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 hypoxa nthine-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 metabo lite of 1-nitropyrene, a ubiquitous environmental carcinogen. In popul ations of T-cells from two unrelated donors, a dose of 1-NOP that redu ced survival to 40% of the untreated cells increased the HPRT mutant f requency 6 to 7 times over the background frequency of 5 x 10(-6). The coding region of 35 independent mutants was amplified by polymerase c hain reaction and sequenced. Single base substitutions were found in 6 3% of the mutants (22 of 35). These were distributed randomly througho ut 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 mu tants 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 result ed from V(D)J recombinase acting illegitimately to recombine sites loc ated in introns 1 and 3 of the HPRT gene. T-cells from a second unrela ted 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 pre sent study is the first report showing that exposure to an environment al carcinogen can cause mutations induced by the action of this enzyme .