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

Reactive oxygen species (ROS) produced by intracellular metabolism are beli eved to contribute to spontaneous mutagenesis in somatic cells. Hydrogen pe roxide (H2O2) has been shown to induce a variety of genetic alterations, pr obably by the generation of hydroxyl radicals via the Fenton reaction. The kinds of DNA sequence alterations caused by H2O2 in prokaryotic cells have been studied extensively, whereas relatively little is known about the muta tional spectrum induced by H2O2 in mammalian genes, We have used the T-cell cloning assay to study the ability of H2O2 to induce mutations at the hypo xanthine guanine phosphoribosyltransferase (HPRT) locus in primary human ly mphocytes. Treatment of cells for 1 h with 0.34-1.35 mM of H2O2 caused a do se dependent decrease of cell survival and increase of the HPRT mutant freq uency (MF). After 8 days of expression time, the highest dose of H2O2 cause d a 5-fold increase of MF compared to the untreated control cells. Mutant c lones were collected and the genomic rearrangements at the T-cell receptor (TCR) gamma-locus were studied to identify independent mutations. RT-PCR an d 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 H2O2, treated cells. In both sets, single base pair substitutions were the most common ty pe of mutation (5/6 and 13/20, respectively), with a predominance of transi tions 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 ne w mutations not previously reported in the human HPRT mutation database. Ov erall, the kinds of mutation occurring in T-cells in vivo and H2O2 treated cells were similar, albeit the number of mutants was too small to allow a m eaningful statistical comparison. These results demonstrate that H2O2 is mu tagenic to primary human T-lymphocytes in vitro and induces mutations of th e same kind that is observed in the background spectrum of HPRT mutation in T-cells in vivo. (C) 2000 Elsevier Science B.V. All rights reserved.