Unveiling Benznidazole's mechanism of action through overexpression of DNA repair proteins in Trypanosoma cruzi

Benznidazole (BZ) is the most commonly used drugfor the treatment of Chagas disease. Although BZ isknown to induce the formation of free radicals andelectrophilic metabolites within the parasite Trypa-nosoma cruzi, its precise mechanisms of action arestill elusive. Here, we analyzed the survival of T.cruzi exposed to BZ using genetically modified par-asites overexpressing different DNA repair pro-teins. Our results indicate that BZ induces oxidationmainly in the nucleotide pool, as heterologousexpression of the nucleotide pyrophosphohydro-lase MutT (but not overexpression of the glycosy-lase TcOgg1) increased drug resistance in theparasite. In addition, electron microscopy indi-cated that BZ catalyzes the formation of double-stranded breaks in the parasite, as its genomicDNA undergoes extensive heterochromatinunpacking following exposure to the drug. Further-more, the overexpression of proteins involved inthe recombination-mediated DNA repair increasedresistance to BZ, reinforcing the idea that the drugcauses double-stranded breaks. Our results alsoshow that the overexpression of mitochondrialDNA repair proteins increase parasite survivalupon BZ exposure, indicating that the drug induceslesions in the mitochondrial DNA as well. Thesefindings suggest that BZ preferentially oxidizes thenucleotide pool, and the extensive incorporation ofoxidized nucleotides during DNA replication leadsto potentially lethal double-stranded DNA breaks inT. cruzi DNA. Environ. Mol. Mutagen. 55:309–321, 2014.