Mutational spectrum of 1,3-butadiene and metabolites 1,2-epoxybutene and 1,2,3,4-diepoxybutane to assess mutagenic mechanisms.

1,3-Butadiene (BD) is a multisite carcinogen and is mutagenic in multiple tissues of B6C3F1 mice. BD is bioactivated to at least three directly mutagenic metabolites: 1,2-epoxybutene (EB), 1,2-epoxy-3,4-butanediol (EBD), and 1,2,3,4-diepoxybutane (DEB). However, the contribution of these individual metabolites to the carcinogenicity and in vivo mutatidnal spectrum of BD is uncertain. To assess the role of two BD metabolites EB and DEB in the in vivo mutagenicity of the parent compound BD, we examined the in vitro mutational spectra of EB and DEB in human and rodent cells. We also examined the in vivo mutagenicity and mutational spectrum of inhaled EB in the lung. In the bone marrow and spleen of B6C3F1 laci transgenic mice, BD-induced an increased frequency of the identical class of point mutations at A:T base pairs: AT-->GC transitions and AT-->TA transversions. BD exposure also induced an increased frequency of GC-->AT transitions in the spleen that was not observed in bone marrow, demonstrating tissue-specific differences in mutation spectrum. Exposure of Rat2 laci transgenic cells and human TK6 lymphoblasts to EB-induced an increased frequency of AT-->TA transversions. DEB exposure induced an increased frequency of AT-->TA transversions and partial deletions at hprt in human cells. In Rat laci transgenic cells, DEB was not mutagenic at laci but induced an increased frequency of micronuclei. In contrast to inhaled BD, inhaled DEB and EB were not mutagenic in the bone marrow or spleen. However, EB was mutagenic in the lungs. In the lung of mice, EB-induced specific increases in GC-->AT transitions, AT-->TA transversions, and deletion events. AT-->TA transversions are the most consistent mutation observed across biological systems following in vivo exposure to BD or in vitro exposures to EB and DEB. Although, BD exposure in mice induces chromosomal alterations and single base substitutions, the specific BD metabolite that induces the genetic events leading to tumors is uncertain. At present, it appears that only DEB can effectively induce this range of mutagenic events at levels of this metabolite that occur in the blood of mice exposed to BD. Detailed investigations to identify relevant biomarkers of BD exposure and response, particularly DNA adducts or lesions, that can be biologically linked to the range of genotoxic events known to occur in mice exposed to BD are needed.

Lung-specific mutagenicity and mutational spectrum in B6C3F1 lacI transgenic mice following inhalation exposure to 1,2-epoxybutene.

1,3-Butadiene (BD) is carcinogenic and mutagenic in B6C3F1 mice. BD inhalation induces an increased frequency of specific base substitution mutations in the bone marrow and spleen of B6C3F1 lacI transgenic mice. BD is bioactivated to at least three mutagenic metabolites: 1,2-epoxybutene (EB), 1,2-epoxy-3,4-butanediol (EBD), and 1,2,3,4-diepoxybutane (DEB), however, the contribution of these individual metabolites to the in vivo mutational spectrum of BD is uncertain. In the present study, lacI transgenic mice were exposed by inhalation (6h per day, 5 days per week for 2 weeks) to 0 or 29.9ppm of the BD metabolite, EB to assess its contribution to the in vivo mutational spectrum of BD. No increase in lacI mutant frequency was observed in the bone marrow or spleen of EB-exposed mice. The lack of mutagenicity in the bone marrow or spleen likely relate to insufficient levels of EB reaching these tissues. The lacI mutant frequency was increased 2.7-fold in the lungs of EB-exposed mice (mean+/-S.D., 9.9+/-3.0x10(-5)) compared to air control mice (3.6+/-0.7x10(-5)). DNA sequence analysis of 65 and 66 mutants from the lungs of air control and EB-exposed mice, respectively, revealed an increase in the frequency of two categories of base substitution mutation and deletions. Like mice exposed to BD, EB-exposed mice had an increased frequency of A:T-->T:A transversions. However, in contrast to the BD mutational spectra, G:C-->A:T transitions at 5'-CpG-3' sequences, occurred with increased frequency in the EB-exposed mice. The increased frequency of deletions as well as the induction of two tandem mutations and a tandem deletion in the lungs of EB-exposed mice are also inconsistent with previous mutational spectra from BD-exposed mice or EB-exposed cells in culture. We hypothesize that the direct in vivo mutagenicity and further in situ metabolism of EB in the lungs of EB-exposed mice played a prominent role in the generation of the current mutational spectrum.

1,3-butadiene: cancer, mutations, and adducts. Part II: Roles of two metabolites of 1,3-butadiene in mediating its in vivo genotoxicity.

1,3-Butadiene (BD) is carcinogenic in mice and rats, with mice being more susceptible than rats to its carcinogenic effects. 1,3-Butadiene is mutagenic in the bone marrow and spleen cells of B6C3F1 lacI transgenic mice. The goal of this research was to assess the roles of two BD metabolites, 1,2-epoxy-3-butene (BDO) and 1,2,3,4-diepoxybutane (BDO2), in the mutagenicity and mutational spectrum of the parent compound BD by determining the mutagenicity and mutational spectra of BDO and BDO2 in human and rodent cells in vitro and in vivo. In human TK6 lymphoblastoid cells (TK6 cells), BDO exposure increased the frequency of G.C-->A.T transitions and A.T-->T.A transversions (Fisher exact test; p < 0.05). The most striking difference in the type of base-substitution mutations between BDO-exposed and BDO-unexposed TK6 cells was the 19-fold increase in A.T-->T.A transversions. 1,2,3,4-Diepoxybutane increased the frequency of A.T-->T.A transversions (Fisher exact test; p < 0.05) and the frequency of deletions in exposed TK6 cells compared with unexposed controls. Exposure of Rat2 lacI transgenic fibroblasts (Rat2 cells) to BDO increased the frequency of three types of base-substitution mutations: G.C-->A.T transitions, G.C-->T.A transversions, and A.T-->T.A transversions. Exposure of Rat2 cells to BDO2-induced dose-dependent increases in micronuclei at exposure levels that apparently did not induce mutagenicity at the lacI transgene. The lack of detectable mutagenicity at the lacI transgene in Rat2 cells exposed to BDO2 probably reflects the poor recovery of large deletions by this lambda phage-based mutagenicity assay. Inhalation exposure of B6C3F1 lacI transgenic mice (lacI mice) and F344 lacI transgenic rats (lacI rats) to BDO (29.9 parts per million [ppm]; 6 hours/day; 5 days/week for 2 weeks) did not increase the lacI mutant frequency (MF) in bone marrow or spleen cells of mice and rats, but in the cells of mouse lung (a tumor target organ for BD), significant mutagenicity was observed. An increased lacI MF was also observed in the bone marrow cells of rats exposed to BDO. Inhalation exposure of lacI mice and lacI rats to BDO2 (3.8 ppm; 6 hours/day; 5 days/week for 2 weeks) did not increase the lacI MF in bone marrow or spleen cells of mice or in the spleen cells of rats. An increased lacI MF was observed in the bone marrow cells of rats exposed to BDO2. In the present study, BDO specifically induced G.C-->A.T and A.T-->T.A transversions in vitro at both the endogenous hypoxanthine phosphoribosyltransferase (hprt) gene and the lacI transgene in Rat2 cells. It also induced an increased frequency of G.C-->T.A transversions in Rat2 cells. These types of mutations also occur at an increased frequency in mice exposed to the parent compound, BD. This finding demonstrates the induction of consistent mutational types across biological systems by BDO and indicates that BDO, but not BDO2, probably has a role in mediating the mutations recovered at the lacI transgene in animals exposed to the parent compound, BD. Therefore, it is apparent that in mice exposed to BD at carcinogenic levels, BDO and BDO2 act in concert to mediate the range of genotoxic responses. These data demonstrate that certain DNA adducts (guanine or adenine) may be useful biomarkers for BD genetic effects. However, other DNA lesions that can account for BDO2-induced deletions and chromosomal alterations also need to be considered as biomarkers for BD-induced genotoxicity.

Molecular analysis of lacI mutants from transgenic fibroblasts exposed to 1,2-epoxybutene.

1,3-Butadiene (BD) is a genotoxic carcinogen that is bioactivated to at least two mutagenic metabolites: 1,2-epoxybutene (EB) and 1,2:3,4-diepoxybutane (DEB). We reported previously that lacI transgenic mice exposed to BD had an increased frequency of specific base substitution mutations in the bone marrow and spleen relative to unexposed controls. In the experiments described here, we determined the mutagenicity and mutational spectrum of EB in Rat2 lacI transgenic fibroblasts as a means of assessing the contribution of this metabolite to the lacI mutational spectrum of BD. Rat2 cells were exposed to 0, 0.4, 0.6, 0.8 or 1.0 mM EB for 24 h, resulting in a range of cell survival from 100 to 15%, respectively. Mutagenicity was assessed at 0, 0.6 and 1.0 mM EB. Unexposed controls had a background mutant frequency of 6 +/- 1 +/- 10(-5), while the mutant frequency in cells exposed to 0.6 and 1.0 mM EB was increased 2- and 3-fold, respectively. DNA sequence analysis of 154 lacI mutants recovered in these experiments revealed an increase in the frequency of specific base substitution mutations in cells exposed to 1.0 mM EB compared with controls. These included G:C-->A:T transitions at non-CpG sites, G:C-->T:A transversions and A:T-->T:A transversions, which have all been observed in lacI mutants isolated from transgenic mice exposed to BD. These results suggest that EB causes mutation primarily by base substitution and that the spectrum of these mutations closely resembles that of BD. These data, along with previous findings from our laboratory, suggest that EB is more likely than DEB to be primarily responsible for the lacI mutational spectrum observed in lacI transgenic mice exposed to BD.

The butadiene metabolite, 1,2:3,4-diepoxybutane, induces micronuclei but is only weakly mutagenic at lacI in the Big Blue Rat2 lacI transgenic cell line.

1,3-Butadiene (BD) is a genotoxic carcinogen that is bioactivated to at least two mutagenic metabolites, 1,2-epoxybutene (EB) and 1,2:3,4-diepoxybutane (DEB). We investigated the mutagenicity and induction of micronuclei by DEB in vitro in Rat2 lambda/lacI transgenic fibroblasts (Big Blue Rat2 cells, Stratagene, LaJolla, CA). Assays for mutagenicity and micronuclei induction were carried out at concentrations of 0, 2, 5, or 10 microM DEB for 24 hours. Exposure of cells to these concentrations of DEB resulted in approximately 100, 50, and 10% survival, respectively, compared with media controls. In independent replicate experiments, no statistically significant increase in lacI mutant frequency was observed in Rat2 cells at any of the DEB exposure concentrations when compared to media or solvent controls. However, regression analyses indicated a trend toward increasing mutant frequency with increasing DEB exposure concentration. Experiments to examine the induction of micronuclei by DEB revealed a concentration-dependent increase in micronuclei in Rat2 cells following exposure to DEB. These results indicate that DEB induces micronuclei in the absence of detectable gene mutation at lacI in Big Blue Rat2 cells. The induction of micronuclei but only weak mutagenicity at the lacI transgene is likely due to the poor recovery of deletions using this lambda shuttle vector system, demonstrating the need to investigate multiple endpoints of genotoxicity when considering the mutational activity of a compound.

Assessment of 1,3-butadiene mutagenicity in the bone marrow of B6C3F1 lacI transgenic mice (Big Blue): a review of mutational spectrum and lacI mutant frequency after a 5-day 625 ppm 1,3-butadiene exposure.

1,3-Butadiene (BD) is a carcinogen that is bioactivated to at least two genotoxic metabolites. In the present article, we review briefly our previous studies on the in vivo mutagenicity and mutational spectra of BD in bone marrow and extend these studies to examine the effect of exposure time (5-days vs. 4-week exposure to 625 ppm BD used in previous studies) on the lacI mutant frequency in the bone marrow. Inhalation exposure to BD at 625 ppm and 1,250 ppm mutagenic in vivo, inducing an increase in the transgene mutant and mutation frequency in the bone marrow. Analysis of the mutational spectrum in BD-exposed and air control mice demonstrated that BD exposure induced an increased frequency of mutations at A:T base pairs. There was no difference in the lacI mutant frequency determined in the bone marrow between a short-term exposure to BD (5 days) and a longer-term exposure (4 weeks). These data taken together demonstrate that inhalation exposure to BD induces in vivo somatic cell mutation.