Kinds and locations of mutations induced in the hypoxanthine-guanine phosphoribosyltransferase gene of human T-lymphocytes by 1-nitrosopyrene, including those caused by V(D)J recombinase.
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.
McGregor
W G
WG
Department of Microbiology, Michigan State University, East Lansing 48824-1316.
Maher
V M
VM
McCormick
J J
JJ
D000595:Amino Acid Sequence; D004254:DNA Nucleotidyltransferases / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D004351:Drug Resistance; D017353:Gene Deletion; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D008297:Male; D008969:Molecular Sequence Data; D017354:Point Mutation / Q000235:genetics*; D011721:Pyrenes / Q000633:toxicity*; D013601:T-Lymphocytes / Q000187:drug effects*; D013866:Thioguanine / Q000494:pharmacology; D045567:VDJ Recombinases
D011721:Pyrenes; C044707:1-nitrosopyrene; D007041:Hypoxanthine Phosphoribosyltransferase; D004254:DNA Nucleotidyltransferases; D045567:VDJ Recombinases; D013866:Thioguanine