Mutagenicity and Mutational Spectrum of 1,3-Butadiene and its Epoxide Metabolites at the Hprt Locus in Cultured Human Lymphoblasts and in Splenic T-cells from Exposed B6C3F1 Mice
Creators: name: Cochrane, Judith E.
Publisher: name: The University of North Carolina at Chapel Hill University Libraries
Year: 1993

description: The objective of this research was to examine the mutagenic potential of butadiene and its epoxide metabolites in a human lymphoblastoid cell line (TK6) and in splenic T-cells isolated from exposed B6C3F1 mice. TK6 cells were exposed for 24 hours to 1,2-epoxybutene (EB), 3,4-epoxy-1,2-butanediol (EBD), or diepoxybutane (DEB) and then plated to determine the toxicity of treatment and to measure mutation frequency at the hypoxanthine-guanine phosphoribosyl transferase (hprt) and thymidine kinase (tk) loci (Liber and Thilly, 1982). Treatments with 2.6 μM DEB, 400 μM EB, and 800 μM EBD resulted in 37% culture survival. At the hprt locus, an induced mutation frequency of 5 x 10[-6] was produced by 3.5 μM DEB, 150 μM EB, and 450 μM EBD; at the tk locus, an induced mutation frequency of 5 x 10[-6] (normal growth mutants) was produced by 2.6 μM DEB, 250 μM EB, and 450 μM EBD. EB, EBD, and DEB also induced slow-growth tk mutants at approximately the same frequency as normal-growth tk mutants. Thus, all three metabolites of butadiene were mutagenic at both the hprt and tk loci, with DEB exhibiting mutagenic activity at concentrations approximately two orders of magnitude lower than EB or EBD. The method of Southern blot was used to investigate the occurrence of deletions induced in vitro at the hprt gene of human lymphoblastoid cells (TK6) after exposure to 1,2-epoxybutene (EB) and diepoxybutane (DEB). Thirty-five EB-induced mutants and 39 DEB mutants were analyzed by the method of Southern blot. Fifty-four percent (54%) of DEB-induced mutants showed altered banding patterns, compared to 11.4% for EB mutants. The mutagenic response at the hprt locus in splenic T-cells of exposed B6C3F1 mice (Skopek et al, 1992) was also studied. Twelve-day-old male mice were exposed to ambient air (controls) or 625 ppm butadiene for 2 weeks (6 hr/day, 5 days/week). Mice were sacrificed two weeks after the end of exposure. Hprt mutation frequencies in 8 exposed mice (range 4-9 x 10[-6]; average 6x10[-6]) were significantly elevated relative to frequencies observed in 7 control animals (range 0.2-3 x 10[-6]; average 1 x 10[-6]). In another experiment, preweanling mice were exposed to 3 doses, every other day, of 60, 80, and 100 mg/kg EB via intraperitoneal injection. Animals were sacrificed two weeks later. Average hprt mutation frequencies of 5 x 10[-6] (range 1-9 x 10[-6]; 7 mice), 4x10[-6] (range 3-6 x 10[-6]; 6 mice) and 8x10[-6] (range 4-15 x 10[-6]; 6 mice) were seen in these groups, respectively, compared to 1 x 10[-6] (range 0.1-4 x 10[-6]; 7 mice) in controls. Twelve-day-old mice were also exposed to i.p. doses of DEB every other day, totalling 21, 28 and 42 mg/kg, with sacrifice two weeks later. Average hprt mutation frequencies observed in these groups were 5x10[-6] (range 2-8 x 10[-6]; 3 mice), 9x10[-6] (range 6-14 x 10[-6]; 4 mice) and 12 x 10[-6] (range 5-22 x 10[-6]; 4 mice) respectively, compared to 1 x 10[-6] (range 0.6-2 x 10[-6]; 4 mice) among control animals. Thus, butadiene and its epoxide metabolites, EB and DEB, are mutagenic in vivo at the hprt locus in splenic T-cells of mice. By the technique of denaturing gradient gel electrophoresis, we have also investigated the mutational spectrum induced in exon 3 of hprt in splenic T-cells of male B6C3F1 mice after inhalation exposure to 1,3-butadiene or intraperitoneal administration of 1,2-epoxybutene or diepoxybutane. Thirteen of the 77 mutants (17%) from 1,3-butadiene exposed animals contained mutations in exon 3. Twenty-six of the 276 mutants (9.4%) from animals exposed to EB contained mutations in exon 3 . Treatment of mice with diepoxybutane (DEB) resulted in 11 of 205 mutants (5.4%) analyzed which contained mutations in exon 3. Mutant DNA was PCR amplified and purified from contaminating wild-type DNA through a series of denaturing gradient gels and then sequenced by an automated DNA sequencer. Similarities in mutational spectrum of the three compounds suggest that the generation of apurinic sites may play a role in mutagenesis by the metabolites of 1,3-butadiene.