thioguanine-anhydrous and 1-nitrosopyrene

The detection of an increase in the frequency of mutants in the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene of circulating T-cells has been proposed as a method to evaluate the biological effects of human exposure to environmental mutagens. We exposed adult human T-cells in vitro to 1-nitrosopyrene (1-NOP), a partially reduced metabolite of 1-nitropyrene, a ubiquitous environmental carcinogen. In populations of T-cells from two unrelated donors, a dose of 1-NOP that reduced survival to 40% of the untreated cells increased the HPRT mutant frequency 6 to 7 times over the background frequency of 5 x 10(-6). The coding region of 35 independent mutants was amplified by polymerase chain reaction and sequenced. Single base substitutions were found in 63% of the mutants (22 of 35). These were distributed randomly throughout the gene. Most of the substitutions (82%) involved G-C base pairs, mainly G.C-->A.T transitions and G.C-->T.A transversions. Fifteen mutants were lacking one or more exons; 9 of the 15 were lacking exons 2 and 3. Examination showed that at least four of the latter had resulted from V(D)J recombinase acting illegitimately to recombine sites located in introns 1 and 3 of the HPRT gene. T-cells from a second unrelated donor were exposed to 1-NOP and 38 additional independent mutants were analyzed. The results indicated that such mutations occurred at a frequency of 2.4 x 10(-6) compared to a background frequency of less than 0.3 x 10(-6). This recombinase, which plays an important role in leukemogenesis, is normally present in developing, but not mature, B- and T-cells such as those used here as target cells for 1-NOP. The present study is the first report showing that exposure to an environmental carcinogen can cause mutations induced by the action of this enzyme.

Topics: Amino Acid Sequence; DNA Nucleotidyltransferases; Dose-Response Relationship, Drug; Drug Resistance; Gene Deletion; Humans; Hypoxanthine Phosphoribosyltransferase; Male; Molecular Sequence Data; Point Mutation; Pyrenes; T-Lymphocytes; Thioguanine; VDJ Recombinases

The cytotoxic and mutagenic effects of 1-nitropyrene (1-NP) and its reduced metabolite 1-nitrosopyrene (1-NOP) were determined in diploid human fibroblasts. Conditions for the metabolic activation of the parent compound (1-NP) by human cells in culture were developed. The cytotoxic effect of 1-NP in normal cells was compared with that for repair-deficient xeroderma pigmentosum (XP) cells, and cells from a patient with hereditary cutaneous malignant melanoma (HCMM), which we have shown earlier are abnormally sensitive to 4-nitroquinoline-1-oxide. The slope of the survival curve for XP cells was 2.5 times steeper than that of normal cells; that of HCMM cells was intermediate. When these cells were exposed to 1-NOP, the slope of the survival curve for the XP cells was also 2.5 times steeper than normal but the HCMM cells showed a normal response, suggesting that their defect is not in repair of DNA adducts, but in activation. 1-NP and 1-NOP also proved to be mutagenic in the human cell assay. When compared on the basis of concentration, 1-NOP was much more mutagenic than 1-NP. But when the compounds were compared on the basis of equal cell killing or equal number of DNA adducts initially bound to DNA, they were very similar. An equal number of residues covalently bound to DNA caused approximately the same amount of cell killing for either compound. XP cells were killed by a 7-fold lower number of bound adducts, suggesting that the increased survival and decreased mutation induction in the normal cells reflects their ability to remove potentially cytotoxic and mutagenic lesions.

Topics: Cell Survival; Cells, Cultured; Diploidy; Drug Resistance; Fibroblasts; Humans; Infant, Newborn; Kinetics; Male; Mutagens; Mutation; Pyrenes; Skin; Thioguanine