Oxidative Stress and DNA Lesions: The Role of 8-Oxoguanine Lesions in Trypanosoma cruzi Cell Viability

Abstract

The main consequence of oxidative stress is the formation of DNA lesions, which can 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 common lesion. These characteristics make 8-oxoG a good cellular biomarker to indicate the extent of oxidative stress. If not repaired, 8-oxoG can pair with adenine and cause a G:C to T:A transversion. When 8-oxoG is inserted during DNA replication, it could generate double-strand breaks, which makes this lesion particularly deleterious. Trypanosoma cruzi needs to address various oxidative stress situations, such as the mammalian intracellular environment and the triatomine insect gut where it replicates. We focused on the MutT enzyme, which is responsible for removing 8-oxoG from the nucleotide pool. To investigate the importance of 8-oxoG during parasite infection of mammalian cells, we characterized the MutT gene in T. cruzi (TcMTH) and generated T. cruzi parasites heterologously expressing Escherichia coli MutT or overexpressing the TcMTH enzyme. In the epimastigote form, the recombinant and wild-type parasites displayed similar growth in normal conditions, but the MutT-expressing cells were more resistant to hydrogen peroxide treatment. The recombinant parasite also displayed significantly increased growth after 48 hours of infection in fibroblasts and macrophages when compared to wild-type cells, as well as increased parasitemia in Swiss mice. In addition, we demonstrated, using western blotting experiments, that MutT heterologous expression can influence the parasite antioxidant enzyme protein levels. These results indicate the importance of the 8-oxoG repair system for cell viability.

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

“…T. cruzi cell lineages stably overexpressing the genes EcMUTT , TcOGG1 , TcMUTY , TcPOLB , TcPOLH , TcPOLK , or TcRAD51 (Aguiar et al, ; Furtado et al, 2012; Lopes et al, ; Moura et al, ; Rajão et al, ; Regis‐da‐Silva et al, ; Schamber‐Reis et al, ) were generated by cloning the respective genes into the pROCK vector. This vector is capable of integrating into the β‐TUBULIN locus of the T. cruzi genome, and gene overexpression is achieved by the presence of a ribosomal promoter upstream the inserted gene (DaRocha et al, ).…”

“…Our research group has been studying different T. cruzi proteins that are involved in DNA metabolism (replication, repair, and recombination), describing its properties using both in vitro and in vivo approaches (Aguiar et al, ; Campos et al, ; Furtado et al, 2012; Lopes et al, ; Moura et al, ; Rajão et al, ; Regis‐da‐Silva et al, ; Schamber‐Reis et al, ). To investigate whether BZ efficacy against T. cruzi is due to its action on DNA, we challenged our genetically modified strains with BZ.…”

Coordination of DNA single strand break repair

“…Also common is hydroxyl addition to the C8-position of guanine, which can further undergo reduction to a ring-opened FaPy-G or oxidation to 8-oxo-7,8-dihydroguanine (8-oxo-G), a mutagenic base lesion often used as a marker of oxidative stress. Common forms of endogenous DNA damage and their consequences are summarized in Table 1 [4, 12-20]. …”

Human articular cartilage repair: Sources and detection of cytotoxicity and genotoxicity in photo-crosslinkable hydrogel bioscaffolds

“…Higher wavelength UV‐A (315‐400 nm) induced cytotoxicity occurs mostly via indirect mechanisms, whereby cellular chromophores act as photo‐sensitizers to generate reactive oxygen species (ROS) which causes insult to proteins, lipids and DNA, the main lesion being the oxidized base 8‐oxo‐7,8‐dihydroguanine (8‐oxoG) . Similarly to CPDs, 8‐oxoG can pair with adenine and cause a guanine:cytosine to thymine:adenine transversion, but can also result in DSBs if inserted during DNA replication . Other studies identified an action spectra to determine cell killing and mutations by monochromatic ultraviolet and visible radiations (254‐434 nm) in human epithelial cells…”