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

Reactive oxygen species (ROS) produced by intracellular metabolism are believed to contribute to spontaneous mutagenesis in somatic cells. Hydrogen peroxide (H(2)O(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(2)O(2) in prokaryotic cells have been studied extensively, whereas relatively little is known about the mutational spectrum induced by H(2)O(2) in mammalian genes. We have used the T-cell cloning assay to study the ability of H(2)O(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(2)O(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(2)O(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) gamma-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(2)O(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(2)O(2) treated cells were similar, albeit the number of mutants was too small to allow a meaningful statistical comparison. These results demonstrate that H(2)O(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.