Objetivo Avaliar o perfil da exposição ocupacional a agentes químicos, em laboratórios, e verificar os impactos na avaliação de risco decorrentes das mudanças nos limites de exposição ocupacional (LEO) e na legislação federal brasileira. Metodologia Revisão de literatura relativa à exposição ocupacional a substâncias químicas, referentes a: conceituação de risco; desenho das curvas dose-resposta; identificação dos fatores que interferem na relação dose-efeito; conceitos de exposição por longo período, da exposição múltipla e em baixas concentrações; e fatores de confusão na monitoração ocupacional. Realização de estudo transversal no Centro de Pesquisas da Petrobras, para avaliar o perfil de exposição a agentes químicos de 3.000 trabalhadores, com atividades preponderantes em laboratórios, metade das quais com exposição em baixas concentrações e por longo período, tendo por base as avaliações ambientais do ano de 2004. Análise temporal dos valores de LEO da American Conferrence of Governmental Industrial Hygienists (ACGIH) e levantamento das mudanças nas legislações federal previdenciária, trabalhista e de saúde nos aspectos relacionados à exposição a agentes químicos. Resultados Foram identificadas 484 substâncias químicas em 243 postos de trabalho, resultando em 2.550 situações de exposição, com média de 3,73 substâncias químicas por local. Os 1.563 trabalhadores com exposição formaram 168 Grupos Homogêneos de Exposição (GHE), cuja composição variou de 1 a 44 trabalhadores (média de 4,55, mediana de 3 e moda de 1). Em cada local de trabalho foram identificados, em média, 4,91 GHE. Foram medidas 977 amostras. Com relação ao GHE, foi notado que em 91,9% das avaliações ocorreram resultados abaixo do nível de ação, correspondendo a 92,5% dos empregados, configurando a exposição a baixas concentrações. Verificou-se ainda que 49,6% dos GHE (49,9% dos empregados) tinham concentrações inferiores ao limite de detecção das técnicas analíticas, enquanto que em 8,1% dos GHE e 7,5% dos empregados, as concentrações estavam em nível igual ou acima do nível de ação. Comprovou-se que nos últimos dez anos foram implantados ou revisados 135 (18,5%) LEO. O número de substâncias cuja redução do LEO é igual ou superior a 50% corresponde à quase totalidade das reduções e, em todos os períodos, as maiores são iguais ou superiores a 80% ultrapassando, portanto, o nível de ação. Ainda que não exista concordância nos valores dos LEO foi constatado que, entre as diferentes agências têm ocorrido freqüentes mudanças na legislação. Estes fatos têm obrigado os profissionais a incorporarem estes conhecimentos a sua prática de trabalho. Conclusões A exposição a substâncias químicas em laboratórios é predominantemente em baixa concentração, variada e múltiplas. Na proteção da saúde de trabalhadores, a aceitação do risco quando a concentração ambiental da substância química é abaixo do nível de ação, deve ser usado com parcimônia, pois os valores de LEO têm apresentado tendência de redução e exclui parcela significativa da população. Nesta se incluem os hipersensíveis, os expostos a substâncias com efeito estocástico, misturas com efeitos aditivos e com curva dose-resposta bifásica / Objective The aim of this study was to assess the profile of occupational exposure to chemicals in laboratories and check the impacts on risk assessment that derive from changes in occupational exposure limits (OEL) and in Brazilian federal laws applicable thereto. Methodology Review of publications on occupational exposure to chemicals concerning the following: risk concept, dose-response curve drawing, identifying factors that interfere with dose-effect relationship; concepts of lengthy exposure, multiple exposure, and low concentration exposure; confusing factors in occupational monitoring. A transversal study was carried out at the Petrobras Research Center in order to assess the profile of exposure to chemicals among 3,000 employees whose job was predominantly performed inside a laboratory, half of which were low concentration, lengthy exposures (the study was based on environmental assessments carried out in 2004). Time analysis of American Conference of Governmental Industrial Hygienists (ACGIH) OEL values. Survey on the changes made in security, labor, and health federal laws as relates to exposure to chemicals. Results 484 chemicals were identified in 243 workplaces. This resulted in 2,550 exposure situations with an average of 3.73 chemicals per location. The 1,563 workers under exposure were divided into 168 Homogeneous Exposure Groups (HEG) comprised by 1 to 44 subjects (mean = 4.55; median = 3; mode = 1). On average, 4.91 HEG were identified in each workplace. 977 samples were measured. Regarding the HEG, it was observed that 91.9% of the assessments showed results below action level. This is equivalent to 92.5% of the amount of workers and fits into the low concentration exposure category. It was also observed that 49.6% of HEG (i.e. 49.9% of workers) showed concentrations lower than detection limit in analytical techniques, whereas concentrations were equal to or greater than action level among 8.1% of HEG and 7.5% of workers. 135 (i.e. 18.5%) OEL were proven to have been implemented or revised. The amount of chemicals whose OEL decrease is equal to or greater than 50% is equivalent to nearly all decreases. Moreover, the highest decreases are equal to or greater than 80%, and therefore exceeded action level. Although there is no common agreement on OEL values, it was observed that applicable laws have been changed by several agencies on a regular basis. These facts have led professionals into considering such information in their work practice. Conclusions Exposure to chemicals in laboratories occurs basically under low, varied, multiple concentration. In the field of workers healthcare, one should be careful while considering a risk for environmental concentration of a chemical below action level. This is because OEL levels have shown to be prone to decrease and thus exclude a significant part of the population. Such part includes hypersensitive individuals, people exposed to chemicals with stochastic effect, mixtures with additive effects and biphasic dose-response curve

Le risque potentiel de cancer induit par les irradiations mammographiques est sujet de santé publique majeur, d’intérêt médical et scientifique. Le but de ce travail a été de quantifier les cassures double-brin (CDB) de l’ADN en conditions exactes d’irradiations mammographiques. Cette quantification a été effectuée sur des cellules épithéliales mammaires non transformées issues de biopsies échoguidées en tissu sain en utilisant l’immunofluorescence de la protéine histone H2AX phosphorylée (γH2AX), avant, 10 min et 24 h après irradiation . Deux populations de patientes ont été incluses dans l’étude 19 sans antécédent familial de cancer du sein et/ou de l’ovaire (faible risque, FR) et 11 à haut risque identifié par le généticien avec ou sans mutation (haut risque, HR). En effet, les gènes mutés suppresseurs de tumeurs (BRCA1, BRCA2, CHK2, ATM, p53, PTEN) sont également impliqués dans la signalisation et/ou réparation des CDB. Spontanément, les patientes HR ont montré significativement plus de CDB spontanées que les LR. Trois effets radiobiologiques majeurs ont été mis en évidence : 1) Un effet de la dose, plus important chez les HR ; 2) Une augmentation significative du nombre de foci γH2AX entre 10 min et 24 h après irradiation ; 3) Un effet de répétition de dose, plus marqué chez les HR. Ces constatations devraient conduire à la ré-évaluation des séquences de dépistages mammographiques dans les populations où le bénéfice en terme de mortalité n’a pas été prouvé, comme dans la tranche d’âge des 40-49 ans et dans la surveillance des patientes où sont recommandées de façon annuelle IRM et mammographie dès l’âge de 30 ans ou 35 ans, les hauts risques et les femmes aux antécédents d’irradiation thoracique dans l’enfance, l’adolescence ou jeune adulte. Une seule incidence mammographique en dépistage pourrait être préconisée en dépistage dans l’attente de travaux radiobiologiques complémentaires évaluant la carcinogenèse éventuelle des irradiations mammographiques / The potential risk of cancer induced by radiation mammography is a major public health issue, medical and scientific interest. The purpose of this study was to quantify the double-strand break (DSB) DNA in exact terms of mammographic radiation. This quantification was performed on untransformed mammary epithelial cells from ultrasound-guided biopsies in healthy tissue using fluorescent protein phosphorylated histone H2AX (γH2AX) before, 10 min and 24 h after irradiation. Two patient populations were included in the study : 19 with no family history of breast cancer and/or ovarian cancer (low risk, LR) and 11 high-risk identified by the geneticist with or without mutation (high risk, HR). Indeed, mutated tumor suppressor genes (BRCA1, BRCA2, CHK2, ATM, p53, PTEN) are also involved in signaling and/or repair of DSBs. Spontaneously, patients showed significantly higher HR of DSBs that spontaneous LR. Three major radiobiological effects were highlighted : 1) A dose low effect, higher in HR; 2) A significant increase in the number of γH2AX foci from 10 min to 24 h after irradiation; 3) An effect of repeated doses more pronounced in HR. These findings should lead to re-evaluate mammographics procedures in screnning in populations where the benefit in term of mortality has not been proved, as women with high familial risk, in the age of group of 40-49 years, and in women treated with chest radiation for childhood, adolescent, or young adult cancer. A single mammographic view could be indicated. Further works assessing the possible carcinogenesis effects of mammographic irradiations will be necessary

Los efectos biológicos producidos por dosis bajas de radiación continúan siendo objeto de controversia. Cada cierto tiempo aparecen nuevos estudios a escala molecular y celular que arrojan luz sobre los mecanismos moleculares básicos que subyacen en las respuestas de los sistemas vivos a las radiaciones ionizantes. En esta tesis se muestran tanto la metodología empleada como los resultados obtenidos tras la irradiación in vitro de diferentes líneas celulares tumorales y no tumorales. Para irradiar los cultivos con dosis bajas (del orden del mGy) de radiación alfa se ha puesto a punto un dispositivo para disolver el gas radiactivo 222Rn en el medio de cultivo celular.Los resultados más interesantes han sido los obtenidos tras la irradiación de la línea MCF-7 de cáncer de mama humano metastásico. Junto con la observación de cambios en el crecimiento (no relacionadas linealmente con la dosis), los análisis genéticos revelaron modificaciones en la expresión de genes de la familia Bcl-2, reguladores de la muerte celular por apoptosis. En concreto, el gen bcl-xS (relacionado con quimiorresistencia en esta línea celular) sólo se expresó en los cultivos irradiados. Tras este resultado se analizó la influencia de la radiación sobre la sensibilidad de las MCF-7 a los fármacos Taxol y VP-16, observándose que los cultivos previamente irradiados respondían mejor al tratamiento con ambos quimioterápicos. / Biological effects due to low doses of radiation still remain as a controversial issue. Every certain time new studies on a cellular and molecular basis give out light on the basic mechanisms underlying the response of living systems to ionising radiation. In this thesis the methodology and results obtained after in vitro irradiation of tumour and normal cell lines are described. In order to expose cells to low doses of alpha particles, a new device for dissolving radioactive gas 222Rn in the culture medium was set up.The most remarkable results are those obtained after the irradiation of human breast metastasic cells of the line MCF 7. Together with influences on the growth rate (non linearly related with the dose of radiation), also genetic changes were observed in the expression of some Bcl 2 gene family members related with apoptotic cell death. In short, bcl xs (related with multidrug resistance phenomena in this cellular line) was only expressed in irradiated cell cultures. After this result, the influence of alpha irradiation on the sensibility of MCF 7 to the chemotherapeutic drugs Taxol and VP 16 was analysed, obtaining that those cell cultures previously irradiated showed an improved response to the action of the drugs.

L’évaluation des risques des produits chimiques utilise pour les effets non mutagènes un seuil de dose sans effet. L’objectif est d’élaborer une démarche qui permette de quantifier les risques non mutagènes. Elle s’inspire de celle utilisée pour les effets cancérigènes mutagènes. L’intérêt d’une approche sans seuil, est illustré par le cas du chlordécone en Guadeloupe. L’évaluation officielle indique 1 à 3 % de la population exposée au-dessus du seuil de dose toxique (atteintes rénales). Personne n’a quantifié les risques lorsque ce seuil est dépassé. Cependant, plusieurs millions d’euros (M€) sont investis chaque année pour la prévention des expositions.L’étude s’appuie uniquement sur des données disponibles en 2013. Les connaissances sur les modes d’actions toxiques du chlordécone permettent d’identifier les effets possibles à faible dose. Les études expérimentales multidoses ou les études épidémiologiques fournissent les données pour dériver des fonctions expositions risques (FER). Les données d’expositions internes (chlordéconémies) sont disponibles avant et après la mise en place en 2003 des actions de prévention. Les risques, estimés en multipliant les expositions par les FER, sont convertis en impacts via le nombre de personnes exposées. Les impacts sont monétarisés avec les DALYs et VOLY.Les effets possibles du chlordécone à faible dose chronique sont : cancers de la prostate et du foie, atteintes rénales et développement cognitif. Evaluer sans seuil et avec les expositions internes, le nombre de décès par cancer du foie avant 2003 est de 5,4 /an et après 2003 de 2,0 /an. Soit 3,4 décès/an évités grâce à la baisse des expositions. Pour les cancers de la prostate on compte respectivement : 2,8 et 1,0 (gain : 1,8 décès/an), pour les atteintes rénales : 0,10 et 0,04 (gain : 0,06 décès/an). Les pertes de points de QI sont de 1 173 pts/an et 1 003 pts/an (gain : 168 pts/an). Avant 2003, le coût total de ces impacts s’élève à 31,8 M€2006/an [10,6-64,3]. Les pertes de points de QI contribuent à 62 % du coût total, les cancers du foie 27 %, les cancers de la prostate 11 % et les atteintes rénales 0,6 %. Après 2003, le coût des impacts est de 21,3 M€2006/an [5,8-42,8]. Les bénéfices dus à la baisse des expositions sont de 10,5 M€2006/an. Les dépenses de préventions étant de 3,25 M€2006/an, le bénéfice est 3 fois plus élevé. La prise en compte d’un seuil de dose sans effet ne change pas significativement ces résultats. Evalué avec les expositions externes, le coût total des impacts ne représente plus que 4 % du coût avec les expositions internes. Une analyse de sensibilité par simulation Monté Carlo, montre que la variable « exposition » est la plus influente sur les résultats.Ce travail est le premier à prendre en compte un scénario d’évaluation sans seuil, ce qui est de plus en plus souvent recommandé pour les perturbateurs endocriniens. C’est aussi le premier à monétariser les impacts sanitaires du chlordécone. Cette approche est féconde d’informations utiles à la décision. Elle permet de comparer des options fondamentales comme l’existence ou non d’un seuil, la prise en compte d’expositions externes ou internes. Elle permet aussi de comparer le coût des actions de prévention aux bénéfices sanitaires qu’elles engendrent. Les résultats obtenus facilitent la hiérarchisation des priorités de sécurité sanitaire. On recommande cette démarche quantitative pour les facteurs d’environnement auxquels sont déjà exposées les populations. Les actions de prévention en Guadeloupe sont efficientes et justifiées au plan du coût social. Des enquêtes épidémiologiques sur le rôle du chlordécone dans les pathologies qui n’ont pas pu être prises en compte (neurotoxicité adulte, maladies auto immunes, autres effets sur le développement) seraient nécessaires. Il serait utile de prévoir des études mécanistiques avant les études épidémiologiques. Prolonger la cohorte TIMOUN permettrait de réduire l’incertitude sur le développement cognitif. / Risk assessment of chemicals uses for non-mutagenic effects a threshold dose without effect. The aim is to develop a risk assessment framework that allows quantification of non-mutagenic risks. It is inspired by the approach used for genotoxic carcinogens. The added value for safety decision making is shown on the case of chlordecone exposure in Guadeloupe. The official assessment is 1 to 3% of the population are exposed above the threshold (kidney damage). No one has quantified the risks when the threshold is exceeded. However, several million euros (M€) are invested annually for the prevention of exposure to chlordecone in Guadeloupe.The study is based only on published data available in 2013. The analysis of modes of action will identify the possible effects of chlordecone at chronicle low doses. Multidoses experimental studies or epidemiological studies provide data to derive exposure-response functions (ERF). The internal exposure data (blood chlordecone concentration) are available for Guadeloupe before and after the introduction in 2003 of preventive actions. The external exposure data are available only after 2003. Risks, estimated with exposures and ERF, are converted into impacts through the number of people exposed. The impacts are monetized via DALYs and VOLY.Four effects of chlordecone can occur at chronic low dose: prostate and liver cancers, kidney damages and cognitive development impairments. Without threshold and with internal exposure data, the estimated numbers of deaths from liver cancer are respectively before and after 2003: 5.4 /year and 2.0 /year. That is 3.4 deaths avoided per year by reducing exposure. For prostate cancer there are respectively 2.8 and 1.0 /yr (gain: 1.8 /yr) and for kidney damages 0.10 and 0.04 /yr (gain: 0.06 /yr). Loss of IQ points are 1 173/yr and 1 003 /yr (gain = 168 pt./yr). Before 2003, the total annual cost of these impacts is 31.8 million €2006 [10.6 to 64.3]. Impacts on cognitive development account for 62% of the total, liver cancer 27%, prostate cancer 11% and kidney damage 0.6%. After 2003, the cost of impacts is 21.3 M€/yr [5.8 to 42.8]. The benefits due to the reduction of exposures (avoided impacts) are 10.5 M€/yr [4.8 to 21.5]. Compared to annual spending estimated at 3.25 M€/yr, the annual benefit is three times higher. Including a threshold dose in the model do not significantly change the results. Use of the external exposure data, dramatically decreased the costs of impacts down to 4% of the costs estimated with internal exposures data. A sensitivity analysis with Monte Carlo simulation show that the "exposure" variable is the one that most influences the results.This work is the first to consider a quantitative assessment of health risks scenario without threshold, which is more and more often recommended for endocrine disrupters. It is also the first to monetize health impacts of chlordecone in Guadeloupe. This approach proves fruitful of useful information for decision making. It enables to compare basic options such as whether or not a threshold should be used in the model, taking into account any external or internal exposures. It also allows a comparison of the cost of preventive actions with the health benefits they generate. The results obtained facilitate the prioritization of actions for health safety. This quantitative approach is recommended for environmental factors to which are already exposed populations. Actions for reducing exposure to chlordecone in Guadeloupe are justified and efficient in terms of social cost. Epidemiological surveys to study the role of chlordecone in diseases that could not been taken into account for lack of knowledge (adult neurotoxicity, autoimmune diseases, and other developmental effects) would be needed. It would be useful to provide mechanistic studies before epidemiological studies implementation. Extend follow-up of the TIMOUN cohort could reduce uncertainty about cognitive development.

Les facteurs environnementaux que sont le Bisphénol A (BPA), un perturbateur endocrinien, et le Benzo[a]pyrène (B[a]P), un agent génotoxique, représentent un véritable enjeu parmi les facteurs de risques du cancer du sein associés à l'exposition environnementale. Nos objectifs sont d'explorer l'impact d'une exposition chronique à de faibles doses (10-10 M) de BPA et/ou de B[a]P sur l'agressivité de la lignée cellulaire pré-cancéreuse mammaire MCF10AT1 (immortelle, transformée, ER-, PR-, HER2-). Les buts principaux sont : (i) d'explorer si la combinaison de deux molécules possédant deux mécanismes d'action distincts conduit à un effet potentialisé par rapport à l'exposition aux molécules seules ; (ii) d'identifier et de prévenir les mécanismes moléculaires et cellulaires associés à cette exposition multiple. Nos résultats démontrent que l'exposition chronique des MCF10AT1 à de faibles doses de BPA et/ou de B[a]P induit un phénotype cellulaire agressif de façon temps-dépendant, avec un effet potentialisé pour la combinaison BPA+B[a]P, comparée aux molécules seules. Le phénotype observé est un phénotype acquis, car toujours présent 30 jours post-exposition. Nous avons également déterminé dans les cellules MCF10AT1 la présence et la fonctionnalité de deux récepteurs : le récepteur Aryl hydrocarbon (AhR) liant le B[a]P et le récepteur couplé à la protéine G (GPER1/GPR30) liant le BPA. D'un point de vue mécanistique, ces deux récepteurs sont impliqués dans le phénotype cellulaire agressif BPA et/ou B[a]P-dépendant, et notre travail révèle un nouveau cross-talk fonctionnel entre ces deux récepteurs. L'inhibition in vitro d'AhR et de GPR30 (inhibiteurs chimiques ou siRNA) permet de bloquer les effets délétères de l'exposition chronique au BPA et/ou au B[a]P. Enfin, l'analyse rétrospective de tumeurs primitives du sein ER-négatives démontre que la signature moléculaire GPR30/AhR possède une valeur de mauvais pronostic, alors que l'expression de GPR30 ou d'AhR n'en possède pas. L'ensemble de ces résultats souligne donc le rôle particulier que joue la présence concomitante d'AhR et de GPR30 dans des cellules précancéreuses/cancéreuses mammaires. En conclusion, nos résultats permettent d'identifier des cibles potentielles pour le développement de nouvelles stratégies préventives capables de bloquer la carcinogenèse mammaire due à l'exposition chronique au BPA et/ou B[a]P, et d'envisager des nouveaux biomarqueurs de cette exposition environnementale / Environmental factors such as Bisphenol A (BPA), an endocrine disruptor, and Benzo[a]pyrene (B[a]P), a genotoxic agent, represent a real issue among the environmental risk factors for breast cancer. Our objectives are to investigate the impact of chronic and low doses exposure to BPA (10-10 M) and/or B[a]P on the aggressiveness of the mammary pre-cancerous cell line MCF10AT1 (immortal, transformed, ER-, PR-, HER2-). The main aims are: (i) explore whether an exposure to the combination of two molecules with two distinct mechanisms of action has a greater impact than the molecules tested alone; (ii) identify and prevent the associated molecular and cellular mechanisms. Our results demonstrate that chronic exposure of MCF10AT1 to low doses of BPA and/or B[a]P induces an aggressive cell phenotype in a time-dependent manner, with a greater effect for (BPA + B[a]P) combination compared to single molecules. The observed phenotype is an acquired phenotype, as it still persists 30 days post-exposure. We also determined the presence and functionality of two receptors in the MCF10AT1 cells: the Aryl hydrocarbon receptor (AhR) binding B[a]P and the G binding protein receptor (GPER1 / GPR30) binding BPA. Mechanistically, these two receptors are involved in the BPA and/or B[a]P-induced aggressive phenotype, and our study reveals a new functional cross-talk/interplay between these two receptors. In vitro, the inhibition of AhR and GPR30 (chemical inhibitors or siRNA) can block the deleterious effects of chronic exposure to BPA and/or B[a]P. Finally, a retrospective analysis of primary ER-negatif subclass breast tumors demonstrates that the GPR30/AhR gene expression signature has a poor prognosis value, whereas GPR30 or AhR mRNA levels were poorly informative. All these results underline the particular role played by the concomitant presence of AhR and GPR30 in mammary precancerous/cancerous cells. In conclusion, our results allow us to identify potential targets for the development of new preventive strategies capable of blocking mammary carcinogenesis due to chronic exposure to BPA and/or B[a]P, and to consider new biomarkers for environmental exposure