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1,3-Butadiene (BD) is a rodent carcinogen that is bioactivated to at least two genotoxic metabolites, 1,2-epoxybutene (EB) and 1,2,3,4-diepoxybutane (DEB). The mutational spectrum for DEB at hprt in human TK6 lymphoblasts (TK6 cells) was determined and compared with the mutational spectrum from spontaneous mutants. A DEB exposure of 4 microM 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 exposed to 4 microM DEB for 24 h (51 DEB-induced mutants) and from a set of spontaneous mutants (n = 43) isolated from the same TK6 stock cell cultures. Molecular analyses of hprt mutations were done by reverse transcription-polymerase chain reaction (RT-PCR) of hprt mRNA or exon-specific 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 < or = 0.05). DEB-induced hprt mutants also had an increased frequency of genomic deletions affecting the 5' region of hprt (7/51; 14% DEB-induced compared to 1/43; 2% in spontaneous). Therefore, DEB is a mutagenic carcinogen that can induce genotoxicity by large deletions, rearrangements or single base substitution mutations.

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A nonsense mutation at the CpG-site in the codon for Arg(169) in the gene for hypoxanthine phosphoribosyltransferase (hprt) was identified by genomic polymerase chain reaction (PCR) and DNA sequencing in cultured fibroblasts from two brothers with Lesch Nyhan's syndrome. The recurrence of mutation at this CpG-site in several unrelated Lesch-Nyhan families suggests that deamination of 5-methylcytosine is a possible mechanism for mutagenesis. The level of hprt-mRNA in the fibroblasts of the patients was similar to that in healthy controls, whereas hprt-enzyme activity was not detectable. The mutation in this family was also identified in five female relatives and prenatally in a male fetus. Unexpectedly, results from hair follicle analyses and fibroblast selection studies in 8-azaguanine and 6-thioguanine medium showed a non-carrier phenotype in three of the female heterozygotes, whereas X-inactivation mosaicism was demonstrated in one heterozygote. A possible explanation for the apparent non-random X-inactivation in this family is the co-existence of the hprt mutation with an undefined X-linked lethal mutation. This observation is of practical relevance for carrier detection in other Lesch-Nyhan families.

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1,3-Butadiene (BD) is an indirect-acting mutagen that is bioactivated in laboratory animals to at least two mutagenic metabolites, 1,2-expoxy-3-butene (EB) and 1,2,3,4-diepoxybutane (DEB). In the present study, the cytotoxicity, mutagenicity and mutational spectrum at hprt were determined after EB-exposure of human TK6 lymphoblastoid cells (TK6 cells). EB was cytotoxic at concentrations ranging from 200 to 1000 microM x 24 h; at 400 microM x 24 h, the cell survival relative to unexposed controls was approximately 10%. Exposure of TK6 cells to EB (400 microM x 24 h) resulted in a 5-9-fold increase in the hprt mutant frequency. Molecular characterization of EB-induced hprt mutants indicated that 78% of the mutations at hprt were single base substitutions. A significant (P < 0.05) increase in A:T-->T:A transversions was observed compared with spontaneous hprt mutants isolated during these studies. All of the A:T-->T:A transversions in EB-induced mutants occurred with the A in the non-transcribed strand. These data indicate that a primary mode of genotoxicity induced by EB in human TK6 cells is the induction of single base substitutions.

The molecular basis of somatic mutation at the hypoxanthine-guanine phosphoribosyl-transferase (hprt) locus in human 6-thioguanine resistant T-cell clones from 17 individuals has been studied by Southern blot analysis, multiplex PCR (polymerase chain reaction) and direct sequencing of PCR amplified hprt cDNAs or genomic DNA. Twenty-three novel mutations were detected, which in addition to previously described mutations provide a background mutational spectrum based on a total of 45 hprt mutations in human T-cells. Twenty T-cell mutants had base substitutions in the coding region leading to 15 missense and five nonsense mutations. In addition to five frameshift mutations caused by four small deletions and one duplication, seven splice mutations, three of them with skipping of exon 8, were detected. Thirteen genomic structural alterations have also been identified; one of these had a genomic exon 1 deletion with a GGCCGG-hexamer in both breakpoints.

The T-cell cloning assay which combines mitogen- and growth factor-dependent expansion of lymphocyte clones with thioguanine selection of hypoxanthine-guanine phosphoribosyl transferase (hprt)-negative cells has been extensively used for studying human somatic gene mutation in vivo. However, large interindividual variations in the hprt mutant frequency (MF), much of which is not explained by donor attributes such as age and smoking habit, and interlaboratory variations in the experimental methodology, including cloning efficiency (CE), call for further developments of the cloning protocol and additional population studies. Using an improved T-cell cloning method, we have studied in vivo hprt MF of 76 non-smoking healthy males aged 23-77 years. The addition of 5% human serum to the growth medium was found to produce a consistently high CE of 61% in average. The MF, ranging from 1.4 to 22.6 x 10(-6) with a mean of 8.6 x 10(-6), increased significantly (P < 0.0001) with age, by 2% per year. A significant (P = 0.002) inverse relationship between MF and CE was observed. Using a PCR-based technique for GSTM1-genotyping, we also studied the relationship between MF and GSTM1 polymorphism. The 38 (50%) GSTM1-negative individuals showed a 20% higher mean MF than the 38 (50%) GSTM1-positive individuals. The difference was however not significant, neither before (P = 0.1) nor after (P = 0.5) correction for CE and the significantly (P = 0.04) higher mean age in the GSTM1-negative group. This study shows that age contributes more than GSTM1 polymorphism to the large interindividual variation in the hprt MF of non-smokers. The relationship between GSTM1 polymorphism and hprt MF in smokers remains to be investigated.