Estresse oxidativo e lesões no DNA: papel da 8-oxoguanina na viabilidade do Trypanosoma cruzi

Abstract

The formation of DNA lesions is one of the consequences of oxidative stress, which might result in genomic instability and lead to cell death. Guanine is the base that is most susceptible to oxidation due to its low redox potential, and 8-oxoguanine (8-oxoG) is the most abundant lesion. This characteristic makes 8-oxoG a good cellular biomarker to indicate the extent of oxidative stress. If not repaired, the 8- oxoG could pair with adenine and cause G:C to T:A transversion. When 8-oxoG is inserted during the DNA replication it could generate double strand breaks, which makes this lesion particularly deleterious. All organisms evolved repair mechanisms to deal with 8-oxoG. In higher eukaryotes, the enzymes OGG1, MUTYH and MTH (MutT Homologous) constitute the 8-oxoG repair pathway. Trypanosoma cruzi needs to deal with various oxidative stress situations that it is exposed to, such as the mammalian intracellular environment and the triatomine insect gut where it replicates. Since the publication of the parasites genome, some of this DNA repair enzymes have been characterized. However, some important elements of the DNA Repair machinery, such as a MutT homolog, have not been identified. The MutT enzyme is responsible for the hydrolysis of 8-oxo-dGTP to 8-oxo-dGMP in the nucleotide pool, preventing the incorporation of these oxidized nucleotides in DNA. This prompted our group to transform T. cruzi CL Brener clone with the Escherichia coli mutT gene to investigate the importance of the 8-oxoG during the parasites life cycle. In the epimastigote form, the recombinant and wild type strains showed similar growth in normal conditions, but MutT expressing cells were more resistant to hydrogen peroxide treatment. Furthermore, we demonstrated by QPCR experiments that E. coli MutT expression in T. cruzi reduces the amount of nuclear DNA lesions, in comparison to control cells. The recombinant strain also showed statistically significant increase in growth after 48 hours of infection in fibroblasts when compared to wild type cells, as well as increased parasitemia in Swiss mice. Additionally, we demonstrated by western blotting experiments that MutT heterologous expression can influence the parasites mitochondrial and cytosolic peroxidase enzymes (MPX and CPX) protein levels. These results show the importance of the 8-oxoG repair system for T. cruzi cell viability. This study also accomplished the characterization of TcMTH, the T. cruzi MutT homolog. The gene sequence was studied in silico, and parasites overexpressing TcMTH showed increased resistance to hydrogen peroxide treatment.