),
concept which made its proof broadly in the industries of production of consumer goods. The transformation of scientific
activity could be the result of a will of orientation of researches with the aim of certain goals (cf. supra). Then, what
Science win in power of transformation of the real, Science risks to lose its critical rigor and a part of its freedom of
thought disconnected from any ideology. By thinking to Albert Einstein (a scientific winner) and Giordano Bruno (a
scientific looser relatively to the social/religious acceptance of scientific novelties), P. Nouvel in the Banquet (15) writes:
“Glory to the one who provides the most distinguished face of the ideology of the instant, dead the one who would try to
put it in danger”. But, do the scientific researchers think that the research is an ordinary good of consumption? Must they
wait for existent creative investigations of other developed countries before knowledge production? It is possible to
understand the interest for decision-makers to use a model easily applicable to the policy of research and to apply it to
allow an annual budget founded on quantifiable indicators, often linked with mono-disciplines and “classical” scientific
peers review evaluation. And then, in facts, the ambivalence of the scientist appears more than never in its relations with
socio-economy, at the same time consenting and reticent, manipulated and manipulative… Hired in the complex of
“technical delight” (remember Robert Oppenheimer), it represents at least two roles, two forms of commitment, two
visions of the ethics of knowledge the swing of which between firm belief and responsibility concerns rather a
contradictory and complex loyalty…
The act of creation has a descriptive aspect and a building aspect (16). It is
necessary that it breaks structures of the mental organization to rank a new synthesis. It is therefore a way of break of
inertial intellectual processes, which let think that tomorrow will enter in a reassuring continuity with the past, what
justifies the principles of optimization which have just been mentioned (17). Stimulated interdisciplinary research,
important contribution in the development of the sciences of action must be analyzed as a complex system and thought
also with a fecundity of responsibilities and must allow thinking about cultural changes linked to a research of social
usefulness. It is not therefore a “cosmetic”, fair “presentable” argument to apparently satisfy the society, it is an overhaul
of the system and the frame of human interactions which must be envisaged; responsibility is worth for complex system,
to think of the incorporation of sciences into the social reality, it is to think of the social responsibility of research (and
reciprocally). It is definitely on such important foundations that the researchers will be considered as legitimist actors in
an interdisciplinary but also socially responsible research. And, as underlines it Hannah Arendt (18): “Finally, the
powerless truth is not also miserable as the unconcerned power of the truth?”. Should we able to re-examine, in a serene
way, the elements of responsibility and opening of the researchers in the present complex social system? At the same
time, the Science would cease being a philosophical humanistic adventure to be only a “cold” implement supporting only
the technological development? And, in spite of recurrent political discourses and significant financings, the present
innovation issued from the Science remains difficult (at least in France in view of quantitative results). We do not turn
intentionally (or not) as held by a thong around an inscribed fixed axis, in a more or less indelible way, in the culture of
the National research system? It is possible to settle the question of the defense of the existent paradigms by peers,
anxious with being able them and of their role as “security guard” of a certain conservative coherence? However, it is
possible to think that every researcher has, at various degrees, minimums of “still” aptitude in terms of creativity and
ingenious resolution of perceptible problems. But cultural, social, external pressures, career, funding, etc. can be
conjugated and heavily restrain this useful potential for an effective societal innovation. The development of
interdisciplinary research cannot probably be translated by a catalogue of “good” practices applied as a formal checklist. It
needs to experiment (location of difficulties and problems to be solved) and to complete organizational methodologies
(elaboration of action plans to be used as working hypotheses to get involved on spotted problems; towards learning
organizations,…). It must move away from the “explosive” accumulation of the encysting models used by disciplines, while
knowing how to work positively with and, contrariwise, try to promote a real exchange between disciplines, vector of
cultural change, to get involved in fecundation and crossed studies of public utility (7). In fact, with complexity, it is the
barriers between disciplines which have to disappear (without of course wanting therefore to abolish the disciplines of
deepening which have their own usefulness). At the same time, flexible organization and cooperation of points of view
have to be implemented allowing the bet compromise between disciplinary competences and of behaviors for action (19).
3
P&R Scientific
It is probably necessary to come back to reasoning of (20) Norbert Wiener (1992), that of the engineer whose look does
not stop in the understanding of only material facts, but stretches up to human facts (actual and perceptible for
tomorrow). But, simultaneously, must we accept that Science can work only by elaborating itself its own questions,
sheltered from emergency and inherent distortion of economic contingencies? How then to act in confident relation with
the Society? How to act together with researchers who do not know each other and who do not speak the same language?
II-2 - And, what about nano-drugs?
The concept of using nanotechnology in medical research and clinical practice holds great values and opens real
perspectives for effective innovations in medical sciences (21). A broad spectrum of technologies can be used individually
or in combination to make products and applications and to better understand science. One way of characterizing
nanotechnology is by utilizing the following 4 segments: tools, materials, devices & intelligent materials and machines.
The applications, which pertain to both medical and surgical diagnosis and therapeutic techniques, are as diverse as the
development of nano-particles for diagnostic and screening purposes, artificial receptors, DNA sequencing using nanopores, manufacture of unique drug delivery systems, gene therapy, and enablement of tissue engineering, single-virus
detection. Indeed, nanostructures, whether still under development or already in use, have the potential to play a critical
role in the future of medicine, as they can be carriers for drugs, genes, and imaging agents as well as targeting units.
The need for the development of a new framework, including regulations, procedures and mechanisms, adapted
specifically to govern research in nanotechnology, thus nano-medicine, is as salient in OECD Countries (22).
Heightened uncertainty regarding risks, fast-evolving science yielding complex and increasingly active materials, likelihood
of research on vulnerable participants including patients, and potential risks to others beyond the research participant are
identified as relevant confluent factors in support of the need for an exceptional oversight beyond the existing “Common
Rules” supervised by either local or federal institutions. Concern regarding potential risks does not involve only the
volunteers and candidates recruited to participate to clinical trials, but also workers employed in the manufacturing
industries, as well as, during the fundamental research stage, scientists conducting experiments in public or private
Laboratories. In this last domain, the researcher’s culture is of great importance (23).For nanotechnologies, not only used
in the medical domain, more and more enthralled debates develop on the toxic potential of certain nano-materials; active
groups offer interpretative schemes for industrial developments rejections or control (24). The carcinogenic effects of
asbestos and time delay to take into account them often act as negative memory. In this respect, the analogy asbestos –
nano-particles (case of carbon nano-tubes) is preeminent; it operates two modes of thought (25):
-
Responsibility of the scientists in relation to the public, with health polemics which it would be necessary to
anticipate;
-
The existence of a “Pandora box” not to be opened because of unknown health and ethical effects in the long term.
Even if nobody stress on the possible progress which will be allowed thanks to nanotechnologies, deep and converging
questionings manifest themselves; they seed confusion for medical innovations to treat, as an example, on certain
cancers (as it already occurs, to a lesser degree, for the use of certain more classical chemical anti-cancers drugs which
are dangerous for health services staff and for some extent for patients) (26).
To get legitimately involved in this ambivalent and new context, in the absence of toxicological robust knowledge, the
exploration of the precautionary principle (27) in this research area is normally necessary. To reach this initial target, it is
possible to lean on a charter of “Socially Responsible Research” published recently by certain Western Governments and
National Research Organizations (7,28). In this frame of thinking, responsibility is not a disposition which prevents science
from following its own search. It is necessary to favor the multiplicity of points of view, in other words to have an
approach more planed by science, more controversial, to play all in all new forms of communicational rationalism because
collective: learn to be able of constructing with other experts an object and a scientific problem by minimizing risks
pacifically to link up better Sciences, Technologies and Society (for today and, if possible, for the future).
In this respect, several domains of actions, in an interdisciplinary research linked on the development of new nano-drugs
can be proposed (21):
1.
Scientific (mono-disciplinary) approach: synthesis of new chemical compounds, relationship between chemistry and
therapeutics, chemical linkage of drugs to nano-particles, dose effect relationships, test on animals, etc.
2.
Responsible and/or ethical approach: On the basis of actual knowledge, it is possible to define the more safe
conditions of synthesis, without risk of exposure of the operators, of grafted nano-particles, their transport as well as
their clinical use by taking into account hospital best practices and elimination of wastes in satisfactory conditions
(29,30). By linking through an interdisciplinary approach specialists of complementary domains (engineering
4
P&R Scientific
sciences, chemists, biologists, medical scientists, in association with jurists and of referents in hygiene and safety), it
is possible in principle to define satisfactory technical criteria issued for “hard scientists”. However, if the insertion of
disciplines involved in the scientific mastering of correlation between nano-particle used as a drug and medical
treatment is satisfactory, it is possible to transfer the scientific knowledge into the technological world leading to
potential medical applications. But, the central question of social agreement on the positive use of suspicious
materials (coming from the nano-world…) is important and has to be solved. In situation of uncertainty, several
criteria should be estimated on realization and impact of researches: “social acceptance”, easiness of observation,
reducibility on one hand, irreversibility, strictness, plausibility on the other hand. These elements of oriented thinking
have, as much as possible, to be associated with all validated knowledge showing the “consistency” of hazard and risk
(potentially supported, credible, hypothetical, potential, etc.) (31). Leaving with a vision shared by the participants in
the research operation, the exchange with scientists of other domains must be envisaged in order to test the
robustness of the first one internal expertise. Approach should be then enlarged to “less informed” representatives
(general population) allowing a first approach of the possible influence of militant members playing with the social
control of decisions (militant associations for instance) to hire the development of researches in terms of application.
3.
Toxicological approach: One of the credible means to reduce uncertainty on hazards consists on the development of
research in toxicology in order to avoid the use of the precautionary principle, leading to come back in a more serene
way on foundations of mastering of risks with stabilized methodologies of risk prevention. This third axis coupled with
the two others constitutes therefore an essential element of facilitation of a social agreement on the use of new types
of nano-particles medical probes.
III-For what kind of results?
The responsible development for research on nano-drugs leaning on interdisciplinary approaches must allow:
1.
The
mutual association of
interests between specialists
issued from different
domains for
a “true”
interdisciplinarity; how to act in an efficient way?
2.
The definition of the disciplinary research axis to be hired, place and nature of the expertise to allow them;
3.
The learning for the researchers included in an interdisciplinary project for a better understanding of the
correlation between science and society; the place of toxicology in the process of social stabilization of new and
emerging techniques of medical treatment;
4.
The development of methodology(ies) of building of an expertise in field of hazards and badly identified risks;
how to reduce it by a better knowledge of hazards?
5.
Etc.
In this understanding on ignorance and uncertainty, it must be possible to evaluate the level of the present uncertainties
of scientific and technical knowledge, of approximations of knowledge, abuses of interpretation, limits of competence, to
measure their perception, at least the expanse of undecided questions, and interrogation marks.
IV-Methodology
It is offered to use the competences of the readers of “Prevention and Research” to help the author to improve, modify
this general vision about the entire question recalled above. On the basis of transmitted answers, it will be possible to
translate the synthesis of comments as proposal for decision-makers to locate interdisciplinarity as means of
redeployment of activities centered on new medical applications, domain where nano-drugs act here as focal object:
1.
How to define the frame of interdisciplinarity in the field? Is it of existent spontaneous origin linked with the
“hazardous” association of disciplines or contrariwise the result of top-down stimulation by getting involved via
forecasting approaches? How to produce added values during the development of new (nano) drugs?
2.
How to define again the place of interdisciplinarity for innovation in nano-medicine? For what type of innovation? How
to create favorable conditions to the blossoming of new ideas and to their support for innovation and creativity in
domain? How to settle “good questions”? What is to be done? How in processes of innovation associate freedom,
initiatives and social and economic responsibilities? At the bottom, how to construct a collective intelligence leaning
on interdisciplinary foundations?
3.
How to go out of the disciplinary work division which can separate the scientific idea of its medical, social (societal)
links? How to elaborate an interdisciplinary expertise for responsible action, mastering risks? What are the
deontological foundations which support such action? How to links up Science-Innovation and Society?
4.
How to assess/evaluate a scientific activity opened on / for the Society? How to define the interdisciplinary excellence
in nano-medicine? How to evaluate researchers in case of a fiasco linked to the catch of risk? How to go out of a
5
P&R Scientific
“vicious circle” of the evaluation of an innovation by using performance criteria linked to the pre-existing disciplinary
system (32)? How to construct a methodology to help the researchers to ameliorate their own practices with the aim
of the social usefulness of the projects?
5.
How to include long temporalities in scientific activities (problem of the short term contracts in relation with the time
scale for success and excellence in research)? How to satisfy the social needs through the incorporation of different
disciplinary knowledge? How to manage irreversibility led by “strategically“choices coming from the top management?
6.
How to develop a collective long-term forecasting in nano-medicine and a more individual activity in a context of
economic competitiveness and of competition between researchers? How to pilot actions open to the social needs?
How to insert principles of organizational flexibility to reach objectives faster?
7.
How to go out of the travesty of ideas induced by a possible control from certain stakeholders and/or decisionmakers? How take out of (or know how to work with) protection on behalf of the “predominant”? How to raise central
inflexibility by risk of disturbance of the status quo? How to go beyond the factors which stifle innovation? What do
we hear by values?
8.
How to optimize the dialogue of the “scientists” with the real, with all the concerned different bodies of the Society?
How to generate trust in the activities of interdisciplinary research and innovation, while this concept starts with a
reduction of medical and/or social complexity? How we can train a part of the research body for a better social
responsibility (of the researcher in his entire environment)?
9.
How to develop partnerships with other researchers and with the socioeconomic system? How to federate all
energies? How to promote a shared research? How to support determination, firm belief and enthusiasm of the
researchers in sciences of action acting for the service of the Society? How to “fabricate” interest for what we do?
10. How to explore “intelligently” the precautionary principle (27) on researches with potential and uncertain risks? How
to manage through heuristic approaches a satisfactory prevention of possible risks for the operators and the citizens
in the field of nanotechnologies and, in association, in nano-medicine? How to be sure of the respect of the present
regulation? How to go out of “conspiracy” ideologies issued from militant groups? Can the biomedical research linked
to topic be completely neutral?
V- Provisory closure: For an operational proposal
In France, the author try to follow this search of information to deepen the present subject. He expects comments and
suggestions on behalf of the active readers of “Prevention and Research” before for 31-01-2012 (to be transmitted
under free form to [email protected]). On this deepening basis, the filled reservoir of knowledge will be able to
serve for making useful proposals in form of scenarios (using SWOT analysis for instance).
In the complex domain of nano-biosciences or of nano-medicine, it will be necessary to think on how crossing ideas, how
to link the scientific and medical actors positively to such project, how to bring added values in interdisciplinary research
proposal in the field, how to build a network of experts to confer a theoretical substrate on approaches offered by
experiments, counter-proposals to adjust reasoning and methodologies in disputations and in resistance of scientists
(corresponding to the wish of a real common understanding). Of course, this remark is related to the essential question in
interdisciplinarity to positively gather heterogeneous talents between science, medicine and society, between innovation
and prevention of risks, and to the development of existent expertise issued from different domains in order to try to
resolve together problems linked to forms of “imprisonment” of Science. It is therefore necessary to write together a
“history” shared by those who make it and by those for whom it is intended. It is on this foundation that it will be possible
to construct honestly forms of collective imagination allowing action (and valid proposals for some time). Conclusions of
this work of synthesis will be the object of a publication in “Prevention and Research” in 2012.
Reference
1 Benkiran R. La complexité ; vertiges et promesses. Le Pommier Ed, Paris, 2000.
2 Weingart P. Interdisciplinarity: The paradoxical discourse. Weingart and P. Practising Interdisciplinarity Ed. Toronto
Univ. Press , Toronto – Canada, 2000.
3 Alvarez-Pereyre F. L'exigence interdisciplinaire. MSH Ed. Paris- France, 2003.
4 Camacho-Hübner E. De l'interdisciplinarité comme paradigme de recherche. Espaces temps.net , 2007.
http://espacetemps.net/document3842.html
5 Gramaccia G. Démocratie participative et communication territoriale. Vers la micro – représentativité. L’Harmattan Ed.Paris, 2008.
6
P&R Scientific
6 Vinck D. Pratiques de l'interdisciplinarité. Presses Universitaires de Grenoble, Ed. Grenoble- France, 2000.
7 André J.C. Vers le développement d’une recherche durable…. Ou vers la (ré) humanisation des sciences des artefacts.
Env. Risques et Santé. 2008 ; 7:47-54.
8 Weber M. le savant et le politique. 1919. http://classiques.uqac.ca/classiques/Weber/savant_politique/Le_savant.doc
9 Hirsch J.E. An index to quantify an individual's scientific research output. Proc. Natl. Acad. Sci. 2005; 102:1656916572.
10 Nowotny H. et al. la Science. Belin Ed. – Paris France, 2003 .
11 Nowotny H. et al. The Public Nature of Science under Assault: Politics, Markets, Science and the Law. Springer Ed. Heidelberg –Allemagne et New-York – USA, 2005 .
12 Nowotny H. Interdisciplinarity research – why does it matter? NEST conference, 21 sept, 2005.
http://www.nestconference.com/pdf/nowotny.pdf
13 EU. The European Charter for Researchers. 2005.
14 Taylor F. W. The principles of scientific management. Harper & Brothers Ed, New-York-USA, 1911.
15 Nouvel P. De l’idéologie scientifique. Le Banquet, revue du CERAP, Paris – France, 1997 ;125-132.
16 Koestler A. The Act of Creation. Pan Books Ed, London – UK , 1964.
17 Helie S, Sun R. Incubation, insight, and creative problem solving: A unified theory and a connectionist model.
Psychological Review 2010; 117: 994-1024.
18 Arendt H. The Human condition. Chicago Univ. Press – USA, 1958.
19 Le Moigne J.L. Agir-penser en complexité ; le discours de la méthode de notre temps. 2011. Leroi-Gourhan A. Milieux
et techniques. Michel A Ed, Paris, 1973. http://www.intelligence-complexite.org/fileadmin/docs/1107-dossier27-2.pdf
20 Wiener N, et al. L'utopie de la communication- L’émergence de l’homme sans intérieur. La Découverte Ed. Paris –
France, 1992.
21 Manigat R. Preventing occupational, environmental and more broadly societal health risks emerging in the
development of nano-drugs - What should prevail? Intech Ed. under press, 2011-2012.
22 EU. Responsible nano code. 2008. http://www.responsiblenanocode.org/
23 Couleaud P, Faure M, Verhille M, et al. From public to occupational health: towards an inverse push-pull paradigm in
nanotechnologies innovation. G. Ital.
Med.
Lav. Erg. 2010; 32: 400-402.
24 Ponce Del Castillo A. M. Nanogouvernance : Comment l’Union Européenne doit-elle mettre en place la traçabilité des
nanomatériaux. ETUI Policy Brief 2011 ; 2 :1-5. http://www.etui.org/
25 CNDP, French National Commission for Public Debate. Débat public nanotechnologies. 2010.
http://www.debatpublic-
nano.org.
26 Ndaw S. Exposition professionnelle aux médicaments cytotoxiques en secteur hospitalier - Biométrologie et
contamination environnementale. 31th National Congress of occupational medicine – Toulouse – France, 2010 .
27 UNESCO . Precautionary Principle. 2005. http://unesdoc.unesco.org/images/0013/001395/139578e.pdf
28 French UK Embassy. Responsible innovation Report. 2011. http://www.ambascience.co.uk
29 Bechet D, Couleaud P, Frochot C. Nanoparticles as vehicles for delivery of photodynamic therapy agents. Trends
Biotechnol. 2008; 26,: 612-621.
30 Brevet D,
Gary-Bobo M, Raehm L. Mannose-targeted mesoporous silica nanoparticles for photodynamic therapy.
Chem. Commun. 2009;12: 1475-1477.
31 Chevassus au Louis B. L’analyse des risques : l’expert, le décideur et le citoyen. Editions Quae, Collection “Sciences en
question“ Paris – France, 2007.
32 Joulian F, de Cheveigné S, Le Marec J. Evaluer les pratiques interdisciplinaires. Natures, Sciences et Sociétés, 2005 ;
13, :284-290.
Corresponding Author: Jean-Claude Andrè
LRGP-CNRS, 1, rue Grandville – 54000 Nancy – France
email: [email protected]
7
P&R Scientific
RISCHIO BIOLOGICO LEGIONELLA PRESSO STRUTTURE
SANITARIE DI ROMA E PROVINCIA
BIOHAZARD LEGIONELLA IN HEALTH CARE FACILITIES IN ROME AND PROVINCE
Giorgi DA 1, Palmieri S 1, Renzi G 1, Massoni F 2, Ricci S
2
1
ARPALazio – Servizio Ambiente e Salute – Sezione provinciale di Roma
2
Dipartimento di Scienze Anatomiche, Istologiche, Medico Legali e dell’Apparato Locomotore –
Università degli studi “Sapienza” di Roma
1
ARPALazio – Environment and Health Service – Provincial section of Rome
2
Department of Anatomical Sciences, Histological, Forensic and of the Locomotor System –
“Sapienza” University of Rome
Citation: Giorgi DA, Palmieri S, Renzi G, et al. Rischio biologico Legionella presso strutture sanitarie di Roma e
provincia. Prevent Res 2011; 1 (1): 08-15
Parole chiave: Legionella, Pneumonia, Biohazard
Key words: Legionella, Pneumonia, Biohazard
Abstract
Introduzione: La legionella è un agente eziologico di polmonite batterica, un fattore di rischio di polmonite per i
pazienti, ma anche per lavoratori.
Obiettivi: Gli Autori procedono ad una valutazione del rischio infezione da legionella mediante una descrizione
epidemiologica dei casi di positività derivanti dall’attività di prevenzione e controllo operata in provincia di Roma
dall’Agenzia Regionale Protezione Ambiente Lazio (ARPALazio).
Metodi: Sono stati analizzati 4354 campioni, così ripartiti: 27,65% nel 2007, 35% nel 2008 e 37,35% nel 2009.
L’attività di prelievo è stata uniformemente distribuita, tra 25% e 30%, nei singoli trimestri.
Risultati: Nel 2007 i campioni non conformi risultano il 10,60%, nel 2008 diventano il 24,57% ed infine il 26,63% nel
2009. Il livello di contaminazione risulta basso nel 13%, medio nell’11% ed alto nel 3% del totale.
Conclusioni: La contaminazione da Legionella non presenta una specificità di distribuzione temporale nei quattro
trimestri considerati per ogni anno o una specificità di distribuzione per quanto riguarda il tipo di struttura sanitaria, ed il
livello di contaminazione nel complesso si assesta su valori bassi-medi.
8
P&R Scientific
Abstract
Background: Legionella is a causative agent of bacterial pneumonia, a risk factor for pneumonia for patients, but also
for workers.
Objectives: The authors describe the cases arising from the activities of prevention and control operating within the
province of Rome by the Lazio Regional Environmental Protection Agency (ARPALazio).
Methods: 4354 samples were analyzed, as follows: 27.65% in 2007, 35% in 2008 and 37.35% in 2009. The activity
sampling was evenly distributed between 25% and 30% in each quarter of year.
Results: In 2007 the non-compliant samples are 10.60%, to become 24.57% in 2008 and finally 26.63% in 2009. The
level of contamination is low in 13%, 11% medium and high in 3% of the total.
Conclusions: The Legionella contamination does not present a specific distribution in the four quarters of time
considered for each year or a specific distribution for the type of health facility, and the level of contamination settles on
medium-low values.
Introduzione
La Legionella è un agente eziologico di polmonite batterica. Un bacillo Gram-negativo, lungo fino a 20 μm, aerobio,
asporigeno, mobile in quanto munito di uno o più flagelli. Comprende 53 specie ripartite in oltre 70 sierogruppi. La
specie pneumophila comprende 16 sierogruppi (1) e rientra nell’eziologia di oltre il 90% dei casi di polmonite batterica
da legionella, più precisamente il sierogruppo 1 di oltre l'84%. Segue il 3,9% di polmoniti da L. longbeachae ed il 2,4%
da L. bozemanae.
Il 2,2% si devono a L. micdadei, L. dumoffii, L. feeleii, L. wadsworthii e L. anisa (2). Uno studio ha evidenziato che L.
pneumophila sierogruppo 1 si ritrova nell'ambiente in misura ridotta (dal 18 al 45%), mentre gli altri sierogruppi sono
più frequenti (3). I casi notificati rappresentano il 2-15% di tutte le polmoniti comunitarie ed il 15-20% di quelle
nosocomiali, con una letalità rispettivamente del 20 e 40% (4, 5).
In Italia, fino al 1998 erano notificati non più di 100 casi l'anno, poi le segnalazioni sono aumentate con 325 casi nel
2001, 639 nel 2002 e 600 nel 2003 (6, 7).
La via di trasmissione è quella aerea, ovvero goccioline di aerosol contenenti i batteri generate dall’apertura di rubinetti
come la doccia o lavandino, oltre che da vasche idromassaggio e piscine, bagni turchi e saune, etc.. Soprattutto in
ambiente sanitario l’infezione da Legionella è particolarmente sentita.
Tra i fattori predisponenti la malattia, infatti, risultano l’età avanzata, il fumo di sigaretta, la presenza di malattie
croniche, l’immunodeficienza, ma anche il grado di intensità dell’esposizione, rappresentato dalla quantità di legionelle
presenti e dal tempo di esposizione.
In questo senso gli operatori sanitari e figure operanti in strutture sanitarie, ad esempio gli addetti ai servizi di igiene e
manutenzione, possono costituire una categoria a rischio. Un’indagine sulla prevalenza di anticorpi anti-Legionella in
soggetti che lavorano in strutture contaminate ha visto una positività sierologica, riguardante i sierogruppi 7-14,
maggiormente presenti nell'acqua calda delle strutture indagate (8). L’Istituto Superiore di Sanità (ISS) il 5 maggio
2000 ha elaborato le Linee guida per la prevenzione ed il controllo della legionellosi (G.U. n. 103 del 5 maggio 2000).
Tale strumento costituisce un valido punto di riferimento in materia di prevenzione e sorveglianza per ospedali e case di
cura. Sono seguite, nel 2005, le Linee guida recanti indicazioni ai laboratori con attività di diagnosi microbiologica e
controllo ambientale della legionellosi (GU n. 29 del 5 febbraio 2005).
L’obiettivo dello studio è quello di presentare i risultati delle analisi effettuate dall’Agenzia Regionale Protezione
Ambiente Lazio (ARPALazio) su prelievi eseguiti presso strutture sanitarie dislocate sul territorio di Roma e provincia nel
corso del triennio 2007-2009 e, quindi, una valutazione del Rischio Relativo rispetto alle altre strutture interessate
dall’attività di prevenzione e controllo dell’ARPA.
9
P&R Scientific
Materiali e metodi
Nelle strutture sanitarie dislocate sul territorio di Roma e provincia sono stati raccolti 283 campioni, rappresentati da
acqua e tamponi su superficie, nel 2007, nel 2008 sono stati 297 e 214 nel 2009. Le strutture sanitarie coinvolte sono
state suddivise per tipo: 113 prelievi presso case di cura, 634 presso ospedali, 11 negli studi odontoiatrici, 10 negli studi
medici e 26 nei poliambulatori medici.
Ed i campioni in base ai punti di prelievo: docce e lavandini. Terreni di coltura impiegati per la semina sono GVPC-agar,
BCYE-agar e CYE agar base in confezione originale che, forniti di certificato di qualità, rispondono alle esigenze di qualità
più rigorose.
Infatti, da parte della ditta produttrice sono sottoposti a controlli qualitativi su ciascun lotto. Mentre i reagenti per la
preparazione del campione sono una soluzione di Ringer o compresse di Ringer, per il trattamento di decontaminazione
è stato usato tampone acido HCl/KCl a pH 2,2, ed infine per l’identificazione definitiva di Legionella pneumophila (siero
gruppi 1, 2-14) e species il Legionella Latex Test ed il Slidex Legionella Kit.
I terreni ed i reagenti sono conservati, al riparo dalla luce, in frigorifero a T°C=4±4 o nell’armadio a temperatura
ambiente secondo le relative caratteristiche, fino alla data di scadenza indicata sull’etichetta (1 settimana nel caso in cui
il terreno CYE agar base dovesse essere preparato in laboratorio).
Il procedimento utilizzato è il seguente.
Dal campione originale si prelevano 10 ml che vengono messi in un contenitore sterile con tappo a vite. Il resto del
campione viene sottoposto a filtrazione per mezzo di rampe filtranti, con l’uso di imbuti e bicchieri sterilizzati mediante
flambatura prima e dopo il loro utilizzo.
Essendo le rampe di filtrazione costituite da più stazioni filtranti collegate in serie e munite di rubinetto autonomo, è
possibile la filtrazione simultanea di più campioni d’acqua. Dopo la filtrazione, la membrana (o le membrane nel caso
fosse necessario utilizzarne più di una in successione), viene posta nel contenitore sterile contenente i 10 ml prelevati
all’inizio e sottoposta all’azione dell’agitatore (15 minuti a 150 rpm: formazione del concentrato) per consentire il
distacco dei batteri catturati e la loro risospensione.
Il concentrato, prima di essere conservato al buio, in frigorifero a T°C=6±2 per non oltre 14 giorni, viene analizzato
come tal quale (si tratta di una semina diretta su terreno di coltura selettivo che non prevede alcun trattamento
particolare del concentrato) e dopo trattamento a caldo (si tratta di una semina su terreno di coltura selettivo dopo il
trattamento di decontaminazione che prevede il trasferimento di 1,5 ml in contenitore sterile, in bagnomaria a T°C
=50±1, per 30±2 min.). In caso d’intervento di campioni che provengono da circuiti di acqua calda, il trattamento a
caldo viene sostituito dal trattamento di decontaminazione con acido. Tale trattamento prevede: il trasferimento di 4ml
in provetta sterile, la centrifuga a 3000 giri per un tempo di 30±1 min. o 6000 giri per un tempo di 10±1 min.,
l’estrazione di 3 ml di sovranatante, l’aggiunta di 3 ml di tampone acido HCl/KCl a ph 2,2, l’agitazione delicata e la posa
a temperatura ambiente per 5±0.5 min., la semina su terreno di coltura selettivo. La semina del campione d’acqua
concentrato viene effettuata su un terreno colturale di tipo selettivo.
Si tratta del GVPC-agar che, per la presenza di L-cisteina ed alcuni antibiotici, favorisce la crescita della Legionella e
l’allontanamento della flora batterica concomitante. La quantità d’inoculo da seminare è pari a 0,1 ml. L’incubazione
delle piastre avviene in aerobiosi, in termostato a T°C =37±1, con 2,5% di CO2 e in ambiente umido per un tempo
massimo di 10 giorni.
La crescita batterica viene monitorata dal personale tecnico ogni 2/3 giorni. In caso di piastre molto contaminate
(crescita batterica >200), che ostacolerebbero la corretta identificazione ed enumerazione delle colonie, si procede con
diluizioni in base 10 del concentrato, precedentemente conservato, mediante acqua distillata sterile o soluzione di Ringer
ed alla semina delle relative piastre di GVPC-agar.
Dalla lettura delle piastre, le colonie che già dopo 2-3 giorni di incubazione crescono su terreno selettivo GVPC-agar e si
presentano di piccole dimensioni, di colore bianco-grigio, leggermente convesse e con margini regolari, passano prima
attraverso un’identificazione presuntiva e poi attraverso un’identificazione definitiva.
Le colonie cresciute su terreno selettivo GVPC-agar, con le caratteristiche sopra descritte, sono considerate Legionelle
spp “SOSPETTE”(S) e vengono isolate su due terreni colturali di tipo non selettivo quali il BCYE-agar (con L-cisteina) che
sviluppa Legionella, e CYE agar base (o BCYE-Cys: privo di L-cisteina) sul quale, invece, le colonie di Legionella non
crescono per assenza del supplemento di crescita.
10
P&R Scientific
La semina avviene nel seguente modo: dalle piastre di terreno GVPC-agar più rappresentative per la presenza di
maggiore crescita di colonie sospette, si prelevano 5 colonie e si trapiantano su entrambi i terreni BCYE-agar e CYE Agar
Base (il numero di colonie da testare si riduce a 2 per tipo nel momento in cui sono presenti differenti tipi di colonie
sospette). Dopo 2 giorni (24h+24h) di incubazione a T°C =37±1, se si osserva una crescita differenziale con sviluppo
delle colonie esclusivamente su terreno contenente cisteina, allora le colonie che inizialmente erano delle sospette
Legionelle vengono identificate come “PRESUNTE”(P) (Legionella spp) Le colonie riconosciute come presunte Legionelle e
con un diametro di almeno 1 mm sono sottoposte al test di agglutinazione al lattice.
Si tratta di un metodo di screening semplice e rapido che sfrutta particelle di lattice blu sensibilizzate con anticorpi
specifici di coniglio che reagiscono con gli antigeni presenti sulla parete cellulare di Legionella pneumophila e Legionella
species, formando un evidente precipitato.
Le particelle che agglutinano in non più di 1 minuto confermano la presenza di Legionella pneumophila, sierogruppi 1 o
2-14, o Legionella species (Legionelle “DEFINITIVE”(D)).
Risultati
Nel 2007 i campioni non conformi risultano il 10,60%, nel 2008 diventano il 24,57% ed infine il 26,63% nel 2009.
Figura 1
Il livello di contaminazione risulta basso (tra 100 ufc e 1000 ufc) nel 13%, medio (tra 1000 ufc e10000 ufc) nell’11% ed
alto(>10000 ufc) nel 3% del totale.
11
P&R Scientific
Figura 2
Nel 2007 l’indagine è stata effettuata esclusivamente su strutture ospedaliere, di cui l’89% dei prelievi è risultato
conforme, mentre l’11% non conforme. Nel 2008 i non conformi sono diventati il 31%, che si accompagnano ad un 5%
nelle case di cura esaminate. I prelievi effettuati presso odontoiatri e poliambulatori sono risultati conformi nella totalità.
Infine nel 2009 i prelievi non conformi hanno interessato il 23% dei prelievi ospedalieri, il 30% delle case di cura, ed
infine l’80% dei poliambulatori.
Figura 3
12
P&R Scientific
I punti di prelievo sui quali si è concentrata l’attenzione degli Autori sono nel particolare docce e lavandini.
Nel 2007 un livello di contaminazione basso ha riguardato il 7% dei prelievi da lavandino ed il 10% da doccia; un livello
medio il 4% da lavandino ed un 7% da doccia; alto solo il 3% da lavandino.
Figura 4
Nel 2008 un livello basso di contaminazione ha riguardato il 16% dei lavandini campionati ed il 13% delle docce. I livelli
medi di contaminazione il 13% dei lavandini e l’11% delle docce. Alto l’1% dei lavandini ed il 2% delle docce.
Figura 5
Nel 2009 il 13% dei prelievi da lavandino sono risultati con un basso livello di contaminazione, il 17% delle docce
esaminate. Il 13% dei lavandini un medio livello di contaminazione, il 7% delle docce. Il 2% dei lavandini ha presentato
un alto livello di contaminazione, il 7% delle docce.
Figura 6
Il Rischio Relativo di Legionella presso strutture sanitarie rispetto a strutture ricettive (camping, hotel, residenza
religiosa), abitazioni private, ed altre strutture (aereo, cantiere, carcere, caserma, circolo sportivo, nave, residenza
13
P&R Scientific
studenti, ristorazione, scuola, spogliatoio, ufficio, etc.) risulta essere 2.148 (intervallo di confidenza al 95%: 1.748 2.639).
Conclusioni
La contaminazione da Legionella non presenta una specificità di distribuzione temporale nei quattro trimestri considerati
per ogni anno e l’incremento del numero di campioni non conformi va interpretato considerando il contestuale aumento
dell’attività di campionamento nel biennio 2008-2009 rispetto al 2007. Il livello di contaminazione nel complesso si
assesta su valori bassi-medi.
Anche il coinvolgimento sostanziale delle strutture ospedaliere e case di cura, quindi di ambienti contraddistinti da un
contesto di persone più numerose rispetto a studi odontoiatrici o poliambulatori, risente di una attività di controllo e
campionamento decisamente più significativi.
Stessa conclusione si può trarre per quanto concerne il punto di prelievo, anche se, a riguardo, è presumibile una
maggiore contaminazione dei lavandini rispetto alle docce, anche in funzione del diverso indice di utilizzazione di questi,
probabilmente maggiore nell’uno, il lavandino, piuttosto che nell’altro, la doccia.
Tuttavia questi risultati, che comunque rilevano una positività del campionamento e delle successive analisi, hanno
indotto gli Autori ad approfondire lo studio valutando un rischio relativo di positività alla Legionella.
E da questa valutazione emerge una associazione, statisticamente significativa, tra Legionella e strutture sanitarie,
rispetto ad altri tipi di sedi quali strutture ricettive ed abitazioni private.
Le motivazioni vanno ricercate nelle circostanze, quali la carica batterica, il periodo di esposizione e lo stato di salute
degli utenti esposti, che rendono tale habitat un contesto favorevole alla contaminazione, giustificando in questo modo
le sollecitazioni, rivolte agli operatori sanitari, di ricorrere a tutte le necessarie misure di sorveglianza nei riguardi del
rischio biologico da Legionella.
In questo senso, le Linee guida elaborate dall’Istituto Superiore di Sanità il 5 maggio 2000 costituiscono un efficace e
valido strumento a disposizione dei soggetti coinvolti.
Innanzitutto si parla di casa presunto nell’eventualità di segni di polmonite rilevabili all’esame clinico e/o esame
radiologico, con almeno uno di questi parametri aggiuntivi: incremento di almeno 4 volte o superiore a 1:256 del titolo
anticorpale specifico per il sierogruppo 1 ovvero immunofluorescenza diretta condotta su materiale patologico positiva.
Si definisce, invece, caso accertato qualora all’esame clinico e/o radiologico positivi fanno seguito o l’isolamento del
microrganismo da materiale organico quale secreto respiratorio, broncolavaggio, campione tissutale polmonare,
essudato pleurico, essudato pericardico, sangue, etc., o l’immunofluorescenza diretta o microagglutinazione positive per
un incremento di almeno 4 volte del titolo anticorpale specifico e condotte su due sieri prelevati a distanza di almeno 10
giorni oppure la positività delle urine per l’antigene specifico.
La legionellosi è soggetta a notifica obbligatoria in classe II in base al D.M. 15/12/90 da parte del medico segnalatore,
entro 48 ore, al Servizio di Igiene e Sanità Pubblica (SISP) dell’Azienda USSL. Successivamente il SISP provvederà, una
volta validata la diagnosi, a trasmettere il modello 15 alla Regione che informerà della notifica individuale il Ministero
della Sanità ed all'ISTAT.
Nell’eventualità di focolai epidemici, ovvero qualora fossero accertati almeno due casi ricollegabili ad una stessa
esposizione nell’arco di sei mesi, si dovrà procedere alla notifica obbligatoria in classe IV. Il medico segnalatore entro 24
ore dovrà notificare il caso al SISP della ASL di diagnosi, che trasmetterà il modello 15 alla Regione, al Ministero della
Sanità, all'Istituto Superiore di Sanità ed all'ISTAT.
Inoltre il medico che accerta la diagnosi dovrà provvedere alla compilazione della scheda di sorveglianza (Circolare
400.2/9/5708 del 29/12/93) da inviare al SISP dell’Azienda USSL da parte della Direzione Sanitaria dell’Ospedale ed
all’I.S.S. da parte o della Direzione Sanitaria dell’Ospedale o dal SISP dell’Azienda USSL di competenza. Il SISP
provvederà alla trasmissione mensile delle schede alla Regione.
I ceppi isolati e sospetti di Legionella dovranno essere inviati per la conferma al Laboratorio di Batteriologia e Micologia
Medica dell’ISS, che è il laboratorio nazionale di riferimento per la legionellosi.
14
P&R Scientific
Bibliografia
1. Diederen BMW. Legionella spp and Legionnaires' disease. J. Infect 2008;56(1):1–12.
2. Yu VL, Plouffe J F, Pastoris M C et al. Distribution of Legionella species and serogroups isolated by culture in patients
with sporadic community- acquired legionellosis: an international collaborative survey. J. Infect Dis. 2002;186(1):127128.
3. Borella P, Mazzini E, Bargellini A, et al. Sieropositività per Legionella spp. in gruppi di popolazione a diverso rischio
espositivo. Journal of Preventive Medicine and Hygiene 2004; 45(4): 167.
4. Fields BS, Benson RF, Besser RE et al. Legionella and Legionnaires’ Disease: 25 year of Investigation. Clin. Microbiol.
Rev. 2002; 15(3):506-26.
5. Roig J, Sabria M, Pedro-Botet ML et al. Legionella spp.: community acquired and nosocomial infections. Curr. Opin.
Infect. Dis. 2003; 16(2):145-51.
6. Rota MC, Pastoris MC, Ricci ML, et al. Rapporto annuale sulla legionellosi in Italia nel 2002. Notiziario ISS 2003;
16(12):3-8.
7. Rota MC, Ricci ML, Caporali MG et al. La legionellosi in Italia nel 2003. Rapporto annuale. Notiziario ISS 2004;
17(10):8-13.
8. Borella P, Montagna MT, Stampi S et al. Indagine multicentrica sui fattori di rischio correlati alla presenza di
Legionella spp. nell’acqua di
strutture pubbliche e private. Journal of Preventive Medicine and Hygiene 2004; 45
(4):328.
Autore di riferimento: Serafino Ricci
"Sapienza", Università di Roma, Medicina Sociale, Dipartimento di Scienze Anatomiche, Istologiche, Medico Legali e
dell’Apparato Locomotore
Viale Regina Elena, 336 - 00161 Roma
tel. 0649912547
e-mail: [email protected]
Corresponding Author: Serafino Ricci
"Sapienza", University of Rome, Social Medicine
Viale Regina Elena, 336 - 00161 Rome – Italy
tel. +39.0649912547
15
P&R Scientific
IL VALORE GIURIDICO E MEDICO-LEGALE DELLE LINEE
GUIDA
THE LEGAL AND FORENSIC VALUE OF THE GUIDELINES
Angioni C 1, Montanari Vergallo G
1
1
1
, Catarinozzi I
1
, Iovenitti L 1, Frati P 1
“Sapienza” Università di Roma, Scuola di Specializzazione in Medicina Legale
“Sapienza” University of Rome, School of Specialization in Forensic Medicine
Citation: Angioni C , Montanari Vergallo G, Catarinozzi I, et al. Il valore giuridico e medico-legale delle linee
guida. Prevent Res 2011; 1 (1): 16-21
Parole chiave: diritto alla salute, responsabilità medica, trattamento sanitario
Key words: right to health care, medical responsibility, medical treatment
Abstract
Scopo della ricerca: In questo studio gli Autori esaminano l’attuale quadro giurisprudenziale relativo al valore giuridico e
medico-legale delle linee guida con lo scopo di illustrare quali sono le norme che regolano il loro utilizzo durante la diagnosi e
la terapia.
Materiali e metodi: Gli Autori analizzano il caso deciso dalla sentenza n. 8254/2011 della Suprema Corte tenendo conto
delle sue peculiarità cliniche in relazione al principio giuridico affermato. Viene valutata la compatibilità di tale sentenza con i
principi sanciti in giurisprudenza e con le regole deontologiche.
Risultati e conclusioni: Il principio affermato dalla Suprema Corte nella sentenza esaminata, secondo cui le linee guida
ispirate da esigenze di natura economica non possono influenzare le scelte terapeutiche del medico, si scontra con la
giurisprudenza della Corte costituzionale, la quale sottolinea la necessità di considerare in ambito sanitario sia l’aspetto legale
sia quello riguardante le risorse finanziarie, ai fini di una completa protezione del paziente.
Inoltre, la decisione in esame introduce una norma che non permette ai medici di esercitare pienamente la propria libertà
professionale nell’interesse dei pazienti senza correre il rischio di ricevere sanzioni amministrative o di dover rispondere a
responsabilità di ordine legale.
Abstract
Scope: The Authors examine the current jurisprudential context relative to the juridical and forensic value of the guidelines
in order to illustrate what are the rules that regulate their use in diagnosis and therapy.
16
P&R Scientific
Materials and Methods: The Authors analyze the case decided by the sentence no 8254/2011 of Supreme Court of Appeals
considering its clinical characteristics in relation to the legal principle affirmed. It is evaluated the compatibility of this
sentence with the principles enshrined in law and the rules of professional conduct.
Results and conclusions: The Authors emphasize this decision clashes with the Constitutional Court that judges the law to
health has a limited protection, because also financial resources have to be taken into account. Moreover, this decision
introduces a principle that doesn’t permit physicians to fully exercise their professional independence in the interest of their
patients, without running the risk of administrative sanctions or legal liability.
Introduzione
La salute è considerata un diritto inalienabile dell’individuo, appartenente all’uomo in quanto tale, dal momento che
deriva dall’affermazione del più universale diritto alla vita e all’integrità fisica di cui rappresenta una delle declinazioni
principali. A partire, infatti, dalle direttive fondamentali stabilite dalla Conferenza Internazionale della Sanità (New York,
1946) e fatte proprie dall’Organizzazione Mondiale della Sanità (OMS), la salute è definita come: “uno stato di completo
benessere fisico, mentale, sociale e non consiste soltanto nell’assenza di malattie o infermità. Il possesso del migliore
stato di sanità che si possa raggiungere costituisce uno dei diritti fondamentali di ciascun essere umano (1).
In linea con la dichiarazione dell’OMS, le principali Convenzioni Internazionali sanciscono il diritto alla salute come uno
dei diritti fondamentali dell’individuo e delle collettività e la sua tutela come uno dei doveri degli Stati. Ai sensi della
Dichiarazione Universale dei Diritti Umani: “ogni individuo ha diritto ad un tenore di vita sufficiente a garantire la salute
e il benessere proprio e della sua famiglia” (2).
Nell’articolo 32 della Costituzione si legge: “la Repubblica tutela la salute come fondamentale diritto dell’individuo e
interesse della collettività, e garantisce cure gratuite agli indigenti. Nessuno può essere obbligato a un determinato
trattamento sanitario se non per disposizione di legge. La legge non può in nessun caso violare i limiti imposti dal
rispetto della persona umana” (3).
Tale norma costituzionale si presenta con un duplice profilo di genericità e di relativa generalità, garantendo il diritto alla
salute, sia nel suo profilo individuale che nella dimensione collettiva, e consentendo la fruizione dei diritti sociali secondo
criteri e valori di omogeneità e uniformità (4). Tale diritto si concretizza in un complesso di regole - che stabiliscono il
finanziamento e l’organizzazione delle prestazioni sanitarie, imponendo un uso corretto dei fondi, allo scopo di sfruttare
efficientemente le risorse (5) - e di funzioni, strutture, servizi e attività - utili alla promozione, al mantenimento e al
recupero della salute fisica e psichica di tutta la popolazione, senza distinzione e secondo modalità che assicurino
l’eguaglianza dei cittadini (6).
Tuttavia il Sistema Sanitario Nazionale non ha saputo far fronte all’aumento vertiginoso dei costi per carenza strategica
nella utilizzazione delle risorse e per inefficienza e sprechi risultanti da una gestione non ottimale e spesso obsoleta (7).
Per ovviare a queste carenze organizzative, con i D.lgs. 502/92 e 517/95 il Sistema Sanitario Nazionale è stato riformato
sulla base degli indirizzi di aziendalizzazione, regionalizzazione del servizio e concorrenza tra strutture pubbliche e
private.
A partire dall’istituzione delle Aziende (8), sono state adottate prevalentemente politiche di razionalizzazione e di
compatibilizzazione finalizzate al risparmio e all’economicità. Siffatte politiche hanno avuto l’effetto di ridurre il tasso di
crescita della spesa, di eliminare sprechi e di raggiungere buoni risultati di efficienza (9).
Tale riduzione di spesa, a fronte di un incremento dei consumi sanitari, ha comportato un cambiamento della natura,
una volta inviolabile, del diritto alla salute, trasformandolo in un diritto subordinato alle regole finanziarie. Per cui, nel
corso degli ultimi anni, si è verificato un cambiamento del ruolo del medico: da dominus indiscusso della vita sanitaria
dei pazienti a dipendente qualificato di un’azienda sempre più attenta a costi e problematiche burocraticoamministrative. Pertanto si può evidenziare come le problematiche economiche possano influire sulle scelte del medico,
il quale si trova spesso ad operare legato a logiche e direttive aziendali da un lato e alla necessità di tutelare il diritto alla
salute del paziente dall’altro.
Di conseguenza, il diritto alla salute si trova ad essere sempre più oggetto di contenzioso medico-legale: la
responsabilità professionale del medico si è trasformata dunque in un fenomeno di ampie proporzioni e forte impatto
sociale, tale da condizionare l’operato dei medici e delle stesse strutture (10).
17
P&R Scientific
Quindi la fattispecie giuridica della responsabilità professionale medica in Italia e ovviamente nel resto d’Europa, è
divenuta un tema di grande attualità per la crescente considerazione che, a livello sociale, viene ormai dedicata al diritto
alla salute posto fra i diritti fondamentali degni della massima tutela.
Contemporaneamente, però, la crescente domanda e offerta, pubblica e privata, di prestazioni sanitarie determina
l’incessante incremento di fattispecie dannose, paragonabili come causa di morte e danno alla persona a quelle proprie
della circolazione stradale (11). Il quadro complessivo in prospettiva è destinato a complicarsi a causa di due fenomeni
che caratterizzano il contesto sanitario quale da un lato l’evoluzione della tecnica e della ricerca scientifica con la
tendenza a sfruttarne i risultati in termini di profitto e dall’altro la necessità di contemperare la tutela del diritto alla
salute con l’inevitabile problema della scarsità e limitatezza delle risorse economiche.
Se da una parte, infatti, le nuove acquisizioni nel campo della diagnosi e della terapia hanno contribuito a migliorare la
qualità dei risultati e a diminuire i rischi connessi a determinati atti medico-chirurgici, dall’altra si sta assistendo ad un
aumento della richiesta risarcitoria per presunti errori medici o per episodi attribuibili alle innumerevoli carenze emerse
nella gestione e nella funzionalità del Servizio Sanitario Nazionale (12).
Poiché la normativa italiana, in tale ambito, risulta generica e carente, la giurisprudenza penale, sia di merito che di
legittimità, si è espressa attraverso alcune fasi evolutive ed a volte antitetiche tra loro (13). Inizialmente è stata a
favore dei medici, quindi progressivamente più critica verso i sanitari (14, 15), fino ad un completo sbilanciamento in
senso opposto.
Ad oggi, dunque, la giurisprudenza è diventata un “sottosistema” rispetto alle scarne norme dei Codici penale (16, 17,
18) e civile (19) in materia di colpa: la magistratura si trova a rivestire il ruolo di mediatore tra la carenza ed
inadeguatezza della normativa vigente da un lato, e una casistica sempre più ricca ed eterogenea dall’altro (20).
Infatti il legislatore italiano non sembra intenzionato ad intervenire positivamente in tale contesto, determinando una
sempre maggiore “responsabilizzazione” della magistratura che sta assumendo progressivamente il ruolo di mediatore
fra norme non specifiche di settore e pertanto inadeguate e realtà sociali che necessitano comunque di una cornice
giuridica di riferimento. In quest’ottica giuridica di progressiva affermazione del ruolo delle Corti da semplice applicatore
della legge, tipico dei paesi di civil law come appunto l’Italia, a quello di veri e propri interpreti, s’inserisce la discussione
del caso che intendiamo presentare e commentare, in cui il binomio tra necessità di tutelare il diritto alla salute e i limiti
imposti dai DRG e dalle linee guida non trova facile soluzione, suscitando così un vivace dibattito sia a livello medico che
giuridico.
Descrizione del caso
La vicenda risale al 9 giugno 2004, epoca in cui Il sig. X veniva trasportato in ospedale dove in urgenza, durante
l’esecuzione di una coronarografia, era sottoposto ad una angioplastica coronarica con applicazione di uno stent
medicato perché colpito da infarto miocardico con grave insufficienza respiratoria. Successivamente e precisamente il 14
giugno era quindi trasferito dal reparto di terapia intensiva a quello di cardiologia con diagnosi di edema polmonare,
infarto miocardico acuto con patologie preesistenti quali l’ipertensione arteriosa in soggetto fumatore. Nei giorni
successivi veniva sottoposto a molteplici accertamenti tar cui l’ECG holter.
Il giorno 18 e precisamente 9 giorni dopo il ricovero mostrandosi il paziente asintomatico, con obbiettività negativa, con
scomparsa dell’eritema precedentemente manifestato, veniva dimesso con la consegna di una lettera per il medico
curante nella quale si segnalava la diagnosi di infarto al miocardio acuto anteriore esteso e di edema polmonare acuto,
con la raccomandazione di eseguire la prescritta terapia farmacologica con l’esecuzione di un test ergonometrico dopo
due mesi ed una scintigrafia miocardica dopo sei mesi. Preme però sottolineare come dall’anamnesi risultasse che il
soggetto in questione fosse fumatore, iperteso, affetto da ipercolesterolemia grave, da ipertrigliceridemia, obesità, tutti
indicatori di una sindrome dismetabolica.
Nella notte successiva alle dimissioni veniva colto da tosse e dispnea e, trasportato in ospedale, vi giungeva in arresto
cardio-circolatorio. Causa della morte, accertata tramite autopsia, era una aritmia tipo tachicardia-fibrillazione.
Nei confronti del medico che aveva proceduto alle dimissioni è stato mosso l’addebito di aver agito con colpa avendo
dimesso il paziente con esiti di recente infarto esteso del miocardio a nove giorni di distanza dall’intervento di
angioplastica all’arteria interventricolare causandone la morte a seguito di attacco cardiaco intervenuto a poche ore dalle
dimissioni.
18
P&R Scientific
Fondamentale nell’accertamento della responsabilità si è palesato il ruolo rivestito dalle linee guida richiamate dallo
stesso perito del giudice, a detta del quale il professionista si era attenuto “scrupolosamente” nel decidere le dimissioni.
Per il giudicante di primo grado tale condotta non è stata considerata scevra da colpa poiché fermo restando il valore di
tali regole o protocolli come indicazioni generali riferibili ad un caso astratto, permaneva comunque per il medico, la
necessità di valutare specificamente il caso affidato al suo giudizio, di rilevarne ogni particolarità, di adottare le decisioni
più opportune anche discostandosi da quelle regole (21).
Dall’anamnesi del paziente era, infatti, ricavabile la condizione di soggetto a rischio coronarico, situazione aggravata
dalla severità dell’infarto. Nel successivo grado di giudizio, il professionista veniva invece assolto in quanto il magistrato
considerava non provata l’incompatibilità fra il suo ossequio alle linee guida e il mancato rispetto dei canoni di diligenza,
prudenza e perizia caratterizzanti la professione medica. Del resto, a sostegno di tale teoria ricorreva sia il livello di
funzione meccanica cardiaca residua (29% frazione di eiezione) perfettamente rientrante nei parametri previsti dagli
standards sia l’assenza di segnali predittivi di eventi elettrici avversi, il tutto avvalorato dalle condizioni del paziente al
momento delle dimissioni: asintomatico da giorni con markers di necrosi normalizzati.
Il giudice concludeva per l’assoluzione poiché le condizioni del paziente seppur critiche non erano diverse né più gravi
rispetto a quelle di altri pazienti che rientravano nel campo di applicazione delle linee guida e quindi tali da richiedere un
diverso trattamento.
Con la sentenza n. 8254 del 2011, la Corte Suprema di Cassazione Penale ha annullato la sentenza impugnata con rinvio
ad altra sezione della Corte d’Appello di Milano. Infatti, la Corte di Cassazione nel successivo grado di giudizio annullava
la sentenza motivando come segue: “nel praticare la professione il medico deve, con scienza e coscienza, perseguire
l’unico fine della cura del malato utilizzando i presidi diagnostici e terapeutici di cui al tempo dispone la scienza medica,
senza farsi condizionare da disposizioni o direttive che non siano pertinenti ai compiti affidatigli dalla legge e alle
conseguenti relative responsabilità”.
Ciò vale, in particolare, per le linee guida dettate dall’amministrazione sanitaria per garantire l’economicità della
struttura ospedaliera onde il medico che ha il dovere deontologico di anteporre la salute del malato a qualsiasi altra
diversa esigenza e si pone rispetto a questo in una posizione di garanzia, non sarebbe tenuto al rispetto di tali direttive,
laddove risultino in contrasto con le esigenze di cura del paziente e non potrebbe andare esente da colpa ove se ne lasci
condizionare, senza adottare le decisioni più opportune a tutela della salute del paziente.
Discussione
La progressiva diffusione negli ultimi anni di raccomandazioni, linee guida e protocolli testimonia l’esigenza della
comunità scientifica di ordinare il sapere medico in sistemi conoscitivi, operativi e strategici finalizzati all’innalzamento
degli standards di qualità. Tale tendenza che accomuna la maggior parte dei paesi europei incontra la prima difficoltà
laddove non è possibile rinvenire una definizione universale di linee guida anche se generalmente si considerano alla
stregua di “asserti sviluppati in modo sistematico allo scopo di aiutare le decisioni del medico e del paziente riguardo alle
cure sanitarie più adatte” (OMS) ovvero raccomandazioni di comportamento clinico prodotte attraverso un processo
sistematico, allo scopo di assistere medici e pazienti, nel decidere quali siano le modalità di assistenza più appropriate in
specifiche circostanze cliniche. Il loro carattere non vincolante che le differenzia profondamente dai protocolli determina
sovente ricadute giuridiche e medico-legali di rilevante entità laddove se ne faccia uso per procedere all’accertamento di
una presunta responsabilità professionale. Le innumerevoli varianti che interagiscono con la loro applicazione pratica
come ad esempio gli ostacoli inevitabili di carattere organizzativo, comportamentale, la necessità del razionamento delle
risorse economiche o ancora le aspettative difficilmente codificabili dei pazienti fanno sì che debbano essere considerate
un utile strumento di indirizzo e sostegno applicabile in determinate e precise situazioni ( ma non sempre comunque e
ovunque), lasciando necessariamente spazio alla abilità, preparazione culturale, buon senso e sensibilità del singolo
professionista. In un’ottica medico-legale, le raccomandazioni contenute nelle linee guida pur importanti per garantire
uniformità ai comportamenti professionali alla luce delle migliori conoscenze ed evidenze disponibili, devono essere
tradotte nella pratica clinica quotidiana dal singolo medico e centrate sullo specifico paziente, in considerazione delle sue
peculiarità individuali e delle sue richieste.
A tale logica si è sostanzialmente ispirata la decisione della Corte di Cassazione oggetto di commento, laddove ha
considerato comunque colpevole il medico che si era attenuto alle linee guida in uso nella propria struttura sanitaria, non
esercitando la propria autonomia culturale su un paziente considerato “particolare”, omettendo cioè la verifica della
19
P&R Scientific
rispondenza delle dimissioni alle specifiche condizioni di salute del paziente tali da configurarlo come soggetto a rischio.
Per il giudicante, infatti, l’osservanza delle linee guida nulla può aggiungere o togliere al diritto del malato di ottenere le
prestazioni mediche più appropriate né all’autonomia e alla responsabilità del medico nella cura del paziente soprattutto
se esse rappresentino uno strumento improntato a criteri di economicità di gestione e non alle effettive esigenze del
singolo paziente. Si segnala ovviamente una certa incongruità con l’attuale riordino del Servizio Sanitario Nazionale
italiano che a decorrere dal 1992 è fortemente improntato invece a criteri di privatizzazione e contenimento dei costi
sanitari. La stessa evoluzione della giurisprudenza della Corte Costituzionale segna tale tendenza tanto è vero che più
volte ha affermato come il diritto alla salute “sotto il profilo assistenziale e cioè del diritto ai trattamenti sanitari è
soggetto alle determinazioni del legislatore ordinario ovvero si configura come un diritto costituzionalmente condizionato
all’attuazione che il legislatore ne dà attraverso il bilanciamento con gli altri interessi costituzionalmente protetti fra i
quali è compresa la considerazione delle risorse organizzative e finanziarie disponibili” (22) o ancora come “in presenza
di limitatezza delle risorse non è pensabile di poter spendere senza limite perché è la spesa a dover essere commisurata
alle effettive disponibilità finanziarie, le quali condizionano la quantità ed il livello delle prestazioni sanitarie”. Tale
sentenza ha la finalità di evitare “gli sprechi di risorse nonché ingiustificate sacche di privilegio locale, del tutto
incompatibili con l’interesse nazionale che, nell’attuale difficile situazione della finanza pubblica, non può che concretarsi
nella ottimale utilizzazione delle risorse disponibili” (23).
Opinione ormai condivisa è che pur trovando la garanzia della tutela della salute un fondamento costituzionale sia
comunque soggetta ad ampi spazi di discrezionalità politica e amministrativa per quanto concerne il duplice profilo del
contenuto delle prestazioni e delle modalità di attuazione dei servizi.
Il diritto alla salute al pari degli altri diritti sociali ha subito un processo di relativizzazione che lo ha caratterizzato come
“diritto finanziariamente condizionato” (24).
Tale impostazione non risulta facilmente coniugabile con quanto affermato viceversa dalla Corte di Cassazione laddove
sottolinea come a nessuno sia consentito anteporre la logica economica alla logica della tutela della salute, aggiungendo
che la valutazione di dimissibilità deve essere di ordine medico, non statistico e deve essere rapportata alle condizioni
psicofisiche del malato, alla prognosi circa il decorso successivo ed alla possibilità di proseguire le cure a domicilio. La
logica dell’assistenza deve essere improntata alla tutela della salute e la direttrice del medico non può che essere quella
di rapportare le proprie decisioni solo alle condizioni del malato del quale è comunque responsabile.
Ancora più perentoria è l’ulteriore affermazione del giudicante avvalorata da un malcostume processuale quale quello di
non produrre con cura la documentazione probatoria laddove afferma che “nulla peraltro si conosce dei contenuti di tali
linee guida né dell’autorità dalle quali provengono né del loro livello di scientificità né delle finalità che con esse si
intende perseguire né è dato di conoscere se le stesse rappresentino un’ulteriore garanzia per il paziente ovvero come
sembra di capire dalla lettura delle sentenze in atto, altro non siano che uno strumento per garantire l’economicità della
gestione della struttura ospedaliera”.
Che a farne le spese non sia il medico, imprigionato fra logiche e direttive aziendalistiche e la sua reale possibilità di
poter svolgere con professionalità il proprio operato!
Bibliografia
1. OMS. Definizione di Salute secondo l’Organizzazione Mondiale della Sanità.
2. Dichiarazione Universale dei Diritti Umani, 1948.
3. Art. 32 Cost., Titolo II Rapporti etico-sociali, 1948.
4. Ferrara R. Trattato di biodiritto. Salute e sanità, Giuffrè, Milano, 2011.
5. Dlgs 30 dicembre 1992, n. 502: “Riordino della disciplina in materia sanitaria, a norma dell’art.1 della legge 23
ottobre 1992, n. 421”.
6. Alfano A. L’integrazione socio-sanitaria:lo scenario internazionale. Salute e territorio, ETS, 2006.
7. Collicelli C. L’integrazione socio-sanitaria:i costi della mancata integrazione. Salute e territorio, ETS, 2006.
8. Comma modificato art. 1 Dlgs 7 giugno 2000, n. 168, : “Disposizioni integrative e correttive del decreto legislativo 19
giugno 1999, n. 229, in materia di principi e criteri per l’organizzazione delle Aziende sanitarie locali e di limiti
dell’esercizio del potere sostitutivo statale, nonché di formazione delle graduatorie per la disciplina dei rapporti di
medicina generale”.
9. CAVICCHI I. L’integrazione socio-sanitaria: la crescita della spesa sanitaria. Salute e territorio, ETS, 2006.
10. PICCHIONI D.M, MOLINELLI A, CELESTI R. La responsabilità professionale del medico. Zacchia 2005; 23:78.
20
P&R Scientific
11. Merry A, Miccal Smith A. L’errore la medicina e la legge, vol. XVII, Giuffrè, Milano, 2004.
12. IAPICHINO F.P, VENTURINI E. Ulteriore contributo in tema di responsabilità professionale. Zacchia, 2000;1:73.
13. Avecone P. La responsabilità penale del medico. Vallardi, Padova, 1981.
14. Crespi A. Il grado della colpa nella responsabilità professionale del medico chirurgo. Scuola Positiva, 1960.
15. Cass. 26 gennaio 1968, n. 124.
16. Offidani C. Sentenza n. 27 del 10 luglio 2002 della Corte di Cassazione Sezioni Unite Penali: una pronuncia dirimente
nella valutazione della responsabilità professionale medica. Jura Medica, 47:16, 2003.
17. Art. 42 c.p. “Nessuno può essere punito per un'azione od omissione preveduta dalla legge come reato, se non l'ha
commessa con coscienza e volontà ”.
18. Art. 43 c.p. “Elemento psicologico del reato”.
19. Art. 2236 c.c.”Se la prestazione implica la soluzione di problemi tecnici di speciale difficoltà, il prestatore d’opera non
risponde dei danni, se non in caso di dolo o colpa grave”.
20. Introna F. Responsabilità professionale. Jura Medica,2002; 335:15.
21 Art. 12 Codice Deontologico del medico.
22. Corte Costituzionale sentenza n. 455 del 16 ottobre 1990.
23. Corte Costituzionale sentenza n. 416 del 1995.
24. Merusi V. Servizi pubblici instabili, Bologna, il Mulino, 1990; Molaschi V. I Livelli essenziali delle prestazioni nella
sanità. Ferrara R.: Trattato di biodiritto. Salute e sanità, Giuffrè, Milano, 2011; 459.
Autore di riferimento: Gianluca Montanari Vergallo
Sapienza” Università di Roma, Scuola di Specializzazione in Medicina Legale
Viale Regina Elena, 336 – 00161 Rome – Italy
Corresponding Author: Gianluca Montanari Vergallo
“Sapienza” University of Rome, School of Specialization in Forensic Medicine
Viale Regina Elena, 336 – 00161 Rome – Italy
21
P&R Scientific
PAPILLOMA VIRUS E LESIONI DELLA MUCOSA ORALE.
INFEZIONE E AUMENTO DEL POTENZIALE DI
DEGENERAZIONE MALIGNA
PAPILLOMA VIRUS AND ORAL POTENTIALLY MALIGNANT DISORDERS
Palaia G 1, Lo Giudice R 1, Cavallini C 1, Gaimari G 1, Tenore G
1
1
Dipartimento di Scienze Odontostomatologiche e Maxillo Facciali, Facoltà di Medicina e Odontoiatria, Sapienza
Università di Roma. Direttore: Prof. Antonella Polimeni
1
UOC di Clinica Odontostomatologica. Dirigente II livello: Prof. Massimo De Luca
1
Master EMDOLA (European Master Degree in Oral Laser Applications ); Direttore: Prof. Umberto Romeo
1
Department of Odontostomatologic and Maxillofacial Science, Faculty of Medicine and
Odontology, “Sapienza” University of Rome. Director: Prof. Antonella Polimeni
1
UOC Odontostomatologic Clinic. Director 2nd level: Prof. Massimo De Luca
1
Master EMDOLA (European Master Degree in Oral Laser Applications ); Director: Prof. Umberto Romeo
Citation: Palaia G, Lo Giudice R, Cavallini C, et al. Papilloma virus e lesioni della mucosa orale. Infezione e aumento del
potenziale di degenerazione maligna. Prevent Res 2011; 1 (1): 22-28
Parole chiave: Papilloma virus umani, lesioni potenzialmente maligne, prevenzione del carcinoma orale
Key words: Human papilloma viruses, potentially malignant lesions, prevention of oral cancer
Abstract
Introduzione: L'insorgenza del cancro orale è stata associata a diversi fattori di rischio, quali tabagismo, alcool,
sostanze irritanti e radiazioni.
Obiettivi: E' stata evidenziata una significativa associazione tra infezione da papillomavirus umano (HPV), carcinoma
orale a cellule squamose e, anche se in modo variabile, lesioni potenzialmente maligne della cavità orale, quali
leucoplachia, eritro-leucoplachia eritroplachia. Questo articolo ha lo scopo di descrivere le caratteristiche principali
dell’HPVe analizzare il suo ruolo nello sviluppo del carcinoma.
22
P&R Scientific
Metodi: Dall’analisi critica della letteratura corrente, sono stati selezionati diversi studi volti a valutare il grado di
esposizione e l’attività virale in alcuni tumori. Diversi studi hanno analizzato la prevalenza dell’HPV in questi tumori, ma i
risultati sono contrastanti, essendo collegati alla popolazione dello studio, al tipo di analisi e di prelievo.
Risultati: La maggior parte delle neoformazioni maligne correlate ad infezione da HPV, contengono DNA virale integrato
nel corredo genetico della cellula ed esprimono due geni virali, E6 ed E7, che codificano entrambi per oncoproteine. Dati
presenti in letteratura, hanno evidenziato che l’HPV possa essere l’agente causale di uno specifico tipo di tumore della
testa e del collo e anche un indicatore di prognosi.
Conclusioni: Mentre il ruolo dell'infezione da HPV nell'insorgenza della cervice uterina è oggi ben accertato, scarse sono
le informazioni sulla prevalenza, i determinanti e la storia naturale dell'infezione della mucosa orale, e altri studi sono
necessari per chiarire il potenziale ruolo oncogeno dell'HPV nell'insorgenza del carcinoma orale.
Abstract
Background: Oral cancer has been associated with several risk factor such as smoking, alcohol, irritants and irradiation
The presence of HPV in oral cancers suggests that HPV may play a similar role in transforming the oral epithelia.
Objectives: A significant association was appreciated between infection with human papillomavirus (HPV), squamous
cell carcinoma and potentially malignant disorder of the oral cavity, such as leukoplakia, erythro-leukoplakia
erythroplakia. This review will attempt to focus on relevant characteristics of HPV, analyse its role in oral cancer and
discuss some emerging developments.
Methods: From the critical analysis of the current literature, many studies evaluating different markers of exposure and
viral activity in tumors were collected. Several studies have investigated the prevalence of HPV in these cancers, but the
prevalence of HPV detection varies broadly, depending on the population, combination of sub-sites, typology of
specimen, and method of detection.
Results: The majority of HPV-related cancers contain HPV DNA integrated into the host cell genome and express only
two viral genes, E6 and E7, both of which encode oncoproteins. Data appearing in the literature have provided strong
evidence that HPVs may be the cause of a defined subset of head and neck cancers and also an indicator of improved
survival.
Conclusions: While the role of HPV infection in the onset of cervix cancer is now well established, there is little
information on the prevalence, determinants and natural history of the infection in the oral mucosa, and further studies
are needed to clarify the potential role of HPV in the onset of oral cancer.
Introduzione
Il carcinoma orale ed orofaringeo è uno dei maggiori problemi di salute pubblica a livello mondiale. In Italia, con circa
4000 nuovi casi l'anno il trend epidemiologico si è attestato su valori costanti, senza miglioramenti negli ultimi due
decenni.
Alla base della patologia, il tabagismo e l'abuso di alcol con un'incidenza di circa il 90% dei casi. Altri fattori causali
accertati, sono le carenze alimentari (soprattutto per quanto riguarda la mancanza di un idoneo apporto di vitamine,
antiossidanti e micronutrienti) e la scarsa igiene orale (1-10).
23
P&R Scientific
Obiettivi
Negli ultimi anni, si è investigato sul ruolo etiopatogenetico del Papilloma virus umano (HPV) nell'oncogenesi orale. I
papilloma virus umani (HPV) sono un gruppo eterogeneo di agenti virali appartenenti alla famiglia Papillomaviridae. Si
conoscono circa 200 genotipi diversi di HPV umani che vengono classificati in genotipi ad alto-rischio (HPV 16, 18, 31,
33, ecc.), associati a lesioni potenzialmente ed effettivamente maligne e in genotipi a basso-rischio (tra cui HPV 2, 4, 6,
11, 13, 32) associati più comunemente alle manifestazioni benigne (verruche volgari, condilomi)(2-3).
Questo articolo ha lo scopo di descrivere le caratteristiche principali dell’infezione da HPV nel cavo orale e analizzare il
suo ruolo nello sviluppo del carcinoma.
Metodi
Dall’analisi critica della letteratura corrente, sono stati selezionati diversi studi volti a valutare il grado di esposizione e
l’attività virale in alcuni tumori orali.
Diversi studi hanno analizzato la prevalenza dell’HPV in questi tumori, ma i risultati sono contrastanti, essendo collegati
alla popolazione dello studio, al tipo di analisi e di prelievo (16,17-20).
L'azione oncogena di questo gruppo di virus, ed in particolare di alcuni tipi, definiti appunto ad alto rischio, è stata ormai
comprovata nell'ambito di tutti i carcinomi della cervice uterina; in particolare le proteine virali E6 ed E7 sono state
indicate responsabili della azione oncogena attraverso l'inattivazione di elementi proteici alla base della regolazione del
ciclo di replicazione cellulare come la p53 e la pRB (4-8).
L’associazione tra HPV e carcinoma a cellule squamose è stata inizialmente descritta da Syrjänen et al, nel 1983. I
risultati hanno dimostrato che l’HPV poteva essere coinvolto nell’insorgenza di alcuni tipi di carcinoma. Negli anni
successivi, la letteratura ha fornito prove evidenti che l’HPV possa rappresentare un fattore di rischio indipendente per il
carcinoma a cellule squamose (15,16).
Le lesioni orali HPV-associate possono essere di natura benigna, potenzialmente maligna o francamente maligna. Le
lesioni benigne sono esofitiche, sessili o peduncolate, con una superficie liscia o “a cavolfiore” di colore bianco o roseo
(Fig.1 e 2).
Figura 1. Lesione benigna da HPV situata sul dorso linguale. La lesione si presenta rilevata, di colorito biancastro e
sessile.
24
P&R Scientific
Figura 2. Lesione benigna da HPV situata sulla gengiva aderente in zona premolare.
Possono essere singole, multiple o raggruppate e sono asintomatiche, croniche e a volte regrediscono spontaneamente.
In letteratura si riferisce la prevalenza dei genotipi HPV 6 e 11 nella mucosa normale, così come in lesioni benigne
associate all'HPV quali il papilloma squamoso e il condiloma acuminato, mentre i genotipi HPV 2 e 57 sono
maggiormente presenti nelle verruche volgari. Le lesioni epiteliali potenzialmente maligne a cui può essere associata
l’HPV sono principalmente la leucoplachia e il lichen planus, due patologie della mucose in cui si riscontra un
ipercheratosi dell’epitelio, con caratteristico aspetto biancastro non asportabile (Fig.3).
Figura 3. Leucoplachia a placca della gengiva aderente.
I genotipi HPV caratteristicamente associati a questa tipologia di lesioni sono HPV 16 e 18 ed è possibile che la superinfezione di cellule epiteliali inizialmente degenerate possa promuovere la progressione della trasformazione maligna,
attraverso meccanismi tuttora sconosciuti.
Il carcinoma orale è associato principalmente all’HPV 16 (12), non ha un unico aspetto clinico ma in generale, qualunque
lesione della mucosa persistente, di consistenza duro lignea e ricoperta da mucosa ulcerata o atrofica deve essere
considerata sospetta (Fig.4).
25
P&R Scientific
Figura 4. Eritroleucoplachia collocata sulla mucosa geniena. Notare le chiazze biancastre alternate ad aree con
caratteristiche prettamente atrofico-erosive.
In generale, non è possibile eseguire una diagnosi certa con l’esclusivo esame clinico di eventuali lesioni. È pertanto
indispensabile rivolgersi ad uno specialista per verificare tramite esami più approfonditi la natura della lesione,
eseguendo dove opportuno dei prelievi tissutali per le analisi istologiche.
La presenza di infezione da HPV è facilmente determinabile eseguendo un prelievo cellulare tramite brush, uno
spazzolino sterile che raccoglie in maniera totalmente indolore le cellule degli strati epiteliali più superficiali. Una volta
fissati, si ricerca nei campioni prelevati la presenza di DNA virale.
L’infezione da HPV si trasmette tramite contatto con individui o con aree del corpo infette (ad esempio le verruche nelle
mani). Il virus si trasmette soprattutto attraverso rapporti sessuali vaginali o anali con partner portatori del virus. Il
rischio di contrarre il virus quindi aumenta con l’aumentare del numero dei partner sessuali. Anche altri tipi di rapporti
sessuali (orali o manuali) possono essere vie di trasmissione, ma molto più raramente.
Risultati
Le ultime osservazioni in merito all’infezione da HPV delle mucose orali rivelano un rischio tendenziale aumentato di ben
14 volte di insorgenza di carcinoma orofaringeo nei soggetti sieropositivi all'HPV-16, avvalorando l'interessante ipotesi
che l'infezione virale da HPV possa precedere l'insorgenza del carcinoma orofaringeo di 10 o più anni (9,13).
L'importanza di tali osservazioni è ancor maggiore se consideriamo le caratteristiche della oncogenesi orale; infatti nel
processo evolutivo di un carcinoma orale, in circa il 50% dei casi la patologia insorge su pregressi quadri clinici che per
tale motivo vengono definiti Lesioni Epiteliali Potenzialmente Maligne, il cui decorso è spesso subclinico ed aspecifico. Il
carcinoma orale a cellule squamose associate all’HPV-16 presenta una prognosi nettamente migliore, indipendentemente
dal tipo di trattamento a cui si sottopone, con bassa incidenza di mortalità e recidiva (6,11,19).
L’utilizzo del vaccino sembra poter prevenire l’infezione da HPV non solo nella mucosa genitale, ma anche nella mucosa
orale. L’impatto degli attuali vaccini contro l’HPV sull’incidenza delle infezioni orali persistenti deve essere ancora
identificato.
Sebbene la vaccinazione coinvolga al momento solo la popolazione femminile, studi immunogenetici hanno dimostrato
che il vaccino induce una forte risposta immune anche nell’uomo, dato fondamentale se si considera che il cancro
associato all’HPV è prevalente nella popolazione maschile.
26
P&R Scientific
Conclusioni
Circa il 75% della popolazione mondiale è stata o sarà infettata dall’HPV (14). Laddove si notino lesioni precancerose o
francamente maligne anche in pazienti privi dei consueti fattori di rischio (giovani, non fumatori) è di fondamentale
importanza investigare sulla possibile sovrainfezione da HPV, per poter trarre da questa eventuale compresenza
un'ipotesi predittiva del decorso di tali lesioni, permettendo di orientarne le terapia ed il follow up in modo più consono
all'elevato rischio degenerativo.
Bibliografia
1.
Auluck A, Hislop G, Bajdik C, et al. Trends in oropharyngeal and oral cavity cancer incidence of human
papillomavirus (HPV)-related and HPV-unrelated sites in a multicultural population: the British Columbia experience.
Cancer 2010 Mar 24.
2.
Badaracco G, Rizzo C, Mafera B, et al. Molecular analyses and prognostic relevance of HPV in head and neck
tumours. Oncol. Rep. 2007 Apr;17(4):931-9.
3.
Castro TP, Bussoloti Filho I. Prevalence of human papillomavirus (HPV) in oral cavity and oropharynx. Braz J
Otorhinolaryngol. 2006 Mar-Apr;72(2):272-82.
4.
Chaudhary A K, Singh M, Sundaram S, Mehrotra R. Role of human papillomavirus and its detection in potentially
malignant and malignant head and neck lesions: updated review. Head & Neck Oncology 2009; 1:22
5.
Chocolatewala NM, Chaturvedi P. Role of human papilloma virus in the oral carcinogenesis: an Indian perspective. J
Cancer Res Ther. 2009 Apr-Jun;5(2):71-7.
6.
de Jong MC, Pramana J, Knegjens JL et al. HPV and high-risk gene expression profiles predict response to
chemoradiotherapy in head and neck cancer, independent of clinical factors. Radiother. Oncol. 2010 Mar 24.
7.
Feller L, Khammissa R AG, Wood N H, Lemmer J. Epithelial maturation and molecular biology of oral HPV.
Infectious Agents and Cancer 2009; 4:16
8.
Furniss CS, McClean MD, Smith JF et al. Human papillomavirus 16 and head and neck squamous cell carcinoma.
Int J Cancer. 2007 Jun 1;120(11):2386-92.
9.
Goon P KC, Stanley M A, Ebmeyer J, et al. HPV & head and neck cancer: a descriptive update. Head & Neck
Oncology 2009; 1:36.
10.
Hennessey PT, Westra WH, Califano JA. Human papillomavirus and head and neck squamous cell carcinoma:
recent evidence and clinical implications. J Dent Res. 2009 Apr;88(4):300-6.
11.
Jo S, Juhasz A, Zhang K, Ruel C, et al. Human papillomavirus infection as a prognostic factor in oropharyngeal
squamous cell carcinomas treated in a prospective phase II clinical trial. Anticancer Res. 2009 May;29(5):1467-74.
12.
Kingsley K, Johnson D, O'Malley S. Transfection of oral squamous cell carcinoma with human papillomavirus-16
induces proliferative and morphological changes in vitro. Cancer Cell Int. 2006 May; 22:6-14.
13.
Lohavanichbutr P, Houck J, Fan W, et al. Genomewide gene expression profiles of HPV-positive and HPV-negative
oropharyngeal
cancer:
potential
implications
for
treatment
choices.
Arch
Otolaryngol
Head
Neck
Surg.
2009Feb;135(2):180-8.
14.
Mannarini L, Kratochvil V, Calabrese L, et al. Human Papilloma Virus (HPV) in head and neck region: review of
literature. Acta Otorhinolaryngologica Italica 2009;29:119-126
15.
Pintos J, Black MJ, Sadeghi N, et al. Human papillomavirus infection and oral cancer: a case-control study in
Montreal, Canada. Oral Oncol. 2008 Mar;44(3):242-50.
16.
Reddout N, Christensen T, Bunnell A, et al. High risk HPV types 18 and 16 are potent modulators of oral squamous
cell carcinoma phenotypes in vitro. Infectious Agents and Cancer 2007; 2:21.
17.
Romanitan M, Näsman A, Ramqvist T, et al. Human papillomavirus frequency in oral and oropharyngeal cancer in
Greece. Anticancer Res. 2008 Jul-Aug;28(4B):2077-80.
18.
Ryerson AB, Peters ES, Coughlin SS, et al. Burden of potentially human papillomavirus-associated cancers of the
oropharynx and oral cavity in the US, 1998-2003. Cancer. 2008 Nov 15;113(10 Suppl):2901-9.
19.
Schlecht NF. Prognostic value of human papillomavirus in the survival of head and neck cancer patients: an
overview of the evidence. Oncol Rep. 2005 Nov;14(5):1239-47.
27
P&R Scientific
20.
Smith EM, Ritchie JM, Pawlita M, et al. Human papillomavirus seropositivity and risks of head and neck cancer.Int J
Cancer. 2007 Feb 15;120(4):825-32.
Autore di riferimento: Gaspare Palaia
"Sapienza" Università di Roma, Facoltà di Medicina e Odontoiatria, Dipartimento di Scienze
Odontostomatologiche e Maxillo-Facciali; UOC di Clinica Odontostomatologica. Dirigente di II
livello: Prof. Massimo De Luca; Master EMDOLA (European Master Degree in Oral Laser Applications)
Viale Regina Elena, 287/A, Via Caserta, 6 00161 - Roma - Italia
Corresponding Author: Gaspare Palaia
"Sapienza" University of Rome, Faculty of Medicine and Odontology, Department of
Odontostomatologic and Maxillofacial Science; UOC Odontostomatologic clinic. Director 2nd
level: Prof. Massimo De Luca; Master EMDOLA (European Master Degree in Oral Laser Applications)
Viale Regina Elena, 287/A, Via Caserta, 6 00161 - Rome – Italy
28
P&R Scientific
BIOPSIA NELLE LESIONI DEL CAVO ORALE: MEZZO PER LA
CERTEZZA DELLA DIAGNOSI
BIOPSY IN ORAL LESIONS: TOOL FOR A CERTAIN DIAGNOSIS
Tenore G 1, Carpenteri F 1, Lo Giudice R 1, Cavallini C 1, Gaimari G 1, Palaia G
1
1
Dipartimento di Scienze Odontostomatologiche e Maxillo Facciali, Facoltà di Medicina e
Odontoiatria, Sapienza Università di Roma. Direttore: Prof. Antonella Polimeni
1
UOC di Clinica Odontostomatologica. Dirigente II Livello: Prof. Massimo De Luca
1
Master EMDOLA (European Master Degree in Oral Laser Applications ): Direttore: Prof. Umberto Romeo
1
Department of Odontostomatologic and Maxillofacial Science, Faculty of Medicine and
Odontology, “Sapienza” University of Rome. Director: Prof. Antonella Polimeni
1
UOC Odontostomatologic Clinic. Director 2nd level: Prof. Massimo De Luca
1
Master EMDOLA (European Master Degree in Oral Laser Applications): Director: Prof.Umberto Romeo
Citation: Tenore G, Carpenteri F, Lo Giudice R, et al. Biopsia nelle lesioni del cavo orale: mezzo per la
certezza della diagnosi. Prevent Res 2011; 1 (1): 29-35
Parole chiave: biopsia orale, patologia orale, biopsia laser
Key words: oral biopsy, oral pathology, laser biopsy
Abstract
Introduzione: La biopsia è una procedura diagnostica che consiste nel prelievo, da un organismo vivente, di un
frammento tissutale, allo scopo di sottoporlo ad un esame istologico e, quindi, ottenere una diagnosi di certezza che
possa o meno confermare il sospetto diagnostico clinico.
Obiettivi: Scopo del presente lavoro è stato quello di analizzare i margini dei frammenti tissutali prelevati mediante
laser chirurgici, per valutare gli eventuali danni, dovuti all’effetto termico, presenti in sede epiteliale e connettivale.
Metodi: Sono stati esaminati 17 campioni tissutali prelevati in pazienti che, all’esame obiettivo, presentavano segni
clinici di patologie orali che richiedevano un accertamento istologico. Le biopsie sono state eseguite con due diversi
strumenti: Laser a diodi (Laser Innovation, Italia); Laser KTP (SmartLite, DEKA, Italia). I frammenti tissutali asportati
dalla mucosa orale, sono stati conservati in formalina e, quindi, inviati all’anatomo patologo.
29
P&R Scientific
Risultati: La biopsia laser può essere eseguita con strumenti a diversa lunghezza d’onda, caratterizzati dalla capacità di
interagire con i tessuti molli e di permettere il prelievo di campioni degli stessi. Grazie all’interazione laser-emoglobina, il
campo operatorio risulterà essere esangue, permettendo una maggiore visibilità e consentendo di effettuare interventi
chirurgici in pazienti con turbe della coagulazione; in alcuni casi è possibile procedere senza l’utilizzo di anestetici locali,
o comunque con un uso ridotto degli stessi.
Conclusioni: Lo studio dimostra che i margini incisionali di prelievi bioptici realizzati nei tessuti molli del cavo orale con
laser a diodi e KTP non presentano alterazioni tali da comprometterne l’esame istologico.
Abstract
Background: Biopsy is a diagnostic procedure that involves taking a tissue sample from a living organism in order to
submit it to a histological examination to obtain a definitive diagnosis, which may or may not confirm the clinically
suspected diagnosis.
Objectives: The purpose of this study was to analyze the margins of the tissue fragments taken by laser surgery, to
assess the damage, caused by thermal effect, present in the epithelial and connective tissue.
Methods: 17 tissue samples have been examined, taken from patients that at objective examination showed clinical
signs of oral disease that required histological verification. Biopsies were performed with two different instruments:
diode laser (Laser Innovation, Italy) and KTP laser (SmartLite, DEKA, Italy). The tissue fragments excised from the
buccal mucosa, were preserved in formalin and then sent to the anatomic pathologist.
Results: Laser biopsy can be performed using instruments with different wavelengths, characterized by their capacity to
interact with soft tissues and to allow sampling of the same tissues.Thanks to laser-hemoglobin interaction, the
operative field was bloodless, allowing greater visibility and furthermore the performance of surgery in patients with
coagulation disorders. Finally, in some cases it is possible to proceed without the use of local anesthetics, or with a
reduced use of them.
Conclusions: The study shows that the incisional margins of biopsies carried out in the soft tissues of the oral cavity
with KTP and diode laser do not present alterations that may compromise the histological examination.
Introduzione
Le lesioni dei tessuti molli del cavo orale, alla luce delle classificazioni più recenti, possono essere distinte in lesioni
sospette e lesioni non sospette(1). Le lesioni sospette, comprendono le lesioni epiteliali potenzialmente maligne (LEPM)
(Fig. 1), i carcinomi e i melanomi.
30
P&R Scientific
Fig. 1: Lesioni epiteliali potenzialmente maligne (LEPM) del cavo orale
Leucoplachia plicata della lingua
Cheilosi solare sul vermiglio labiale
Forma reticolare di Lichen
Planus Orale
Le lesioni non sospette comprendono le lesioni fibroepiteliali, le lesioni ad alta componente vascolare, le lesioni
pigmentate, le lesioni virali, le lesioni delle ghiandole salivari minori, le lesioni neoplastiche clinicamente benigne. (Fig.
2)
Fig.2: Lesioni non sospette del cavo orale.
Iperplasia della mucosa gengivale
che ricopre gli elementi dentari
Papilloma a cellule squamose
Mucocele
La biopsia, in genere, è indicata per:
•
Diagnosticare lesioni neoplastiche, pre-neoplastiche ed altre patologie dei tessuti molli;
•
Chiarire l’origine di tumefazioni persistenti non diagnosticabili clinicamente;
•
Individuare l’origine di ulcerazioni che non guariscono entro due settimane;
•
Definire la natura di lesioni che non regrediscono dopo terapia;
•
Eliminare lesioni di dimensioni opportune e verificarne la natura;
•
Porre diagnosi di malattia sistemica(2).
Normalmente, vale la regola che “Qualunque lesione che non migliori sensibilmente entro 14 giorni dalla rimozione dei
possibili agenti irritanti, deve essere considerata potenzialmente maligna e sottoposta a biopsia ed esame
istologico”(OMS).
Le biopsie possono essere classificate in base alla tecnica utilizzata, al materiale impiegato, al timing clinico, alla
posizione della lesione. La scelta del tipo di biopsia da effettuare, dipende da diversi fattori riguardanti la lesione, quali:
sede, dimensioni, rapporti con i tessuti circostanti, tipologia dei tessuti circostanti, sospetto diagnostico. Relativamente
alla tecnica utilizzata, possiamo distinguerle in incisionali ed escissionali.
La biopsia escissionale consiste nella rimozione in toto della lesione, consentendo, allo stesso tempo, di effettuare sia
una procedura diagnostica che terapeutica(2,3,4).
La nostra Scuola prevede l’esecuzione della biopsia escissionale nel caso di lesioni clinicamente benigne di diametro
minore o uguale a 2 cm, in caso di lesioni peduncolate, o di lesioni clinicamente maligne di diametro inferiore a 1 cm. La
31
P&R Scientific
biopsia incisionale prevede il prelievo di uno o più frammenti rappresentativi della lesione, unitamente ai tessuti
adiacenti, profondi e circostanti ad essa e, solo dopo l’esame istologico, è possibile stabilire il trattamento della lesione
residua.
Essa è indicata nel caso di lesioni maligne o sospette di diametro superiore a 1 cm, o nel caso di lesioni clinicamente
benigne di diametro superiore a 2 cm, o localizzate in aree di particolare rilievo estetico o funzionale (lingua, palato
molle, lingua, trigono retromolare), in cui, quindi, è meglio accertarsi della reale natura della lesione, prima di procedere
alla rimozione in toto della stessa(2,3).
In letteratura esistono delle controversie relative alla possibilità che la biopsia incisionale effettuata su lesioni
potenzialmente maligne possa aumentare il rischio di metastasi(2). Per quanto riguarda le tecniche bioptiche incisionali,
è di assoluta importanza la sede del prelievo.
La scelta di tale parametro è fondamentale ai fini della rappresentatività del campione nel corso dell’esame istologico.
E’ una decisione legata all’aspetto clinico della lesione e all’esperienza del chirurgo, ma può essere guidata, ad esempio,
da una precedente citologia esfoliativa o dall’utilizzo di un colorante vitale come il blu di toluidina(2,4). La colorazione
con tale sostanza, consente di evidenziare displasie e piccole lesioni maligne che spesso non vengono individuate
all’esame clinico.
Il blu di toluidina, infatti, colora le aree corrispondenti a maggiore attività mitotica delle cellule grazie alle sue proprietà
spiccatamente acidofile. E’ un test altamente sensibile, con possibilità di falsi positivi solo in caso di lesioni erosivoulcerative o cheratosiche(2)(Fig. 3).
Fig.3: Lesione leucoplasica linguale “mappata” con blu di toluidina.
Il frammento di tessuto prelevato, deve comprendere, quindi, nel caso in cui sia guidato dal giudizio del clinico, noduli,
aree eritematose, ulcere ed erosioni; nel caso in cui sia guidato dai coloranti vitali, le aree che hanno trattenuto
maggiormente il colore; nel caso in cui sia stato preceduto dalla citologia esfoliativa, le aree di prelievo delle cellule più
alterate(1,5).
Obiettivi
È possibile realizzare prelievi di campioni istologici utilizzando due diverse procedure, che vedono impiegati,
rispettivamente, il bisturi ed il laser. Le biopsie eseguite con il bisturi, vengono definite “a lama fredda”. Tale metodica
consente di ottenere un frammento tissutale caratterizzato dalla presenza di margini periincisionali netti, ben definiti,
privi di alterazioni strutturali; tuttavia, tale intervento chirurgico, necessita di anestesia e di sutura, e, il campo
operatorio, sarà sempre sanguinante.
La biopsia laser può essere eseguita con strumenti a diversa lunghezza d’onda, caratterizzati dalla capacità di interagire
con i tessuti molli e di permettere il prelievo di campioni degli stessi.
I prelievi bioptici realizzati con i laser presentano alcuni vantaggi rispetto a quelli realizzati con il bisturi: generalmente
questi interventi non necessitano di anestesia, né di sutura e la guarigione del sito di prelievo, almeno nelle fasi iniziali,
è più rapida(6). Tuttavia, a causa degli effetti termici del laser, i margini incisionali dei campioni tissutali risultano essere
alterati, creando dubbi sulla reale efficacia di questa metodica.
I laser più comunemente utilizzati a questo scopo sono il laser a Diodi, il laser KTP, il laser CO2, il laser Nd:YAG, il laser
Er:YAG. Dalla letteratura italiana ed internazionale emergono interessanti studi(7,8,9,10,11) riguardo le applicazioni
32
P&R Scientific
laser nel campo della biopsia dei tessuti molli su lesioni clinicamente benigne, ma pochi di essi valutano gli effettivi
danni causati da tali strumenti a livello dei margini incisionali dei frammenti tissutali. L’integrità dei margini
periincisionali di un tessuto, infatti, è un parametro di fondamentale importanza nella valutazione di uno strumento
utilizzato per effettuare biopsie.
Da essa può dipendere sia l’esito dell’esame istologico che la corretta guarigione del sito del prelievo. Dallo studio di
Romeo et al.(10), nel quale vengono valutati gli effetti di laser Er,Cr:YSGG, Nd:YAG, e di due laser a diodi (di lunghezza
d’onda rispettivamente pari a 808 nm e 980 nm) su lingua di maiale, emerge che tutti i laser esaminati possono essere
utilizzati per effettuare prelievi bioptici.
Infatti, pur causando tutti delle lesioni a livello marginale del tessuto prelevato, nessuno di essi crea compromissione
dell’esame istologico. In particolare, i laser che creano minor carbonizzazione marginale sono il diodi 808 nm pulsato,
l’Er,Cr:YSGG, nei quali il danno stimato è inferiore al millimetro. Lo studio sottolinea, infine, la necessità di aumentare la
distanza dei margini di incisione del prelievo bioptico di 2-3 mm rispetto alla lama fredda, per evitare che gli effetti
termici del laser compromettano i risultati dell’esame istologico dello stesso.
Dallo studio presentato nell’articolo di Rizoiu et al(12), realizzato su conigli, emerge che non ci sono sostanziali
differenze a livello istologico tra i margini incisionali nei frammenti bioptici escissi con il laser e quelli effettuati a lama
fredda. Nell’articolo di Dalrymple e Russell(13), in cui viene presentato lo studio sulla valutazione dei margini
periincisionali di prelievi bioptici incisionali ed escissionali eseguiti con laser su lesioni della cervice uterina, emerge che
le alterazioni marginali sono in media di 0,3 millimetri.
Tuttavia, a causa dei danni termici, nel 12% dei casi l’esame istologico ha dato esito incerto. Dalle diverse pubblicazioni
sull’argomento non emerge la possibilità di valutare, in vivo, le alterazioni marginali di campioni bioptici prelevati con il
laser. Tale considerazione ci ha indicato la necessità di avviare uno studio relativo all’esame istologico, eseguito in vivo,
dei margini periincisionali di frammenti tissutali ricavati mediante biopsia laser. Lo scopo del nostro studio è quello di
confrontare la validità di campioni bioptici dei tessuti molli del cavo orale, prelevati mediante laser o bisturi.
Materiali e Metodi
Sono stati esaminati 17 campioni tissutali prelevati in pazienti che, all’esame obiettivo, presentavano segni clinici di
patologie orali che richiedevano un accertamento istologico.
In
alcuni
casi,
la
lesione
è
stata
trattata
con
una
biopsia
escissionale,
che
ha
consentito
di
attuare,
contemporaneamente, una procedura diagnostica e terapeutica. Nei casi in cui, per motivi legati alla sede o alla
dimensione della lesione non è stato possibile rimuoverla in toto, si è optato per l’esecuzione di una biopsia incisionale,
che ha consentito di effettuare l’esame istologico della stessa, e, in base all’esito di questo, decidere come trattarla.
Tutti i prelievi tissutali sono stati eseguiti dallo stesso operatore, in maniera tale che, i risultati emersi nello studio siano
privi della variabilità operatore-dipendente. E’ chiaro, infatti, che l’esperienza e la conoscenza dei parametri degli
strumenti laser e delle caratteristiche biologiche dei tessuti con cui entrano in contatto da parte di colui che esegue
l’intervento, siano alla base della corretta esecuzione della biopsia e che da essi dipenda l’assenza di danno
periincisionale.
Le biopsie sono state eseguite con due diversi strumenti: laser a diodi (Laser Innovation, Italia); laser KTP (SmartLite,
DEKA, Italia). I frammenti tissutali asportati dalla mucosa orale, sono stati conservati in formalina e, quindi, inviati
all’anatomo patologo. Successivamente sono stati colorati con ematossilina-eosina, fissati e tagliati seguendo le
metodiche tradizionali. I 17 prelievi sono giunti all’osservazione del medesimo specialista che, mediante microscopio
ottico ha elaborato, per ciascuno di essi, la diagnosi istologica.
La presenza di alterazioni a livello dei margini periincisionali non ha creato particolari problemi nella valutazione
istologica: per tutti i campioni è stato possibile ottenere una diagnosi di certezza. In seguito i frammenti tissutali sono
stati nuovamente osservati al microscopio ottico, ad ingrandimento 5X e 10X.
Con l’ausilio di un appropriato software sono state valutate quantitativamente e qualitativamente le alterazioni marginali
dovute all’azione termica dei laser sugli istologici. Dal punto di vista quantitativo è stata eseguita una misurazione in
millimetri; per quanto riguarda l’aspetto qualitativo, è stato valutato il coinvolgimento del tessuto epiteliale e del
connettivo nelle alterazioni termiche.
33
P&R Scientific
Risultati
L’uso del laser e le interazioni che esso può avere con i tessuti sono sostanzialmente regolati da due tipi di fattori. I
fattori operatore-dipendente riguardano sia le modalità di utilizzo del laser, sia il tempo di applicazione, sia la scelta
della distanza di taglio dai margini lesionali; i fattori operatore-indipendenti riguardano invece la lunghezza d’onda
specifica del laser e le proprietà ottiche del tessuto.
I risultati ottenuti mostrano come il danno dovuto a carbonizzazione e/o coartazione sia più limitato in alcuni tipi di
lesioni (mucocele) piuttosto che in altri (lichen planus), dimostrando come cellule in condizioni diverse rispondono in
maniera diversa al laser. Bisogna sottolineare che l’uso del laser non è consigliabile per la realizzazione di biopsie su
lesioni sospette.
Nella valutazione istologica di alterazioni tissutali che già clinicamente mostrano segni di malignità, è fondamentale,
infatti, l’analisi dell’infiltrazione cellulare nei tessuti adiacenti.
Gli effetti termici del laser potrebbero compromettere la possibilità di realizzare un’accurata analisi dei margini della
lesione, e quindi di stabilire le reali dimensioni del tumore; anche se, ampliando i margini incisionali di soli 1-2
millimetri, gli effetti termici del laser non creano alcun tipo di alterazione istologica nei tessuti prelevati.
Conclusioni
Nel nostro studio è emerso che la biopsia dei tessuti molli del cavo orale, effettuata con laser a diodi o KTP, non crea
alterazioni marginali di rilevante importanza ai fini della diagnosi istologica.
Il laser, utilizzato da un odontoiatra esperto, consente di ottenere frammenti istologici con importanti vantaggi per
l’operatore e il paziente.
Grazie all’interazione laser-emoglobina, il campo operatorio risulterà essere esangue, permettendo una maggiore
visibilità e consentendo di effettuare interventi chirurgici in pazienti con turbe della coagulazione; in alcuni casi è
possibile procedere senza l’utilizzo di anestetici locali, o comunque con un uso ridotto degli stessi; la guarigione postoperatoria risulterà essere più rapida, soprattutto nelle fasi iniziali.
Ampliando i margini incisionali di soli 1-2 millimetri, gli effetti termici del laser non creano alcun tipo di alterazione
istologica nei tessuti prelevati. L’uso dei dispositivi laser nelle biopsie dei tessuti molli del cavo orale è una realtà che
attualmente deve essere sempre considerata dagli odontoiatri come valida alternativa all’uso della lama fredda,
comportando, rispetto a quest’ultima notevoli vantaggi.
Bibliografia
1.
García-Peñín A. Biopsia en Cirugía Bucal. Cirugía Bucal: patología y técnica. Donado M ed. Madrid, Masson, 1990;
119-31.
2.
Mota-Ramírez A, Javier Silvestre F, Simó J M. Oral biopsy in dental practice. Med Oral Patol Oral Cir Bucal. 2007
Nov 1.
3.
Oliver R J, Sloan P, Pemberton M N. Oral biopsies: methods and applications. British dental journal 2004 march;
196.
4.
Romeo U, Libotte F, Palaia G et al. Histological in vitro evaluation of the effects of Er:YAG laser on oral soft tissues.
Lasers Med Sci. 2011 Jul 28.
5.
Lodi G, Sardella A, Demarosi F, et al. Oral biopsy. A prospective study on 286 consecutive procedures. Minerva
Stomatol. 2007 May;56(5):241-51.
6.
Fonseca, Oral and maxillofacial surgery, Sounders, 2000.
7.
Bornstein MM, Winzap-Kälin C, Cochran DL, Buser D. The CO2 laser for excisional biopsies of oral lesions: a case
series study. The International journal of periodontics & restorative dentistry 2005;25(3):221-9.
8.
Hazem Mohammad S, Ali Mohammad S. Excision Biopsy of Tongue Lesions by Diode Laser. Photomedicine and
Laser Surgery. 2007 February 1; 25(1): 45-49.
9.
Antonio L.B, PINHEIRO,John W. FRAME. Surgical Management of Premalignant Lesions of the Oral Cavity with the
CO2 Laser. Braz Dent J 1996; 7(2): 103-108.
10.
Romeo U, Del Vecchio A, Ripari F et al. Effects of Different Laser Devices on Oral Soft Tissues: In Vitro Experience.
J Oral Applications,2007.
34
P&R Scientific
11.
Romeo U, Palaia G, Del Vecchio A et al. Effects of KTP laser on oral soft tissues. An in vitro study. Lasers Med Sci.
2010 Jul;25(4):539-43.
12.
Rizoiu IM, Eversole LR, Kimmel AI. Effects of an erbium, chromium: yttrium, scandium, gallium, garnet laser on
mucocutanous soft tissues. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 1996 Oct;82(4):386-95.
13.
Dalrymple & Russell. Thermal artefact after diathermy loop excision and laser excision cone biopsy. International
Journal of Gynecological Cancer.Volume 2001 Dec; 9 Issue 3: 238 – 242.
Autore di riferimento: Gaspare Palaia
"Sapienza" Università di Roma, Facoltà di Medicina e Odontoiatria, Dipartimento di Scienze
Odontostomatologiche e Maxillo-Facciali; UOC di Clinica Odontostomatologica. Dirigente di II
livello: Prof. Massimo De Luca; Master EMDOLA (European Master Degree in Oral Laser Applications)
Viale Regina Elena, 287/A, Via Caserta, 6 00161 - Roma - Italia
Corresponding Author: Gaspare Palaia
"Sapienza" University of Rome, Faculty of Medicine and Odontology, Department of
Odontostomatologic and Maxillofacial Science; UOC Odontostomatologic clinic. Director 2nd
level: Prof. Massimo De Luca; Master EMDOLA (European Master Degree in Oral Laser Applications).
Viale Regina Elena, 287/A, Via Caserta, 6 00161 - Rome – Italy
35
P&R Scientific
LA DEVIANZA COME PROBLEMA SOCIALE: LE RISPOSTE
DELLA PSICOLOGIA
DEVIANCY AS SOCIAL PROBLEM: THE ANSWERS OF PSYCHOLOGY
Tomei G 1, Sancini A
1
2
1
“Sapienza” Università di Roma, Dipartimento di Neurologia e Psichiatria
2
“Sapienza”Università di Roma, Scuola di Specializzazione in Medicina del Lavoro
"Sapienza", University of Rome, Department of Neurology and Psychiatry
2
"Sapienza", University of Rome, School of Occupational Medicine
Citation: Tomei G, Sancini A. La Devianza come problema sociale: le risposte della Psicologia.
Prevent Res 2011; 1 (1): 36-43
Parole chiave: devianza, anomia, potere, autorità, conformismo
Key words: deviancy, anomie, governance, power, authority, conformism
Abstract
Scopo: Il comportamento deviante è tale in quanto infrange una serie di norme sociali più o meno consapevolmente
riconosciute dai più. Scopo dello studio è descrivere e analizzare le caratteristiche di tale comportamento.
Materiali e metodi: Si è tentato di individuare le cause della devianza in un rapporto complesso con le figure
genitoriali, con l’Autorità generalmente intesa, con i Gruppi sociali che detengono il Potere ecc.. valutando teorie a
partire dalla psicoanalisi fino alla più recente sociologia.
Risultati e conclusioni: Pur ammettendo la possibile presenza di un certo tipo di disturbi di personalità nella struttura
psichica del deviante, non si può non puntare l’attenzione sulle metodiche che le varie società utilizzano per
l’integrazione dei cittadini, soprattutto nelle agenzie fondamentali preposte all’educazione del minore: famiglia e scuola.
Metodi didattici all’avanguardia, che senz’altro forniscano al discente griglie comportamentali e regole di condotta, che
però al tempo stesso non dimentichino la dimensione fondamentale del gioco, dello svago e della ricerca personale, sono
da incentivare fortemente. Con la consapevolezza che, nel bambino e nell’adolescente, “trasgredire” determinate regole
con coscienza critica e capacità di discernimento, aiuta a formare un cittadino consapevole, responsabile e rivolto
all’innovazione di paradigmi comportamentali spesso datati e inadeguati, anche se comunemente accettati con passività
dai più.
36
P&R Scientific
Abstract
Scope: Deviant behaviour is the one that breaks those rules most people regard as social. The study describes and
analyzes the characteristics of this behavior.
Materials and methods: Psychology and also the latest Sociological Theories have tried to find the causes of deviance
in the complex and difficult relationship with parental figures, with Authority in general, with the Part of society that
holds Power etc.
Results and conclusions: While admitting the possible presence of some kinds of personality disorders in the deviant’s
psychic structure we cannot avoid focusing on the methodologies used for the integration of citizen above all in those
fundamental units in charge of minors’ education: Family and School.
Advanced teaching methods which can provide behavioural models and rules are to be strongly encouraged, without
forgetting the essential dimension of playing, of research and also of individual personal growth.
Nevertheless we must be aware that ‘breaking’ the rules with a sense of responsibility and discernment helps a young
man to grow informed and responsible, able to renew his behavioural patterns often dated and deficient albeit mainly
passively accepted.
Introduzione
Il comportamento deviante è quello che si verifica quando un individuo, o un gruppo di individui, si discosta nelle sue
parole o atteggiamenti da una presunta Norma comportamentale che in una data società o in un dato contesto è
condivisa dai più.
Il cosiddetto deviante non accetta, o semplicemente non ha interiorizzato, le regole che vigono in un determinato
contesto aggregativo e ne viola i presupposti, in maniera più o meno consapevole: la delinquenza o criminalità, la
malattia mentale, l’abuso di droghe o alcool, alcuni comportamenti sessuali ecc. sono considerati forme di devianza che
connotano l’individuo come “diverso” rispetto ad uno standard comunemente accettato in quel dato contesto, e lo
rendono bersaglio di riprovazione sociale, condanna e spesso di ritorsioni punitive o denigratorie. La società fa sentire le
sue norme specifiche all’individuo fin dalla prima infanzia, e tutta la vita dell’individuo si configura così come un
“addestramento alla società”.
Le prime norme sono interiorizzate nel rapporto “madre-bambino” e ancor di più “padre-bambino”. La cosiddetta
“socializzazione primaria” avviene nei primi anni di vita del bambino, quando la madre gli insegna ad es. ad usare
forchetta e coltello per mangiare, per poi passare alla “socializzazione secondaria”, che avviene quando si entra a scuola
e si iniziano ad apprendere le competenze per svolgere un proprio ruolo sociale.
Le varie forme di socializzazione avvengono secondo meccanismi di punizione e ricompensa. Non sempre la devianza
può essere considerata oggettiva: spesso un dato comportamento risulta deviante in un paese e non in un altro, o in un
dato periodo storico piuttosto che in un altro. Può capitare a volte, dato che la Società, o le varie società pongono
sempre delle norme a difesa del proprio assetto, che il deviante, infrangendo dette norme, si ponga come una variante
“X”, che sia cioè portatore di novità, di nuovi paradigmi che più avanti, quando il gesto deviante sia stato assimilato,
diventino nuova norma essi stessi.
E’ altrettanto vero però che, se molti giudizi possono essere percepiti diversamente nello spazio e nel tempo, diverse
norme sociali, e l’infrazione delle stesse, sono giudicate allo stesso modo nella maggior parte dei contesti: è il caso ad
es. del divieto dell’incesto, dell’infanticidio, dell’uccisione del genitore ecc.. Anche i mezzi di comunicazione di massa
possono essere veicolo di normatività e socializzazione.
Ne è un esempio la Teoria della Spirale del Silenzio, formulata da Elisabeth Noelle-Neumann (1): secondo questa teoria
gli individui si conformano, nelle conversazioni sociali, ai modelli e alle informazioni formulate e proposte loro dai massmedia.
37
P&R Scientific
Chi non vuole o non può conformarsi a questi modelli, piuttosto che apparire estraneo e in disaccordo con gli argomenti
e i modi della conversazione, resta in silenzio. Per cui la spirale del silenzio è il meccanismo di integrazione che costringe
appunto al silenzio coloro che dissentono dai messaggi e dagli stili di vita proposti a livello mediale.
Una prospettiva storica
Il Controllo Sociale è elemento integrante di ogni società: ogni società è generatrice di forme di conformismo, di
irreggimentazione, di meccanismi di persuasione e sorveglianza e di conseguenza di tecniche di sanzionamento e
punizione della difformità sociale. La Psicoanalisi postula nell’individuo la presenza di un Super-Io che detta le regole del
comportamento, il quale è il risultato dell’”introiezione” di regole sociali, delle quali i primi latori sono le figure genitoriali,
in genere la figura paterna. L’individuo appunto introietta le norme sociali impartitigli dai genitori, e in seguito le fa sue,
accorpandole nel Super-Io (2).
Queste regole del Super-Io si scontrano contro l’essenza pulsionale dell’individuo, il cosiddetto Es, contro cioè la forza
degli istinti primari, riconducibili alla spinta del Principio di Piacere, il quale tende al soddisfacimento immediato e
inderogabile di ogni impulso. Dallo scontro fra l’Es e il Super-Io, e delle rispettive istanze, si forma, secondo i principibase della psicoanalisi, l’Io, che nell’individuo sano si riesce a conformare al Principio di Realtà, cioè al differimento e
all’attesa nel soddisfacimento delle pulsioni primarie.
L’individuo malato o immaturo, al contrario, non riesce a tenere in equilibrio queste due forze contrastanti, è mosso
inesorabilmente dal Principio di Piacere, e da qui nasce la sua tendenza ad infrangere le norme comunemente accettate
e a “delinquere” da un ordine sociale prestabilito. Questo secondo appunto la psicoanalisi. L’individuo senza norme,
isolato, preda dei suoi impulsi più profondi risulta vittima di quella che in sociologia viene chiamata Anomia (dal lat. Anomos: assenza di leggi, di regole).
Gli studi sull’anomia sono fondamentali per le scienze sociologiche, e sull’individuo alienato, escluso dal tessuto
connettivo della società e dell’aggregazione con i propri simili, sono sorte numerose teorie, fra cui quella sull’incidenza
dei casi di suicidio rapportati alle caratteristiche del contesto sociale circostante (3). Sulla scia del testo di Talcott
Parsons “The Social System” (4), una vasta parte di sociologia nord-americana (riunitasi intorno alla cosiddetta Scuola di
Chicago) ha affrontato il problema della devianza, collegandola e rapportandola allo sviluppo incontrollato delle
metropoli occidentali, con le conseguenze di disorganizzazione sociale, urbanizzazione selvaggia ecc. E’ stato
sottolineato il formarsi e crescere, sull’onda della perdita di valore di alcune norme etico-sociali e dell’indebolirsi
dell’influenza di alcuni gruppi primari, di un vasto strato di popolazione povera o indigente e con scarsa coscienza eticocomportamentale (5).
L’accorpamento di una working-class scarsamente o per niente istruita agglomerata in centri-dormitorio anti-igienici e
iperaffollati ha causato il diffondersi di malessere e disagio sociale e l’emergere di bande di giovani emarginati
delinquenti e border-line senza alcuna prospettiva occupazionale e quindi di integrazione. La Scuola di Chicago ha
affrontato questi problemi dal punto di vista teorico e ha postulato la presenza del “relativismo” culturale alla base della
devianza, per cui gruppi ed etnie sociali diverse stabiliscono il proprio rapporto con lo Stato e con le etnie dominanti in
base ad un complesso codice che fa riferimento, per ciascuno, alla propria cultura di appartenenza, e quindi la tendenza
a delinquere è presente tanto più quanto più è percepita la lontananza dai centri di potere e di elaborazione delle
decisioni.
Ciò non toglie che la Scuola di Chicago si sia soffermata anche sugli atti di devianza realizzati dalla upper-class, la
cosiddetta devianza dei colletti bianchi, in special modo per quanto riguarda i reati finanziari, le truffe bancarie e le
speculazioni di borsa (6).
Secondo Robert Merton (7) le principali fonti di tensioni e frustrazione per i giovani, sia di classe povera che di classe
elevata, sono le difficoltà a raggiungere uno status di vita elevato, o a mantenere quello riservato loro dai genitori.
Inoltre le modalità con cui, a livello di gruppo e non ufficiale, viene sanzionato il comportamento del presunto deviante
sono riconducibili ad altre forme di comportamento negativo e censurabile, riconducibili alle dinamiche del bullismo o
altrimenti dette “dinamiche del Capro Espiatorio” (8). Secondo la Labelling Theory (Teoria dell’Etichettamento), sono i
gruppi sociali dominanti che definiscono gli individui integrati e non, che danno la patente di outsider a seconda dei loro
scopi per mantenere e difendere lo status-quo. La definizione di norma e devianza diventano dei concetti relativi
attraverso cui i Gruppi al potere gestiscono il loro predominio. Secondo la labelling theory un individuo inizia la sua
carriera deviante con un atto, un gesto, un comportamento, a volte del tutto accidentale, che subisce la disapprovazione
sociale.
38
P&R Scientific
Poi, nel caso persista nelle sue modalità comportamentali, ne consegue l’isolamento, la riprovazione e la conseguente
degradazione in termini di reputazione e ruolo percepito. In seguito il comportamento deviante si “fissa” e l’individuo
diventa outsider in maniera definitiva. Chiaramente in questo caso i gruppi dominanti hanno buon gioco per inserire in
quest’area tutti i soggetti o gruppi dissidenti o non conformi al loro modo di gestire i rapporti di forza e di pensiero. E’
evidente che da qui inizia tutto quel filone della sociologia che si occupa di analizzare i meccanismi di funzionamento del
Potere, e che oltre a proporre punti di vista interessanti e fecondi, spesso ha portato ad una accettazione acritica e
tollerante del punto di vista del deviante, altrettanto fuorviante e giustificatoria.
Erving Goffman (9) analizza i rapporti fra ruolo sociale e identità: quando il ruolo che si è assunto, o che si è stati
costretti ad assumere in un dato contesto associativo, è troppo discrepante dalla propria identità, o da quella che si
auto-percepisce o che è percepita dagli altri, questa discrepanza provoca disagio, senso di costrizione ed è un principio
di incubazione di forme più o meno eclatanti di trasgressione. Altri autori hanno sottolineato il mutare del rapporto
Repressione-Permissivismo, sbilanciatosi a favore di quest’ultimo, che è in realtà un falso permissivismo che impone
invece una cultura materialistica e forzatamente e nevroticamente edonistica (10). E’ da queste analisi che provengono
tutte quelle teorie che inscrivono la figura del deviante in un’ottica dialettica nei confronti dei sistemi di Potere
dominanti.
In questo caso la devianza è solo una delle forme con le quali viene stigmatizzato il “pensiero divergente” o che
comunque non si allinea con quello che viene ritenuto il pensiero globale. Prima in “Sorvegliare e Punire”, poi nella
“Microfisica del Potere” Michel Foucault identifica il potere in una specie di reticolo di punti in vorticoso movimento,
ciascuno dei quali fa sentire la sua influenza sugli altri: alcune categorie sociali e lavorative vengono quindi
marginalizzate più di altre, ma non esiste chi è completamente escluso dalla rete di disseminazione e distribuzione del
potere. Ciascuno subisce un potere da qualcuno e lo fa sentire su qualcun altro, perciò non esistono per natura classi più
giustificate nell’applicare meccanismi di devianza e altre meno (11,12).
E’ però vero che esistono classi che detengono più potere in assoluto rispetto ad altre, e che modellano in generale la
percezione delle opinioni comuni. La Sinistra critica e il pensiero ad esempio di Herbert Marcuse o Erich Fromm
sottolineano il processo di omologazione che subiscono tutti i soggetti portatori di una visione personale e dissonante. Le
classi al potere, attraverso i megafoni costituiti dai mezzi di informazione e dalle reti del controllo sociale, tentano e
spesso riescono a ricondurre all’ordine ogni dissidenza e divergenza, anche attraverso i sistemi di detenzione coatta e di
rieducazione comportamentale(10, 13). I movimenti cosiddetti dell’Antipsichiatria hanno origine da qui (14).
Classificazione
Attraverso l’analisi dei disturbi di personalità si può avere un quadro dei disturbi psicologici che, accentuando certi
caratteri stabili dell’essere umano, rappresentano variazioni più o meno accentuate da una presunta Norma
comportamentale riconosciuta:
Il DSM IV (Diagnostic and Statistical Manual of Mental Disorders) raccoglie in tre gruppi i vari disturbi di
personalità;
Il gruppo A include i disturbi di personalità caratterizzati dal comportamento bizzarro, strano o eccentrico:
•
Disturbo paranoide: chi ne soffre tende a interpretare il comportamento degli altri come malevolo,
•
Disturbo schizoide: chi ne soffre non è interessato al contatto con gli altri, preferendo uno stile di vita
comportandosi in modo sospettoso;
appartato e distaccato. E’ caratterizzato anche da distorsioni del pensiero razionale;
•
Disturbo schizotipico: rilevato in persone considerate eccentriche nel comportamento e nell’abbigliamento,
con scarso contatto con la realtà e che danno molto peso e rilevanza a intuizioni magiche, sovrannaturali o
paranormali.
39
P&R Scientific
Il gruppo B include i disturbi caratterizzati da un’emotività amplificata e imprevedibile e dall’instabilità delle relazioni
affettive:
•
Disturbo border-line: presente in chi soffre di impulsività, instabilità emotiva e relazionale e nell’idea di se stesso,
•
Disturbo istrionico: chi ne soffre tende a ricercare in modo morboso e patologico l’attenzione degli altri, a mettere
oscillando tra posizioni estreme e contrastanti;
in atto comportamenti seduttivi e manipolatori e a manifestare in modo teatrale ed eccessivo le proprie emozioni
•
Disturbo narcisistico: chi ne soffre pretende in modo smodato l’ammirazione degli altri, pone se stesso al di sopra
di tutto e patisce la mancanza di soddisfazione a questo impulso. Spesso sviluppa comportamenti sadici e autoetero distruttivi;
•
Disturbo anti sociale: chi ne soffre non sente il bisogno di rispettare le leggi, tende a violare i diritti degli altri,
non prova senso di colpa per i danni commessi.
Il gruppo C include i disturbi caratterizzati da una forte ansia e da paure spesso immotivate:
•
Disturbo evitante: il paziente tende ad evitare le situazioni sociali per paura di essere giudicato o osservato.
•
Disturbo dipendente: chi ne soffre sente il forte bisogno di essere accudito, protetto e difeso. Delega le proprie
Presenta marcata timidezza e forte senso di inadeguatezza e inferiorità;
responsabilità ed è fortemente inaffidabile;
•
Disturbo ossessivo-compulsivo: ha una forte tendenza al perfezionismo e alla precisione. Forte preoccupazione
per l’ordine e per il controllo sulle cose e le persone. Tende ad eseguire i suoi compiti con maniacalità e soffre
quando le cose non sono sotto il suo completo controllo.
Ammesso che detti disturbi siano niente altro che un’accentuazione di caratteristiche di personalità comunemente
riscontrabili nella media dei soggetti della popolazione generale, è sicuramente vero che quando si configurano come
disturbi di personalità generano nel soggetto una patologia più o meno marcata, che li inquadra come “soggetti
devianti”.
La Devianza come rifiuto del conformismo
Si può quindi argomentare che la devianza costituisca, nelle sue basi, un Rifiuto dell’Autorità in senso generico:
genitoriale, sociale, didattica ecc. E’ evidente che la formazione della personalità passa anche attraverso il rifiuto di una
norma assodata per certa, e le migliori intelligenze spesso non si sono assoggettate ad un sapere consolidato ma hanno
forzato, con i loro comportamenti e atteggiamenti, i limiti e la cornice delle usanze del tempo. Il filosofo e psicoanalista
Wilhelm Reich (15, 16) ipotizza che la genesi e il consolidarsi dei movimenti di stampo fascista nelle varie nazioni a
livello mondiale sia da ricondurre alla formazione, a partire dal livello atomistico della psiche dei singoli individui, della
cosiddetta “corazza” caratteriale: uno scudo emotivo che il soggetto costruisce per difendersi dall’ansia causata dallo
scontro fra pulsioni interne e risposte dell’ambiente.
Questo può verificarsi quando subentra un eccessivo tentativo di irreggimentazione dei meccanismi psicologici, e che
causa una contrattura del libero fluire delle energie pulsionali, sia a livello corporeo che mentale. La devianza può quindi
configurarsi attraverso molteplici modelli interpretativi, ed è chiaro come sia un argomento che possa anche essere
oggetto di fraintendimenti e categorizzazioni scarsamente scientifiche e credibili.
E’ noto come in Usa e in altre nazioni occidentali spesso venga diagnosticato il cosiddetto Deficit dell’Attenzione (ADDAttention Deficit Disorder), e anche somministrati farmaci, a bambini semplicemente vivaci, curiosi, sicuramente
iperattivi ma con al contempo spiccate doti di creatività ecc. Lo scrittore Pier Paolo Pasolini (17) nella raccolta di articoli
giornalistici “Lettere Luterane” elabora un breve trattato di pedagogia, che è costituito dalle “Lettere a Gennariello”. In
questi scritti, pubblicati postumi sul Corriere della Sera e poi in volume, l’autore ipotizza un colloquio fra lui e un giovane
“guaglione” napoletano, al quale Pasolini finge di rivolgersi direttamente e del quale ne descrive le caratteristiche fisiche
e morali: si tratta di un giovane partenopeo allegro e irriverente, dai comportamenti un po’ disinvolti (è un “mariuolo”)
ma non privo di intelligenza e in fondo di bontà d’animo.
40
P&R Scientific
Scrive Pasolini: “… il fatto che tu sia napoletano esclude che tu, pur essendo borghese, non possa anche essere
interiormente carino. Napoli è ancora l’ultima metropoli plebea, l’ultimo grande villaggio (…) questo fatto generale e
storico livella fisicamente e intellettualmente le classi sociali.
La vitalità è sempre fonte di affetto e ingenuità. A Napoli sono pieni di vitalità sia il ragazzo povero che il ragazzo
borghese.” Napoli è perciò rimasta, secondo l’autore (che in seguito sarà costretto a ritrattare questa dichiarazione),
miracolosamente intatta da secoli, uno degli ultimi avamposti quindi non aggrediti delle infiltrazioni piccolo-borghesi e
moderniste che per Pasolini deturpano antropologicamente e irrimediabilmente le altre città italiane e i loro abitanti.
Pasolini illustra all’immaginario giovane interlocutore l’Italia degli anni di piombo, il conformismo imperante e l’ideologia
consumistica che si sono impadroniti della nazione.
E a questo ragazzo immaginario, simpatico, vitale, per nulla odioso come la maggior parte dei suoi coetanei, l’autore
dedica delle righe assai illuminanti sul suo pensiero. In tutti i suoi lavori, dai romanzi “Ragazzi di vita” e “Vita violenta”,
per passare ai film e agli articoli corsari, fino al libro-testamento “Petrolio”, Pasolini si rivolge a ragazzi “devianti”, che
vivono valori diversi rispetto a quelli che la non-etica del consumismo e le false risposte del perbenismo propongono
loro. Il suo sguardo si sofferma sulla loro mancanza di malizia, sul loro selvaggio anticonformismo e tenta di indicare
loro una strada per non farli inglobare da un sistema corrotto e pervasivo.
Il discorso sulla devianza in Pasolini si fa complesso e privo di risposte banali e pacificatorie, alla ricerca di una libertà
che sia vera, piena e non inquadrata in omologazioni e conformismi molto più deleteri e immorali di quella che lui
considera, in questi ragazzi, una forma di “trasgressione” spontanea, vitalistica e in fondo innocua.
Il pensiero di Pasolini arriva ad una radicale condanna delle agenzie attraverso cui è veicolata la formazione in Italia e
che sono preposte alla formazione del minore alla vita adulta: in un altro celebre articolo propone, in maniera un po’
provocatoria e paradossale, ma non priva di accenni sensati, l’abolizione della scuola dell’obbligo e della televisione per
come erano intesi in Italia in quel periodo storico (anni ’60-’70), portatrici entrambe di valori conformisti e involutivi. E’
senz’altro vero che Famiglia-Scuola sono oggi in grave ritardo in confronto all’evolversi degli stili di vita della presunta
modernità, mentre la società muta vorticosamente, e così il mondo aziendale e delle multinazionali, antitetico alle visioni
provincialistiche e ingessate con cui è impostato il modello didattico-formativo nel nostro paese.
Proposte
Si può quindi concludere che la rete delle norme sociali e del controllo sono strumenti indispensabili per la vita
associativa ma spesso imbrigliano e etichettano i comportamenti di individui che portano in sé una quota parte di
innovatività e originalità.
Si rischia così di stigmatizzare chi è portatore di idee e pensieri nuovi, originali e moderni, in virtù della egemonia di una
cultura conservatrice e conformista. Nella maggior parte dei casi l’individuo deviante è senz’altro un individuo nella cui
struttura psichica è possibile riscontrare la presenza di un certo tipo di disturbi di personalità. Le norme sociali, e il
sanzionamento della devianza sono quindi strumenti e metodiche inevitabili in qualsiasi tipo di società che voglia essere
efficacemente funzionante.
Non si può però non rilevare la forza innovativa di certi comportamenti e modi di agire e pensare, e puntare l’attenzione
sull’inadeguatezza delle metodiche educative e di integrazione che fanno capo, in tempi attuali, alle agenzie
fondamentali preposte alla formazione del minore: famiglia e scuola. Nell'opera “Homo ludens” (18) il filosofo olandese
Johan Huizinga concentra la sua attenzione sul gioco come complesso sistema culturale: «(...) ciò non significa che il
gioco muta o si converte in cultura, ma piuttosto che la cultura, nelle sue fasi originarie, porta il carattere di un gioco;
viene rappresentata in forme e stati d'animo ludici: in tale "dualità-unità" di cultura e gioco, gioco è il fatto primario,
oggettivo, percepibile, determinabile concretamente; mentre la cultura non è che la qualifica applicata dal nostro
giudizio storico dato al caso.» Gregory Bateson, invece, individua l'essenza del gioco nel suo essere metalinguaggio:
dato che i giochi sono qualcosa che "non è quello che sembra", perché un'attività ludica sia veramente tale ogni
giocatore deve poter affermare: "Questo è un gioco", cioè ci deve essere la consapevolezza che l'azione è fittizia e che
"meta-comunica" questa sua finzione. Un tipo di percezione della ambivalenza e del carattere contingente circa il proprio
ruolo simile a quanto espresso da Goffman (9). La metacomunicazione, quindi, per Bateson serve per rivelare la natura
del "come se" del gioco, e la sua creazione di un mondo irreale in cui azioni fittizie simulano azioni reali.
Il gioco è quindi un elemento fondamentale dell’esplorazione del mondo da parte del bambino e dell’adolescente. Nel
gioco si simulano regole diverse e alternative a quelle che sottendono al mondo reale. Nel gioco si impara a infrangere
41
P&R Scientific
regole consolidate, a “carnevalizzare” il reale e a sottoporlo ad operazioni di sovvertimento, ribaltamento e
contraddizione.
In molti giochi si incarnano identità fittizie (i role-playing games) e si infrangono e si smontano le regole di
funzionamento della realtà di gioco e anche della realtà percepita, in virtù di una maggiore consapevolezza del proprio
agire e di una più chiara e consapevole comprensione dei meccanismi di interazione con gli altri. Metodi didattici
all’avanguardia, che senz’altro forniscano al discente griglie comportamentali e regole di condotta, che però al tempo
stesso non dimentichino la dimensione fondamentale del gioco, dello svago e della ricerca personale, sono da incentivare
fortemente.
Con la consapevolezza che, nel bambino e nell’adolescente, “trasgredire” determinate regole con coscienza critica e
capacità di discernimento, aiuta a formare un cittadino consapevole, responsabile e rivolto all’innovazione di paradigmi
comportamentali spesso datati e inadeguati, anche se comunemente accettati con passività dalla maggioranza (19).
Bibliografia
1.
Noelle-Neumann E. La Spirale del Silenzio – per una teoria dell’opinione pubblica. Meltemi, Roma, 2002.
2.
Barbagli M, Asher C, Savona E. Sociologia della Devianza, Il Mulino, Bologna, 2003.
3.
Durkheim, E. Les règles de la méthode sociologique, Alcan, Paris, 1895 ; Le regole del metodo sociologico, Edizioni
di Comunità, Milano, 1963.
4.
Parsons, T. The Social System, Free Press, Glencoe, Ill. 1951; Il sistema sociale, Edizioni di Comunità, Milano, 1965.
5.
Cohen A.K. Delinquent Boys. The Culture of the Gang, Free Press, New York, 1955; Ragazzi delinquenti, Feltrinelli,
Milano, 1963.
6.
Sutherland E.H. White Collar Crime, Holt, New York, 1949; Il crimine dei colletti bianchi: la versione integrale,
Giuffrè, Milano, 1987.
7.
Merton R.K. Social Structure and Anomia. American Sociological Review, 1938; III, 5:672-682.
Teoria e struttura sociale, Il Mulino, Bologna, 1966.
8.
Gemmill G. The dynamics of scapegoating in small groups, in Small Group Research, 1989;Vol. 20 n.4.
9.
Goffman E. The Presentation of Self in Everyday Life, Anchor Books, Doubleday, 1956; La vita quotidiana come
rappresentazione, Il Mulino, Bologna,1969.
10. Marcuse H. L’uomo a una dimensione, Beacon Press, Boston, 1966;Einaudi, 1967.
11. Foucault M. Surveiller et punir. Naissance de la prison, Gallimard, Paris, 1974 ; Sorvegliare e punire. La nascita
della prigione, Einaudi, Torino, 1976.
12. Foucault M. Microfisica del Potere, Einaudi, Torino,1977.
13. Marcuse H. Eros e Civiltà, Beacon Press, Boston, 1955; Einaudi, 1964.
14. Mead G.H. Mind, Self and Society, University of Chicago Press, Chicago, 1934. Mente, sé e società, Giunti, Firenze,
1992.
15. Reich W. Psicologia di massa del fascismo, 1933. Belfiore F, Wolf A, SugarCo, Milano 1971; Mondadori, Milano 1974.
16. Reich W. Analisi del carattere 1933. Belfiore F, Wolf A, SugarCo, Milano 1973.
17. Pasolini P P. Lettere Luterane. Garzanti, Milano 1975.
18. Huizinga J. Homo ludens. Amsterdam ,1938. Einaudi, Torino, 1972.
19. Bateson G. Verso un’ecologia della mente1972. Adelphi, Milano, 1977.
42
P&R Scientific
Autore di riferimento: Gianfranco Tomei
"Sapienza" Università di Roma, Dipartimento di Neurologia e Psichiatria
Viale dell'Università, 30 00185 Roma – Italia
Corresponding Author: G. Tomei
"Sapienza", University of Rome, Department of Neurology and Psychiatry
Viale dell'Università, 30 00185 Rome – Italy
43
P&R Scientific
HUMAN HERPES VIRUS 8 (HHV-8): SALIVARY SHEDDING IN
MOTHERS AND CHILDREN FROM UGANDA: RISK FACTORS
AND CLUES ABOUT TRANSMISSION
HERPESVIRUS UMANO 8 (HHV-8) NELLA SECREZIONE SALIVARE DI MADRI E
BAMBINI DELL’UGANDA: FATTORI DI RISCHIO PER LA TRASMISSIONE
Romano R 1, Gramolelli S 1, Tabacchi F 1, Russo G 1, Verzaro S 1, Marinucci F 2, Paganotti GM1,
Gaeta A 1, Coluzzi M 1
1
Department of Public Health and Infectious Diseases, “Sapienza” University of Rome, Piazzale A.
Moro 5, 00185 Rome – Italy
2
Institute of Human Virology, University of Maryland School of Medicine, 725 Lombard street,
Baltimore, MD 21201, USA
1
"Sapienza", Università di Roma, Dipartimento di Sanità Pubblica e Malattie Infettive,
Piazzale A. Moro 5, 00185 Roma - Italia
2
Università del Maryland, Facoltà di Medicina, Istituto di Virologia Umana, 725 Lombard street, Baltimora,
MD 21201, USA
Citation: Romano R, Gramolelli S, Tabacchi F, et al. Human herpes virus 8 (HHV-8): salivary shedding in
mothers and children from Uganda: risk factors and clues about transmission. Prevent Res 2011; 1 (1): 44-52
Key words: HHV-8 mother-to-child transmission, saliva, promoter arthropod, Real Time
PCR, skin reaction
Parole chiave: HHV-8 mother-to-child transmission, saliva, promoter arthropod, Real Time
PCR, skin reaction
Abstract
Background: In Africa, the increase of Kaposi Sarcoma-associated herpesvirus (KSHV/HHV-8) seroprevalence during
childhood suggests an horizontal intrafamilial transmission. However, the exact transmission modes are yet unknown,
but there is evidence that the virus is intermittently spread through the saliva of seropositive subjects. Furthermore, a
significant correlation was found between the geographical distribution of bloodsucking arthropods and incidence rates of
Kaposi Sarcoma.
In this view, key roles can be played by: i) the inflammatory reaction due to the arthropod bites which create a microenvironment favourable to viral replication, and ii) the use of saliva as first-aid medication in several circumstances.
According to that, the viral transmission could occur through the application of mothers’ saliva on children’s skin to
relieve itching and scratching after a bloodsucking arthropod bite. This work investigated the risk factors involved in
HHV-8 transmission based on the “promoter arthropod hypothesis”
Methods: One hundred and thirty questionnaires were administered to Ugandan children (54 from settled and 76 from
nomad communities) to evaluate the skin inflammatory reaction to the arthropod bites and the frequency of behavioural
44
P&R Scientific
practices associated with saliva usage. At the same time saliva samples were collected from the children and respective
mothers to detect the presence of HHV-8 DNA by Real-Time PCR.
Results: We detected HHV-8 DNA in 23.85% of mothers and in 11.54% of children. The use of saliva is significantly
more common in settled than in the nomad communities (p<0.001). The statistical analysis showed significant
association between: i) skin inflammatory reaction and use of saliva in both groups; ii) environmental factors which
increase the presence of bloodsucking arthropods and skin inflammatory reaction.
Conclusions: From our data we suppose that HHV-8 transmission could occur through non-sexual routes and
that the infected saliva applied at child bite site could represent a favourable condition for the acquiring of the infection.
Abstract
Introduzione: In Africa, l’aumento della sieroprevalenza del sarcoma di Kaposi associato all’herpesvirus (KSHV/HHV-8)
durante l’infanzia, suggerisce una trasmissione orizzontale intrafamiliare.
Anche se, le esatte modalità di trasmissione sono ancora sconosciute, ci sono prove evidenti che il virus è diffuso in
modo intermittente attraverso la saliva di soggetti sieropositivi. Inoltre, è stata trovata una correlazione significativa tra
la distribuzione geografica degli artropodi ematofagi e i tassi di incidenza del sarcoma di Kaposi.
Alla luce di ciò, ruoli chiave possono essere svolti da : i) la reazione infiammatoria dovuta alle punture dell’artropode che
creano un micro-ambiente favorevole alla replicazione virale; ii) l’uso della saliva come prima medicazione in diverse
circostanze. In accordo con questo, la trasmissione virale potrebbe avvenire attraverso l’applicazione della saliva
materna sulla pelle dei bambini per lenire il fastidio e il prurito provocati dalla puntura dell’artropode promotore. Questo
lavoro si è occupato dei fattori di rischio coinvolti nella trasmissione dell’HHV-8 basata “sull’ipotesi dell’artropode
promotore”.
Metodi: Centotrenta questionari sono stati somministrati a bambini dell’Uganda (54 provenienti da una comunità
stanziale e 76 da una comunità nomade) per valutare la reazione infiammatoria cutanea provocata dalle punture degli
artropodi e la frequenza delle pratiche comportamentali associate all’uso della saliva. Contemporaneamente sono stati
prelevati campioni di saliva dai bambini e dalle rispettive madri per determinare la presenza del DNA dell’HHV-8
attraverso la Real-Time PCR.
Risultati: Abbiamo trovato DNA dell’HHV-8 nel 23.85% delle madri e nell’11.54% dei bambini. L’uso della saliva è
significativamente più utilizzata nella comunità stanziale rispetto a quella nomade (p<001). Le analisi statistiche hanno
mostrato un’associazione significativa tra: i) la reazione infiammatoria cutanea e l’uso della saliva in entrambe le
comunità; ii) i fattori ambientali che incrementano la presenza di artropodi ematofagi e la reazione infiammatoria
cutanea.
Conclusioni: Dai nostri dati possiamo suggerire che la trasmissione dell’ HHV-8 possa avvenire anche attraverso
modalità non sessuali e che la saliva infetta, applicata al bambino sul sito della puntura, possa rappresentare una
condizione favorevole alla trasmissione dell’infezione.
45
P&R Scientific
Background
The Human herpesvirus 8 (HHV-8) is the causal agent of Kaposi sarcoma (KS) [7] and is also involved in the
pathogenesis of two rare lymphoproliferative disorders: Multicentric Castleman disease (MCD) [18] and Primary Effusion
Lymphoma (PEL) [6]. In sub-Saharan Africa, after the onset of AIDS epidemic, KS became the most common
malignancy among adults and the second most prevalent cancer type in childhood [15]. Thus, KS emerges as an
important problem in sub-Saharan African public health; the investigation of HHV-8 transmission route in these areas
represents a key factor in the reduction of KS incidence.
HHV-8 seroprevalence in sub-Saharan Africa ranges from 40 to 70% in adults [10,13,17] while in Mediterranean
countries is reported to be 16.5-18.7%[17]. Interestingly, in Amerindian tribes HHV-8 is hyperendemic showing the
highest infection rates ever reported (about 80% in adults) [12,9].
In Uganda HHV-8 seroprevalence ranges from 26.6% in children to 40.1% in adults showing an age dependent increase
during childhood [13,5,14]. In several studies is reported that saliva is the major reservoir of HHV-8 [12,14]. Salivary
secretions of seropositive subjects intermittently harbour the virus at greatest concentrations [11,19].
Despite in adulthood the transmission is associated both to unprotected sexual activity and drug injection [16], among
children a primary role could be played by salivary transmission [5,14,11]. In particular, the contagion could occur
through exposure to infected saliva from mothers or other family members [14], even if little is known about the
behavioural practices that expose children to saliva [4]. In African countries the use of traditional methods is very
common, this practices mainly consist on the application of either saliva or premasticated herbs on child’s skin as firstaid medication in several circumstances [4,20].
One hypothesis is that HHV-8 could be transmitted by a seropositive mother to the child through the application of the
infected saliva on child's skin to relieve itching and scratching due to a bloodsucking arthropod bite. These arthropods
does not inject the virus, prepare instead the cellular and biochemical skin environment promoting the virus infection
[8].
During the blood meal, an arthropod inoculates antihaemostatic and immunogenic molecules which evoke an immune
response at the bite site with release of Th2 cytokines [1]. As HHV-8 is avid in these cytokines, viral transmission could
be facilitated by a strong inflammatory reaction [8,3,2].
Furthermore, several works underlined a significant correlation between the geographical distribution of bloodsucking
arthropods and incidence rates of KS [3,2]. According to that, the arthropod, through its biting, increases HHV-8
transmission risk, acquiring the role of “promoter of the infection” [8]. In this study, according to this hypothesis, the
risk factors involved in HHV-8 transmission had been investigated: the presence of HHV-8 DNA in saliva of mothers and
respective children, the use of maternal saliva to soothe children’s itchy bites, the skin inflammatory reaction at
children’s bite site as well as the presence of environmental factors which could increase the presence of bloodsucking
arthropods.
Furthermore, to reveal differences in HHV-8 spreading and saliva-based behavioural practices, we included in the study
two groups: settled and nomad communities from Uganda. The former group lived mainly in urban contexts showing
sedentary habits, whereas, the latter owned to a nomadic community which was moving throughout Karamoja region to
feed livestock.
Methods
Study area and subjects
One hundred and thirty Ugandan children and respective mothers had been enrolled in the study in 2007 and 2008. The
questionnaires were administered at children (3-13 years old) in schools or health care centres. Moreover, saliva
samples from the interviewed children and mothers had been collected, spotted on filter paper, air dried and then
separately stored in plastic envelopes.
Formal permission and signed informed consent were respectively obtained from the local authorities (school and health
care service directors) and from parents or caregivers of the children.
Study participants were recruited in settled (54 children and 54 mothers) and nomad (76 children and 76 mothers)
communities, the former from Kampala-Great Valley children centre, Moroto-Kakoliye and Kasimeri primary schools,
Rupa-Kidepo health centre and the latter from Karamoja-Namalu health centre.
Questionnaires
Questionnaires were developed by one of the authors (Romano R). The first section included sociodemographic
46
P&R Scientific
questions about age, gender, lifestyle (i.e. nomad or settled community, access to water facilities and presence of
stagnant or running water in the neighbourhood), presence of domestic animals (i.e. hens, cows, sheep, goats,
donkeys). The second section included questions about possible promoter arthropods (i.e. perception of insects in the
environment, bite frequency, part of the body most bitten). The third section included questions on bitten skin reaction
(i.e. itchiness, pain, irritation, swelling, skin mark, lasting of reaction). The last section concerned treatment received
(i.e. traditional methods including saliva and premasticated herbs or pharmaceutical products).
DNA extraction.
Each sample had been observed through an UV lamp to detect the exact position of the salivary fluid on filter paper,
then the spot (e.g. circle) was excised and placed into 2.0 ml lysis buffer for elution from the filter card by overnight
gently rocking at room temperature. Then the paper was removed from the tube and the samples were immediately
processed. DNA was extracted by an automatic nucleic acid extractor (Nuclisens EasyMag – Biomerieux). Each specimen
samples added with 65 µl of magnetic silica, 10 µl of internal extraction control (CPE, Nanogen Advanced Diagnostics),
represented by purified beta globin sequence, and 55 µl of wash reagent was loaded in the instrument; DNA was
automatically extracted, eluted in 55 µl of specific buffer and immediately used in Real Time PCR, an aliquot was stored
at -80°C.
Real Time PCR
Extracted DNA was analyzed for the presence of HHV8 by Qualitative TaqMan Real Time PCR using a commercially
available kit (Nanogen Advanced Diagnostics, Italy). The assay was performed by a multiplex format targeting both the
sequence of HHV8 capsid protein gp27 and beta globin control gene by using 5’ reporter dye 6-carboxyfluorescein
(FAM)-labelled probe for viral gene and 5’ fluorescein (VIC)-labelled probe for human beta globin respectively. Each PCR
reaction was carried out with ABI PRISM 7000 (Applied Biosystem, USA) in 96-well plate, adding 5 µl of extracted DNA
to 20 µl of amplification mixture. In each run HHV8 specific standard constructed with dilutions of plasmids carrying the
specific viral gene was added. The standard was used as positive amplification control. The real time PCR thermal profile
consisted of a first cycle at 50°C for 2 minutes, a second cycle at 95°C for 10 minutes, and a third step which included
45 cycles at 95°C for 15 seconds and 60°C for 1 min. The analysis was performed in two hours for single run and, at the
end of the process, computer reported as “positives” the samples that showed HHV-8 DNA presence.
Data analysis
SPSS, Version 16, was used for statistical analyses. In both communities descriptive statistics were calculated for each
question related to the type of treatment received (saliva and premasticated herbs or pharmacological products) and
risk factors. Yates-corrected χ2 test, calculated odds ratios (OR) with their corresponding 95% confidence intervals (CI),
were used to investigate factors associated with traditional methods.
Furthermore, binary logistic regression analysis (BLR) was used to investigate the impact of different independent risk
factors on the dependent dichotomous variable “use of traditional methods”. Risk factors included symptoms related to
the bite (itchiness, pain, irritation, swelling, skin mark and duration of reaction) and environmental factors (stagnant
water and domestic/courtyard animals) which increase the presence of bloodsucking arthropods. These factors represent
the most important ones related to the HHV-8 transmission route through promoter arthropods. We report only
significant associations (p<0.05). Sample sizes differ slightly between tests because of missing values due to lack of
answers.
Results
The median age of the children was 6.95 years (range, 3-13 years) and 43% (56/130) were male. Children from settled
and nomad communities did not significantly differ in the distribution of age and gender. Data about mother’s age were
not collected.
Frequencies of questionnaires answers directed to children and related to risk factors involved in HHV-8 transmission are
shown in table 1. The majority of children (99.6% = 129/130) declared to have been bitten (data not shown) and
35.45% (39/110) of them receive traditional methods as treatment. Furthermore, among the symptoms of the
inflammatory skin reaction, itching is reported to be the most widespread (61.42%) and more than a half of children
were exposed to both selected environmental factors (Table1)
47
P&R Scientific
In the total sample of children enrolled, the BLR analysis showed that the use of traditional method is directly associated
with symptoms of skin reaction such as irritation (OR: 2.96; 95% CI: 2.41-153.91; p= 0.04) and swelling (OR: 3.17;
95% CI: 2.95-191.18; p= 0.03). Moreover, statistical analysis revealed a direct relationship between selected
environmental factors and symptoms of skin reaction.
In particular, the presence of either domestic or courtyard animals is associated with skin pain due to the arthropod bite
(OR: 5.25; 95% CI: 2.33-28.81; Yates corrected χ2= 13.68; p<0.001); in addition, the presence of running or stagnant
water nearby home location is directly related to skin swelling (OR: 5.69; 95% CI: 2.27-14.25; Yates corrected χ2=
13.89; p<0.001).Comparing the frequencies of the risk factors between the settled and the nomad communities,
statistically significant differences had been revealed. In particular, in the settled community are more widespread: i)
the use of traditional methods (Yates-corrected χ2 : 12.98; OR: 3.05; 95% CI: 1.69-5.53; p< 0.001); ii) swelling
(Yates-corrected χ2 : 36.98; OR: 8.54; 95% CI: 4.12- 17.68; p< 0.001); iii) itchiness (Yates-corrected χ2 : 60.55; OR:
18.90; 95% CI: 8.14- 43.88; p< 0.001). Furthermore, statistical analysis revealed that in the nomad community more
frequently recur: i) pain (Yates-corrected χ2 : 18.47; OR: 4.07; 95% CI: 2.15- 7.68; p< 0.001); ii) presence of running
or stagnant water nearby domestic location (Yates-corrected χ2 : 60.55; OR: 18.90; 95% CI: 8.14- 43.88; p< 0.001);
iii) presence of domestic/courtyard animals (Yates-corrected χ2 : 25.79; OR: 4.84; 95% CI: 2.63- 8.90; p< 0.001).
There were no statistically significant differences between the settled and nomad communities in the frequencies of both
use of pharmacological products and irritation.
Frequencies of the HHV-8 DNA in mothers’ and children’s saliva are reported in Table 2.
48
P&R Scientific
Table 2 HHV-8 DNA in saliva
CI: confidence interval; OR: odds ratio.
Overall, we detected HHV-8 DNA in 31 mothers and 15 children. Statistically significant differences in the presence of
HHV-8 DNA in mothers’ and children’s saliva emerged just in the nomad group, whereas in the settled one this
difference had not been revealed.
Out of 15 HHV-8 DNA-positive children, 7 (1 male and 6 females) belonged to the nomad community and 8 to the
settled one (1 male and 7 females). Overall, the sex ratio among children was 13 females vs. 2 males (p=0.03) as
shown in Table3. Moreover, we divided children according to age into two groups: the former included children between
3 and 6 years of age and the latter those between 7 and 13 years. Results indicated that the majority of positive
children (11/15) were aged between 7 and 13 years (p=0.01), included both HHV8 positive male children. There is no
statistically significant relationship between the use of traditional methods and the children’s positivity to HHV-8
presence in saliva, as reported in Table 3.
Table 3 Relationship between HHV-8 DNA in children's saliva and risk factors
#: use of saliva and/or premasticated herbs; CI: confidence interval; OR: odds ratio.
49
P&R Scientific
Discussion
In sub-Saharan Africa, several studies proposed that an horizontal mother-to-child HHV-8 transmission through saliva
could occur [5,14]. We suggest that, according to the promoter arthropod hypothesis, the virus could be transmitted
through the application of mothers' infected saliva on child's skin (traditional methods) to relieve itching and scratching
due to a bloodsucking arthropod bite [8].
Traditional practices as treatment of insect bites are quite widespread in the selected communities: about one third of
the interviewed children declared to receive them after a bloodsucking arthropod bite. Furthermore, this habit is
significantly more common among mothers from settled community rather than among those from the nomad one, this
difference is probably due to the different ways of life of the two populations.
The nomad group, in fact, belongs to the Karimojong tribe which exhibits semi-nomadic habits and remained culturally
isolated from the rest of Ugandan urban population.
The symptoms of the skin inflammatory reaction after a bloodsucking arthropod bite, in particular skin irritation and
swelling, are directly associated with the use of traditional methods. Thus, child's immune response to the bite induces
mothers to apply their own saliva at the bite site. In addition, this data underlines the promoting role of the arthropod
bite: its blood meal on the young host, which evokes irritation and swelling, increases the children's exposure to
maternal saliva and the risk of HHV-8 mother-to-child transmission. This is one of the first studies which aims to reveal
HHV-8 DNA in healthy mothers' and children's saliva living in HHV-8 endemic areas.
The prevalence rates of HHV-8 DNA in saliva represent an underestimation of the real seroprevalence because of the
intermittent characteristics of the viral salivary shedding.
The presence of HHV-8 DNA in not-yet-sexually-active children's saliva confirms that in the selected area intrafamilial
horizontal transmission could occur. Moreover, the percentage of positive children increases with age, according to
previous seroprevalence studies in this area. [17,5,14] However, unexpectedly, we found a significant higher prevalence
of the virus in female children's saliva. Despite it is known that African Kaposi Sarcoma affects mainly male population,
the infection in endemic areas seems to occur with the same frequency in both gender. [15,10,13,5,14] Thus, the data
we found are not in agreement with previous studies. One explanation is that female children, who live closer to the
mother than their male counterpart, are more exposed to maternal saliva. However, this fact needs to be confirmed by
further large-scale studies.
The association between the symptoms of skin inflammatory reaction and environmental factors indicates that
domestic/courtyard animals and water sources could increase the presence of bloodsucking arthropods promoter of
HHV-8 infection. In particular, water reservoirs represent micro-environment favourable to life and reproduction of
several bloodsucking arthropods which at least for one phase of their lifecycle depend on water (Coquillettidia, Aedes,
Ochlerotatus, Culicoides and Leptoconops) [8,3,2].
Moreover, the presence of domestic/courtyard animals is directly related to skin pain, indicating that this environmental
factor could increase the presence of zoophilic bloodsucking arthropods (Phlebotomus and Simulium). [8]. These insects
are not completely adapted to bite the human host and are able to produce a more intense inflammatory reaction at the
bite site. Comparing data from settled and nomad communities, a statistically significant difference in the use of
traditional methods emerges.
In line with these data, the use of saliva is more widespread in the settled population than in the nomad one. Moreover,
significant difference in the presence of viral DNA in mothers and their children had been recorded just in the nomad
community. On the other hand, in the settled group, where a half of children declare to receive their mother’s saliva at
the bite site, there is no difference in the presence of viral DNA between mothers and respective children.
Thus, in the settled community non-sexual transmission of HHV-8 represents the main source of contagion. In this
context the infection is essentially acquired during childhood, whereas in the nomad group the infection seems to occur
primarily during adulthood, probably through a sexual route.
Conclusions
HHV-8 DNA was found in both children’s and mothers’ saliva.
Symptoms of skin reaction due to a bloodsucking arthropod bite and some environmental factors associated with
bloodsucking arthropod presence are associated to the use of mother’s saliva on children skin to reduce itching and
scratching, and thus increase the risk of HHV-8 intrafamilial horizontal transmission. Anyway, further large-scale
epidemiological surveys are needed to confirm these findings.
50
P&R Scientific
Authors’ contributions
RR organised the field survey, analyzed data and edited manuscript. GS performed molecular and statistical analysis,
drafted the manuscript. TF performed the database, statistical analysis and drafted the manuscript. RG carried out the
field survey. VS performed molecular analyses. MF organised and supervised the field survey. PGM performed statistical
analyses and drafted the manuscript. GA supervised the molecular analyses and edited the manuscript. CM conceived
the study and edited the manuscript. All authors read and approved the final manuscript.
Acknowledgements
We are particularly grateful to all the children interviewed, the technical staff for their support, the schools and health
care service directors for their collaborative attitude throughout the investigation.
Competing interests
The authors declare that they have no competing interests.
References
1. Andrade BB, Teixeira CR, Barral A, Barral-Netto M. Haematophagous arthropod saliva and host defense system: a tale
of tear and blood. Ann Braz Ac Sciences 2005; 77:665-693.
2. Ascoli V, Facchinelli L, Valerio L, et al. Kaposi's sarcoma, human herpesvirus 8 infection and the potential role of
promoter-arthropod bites in northern Sweden. J Med Virol 2006;11:1452-1455.
3. Ascoli V, Senis G, Zucchetto A, et al: Distribution of “promoter” sandflies associated with incidence of classic Kaposi’s
sarcoma. Med Vet Entomol 2009; 3:217-225.
4. Butler LM, Neilands TB, Mosam Mzolo S. A population-based study of how children are exposed to saliva in KwaZuluNatal Province, South Africa: implications for the spread of saliva-borne pathogens to children. Trop Med Int Health
2010; 15:442-453.
5. Butler LM, Were WA, Downing R, et al. Human Herpesvirus 8 infection in children and adults in a population-based
study in rural Uganda. J Infect Dis 2011; 203:625-634.
6. Cesarman E, Chang Y, Moore PS et al. Kaposi's sarcoma-associated herpesvirus-like DNA sequences in AIDS-related
body-cavity-based lymphomas. N Engl J Med 1995; 332:1186-1191.
7. Chang Y, Cesarman E, Pessin MS et al. Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi’s
sarcoma. Science 1994; 266:1865–1869.
8. Coluzzi M, Manno D, Guzzinati S et al. The bloodsucking arthropod bite as possible cofactor in the transmission of
human herpesvirus-8 infection and in the expression of Kaposi's sarcoma disease. Parassitol 2002; 44:122-123.
9. Cunha AM, Caterino-de-Araujo A, Costa SC et al. Increasing seroprevalence of human Herpesvirus 8 (HHV-8) with
age confirms HHV-8 endemicity in Amazon Amerindians from Brazil. J Gen Virol. 2005; 9: 2433-2437.
10. Dedicoat M, Newton R. Review of the distribution of Kaposi' s sarcoma associated herpesvirus (KSHV) in Africa in
relation to the incidence of Kaposi' s sarcoma. Br J Cancer 2003; 88:1-3.
11. De Franca TR, De Araùjo RA, Ribeiro CM. Salivary shedding of HHV-8 in people infected or not by human
immunodeficiency virus 1. J Oral Pathol Med 2011; 40: 97-102.
12. de Souza VA, Sumita LM, Nascimento MC et al: Human Herpes virus- 8 infection and oral shedding in Amerindian
and non Amerindian populations in the Brazilian Amazon region. J Infect Dis 2007; 96: 844-852.
13. Dollard SC, Butler LM, Jones AM et al. Substantial regional differences in human herpesvirus 8 seroprevalence in
sub-Saharan Africa: insights on the origin of the "Kaposi's sarcoma belt". Int J Cancer 2010; 127:2395-2401.
14. Mbulaiteye SM, Pfeiffer RM, Whitby D et al. Human herpesvirus 8 infection within families in rural Tanzania. J Infect
Dis 2003; 187: 1780-1785.
15. Parkin DM, Sitas F, Chirenje M et al. Part I: Cancer in Indigenous Africans-burden, distribution and trends. The
Lancet Oncol 2008; 9:682-692.
16. Perna AM, Bonura F, Vitale F et al. Antibodies to human Herpesvirus 8 (HHV8) in general population and in
individuals at risk for sexually transmitted diseases in Western Sicily. Int J epidemiol 2000; 1:175-179.
17. Serraino D, Toma L, Buttò S et al. A seroprevalence study of humanherpesvirus type 8 (HHV8) in eastern and
Central Africa and in the Mediterranean area. Eur J Epidemiol 2001; 17: 871-876.
51
P&R Scientific
18. Soulier J, Grollet L, Oksenhendler E et al. Kaposi's sarcoma-associated herpesvirus-like DNA sequences in
multicentric Castleman's disease. Blood 1995; 86:1276-1280.
19. Taylor MM, Chohan B, Lavreys L et al. Shedding of human herpesvirus 8 in oral and genital secretions from HIV-1seropositive and -seronegative Kenyan women. J Infect Dis 2004; 190: 484-488.
20. Wojcicki JM. Traditional behavioural practices, the exchange of saliva and HHV-8 transmission in sub-Saharan
African populations. Br J Cancer 2003; 89:2016-2017.
Corresponding Author: Rita Romano
"Sapienza", University of Rome, Department of Public Health and Infectious Diseases
Piazzale A. Moro 5, 00185 Rome – Italy
Autore di riferimento: Rita Romano
"Sapienza", Università di Roma, Dipartimento di Sanità Pubblica e Malattie Infettive
Piazzale A. Moro 5, 00185 Roma – Italia
52
P&R Scientific
PREDICTIVE MEDICINE IN CARDIOVASCULAR DISEASES.
WHAT NEXT?
LA MEDICINA PREVENTIVA NELLE PATOLOGIE CARDIOVASCOLARI. COSA CI
RISERVA IL FUTURO?
Andreozzi P 1, Viscogliosi G 1, Servello A 1, Marigliano B 1, Ettorre E 1, Marigliano V
1
1
“Sapienza” University of Rome . “Predictive Medicine Unit”. Department of Cardiovascular, Respiratory,
Nephrologic and Geriatric Sciences”
1
"Sapienza" Università di Roma. “Unità di Medicina Predittiva”, Dipartimento di Scienze
Cardiovascolari, Respiratorie, Nefrologiche e Geriatriche”
Citation: Andreozzi P, Viscogliosi G, Servello A, et al. Predictive medicine in Cardiovascular Diseases.
What next? Prevent Res 2011; 1 (1): 53-59
Key words: Prediction, Permissive genotypes, Cardiovascular diseases
Parole chiave: Previsione, genotipo Permissivo, malattie Cardiovascolari
Abstract
Background: The full knowledge of the human genome, derived from its sequencing in 2001’s has led to increasingly
understand the importance of the genes/environment interactions and has allow to characterize several genetic factors that
can determine the individual susceptibility to certain diseases.
Objectives: The emergence of predictive medicine is a consequence of this knowledge, and it plays an important role in
chronic-degenerative diseases.
Methods: We selected several studies by the critical analysis of the current literature about the predictive methods in
cardiovascular diseases.
Results: In cardiovascular diseases the predictive approach allows to screen high-risk subjects and to implement a
personalized therapeutic approach.
Conclusions: In the future the personalized predictive medicine will be the best approach in pre-clinical diagnosis and
management of chronic-degenerative diseases.
53
P&R Scientific
Abstract
Introduzione: La conoscenza ormai completa del genoma umano, derivate dal suo sequenziamento nel 2001, ha
permesso di comprendere sempre più l’importanza delle interazioni gene/ambiente e la caratterizzazione di fattori genetici
individuali che possono determinare suscettibilità ad ammalare per determinate patologie.
Obiettivi: Da queste conoscenze è nata la medicina predittiva, la cui importanza si riflette soprattutto nell’ambito delle
malattie cronico-degenerative.
Materiale e metodo: Dall’analisi critica della letteratura corrente, sono stati selezionati diversi studi circa la validità
dell’approccio predittivo nelle malattie cardiovascolari.
Risultati: Nelle malattie cardiovascolari la medicina predittiva offre innumerevoli possibilità, sia per lo screening dei
soggetti a rischio sia per un approccio terapeutico personalizzato.
Conclusioni: La medicina predittiva personalizzata rappresenterà in futuro il modello più adatto per la diagnosi pre-clinica
e la gestione delle malattie cronico-degenerative.
Background
The “Omics” era
In 2001 was completed the human genome sequencing by the Human Genome Project, and the new knowledge on genetics
have allowed us to understand the genetic basis of several diseases. Diseases and the individual genetic are viewed in a new
integrated perspective: DNA can not be considered a closed system that works independently, but it has complex
relationships with the environment at many levels. Gene expression is highly unstable and continuously influenced by
external factors, and the plasticity of the genome can be seen when it must cope to various kind of exogenous stressors. In
this post-genomic era born the concept of functional genomics that concerns the understanding of how genes work and how
they interact in complex pathways (1). Emerging high-throughput technologies make it possible to analyze genes, proteins
and metabolites in a holistic and integrated way. In this context have developed the concepts of proteomics, metabolomics
and transcriptomics (2). “Omics” refers to comprehensive methodologies that attempt to analyse the complete output of an
organism’s genes (genomics), transcripted RNA (transcriptomics), metabolites (metabolomics) and proteins (proteomics).
The recent increasing availability of integrated data and development of computational analyses have make it possible to
translate these aforementioned concepts into practice. The functional
or dynamic genomics is based on the integration of clinical informations, biology, informatics engineering and etics (see
Figure 1.).
Permissive genotypes and gene/environment interactions
“Permissive genotype” is the most important issue derived from the genomics for its great practical relevance. Diseases
occur either due to innate constitutional factors as well as to environmental factors: the genetics can be individually set up to
produce certain disease but also to protect against other, and the environment comes into play on this given genotypic set.
However not all individuals who share the same unfavorable environment develop the disease, and not all individuals who
share the same favorable environment are immune from the disease. Genomics has taught us that almost all diseases
require a permissive genotype to rise up. So permissive genotypes are the biological basis of disease susceptibility and codify
the way in which each person interacts with the environment through its genetics.
Genes and environment can interact mainly in 3 ways: physical and chemical external factors can directly affect DNA;
epigenetic modulation of DNA may lead to genes silencing or expression; individual genetic variations may direct the
response to the environment. The individual genetic variability may consist in more or less evident alterations in DNA
structure. Individuals are distinguished from one another by 0.1% difference in the genome nucleotide sequence. The most
striking examples are the loss or gain of entire chromosomes (monosomy, trisomy) or deletions and translocations. However
the inter-individual variance in DNA sequence is mainly due to single nucleotide polymorphisms (SNP), characterized by the
54
P&R Scientific
substitution of a single base pair in gene sequence. SNPs occur in the population with an allele frequency of 1% or more
(1,3,4). The base pair substitution may result in the synthesis of the same aminoacid on the polypeptidic translated chain
(synonymous encoding), or in a different aminoacid (not-synonymous encoding) or in the non-codification when the mutation
involves a transcriptional gene region. It is clear that when genetic variation is pervasive and clinically relevant, it leads to a
morbid phenotype. Isolated SNP generally causes poor alteration in the wild protein concentration and function (3,4).
However the presence of many SNPs in the same genome is more able to determine innate frailty. SNPs occurrence is often
the determinant of susceptibility to disease in many polygenic human conditions such as the cardiovascular disease. Complex
genetic disease depends at last on the interaction between several different genes with environmental factors.
The predictive medicine
Definition
Modern medicine has the opportunity to use the genomics knowledge about molecular phenotypes and genetic background
and biomarkers. Predictive medicine is the direct consequence of this. It is a new model of medicine that applies to healthy
individuals and aims the preservation of the health state rather than the treatment of disease. While preventive medicine is
based on epidemiology and applies to whole population, the predictive medicine is personalized and it is based on genetics.
Genetic screening allows to detect “healthy” genetically frail individuals at risk of developing
a particular disease in time. The knowledge of an individual genetic background surely allows us to detect an individual
genetic predisposition to a certain disease through the evaluation of selected polymorphysms of genes potentially associated
with a morbid condition (4,5).
Through the genetic characterization we are able to distinguish clinical subtypes of a single disease to better implement
prevention and/or early intervention. The predictive diagnosis can then reveal the genetic predisposition and quantifies the
risk of a disease development in life time (1,4-8). The current risk factor profiling derives from epidemiology, and it is based
on large populations studies. Predictive medicine will change this approach by the introduction of genomics profile upon
epidemiological investigations, focusing on individuals.
Predictive medicine is a complex and integrated approach to the patient. It results from laboratory technologies, statistics,
genetic and environmental risk factors detection, in order to outline the possible clinical history of the individual and to
interfere if it is possible through the implementing of personalized lifestyle and therapy (5-11): since we know the individual
predisposition to ill, we can easily work on known risk factors to eliminate or delay the disease onset. At this time we are not
able to operate on the genotype, but only on the modifiable risk factors. The knowledge of an individual frailty allow us to
avoid certain risk factors and get in protective factors. Some polymorphisms are currently typified, overall in the fields of
atherotrombosis, inflammation, hypertension and oncology (11-13). However many potential clinical useful polymorphisms
are not still available.
Applications: the pharmacogenomics and the nutrigenomics
The greatest areas of theorical and clinical application of genomics are clinical nutrition and pharmacotherapy (1,3,14). The
genomic knowledge have given new basis to pharmacotherapy. Both drug efficacy and safety may be potentially improved by
the genotype-based pharmacotherapy, according to the paradigm of “the right drug for the right patient at the right dose at
the right time”. Pharmacogenomics analyse the way which an individual genetics modulates the interaction with an external
molecule (the drug). The response to pharmacotherapy can be highly variable among subjects, and pharmacogenomics
explain these differences in term of individual genetic variations. To better understand the individual response to a drug
should be targeted the entire metabolic pathways involved in drugs dynamics and kinetics, rather than the single gene or
protein characterization. Adverse drug reactions may also be predicted or closely monitored by genomic and proteomic
profiling. The examples of characterized polymorphisms involved in drug response are numerous (14,15). For example
variants of CYP2C9, CYPC9*2 and CYPC9*3, involved in warfarin metabolism, confer less efficiency than wild allele in drug
clearance, increasing the risk of bleeding. Polymorphisms Arg389 and Gly389 of the β1-adrenergic receptor gene confers a
differential response to β-blockers. Current limitations of pharmacogenomics are the lack of integrated clinical trials (15,16).
Through the future complete characterization of the individual differences in the entire metabolic pathways that underlie the
drug effects and metabolism, and the application of pharmacogenomics to large clinical trials we can reach the goal of the
personalized prescription, in order to maximize the drugs effectiveness and safety and to minimize the side effects. The
Nutrigenomic is an other example of integrative "-omic".
55
P&R Scientific
The nutrient-gene interactions and the relations between nutrition and physiology have been better characterised by the
developing of DNA sequencing techniques and protein analysis. The concept of nutrigenomics refers to the gene expression
regulation by nutrients (17-20).
The aim of the nutrigenomic approach is the identification of nutritional status and disease biomarkers and to define an
individualise nutrient requirement (18,19). Nutrigenomics studies how the diet may modulate the metabolism physiology and
it assesses the potential role of diet in disease prevention.
The different responses to nutrients (= the environment) depends on the individual genomic setting, and each subject may
respond in a different way to the same diet. Many SNP are involved in these differencies (18-10). The main goal of genomic
nutrition is to understand the way which nutrients modulates genes and how these affect polymorphisms in the leading to a
morbid phenotype. This could explain many of the different phenotypes that are observed for the same genetic variant.
The limits of the prediction
Predictive medicine still has many limitations. First of all the results of predictive tests rarely give certainty. The test not
always allow to understand when and with what characteristics the subject develop the disease: in most cases it reveals only
the individual susceptibility.
Besides this it could happen to identify a susceptible subject in the context of a disease for which no interventions can be
taken about its onset and natural history. There are also ethical implications. Healthy subjects receiving the diagnosis of
susceptibility could be pre-patients for many years before developing the disease, with psychological, employment and social
consequences.
The cardiovascular diseases
Cardiovascular diseases and prediction
In the past 3-4 years, the discovery of genetic associations for complex diseases and complex traits has been implemented
by the advent of quick genotyping platforms and by improved quality control measures in genetic epidemiology studies. The
interaction between epidemiology and public health and genetics consist in the understanding of how genes and the
environment act together to produce disease, and how the environment can be modified in a personalized wise to prevent or
delay the onset of disease. The current demographic picture is characterized by chronic age-related and degenerative
conditions. It reflects the dramatic increase in life span and the parallel reduction in early mortality rate with consequent
growth of the elderly population.
Most chronic and degenerative diseases have a complex pathogenesis that involves genetic, epigenetic and environmental
factors.
Cardiovascular disease is the main cause of mortality in the western countries, and it is a consequence of a complex
interplay of genetics and environment. There are several evidence that heritable factors underlie the variation in clinical and
subclinical cardio-vascular disease and its risk factors in the populations (21,22).
The heritability can be defined as the amount of interindividual phenotypic variation due to genetic variations among
individuals. There is substancial evidence that several heritable factors underlie the phenotypic variation in clinical cardiovascular diseases. The impact of a familial predisposition is not apparent in many studies about complex CVD such as
myocardial infarction and stroke (21,22).
Only few models of CVD having a Mendelian transmission exist. Monogenic causes have been identified for some
cardiovascular diseases, for example some forms of dilated cardiomyopathy, long QT syndrome, arrhythmogenic right
ventricular cardiomyopathy.
Evidence for a genetic basis to complex diseases without Mendelian transmission has growth in recent years. Several trials
(23-25) suggest the presence of a low-moderate heritability in subclinical atherosclerosis measures like arterial calcifications,
wall artery thickness, anckle-brachial index and left ventricular hypertrophy. A moderate heritability has been also shown for
heart failure, blood pressure, blood cholesterol, body mass index and tobacco dependence (26,27).
Examples of SNPs involved in cardiovascular frailty
SNPs, the most common source of inter-individual variability, may underlie cardiovascular frailty. Many SNPs involved in
cardiovascular susceptibility have been characterized, and they are involved in several metabolic pathways. Examples are
genes for integrin 3 beta (ITGbeta3) (28), a structural endotelial protein; cholesterol ester transfer protein (CETP) and CILP2
(29), involved in lipid metabolism; plasminogen activation inhibitor 1 (PAI-I) (30), involved in haemostasis; matrix metallo
56
P&R Scientific
proteinase 3 (MMP3) (28); coagulation factor VII (31); genes involved in inflammation like tumor necrosis factor (TNF) and
interleukin-6 (IL-6) (28), and genes involved in arachidonic acid metabolism such as arachidonate 5-lipoxygenase-activating
protein (ALOX5AP), leukotriene A4 hydrolase (LTA4H) and prostaglandin-endoperoxide synthase 2 (PTGS2) (32).
Others examples are genes for CDKN2, PITX2, NOS1AP and NOTCH2. SNPs affecting those genes have been correlated with
cardiovascular outcomes.
For example SNPs -668/4G--5G of PAI-I, 804C--A of LTA4H and rs 1333049-C of CDKN2 have shown to increase
susceptibility for myocardial infarction (28-30,33); -1171/5A--6A of MMP3 and -634C--G of IL-6 have been related to
coronary spasm (28); -863C--A of TNF has been related to increased risk of restenosis after stenting (28,33);
rs10494366-G of NOS1AP has shown to increase QT prolungation risk, and rs10923931-T of NOTCH2 seems to increase
risk for Type II diabetes onset (33).
Many studies have shown a sex difference in the outcome pattern and occurrence according to the same SNPs (34). For
example -668/4G--5G of PAI I seems to increase myocardial infarction risk overall in women ( -1171/5A--6A of MMP3
correlates with coronary spasm mainly in men, while -634C--G of IL6 does in women (35).
By the analysis of clinical studies is clear that in women aged <65 years is increased the global CHD risk then in men. These
sex differences in the association of polymorphisms with myocardial infarction in women <65 years could be due to genetic
factors, beyond the differences in estrogen/estrogen receptor signaling (34).
Discussion
The cardiovascular diseases prevention is a major goal of public health. The classical preventive medicine have founded on
classical risk factors such as hypertension, smoking, overweight, hyperlipemia and diabetes.
The predictive approach uses patient-centered strategies.
The characterization of the individual genetic profile allow us to better understand the personal disease risk, by the
knowledge of the individual environment and life-style.
The new genomic knowledge and the new high-throughput tecniques will permit to characterize genes and proteins of
interest and to understand the systematic interactions between genes, proteins and environment.
By the combination of this approach with genomic variance knowledge, we can optimize the identification of targets in
disease pathways.
The goal of predictive medicine in general will be the identification of genetically high-risk individuals that may really benefit
from screening and person-centered interventions, and also the responseness to preventive interventions should be
predicted.
The greatest limitation of current predictive medicine is the inability to accurately characterise the individual risk, overall in
cardiovascular diseases. It is to underline that the effects of SNPs on the development of CHD are small per se, and the
association is complex and highly influenced by environmental factors and by age, sex and by the presence of classical
vascular risk factors.
Predictive medicine can be applied at every stage of disease. It can identify pre-clinical frail subjects and implement a whole
person-centered health plan to enhance disease onset and progression, but it can also be applied in secondary and tertiary
prevention, in the context of a late and irreversible chronic disease such as heart failure. The personalized pharmacotherapy
will allow to implement the management even in late stages of disease.
The intervention on editable environmental risk factors remain the miliarstone of current predictive medicine. It is to
underline that the current predictive medicine is still based on the environmental risk factors control. Beyond the risk factors
personal profile characterization, it is fundamental the familiar history of patients and the delineation of family tree, to
understand the subject frailty and to extend the prediction and prevention to its familiars. Currently the person-centered
modifications in diet and lifestyle are integrated in the Evidence Based Medicine.
For example the American Diabetes Association indicates that a mild weight loss is per se sufficient to prevent the diabetes
onset in subjects at high risk for type II diabetes through a nutritional individual plan and physical activity, while any
medication have shown to be able to prevent diabetes.
The risk factor modification and changes in lifestyle should be extended to the whole population, in particular to high risk
individuals. In the future the application of genetics knowledge to all individuals and the development of personalized
interventions such as nutrigenomics and pharmacogenomics will permit the full gene-based presymptomatic prediction of
diseases and finer diagnostic subclassifications.
57
P&R Scientific
Earlier and more targeted interventions will be implemented by the improvement of risk assessment tools, while
pharmacogenomics will guide therapeutic decisions and monitor response to therapy.
References
1. Hocquette JF, Cassar-Malek I, Scalbert A, Guillou F. Contribution of genomics to the understanding of physiological
functions. J Physiol Pharmacol 2009; 60:5-16.
2. International HapMap 3 Consortium. Integrating common and rare genetic variation in diverse human populations. Nature
2010; 467: 52-8.
3. Manolio TA. Genomewide association studies and assessment of the risk of disease. N Engl J Med 2010; 363: 166-76.
4. Sandhu M, Wood A, Young E. Genomic risk prediction. Lancet 2010; 376: 1366-7.
5. Ashley EA, Butte AJ, Wheeler MT et al. Clinical assessment incorporating a personal genome. Lancet 2010; 375: 1525-35.
6. Davey Smith G, Ebrahim S, Lewis S et al. Genetic epidemiology and public health: hope, hype, and future prospects.
Lancet 2005; 366: 1484-98.
7. Licastro F, Caruso C. Predictive diagnostics and personalized medicine for the prevention of chronic degenerative diseases.
Immun Ageing 2010; 7:S1.
8. Ouzounian M, Lee DS, Gramolini AO et al. Predict, prevent and personalize: Genomic and proteomic approaches to
cardiovascular medicine. Can J Cardiol 2007; 23: 28A-33A.
9. Hirschhorn JN, Gajdos ZK. Genome-wide association studies: results from the first few years and potential implication for
clinical medicine. Annu Rev Med 2011; 62: 11-24.
10. Offit K. Genomic profiles for disease risk: predictive or premature? JAMA 2008; 299: 1353-5.
11. Amici A, Pecci MT, Linguanti A et al. Self-administrated test based on the Marigliano-Cacciafesta Polypathological Scale
(MCPS), as a screening tool for early identification of frailty in the elderly: a cohort study. Arch Gerontol Geriatr 2011;
52:60-5.
12. Varmus H. Ten years on the human genome and medicine. N Engl J Med 2010; 362: 2028-9.
13. Botstein D, Risch N. Discovering genotypes underlying human phenotypes: past successes for mendelian disease, future
approaches for complex disease. Nat Genet 2003; 33: 228-37.
14. Evans WE, Mc Leod HL. Pharmacogenomics-drug disposition, drug targets, and side effects. N Engl J Med 2003; 348:
538-49.
15. Burns DK. Developing pharmacogenetic evidence throughout clinical development. Clin Pharmacol Ther 2010; 88: 86770.
16. Daly AK. Pharmacogenetics and human genetic polymorphisms. Biochem J 2010; 429: 435-49.
17. Zeisel SH. Nutrigenomics and metabolomics will change clinical nutrition and public health practice: insights from studies
on dietary requirements for choline. AmJClinNutr 2007; 86: 542-48.
18. Strobush L, Berg R, Cross D et al. Dietary intake in the Personalized Medicine Research Project: a resource for studies of
gene-diet interaction. Nutr J 2011; 10:13.
19.
Lee
WN,
Go
VL.
Nutrient-gene
interaction:tracer-based
metabolomics.
J
Nutr
2005;135:3027-32.
20. Ferguson LR. Genome-wide association studies and diet. World Rev Nutr Diet 2010; 101: 8-14.
21. Thanassoulis G, Vasan RS. Genetic cardiovascular risk prediction: will we get there? Circulation 2010; 122: 2323-34.
22. Ripatti S, Tikkanen E, Orho-Melander M et al. A multilocus genetic risk score for coronary heart disease: case-control and
prospective cohort analyses. Lancet 2010; 376: 1393-400.
23. Wilson PW, D'Agostino RB, Levy D et al. Prediction of coronary heart disease using risk factor categories. Circulation
1998; 97: 1837-1847.
24. Morita H, Larson MG, Barr SC et al. Single-gene mutations and increased left ventricular wall thickness in the community.
Circulation 2006; 113: 2697-2705.
25. Manolio TA, Boerwinkle E, O'Donnell CJ et al. Genetics of ultrasonographic carotid atherosclerosis. Arterioscler Thromb
Vasc Biol 2004; 24: 1567-1577.
26. Kathiresan S, Melander O, Anevski D et al. Polymorphisms associated with cholesterol and risk of cardiovascular events.
N Engl J Med 2008; 358: 1240-1249.
27. The Wellcome Trust Case Control Consortium. Genome-wide association study of 14.000 cases of seven common disease
controls. Nature 2007; 447: 661-678.
58
P&R Scientific
28. Belfer I, Wu T, Hipp H et al. Linkage of large-vessel carotid atherosclerotic stroke to inflammatory genes via a systematic
screen. Int J Stroke 2010; 5: 145-51
29. Stankovic S, Majkic-Singh N. Genetic aspects of ischemic stroke: coagulation, homocysteine, and lipoprotein metabolism
as potential risk factors. Crit Rev Clin Lab Sci. 2010;47:72-123.
30. Bentley JP, Asselbergs FW, Coffey CS et al. Cardiovascular risk associated with interactions among polymorphisms in
genes from the renin-angiotensin, bradykinin, and fibrinolytic system. PLoS One 2010; 5: 12757
31. Iacoviello L, Di Castelnuovo A, De Knijff P et al. Polymorphisms in the coagulation factor VII gene and the risk of
myocardial infarction. N Engl J Med 1998; 338: 79 –85.
32. Crosslin DR , Shah SH, Nelson SC et al. Genetic effects in the leukotriene biosynthesis pathway and association with
atherosclerosis. Hum Genet 2009;125:217-29.
33. Marenberg ME, Risch N, Berkman LF et al. Genetic susceptibility to death from coronary heart disease in a study of twins.
N Engl J Med 1994; 330: 1041– 1046.
34. Arain FA, Kuniyoshi FH, Abdalrhim AD, Miller VM. Sex/Gender medicine: the biological basis for personalized care in
cardiovascular medicine. Circ J 2009; 73: 1774-1782
35. Murase Y, Yamada Y, Hirashiki A et al. Genetic risk and gene – environmental interaction in coronary artery spasm in
Japanese men and women. Eur Heart J 2004; 25: 970 – 977.
Corresponding Author: Evaristo Ettorre
"Sapienza" University of Rome. “Predictive Medicine Unit”. Department of Cardiovascular, Respiratory, Nephrologic and
Geriatric Sciences”
Viale del Policlinico 155, I-00161, Rome - Italy
Autore di riferimento: Evaristo Ettorre
“Sapienza” Università di Roma. “Unità di Medicina Preventiva”. Dipartimento di Scienze Cardiovascolari, Respiratorie,
Nefrologiche e Geriatriche
Viale del Policlinico 155, I-00161, Roma, Italia.
59
P&R Scientific
ESTIMATED RISK ASSESSMENT OF THE EXPOSED TO ASBESTOS
VALUTAZIONE DEL RISCHIO STIMATO NEGLI EX ESPOSTI ALL'AMIANTO
Sancini A 1, De Sio S 1, Ciarrocca M 1, Fioravanti M 2, Andreozzi G 1, Sarlo O 3, D’Amelio R 3, Anselmi
A 4, Mascia E 8, De Lorenzo G 5, Ferrante E 6, Gaudioso F 7, Rauccio A 9, Zelano V 3, Tomei F 1,
Tomei G
1
2
University of Rome “Sapienza”, Department of Anatomy, Histology, Medical-Legal and the Orthopaedics, Unit of
Occupational Medicine (Dir: Prof. F. Tomei), Viale Regina Elena 336, 00161 Rome, Italy
2
University of Rome “Sapienza”, Department of Psychiatric and Psychological Science, Piazzale Aldo Moro 5, 00185
Rome, Italy
3
General Direction of Military Health
4
Aesenal M.M. – La Spezia (CO.CE. R – Interforce)
5
Armed Force General Command CC – Health Departement
6
Respiratory Pathophysiology Service-Cardiorespiratory Disease Departement – Policlinico Militare Rome
7
Sanitary Departement naval Base –Augusta
8
Second Istance Medical Commission MM – Bari
9
Primary Nursing A.M.
1
Dipartimento di Anatomia, Istologia, Medicina Legale e Ortopedia, Unità di Medicina del
Lavoro(Dir: Prof. F. Tomei), Università di Roma “Sapienza”,Viale Regina Elena 336, 00161 Roma, Italia
2
Dipartimento
Scienze
Psichiatriche
e
Medicina
Psicologica,
Università
di
Roma“Sapienza”
Piazzale Aldo Moro 5, 00185 Roma, Italia
3
Direzione Generale della Sanità Militare
4
Arsenale M.M. – La Spezia (CO.CE. R – Interforze)
5
Comando Generale Arma CC – Direzione di Sanità
6
Servizio di Fisiopatologia Respiratoria Dipartimento di Patologia Cardio-Respiratoria – Policlinico Militare Roma
7
Sezione Sanitaria Base Navale–Augusta
8
Commissione Medica di Seconda Istanza MM – Bari
9
Infermeria Principale A.M.
Citation: Sancini A, De Sio S, Ciarrocca M, et al. Estimated risk assessment of the exposed to asbestos. Prevent Res 2011; 1
(1): 60-71
60
P&R Scientific
Key words: asbestos, algorithm, exposure, environment
Parole chiave: asbesto, algoritmo, esposizione, ambiente
Abstract
Objectives:
The purpose of this study is the develop an algorithm based on the findings of occupational anamnestical
information provided by a group of workers.
Methods: Four dimensions are analyzed and described: 1) present and/or past occupation, 2) type of materials and
equipment used in performing working activity 3) environment where these activities are carried out, 4) period of time when
activities are performed.
Results: From the combination of the four identified dimensions it is possible to have 108 ELSA codes divided in three
typological profiles of estimated risk of exposure.
Conclusions:
The application of the algorithm offers some advantages compared to other methods used for identifying
individuals exposed to asbestos because the ELSA Code takes in account other indicators of risk besides those considered in
the Job-Exposure Matrix (JEM).
Abstract
Obiettivi : Lo scopo di questo studio è di sviluppare un algoritmo basato sui risultati della raccolta di notizie clinicheanamnestiche fornite da un gruppo di lavoratori.
Metodi: Vengono analizzate e descritte quattro dimensioni: 1) la mansione (attuale e/o pregressa) svolta, 2) il tipo di
materiali e macchinari utilizzati nell’espletamento di suddette attività lavorative, 3) l’ambiente in cui tali operazioni vengono
svolte, 4) il periodo di tempo in cui effettivamente sono state svolte.
Risultati: Dalla combinazione delle quattro dimensioni individuate è possibile ottenere 108 codici ELSA che sono stati
assegnati in tre profili tipologici di rischio stimato di esposizione.
Conclusioni: L'applicazione dell'algoritmo offre alcuni vantaggi rispetto ad altri metodi utilizzati per identificare gli individui
esposti ad amianto in quanto il codice ELSA prende in considerazione altri indicatori di rischio oltre a quelli considerati nel
Job-Exposure Matrix (JEM).
61
P&R Scientific
Introduction
The term asbestos indicates a number of fibrous silicates that belong to two different metamorphic rocks: serpentine and
amphibole (1,2). Both the serpentine and the amphibole can release fibers that are pathogenic to humans, mostly causing
non-neoplastic lung diseases such as pleural plaques, pleural effusions, rounded atelectasis, specific fibrotic disease
(asbestosis), damaged lung function but also cancers in different areas (eg. pleural mesothelioma, lung cancer) (3-10).
Asbestos was declared a carcinogenic agent to humans by the U.S Environmental Protection Agency (11), and the
International Agency for Research on Cancer (12), its dangerous effect has been strongly documented in scientific literature.
After these evidences, many countries have banned the use of such material (13).
Different methods are used in literature about the risk assessment of exposure to asbestos, but none of these appears to be
universally shared by researcher on this issue. These methods consist of 1) assessment by an expert, 2) evaluation of aerodispersed fibers in the workplace (14-16), 3 ) administration of aimed questionnaires (17-19) 4) analysis of the working life
by the construction of exposure matrices (20,21).
1) The expert evaluation is based on different parameters like the performance of a task in a specific industry (18,22), the
arbitrary assigning, often not supported by reliable data, of a score for the intensity of exposure during the task (23), the
duration of the job at risk (24).
2) As to the procedures for the evaluation of aero-dispersed fibers in the workplace, although objective, they show
restrictions linked to the time and to the space of execution and they do not include individual exposures of subjects. While
taking into account individual exposures, individual dosimetry is the measure of a limited period of time and is not useful to
observe long exposures such as the ones asbestos requires (15,25,26).
3) As to questionnaires, they appear to be highly heterogeneous both for the modalities and
accuracy of collecting
information (14,17,27-30). In studies using questionnaires, the classification of acquired information is usually based on the
individual expert evaluation which is a procedure poorly reproducible and repeatable. (27,30-33).
4) The assessments based on the construction of the employment matrices (Job-Exposure Matrix JEM-) correlate the level of
asbestos exposure not only to the task, but also to the productive sector where it is performed and they are typically
constructed around two dimensions: the specific job and the exposure.
The exposures considered may be expressed in different ways, by exposure class or by another simple criteria that is the
“present / not present” exposure, depending on the purpose for which any matrix is constructed. In literature, the
professions and the economic activities are often classified by using international standard codes (eg. ISIC International
Standard Industrial Classification). JEM are usually made on these codes and associate each occupation with the
corresponding evaluation of exposure to any particular substance: all this in order to facilitate the use of JEM in the
international standard method. The exposure assessments of each substance are based on measurements and/or data
present in the literature, studied and evaluated by special groups of occupational medicine experts.
There are different JEM, created and available from various organizations of different countries, their use is very common for
the identification of exposed and unexposed personnel, in order to execute epidemiological studies, which are an instrument
ready to use, providing an initial estimation of the correlation between the profession and exposure to each specific
substance, in the absence of data on concentration measures (environmental sampling and / or personal) (6,8,21,34-37). It
should be noted that JEM take into account only marginally other risk factors such as materials and equipment used while
performing the work.
In addition, it is important to notice that: a) the revision of data present in scientific literature indicates that the latent period
for asbestos-related disease onset is variable, but anyway long-term, even 30-40 years for the onset of mesothelioma of the
pleura (1,9,10,38) b) there are many data reported in scientific literature about the excess of diseases in workers exposed to
asbestos (39-41) c) the Italian Armed Forces sensitive to these issues, have thought it necessary to undertake and finance a
research project with the University of Rome "La Sapienza" for monitoring the effects on the health of its workers exposed to
asbestos.
This research project entitled "Study of indicators for Asbestos Related Diseases In the Armed Forces working categories” is
addressed to the military and civilian staff who voluntarily, want to join; d) considering the length of latency periods the
attention of researchers, doctors and everyone involved in this issue, is aimed to find a method to evaluate the probability
and the amount of exposure over the years, in order to carry out an adeguate "monitoring" the exposed workers’ health.
On the base of critical review of the works on exposure matrices we thought it necessary to develop a method which could
include other elements of risk assessment gathered from the examination of international scientific literature already
published (42).
62
P&R Scientific
The aim our work is to build an algorithm, constructed on the results of anamnestic information provided by workers through
a specially designed questionnaire. The algorithm is intended to discriminate in a probabilistic way, the risk of exposure by
assigning a code to every worker (ELSA code - Exposure to Asbestos Estimated Work). The construction of the algorithm has
been performed in order to minimize the possibility that a more exposed subject is classified as less exposed and vice versa.
This algorithm will be useful in conducting retrospective studies on previous exposures and in the estimated evaluation of
current exposures.
Although the construction of this algorithm comes from the project sponsored by the Armed Forces it can be adapted to
evaluate the risk of asbestos in other working situations.
Methods
We conducted a search of articles published from 1953 until July 2008, on the major on line search engines available, such
as Pubmed, Toxline, EMBASE, Scopus, Google Scholar, Biomed Central, Nioshtic-2, and consulted the conference proceedings
organized by the national SIMLII (Italian Society of Occupational Medicine and Industrial Hygiene), INAIL (National Institute
for Insurance against Accidents at Work) and ReNaM (National Register of Mesothelioma), in order to establish a database
containing all studies present in literature on occupational exposure to asbestos. This search, of more than thousand articles,
allowed to create a database containing about 650 articles.
Later, we made two meta-analysis and a synthetic-critical analysis of the information included in the database (9,10). All
elements were synthesized in a questionnaire made of 90 anamnestic and clinical items, administered to the employees of
the Armed Forces joining the project.
Most of these items were aimed to get information on health status, on habits and lifestyle, on working history, etc., and 9
items were specific to the construction of the algorithm.
To organize the algorithm, we investigated risk factors such as: 1) the task, 2) the risk concerning the equipment and the
materials used for performing the task, 3) the risk in the environment where the task was carried out and 4) the duration of
task.
This information is obtained with administration of a specially designed questionnaire and is organized in the four different
areas identified above. Each information obtained from one of the four areas, is summarized in the four dimensions which
compose the ELSA code. A binary or ternary code to fix each dimensions, was used according to the nature of the variables
that determine the size of the risk.
The ELSA code thus obtained, is allocated in one of the following 3 profiles of estimated exposure risk:
1. Present Systematic risk;
2. Present Unsystematic risk;
3. Little risk or no risk at all.
In the typological profile " present Systematic risk ", we will include only ELSA codes for which the risk of the presence of
asbestos in each dimension cannot be defined to be low or absent, in the typological profile "no risk or low-density" we will
include ELSA codes where the risk of the presence of asbestos in each of the dimensions can not be considered high.
The code is unique for each current and/or previous task carried out, and for this reason if a worker carries out more than
one work, he will be associated to as many ELSA code as the number of tasks performed. To protect the health from the
effects induced by exposure to asbestos the most risky ELSA code will be considered in giving the worker his typological
profile.
Each of the four dimensions of the ELSA code are now analyzed and described.
2.1 First dimension of the ELSA code – task.
To identify the first dimension of ELSA code, we carried out the analysis of the information in our database and the
consultation of the occupational exposure matrix (JEM Job Exposure Matrix).
The JEM are databases designed to evaluate and quantify the correlations between occupations, work activities and tasks
with the risks of exposure to any substances.
They are typically constructed around two dimensions, the occupation and the exposure, and are represented by
alphanumeric codes.
Among the various available JEM, we consulted:
• Evalutil, developed by the French Institute of Public Health, Epidemiology and Development (Institut de Santé Publique de
Developpement et d'Epidemiology ISPED) with the University Victor Segalen Bordeaux 2, in Bordeaux (43,ISPED; INVS),
63
P&R Scientific
which analyzes in the specific asbestos section thousands of literary studies, evaluating for each task the percentage of
exposed,
exposure
intensity
and
duration
of
exposure
as
a
percentage
of
total
working
time;
• FINJEM, developed by the Finnish Institute of Occupational Health (Finnish Institute of Occupational Health FIOH) with the
aim of assessing occupational exposures within epidemiological studies (DORS, 44, FIOH);
• ALOHA, developed by the Institute for Risk Assessment Sciences, Utrecht University, to evaluate exposure to various
substances involved in the onset of respiratory disorders (DORS);
• CAREX, prepared by the European Union to provide an estimation of the number of workers exposed to carcinogenic agents
in the different EU countries (45,46);
• Matlin PRIOR, financed by the Regional Centre for Epidemiology and Environmental Health of the Region of Piemonte
(DORS; Matlin);
• AMYANT, prepared by INAIL CONTARP, and developed to calculate a presumed annual exposure in ff / cc according to two
dimensions: exposure time and specific work carried out. The exposure for each process is derived from scientific literary
data or from environmental assessments “in the field", often conducted by INAIL-CONTARP.
• U.S. N.O.E.S. (National Occupational Exposure Survey- USA) by the NIOSH, aimed to provide an estimation of the number
of workers for each economic activity and exposed to each detected substance (DORS; NOES).
It is evident that the consultation of the JEM is useful to estimate "theoretical" risk of exposure for each work task. In our
study we decided to use the information of the JEM together with the news of our database, in order to construct the first
dimension of ELSA code. For istance, the task "administrative," which is a “not risky function”, according to JEM analysis and
to our database, was classified as "1".
Same criteria were adopted for the classification of other tasks.
In the questionnaire, the worker is asked, the type of task present and/or past carried out; then tasks are classified by
assigning number (1-2-3-4) increasing according to the probability of exposure to asbestos. So, for instance, a worker who is
or was assigned to administrative activities of VDT is rated "1", a worker who is or was assigned to the activity of stocking
and inventory of goods is rated "2", a worker who is or has been assigned to the task of mechanical engines of heavy
vehicles"3", a worker who is or has been assigned to manufacturing thermal insulating material for thermal insulation"4".
The actual exposure will be evaluated: e.g. If a worker is or was a mechanic of heavy vehicles produced after asbestos was
banned, his task will be classified as "1", "2" instead of "3" or vice versa.
To determine the first dimension of the code, the number correlated to the job (or tasks) with the highest risk class will be
taken into account. (If a worker carries out the job of Employee of the administrative department in charge of
correspondence - risk class 1 - and also the task of insulated tubing Employee - risk class 4 -, the first dimension of the code
that will be assigned to employee will be number 4).
Later on the Technical Committee, in order to adapt the algorithm to the working environment of the Armed Forces, made
the correspondence of the task of military personnel to those classified as above, for instance the mansion administrative
officer of employee VDT was paired to the task of computer operator and classified "1"; similarly personnel employed in the
manufacturing of thermal insulating materials was paired the task of “equipment technician ”, and classified "4". Tasks
classified as "4" are those where the exposure to asbestos can not be excluded, and therefore, they are the highest category
of risk.
2.2 The Second dimension of ELSA code-RISK “environmental”
To the identify the second dimension of ELSA code, the questionnaire includes items that provide information about:
• work environment with possible presence of asbestos known by the employee (answer YES): high probability of exposure
(E +) as in Shipyard (47-49) and work environment with the possible presence of asbestos known by the employee (answer
DON’T REMEMBER or NO) : low probability of exposure (E-) such as Office (50 ). This classification was made according to a
review and a synthesis of data present in literature included into our database;
• for any environment / room designated by the employee you can tick the "dust-yes or no, the presence (P +), absence or
minimal presence of dust (P-);
• Evaluation of the conservation status of the work environment and equipment provided . For any"material" identified one of
these four items can be selected: intact environment and equipment not susceptible to damage: low risk (B); intact
environment and materials susceptible to damage (B); damaged environment and materials with not extended damaged
area: medium Risk (M), damaged environment and materials with extended damaged area: high Risk (A).
64
P&R Scientific
Through the different combinations of these parameters it is possible to identify three classes of environmental risk: A (high
risk), M (medium risk), B (low risk): see Table 1.
Table1
E+ (high
probability of
exposure)
E(low probability of
exposure)
P+ (presence of
dust)
E+ P+ (A)
E- P+ (B)
P- (absence /
minimal
presence of
dust)
E+ P- (M)
E- P- (B)
According to typological profile of risk , in the "present systematic risk" profile, no ELSA code of environmental risk with class
"B" appears . For the same criterion ELSA codes of environmental risk with class" A ", is not present in the profile "risk
absent or minimum density".
2.3 The third dimension of ELSA code - risk of equipment, materials and maintenance
To identify the code of the third dimension of ELSA, the questionnaire contains questions related to the description of any
machinery and / or work equipment, the materials used to perform the task and carry out the maintenance containing
asbestos, we evaluated:
•
machinery, classified with the number 2 (perforating equipment or machinery producing dust) and 1 (non-perforating
equipment or machinery which don’t produce dust);
• materials, identified Am + the ones with high probability of release of asbestos according to DM September 6, 1994 (such
as insulating coatings of pipes or boilers) and according to the Act of Union No Inspection 4-00010, No Seat 4, 2006 SENATE
OF THE REPUBLIC Legislature XV Am -and all other types of equipment not found in this classification;
• For maintenance and / or repair of equipment or materials containing asbestos such as crushing for elimination of
asbestos, or containment and encapsulation activities classified M+;
• No maintenance carried out, classified M-;
Through the different combinations of these parameters it is possible to identify three classes of risk of machinery and
equipment: A (high risk), M (medium risk), B (low risk): see Table 2.
Table 2. The third dimension of ELSA code - risk of equipment,
materials and maintenance
Machinery
Maintenance
Materials
M+
M-
1
1M+Am+ (A)
1M+Am- (B)
1M-Am+(M)
1M-Am- (B)
Am+/Am-
2
2M+Am+(A)
2M+Am- (B)
2M-Am+(M)
2M-Am- (B)
Am+/Am-
65
P&R Scientific
In the profile "present systematic risk" no ELSA code with class of risk "B" for machinery and equipment is included,
similarly, no ELSA code with risk "A" for machinery and material. is present in profile "little risk or no risk at all".
2.4The fourth dimension of time-code ELSA
Once the first 3 dimension codes are fixed the algorithm requires the identification of the fourth dimension, that is the
duration of exposure. The exposure period is calculated in months, summing exposure time of the tasks performed with the
highest risk class (corresponding to the code assigned to the employee for his task, according to the criteria described
above, the same is done, for the second dimension (work environment) and the third dimension (Machinery and Materials).
E.g. If an employee with ELSA code 4A1M2- performed 3 tasks (ONE SIZE): the first at risk 4 for 24 months, the second
at
risk 1 for 10 months and a third one at risk 4 for 12 months, the duration of exposure on the first dimension (work) is the
sum of tasks performed at risk class 4 - in this example the duration will be 36 months), in the same way all months he
spent in a workplace with risk class A (second dimension) (eg 24 months) and used equipment and materials with M class of
risk (third dimension) (eg 12 months) are summed.
At the end of this evaluation process, we have "3 times" duration , 36 months for the job, 24 months for the environment;
12 months for machinery and materials. Only the longest dimension at the highest risk (36 months) will be taken into
consideration. This period determines the FOURTH DIMENSION according to these principles: Class A3 for duration> 10
years (high duration), Class M3 for duration between 2 and 10 years (average), Class B3 for exposure duration <2 years
(low duration).
In this case it is class M3. So the final code assigned to the employee of the example above is: 4AMM.
In case the duration taken into consideration for the assignment of the code is classified on a minimum level of risk
dimension, the code assigned to this duration is always B.
For the time used in performing the task (for short "duration of task"), we identified three conventional classes: Class A job
length> 10 years (high duration of the task) (Decree Ministry of Labour and Social Policy No. 16179 of October 27, 2004;
51), Class M job length between 2 and 10 years (average duration of job), Class B job length <2 years (low duration of the
task) (52-54).
No ELSA code class of duration of exposure "B" Is included in the profile "present systematic risk", differently, the profile
"little risk or no risk at all" can included ELSA codes with class duration of exposure "A", as long as it is associated to risk of
the task "1" and no risk class "A" is in the other two dimensions.
Furthermore, for subjects with more tasks, the duration of exposure becomes cumulative for the tasks in the same class of
risk, (that is the durations of exposure of each task are to be summed).
To identify the fourth dimension of ELSA code it is necessary to make some considerations about the mesothelioma. Some
studies in literature say that the process by which the inhalation of fibers influences the development of mesothelioma occurs
shortly after the beginning of exposure, that subsequent exposures give a relatively little increment to the risk, and that the
first two years of working exposure are sufficient to trigger the mesothelioma; (52-54).
Other authors suggest, on the contrary, that with the increasing of exposure incidence of mesothelioma grows too; that is
doses of exposure are cumulative (55,56).
At present, only for the pleural mesothelioma disease
some authors give no importance to duration exposure.
Epidemiological data is underline how important the duration of the interval between exposure and the detection of the
clinical condition of the employee is as a risk factor.Latency time between the contact with asbestos fibers and the onset of
mesothelioma is quite long (51).
Results
From the combination of the four dimensions 108 ELSA codes can be identified. These codes are divided in three typological
profiles of estimated risk of exposure (see Table 3): little risk or no risk at all, non-systematic risk, systematic risk present.
66
P&R Scientific
Discussion
There is not a generally shared method in literature by which it is possible the identification of individuals exposed to
asbestos and the classification into classes of exposure intensity. Most of the studies identify exposed to asbestos through
questionnaires and through assessment of an expert (27,30,31,57).
ELSA code was obtained through a synthesis of information that international scientific literature identifies as most predictive
for the onset of asbestos-related disorders such as type of job performed (present and/or past), type of materials and
equipment used at work, environment where work takes place
and span of time when work is done. Although this
information is obtained subjectively by administering a specially constructed questionnaire to the employee, the process by
which a worker is allocated in one of three categories of risk exposure is objective because based on a systematic evaluation
of the presence of asbestos in each of the four investigated areas of risk.
A worker is placed in the category " systematic risk" only if each one of the four dimensions can be considered positive with
reasonable certainty.
So a worker is classified in the category " little risk or no risk at all " when exposure is little, with absolute certainty, for each
of the four dimensions considered. Therefore we consider the first subject "positive" for exposure and the second "not
positive" for exposure.
The algorithm allows to identify ELSA codes for any different task performed by the worker and therefore to attribute the
highest risk code to the worker.
According to further studies, for the validation of the algorithm, it could be possible a graduation of risk and the possibility to
give different weight to the four dimensions that make up the algorithm.
67
P&R Scientific
Conclusions
We believe that the application of our algorithm allows to obtain some advantages over methods that are currently used in
scientific literature for the identification of people exposed to asbestos and can be summarized as follows:
Examples of application of the ELSA code: a code 4AAA is a worker who carried or has carried out a job at high risk of
exposure to asbestos (a
maintenance and custody Officer of structures containing asbestos), with a high degree of
environmental risk (a worker in shipyard, with the presence of dust), with a high degree of risk associated with materials and
equipment used (containing asbestos) and whose activity has gone on for more than 10 years;
1.
the application of the algorithm can be made in situations of exposure to asbestos through retrospective evaluation of
occupational anamnesis for present or previous expositions;
2.
the classification of workers exposed to asbestos by assigning ELSA code is more detailed than that obtained through
the use of the JEM, which we integrated from our database, because ELSA code takes in account other risk descriptors a part
from task such as materials and equipment used, environmental risk and duration of exposure;
3.
once validated and confirmed, ELSA code can be used for the purposes of epidemiological research on the development
of diseases and / or asbestos-related disorders.
References
1.
Tomei F, Giuntoli P, Bacaloni A et al. Inquinamento da amianto. Dif Soc. 1997; 76: 49-58.
2.
Bernstein DM, Hoskins JA. The health effects of chrysotile: current perspective based upon recent data. Regul Toxicol
Pharmacol. 2006; 45: 252-264
3.
Tomei F, Giuntoli P, De Sio S et al. La patologia da amianto. Dif soc. 1998; 77:105.
4.
Pass HI, Liu Z, Wali A et al. Asbestos exposure, pleural mesothelioma, and serum osteopontin levels. N Engl J Med.
2005; 353: 1564-1573.
5.
Tomei F, Giuntoli P, Tomao E. Diagnosi della patologia da amianto. Prevenzione Oggi 1999; 3: 13-23.
6.
Berry G, Newhouse ML, Wagner JC. Mortality from all cancers of asbestos factory workers in east London 1933-80.
Occup Environ Med. 2000; 57: 782-785.
7.
Brown SC, Schonbeck MF, Mcclure D et al. Lung cancer and internal lung doses among plutonium workers at the rocky
flats: a case-control study. Am J Epidemiol. 2004 ;160: 163-172.
8.
Batra P, Brown K, Hayashi K. Rounded atelectasis. J Thorac Imaging. 1996; 11: 187-197.
9.
Matrat M, Pairon JC, Paolillo AG et al. Asbestos exposure and radiological abnormalities among maintenance and
custodian workers in buildings with friable asbestos-containing materials. Int Arch Occup Environ Health. 2004; 77: 307-312
10.
Tomao E, Palitti T, Rosati MV et al. Prove di funzionalità respiratoria in lavoratori con alterazioni radiologiche asbesto-
correlate : meta-analisi. G Ital Med Lav Ergon. 2008; 30 3 suppl. 2: 157-158.
11.
Filippelli C, Martines V, Palitti T et al. Meta-analisi sulla funzionalità respiratoria nei lavoratori esposti ad amianto. G Ital
Med Lav Ergon. 2008; 30: 142-5.
12.
Environmental Protection Agency. Airborne asbestos health assessment update. 1986. Ed. EPA EPA/6000/8-84/003°
Washinghton DC.
13.
International Agency for Research on Cancer. IARC monographs on the evaluation of carcinogenic risks to humans.
1987. IARC Ed. Suppl. 7: 106-116 Lyone.
14.
Landigran PJ, Soffritti M. Collegium Ramazzini call for an international ban on asbestos. Am J Ind Med. 2005; 47: 471-
474.
15.
Wang X, Yano E, Wang Z et al. Adverse effects of asbestos exposure and smoking on lung function. Am J Ind Med.
2006 ; 49: 337-342.
16.
Seldèn AI, Berg NP, Lundgren EAL et al. Exposure to tremolite asbestos and respiratory health in Swedish dolomite
workers. Occup Environ Med. 2001; 58 :109: 670-677.
17.
Reid A, De Klerk N, Ambrosini GL et al. The effect of abestosis on lung cancer risk beyond the dose related effect of
asbestos alone. Occup Environ Med 2005; 62:885-889.
68
P&R Scientific
18.
Ulvestad B, Kjaerheim K, Martinsen JI et al. Cancer incidence among members of the Norwegian Trade Union of
Insulation workers. J Occup Environ Med. 2004; 46 (1): 84-89.
19.
LeVan TD, Koh WP, Lee HP et al. Vapor, dust and smoke exposure in relation to adult onset asthma and chronic
respiratory symptoms. Am J Epidemiol. 2006; 163(12): 1118-1128.
20.
Lange JH, Thomulka KW, Sites SLM et al. Personal airborne asbestos exposure levels associated with various types of
abatement. Bull Environ Contam Toxicol. 2006; 76: 389-391.
21.
Magnani C, Agudo A, Gonzales CA et al. Multicentric study on malignant pleural mesothelioma and non-occupational
exposure to asbestos. Br J Cancer. 2000; 83 (1): 104-111.
22.
Paris C, Benichou J, Bota S et al. Occupational and non occupational factors associated with high grade bronchial pre-
invasive lesions. Eur Respir J. 2003 ; 21: 332-341.
23.
Merler E, Buiatti E, Vainio H. Surveillance and intervention studies on respiratory cancers in asbestos-exposed workers.
Scand J Work Environ Health. 1997; 23: 83-92.
24.
Lafuente MJ, Casterad X, Laso N et al. Alpha 1 antitripsin polymorphism and the risk for asbestosis in occupational
exposure to asbestos. Toxicol Lett.2002; 136: 9-17.
25.
Koskinen K, Pukkala E, Martikainen R et al. Different Measures of Asbestos Exposure in Estimating Risk of Lung Cancer
and Mesothelioma Among Construction Workers. J Occup Environ Med. 2002; 44: 1190-1196.
26.
Fikfak DM, Kriebel D, Quinn MM et al. A case control study of lung cancer and exposure to chrysothile and amphibole at
Slovenian asbestos-cement plant. Ann Occup Hyg 2007; 51 (3): 261-268.
27.
McDonald JC, Harris J, Armstrong B. Mortality in a cohort of vermiculite miners exposed to fibrous amphibole in Libby,
Montana. Occup Environ Med. 2004; 61: 363-366.
28.
Welch LS, Yair IA , Haile E et al. Asbestos and peritoneal mesothelioma among college-educated men. Int J Occup
Environ Health. 2005 ; 11:254-258.
29.
Loewen G, Natarajan N, Tan D et al. Autofluorescence bronchoscopy for lung cancer surveillance based on risk
assessment. Thorax. 2007; 62: 335-340.
30.
Maule MM, Magnani C, Dalmasso P et al. Modelling mesothelioma risk associated with environmental asbestos
exposure. Environ Health Perspect. 2007; 115: 1066-1071.
31.
Dement JM, Welch L, Bingham E et al. Surveillance of respiratory diseases among construction and trade workers at
department of energy nuclear sites. Am J Ind Med. 2003; 43: 559-573
32.
Dianzani I, Gibello L, Biava A et al. Polymorphisms in DNA repair genes as risk factors for asbestos-related malignant
mesothelioma in a general population study. Mutat Res. 2006; 599: 124-134.
33.
Dietz A, Ramroth H, Urban T et al. Exposure to cement dust, related occupational groups and laryngeal cancer risk:
results of a population based case-control study. Int J Cancer. 2004; 108: 907-911
34.
Marinaccio A, Nesti R. Analysis of survival of mesothelioma cases in the Italian register (ReNaM). Eur J Cancer. 2003;
39: 1290-1295.
35.
Guénel P, Imbemon E, Chevalier A et al. Leukaemia in relation to occupational exposures to benzene and other agents:
a case-control study nested in a cohort of gas and electric utility workers. Am J Ind Med 2002; 42: 87-97.
36.
Mas S, Casterad X, Laso N et al. Concentration of hydroxyproline in blood. A biological marker in occupational exposure
to asbestos and its relationship with Pi*Z and Pi*S polymorphism in the alpha-1 antitrypsin gene. Am J Ind Med. 2004; 45:
186-193.
37.
Melchior M, Goldberg M, Krieger N et al. Occupational class, occupational mobility and cancer incidence among middle-
aged men and women: a prospective study of the French GAZEL cohort. CCC. Cancer Causes and Control. 2005; 16: 515524
38.
Purdue MP, Järvholm B, Bergdahl IA et al. Occupational exposures and head and neck cancers among Swedish
construction workers. Scand J Work Environ Health. 2006; 32 (4): 270-275.
39.
Cugell DW, Kamp DW. Asbestos and the pleura a review. Chest. 2004; 125: 1103-1117.
40.
Attanoos RL, Thomas DH, Gibbs AR. Synchronous diffuse malignant mesothelioma and carcinomas in asbestos-exposed
individuals. Histopathology. 2003; 43: 387-392.
41.
Neri M, Filiberti R, Taioli et al. Pleural malignant mesothelioma, genetic susceptibility and asbestos exposure. Mut Res.
2005; 592: 36-44.
69
P&R Scientific
42.
Cullen MR, Barnett MJ, Balmes JR et al. Predictors of lung cancer among asbestos-exposed men in the β-carotene and
retinol efficacy trial. Am J Epidemiol. 2005; 3 (161): 260-270.
43.
Curin K, Saric M, Strnad M. Incidence of malignant pleural mesothelioma in costal and continental Croatia:
epidemiological study. Croat Med J. 2002; 43 (4): 498-502.
44.
Martines V, FioravantiM, Anselmi A et al. Proposta di algoritmo per la valutazione stimata dell’esposizione lavorativa ad
amianto. G Ital Med Lav Erg 2010; 32:2, 154-161
45.
Orlowski E. EVALUTIL: base de données pour l’évalutation des expositions à l’amiante des utilisateurs de matériaux en
contenant. Cahiers de notes documentaries- Hygiène et sécurité du travail 1997; 166:5-16.
46.
Kauppinen T, Toikkanen J, Pukkala E. From cross-tabulations to multipurpose exposure information systems: a new
job-exposure matrix. Am J Ind Med 1998; 33: 409-417.
47.
Kauppinen T, Toikkanen J, Pedersen et al. Occupational exposure to carcinogens in the European Union. Occup Environ
Med 2000; 57: 10-18.
48.
Mirabelli D, Kauppinen T. Occupational exposures to carcinogens in Italy: an update of CAREX database. Int J Occup
Environ Health 2005; 11: 53-63.
49.
Ascoli V, Cavone D, Merler E et al. Mesothelioma in blood related subjects: report of 11 clusters among 1954 Italy cases
and review of the literature. Am J Ind Med. 2007; 50: 357-369.
50.
Hilliard AK, Lovett JK, McGavin CR. The rise and fall in incidence of malignant mesothelioma from a British Naval
Dockyard, 1979-1999. Occup Med 2003; 53: 209-212.
51.
Koskinen K, Pukkala E, Reijula K, Karjalainen A. Incidence of cancer among the partecipants of the Finnish Asbestos
Screening Campaign. Scand J Work Environ Health 2003; 29 (1): 64-70.
52.
Fedi A, Blagini B, Melosi A et al. Assessment of asbestos exposure, mortality study, and health intervention in workers
formerly exposed to asbestos in a small factory making drying machines for textile finishing and the paper mill industry in
Pistoia. Med Lav. 2005; 96(3):243-249.
53.
Boffetta P. Health effects of asbestos exposure in humans: a quantitative assessment. Med Lav. 1998; 89: 471-480.
54.
Chiappino G. Mesotelioma: il ruolo delle fibre ultrafini e conseguenti riflessi in campo preventivo e medico-legale. Med
Lav. 2005 ; 96:3-23.
55.
Peto J, Seidman H, Selikoff IJ. Mesothelioma mortality in asbestos workers: implications for models of carcinogenesis
and risk assessment. Br J Cancer. 1982 ; 45: 124-136.
56.
Parkes WR. Occupational lung disorders - Oxford - Butterworths – Heinemann 1994.
57.
Hansen J, De Lerke NH, Musk AW, Hobbs MST. Environmental exposure to crocidolite and mesothelioma. Exposure-
response relationship. Am J Respir Crit Care Med. 1998; 157: 69-75.
58.
Iwatsubo Y, Patron JC, Boutin C et al. Pleural Mesothelioma: dose-response relation and low levels of asbestos
exposure in a French population-based case-control study. Am J Epidemiol. 1998; 148(2): 133-142.
59.
Nam J , Rice G, Gail MH. Comparison of asbestos exposure assessments by next-of-kin respondents, by an occupational
hygienist, and by a job-exposure matrix from the national occupational hazard survey. Am J Ind Med. 2005; 47: 443-450.
70
P&R Scientific
Corresponding Author: Gianfranco Tomei
Address: Via Monte delle Gioie 13
Zip Code: 00199 Rome, Italy
Phone: +390649912540
Fax: +390686203178
Autore di riferimento: Prof. Gianfranco Tomei
Via Monte delle Gioie 13, 00199 Roma, Italia
Telefono: +390649912540
Fax: +390686203178
71
P&R Scientific
NEW PERSPECTIVES IN HEMOGLOBIN ADDUCTS ANALYSIS:
SELECTIVE DIGESTION WITH CALPAIN I
NUOVO APPROCCIO ALL’ANALISI DEGLI ADDOTTI EMOGLOBINICI: DIGESTIONE
SELETTIVA CON CALPAINA I
Pieri M
1
1,2
, Miraglia N
2,*
, Genovese G 2, Guadagni R 2, Acampora A 1, Sannolo N
2
Department of Public Medicine and Social Safety, University of Naples “Federico II”, Via Pansini, 5. 80131, Naples,
Italy
2
Department of Experimental Medicine – Section of Occupational Medicine, Hygiene and Industrial Toxicology,
Second University of Naples, Via L. De Crecchio, 7. 80138, Naples, Italy
1
Dipartimento di Medicina Pubblica e Sicurezza Sociale, Università di Napoli “Federico II”, Via Pansini, 5. 80131,
Napoli, Italia
2
Dipartimento di Medicina Sperimentale – Sezione di Medicina del Lavoro, Igiene e Tossicologia Industriale,
Seconda Università di Napoli, Via L. De Crecchio, 7. 80138, Napoli, Italia
Citation: Pieri M, Miraglia M, Genovese G, et al. New perspectives in hemoglobin adducts analysis:
selective digestion with Calpain I. Prevent Res 2011; 1 (1): 72-86
Key words: hemoglobin adducts, Calpain I, mass spectrometry
Parole chiave: addotti emoglobinici, Calpaina I, spettrometria di massa
Abstract
Background: Hemoglobin adducts are considered suitable biomarkers to evaluate human exposure to electrophile
agents.
One of the procedures used for the molecular dosimetry of hemoglobin adducts is the enzymatic digestion of the
protein and the subsequent quantification of modified peptides by mass spectrometry.
Due to the low level of modified hemoglobin peptides with respect to the corresponding unmodified ones, for the
detection of adducts, samples must be enriched for them.
Objectives: Within this item, hemoglobin was digested with Calpain I and the study was divided into two phases.
Firstly we characterized proteolytic sites on pure hemoglobin, then we evaluate the ability of Calpain to discriminate
between normal and alkylated globin chains.
72
P&R Scientific
Methods: Digestions of pure hemoglobin sample and of an alkylated one with Calpain I at 200:1 and 25:1 weight
ratios, for 1, 5 and 18 hrs were carried out.
Results: By using a 200:1 weight ratio only peptides
α(1-137), α(1-138)
that the first proteolytic sites are located at the C-terminus ends of both
and
β(1-142)
were produced, pointing out
α- and β-chains.
The identification of peptides obtained from the 25:1 digestions allowed the determination of internal secondary
proteolytic sites, not reported in literature.
In both cases, a non-negligible proteolysis was observed only when a reaction time of 18 hrs was used.
Conclusions: Results showed a progressive enrichment of modified hemoglobin with respect to the unmodified one,
particularly for the
α-chain
and represent a promising initial step in the development of a selective purification
procedure to be applied for protein adducts analysis.
Abstract
Introduzione: Gli addotti emoglobinici rappresentano una classe di biomarcatori utilizzati per valutare l’esposizione
umana ad agenti elettrofili.
Una delle procedure analitiche utilizzate per la dosimetria molecolare degli addotti emoglobinici prevede la digestione
enzimatica della proteina e la successiva identificazione dei peptidi modificati mediante spettrometria di massa.
Prima dell’analisi è necessario purificare i campioni, per concentrare la frazione di peptidi modificati.
Obiettivi: Il lavoro illustra i risultati ottenuti dalla digestione di campioni emoglobinici con Calpaina I. Lo studio si è
articolato in due fasi.
Inizialmente sono stati caratterizzati i siti di digestione proteolitica; successivamente, è stata valutata la capacità della
Calpaina I di discriminare tra catene globiniche native ed alchilate.
Metodi: Campioni di emoglobina nativa ed alchilata sono stati proteolizzati con Calpaina I, variando i rapporti di
incubazione (200:1 e 25:1, in peso) e i tempi di reazione (1, 5 e 18 ore).
Risultati: Utilizzando un rapporto di incubazione 200:1 sono stati evidenziati i soli peptidi
β
α (1-137), α (1-138) e
(1-142), a testimonianza del fatto che i siti proteolitici principali sono localizzati alle estremità C-terminali di entrambe
le catene
α e β.
Utilizzando un rapporto di incubazione 25:1 sono stati evidenziati siti proteolitici secondari, non riportati in letteratura.
In entrambi i casi è stato necessario condurre le reazioni per 18 ore.
Conclusioni: I risultati hanno evidenziato un progressivo arricchimento della porzione globinica alchilata, soprattutto
per la catena
α
e rappresentano un promettente punto di partenza per lo sviluppo di una procedura di purificazione
selettiva da applicare all’analisi degli addotti proteici.
73
P&R Scientific
Background
The potential health risk associated with the absorption of genotoxic agents within the organism, led to an increasing
interest in the evaluation of human exposure to these agents during the last decades.
Most
chemical
carcinogens
require
metabolic
activation
to
reactive
electrophiles
that
can
modify
cellular
macromolecules, such as DNA and proteins. As a consequence of the exposure to a lot of chemical carcinogens, a
damage of the structural integrity of DNA was observed, with the formation of a covalent carcinogen binding
(carcinogen-DNA adduct) (1, 2).
In some cases, DNA damage is considered to be causative and directly related to tumour formation (1, 3-6); while
modifications of proteins can be used to provide information about exposure, if the adduct is stable and the protein has
a known life-time (7-11).
In fact, protein adducts are considered a valid surrogate for DNA adduct formation, since many chemical carcinogens
bind to both DNA and protein in blood with similar dose-response kinetics (7, 9, 12). Hemoglobin (Hb) adducts, in
particular, have been found to be suitable biomarkers for evaluating exposure to toxicants (13-19), due to the fact that
hemoglobin is easy to sample, forms stable adducts, which are not subject to repair processes, and has a long life time
(approx. 120 days in humans).
Peptide mapping and tandem mass spectrometry, coupled with classical biochemical strategies, as enzymatic digestions
and the modified Edman degradation, have been extensively applied to investigate protein adducts and to evaluate
human exposure to toxicants (14). Another method, based on the quantification of modified hemoglobin peptides, has
been proposed, and in some cases successfully applied, for the biological monitoring of workers occupationally exposed
to different classes of toxicants such as epichlorohydrin, methyl bromide and butadiene (20-22), putting in evidence the
potentiality of this strategy.
Such a procedure implies a structural characterization of the hemoglobin-carcinogen interaction, in order to identify the
most reactive amino acid residue within the hemoglobin sequence. Then a tryptic digestion of hemoglobin samples is
performed and the resulting mixture is analysed by Liquid Chromatography/Electrospray-Tandem Mass Spectrometry
(LC/ESI-MSMS).
The major limitation of this technique is related to the low level of modified hemoglobin peptides with respect to the
corresponding unmodified ones. In fact, nowadays, the levels of hemoglobin adducts found in subjects exposed to
different carcinogens are in the range 2-200 pmol/g globin (14), i.e. in 1 g of hemoglobin there are only 2-200 pmol of
alkylated hemoglobin modified by the carcinogen.
The LC/ESI-MSMS technique actually allows to achieve high instrumental sensitivity (fmol) but the alkylated peptide has
to be pure, namely for the detection of hemoglobin adducts, samples must be enriched for adducts or adducts must be
removed from the protein, before analysis (15).
Objectives
The present study was focused on the identification of an enzyme able, in principle, to discriminate between normal and
alkylated globin chains, so that a selective digestion can be carried out.
The basis of this choice is related to the nature of the most frequently alkylated amino acid residues within Hb. Several
amino acids possess side-chains containing nucleophilic sites with reactivity towards electrophiles; this reactivity
depends on the relative amount of the non-protonated form, all things considered related to the blood pH and the pKa of
the group (14).
Three amino acids in Hb show the pKa values of the nucleophilic centres in close to the blood pH: the nitrogen atoms of
N-terminal Valine, the imidazole nitrogens of Histidine residues and the thiolic sulphur of Cysteins. In previous studies it
has been reported that at low level of concentration of the alkylating agent, Histidine and Cysteins residues are even
more reactive with respect to the N-terminal Valine (18-22).
So in the development of a selective digestion procedure, Calpain I, a neutral calcium-dependent thiol protease (EC
3.4.22.17), seemed to be a promising choice. In particular, the
μ-isozyme was preferred to the m-one, due to the minor
Ca2+ demand (μM with respect to mM amounts, respectively), that is a useful feature for the subsequent LC/ESI-MS
and MSMS analyses. Cysteine proteases have mechanistic similarities with serine proteases but are better nucleophiles
due to the extra shell of electrons present in the sulphur of the thiol group.
74
P&R Scientific
They require an essential Cysteine residue in the active site for hydrolysis, whose nucleophilicity is enhanced by the
close proximity of an active site Histidine, which acts as a proton donor/general base. So a thiolate-imidazole charge
relay diade is present, allowing a broad pH range of enzymatic activity - from 4.0 to 8.5, relating to the pKa values of
Cysteine and Histidine, respectively (23).
Literature studies report that Calpain was used with many proteins as substrates, with various purposes (24-28) but the
structural clues of substrate recognition by Calpain are still aìunder debate (29).
The starting point of the present work is the idea that, if Cysteine and Histidine residues of the substrate were implied in
the recognition process, when alkylation occurs on these residues, Calpain would not recognize the alkylated substrate,
thus permitting a selective digestion.
Namely, in the case of hemoglobin, the unmodified hemoglobin would be digested, giving rise to low molecular weight
peptides, while the modified one (Hb adduct) would not be digested and could be purified from the peptide mixture.
Since there are few reports concerning the characterization of proteolitic sites and the reaction conditions for an optimal
digestion of hemoglobin chains (and none of which involve mass spectrometry as detection technique), the first part of
the work consisted of the digestion of native hemoglobin with different amount of Calpain and different reaction time.
Once identified the main proteolitic sites, during the second part of the work a globin sample previously alkylated in
vitro with epichlorohydrin (1-chloro-2,3-epoxypropane; ECH) was digested.
Epichlorohydrin is widely used as raw material in the industrial synthesis of a number of glycerol and glycidiol
derivatives and epoxy resins (30). ECH is able to induce DNA damage (31, 32) and is considered as probably carcinogen
to humans (33). The choice of ECH as alkylating agent in the development of the study here reported was motivated by
the fact that the reaction conditions for in vitro over-alkylation, as well as a punctual characterization of the alkylated
amino acid residues were well known and previously reported (18-20).
The selective enrichment of the alkylated globin portion with respect to the unmodified one was evaluated through an
estimation of alkylated
α-
and
β−chains
percentages in comparison with the normal ones by analysing samples before
and after digestion with different amounts of Calpain I and different reaction times.
Materials and methods
Caution: Epichlorohydrin is probably carcinogen to humans; TFA is
harmful by inhalation and causes severe burns.
They should be handled carefully according to appropriate environmental safety and health protocols.
Epichlorohydrin was from Acros, Carlo Erba Reagents (Milan, Italy).
Calpain I, Human Erythrocytes, was from Calbiochem (Merck Biosciences, Darmstadt, Germany). All other reagents
were from Sigma-Aldrich S.r.l. A Jupiter C18 (250 X 2.0 mm, 5
μm,
300
Å)
column (Phenomenex, St. Torrance, CA,
USA) was used for peptide separations.
HPLC grade-solvents were from Carlo Erba (Milan, Italy). LC/ESI-MSMS analyses were carried out using an Agilent 1100
series HPLC modular system (Palo Alto, CA, USA) and a LCQTMDECA (ThermoQuest, Finnigan, San José, CA, USA) ion
trap mass spectrometer, equipped with an electrospray ion source.
Hemolysis
Red cells from a healthy volunteer were washed three times with isotonic 0.9% NaCl solution and bidistillated water was
added to erythrocytes to achieve a hemoglobin concentration of 5 g/100 mL.
The hemolysed solution was centrifuged at 5000 rpm for 5 min in order to eliminate cellular membranes. Aliquots of 1ml
were stored in freezer at –20 °C.
In vitro incubation of hemolysed red cells with ECH and globins precipitation
Hemolysed solution aliquots were suspended with 2 mL of 10mM sodium phosphate buffer (pH 7.0). ECH was added to
the solution in order to have an Hb to ECH molar ratio of 1:30. The reaction mixture was left at 37 °C for 3 h, and then
the solution was stored at –20 °C.
The obtained globin mixture, consisting of Hb and alkylated Hb (HbECH) were precipitated with cold 5.4% HCl in acetone
and washed with cold pure acetone.
75
P&R Scientific
Then they were dried under a nitrogen stream and stored at –20 ºC. The same precipitation procedure was applied to
hemolysis solution aliquots in order to obtain unmodified precipitated globins (Hb).
Enzymatic digestion of Hb and HbECH with Calpain I
Aliquots of 1 mg of pure Hb or Hb alkylated as described above were digested with Calpain I at 37 °C using a solution of
0.1% TFA:(100μM CaCl2 + 100mM H3BO4) = 1:1 (v:v) as buffer, whose pH was adjusted to 7.0 by adding 30% NH4OH
solution.
Digestion with Calpain I was carried out by varying enzyme amounts and reaction times. In particular, globins-Calpain
ratios of 200:1 and 25:1, w:w (i.e. 1 mg of globin samples was digested with 5
μg
and 40
μg
of Calpain, respectively)
and reaction times of 1 h, 5 h and 18 h were used. Reactions were stopped by freeze drying and samples stored at –20
°C until analysis.
LC/ES-MS and MSMS analysis of Calpain-digested globins
Calpain-digested globins were fractionated by HPLC; details of chromatographic separation are reported elsewhere (22).
Liquid chromatography was performed using a Phenomenex C18 (250 × 2.0 mm) column at a flow rate of 0.2 mL/min
directly connected to the electrospray ion source of the mass spectrometer (heated capillary temperature = 300 °C).
Data were acquired and processed using the Xcalibur program (version 1.1, ThermoQuest).
Peptide mixtures were analysed in the MS mode with an acquisition range from m/z 300 to 2000. Signals of singly,
doubly and triply charged ions corresponding to each peptide of interest were measured and the peptide molecular
weights were worked out by the Xcalibur Biowork software (Turbosequest 1.2, ThermoQuest).
The
hemoglobin
α-
and
β−chains
sequences
were
searched
by
means
of
the
Expasy
Proteomics
Server
(http://us.expasy.org), the measured peptide masses were introduced, so that peptides matching the measured
molecular weight values are determined.
In particular, the FindPept tool was used, setting the following research parameters: average peptide masses, mass
tolerance of ± 2.0 Da, Cysteins in reduced forms, without Methionines oxidized, no acidic and C-terminal residues
esterified, no possible N-Acetylation or N-Formylation, without any specified proteases. When more sequences were
possible, LC/ESI-MSMS analyses were carried out so that any ambiguousness was avoided in the identification of
proteolytic peptides.
Tandem mass spectrometry experiments were performed by using singly or doubly charged ions as first precursor ions.
They were isolated in the ion trap (trapping window = 1 u) and fragmentation was induced by the application of a
supplementary voltage (30%, arbitrary units) for 10 msec.
Product ions mass spectra were acquired in variable mass ranges, according to the analysed peptide.
Results
Digestion of Hb: main proteolytic sites at different reaction times and Calpain amounts
Figure 1 reports the LC/ESI-MS full scan total ion chromatograms concerning the digestion of hemoglobin with Calpain
at 200:1 weight ratio, for the three considered reaction times, i.e. 1, 5 and 18 hrs (panels a, b and c, respectively).
76
P&R Scientific
Figure 1. LC/ESI-MS total ion chromatograms of hemoglobin digested with Calpain I at 200:1 (w:w) ratio at 37 °C for
1h (panel a), 5 hrs (panel b) and 18 hrs (panel c).
β
Relative Abundance (%)
a)
α
α (1-137)
α (1-138)
β (1-142)
b)
c)
Time (min)
In the first case only peptides
α(1-137), α(1-138)
and
proteolytic sites are located at the C-terminus ends of both
β(1-142)
were produced, thus pointing out that the first
α− and β−chains.
On the same enzyme amount, the increment of the reaction time resulted in an enhanced globins degree of digestion,
but only when an 18 hrs reaction time was used a non-negligible proteolysis was observed.
In fact, even if
α
and
β globins
were the most abundant ones in the chromatogram (Figure 1 panel c), a lot of peptides
were detectable.
They were identified as previously described, thus secondary proteolytic sites were identified.
The complete analysis of the chromatogram allowed the identification of complementary peptides with respect to the
cleavage site so that about the 100% of both
α
and
β chains
sequences was matched. The only exceptions were
represented by dipeptides, which were reasonably not retained by the column. The Figure 2 reports the primary
proteolytic sites pointed out by the18 hrs reaction time experiment. As evident, the
marked digestion if compared to the
α-chain
undergoes to a more
β−chain.
77
P&R Scientific
Figure 2. Aminoacidic sequences of
α
and
β-chains. Identification of primary
(1) and secondary (2) proteolytic sites of
hemoglobin digested by Calpain.
Hemoglobin α-chain
2
Val Leu Ser Pro Ala Asp Lys Thr Asn Val Lys Ala Ala Trp Gly Lys Val Gly | Ala His Ala Gly Glu Tyr Gly Ala Glu Ala
Leu Glu
1
Arg Met Phe Leu Ser Phe Pro Thr Thr Lys Thr Tyr | Phe Pro His Phe Asp Leu Ser His Gly Ser Ala Gln Val Lys Gly His
Gly Lys
2
Lys Val Ala Asp Ala Leu Thr | Asn Ala Val Ala His Val Asp Asp | Met Pro Asn Ala Leu Ser Ala Leu Ser Asp Leu His |
Ala His Lys
2
1 1
1
Leu Arg Val Asp Pro Val Asn Phe Lys Leu Leu Ser | His Cys Leu Leu Val | Thr Leu | Ala | Ala His Leu Pro Ala Glu Phe
Thr Pro Ala
1
1
1
Val His | Ala Ser Leu Asp Lys Phe Leu Ala Ser Val Ser Thr Val Leu Thr | Ser | Lys Tyr Arg
Hemoglobin β-chain
1
Val His Leu Thr Pro Glu Glu Lys Ser Ala Val Thr Ala Leu Trp | Gly Lys Val Asn Val Asp Glu Val Gly | Gly Glu Ala
Leu Gly | Arg
2
2
2
Leu Leu Val Val Tyr Pro Trp | Thr Gln Arg Phe Phe Glu Ser Phe | Gly Asp Leu | Ser Thr Pro Asp Ala Val Met Gly Asn
Pro Lys Val
2
Lys Ala His Gly Lys Lys Val Leu Gly | Ala Phe Ser Asp Gly Leu Ala His Leu Asp Asn Leu Lys Gly Thr Phe | Ala Thr
Leu Ser Glu
2
Leu His Cys Asp Lys Leu His | Val Asp Pro Glu Asn Phe Arg Leu Leu Gly Asn Val Leu Val Cys Val Leu Ala His His
Phe Gly Lys
1
1
1
Glu Phe Thr Pro Pro Val Gln Ala Ala Tyr Gln Lys Val Val Ala | Gly Val Ala Asn | Ala Leu Ala | His Lys Tyr His
In general, a weight ratio between Hb and Calpain of 200:1 was not enough for obtaining a satisfactory degree of globin
digestion, as stressed by the persisting of intact
α−
and
β−globins
as major compounds, even in the 18 hrs experiment
(Figure 1, panel c).
Digestions were repeated by increasing the amount of Calpain (25:1, weight ratio) using reaction times of 1, 5 (data not
shown) and 18 hrs. As in the previous case, only when an 18 hrs reaction time was used a strong degree of proteolysis
was observed (Figure 3).
78
1
P&R Scientific
Figure 3. LC/ESI-MS total ion chromatograms of hemoglobin digested with Calpain I at 25:1 (w:w) ratio at 37 °C for 18
hrs.
Time (min)
The complete list of the obtained peptides is reported in Table 1.
Table 1. Identification of haemoglobin peptides from digestion with Calpain (25:1, w:w)
RT
(min)
6.37
Calculated
Mass (Da)
359.1 ± 0.1
Peptide
β(136-139)
Teorical
Mass (Da)
359.4
RT
(min)
39.9
13.52
410.1 ± 0.1
α(40-42)
410.5
40.3
16.36
54.2 ± 0.1
β(123-127)
540.6
41.4
17.95
18.55
542.1 ± 0.2
623.4 ± 0.2
α(93-97)
α(88-92)
542.6
623.8
42.0
42.5
18.9
417.9 ± 0.1
α(64-67)
418.5
43.0
22.0
23.2
839.2 ± 0.1
808.6 ± 0.2
α(68-75)
β(62-69)
839.9
809.0
43.8
44.6
24.56
1073.3 ± 0.1
β(3-12)
1074.2
44.9
25.3
1170.9 ± 0.3
α(1-11)
1171.4
45.3
26.6
915.4 ± 0.1
β(16-24)
916.0
45.9
79
Calculated
Mass (Da)
1399.9 ± 0.1
2647.3 ± 0.2
863.5 ± 0.3
1286.1 ± 0.2
863.7 ± 0.2
827.5 ± 0.1
1487.1 ± 0.3
1368.9 ± 0.1
1158.8 ± 0.5
861.3 ± 0.3
944.0 ± 0.4
3469.3 ± 0.2
831.3 ± 0.1
1060.7 ± 0.3
1130.7 ± 0.27
868.8 ± 0.2
3461.3 ± 0.4
1444.2 ± 0.2
1705.3 ± 0.4
1680.2 ± 0.3
1165.9 ± 0.3
797.8 ± 0.4
Peptide
β(70-82)
α(43-67)
α(123-130)
β(13-24)
α(123-130)
β(25-32)
β(72-85)
α(118-130)
β(98-107)
α(43-49)
β(120-127)
α(43-75)
β(40-45)
β(38-45)
α(93-102)
α(103-110)
β(38-69)
β(3-15)
β(70-85)
β(1-15)
α(26-35)
α(103-109)
Teorical
Mass (Da)
1400.5
2647.9
864.0
1286.5
864.0
827.9
1487.6
1369.6
1159.3
861.9
944.1
3469.8
831.9
1061.2
1131.3
869.1
3461.8
1444.6
1705.9
1680.9
1166.4
798.0
P&R Scientific
1267.8 ± 0.3
1098.9 ± 0.2
1651.4 ± 0.1
1274.3 ± 0.2
1116.8 ± 0.2
1345.7 ± 0.1
1307.8 ± 0.4
1701.7 ± 0.1
1044.6 ± 0.2
1851.8 ± 0.4
1712.6 ± 0.5
α(76-87)
α(31-39)
β(19-33)
β(30-39)
β(40-48)
β(38-48)
β(108-119)
α(36-49)
β(30-37)
α(19-35)
β(108-122)
1268.4
1099.3
1651.8
1274.5
1117.3
1346.5
1308.5
1701.9
1045.3
1852.1
1713.0
1472.5 ± 0.3
1724.8 ± 0.5
1551.6 ± 0.2
β(25-37)
β(30-42)
α(123-137)
1472.8
1725.1
1551.8
β
β(1-139)
β(1-135)
β
15867.2
15046.3
14704.9
15867.2
β(128-135)
α(82-87)
α(19-25)
848.9
654.7
632.6
47.0
27.45
848.4 ± 0.2
654.4 ± 0.1
632.2 ± 0.3
27.88
28.11
1255.4 ± 0.1
631.2 ± 0.1
β(50-61)
β(19-24)
1256.5
631.6
50.4
50.9
28.3
1803.0 ± 0.2
630.9 ± 0.3
1309.7 ± 0.2
614.2 ± 0.3
1147.7 ± 0.2
769.4 ± 0.1
687.3 ± 0.2
α(50-67)
α(76-81)
β(1-12)
β(93-97)
α(88-97)
β(86-92)
α(12-18)
1804.0
631.7
1310.5
614.7
1148.3
769.8
687.8
52.3
2133.7 ± 0.2
2046.6 ± 0.2
792.3 ± 0.1
1288.9 ± 0.3
1359.9 ± 0.3
2246.9 ± 0.3
853.4 ± 0.2
607.3 ± 0.4
2646.6 ± 0.4
β(49-69)
β(50-69)
α(131-138)
α(111-122)
α(110-122)
α(43-63)
α(110-117)
α(98-102)
α(43-67)
2134.5
2047.5
792.8
1289.5
1360.5
2247.5
854.9
607.4
2647.9
66.1
15866.8 ± 0.7
15045.7 ± 0.3
14704.6 ± 0.7
15866.5 ± 0.5
66.6
67.0
69.8
70.5
15866.5 ± 0.8
15301.0 ± 0.5
15126.0 ± 0.5
14591.1 ± 0.5
β
β(1-142)
α
α(1-137)
15867.2
15301.6
15126.4
14591.7
71.8
14678.2 ± 0.8
α(1-138)
14678.8
26.9
28.8
29.7
30.5
31.2
33.36
36.4
37.0
37.8
38.6
39.6
47.5
54.2
54.6
65.4
For each identified peptide, the relative retention times (RT), experimental and theoretical masses are specified. Further
secondary proteolytic sites were identified and the Figure 2 shows some of them, that correspond to complementary
peptides with higher molecular weight; their further digestion gave rise to all the other smaller peptides reported in
Table 1.
In this case a higher degree of digestion for the
β−chain
if compared to the 200:1 (w:w) ratio experiment; on the
contrary, almost the same degree of digestion was found for the
α−chain.
Digestion of Hb and HbECH mixture: selective enrichment evaluation
The incubation of hemolysed solution aliquots with epichlorohydrin produces a sample containing both alkylated and
unmodified hemoglobin (HbECH and Hb), whose molecular weights differ of 92 amu (or multiples, in case of higher
alkylation).
This sample was analysed in LC/ESI-MS (Figure 4, panel a); the chromatographic peaks at RT = 66.5 min corresponds
to
β
and
βΕΧΗ, the one at
RT = 70.0 min is constituted by
80
α and αΕΧΗ.
P&R Scientific
Figure 4. LC/ESI-MS total ion chromatograms of an undigested mixture of Hb and HbECH (panel a). Total ion
chromatograms of the same mixture digested with Calpain using a 200:1 (w:w) ratio (panel b) and 25:1 (w:w) ratio
(panel c) at 37 °C for 18 hrs.
a)
b)
c)
Time (min)
The relative amount of alkylated
and
β)
α−
and
β−chains (αΕΧΗ
and
βΕΧΗ,
respectively) with respect to the unmodified ones
(α
was estimated. To this purpose, the ion currents of the most abundant ions of both chains were extracted from
the total ion current chromatograms (TIC).
Ions from [M+9H]9+ to [M+15H]15+ were considered, i.e. ions at m/z values of 1681.7, 1513.6, 1376.1, 1261.5, 1164.6,
1081.5 and 1009.4 in the case of
in the case of
αΕΧΗ;
α;
ions at m/z values of 1691.9, 1522.8, 1384.5, 1269.2, 1171.6, 1088.0 and 1015.6
ions at m/z values of 1764.0, 1587.7, 1443.5, 1323.3, 1221.6, 1134.4 and 1058.8 in the case of
ions at m/z values 1774.2, 1596.9, 1451.8, 1330.9, 1228.6, 1140.9 and 1064.9 in the case of
βΕΧΗ.
The extraction of these ion currents from the TIC gave rise to four chromatographic peaks corresponding to
β, βΕΧΗ.
The estimation of the relative abundance of
αΕΧΗ
with respect to
α
and
βΕΧΗ
with respect to
β
α, αΕΧΗ,
in the in vitro
alkylated sample was obtained through the area ratio between the obtained chromatographic peaks, in particular
= 0.72 and
βΕΧΗ/β
β;
αΕΧΗ/α
= 0.55.
The enzymatic digestion of the same in vitro alkylated sample with Calpain I was performed at two weight ratios, 200:1
and 25:1, and at three different reaction times, 1, 5 and 18 hrs; all samples were analysed in LC/ESI-MS. As expected,
the reactions at 1 and 5 hrs let only a low degree of digestion both for the 200:1 and 25:1 weight ratio; on the contrary
a more efficient digestion was observed when reactions were conducted for 18 hrs.
81
P&R Scientific
The LC/ESI-MS full scan chromatograms are reported in Figure 4, panels b (referred to the 200:1 weight ratio digestion
of the alkylated globin samples) and c (referred to the 25:1 one).
In both cases, all chromatographic peaks correspond to peptides that derived from the digestion of the unmodified
hemoglobin, contained in the analysed sample. On the contrary, no peptides originated from the digestion of HbECH
were found, even when the digestion was carried out for 48 hrs (data not shown).
Hypothetical alkylated peptides were searched not only by analysing the total ion chromatogram but also by extracting
from the TIC the ion currents related to their hypothetical m/z values, given by the m/z values of the peptide under
investigation plus 92 or 46 amu ([M+H]+ and [M+2H]2+, respectively).
Also in this case, no alkylated peptides were identified. The fact that HbECH was not preferentially digested by Calpain
resulted in a progressive enrichment in
αΕΧΗ
with respect to
α,
while the
βΕΧΗ/β
ratio remained almost constant. In
fact, for both chains the ion current of the most abundant ions within mass spectra was extracted from the total ion
current chromatogram as described above.
In the case of the 200:1 experiment, the
αΕΧΗ /α
ratio was 0.79, while
amount (25:1 experiment) resulted in an increasing of
αΕΧΗ /α
βΕΧΗ/β
was 0.50; the increasing of the enzyme
ratio, equal to 1.06, while
βΕΧΗ/β
was equal to 0.45.
Results are schematised in Table 2.
Table 2. Relative amounts of alkylated globin chains with respect to the unmodified ones in ECH-alkylated globin
samples digested with different amounts of Calpain.
Sample
Undigested ECH-alkylated
globins (Hb+HbECH)
Globins:Calpain = 200:1, 1 h
Globins:Calpain = 200:1, 5 hrs
αECH/α βECH/β
0.725
0.550
0.750
0.845
0.790
1.056
1.154
0.543
0.580
0.496
0.446
0.475
Discussion
Proteic adducts are considered a powerful instrument for the evaluation of human exposure to carcinogen agents and a
valid surrogate of the DNA adducts. A certain number of analytical techniques are nowadays applied in the proteic
adduct analysis, as chemical and enzymatic digestions, electrophoresis or capillary liquid chromatography with different
detection techniques, such as mass spectrometry and fluorescence, or immunochemical approaches, as enzyme-linked
immunosorbent assay (ELISA).
The advancement in the instrumental performances allows high sensitivity levels, from 0.1 to 500 fmol of proteic
adduct; nevertheless there are no reports concerning the costs and the time of the unavoidable purification procedure of
the biological sample and the selective enrichment of the macromolecular adduct under study with respect to the
unmodified proteic fraction.
Over the last few years modified Edman degradation and Raney Nickel procedures have been widely used for the
biological monitoring of exposed workers (34-38).
Methods based on modified Edman degradation allow the determination of adducts on the N-terminal Valine of the
globin chains. However, as reported in literature, depending on the toxicant nature, N-terminal adducts could not be the
most abundant ones, because of the presence of eventually more reactive sites within hemoglobin chains. In such cases
the quantification of N-terminal adducts would not reveal low exposure levels.
82
P&R Scientific
It has been demonstrated that in most cases one of the most reactive site of hemoglobin is the cysteine 93 of the
β-
chain (20, 39).
Methods based on Raney Nickel procedure are based on the cleavage of the carbon-sulphur bond in the electrophilecysteine
adduct
to
form
alcohols,
which
are
extracted,
suitably
derivatised
and
analysed
by
gas
chromatography/electron capture detection (GC/ECD) or gas chromatography/negative chemical ionization-mass
spectrometry (GC/NICI-MS) (37, 40).
These techniques are useful in the evaluation of the exposure to aromatic toxicants, as styrene, benzene and their
metabolites, but are not applied to non aromatic electrophyles. Both modified Edman degradation and Raney Nickel
procedure are characterized by high sensitivity, since selective purification procedures have been developed, thus
allowing the recovery and the subsequent analysis of the alkylated N-terminal residue or Cysteine-bound electrophile,
that are purified from the remaining portion of globin.
The determination of the internal modified peptides overcomes in principle the limitation of both modified Edman
degradation and Raney Nickel procedures, since it is able to detect the carcinogen-protein adduct level at the more
reactive amino acid residue and it is generalizable to almost all electrophiles. Nevertheless, the actual applicability of the
proposed procedure is subjected to the development of an efficacy purification procedure, so that the required
sensitivity could be reached. With this respect, the present work illustrates the preliminary results of a selective
enrichment procedure centred on an enzymatic digestion with Calpain I (a cysteine protease) of hemoglobin samples
containing both normal and alkylated fractions.
The characterization of the main proteolytic sites in the case of pure hemoglobin as substrate revealed high
reproducibility in controlled reaction condition, even if there is no specificity, since proteolysis takes place among amino
acid residues of different nature.
Besides, we found cleavage sites within the whole globin sequences, even if primary cleavage sites are preferentially
located at the C-terminus ends. Recently, Tompa et al. investigated the recognition mechanism and sequential
determinants of Calpain cleavage (29).
The authors, even if suggested to investigate the influence of higher order structural elements, based their study on the
primary sequence of substrates, and assigned a score to each amino acid, according to its position around the scissile
bond, the so-called P2-P1 rule (41,42). Scores are summed and the obtained value is related to the attitude for Calpain
to cleave at a given position.
Our results show a notable agreement with literature data as regards the prevalence of Leu and Val in the P2 position,
and Lys, Arg, Tyr in P1. Moreover, the remarkable occurrence of Thr in P2 for the substrate recognition process is
confirmed. On the contrary, cleavage sites found at the globins ends show low scores, as expected since they are
worked out considering 11 positions around the scissile bond.
The obtained data evidenced that the recognition mechanism of Calpain is influenced not only by the primary sequence
of substrates but also by the reaction conditions used during the proteolysis, such as buffer, Calcium amount,
temperature and time reaction. In fact, digestions carried out on precipitated globins in sodium borate at 25 °C for 10
minutes (43) brought to two cleavage sites only, Lys 11-Ala 12 (α-chain) and Lys 8-Ser 9 (β-chain): while the former
was a cleavage site in our condition reaction too, the latter was not detected at all.
We studied if the alkylation of Cysteine residues within the hemoglobin sequence could influence the cleavage activity of
Calpain I.
When digestion experiments were repeated using hemoglobin samples containing not only Hb, but both unmodified and
alkylated globins (Hb and HbECH), the same proteolytic sites, hence the same peptides previously obtained, were
evidenced and no alkylated peptides were found. Hemoglobin
α-chain
contains only a Cysteine residue (Cys 104);
previous studies demonstrated that such amino acid residue represents an alkylation site for ECH (18, 20). This is in line
with the obtained results, which indicate that the
αECH
chain is not recognised as substrate by the Calpain even
increasing the enzyme amount.
As a consequence a selective enrichment of
Hemoglobin
αECH with respect to α was evidenced.
β-chain contains two Cysteine residues (Cys 93
and 112); under physiological condition the Cys 93 is easily
alkylated, while the other one presents weak reactivity, since it lies within the hydrophobic core of the protein. The
83
P&R Scientific
digestions with Calpain were conducted on precipitated globin samples, so, both the substrates, Hb and HbECH, did not
presented the typical native conformation of the protein and
β-chains
could be recognised by the enzyme, not only by
Cys 93 but through Cys 112 too.
The
β
βECH/β
and
ratio remained nearly constant also when increasing amount of Calpain were used, thus indicating that both
βECH
are digested. This seems to be apparently in contrast with the fact that the LC/ESI-MS full scan
chromatograms did not show the presence of peptides deriving from the alkylated globin chains.
Really the problem could concern the sensitivity of the MS full scan analysis itself, since the intensity of the eventually
alkylated peptides could be too low for being distinguished from the chemical noise especially for small peptides, as the
one formed in the Cys 93 neighbourhood.
This hypothesis requires LC/ESI-MSMS experiments to be confirmed.
As mentioned, a part from the substrate primary structure, higher order structural elements could influence Calpain
recognition and cleavage processes. That is why, even if poor selectivity was obtained between digestions of
β
and
βECH
chains, results could be different if proteolysis experiments were conducted on hemolysed samples, i.e. removing only
the cellular membranes and preserving the native conformation of the protein, with the Cys 112 within the hydrophobic
core and not accessible for the enzyme/substrate recognition.
The obtained preliminary results about the possibility of selectively purify the hemoglobin adducts by removing the
unmodified globin fraction let us to suggest that digestion with Calpain could allow an actual enrichment of the alkylated
hemoglobin fraction present in biological samples.
The subsequent purification of the proteolyses mixture by molecular sieves, with a cut off of 10000 amu, could further
improve the analytical sensitivity, thus allowing the actual applicability of the internal modified peptides procedure in the
evaluation of human exposure to carcinogen agents.
Other enzymes than Calpain could be scrutinized for their capability to a better selective degradation of modified
proteins.
Abbreviations
Hb, hemoglobin; ESI, Electrospray ionization; MSMS, Tandem Mass Spectrometry; ECH, epichlorohydrin, 1-chloro-2,3epoxypropane; HbECH, epichlorohydrin alkylated hemoglobin; TFA, trifluoroacetic acid;
α chain; βECH, epichlorohydrin alkylated β
α ECH,
epichlorohydrin alkylated
chain; ECD, electron capture detection; NICI, negative chemical ionization.
References
1.
Hemminki K, Dipple A, Shuker DEG, et al. DNA adducts: identification and biological significance. IARC Scientific
IARC Lyon 1994; 125.
2.
Singer B, Grunberg D. Molecular biology of mutagens and carcinogens. Plenum Press New York 1983.
3.
Lutz WK. Quantitative evaluation of DNA binding data for risk estimation and for classification of direct and indirect
carcinogens. J. Cancer Res. Clin. Oncol. 1986; 112: 85-191.
4.
Otteneder M, Lutz WK. Correlation of DNA adduct levels with tumor incidence: carcinogenic potency of DNA
adducts. Mutat. Res. 1999; 424: 237-247.
5.
Poirier MC. Concepts and mechanisms in carcinogen-DNA interactions. Hengstler JG et al: Control mechanisms of
carcinogenesis, Druckerei Thieme, Meissen, 1996.
6.
7.
Hemminki K. DNA adducts, mutations and cancer. Carcinogenesis 1993; 14: 2007-2012.
Wild CP, Pisani P. Carcinogen-DNA and carcinogen-protein adducts in molecular epidemiology. Toniolo P et al:
Application of biomarkers in cancer epidemiology, IARC Scientific IARC, Lyon, 1997;142.
8.
Tornqvist M, Landin HH. Hemoglobin adducts for in vivo dose monitoring and cancer risk estimation. J. Occup.
Environ. Med. 1995; 37: 1077-1085.
84
P&R Scientific
9.
Kensler TW, Groopman JD, Wogan GN. Use of carcinogen-DNA and carcinogen-protein adduct biomarkers for
cohort selection and as modifiable end points in chemioprevention trials. Stewart BW et al: Principle of
chemioprevention, IARC, Lyon, 1996.
10. Skipper PL, Tannenbaum SR. Protein adducts in the molecular dosimetry of chemical carcinogens. Carcinogenesis
1990; 11: 507-518.
11. Ehrenberg L, Tornqvist M. Human health risk assessment and biological reactive intermediates: hemoglobin
binding. Witmer CM: Biological reactive intermediates Plenum Press, New York, 1990.
12. Skipper PL, Peng X, Soohoo CK, Tannenbaum SR. Protein adducts as biomarkers of human carcinogen exposure.
Drug Metab. Rev. 1994; 26: 111-124.
13. Farmer P. Analytical approaches for the determination of protein-carcinogen adducts using mass spectrometry.
Groopman JD et al: Molecular dosimetry and human cancer: Analytical, Epidemiological and Social Considerations,
CRC press, Boca Raton, 1991.
14. Tornqvist M, Fred C, Haglund J, et al. Protein adducts: quantitative and qualitative aspects of their formation,
analysis and applications. J. Chromatogr. B 2002; 778: 279-308.
15. Poirier MC, Santella RM, Weston A. Carcinogen macromolecular adducts and their measurement. Carcinogenesis
2000; 21: 353-359.
16. Day BW, Naylor S, Gan L-S, et al. Molecular dosimetry of polycyclic aromatic hydrocarbon epoxides and diol
epoxides via hemoglobin adducts. Cancer Res. 1990; 50: 4611-4618.
17. Osterman-Golkar S, Ehrenberg L. Covalent binding of reactive intermediates to hemoglobin as an approach for
determining the metabolic activation of chemicals-ethylene. Drug Metab. Rev. 1982; 13: 647-660.
18. Miraglia N, Basile A, Pieri M, et al. Ion trap mass spectrometry in the structural analysis of hemoglobin peptides
modified by epichlorohydrin and diepoxybutane. Rapid Commun. Mass Spectrom. 2002; 16: 840-847.
19. Miraglia N, Pieri M, Basile A, et al. Exposure to genotoxic agents: modified peptides as suitable biomarkers. Recent
Res. Develop. Peptides. Research Signpost 2002; 1: 49-63.
20. Miraglia N, Pocsfalvi G, Ferranti P, et al. Mass spectrometry identification of a candidate biomarker peptide from
the in vitro interaction of epichlorohydrin with red blood cells. J. Mass Spectrom. 2001; 36: 47-57.
21. Sannolo N, Mamone G, Ferranti P, et al. Biomonitoring of human exposure to methyl bromide by isotope dilution
mass spectrometry of peptide adducts. J. Mass Spectrom. 1999; 34: 1028-1032.
22. Basile A, Ferranti P, Pocsfalvi G, et al. A novel approach for identification and measurement of hemoglobin adducts
with 1,2,3,4-diepoxybutane by liquid chromatography/electrospray ionization mass spectrometry and matrixassisted laser desorption/ionization tandem mass spectrometry. Rapid Commun. Mass Spectrom. 2001; 15: 527540.
23. Sajid M, McKerrow JH. Cysteine proteases of parasitic organisms. Mol. Biochem. Parasitol. 2002; 120: 1-21.
24. Melloni E, Michetti M, Salamino F, et al. Modulation of the calpain autoproteolysis by calpastatin and phospholipids.
Biochem. Biophys. Res. Commun. 1996; 229: 193-197.
25. Perrin BJ, Huttenlocher A. Calpain. Int. J. Biochem. Cell. Biol. 2002; 34: 722-725.
26. Cottin P, Poussard S, Desmazes JP, et al. Free calcium and calpain I activity. Biochim. Biophys. Acta. 1991; 1079:
139-145.
27. Sorimachi H, Ishiura S, Suzuki K. Structure and physiological function of calpains. Biochem. J. 1997; 328: 721732.
28. Schaecher K, Goust JM, Banik NL. The effects of calpain inhibition on IkB alpha degradation after activation of
PBMCs: identification of the calpain cleavage sites. Neurochem. Res. 2004; 29: 1443-1451.
29. Tompa P, Buzder-Lantos P, Tantos A, et al. On the Sequential Determinants of Calpain Cleavage. J. Biol. Chem.
2004; 279: 20775–20785.
30. Landin HH, Grummt T, Laurent C, Tates A. Monitoring of occupational exposure to epichlorohydrin by genetic
effects and hemoglobin adducts. Mutat. Res. 1997; 381: 217-226.
31. Laskin S, Sellakumar AR, Kuschner M, et al. Inhalation carcinogenicity of epichlorohydrin in noninbred SpragueDawley rats. J. Natl. Cancer Inst. 1980; 65: 751-757.
32. Van Duuren BL, Goldschmidt BM, Kats C, et al. Carcinogenic activity of alkylating agents. J. Natl. Cancer Inst.
1974; 53: 695-700.
33. International Agency for Research on Cancer. Monogr. Eval. Carcinogen. Risks Hum. IARC, Lyon, 1999; 71:2.
85
P&R Scientific
34. Tornqvist M.. Epoxide adducts to N-terminal Valine in hemoglobin. Methods Enzymol. 1994; 231: 650-657.
35. Tornqvist M, Mowrer J, Jensen S, Eherenberg L. Monitoring of environmental cancer initiators through hemoglobin
adducts by Edman modified degradation method. Anal. Biochem. 1986; 154: 255-266.
36. Tornqvist M, Kautiainen A. Adducted proteins for identification of endogenus electrophiles. Environ. Health
Perspect. 1993; 99: 39-44.
37. Ting D, Smith MT, Doane-Setzer P, Rappaport SM. Analysis of styrene oxide-globin adducts based upon reaction of
Raney nickel. Carcinogenesis 1990; 11: 755-760.
38. Rappaport SM, Ting D, Jin Z, et al. Application of Raney Nickel to measure adducts of styrene oxide with
hemoglobin and albumin. Chem. Res. Toxicol. 1993; 6: 238-244
39. Mamone G, Malorni A, Scaloni A, et al. Structural analysis and quantitative evaluation of the modifications
produced in human hemoglobin by methyl bromide using mass spectrometry and Edman degradation. Rapid
Commun Mass Spectrom. 1998; 12: 1783-1792.
40. Lindstrom AB, Yeowell-O’Connell K, Waidyanatha S, et al. Formation of hemoglobin and albumin adducts of
benzene oxide in mouse, rat, and human blood. Chem. Res. Toxicol. 1998; 11: 302-310.
41. Sasaki T, Kikuchi T, Yumoto N, et al. Comparative specificity and kinetic studies on porcine calpain I and calpain II
with naturally occurring peptides and synthetic fluorogenic substrates. J. Biol. Chem. 1984; 259: 12489-12494.
42. Hirao T, Takahashi K. Purification and characterization of a calcium-activated neutral protease from monkey brain
and its action on neuropeptides. J. Biochem. 1984; 96: 775-784.
43. Melloni E, Salamino F, Sparatore B, et al. Characterization of the single peptide generated from the aminoterminus end of alpha- and beta-hemoglobin chains by the Ca2+-dependent neutral proteinase. Biochim. Biophys.
Acta 1984; 788: 11-16.
Corresponding Author: *Nadia Miraglia
Department of Experimental Medicine – Section of Occupational Medicine, Hygiene and Industrial Toxicology,
Second University of Naples, Via L. De Crecchio, 7. 80138, Naples, Italy
Phone +39 (0)81 5665900. Fax +39 (0)81 5469185
E-mail: [email protected]
Autore di riferimento: *Nadia Miraglia
Dipartimento di Medicina Sperimentale – Sezione di Medicina Occupazionale, Igiene e Tossicologia Industriale
Seconda Università di Napoli, Via L. De Crecchio 7, I-80138 Napoli, Italia
Phone +39 (0)81 5665900. Fax +39 (0)81 5469185
E-mail: [email protected]
86
P&R Scientific
META-ANALYSIS: CARDIOVASCULAR EFFECTS IN WORKERS
OCCUPATIONALLY EXPOSED TO URBAN POLLUTION
META-ANALISI: EFFETTI SULL'APPARATO CARDIOVASCOLARE IN LAVORATORI
ESPOSTI A INQUINAMENTO URBANO
Sancini A 1, Caciari T 1, Di Pastena C 1, Sinibaldi F 1, Scala B 1, Fiaschetti M 1, De Sio S 1,
Maurizi D 1, Nardone N 1, Scimitto L 1, Miracco P 1, Tomei F 1, Tomei G 2, Ciarrocca M
1
1
University of Rome “Sapienza”, Department of Anatomy, Histology, Medical-Legal and the Orthopaedics, Unit
of Occupational Medicine (Dir: Prof. F. Tomei), Viale Regina Elena 336, 00161 Rome, Italy
2
University of Rome “Sapienza”, Department of Psychiatric and Psychological Science, Piazzale Aldo Moro 5,
00185 Rome, Italy
1
Dipartimento di Anatomia, Istologia, Medicina Legale e Ortopedia, Unità di Medicina del Lavoro
(Dir: Prof. F. Tomei), Università di Roma “Sapienza”,Viale Regina Elena 336, 00161 Roma, Italia
2
Dipartimento Scienze Psichiatriche e Medicina Psicologica, Università di Roma “Sapienza” Piazzale
Aldo Moro 5, 00185 Roma, Italia
Abstract published: G Ital Med Lav Erg 2010; 32:4, Suppl, 352-354
Citation: Sancini A, Caciari T, Di Pastena C, et al. Meta-analysis: cardiovascular effects in workers occupationally
exposed to urban pollution. Prevent Res 2011; 1 (1): 87-100
Key words: cardiovascular system, cardiovascular diseases, urban pollution
Parole chiave: sistema cardiovascolare, malattie cardiache, inquinamento urbano
Abstract
Background: Many studies showed a connection between exposition to high levels of urban pollution (especially to
particulate and traffic noise) and the onset of even deadly cardiovascular diseases.
DESIGN: Meta-analysis of case-control design.
Objectives: The aim of this study is to estimate the association between cardiovascular effects and occupational
exposition to atmospheric pollution in urban environment.
87
P&R Scientific
Methods: DATA SOURCES: Biomedcentral, MEDLINE/ PubMed, MEDLINE/ National Library of Medicine (NLM), MEDLINE
Plus, Nioshtic-2, Scopus, TOXNET/Toxline, unpublished studies known by the authors and acts of national and
internationl conferences between 1988 and May 2010 included. There has not been any kind of language or typological
restriction.
CRITERIA OF ELEGIBILITY: The research on cardiovascular effects includes control cases of workers exposed to urban
pollution, compared with non-exposed subjects.
PARTICIPANTS AND INTERVENTIONS: The selected studies present outdoor workers exposed to urban pollution (drivers
and petrol pump attendants) and a control group of indoor workers (managers, university students and other selected
subjects).
STUDY APPRAISAL AND SYNTHESIS METHODS: The evidences (independently token from two different authors) have
been grouped in two classes, the first one formed by continuous variables (systolic blood pressure, diastolic blood
pressure, heart rate, total cholesterol, HDL cholesterol, LDL cholesterol, triglycerides) and the second one by
discontinuous variables (electrocardiographic abnormalities prevalence, hypertension prevalence, hypercholesterolemia
prevalence).
The studies related to both classes and presenting more than one variable for each class have been included and used,
in order to elaborate the results. We calculated heterogeneity in each variable (ES calculation for continuous variables
and OR calculation for discontinuous variables).
Results: On 378 publications, we have selected and included 16 articles. The variables show statistically irrelevant
differences between exposed group and control group, except regarding the triglycerides. Limitations. The controlled
studies are limited and characterized by a non-homogeneous evaluation of both expositional times of workers to urban
pollution and of professional expositional values.
Conclusions: Considering the heterogeneity and the lack of studies, it is impossible at the moment to document effects
on the cardiovascular system in workers exposed to urban pollution. The results of this meta-analysis only suggest the
association of urban pollution with alteration of triglycerides blood levels (referring to just three studies). As reported in
scientific literature on this subject, it is necessary to conduct a future professional investigation on this subject with
more qualified and homogeneous studies.
Abstract
Introduzione: Numerosi studi hanno dimostrato un’associazione tra esposizione ad elevati livelli di inquinamento
urbano (nella fattispecie l’esposizione al particolato sia a breve che a lungo termine e il rumore da traffico veicolare) e
insorgenza nonché mortalità per patologie cardiovascolari.
DESIGN: meta-analysis of case-control design.
Obiettivi: Valutare l’associazione esistente tra effetti cardiovascolari ed esposizione occupazionale ad inquinamento
atmosferico in ambiente urbano.
Metodi: DATA SOURCES: Biomedcentral, MEDLINE/ PubMed, MEDLINE/ National Library of Medicine (NLM), MEDLINE
Plus, Nioshtic-2, Scopus, TOXNET/Toxline, inclusi gli studi non ancora pubblicati di cui gli autori erano a conoscenza e gli
atti dei Convegni nazionali ed internazionali, dal 1988 al Maggio 2010. Non è stata effettuata nessuna restrizione, di
lingua o tipo di pubblicazione, alla ricerca.
STUDY ELIGIBILITY CRITERIA: sono stati inclusi gli studi caso-controllo in cui i lavoratori esposti ad inquinamento
urbano sono stati comparati con quelli non esposti in relazione agli effetti sull’apparato cardiovascolare.
PARTICIPANTS, AND INTERVENTIONS: sono stati selezionati gli studi in cui la popolazione degli esposti all’inquinamento
urbano era costituita da addetti ad attività in ambiente outdoor, viabilisti, benzinai ed autisti professionisti mentre il
88
P&R Scientific
gruppo dei controlli era rappresentato da addetti ad attività indoor, amministrativi, studenti universitari e soggetti
selezionati tra la popolazione generale.
STUDY APPRAISAL AND SYNTHESIS METHODS: i dati, estratti da due autori diversi in modo indipendente, sono stati
raggruppati in due classi di variabili, la prima costituita da variabili continue (pressione arteriosa sistolica, pressione
arteriosa diastolica, frequenza cardiaca, colesterolo totale, colesterolo HDL, colesterolo LDL, trigliceridi) e la seconda da
variabili discontinue (alterazioni elettrocardiografiche, ipertensione, ipercolesterolemia, ipertrigliceridemia). Gli studi che
riportavano i dati relativi ad entrambe le classi e per ciascuna classe più variabili sono stati inseriti e quindi utilizzati più
volte per l’elaborazione dei risultati. Per ciascuna variabile è stata calcolata l’eterogeneità, per le variabili continue è
stato effettuato il calcolo dell’ES mentre per le variabili discontinue è stato effettuato il calcolo dell’OR e della sua
significatività.
Risultati: Su un totale di 378 pubblicazioni, sono stati selezionati ed inclusi nella meta-analisi 16 articoli. Le variabili
studiate non mostrano differenze statisticamente significative nel gruppo degli esposti rispetto ai controlli tranne che per
i trigliceridi.
LIMITATIONS: gli studi controllati risultano essere limitati e sono caratterizzati da una non omogenea valutazione sia dei
tempi di esposizione dei lavoratori all’inquinamento urbano sia dei valori di esposizione professionale.
Conclusioni: Considerata l’eterogeneità e la scarsità degli studi, soprattutto per alcuni parametri, al momento non è
possibile documentare, nei lavoratori professionalmente esposti all’inquinamento urbano, la presenza di effetti avversi
sull’apparato cardiovascolare. Infatti l’analisi dei risultati ottenuti con la nostra meta-analisi suggerisce la sola
associazione tra inquinamento urbano e alterazioni dei livelli ematici di trigliceridi, il risultato è inoltre relativo perché
riferito a tre studi. A fronte di quanto riportato in materia dalla letteratura scientifica relativamente alla popolazione
generale, è necessario che la ricerca futura approfondisca tale tematica in ambito professionale con studi condotti in
maniera più idonea ed omogenea.
Introduction
Urban pollution is a considerable health problem all-over the world (1).
Many observational and experimental studies in general population found an association between urban pollution and
mortality due to cardiovascular diseases, considering urban pollution a possible joint cause of their onset(2-7).
Even if the process implying this connection is not yet fully explained, it is possible to recognize some risk factors, such
as the exposition to chemical agents short-term (2,8-11) and long-term (2, 5, 12-15) exposition to particulate matter,
lead, (16-21)cadmium (4), carbon monoxide(22) and to traffic noise (12-13,23-24).
The aim of this meta-analysis is to conduct a systematic revision of the scientific evidence on cardiovascular effects in
workers exposed to urban pollution (25).
The intention is to determine possible cardiovascular effects and especially modifications of risk factors (as blood
pressure, heart rate, lipid blood levels) of urban pollution on workers.
Materials
The study has been conducted and reported as in the MOOSE Consensus Statement (26).
We did a systematic research of observational studies (control-cases from January 1988 to May 2010) reporting
cardiovascular effects in exposed (cases) and non-exposed (control) subjects to urban pollution. We also considered
known but not yet published data and acts of national and international conferences. A further examination of the
bibliography in previously published articles, reviews and meta-analysis on this subject has been conducted, in order to
find other useful publications. There has not been any kind of language or typological restriction to the research. The
utilizable works have been identified with a systematic research on the following on-line search engines: Biomedcentral,
MEDLINE/ PubMed, MEDLINE/ National Library of Medicine (NLM), MEDLINE Plus, Nioshtic-2, Scopus, TOXNET/Toxline.
The research has been conducted introducing the following keywords, combined with “Boolean Operators”:
89
P&R Scientific
•
police workers (or police officers or traffic police or traffic wardens) AND cardiac function, cardiovascular
function, heart disease, cardiac diseases, cardiovascular diseases, cardiac system, cardiovascular system, blood
pressure, heart rate, electrocardiogram, electrocardiography, hypertension;
•
mail carriers (or postal workers) AND cardiac function, cardiovascular function, heart disease, cardiac diseases,
cardiovascular diseases, cardiac system, cardiovascular system, blood pressure, heart rate, electrocardiogram,
electrocardiography, hypertension;
•
street vendors (or shop vendors) AND cardiac function, cardiovascular function, heart disease, cardiac diseases,
cardiovascular diseases, cardiac system, cardiovascular system, blood pressure, heart rate, electrocardiogram,
•
bus drivers AND cardiac function, cardiovascular function, heart disease, cardiac diseases, cardiovascular
diseases,
cardiac
system,
cardiovascular
system,
blood
pressure,
heart
rate,
electrocardiogram,
•
taxi drivers AND cardiac function, cardiovascular function, heart disease, cardiac diseases, cardiovascular
diseases,
cardiac
system,
cardiovascular
system,
blood
pressure,
heart
rate,
electrocardiogram,
•
filling station attendants (or petrol station attendants or gas station attendants or gasoline station attendants)
AND cardiac function, cardiovascular function, heart disease, cardiac diseases, cardiovascular diseases, cardiac
system,
cardiovascular
system,
blood
pressure,
heart
rate,
electrocardiogram,
electrocardiography,
hypertension;
•
road sweepers (or street sweepers or road cleaners or street cleaners) AND cardiac function, cardiovascular
function, heart disease, cardiac diseases, cardiovascular diseases, cardiac system, cardiovascular system, blood
pressure, heart rate, electrocardiogram, electrocardiography, hypertension.
Selection process and Data abstraction
Two reviewers independently evaluated title, abstract and keywords in each resulting study and applied criteria of
exclusion and inclusion. The same procedure was applied to full texts and used for the choice of the relevant data, in
order to increase the reliability of the study. We included control-case studies dealing with both outdoor workers (cases
– as local policemen and professional drivers) and indoor workers (controls – as administrative local agents, employees,
university students and other selected subjects). Firemen and street maintenance staff were excluded, considering that
their exposition to urban pollution is occasional and added to other harmful specific pollutions that can have effects on
the cardiovascular system. We only considered the research in which the connection between urban pollution and
cardiovascular effects had been studied according to at least one of the following variables: systolic blood pressure
(SBP) and diastolic blood pressure (DBP) or hypertension prevalence, heart rate (HR), electrocardiographic
abnormalities (ECGA).
We also considered blood lipids levels as total cholesterol (TCHO), LDL cholesterol (LDL) and HDL cholesterol (HDL),
triglycerides (TRY) or prevalence hypertrigliceridemia or hypercholesterolemia, considering their importance in
cardiovascular pathologies. All the articles not concerning our aim were excluded (articles not specifically dealing with
the above-mentioned tasks, not based on the control-case system and with incomplete or lacking data).
Data analysis
The Effects Size (ES), reflecting the strength of the relationship between two variables, was performed by Standardized
Mean Difference (SMD) or Weighted Mean Difference (WMD), at the presence of heterogeneity, when the data were
expressed in the media and Standard Deviation, for continuous variables (Class I). While it was expressed by Odds Ratio
(OR) when the data were expressed as frequency, for the discontinuous variables (Class II). For measures of ES based
on the Odd Ratio (OR), a ratio of 1.0 indicates the lack of differences among the studied groups. The heterogeneity
among the used data, referring to the variability or differences among studies in estimating the effect, was assessed
with the Index of Inconsistency (I2). Using I2 we calculated the percentage of variance due to heterogeneity rather than
the real case. If the value of I2 was close to zero the observed variance was due to chance, if it was higher the variance
was due to several factors that need to be investigated. The calculation of ES was made using the Random Effects Model
(REM) for high values of I2 and the Fixed Effects Model (FEM) for I2 values close to zero (40).
90
P&R Scientific
Results
With the application of this criteria, we chose 378 publications. All the articles not inherent with our research were
excluded. We examined the full texts of 80 potentially acceptable articles and 16 of them (reported on table 1)
respected the criteria for inclusion. All the subjects (7714 workers) were divided into two groups, the exposed (EX –
3699 outdoor workers) and the control group (C – 4015 indoor workers).
Table 1 - Characteristics of included studies
Study
Country
Partecipans
Biava
1992 [27]
Italy
292
traffic Crosssectional
policemen /
60
hospital study
employees
used
as
control group
Bigert
2003 [28]
Swedish 106
Observation
al study
professional
drivers
/
1482
control group
selected from
general
population
Heart
attack
risk
in
professional
drivers
(bus,
taxi and truck
drivers)
Cervone
1993 [29]
Italy
28
men Observation
traffic
al study
policemen /
14
control
group
selected from
general
population
Effects
of
exposure
to
urban pollution
on health and
the activation
of
immune
system
in
traffic
policemen
Hedberg
1993 [30]
Swedish 440
men Observation
al study
professional
drivers /
1000
men
subjects
control group
Risk factors of
cardiovascular
disease
in
professional
drivers.
Study
design
Outcomes
Method
Main results
Evaluated Variables
The
effects
caused
by
exposition
to
urban
pollutants
(chemical
matters,
IPA,
benzene,
asbestos, lead,
monoxide).
Objective
examination
Spirometry
Electrocardiogram
Neurological visit
Determination
of
motor conduction’s
velocity of median
nerve
Psychological
questionnaire
Blood lead levels
Blood
carboxyhemoglobin
levels
Chemistry exam
(blood test)
Valuation of heart
attack risk factors:
•
smoke
cigarette's habit
•
alcohol's
consumption
•
physical
exercise
during
spare time
•
overweight
•
diabetes
mellitus
•
hypertensi
on
•
socialeconomical status
•
work
commitments
Anamnestic
questionnaire
Blood pressure
Chemistry exams
(blood test)
Urinary lead levels
Lymphocyte
subpopulations'
study
There
is
a
statistically
significant
difference between
exposed group and
control group about
musculo-skeletal
disorders and about
blood lead level.
Cardiac disorders
EX Male 2/179 (1.2),
EX Female 0/113 (-)
C Male: 1/36 (2.7), C
Female 1/24 (4.1)
Results
of
this
study show that
Odds
Ratio
for
heart attack in bus
drivers is 2.14 (IC
95% 1.34-3.41), in
taxi drivers heart
attack is 1.88 (IC
95%
1.19-2.98)
and in truck drivers
heart attack is 1.66
(IC 95% 1.22-2.26)
Prevalence
hypertension
EX 30/106
C 23/1482
Urinary lead values
are
significantly
increased
in
exposed
group
compared
to
control
group
(p<0.01).
EX: SBP 123.8 (13.6),
DBP 82.4 (8.6),CHOL
TOT 238 (46), TRY
185 (124), GLY 97
(15)
C: SBP 127.2 (16.1),
DBP 78.6 (9.3),CHOL
TOT 229 (55), TRY
137 (85), GLY 97 (27)
Results
of
this
study show that
Odds Ratio (OR) for
cardiovascular risk
in
professional
drivers is 3.18 (IC
95%
2.41-4.20)
compared
to
control group.
EX: CHOL TOT 6.32
(6.19-6.45), HDL 1.26
(1.23-1.30), TRY 1.85
(1.51-2.19),
Prevalence
hypertension 33/440.
C: CHOL TOT 6.30
(6.22-6.39), HDL 1.28
(1.25-1.30), TRY 1.63
(1.55-1.71),
Prevalence
hypertension 35/1000.
91
Anamnestic
questionnaire
Objective
examination
Blood pressure
Chemistry exams
(blood test)
P&R Scientific
Crosssectonal
study
Mormonto
y
2006[31]
Perù
52
traffic
policemen /
50
police
officers
employed in
indoor
activity
Pala
2002 [32]
Turkey
78
traffic Observation
policemen / al study
21
police
officers
employed in
indoor
activity
Sakata
2007[33]
Nepal
27 men taxi Observation
al study
drivers
/
9
men
university
students and
farmhands
Sancini
2010 [34]
Italy
115
traffic Observation
al study
police
smokers
/
115
police
officers
employed in
indoor
activity
smokers
280
traffic
police
non
smokers
/
280
police
officers
employed in
indoor
activity
non smokers
Sardelli
1995 [35]
Italy
120
traffic Observation
policemen /
al study
Blood lead levels
are
statistically
increased
in
exposed
group
compared
to
control group.
EX:
SBP
113.02
(9.29),
DBP
73.94
(7.75)
C: SBP 114.18 (8.20)
DBP 75.20 (8.35)
Results
of
this
study show that
there is not a
statistically
significant
difference between
exposed group and
control group about
blood pressure and
blood lead levels.
Erythropoietin
The
dose- Blood pressure
Chemistry
exams serum levels are
response
relationship
(blood test)
significantly
between blood Heart rate
reduced in exposed
lead levels and Erythropoietin
group compared to
serum levels
erythropoietin
control group.
Urine analysis
serum
Blood lead levels
concentration
are increased in
in taxi drivers
exposed
group
exposed
to
compared
to
lead
of
control group.
vehicular
emission.
Blood
systolic
Blood pressure Anamnestic
changes
and questionnaire
pressure
average
electocardiogra Objective
value at rest and
examination
phic
electocardiographic
Blood pressure
abnormalities
abnormalities’frequ
in
traffic Electrocardiogram
ency is statistically
policemen
increased in non
exposed
to
smoker
male
urban pollution
subjects of exposed
group compared to
men non smoker
control group
Blood
systolic
pressure
average
value at rest is
statistically
increased in non
smoker
male
subjects of exposed
group compared
to men smokers of
exposed group.
Electocardiographic
abnormalities’frequ
ency is statistically
increased in non
smoker
male
subjects of control
group compared to
men smoker control
group
EX: SBP 119.5 (16.3),
DBP 75.9 (10.2)
C: SBP 116.4 (13.2)
DBP 76.20 (10.50)
Blood
lead
levels and the
effects
of
exposure
to
vehicular
traffic
on
cardiovascular
system
Blood
lead
levels and the
effects
of
exposure
to
vehicular traffic
on
cardiovascular
system
Anamnestic
questionnaire
Blood pressure
Chemistry exams
(blood test)
Blood lead levels
Anamnestic
questionnaire
Blood pressure
Chemistry
exams
(blood test)
Blood lead levels
Health effects Anamnestic
of exposure to questionnaire
92
Blood
levels
EX: SBP 135 (12),
DBP 79.0 (10), HR 78
(10), CHOL TOT 149
(28),
CHOL
HDL
37.9(7.8)
C: SBP 122 (8), DBP
78 (9), HR 71(4),
CHOL TOT 157(26),
CHOL HDL 37.6(9.3)
SMOKERS:
EX. Male:
SBP 126 (13.2),
DBP 78.5 (9.1),
ECG abnormalities 8
(19.0)
C Male:
SBP 129 (14.2),
DBP 81.4 (10.5),
ECG abnormalities 13
(30.9)
EX. Female
SBP 114.4 (13.6),
DBP 72.6 (9.8),
ECG
abnormalities
4(5.4)
C Female
SBP 114.6 (14.2),
DBP 73.2 (9.8),
ECG
abnormalities
7(9.5)
NON SMOKERS:
EX Male:
SBP 134.7 (19.2),
DBP 82.2 (8.9),
ECG Abnormalities 30
(20.8)
C Male:
SBP 128.6 13.6(),
DBP 81.3 (8.4),
ECG
Abnormalities
15(10.4)
EX Female:
SBP 116.2 13.8),
DBP 72.9 (9.5),
ECG
Abnormalities
16(11.7)
C Female:
SBP 116.6 (12.5)
DBP 75.9 (14.7),
ECG Abnormalities 15
(11)
cholesterol EX:
prevalence
are hypercholesterolemia
P&R Scientific
30
administrativ
e workers
Tomao
2002[14]
Italy
Tomei
2004[12]
Italy
Volpino
2004[13]
Italy
urban pollution
and noise in
traffic
policemen
118
men Observation
al study
traffic
policemen
118
police
officers
employed in
indoor
activity
Lipid
metabolism
traffic
policemen
exposed
urban
pollution.
77
non Observation
al study
smokers
traffic
policemen /
87
non
smokers
police
officers
employed in
indoor
activity
43 smokers
traffic
policemen /
29 smokers
police
officers
employed in
indoor
activity
Blood pressure
changes
in
traffic
policemen
exposed
to
urban pollution
through
ambulatory
monitoring of
blood pressure
during
24
hours.
68
men Observation
al study
traffic
policemen /
62
police
officers
employed in
indoor
activity
The effects of
exposure
to
urban pollution
on
cardiovascular
and respiratory
systems
in
traffic
policemen
93
in
to
significantly
increased
in
exposed
group
compared
to
control
group.
Spirometry shows
that
respiratory
obstructive
diseases
are
increased
in
exposed
group
compared
to
control group and
that
mild
hypertension
is
increased
in
exposed
group
more than control
in group.
HDL average values
Anamnestic
questionnaire
are
significantly
Objective
reduced in exposed
examination
group compared to
Chemistry exams
control
group,
(blood test)
instead
triglycerides
average values are
significantly
increased
in
exposed
group
compared
to
control group.
Blood
systolic
Anamnestic
questionnaire
pressure
average
Objective
value
monitored
examination
during 24 hours is
Chemistry exams
statistically
(blood test)
increased in non
Blood pressure
smoker
male
Monitoring of blood subjects of exposed
pressure during 24 group compared to
hours
men non smoker
.
control
group.
Blood
diastolic
pressure
average
values
monitored
during 24 hours,
between 6 am and
11 am and between
10 pm and 6 am,
are
statistically
increased in non
smoker
male
subjects of exposed
group compared to
non smoker male
subjects in control
group.
Objective
examination
Blood pressure
Cardiology visit
Electrocardiogram
Othorinolaryngolog
y visit
Chest radiography
Audiometry
Chemistry exams
(blood test)
Pneumotacograph
Anamnestic
questionnaire
Objective
examination
Chemistry exams
(blood test)
Blood pressure
Heart rate
Respiratory
frequency
Electrocardiogram
Stress
electrocardiogram
Hemogasanalysis
Spirometry
Diastolic
pressure
average
values
during rest period
are
statistically
increased
in
exposed
group
compared
to
control group.
Diastolic
pressure
average
values
during stress are
statistically
increased
in
exposed
group
compared
to
80/120
Prevalence
Hypertension
moderate: N. 14
ECG
Abnormalities
prevalence: N. 28
C:
prevalence
hypercholesterolemia
8/30
Prevalence
Hypertension
moderate: N. 2
ECG
Abnormalities
prevalence: N. 8
EX: CHOL TOT 208.2
(46.4),
HDL
29.9
(7.2),
TRY
173.8
(168.9).
C: CHOL TOT 202.6
(36.8),
HDL
55.3
(9.9),
TRY
138.1
(88.7).
EX:
SBP 135 (12),
DBP 79.0 (10),
CHOL TOT m 225.8
(55.2), CHOL TOT f
188.6 (40.7),
CHOL HDL m 43.4
(5.8), CHOL HDL f
58.7 (13.2), TRY m
181 (198.2) , TRY f
107 (48), LDL m 146
(34.4) , LDL f 108.3
(31.4) .
C:
SBP 122 (8),
DBP 78 (9),
CHOL TOT m 230.4
(26.9), CHOL TOT f
191.1 (26.8),
CHOL HDL m 43.1
(3.65), CHOL HDL f
48.7 (8.9), TRY m
167.8 (56) , TRY f
95.9 (31.5), LDL m
153.7 (26.85) , LDL f
125.3 (30.9) .
EX:
Prevalence
Hypertension 16/68
SBP 130 (12.6),
DBP 85.7 (8.5),
C:
Prevalence
Hypertension 16/62
SBP 127 (8.4),
DBP 81.9 (6.8),
P&R Scientific
Wang
2001 [36]
Taiwan
1761
male Observation
al study
bus drivers
/ 536 male
administrativ
e workers
Cardiovascular
risk factors in
bus
drivers
exposed
to
vehicular
emissions and
urban pollution
Anamnestique
questionnaire
Objective
examination
Chemistry exams
(blood test)
Electrocardiogram
Zefferino
2003[37]
Italy
30
traffic Observation
al study
policemen,
studied
during
a
working day
/
30
traffic
policemen
studied
during a day
off.
The effects of
exposure
to
urban pollution
on heart rate
in
policemen
employed
in
emergency
care
Anamnestique
questionnaire
Objective
examination
PSS
test
(Professional Stress
Scale Test)
Cortisol
salivary
levels
Salivary
IL-1B
levels
Electrocardiogram
Study of heart rate
variability
Zefferino
2006[38]
Italy
30
traffic Observation
al study
policemen
studied
during
a
working day
/ 30 traffic
policemen
studied
during a day
off.
Some salivary
stress markers
in
policemen
employed
in
emergency
care.
Anamnestique
questionnaire
Objective
examination
PSS
test
(Professional Stress
Scale Test)
Cortisol
salivary
levels
Salivary
IL-1B
levels
Electrocardiogram
Blood pressure
94
control group.
Hypertension,
hypercholesterolem
ia,
hypertriglyceridemi
a
and
electrocardiograph
abnormalities
are
statistically
increased
in
exposed
group
compared
to
control group.
Cortisol levels are
significantly higher
at the beginning of
work shift than at
the end. In the
morning
during
holiday,
average
cortisol
concentration
is
statistically
decreased
compared
to
cortisol
concentration in the
afternoon.
T
test
shows
statistically
significant
difference between
average
cortisol
levels at the same
time
during
holidays
and
working days.
IL1-B
average
concentration
at
the beginning of
work shift is higher
than at the end of
work shift (p<0.05)
Statistical analysis
shows statistically
significant
association
between LF/HF (low
frequency/high
frequency) of heart
rate measured at 8
am of day off and
the difference of
salivary
cortisol
levels at work.
Cortisol levels are
significantly higher
at the beginning of
work shift than at
the end (p<0.05).
During holiday in
the
morning,
average
cortisol
concentration
is
statistically
decreased
compared
to
cortisol
concentration in the
afternoon
EX:
Prevalence
Hypertension
986/1761, Prevalence
598/1761
Prevalence
hypertriglyceridemia
376/1761.
C:
Prev. Hyper 164/536
Prevalence
162/536
Prevalence
hypertriglyceridemia
166/536.
EX
HR during work
78.53 (10.47)
C:
HR during holiday
74.46 (8.84)
EX:
SBP/ DBP during work
135.3/89 (14.6/9.9)
C:
SBP/
DBP
during
holiday
131.8/ 84
(14.6/8.7)
P&R Scientific
Zhang
1994[39]
China
36
traffic Observation
al study
policemen
and
277
gasoline
workers /
342
administrativ
e workers
The aim of this
study
is
to
evaluate
the
effects of lead
exposure
on
workers’
health.
(p<0.05).
T
test
shows
statistically
significant
difference between
cortisol
average
levels at the same
hour during holiday
and working days
(p<0.05).
IL1-B
average
concentration
at
the beginning of
work shift is higher
than at the end
(p<0.05).
Anamnestique
Results show no
questionnaire
statistically
Objective
differences
examination
between
exposed
Electrocardiogram
group and control
Urinary lead levels
group
about
Urinary diethyl lead electrocardiographi
c abnormalities.
levels
Triethyl lead levels
EX (Traffic policemen
and
277
gasoline
workers):
ECG
Abnormalities
prevalence 18.5/277,
8.33/36
C:
ECG
Abnormalities
prevalence 6.43/342
We grouped the studies according to the nature of each investigated variable and identified two classes of variables:
continuous variables (systolic blood pressure, diastolic blood pressure, heart rate, total cholesterol, HDL cholesterol, LDL
cholesterol, triglycerides) and discontinuous variables (electrocardiographic abnormalities prevalence, hypertension
prevalence, hypercholesterolemia prevalence).
Even if at first we identified a prevalence of hypertriglyceridemia, we did not conduct statistical analysis on it, because it
was treated in one work only. The studies that reported data for both classes and several variables for each class were
entered and processed more than once for the elaboration of results. Evaluating the parameters for Class I, we included
a total of 3774 workers divided into two groups: 2037 subjects in the exposed group (EX) and 1737 subjects in the
control group (C). In the assessment of parameters related to discontinuous variables we included a total of 11737
workers: 6978 EX and 4759 C. Table 2-3 summarise the results of studies.
Results of Continuous variables: Class I
Systolic and diastolic blood pressure, evaluated in eight studies (12,13,29,31,32,33,34,38), and heart rate, in two
studies (33,37), show no statistically significant differences in the exposed compared to controls. The studies were
heterogeneous between them.
For the lipid parameters, total cholesterol (measured in four studies (12,14,29,33), and LDL cholesterol, measured in
one study (12) show no statistically significant differences and are characterized by a low index of heterogeneity. HDL
cholesterol (measured in three studies, (12,14,33) shows no statistically significant differences, but is characterized by a
high level of heterogeneity. Regarding triglycerides, measured in three studies (12,14,29),
there is a statistically
significant difference in the exposed compared to controls, with a low index of heterogeneity.
95
P&R Scientific
Table 2: The results of Class I (continuous variables)
NUMBER
VARIABLE
OF
P
I2 %
P 0.239
37.4
P 0.126
47.8
P 0.126
47.8
P 0.358
0.00
P 0.43
98.4
P 0.87
0.00
P 0.009
0.00
Meta-analysis index
SUBJECTS
Systolic
blood EX: 518
pressure
C: 417
Diatolic blood EX: 518
pressure
Heart rate
C: 417
EX: 57
C: 39
Total
EX: 293
cholesterol
C: 257
HDL
EX: 265
cholesterol
C: 243
LDL
EX: 120
cholesterol
C:116
Trycliceridaes
EX: 266
C: 248
SMD 0.093
[-0.062; 0.247]
SMD 0.121
[-0.34; 0.275]
WMD 0.121
[-0.34; 0.275]
WMD 0.094
[-0.106;- 0.295]
SMD 0.897
[-3.171; 1.37]
WMD 0.054
[-0. 485; 0.378]
WMD 0.280
[0.070; 0.489]
EX = Experimental Group
C = Control Group
P: = Probability
I² = Heterogeneity Index
SMD = Standardized Mean Difference
WMD =Weighted Mean Difference
Results of Not-continuous variables: Class II
Prevalence of electrocardiographic abnormalities, evaluated in five studies (12,27,35,36,39) took twice and the
prevalence of hypertension (assessed in two studies (35,36), do not give statistically significant differences in the
exposed compared to controls, with a high degree of heterogeneity.
Regarding the data on the prevalence of hypercholesterolemia (evaluated in two studies (35,36), there are not
statistically significant differences in the exposed compared to controls, with a high degree of heterogeneity.
96
P&R Scientific
Table 3. The results of Class II (not-continuous variables)
NUMBER
VARIABLE
OF
P
I2 %
P 0.702
73.0
P 0.169
86.3
P 0.37
87.6
Meta-analyis index
SUBJECTS
Electrocardiographic
EX: 2602
alterations
C: 1083
Hypertension
Hypercholesterolemia
EX: 2495
C: 3110
EX: 1881
C: 566
OR = 1.165
[0.53; 2.55]
OR = 1.39
[0.86; 2.33]
OR = 1.73
[0.51; 5.86]
EX = Experimental Group
C = Control Group
n.s. = not significant
P = Probability
I² = Heterogeneity Index
ES = Effect Size
OR = Odd ratio
Discussion
Many studies in the general population show the association between exposure to urban pollution and cardiovascular
effects, such as hypertension (41), ischemic heart disease, arrhythmias, heart failure, stroke and sudden death (3),
probably for alterations of the cardiac autonomic and myocardial perfusion and electrical instability (3, 5, 42, 43). In
addition, airborne particulates, according to the theory of Twickler et al (44), conveyed by the lipids, would be able to
infiltrate the vessel intima by promoting the formation of atherosclerotic plaques. Hence the importance of early
assessment in subjects exposed to any load changes in heart rate, blood pressure, lipid blood levels and
electrocardiogram.
The meta-analysis shows that the association between occupational exposure to urban air pollution and cardiovascular
effects gives statistically significant values in outdoor workers only for the blood levels of triglycerides. This value is
however relative, as reported in only three studies (12,14,29).
The result is confirmed by the work of Wang, that was not included in the meta-analysis because it is a single work. The
study, conducted on a large number of subjects, reports a statistically significant difference in the prevalence of
hypertriglyceridemia between bus drivers and skilled workers.
We found the same limitation of controlled studies also in the evaluation of other examined variables. Reviewed studies
are heterogeneous, as to the outcomes. Among 16 studies of our meta-analysis, only five focused on the association
between urban pollution and effects on the cardiovascular system (12,13,30,31,34). One of the remaining studies (36),
(analyzing the incidence of coronary heart disease in professional drivers) identified among
the potential risk factors both urban pollution and specific work factors. Four studies assessed the effects of urban
pollution on health, including the cardiovascular system (27,29,35,39).
One study (14) investigated the effects of urban pollution on the lipidic asset; this alteration is a known cardiovascular
risk factor. In their work, Pala and Sakata (32,33), evaluated blood lead levels respectively in policemen and taxi
drivers; the first incorporated blood pressure within the parameters of the assessment. The second incorporated heart
rate and the lipidic asset.
97
P&R Scientific
In conclusion, three more studies (28,37), dealing with subjects professionally exposed to urban pollution, attributed the
cardiovascular effects in outdoor workers to stress.
Even in literature it comes out stress is higher in workers exposed to urban pollution (45-48).
A non-uniform assessment of both time of exposure to pollution of urban workers, sometimes not even mentioned, and
of values of occupational exposure (environmental monitoring and / or organic) is a further problem. With the exception
of Sardelli and Zhang who quoted environmental measures [Sardelli P et al., 1995; Zhang W et al.,1994]) and Volpino,
Tomei and Sancini who evaluated biological indicators (Sancini A et al., 2010; Tomei F at al., 2004, Volpino P et al.,
2004), other authors recruited subjects only considering their work task, simply monitoring the exposure on the base of
questionnaires or assuming Mormontoy (Mormontoy W et al., 2006) that traffic police officers are exposed to pollution
from vehicular traffic.
Therefore, considering data from the scientific literature, related to the general population, which impute adverse
cardiovascular effects to urban pollution and given the data emerging from our meta-analysis on population
occupationally exposed, we believe it very important to carry on research occupationally exposed groups. To increase
the reliability of the parameters relating to the effects of urban pollution on the cardiovascular system, studies should
pay particular attention to procedures ascertaining level and type of pollutants, duration and terms of exposure, age of
the subjects evaluated, type of job and presence of other environmental stressors, as well as personal ones, that can
affect cardiovascular function, such as smoking, incorrect eating habits and excess fat mass (49).
References
Articles marked with an asterisk are those used for the meta-analysis
1. Brunekreef B, Holgate ST. Air pollution and health. Lancet 2002; 360: 1233-1242.
2. Hassing HC, Twickler TB, Kastelein JJ, et al. Air pollution as noxious environmental factor in the development of
cardiovascular disease. Neth J Med 2009; 67: 116-121.
3. Brook RD. Cardiovascular effects of air pollution. Clinical Science 2008; 115: 175-187.
4. Bhatnagar A. Environmental cardiology: studying mechanistic links between pollution and heart disease. Circ Res
2006; 99 (7): 692-705.
5. Hoffmann B, Moebus S, Stang A, Beck EM, Dragano N, Möhlenkamp S, Schmermund A, Memmesheimer M, Mann K,
Erbel R, Jöckel KH. Heinz Nixdorf RECALL Study Investigative Group. Residence close to high traffic and prevalence of
coronary heart disease. Eur Heart J 2006; 27 (22): 2696-702.
6. Pope CA 3rd, Burnett RT, Thurston GD, et al. Cardiovascular mortality and long-term exposure to particulate air
pollution: epidemiological evidence of general pathophysiological pathways of disease. Circulation 2004; 109 (1): 71-7.
7. Dockery DW. Epidemiologic evidence of cardiovascular effects of particulate air pollution. Environ Health Perspect
2001; 109 (Suppl 4): 483-486.
8. Peters A, Dockery DW, Muller JE, et al. Increased particulate air pollution and the triggering of myocardial infarction.
Circulation 2001a; 103: 2810-2815.
9. Peters A, von Klot S, Heier M, et al. Exposure to traffic and the onset of myocardial infarction. N Engl J Med 2004; 1:
1721-1730.
10. Katsouyanni K, Touloumi G, Samoli E, et al. Confounding and effect modification in the short-term effects of ambient
particles on total mortality: results from 29 European cities within the APHEA2 project. Epidemiology 2001; 12: 521531.
11. Mills NL, Donaldson K, Hadoke PW, et al. Adverse cardiovascular effects of air pollution. Nat Clin Pract Cardiovasc
Med 2009; 6: 36-44.
12. *Tomei F, Rosati MV, Baccolo TP, et al. Ambulatory (24 Hour) Blood Pressure Monitoring in Police Officers. J Occup
Health 2004; 46: 235-243.
13. *Volpino P, Tomei F, La Valle C, et al. Respiratory and cardiovascular function at rest and during exercise testing in
a healthy working population: effects of outdoor traffic air pollution. Occup Med 2004; 54: 457-482.
14. *Tomao E, Tiziana PB, Rosati V, et al. The effects of air pollution on the lipid balance of traffic police personnel. Ann
Saudi Med 2002; 22 (5-6): 287-290.
98
P&R Scientific
15. Hoek G, Brunekreef B, Goldbohm S, et al. Association between mortality and indicators of traffic-related air pollution
in the Netherlands: a cohort study. Lancet 2002; 360: 1203-1209.
16. Papanikolaou NC, Hatzidaki EG, Belivanis S, et al. Lead toxicity update. A brief review. Med Sci Monit 2005; 11: 329336
17. Ademuyiwa O, Ugbaja RN, Idumebor F, et al. Plasma lipid profiles and risk of cardiovascular disease in occupational
lead exposure in Abeokuta, Nigeria. Lipids Health Dis 2005; 4: 19.
18. Al-Saleh I, Shinwari N, Mashhour A, et al. Is lead considered as a risk factor for high blood pressure during
menopause period among Saudi women? Int J Hyg Environ Health 2005; 208: 341-56.
19. Karita K, Yano E, Dakeishi M, et al. Benchmark dose of lead inducing anemia at the workplace. Risk Anal 2005; 25:
957-962.
20. Eibensteiner L, Del Carpio Sanz A, Frumkin H, et al. Lead exposure and semen quality among traffic police in
Arequipa, Peru. Int J Occup Environ Health 2005; 11: 161-166.
21. Khalil N, Wilson JW, Talbott EO, et al. Association of blood lead concentrations with mortality in older women: a
prospective cohort study. Environ Health 2009; 8: 15.
22. Maitre A, Bonneterre V, Huillard L, et al. Impact of urban atmospheric pollution on coronary disease. Eur Heart J
2006; 27 (19): 2275-84.
23. Leon Bluhm G, Berglind N, Nordling E, et al. Road traffic noise and hypertension. Occup Environ Med 2007; 64: 1226. 29. Tomei G, Anzani MF, Casale T, et al. Extra-auditory effects of noise. G Ital Med Lav Ergon 2009; 31 (1): 37-48.
24. Tomei G, Anzani MF, Casale T, et al. Extra-auditory effects of noise. G Ital Med Lav Ergon 2009; 31 (1): 37-48.
25. Everrit BS, Howell DC. Encyclopaedia of statistics in behavioural science. Chichester, West Sussex (UK): John Wiley
and Sons, Ltd 2005.
26. Meta-analysis of Observational Studies in Epidemiology. Aproposal for reporting. Consensus Statement. JAMA, April
19, 2000 – Vol 283, No. 15.
27. *Biava PM. Indagine epidemiologica sulle condizioni di salute dei vigili urbani di Milano in rapporto all’inquinamento
da traffico veicolare. Med Lav 1992; 83 (3): 249-258.
28. *Bigert C, Gustavsson P, Hallqvist J, et al. Myocardial Infarction Among Professional Drivers. Epidemiology 2003;
14: 333-339.
29. *Cervone M, Pavone D, Tracanna A, et al. Sottopopolazioni linfocitarie nei vigili urbani di Pescara (studio
preliminare). In Atti 56° Congresso Nazionale della Società Italiana di Medicina del Lavoro e Igiene Industriale. Venezia
21- 23 october 1993, Vol II: 731-734.
30. *Hedberg GE, Jacobsson KA, Janlert U, et al. Risk indicators of ischemic heart disease among male professional
drivers in Sweden. Scand J Work Environ Health 1993; 19: 326-33.
31. *Mormontoy W, Gastañaga C, Gonzales GF et al. Blood lead levels among police officers in Lima and Callao, 2004.
Int J Hyg Environ Health 2006; 209: 497-502.
32. *Pala K, Akis¸ N, Izgi B, et al. Blood lead levels of traffic policemen in Bursa, Turkey. Int J Hyg Environ Health 2002;
205: 361-365.
33. *Sakata S, Shimizu S., Ogoshi K. et al. Inverse relationship between serum erythropoietin and blood lead
concentrations in Kathmandu tricycle taxi drivers. Int Arch Occup Environ Health 2007; 80: 342-345.
34. *Sancini A, Palermo P, Di Giorgio V, et al. Cardiovascular parameters in workers exposed to urban pollutions. G Ital
Med Lav Ergon 2010 Jan-Mar; 32 (1): 32-9.
35. *Sardelli P, D’Aniello MA, Padula M, et al. Sorveglianza sanitaria e rilevazione del rischio ambientale in un campione
di vigili urbani di Napoli. In Atti 58° Congresso SIMLII. Bologna 11-14 october 1995; 1087-1091.
36. *Wang PD. Coronary heart disease risk factors in urban bus drivers. Public health. 2001; 115: 261-264.
37. *Zefferino R, Facciorusso A, Lasalvia M, et al. Salivary markers of work stress in an emergency team of urban police
(1 degree step). G Ital Med Lav Erg 2006; 28: 4,472-477.
38. *Zefferino R, L’Abbate N, Facciorusso A, et al. Assessment of heart rate variability (HRV) as a stress index in an
emergency team of urban police. G Ital Med Lav Ergon 2003; 25: 167-169.
39. *Zhang W et al. Early health effects and biological monitoring in persons occupationally exposed to tetraethyl lead.
Int Arch Occup Environ Health 1994; 65: 395-399.
99
P&R Scientific
40. Borenstein M, Hedges LV, Higgins J, et al. Introduction to metaanalysis. Chichester, West Sussex (UK): John Wiley
and Sons, Ltd 2009.
41. Urch B, Silverman F, Corey P, et al. Acute blood pressure responses in healthy adults during controlled air pollution
exposures. Environ Health Perspect. 2005; 113: 1052-1055.
42. Folino AF, Scapellato ML, Canova C, et al. Individual exposure to particulate matter and the short term arrhythmic
and autonomic profiles in patients with miocardial infarction. European Heart Journal 2009; 30: 1614-1620.
43. Sun Q, Hong X, Wold LE. Cardiovascular effects of ambient particulate air pollution exposure. Circulation 2010; 121
(25): 2755-65.
44. Twickler M, Iinga-Thie G, Cramer MJ. Trojan Horse Hypothesis: Inhaled Airborne Particles, Lipid Bullets, and
Atherogenesis. JAMA, May 24/31, 2006 - Vol 295, No. 20
45. Winkleby MA, Ragland DR, Syme SL. Self-reported stressors and hypertension: evidence of an inverse association.
Am J Epidemiol 1988; 127: 124-134.
46. Schnall PL, Pieper C, Schwartz JE, Karasek RA, Schlussel Y, Devereux RB, Ganau A, Alderman M, Warren K,
Pickering TG. The relationship between job strain, workplace diastolic blood pressure, and left ventricular mass index.
Results of a case-control study. JAMA 1990; 263: 1929-1935.
47. Albright CL, Winkleby MA, Ragland DR, et al. Job strain and prevalence of hypertension in a biracial population of
urban bus drivers. Am J Public Health 1992; 82 (7): 984-989.
48. Nyklícek I, Vingerhoets JJ, Van Heck GL. Hypertension and objective and self-reported stressor exposure: a review. J
Psychosom Res 1996; 40: 585-601.
49. Andreozzi P, Viscogliosi G, Servello A, et al. Predictive medicine in Cardiovascular Diseases. What next?
Prevent Res 2011; 1 (1): 53-59.
Corresponding Author: Gianfranco Tomei
Address: Via Monte delle Gioie 13
Zip Code: 00199 Rome, Italy
Phone: +390649912540
Fax: +390686203178
Autore di riferimento: Prof. Gianfranco Tomei
Via Monte delle Gioie 13, 00199 Roma, Italia
Telefono: +390649912540
Fax: +390686203178
100

volume 1 number 01 - Prevention and Research

Transcript

Documenti analoghi

Seminario Agenda [IT] - Thermo Fisher Scientific

locandina B ils

Invito a Scientific Gaming

L`ultima “scoperta”: la vita non esiste

Scientific Writing - Azienda Ospedaliera San Camillo Forlanini

Luigi Bergamo

Europass Curriculum Vitae - Universitá della Calabria

Uomo e ambiente: nuove strategie di controllo

062941_inglese_scientifico_15_16

la ricerca italiana di punta produce risultati più citati