Exposure to the Fungicide Captan Induces DNA Base Alterations and Replicative Stress in Mammalian Cells.

The classification of the fungicide captan (CAS Number: 133-06-2) as a carcinogen agent is presently under discussion. Despite the mutagenic effect detected by the Ames test and carcinogenic properties observed in mice, the genotoxicity of this pesticide in humans is still unclear. New information is needed about its mechanism of action in mammalian cells. Here, we show that Chinese Hamster Ovary (CHO) cells exposed to captan accumulate Fpg-sensitive DNA base alterations. In CHO and HeLa cells, such DNA lesions require the XRCC1-dependent pathway to be repaired. Captan also induces a replicative stress that activated the ATR signaling response and resulted in double-strand breaks and micronuclei. The replicative stress is characterized by a dramatic decrease in DNA synthesis due to a reduced replication fork progression. However, impairment of the XRCC1-related repair process did not amplify the replicative stress, suggesting that the fork progression defect is independent from the presence of base modifications. These results support the involvement of at least two independent pathways in the genotoxic effect of captan that might play a key role in carcinogenesis. Environ. Mol. Mutagen. 60:286-297, 2019. © 2018 Wiley Periodicals, Inc.

Validation of Gelbond® high-throughput alkaline and Fpg-modified comet assay using a linear mixed model.

Even if the comet assay has been widely used for decades, there is still a need for controlled studies and good mathematical models to assess the variability of the different versions of this assay and in particular to assess potential intra-experimental variability of the high-throughput comet assay. To address this point, we further validate a high-throughput comet assay that uses hydrophilic polyester film (Gelbond®). Experiments were performed using human peripheral blood mononuclear cells (PBMC) either untreated or treated with different concentration of MMS (methyl methanesulfonate). A positive control for the Fpg (Formamidopyrimidine DNA glycosylase)-modified comet assay (Ro 19-8022 with light) was also included. To quantify the sources of variability of the assay, including intradeposit variability, instead of summarizing DNA damage on 50 cells from a deposit by the mean or median of their percentage DNA tail, we analyzed all logit-transformed data with a linear mixed model. The main source of variation in our experimental data is between cells within the same deposit, suggesting genuine variability in the response of the cells rather than variation caused by technical treatment of cell samples. The second source of variation is the inter-experimental variation (day-to-day experiment); the coefficient of this variation for the control was 13.6%. The variation between deposits in the same experiment is negligible. Moreover, there is no systematic bias because of the position of samples on the Gelbond® film nor the position of the films in the electrophoresis tank. This high-throughput comet assay is thus reliable for various applications. Environ. Mol. Mutagen. 59:595-602, 2018. © 2018 Wiley Periodicals, Inc.

Cell resistance to the Cytolethal Distending Toxin involves an association of DNA repair mechanisms.

The Cytolethal Distending Toxin (CDT), produced by many bacteria, has been associated with various diseases including cancer. CDT induces DNA double-strand breaks (DSBs), leading to cell death or mutagenesis if misrepaired. At low doses of CDT, other DNA lesions precede replication-dependent DSB formation, implying that non-DSB repair mechanisms may contribute to CDT cell resistance. To address this question, we developed a proliferation assay using human cell lines specifically depleted in each of the main DNA repair pathways. Here, we validate the involvement of the two major DSB repair mechanisms, Homologous Recombination and Non Homologous End Joining, in the management of CDT-induced lesions. We show that impairment of single-strand break repair (SSBR), but not nucleotide excision repair, sensitizes cells to CDT, and we explore the interplay of SSBR with the DSB repair mechanisms. Finally, we document the role of the replicative stress response and demonstrate the involvement of the Fanconi Anemia repair pathway in response to CDT. In conclusion, our work indicates that cellular survival to CDT-induced DNA damage involves different repair pathways, in particular SSBR. This reinforces a model where CDT-related genotoxicity primarily involves SSBs rather than DSBs, underlining the importance of cell proliferation during CDT intoxication and pathogenicity.