The protective role of the hMTH1 protein against oxidative stress-induced genomic instability in cells exposed to UVA or gamma radiation

By: Sara Shakeri Manesh
Host: Siamak Haghdoost

Humans are constantly exposed to different types of radiation. It has been suggested that low dose and low dose rate of gamma radiation as well as ultra violet A (UVA) radiation induce oxidative stress in cells that may cause cancer and other age related diseases. The mechanisms behind radiation-induced oxidative stress and its relation to genomic instability and cancer induction are not well known. The level of oxidative stress depends on the quality, dose and dose rate of radiation. UVA as well as low dose and low dose rates of gamma radiation trigger genomic instability at least partly through induction of reactive oxygen species (ROS). Increased levels of ROS in the cell can give rise to mutations via base oxidation in DNA or in nucleotide pool (dNTP). The oxidative base damages include 7, 8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG), 7, 8-dihydro-8-oxo-2'-deoxyadenosine (8-oxo-dA) and 2-dihydro-8-oxo-2'-deoxyadenosine (2-OH-dA). To maintain genomic stability, cells have developed different machineries that repair the oxidized DNA bases in the DNA and the nucleotide pool. Base excision repair proteins remove the oxidized bases from the DNA and the hMTH1 protein cleans the cytosol from modified dNTPs, particularly 8-oxo-dGTP and 2-OH-dATP, preventing their incorporation into the DNA. Previous data suggest that nucleotide pool is an important target for oxidative stress where free radicals can be formed and react with dNTPs. Here we aimed to investigate the protective role of hMTH1 against UVA and gamma irradiation-induced genomic instability by transfecting a human lymphoblastoid cell line with shRNA directed against hMTH1. Western blot analysis was performed to check the level of down-regulated hMTH1. Clonogenic cell survival assay was performed to investigate the cytotoxicity of UVA and gamma radiation. The mutant frequency assay and chromosomal aberration assay were applied to study mutagenicity and cytotoxic effects of radiation. These tests were applied to the transfected and non-transfected TK6 cells. Our results indicate that acute exposure to UVA and gamma radiation affects cell survival and also significantly increases the level of mutation in transfected and non-transfected cells. The level of mutant frequency in transfected cells was significantly higher than that in non-transfected cells after UVA exposure while this pattern was not seen after exposure to gamma radiation. No dose rate effect of gamma radiation for mutations and stable type aberrations was observed in both transfected and non-transfected cells. In conclusion, our results suggest that in gamma exposure the total dose rather than the dose rate influences the level of mutant frequency. Moreover, the protective role of hMTH1 protein against oxidative stress-induced mutant frequency is more pronounced after exposure to UVA than after gamma radiation exposure.