Genotoxic response assessment in the Neotropical fish by alkaline unwinding assay using methyl methane sulfonate (MMS) as chemical model

DE SAN BENITO, E.; CARRIQUIRIBORDE, P.; QUELAS, J.I.; GONZALEZ, P.V.

Orlando

Congreso; 7th SETAC World Congress SETAC North America 37th Annual Meeting; 2016

Society of Environmental Toxicology and Chemistry

The aims of this study was to assess the genotoxic response, single-strand (SSB) and double-strand breaks (DBS), in Cnesterodon decemmaculatus by a fluorometric microscale alkaline unwinding assay using MMS as reference compound. Assay was first set up in vitro generating single strand-breaks by enzymatic digestion with calf thymus endonuclease I on DNA samples obtained from control fish skeletal muscle. Then, in vivo assays were performed exposing fish to 0, 3.75, 7.5, and 15 mg L-1 MMS to build the dose-response curve. Sybr Green I was used as fluorochrome and DNA unwinding was performed on a multi-mode microplate reader. The strand scission factor (SSF) was assessed as endpoint in both assays. Good correlation between digestion time and SSF was observed from 0 to 30 min and 0.65 to 2.17 arbitrary DNA damage units. Clear genotoxic induction was also observed in fish muscle during in vivo assays. The SSF value was increased from 0.15 in controls to 0.45, 0.50 and 0.54 in 3.75, 7.5, and 15 mg L-1 MMS expose fish, respectively. Significant effects were observed earlier from 3.75 mg L-1 (LOEC), and the EC50 was estimated between 0 and that value. In conclusion the developed fluorometric microscale alkaline unwinding assay was sensitive to assess genotoxic effects induced by the reference chemical in the tested South American fish. Therefore, it appears as an easy, quick and reliable tool for assessing genotoxicity in the lab, and potentially in the field using a local species.