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.
Department of Microbiology, Michigan State University, East Lansing 48824-1316.
W G
WG
McGregor
V M
VM
Maher
J J
JJ
McCormick
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