Ecotoxicology studies attempt to evaluate the consequences of exposure to environmental contaminants by defining exposure and effects parameters across multiple levels of biological organization. Genetic markers are well-suited for these studies as they can track both somatic and evolutionary effects. In the studies reported here, connections among contaminant levels in environmental media and biota, in vitro bioassay results, and changes in individual- and population-level biomarkers were explored. Sediment and/or wildlife samples were collected from each of three sites of documented environmental contamination, Sumgayit and Baku in the Republic of Azerbaijan and East Fork Poplar Creek in Tennessee. Sumgayit and Baku are heavily contaminated with petroleum and petrochemical wastes. Sediments from several areas and tissues from turtles inhabiting a contaminated wetland contained high levels of several compounds, including mercury, polycyclic aromatic hydrocarbons, polychlorinated biphenyls, and organochlorine pesticides. Sediment extracts produced variable responses in the Salmonella/microsome mutagenicity assay and did not necessarily reflect contaminant burden. Micronucleus counts in European pond turtles were not statistically different from counts in the same species from an uncontaminated reference site. The counts were statistically correlated with tissue levels of mercury, heptachlor, DDD, hexachlorobenzene, and trans-nonachlor. These results confirmed that Sumgayit and Baku are heavily contaminated with a complex mixture of pollutants and demonstrated that genotoxic effects from exposure to contaminated sediments appear to be slight. East Fork Poplar Creek is a stream that receives contaminant influx from a former Department of Energy nuclear weapons production facility and several point and non-point sources around the city of Oak Ridge, Tennessee. In this study, coefficient of variation in cellular DNA content and randomly amplified polymorphic DNA markers were examined in central stonerollers and compared to previous studies in which the same markers were evaluated in red-breasted sunfish from the same sites. While assay responses were attenuated in stonerollers compared to the sunfish, there is some evidence of genetic damage in both species at the most contaminated sampling site. A common problem in the wildlife studies was high within sample variability combined with small sample size, which most likely masked potential contaminant-induced differences in markers used in these studies.

Ecotoxicology studies attempt to evaluate the consequences of exposure to environmental contaminants by defining exposure and effects parameters across multiple levels of biological organization. Genetic markers are well-suited for these studies as they can track both somatic and evolutionary effects. In the studies reported here, connections among contaminant levels in environmental media and biota, in vitro bioassay results, and changes in individual- and population-level biomarkers were explored. Sediment and/or wildlife samples were collected from each of three sites of documented environmental contamination, Sumgayit and Baku in the Republic of Azerbaijan and East Fork Poplar Creek in Tennessee. Sumgayit and Baku are heavily contaminated with petroleum and petrochemical wastes. Sediments from several areas and tissues from turtles inhabiting a contaminated wetland contained high levels of several compounds, including mercury, polycyclic aromatic hydrocarbons, polychlorinated biphenyls, and organochlorine pesticides. Sediment extracts produced variable responses in the Salmonella/microsome mutagenicity assay and did not necessarily reflect contaminant burden. Micronucleus counts in European pond turtles were not statistically different from counts in the same species from an uncontaminated reference site. The counts were statistically correlated with tissue levels of mercury, heptachlor, DDD, hexachlorobenzene, and trans-nonachlor. These results confirmed that Sumgayit and Baku are heavily contaminated with a complex mixture of pollutants and demonstrated that genotoxic effects from exposure to contaminated sediments appear to be slight. East Fork Poplar Creek is a stream that receives contaminant influx from a former Department of Energy nuclear weapons production facility and several point and non-point sources around the city of Oak Ridge, Tennessee. In this study, coefficient of variation in cellular DNA content and randomly amplified polymorphic DNA markers were examined in central stonerollers and compared to previous studies in which the same markers were evaluated in red-breasted sunfish from the same sites. While assay responses were attenuated in stonerollers compared to the sunfish, there is some evidence of genetic damage in both species at the most contaminated sampling site. A common problem in the wildlife studies was high within sample variability combined with small sample size, which most likely masked potential contaminant-induced differences in markers used in these studies.

Cancers that are deficient in BRCA1 or BRCA2 are thought to be hypersensitive to genotoxic agents because they cannot prevent or repair DNA double strand breaks, but observations in patients suggest this dogma may no longer agree with experiment. Here, we propose that single stranded DNA underlies the hypersensitivity of BRCA deficient cancers, and that defects in double strand break repair and prevention do not. Specifically, in BRCA deficient cells, ssDNA gaps developed because replication was not effectively restrained in response to stress. In addition, we observed gaps could be suppressed by either restored fork restraint or by gap filling, both of which conferred therapy resistance in tissue culture and BRCA patient tumors. In contrast, restored double strand break repair and prevention did not confer therapy resistance when gaps were present. Critically, double strand breaks were not detected after therapy when apoptosis was inhibited, supporting a framework in which double strand breaks are not directly induced by genotoxic agents, but instead are created by cell death nucleases and are not fundamental to genotoxic agents. Together, these data indicate that ssDNA replication gaps underlie the BRCA cancer phenotype, "BRCAness," and we propose are fundamental to the mechanism-of-action of genotoxic chemotherapy.

La Cytolethal Distending Toxin (CDT) est un facteur de virulence produit par de nombreuses bactéries pathogènes Gram négatives. Sa production est associée à différentes pathologies, dont le développement de cancers. Un lien de causalité a été établi entre dommages à l’ADN, mutagénèse et cancérogenèse. Or, différentes études ont classé CDT dans la famille des génotoxines bactériennes. L’action génotoxique de CDT repose sur l’activité de sa sous-unité catalytique CdtB, connue pour induire des cassures double-brin (CDBs) de l’ADN génomique eucaryote. Cependant, des travaux de l’équipe ont montré qu’à des doses 1000 fois plus faibles que celles utilisées dans la littérature, CDT induit des dommages primaires (probablement de type cassure simple-brin), qui dégénèrent en CDB lors de la phase S. Afin de mieux documenter ce modèle, nous avons étudié ici les systèmes de réparation impliqués dans la réponse aux dommages à l’ADN induits par CDT. Nous avons ainsi confirmé l’importance des voies de réparations des CDBs (Homologous Recombinaison et Non-Homologous End-Joining). Nous avons également montré que le Nucleotide Excision Repair, impliqué dans la réparation des adduits à l’ADN, n’est pas impliqué dans la prise en charge des dommages induits par CDT. En revanche, nous avons démontré, pour la première fois, l’implication de systèmes de réparation de dommages plus précoces, comme le Single-Strand Break Repair et la voie de l’Anémie de Fanconi. Pour finir, afin de mieux caractériser ces dommages et leur induction, nous avons initié des travaux visant à étudier, in vitro, l’activité catalytique de CdtB. Dans ce but, différents mutants catalytiques ont été générés, purifiés, et leur activité nucléase a été testée. Une activité nucléase similaire entre les CdtB sauvages et mutantes a été obtenue lors d’un test in vitro (digestion d’un plasmide super-enroulé). Cependant, un test cellulaire (expression nucléaire en cellules eucaryotes de la sous-unité CdtB sauvage ou mutante) indique bien la perte de l’activité nucléase de la sous-unité mutante. Nos résultats montrent donc l’importance de tester les différentes sous-unités dans différents contextes. En conclusion, notre travail conforte les données selon lesquelles CDT induit des CSB, et non des CDB directes de l’ADN. De plus, notre travail a permis d’éclaircir les processus cellulaires activés dans la cellule hôte, suite aux dommages à l’ADN induits par CDT. / The Cytolethal Distending Toxin (CDT) is a virulence factor produced by many pathogenic gram-negative bacteria, its production being associated to various diseases, including tumorigenesis. A causal relationship has been established between DNA damage, mutagenesis and cancerogenesis. Different studies classified CDT among the bacterial genotoxins. The CDT-related pathogenicity relies on the catalytic subunit CdtB action, shown to induce double-strand breaks (DSB) on the host genomic DNA. Previously, our team showed that, at doses 1000 times lower than those used in the literature, CDT probably induces single-strand breaks that degenerate into DSB during S-phase. To document this model, we studied the repair systems involved in host-cell in response to CDT-induced DNA damage. Since various repair pathways allow cells to respond different type of DNA damage, we speculated that non-DSB repair mechanisms might contribute to the cellular resistance to CDT-mediated genotoxicity. First, we confirm that HR is involved in the management of CDT-induced lesions, but also Non Homologous End Joining, the second major DSB repair mechanism. Next we show that nucleotide excision repair, involved in adducts repair, is not important to take care of CDT-induced DNA damage, whereas base excision repair impairment sensitizes CDT-treated cells, suggesting that CDT induce single-strand breaks. Moreover, we demonstrate for the first time the involvement and the activation of the Fanconi Anemia repair pathway in response to CDT. Finally, to better characterize CDT-induced damage, we initiate experiments to study CdtB nuclease activity in vitro. For this, different CdtB mutants have been generated, purified and their nuclease activity tested. A similar nuclease activity has been obtained for the wt or mutant CdtB in an in vitro assay (digestion of a supercoiled plasmid). However, a cell assay (nuclear expression of CdtB in eukaryotic cells) confirms the loss of activity for the mutant subunit. Our results thus indicate the importance to test the CdtB subunit in different context. To conclude, our work reinforces a model where CDT induces single-strand damage and not direct DSB. This also underlines the importance of cell proliferation to generate DSB and sheds light on the activated host-cell systems, after CDT-induced DNA damage.