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.

Induction and time-dependent accumulation of micronuclei in peripheral blood of transgenic p53+/- mice, Tg.AC (v-Ha-ras) and parental wild-type (C57BL/6 and FVB/N) mice exposed to benzene by inhalation.

In this study, we determined the induction and time-dependent accumulation of micronuclei in the peripheral blood of transgenic C57BL/6 p53+/- mice (p53+/- mice), FVB/N Tg.AC v-Ha-ras mice (Tg.AC mice) and their isogenic parental strains, FVB/N and C57BL/6 following inhalation exposure to benzene. Our objective was to determine the impact of p53 heterozygosity in p53+/- mice and the v-Ha-ras transgene in Tg.AC mice on micronuclei induction following exposure to inhaled benzene. A flow cytometric technique that distinguishes micronucleated red blood cells (MN-RBC) from micronucleated reticulocytes (MN-RET) was used. Mice were exposed to 0, 100 or 200 p.p.m. benzene using three different exposure regimens that resulted in an equal weekly cumulative exposure (3000 p.p.m.x hours) to benezene: 100 p.p.m. for 6 h/day, 5 days/week, Monday to Friday (M-F); 100 p.p.m. for 10 h/day, 3 days/week, Monday, Wednesday, Friday (MWF); and 200 p.p.m. for 5 h/day, 3 days/week MWF. Significant elevations of MN-RBC and MN-RET were observed from 1 week exposure in all of the benzene-exposed groups that increased in a time-dependent manner for up to 13 weeks exposure. Fewer MN-RBC and MN-RET were induced in the 200 p.p.m. benzene exposure group than in mice exposed to 100 p.p.m. The reduction in the frequency of MN-RBC in the 200 p.p.m.x5 h benzene exposure group is probably due to metabolic saturation resulting in a lower bone marrow dose (concentration x time) than in the 100 p.p.m. exposure groups. No differences were observed in the frequency of MN-RBC or MN-RET in Tg.AC compared with the FVB/N isogenic controls. At certain time points the frequency of micronuclei was less in the heterozygous p53+/- mice than determined in the wild-type C57BL/6 isogenic parental strain. These results indicate that the heterozygous state in p53+/- mice, but not the v-Ha-ras transgene in Tg.AC mice can influence the induction of micronuclei by benzene.

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.

Expression and distribution of cytochrome P450 2E1 in B6C3F1 mouse liver and testes.

Cytochrome P450 2E1 (CYP2E1) is believed to have a significant role in the bioactivation of 1,3-butadiene (BD) to DNA reactive epoxide metabolites that induce somatic and germ cell genotoxicity in mice. To assess the potential role of in situ bioactivation of BD by mouse testes for inducing germ cell genotoxicity, the presence of CYP2E1 in testes has been demonstrated by reverse transcriptase-polymerase chain reaction (RT-PCR), immunoprecipitation-Western blotting methods (IP-Western) and immunohistochemistry of tissue sections. Detection of CYP2E1 in the testes was limited to interstitial cells. In liver a known site of BD bioactivation and a positive control tissue used for these studies, a discrete, zonal staining pattern of liver CYP2E1 expression detected by immunohistochemical staining was shown. These results suggest that in situ bioactivation of BD in testes by CYP2E1 may contribute to BD-induced germ cell 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 in vivo mutagenicity and mutational spectrum at the lacI transgene recovered from the spleens of B6C3F1 lacI transgenic mice following a 4-week inhalation exposure to 1,3-butadiene.

1,3-Butadiene (BD) is carcinogenic and mutagenic in B6C3F1 mice. We determined the lacI mutant frequency and mutational spectrum in spleen following inhalation exposure to BD at levels that are known to induce tumors. B6C3F1 lacI transgenic mice were exposed to air or to 62.5, 625, or 1250 ppm BD for 4 weeks (6 h/day, 5 days/week) and euthanized 14 days after the last exposure. BD increased the lacI mutant frequency in spleen at all levels of BD examined. In BD-exposed mice, an increased frequency of G:C-->A:T transitions occurred at non-5'-CpG-3' sites. Exposure to BD in B6C3F1 lacI transgenic mice also increased the frequency of base substitution mutations that occurred at A:T base pairs when compared to air controls. The increased frequency of specific mutations at G:C base pairs in spleen was not observed in our previous studies in bone marrow and indicates tissue-specific differences in the BD-induced mutational spectrum. These data demonstrate that in vivo transgenic mouse mutagenicity assays can identify tissue-specific mutagenicity and mutational spectrum responses of genotoxic carcinogens at exposure levels that are known to induce tumors.

Long-term mutagenicity studies with chloroform and dimethylnitrosamine in female lacI transgenic B6C3F1 mice.

The weight of evidence indicates that chloroform induces cancer in the female B6C3F1 mouse liver via a nongenotoxic-cytotoxic mode of action. However, it is probable that DNA damage occurs secondary to events associated with cytolethality and regenerative cell proliferation. The purpose of the present study was to evaluate the potential mutagenic activity of chloroform in the B6C3F1 lacI transgenic mouse liver mutagenesis assay including mutagenic events that might occur secondary to cytolethality. The positive control, dimethylnitrosamine (DMN) is a DNA-reactive mutagen and carcinogen. DMN-induced mutations were anticipated to require only a brief exposure and without further treatment were predicted to remain unchanged over time at those frequencies. Chloroform-induced mutations secondary to toxicity were anticipated to require longer exposure periods and to occur only under conditions that produced sustained cytolethality and regenerative cell proliferation. Female B6C3F1 lacI transgenic mice were treated with daily doses of 2, 4, or 8 mg/kg of DMN by gavage for 4 days and then held until analysis 10, 30, 90, and 180 days postexposure. Livers from DMN-treated mice exhibited a dose-related 2- to 5-fold increase over control mutant frequencies and remained at those levels for 10 through 180 days postexposure. Thus, following the initial induction by DMN no selective mutation amplification or loss was seen for this extended period of time. Female B6C3F1 lacI mice were exposed daily for 6 hr/day 7 days/week to 0, 10, 30, or 90 ppm chloroform by inhalation, representing nonhepatotoxic, borderline, or overtly hepatotoxic chloroform exposures. Timepoints for determination of lacI mutant frequency were 10, 30, 90, and 180 days of exposure. No increase in lacI mutant frequency in the liver was observed at any dose or timepoint with chloroform, indicating a lack of DNA reactivity. DNA alterations secondary to toxicity either did not occur or were of a type not detectable by lacI mutant frequency analysis, such as large deletions.

Reverse transcription-polymerase chain reaction-based methodology to quantify differential gene expression directly from microdissected regions of frozen tissue sections.

Quantitative differences in the expression of oncogenes are a critical feature of the cancer process. Several methods are currently available for assessing differential gene expression, but none can be used to determine quantitative changes in gene expression from small numbers of cells. The ability to conduct this type of quantitative analysis would be useful in the study of definable, early stages of carcinogenesis when very few cells are involved. We therefore developed a highly sensitive, slide-based technique that incorporates the benefits of in situ polymerase chain reaction (PCR) and reverse transcription-PCR (RT-PCR) to quantify differential c-myc gene expression from liver tissue sections having either low or high levels of proliferating hepatocytes. To eliminate the need for isolating and quantifying mRNA, cells of interest were microdissected from frozen histological sections and their RNA directly subjected to RT-PCR amplification. These reactions were conducted in the presence of an internal RNA standard that was specifically designed to normalize differential RT and PCR efficiencies between samples. GENESCAN software analysis was used to determine the ratios of the RT-PCR products of the target gene to the RNA standard. These ratios were then normalized to the numbers of cells isolated, as quantified by image analysis, and comparative gene expression values were determined between sample groups. We conclude that this technology can be adapted to study any gene of interest in any type of frozen tissue or isolated cells. This methodology is particularly applicable to the molecular analysis of histopathologically distinct preneoplastic and neoplastic lesions identified on tissue sections.

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.

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.

Molecular analysis of lacI mutants from bone marrow of B6C3F1 transgenic mice following inhalation exposure to 1,3-butadiene.

Chronic exposure to 1,3-butadiene (BD) results in early occurrence and high incidence of lethal lymphomas in male B6C3F1 mice. Male B6C3F1 lacI transgenic mice (BigBlue) were exposed by inhalation to 0, 62.5, 625 or 1250 p.p.m. BD for 4 weeks (6 h/day, 5 days/week). The lacI- mutant frequency and mutational spectrum were evaluated in DNA isolated from bone marrow cells. Two weeks after exposure the lacI- mutant frequency in bone marrow from BD-exposed mice had increased 2- to 3.5-fold over air control mice. DNA sequence analysis of 56 and 54 lacI- mutants from the air control and butadiene-exposed groups, respectively, demonstrated that there was a shift in the spectrum of base substitution mutations at A:T sites in BD-exposed mice (6/26) compared to the air control mice (2/45). A:T-->T:A transversions were found only in BD-exposed animals. Sequence data also indicate that clonal expansion, a natural process in hematopoiesis, can contribute to the lacI- mutant frequency from bone marrow cells such that mutant frequency and mutation frequency are not equivalent. This study shows that BD is mutagenic in B6C3F1 transgenic mouse bone marrow, causing a shift in the mutation spectrum at A:T base pairs in BD-exposed mice compared to air control mice. Sequencing DNA from lacI- mutants from transgenic animals, to determine mutant/mutation frequency and mutational spectra after subchronic bioassay-like exposure conditions, will aid in linking exposure, dose and biological response for genetic risk assessment of BD.

Use of transgenic mice for assessing the mutagenicity of 1,3-butadiene in vivo.

We have examined the mutagenicity of 1,3-butadiene in vivo in transgenic mice constructed with lambda phage shuttle vectors that contain either lacZ or lacI as transgenes. Thus far, we have shown that butadiene is mutagenic to the lung of lacZ mice (CD2F1, 625 ppm butadiene, 6 h/day, 5 days) and to the bone marrow of lacI mice (B6C3F1, 62.5, 625, 1250 ppm butadiene, 6 h/day, 5 days/week, 4 weeks) exposed by inhalation to the same concentrations used previously in bioassays of the carcinogenicity of butadiene. Analysis of the DNA sequences of lacI mutants from the bone marrow of lacI mice demonstrated a shift in the spectrum of mutation from G:C base pairs in the control group to mutation at A:T base pairs in the exposed group. In addition, analysis of DNA sequences of mutations in three animals exposed to butadiene showed evidence of mutant clonal expansion in vivo. These results indicate that butadiene induces mutation in mouse tissues. Analysis of mutations at the level of DNA sequences is useful for examining the relationship between mutation and mutant frequencies in vivo and the spectrum of mutation in transgenic animals after exposures to carcinogens.