Kathy G. Meyer

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.

Assessment of the in vivo mutagenicity of ethylene oxide in the tissues of B6C3F1 lacI transgenic mice following inhalation exposure

In the present study, the lacI mutant frequency was determined in the tissues of B6C3F1 lacI transgenic mice exposed by inhalation to ethylene oxide (EO). Groups of 15 male transgenic lacI B6C3F1 mice were exposed to either 0, 50, 100, or 200 ppm EO for 4 weeks (6 h/day, 5 days/week) and were sacrificed at 0, 2, or 8 weeks after the last EO exposure. The lacI transgene was recovered from lung, bone marrow, spleen, and germ cells for determination of the lacI mutant frequency. The tissues selected for analysis were tumor target site tissues in chronic bioassays (lung tumors and lymphomas) and germ cells. The lacI mutant frequency in lung was significantly increased at 8 weeks post exposure to 200 ppm EO (6.2 +/- 2.2 vs. 9.1 +/- 1.5. p = 0.046). In contrast, the lacI mutant frequency in spleen and bone marrow at 2 and 8 weeks was not significantly increased in mice exposed to 200 ppm EO. The lacI mutant frequencies in male germ cells for 200 ppm EO-exposed mice were not increased compared to air controls at 2 and 8 weeks post-exposure. In a spleen cell fraction two of three EO-exposed mice at the 200 ppm exposure level demonstrated an elevated lacI mutant frequency. The increased lacI mutant frequency in these animals was likely due to mutant siblings that contained background G:C --> A:T transitions at CpG sites. These results demonstrate that a 4-week inhalation exposure to EO is mutagenic in lung. However, EO did not increase the frequency of mutations recovered at the lacI transgene in other tissues examined under the conditions used in the present studies. Since the mutational spectrum for EO in other systems consists of an increased proportion of large deletions, the lack of a mutagenic response in the tissues examined is likely due to the lack of recovery of large deletions in lambda-based shuttle vector systems. These data indicate that a primary mechanism of EO-induced mutagenicity in vivo is likely through the induction of deletions, not specific point mutations.

Mutational specificity: Assessment of 1,3-butadiene mutagenicity in the bone marrow of B6C3F1 lacl transgenic mice (Big Blue®): A review of mutational spectrum and LACL 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 morrow and extend these studies to examine the effect of exposure time (5‐day vs. 4‐week exposure to 625 ppm BD used in previous studies) on the lacl mutant frequency in the bone marrow. Inhalation exposure to BD at 625 ppm and 1,250 ppm was 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 lacl 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.