Nitric oxide-induced mutations in the HPRT gene of human lymphoblastoid TK6 cells and in Salmonella typhimurium

Characterization of mutations induced by NO in different experimental syste ms will facilitate elucidation of mechanisms underlying its genotoxicity. T he mutagenic specificity of NO in human cells is of particular interest in view of its potential role in inflammation-associated carcinogenesis. We co mpared mutagenesis in human lymphoblostoid TK6 cells and in Salmonella typh imurium induced by exposure to NO delivered into the medium at rates approx imating its production by activated macrophages. Exposure of TK6 cells cont inuously for 60 min decreased viability by 88%, and survivors exhibited a s ixfold increase in mutant fraction in the hprt gene. Independent mutants we re isolated and mutations characterized RT-PCR and DNA sequencing. Among a total 68 mutants analyzed, RT-PCR products were obtained in 41 (60%), and c DNA sequencing revealed that 26 (63%) of them contained mutations located i n the hprt coding region. Base substitutions were present in 18 mu tants, 1 2 occurring at A:T base pairs. Seven mutants contained deletions of 1-27 bp and one a 13-bp insertion; the 15 remaining RT-PCR products contained whol e-exon deletions, 14 involving single exons. Six tester strains of S. typhi murium, each containing one of the six possible point mutations in the targ et codon of a gene in the histidine biosynthetic pathway, were similarly tr eated with No and induction of mutation was detected by reversion to histid ine auxotrophy. Significant increases were observed in frequencies of each of the six possible base mutations, with the highest occurring in G:C --> A :T transitions. The pattern of NO-induced hprt mutations in TK6 cells was s imilar to a recently published spectrum in spontaneous mutants, suggesting that reactive species derived From NO may contribute to spontaneous mutagen esis of the endogenous hprt gene in human cells. (C) Wiley-Liss, Inc.