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 bacteria and human TK6 cells and induces deamination of 5-methylcytosine (5meC) 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 cultured human bronchial epithelial cells (BEAS-2B); and (ii) to compare 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-2B cells were either exposed to 4 mM DEA/NO (Et2N[N2O2]Na, an agent that spontaneously releases NO into the medium) or transfected with the inducible nitric oxide synthase (iNOS) gene. The genotypic mutation assay, which has a sensitivity of 1 x 10(-6), showed that 4 mM ENU induces detectable numbers of G --> A transitions in codon 248 of p53 while 5-methylcytosine deamination was not detected in either iNOS-transfected cells or cells exposed to 4 mM DEA/NO. Moreover, ENU was dose-responsively mutagenic in the phenotypic HPRT assay, reaching mutation frequencies of 24 and 96 times that of untreated control cells at ENU concentrations of 4 and 8 mM respectively; by contrast, 4 mM DEA/NO induced no detectable mutations in this assay, nor were any observed in cells transfected with murine iNOS. We conclude that if NO is at all promutagenic in these cells, it is significantly less so than the ethylating mutagen, ENU.
Felley-Bosco
E
E
Pharmacology and Toxicology Institute, Lausanne, Switzerland.
Mirkovitch
J
J
Ambs
S
S
Macé
K
K
Pfeifer
A
A
Keefer
L K
LK
Harris
C C
CC
D001483:Base Sequence; D001980:Bronchi / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D002460:Cell Line; D003062:Codon; D006152:Cyclic GMP / Q000096:biosynthesis; D003596:Cytosine / Q000378:metabolism; D004048:Diethylamines / Q000633:toxicity; D005038:Ethylnitrosourea / Q000633:toxicity*; D016158:Genes, p53 / Q000187:drug effects*; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000187:drug effects / Q000235:genetics*; D008297:Male; D008745:Methylation; D008969:Molecular Sequence Data; D009152:Mutagenicity Tests; D009153:Mutagens / Q000633:toxicity*; D009154:Mutation; D009569:Nitric Oxide / Q000633:toxicity*; D009589:Nitrogen Oxides; D010641:Phenotype; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D014162:Transfection
D003062:Codon; D004048:Diethylamines; D009153:Mutagens; D009589:Nitrogen Oxides; D009569:Nitric Oxide; D003596:Cytosine; C087229:diethylamine dinitric oxide adduct; D007041:Hypoxanthine Phosphoribosyltransferase; D006152:Cyclic GMP; D005038:Ethylnitrosourea