NITRIC-OXIDE AND ETHYLNITROSOUREA - RELATIVE MUTAGENICITY IN THE P53 TUMOR-SUPPRESSOR AND HYPOXANTHINE-PHOSPHORIBOSYLTRANSFERASE GENES

Nitric oxide (NO) is a cellular messenger which is mutagenic in bacter ia and human TK6 cells and induces deamination of 5-methylcytosine (5m eC) residues in vitro. The aims of this study were: (i) to investigate whether NO induces 5meC deamination in codon 248 of the p53 gene in c ultured human bronchial epithelial cells (BEAS-2B); and (ii) to compar e NO mutagenicity to that of ethylnitrosourea (ENU), a strong mutagen. Two approaches were used: (i) a novel genotypic assay, using RFLP/PCR technology on purified exon VII sequence of the p53 gene; and (ii) a phenotypic (HPRT) mutation assay using 6-thioguanine selection. BEAS-2 B cells were either exposed to 4 mM DEA/NO (Et(2)N[N2O2]Na, an agent t hat spontaneously releases NO into the medium) or transfected with the inducible nitric oxide synthase (iNOS) gene. The genotypic mutation a ssay, which has a sensitivity of 1 x 10(-6), showed that 4 mM ENU indu ces detectable numbers of G --> A transitions in codon 248 of p53 whil e 5-methylcytosine deamination was not detected in either iNOS-transfe cted cells or cells exposed to 4 mM DEA/NO. Moreover, ENU was dose-res ponsively mutagenic in the phenotypic HPRT assay, reaching mutation fr equencies of 24 and 96 times that of untreated control cells at ENU co ncentrations of 4 and 8 mM respectively; by contrast, 4 mM DEA/NO indu ced no detectable mutations in this assay, nor were any observed in ce lls transfected with murine iNOS. We conclude that if NO is at all pro mutagenic in these cells, it is significantly less so than the ethylat ing mutagen, ENU.