A comparative analysis of BrdU and EdU genotoxicity shows that trypanosomatids exhibit unique responses to the presence of these thymidine analogs in their DNA

Abstract

The thymidine analogs 5-bromo-2'-deoxyuridine (BrdU) and 5-ethynyl-2'-deoxyuridine (EdU) are widely used to monitor DNA synthesis. Although studies have reported EdU toxicity in certain cell types, no reports to date describe genotoxic effects associated with BrdU/EdU use in trypanosomatids, etiological agents of neglected tropical diseases. This lack of studies raises two possibilities: either these nucleotides are not toxic to these parasites, or the observed genotoxicity was not relevant under the experimental conditions used. Here, we assessed the potential toxicity of BrdU and EdU across three pathogenic trypanosomatids: Trypanosoma cruzi, Trypanosoma brucei, and Leishmania major. Our findings show that EdU has greater genotoxic potential than BrdU. Nevertheless, trypanosomatids respond markedly differently to DNA alterations caused by this analog: T. cruzi cells stop replicating and accumulate in S phase, T. brucei tolerates the DNA lesions generated, while part of the L. major population appears to display an altruistic behavior that benefits the survivors. Furthermore, in silico assays suggest that, once incorporated into DNA, the ethynyl group increases the EdU's polarity, thereby altering DNA structure more strongly than BrdU does after the first round of DNA replication. Together, our findings suggest that using thymidine analogs in trypanosomatids should be preceded by rigorous preliminary analysis, especially when using EdU. Otherwise, the data may be biased by a BrdU/EdU-triggered DNA damage response. We believe this study may serve as an atlas to guide the initial selection of thymidine analog concentrations and exposure periods.