Hydrogen peroxide induced mutations at the HPRT locus in primary human T-lymphocytes

Abstract

Reactive oxygen species (ROS) produced by intracellular metabolism are believed to contribute to spontaneous mutagenesis in somatic cells. Hydrogen peroxide (H 2O 2) has been shown to induce a variety of genetic alterations, probably by the generation of hydroxyl radicals via the Fenton reaction. The kinds of DNA sequence alterations caused by H 2O 2 in prokaryotic cells have been studied extensively, whereas relatively little is known about the mutational spectrum induced by H 2O 2 in mammalian genes. We have used the T-cell cloning assay to study the ability of H 2O 2 to induce mutations at the hypoxanthine guanine phosphoribosyltransferase ( HPRT) locus in primary human lymphocytes. Treatment of cells for 1 h with 0.34–1.35 mM of H 2O 2 caused a dose dependent decrease of cell survival and increase of the HPRT mutant frequency (MF). After 8 days of expression time, the highest dose of H 2O 2 caused a 5-fold increase of MF compared to the untreated control cells. Mutant clones were collected and the genomic rearrangements at the T-cell receptor (TCR) γ-locus were studied to identify independent mutations. RT-PCR and DNA sequencing was used to identify mutations in the HPRT coding region. Due to a relatively high frequency of sibling clones, only six independent mutations were obtained among the controls, and 20 among the H 2O 2 treated cells. In both sets, single base pair substitutions were the most common type of mutation (5/6 and 13/20, respectively), with a predominance of transitions at GC base pairs, which is also the most common type of HPRT mutation in T-cells in vivo. Among the single base pair substitutions, five were new mutations not previously reported in the human HPRT mutation database. Overall, the kinds of mutation occurring in T-cells in vivo and H 2O 2 treated cells were similar, albeit the number of mutants was too small to allow a meaningful statistical comparison. These results demonstrate that H 2O 2 is mutagenic to primary human T-lymphocytes in vitro and induces mutations of the same kind that is observed in the background spectrum of HPRT mutation in T-cells in vivo.