ANALYSIS OF HPRT MUTATIONS OCCURRING IN HUMAN TK6 LYMPHOBLASTOID-CELLS FOLLOWING EXPOSURE TO 1,2,3,4-DIEPOXYBUTANE

1,3-Butadiene (ED) is a rodent carcinogen that is bioactivated to at l east two genotoxic metabolites, 1,2-epoxybutene (EB) and 1,2,3,4-diepo xybutane (DEB). The mutational spectrum for DEB at hprt in human TK6 l ymphoblasts (TK6 cells) was determined and compared with the mutationa l spectrum from spontaneous mutants, A DEB exposure of 4 mu M for 24 h resulted in an average 5-fold increase in the hprt mutant frequency, Hprt mutants for molecular analysis were isolated from TK6 cells expos ed to 4 mu M DEB for 24 h (51 DEB-induced mutants) and from a set of s pontaneous mutants (n = 43) isolated from the same TK6 stock cell cult ures, Molecular analyses of hprt mutations were done by reverse transc ription-polymerase chain reaction (RT-PCR) of hprt mRNA or exon-specif ic genomic PCR amplification of hprt followed by DNA sequencing of PCR products, There was an increased frequency of A:T-->T:A transversions among the DEB-induced mutants compared to spontaneous mutants (9/51; 18% DEB-induced compared to 2/43; 5% in spontaneous (one-way Fisher's exact test; P less than or equal to 0.05), DEB-induced hprt mutants al so had an increased frequency of genomic deletions affecting the 5' re gion of hprt (7/51; 14% DEB-induced compared to 1/43; 2% in spontaneou s), Therefore, DEB is a mutagenic carcinogen that can induce genotoxic ity by large deletions, rearrangements or single base substitution mut ations.