The mutagenic potential of the epoxide metabolites of butadiene (BD) was measured at the tk and hprt loci in TK6 human lymphoblastoid cells. TK6 cells were exposed for 24 h to 0-400 microM 1,2-epoxybutene (EB), 0-800 microM 3,4-epoxy-1,2-butanediol (EBD), or 0-6 microM 1,2,3,4-diepoxybutane (DEB). Treated cells were allowed to grow for several days and then seeded in medium containing either 6-thioguanine or trifluorothymidine to select for hprt- or tk-/- mutants, respectively. All three metabolites were mutagenic at both loci, with DEB exhibiting activity at concentrations approximately 100-fold lower than EB or EBD. At the hprt locus, an induced mutation frequency of 5 x 10(-6) (approximately twice background hprt- frequency) was produced by treatment with 3.5 microM DEB, 150 microM EB and 450 microM EBD. At the tk locus, a similar increase in mutation frequency (total tk-/- frequency) was produced by treatment with 1.0 microM DEB, 100 microM EB and 350 microM EBD. Each epoxide tested was capable of inducing slow growth tk-/- mutants. This mutant phenotype, as shown previously by others, results from large alterations in the tk region which completely remove the active tk allele. In addition, Southern blot analysis revealed that approximately half of DEB-induced hprt- mutants displayed loss of wild-type hprt restriction fragments. No statistically significant increase in the fraction of hprt deletions among EB mutants was observed. The ability of DEB to induce deletions may be related to its ability to form DNA-DNA and DNA-protein cross-links.

The mutagenic potential and mutational spectra of butadiene (BD), 1,2-epoxybutene (EB), and diepoxybutane (DEB) were determined in splenic T cells from exposed B6C3F1 mice. Mice exposed by inhalation to 625 p.p.m. BD for 2 weeks displayed an average hprt- mutation frequency of 6.2 x 10(-6) compared to 1.2 x 10(-6) in controls. Mice were also given three daily i.p. doses of 60, 80 and 100 mg EB/kg or 7, 14 and 21 mg DEB/kg. Average hprt- frequencies of 5.4 x 10(-6), 4.1 x 10(-6) and 8.6 x 10(-6) were seen in the EB groups, respectively, while average frequencies of 4.6 x 10(-6), 9.4 x 10(-6) and 13 x 10(-6) were seen in the DEB groups. DNA sequencing revealed that approximately half of the mutations induced in vivo by BD, EB and DEB were frameshift mutations. A +1 frameshift 'hotspot' in six consecutive guanine bases in exon 3 was observed with all three compounds. The remaining mutations produced by BD, EB and DEB were transition and transversion mutations at both AT and GC base pairs. Base pair substitutions induced by BD were biased in favor of mutation at AT base pairs. The mutational spectra produced by BD, EB and DEB were very similar to that observed previously with ethylene oxide, suggesting that these epoxide agents may be working through a similar mutagenic mechanism.

Mutations in the p53 oncogene are extremely common in human cancers, and environmental exposure to mutagenic agents may play a role in the frequency and nature of the mutations. Differences in the patterns of p53 mutations have been observed for different tumor types. It is not trivial to determine if the differences observed in two mutational spectra are statistically significant. To this end, we present a computer program for comparison of two mutational spectra. The program runs on IBM-compatible personal computers and is freely available. The input for the program is a text file containing the number and nature of mutations observed in the two spectra. The output of the program is a P value, which indicates the probability that the two spectra are drawn from the same population. To demonstrate the program, the mutational spectra of single base substitutions in the p53 gene are compared in (i) bladder cancers from smokers and non-smokers, (ii) small-cell lung cancers, non-small-cell lung cancers and colon cancers and (iii) hepatocellular carcinomas from high- and low-aflatoxin exposure groups. p53 mutations differ in several important aspects from a typical mutational spectra experiment, where a homogeneous population of cells is treated with a specific mutagen and mutations at a specific locus are recovered by phenotypic selection. The means by which p53 mutations are recognized is by the appearance of a cancer, and this phenotype is very complex and varied.

The Predictive Safety Testing Consortium's first regulatory submission to qualify kidney safety biomarkers revealed two deficiencies. To address the need for biomarkers that monitor recovery from agent-induced renal damage, we scored changes in the levels of urinary biomarkers in rats during recovery from renal injury induced by exposure to carbapenem A or gentamicin. All biomarkers responded to histologic tubular toxicities to varied degrees and with different kinetics. After a recovery period, all biomarkers returned to levels approaching those observed in uninjured animals. We next addressed the need for a serum biomarker that reflects general kidney function regardless of the exact site of renal injury. Our assay for serum cystatin C is more sensitive and specific than serum creatinine (SCr) or blood urea nitrogen (BUN) in monitoring generalized renal function after exposure of rats to eight nephrotoxicants and two hepatotoxicants. This sensitive serum biomarker will enable testing of renal function in animal studies that do not involve urine collection.

Dideoxy chain-termination DNA sequencing was used to determine the specific DNA base changes induced after in vivo exposure of Escherichia coli to N-methyl-Nnitrosourea (MNU) and N-ethyl-N-nitrosourea (ENU) using the xanthine guanine phosphoribosyltransferase (gpt) gene as the genetic target. The resultant mutation spectra were compared with the levels of O6-alkylguanine and 04-alkylthymidine in genomic DNA immediately after exposure. All (39/39) of the MNU-induced mutations were G-C-+A-T transitions. In contrast, 24/33 point mutations isolated following ENU treatment were G-C-+AT transitions, 7/33 were A-T-*GC transitions,