Alkaline gel electrophoresis assay to detect DNA strand breaks and repair mechanisms in Escherichia coli

Reactive oxygen species (ROS) can induce lesions in different cellular targets, including DNA. Stannous chloride (SnCl2) is a ROS generator, leading to lethality in Escherichia coli (E. coli), with the base excision repair (BER) mechanism playing a role in this process. Many techniques have been developed to detect genotoxicity, as comet assay, in eukaryotic cells, and plasmid DNA agarose gel electrophoresis. In this study, an adaptation of the alkaline gel electrophoresis method was carried out to ascertain the induction of strand breaks by SnCl2 in bacterial DNA, from E. coli BER mutants, and its repair pathway. Results obtained show that SnCl2 was able to induce DNA strand breaks in all strains tested. Moreover, endonuclease IV and exonuclease III play a role in DNA repair. On the whole, data has shown that the alkaline gel electrophoresis assay could be used both for studying DNA strand breaks induction and for associated repair mechanisms.

Espécies reativas de oxigênio (ERO) podem induzir lesões em diferentes alvos celulares, incluindo o DNA. O cloreto estanoso (SnCl2) é um gerador de ERO que induz letalidade em E. coli, sendo o reparo por excisão de bases (BER) um mecanismo importante neste processo. Técnicas como o ensaio cometa (em eucariotos) e a eletroforese de DNA plasmidial em gel de agarose têm sido utilizadas para detectar genotoxicidade. No presente estudo, uma adaptação do método de eletroforese em gel alcalino de agarose foi usada para verificar a indução de quebras, pelo SnCl2, no DNA de E. coli, bem como a participação de enzimas do BER na restauração das lesões. Os resultados mostraram que o SnCl2 induziu quebras no DNA de todas as cepas testadas. Além disso, endonuclease IV e exonuclease III estão envolvidas na reparação dos danos. Em resumo, os dados obtidos indicam que a metodologia de eletroforese em gel alcalino de agarose pode ser empregada tanto para o estudo de quebras no DNA, quanto para avaliação dos mecanismos de reparação associados.

Stannous chloride participates in the generation of reactive oxygen species

A genotoxic potentiality to stannous salts has been reported. The relevance of these data is due to the wide application of this metal in our society. The biological effect of these salts might depend on the physicochemical conditions and the route of their administration. There are situations in which stannous salts can be directly administered to human beings endovenously and there is not doubt about their absorption into the body. The disparate and largely unexplained differences suggest that stannous salts as a simple poisoning and/or a remarkable genotoxic agent might be a fertile field for additional investigation. Reactive oxygen species scavengers and metal ion chelators can abolish, at least in part, the effect of stannous salts. This suggests that the generation of free radicals by the reducing agent is involved in the biological effect induced by this metal.