Environmental and Molecular Mutagenesis 42:106 –110 (2003)
A Model of Sensitivity: 1,3-Butadiene Increases Mutant Frequencies and Genomic Damage in Mice Lacking a Functional Microsomal Epoxide Hydrolase Gene Jeffrey K. Wickliffe,1* Marinel M. Ammenheuser,1 James J. Salazar,1,2 Sherif Z. Abdel-Rahman,1 Darlene A. Hastings-Smith,1 Edward M. Postlethwait,1,3 R. Stephen Lloyd,2 and Jonathan B. Ward Jr.1 1
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Department of Preventive Medicine and Community Health, University of Texas Medical Branch, Galveston, Texas Sealy Center for Environmental Health and Medicine, University of Texas Medical Branch, Galveston, Texas 3 Department of Environmental Health Sciences, University of Alabama at Birmingham, Birmingham, Alabama
The specific role that polymorphisms in xenobiotic metabolizing enzymes play in modulating sensitivity to 1,3-butadiene (BD) genotoxicity has been relatively unexplored. The enzyme microsomal epoxide hydrolase (mEH) is important in detoxifying the mutagenic epoxides of BD (butadiene monoepoxide [BDO], butadiene diepoxide [BDO2]). Polymorphisms in the human mEH gene appear to affect the function of the enzyme. We exposed mice with normal mEH activity (WT) and knockout mice without mEH activity (KO) to 20 ppm BD (inhalation) or 30 mg/kg BDO2 (intraperitoneal [IP] injection). We then compared Hprt mutant frequencies (MFs) among these groups. KO mice exposed to BD exhibited a significant (P ⬍ 0.05) 12.4-fold increase in MF over controls and a significant 5.4-fold increase in MF over exposed WT mice. Additionally, KO mice exposed to BDO2 exhibited a significant 4.5-fold increase in MF over controls and a significant 1.7-fold increase in MF
over exposed WT mice. We also compared genomic damage in WT and KO mice (comet tail moment) following IP exposure to 3 mg/kg and 30 mg/kg BDO2. KO mice exposed to 3 mg/kg exhibited significantly more DNA damage than controls (7.5–12.1-fold increase) and exposed WT mice (3 mg/kg; 4.8-fold increase). KO mice exposed to 30 mg/kg BDO2 exhibited significantly more DNA damage than all other groups (2.3– 27.9-fold increase). Correlation analysis indicated that a significant, positive relationship (r2 ⫽ 0.92) exists between comet-measured damage and Hprt MFs. The lack of mEH activity increases the genetic sensitivity of mice exposed to BD and BDO2. This model should facilitate a mechanistic understanding of the observed variation in human genetic sensitivity following exposure to BD. Environ. Mol. Mutagen. 42:106 –110, 2003. © 2003 WileyLiss, Inc.
Key words: butadiene; Hprt; comet; microsomal epoxide hydrolase; gene knockout; genetic sensitivity
INTRODUCTION 1,3-Butadiene (BD) is a documented mutagen and probable carcinogen in humans (group 2A; IARC, 1999). The mutagenicity and carcinogenicity of BD have also been demonstrated in laboratory mice (Huff et al., 1985; Cochrane and Skopek, 1994; Meng et al., 1998a, 1999a,b). More recently, research has focused on elucidating the biotransformation of BD and its mutational characteristics (Meng et al., 2000; Recio et al., 2001). Biotransformation studies suggest that mutagenicity is governed by a balance between the oxidation of BD by monooxygenases (CYP450s) to electrophilic epoxides and the hydrolytic detoxification of these epoxides by microsomal epoxide hydrolase (mEH) (summarized in Jackson et al., 2000). Another detoxification pathway is through glutathione con© 2003 Wiley-Liss, Inc.
jugation of metabolites by glutathione-S-transferases. Many of the genes encoding these metabolizing enzymes, includ-
Grant sponsor: the Advanced Research Cooperation in Environmental Health program (ARCH-NIEHS) Grant number: 1 P30-ES06676; Grant sponsor: the NIEHS Center at UTMB; Grant number: S11 ES-10018; Grant sponsor: NIEHS training award to UTMB; Grant number: T32ES07254 (postdoctoral fellowship to JKW). *Correspondence to: Jeffrey K. Wickliffe, Department of Preventive Medicine and Community Health, University of Texas Medical Branch, Galveston, TX 77555-1150. E-mail:
[email protected] Received 15 April 2003; provisionally accepted 3 May 2003; and in final form 2 June 2003 DOI 10.1002/em.10181
mEH Inactivity Increases Genetic Sensitivity
ing mEH, are polymorphic, resulting in inferred amino acid substitutions (Hassett et al., 1994, 1997). Because of the importance of mEH in detoxifying the reactive epoxides of BD, butadiene monoepoxide (BDO), and butadiene diepoxide (BDO2), our research group has recently conducted studies examining human variability in response to occupational exposure (Abdel-Rahman et al., 2001, 2003). These studies suggest that, in BD-exposed humans, associations exist between the frequency of mutation in the HPRT reporter gene and polymorphisms in mEH that presumably alter its function (Hassett et al., 1994, 1997; Abdel-Rahman et al., 2001, 2003). Thus, mEH appears to be important in modulating the genotoxicity attributable to BD exposure in humans. Unfortunately, mechanistic experimental data that quantitatively explain the role of these mEH polymorphisms in BD-induced genotoxicity are currently unavailable. To derive accurate, quantitative estimates of mEH-modulated mutagenic sensitivity to BD, an appropriate model system must be applied. Miyata et al. (1999) developed a mouse model in which exon 2 of the microsomal epoxide hydrolase gene was disrupted, effectively abolishing the function of mEH. Since mEH is one of the enzymes that is required to deactivate the mutagenic epoxides formed by oxidation of BD, we used the mEH knockout mouse to evaluate genetic sensitivity to BD and BDO2. Sensitivity was estimated using an assay for mutagenic effect, the Hprt cloning assay, and a measure of genomic damage, the comet assay. We hypothesized that mEH-deficient mice would exhibit a significantly higher Hprt mutant frequency (MF) and significantly more genomic damage, as measured by the comet assay. MATERIALS AND METHODS Sources and Husbandry of Mice Wild-type (WT) 129 mice were obtained from Charles River Laboratories (Wilmington, MA) and mEH knockout (KO) 129 mice were derived from a breeding stock provided by Frank J. Gonzalez at the Center for Cancer Research, National Cancer Institute (NCI). A subset of WT (n ⫽ 5) and KO mice (n ⫽ 5) were genotyped using DNA isolated from tail tissue and the PCR protocol outlined in Sinal et al. (2000). Primer sequences specific to the disrupted exon in the mEH gene were suggested by NCI and are as follows: WT-mEH forward, 5⬘-AACTCATCCTGGCTTCTGTGCTG-3⬘; KO-mEH forward, CGACTGCATCTGCGTGTTCGAAT-3⬘; both reactions use the reverse primer, 5⬘-GAGCAAAATGCACATGACCTCAGG-3⬘. All WT mice exhibited the amplicon representative of the normal mEH gene (332 basepairs), and all KO mice exhibited the amplicon representative of the disrupted mEH gene (297 basepairs). Mice were 5 weeks old at the initiation of each experiment and were housed in an accredited animal care facility at the University of Texas Medical Branch in Galveston (UTMB). Mice were fed and watered ad libitum. All requirements mandated by the UTMB Institutional Animal Care and Use Committee were followed.
1,3-Butadiene Inhalation Exposure Female mice (n ⫽ 6 WT, 6 KO) were exposed in Hinners-type stainless steel chambers (0.85 1/m3) to ⬃20 ppm BD for 7 hr per day, 5 days per
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week, for 24 days (30 air volume changes/hr). Temperature and humidity were maintained at 24 ⫾ 1°C and 50 ⫾ 5%, respectively. During the exposures the animals were housed in individual stainless steel wire mesh cages within the chambers to ensure uniform delivery of the gas phase BD. The animals were transferred to sterilized enriched, complete mouse cages housed in an HEPA filtered laminar-flow unit during weekend periods. BD, from a 99% pure source (Scott Specialty Gases, Pasadena, TX), was metered through a mass flow controller to achieve the desired concentration. Chamber concentrations were continuously monitored using gas chromatography (GC) and photoionization detection. Chamber atmospheres were continuously drawn through the GC sample loop and periodically introduced into the flow of N2 carrier gas using a computer-controlled, air-actuated valve. The concentration of BD was calculated by integrating the peak areas. The GC was calibrated at the beginning of each day using a 60 ppm BD certified standard (Scott Specialty Gases). Air controls (n ⫽ 5 WT, 5 KO) were maintained in identical chambers. Animals were sacrificed 4 weeks following inhalation exposure to allow for the manifestation of Hprt mutants. Lymphocytes from the spleen were harvested and cultured for Hprt MF analysis following the procedure of Meng et al. (1998b). MFs were calculated as in Skopek et al. (1982).
Butadiene Diepoxide (Racemic Mixture) Intraperitoneal Exposure Female mice (n ⫽ 3 WT, 3 KO) were given two intraperitoneal (IP) injections, at a 24-hr interval, of 15 mg/kg BDO2 (cat. number 20,253-3; Sigma-Aldrich, St. Louis, MO) for a cumulative dose of 30 mg/kg. Specifically, concentrated BDO2 (1.113 g/ml) was diluted with a sterile, injectable 0.9% sodium chloride solution and brought up into sterile 1 ml tuberculin syringes each fitted with a sterile 25 gauge 5/8 in. needle. The stock dilution of BDO2 was prepared such that a dose of 15 mg/kg would be delivered to a mouse with a mass of 20 g in a 0.1 ml volume. The mass of each mouse was determined prior to each injection so that the volume of the BDO2 solution could be properly adjusted to deliver the appropriate dose for each individual. Saline controls (n ⫽ 4 WT, 4 KO) were used for each experimental group. All solutions were prepared in an HEPA filtered laminar-flow hood. Mice were sacrificed 4 weeks after treatment. The Hprt mutation assay was conducted as described above.
Genomic Damage (Comet Assay) Five-week-old male mice (n ⫽ 4 WT, 4 KO) were injected as described above with either 1.5 or 15 mg/kg BDO2 for cumulative doses of 3.0 and 30 mg/kg, respectively. Saline controls (n ⫽ 2 WT, 2 KO) were used for each experimental group. Spleen lymphocytes were collected 18 hr after the second treatment for assessment of DNA damage using the single cell gel electrophoresis (comet) assay. The comet assay was performed by using the Trevigen™ Comet Assay Kit (Trevigen, Gaithersburg, MD). For imaging and data analysis, nuclei were visualized by fluorescence microscopy with an incorporated digital camera, captured and individually analyzed by Euclid comet analysis software (Euclid Analysis, St. Louis, MO). Tail moment (TM), percentage of DNA in the tail, and intensity of 50 lymphocytes for each of two mice per group were quantitatively analyzed for evidence of DNA damage.
Statistical Analyses Hprt MFs and comet TMs were analyzed using a univariate ANOVA followed by post-hoc mean comparisons (Bonferroni-corrected) using the SPSS program (SPSS, Chicago, IL). In addition, correlation analysis (Pearson’s moment, r2) was used to explore the association between acute genotoxicity, as measured by the comet assay in male mice, and Hprt MFs
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Fig. 1. Hprt mutant frequencies (MFs) in mice exposed to 20 ppm 1,3-butadiene (BD) by inhalation. Mice were exposed for 7 hr/day, 5 days/week, for 24 days. “WT” indicates mice with normal microsomal epoxide hydrolase (mEH) activity and “KO” indicates mice lacking mEH activity. Sample sizes are indicated above each bar. Error bars represent standard errors of the mean (SEM).
Fig. 2. Hprt MFs in mice exposed to 30 mg/kg BDO2 by IP injection. Mice were injected twice with 15 mg/kg BDO2 in a 24-hr period. “WT” indicates mice with normal mEH activity and “KO” indicates mice lacking mEH activity. Sample sizes are indicated above each bar. Error bars represent standard errors of the mean (SEM).
in randomly selected female mice, following exposure to a total dose of 30 mg/kg BDO2. An ␣ ⬍ 0.05 was used to determine statistical significance.
had a significantly higher MF than both controls (P ⬍ 0.001; a 4.5-fold increase) and exposed WT mice (P ⬍ 0.05; a 1.7-fold increase).
RESULTS
Genomic Damage (Comet Assay)
1,3-Butadiene Inhalation Exposure
Comet tail moments (TMs) are presented in Figure 3. Spontaneous levels of genomic damage in the WT (TM ⫽ 0.18 ⫾ 0.02) and KO (TM ⫽ 0.29 ⫾ 0.01) saline controls were not significantly different from each other. WT mice in both exposure groups (TM ⫽ 0.45 ⫾ 0.03, 3 mg/kg; TM ⫽ 1.38 ⫾ 0.22, 30 mg/kg) did not exhibit a significant increase in genomic damage when compared to the saline controls. KO mice in the low-exposure group had significantly (P ⬍ 0.04) more genomic damage (TM ⫽ 2.18 ⫾ 0.08; 4.8 –12.1fold increase) than all other experimental groups except the WT and KO mice in the high-exposure group. KO mice in the high-exposure group had significantly (P ⬍ 0.003) more genomic damage (TM ⫽ 5.03 ⫾ 0.57; 2.3–27.9-fold increase) than all other experimental groups. The correlation analysis indicated that for the saline controls and the high exposure group a significant, positive relationship exists between genomic damage, as measured by the comet assay, and Hprt MFs (P ⬍ 0.01, r2 ⫽ 0.92).
The average concentration of BD in the inhalation chambers throughout the exposure period was 22.1 (⫾0.74, SEM) ppm. Hprt cloning efficiencies ranged from 4.9 – 23.6%. MFs (⫾SEM) were estimated to be 0.97 (⫾0.12) ⫻ 10-6, 0.87 (⫾0.22) ⫻ 10-6, 2.1 (⫾0.57) ⫻ 10-6, and 11 (⫾2.1) ⫻ 10-6 for the control WT, control KO, exposed WT, and exposed KO mice, respectively (Fig. 1). MFs from control WT and KO mice were not significantly different from one another. Exposed WT mice did not exhibit a significant increase in MF when compared to saline controls. Exposed KO mice had a significantly higher MF than controls and exposed WT mice (P ⬍ 0.001; a 12.4-fold and 5.4-fold increase, respectively). Butadiene Diepoxide Intraperitoneal Exposure Hprt cloning efficiencies ranged from 5.6 –10.4%. MFs were estimated to be 1.6 (⫾0.23) ⫻ 10-6, 1.6 (⫾0.47) ⫻ 10-6, 4.3 (⫾0.75) ⫻ 10-6, and 7.1 (⫾0.8) ⫻ 10-6 for the control WT, control KO, exposed WT, and exposed KO mice, respectively (Fig. 2). MFs from control WT and KO mice were not significantly different from one another. Exposed WT mice had a significantly higher MF than controls (P ⬍ 0.05; a 2.7-fold increase). Exposed KO mice
DISCUSSION These initial experiments indicate that mice lacking mEH activity are more sensitive to BD-induced genotoxicity than their WT counterparts that retain normal mEH function. KO mice exposed to BD by inhalation and to BDO2 by IP injection had significantly higher Hprt MFs than both saline
mEH Inactivity Increases Genetic Sensitivity
Fig. 3. Comet tail moments (TM) in mice exposed to 3 mg/kg BDO2 and 30 mg/kg BDO2 by IP injection. Mice were injected twice with 1.5 mg/kg and 15 mg/kg BDO2 in a 24-hr period. “WT” indicates mice with normal mEH activity and “KO” indicates mice lacking mEH activity. Sample sizes for each group were n ⫽ 2. Error bars represent standard errors of the mean (SEM).
controls and exposed WT mice of the same strain. Interestingly, the magnitude of the difference between saline controls and exposed KO mice was most pronounced following exposure to 20 ppm BD, time-weighted average (TWA), for 24 days. Exposure to a subacute, high dose of the bifunctional epoxide, BDO2, did induce a significant increase in Hprt MFs but not to the extent that was observed following BD inhalation exposure. Because it is unlikely that a subacute exposure to 20 ppm BD TWA will result in the metabolic formation of an acute exposure of 30 mg/kg (ppm) of BDO2 (IP experiment), other mechanisms must be acting to explain this difference in Hprt MFs. It could simply be due to cytotoxicity in the IP experiment, since the yields and viability of isolated lymphocytes in mice exposed IP to 30 mg/kg of BDO2 were markedly lower than the saline controls. Coupled with cytotoxicity, the difference in the type of exposure (i.e., subchronic BD vs. subacute BDO2) between the two experiments may explain the difference in results. An additional plausible explanation may be related to the metabolism of BD. Because of the biotransformation of BD, mice in the BD-inhalation study were exposed to BDO, BDO2, and 3,4-epoxy-1,2-butanediol (BDO-Diol). Mice in the BDO2 IP study were exposed primarily to the bifunctional epoxide. While BDO2 is considered to be substantially more mutagenic than BDO or BDO-Diol, it is possible that the combination of epoxides, their relative concentrations (i.e., a shift towards BDO2 in KO mice), and their respective longevities near target molecules, such as DNA, result in an increased rate of somatic cell mutation (Cochrane and Skopek, 1994) following subchronic exposure to BD. Experiments designed to address
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these questions in the context of genetic sensitivity are currently ongoing. Finally, the increase in MFs observed in both experiments fundamentally supports the biologically important role mEH plays in detoxifying the reactive epoxides of BD and in moderating their genotoxicity. The significant increase in genomic damage measured by the comet assay suggests that exposure to high levels of BDO2 destabilizes duplex DNA in vivo. Moreover, the acute nature of this exposure implies that BDO2, in this case, is acting as an ultimate clastogen causing both physical and chemical alterations in duplex DNA, resulting in clustered single- and double-strand breaks. This suggestion is somewhat in contrast to the view that BDO2-induced mutations require processing of the DNA lesions (i.e., adducts and chemically modified bases) generated by BDO2. Cellular physiology (e.g., temporal transcriptional profiling) was not measured in this study, and it is possible that stress responses are rapidly activated, producing the complement of machinery required for the processing of BDO2-induced lesions. Our results, however, provide a possible explanation for the earlier discovery that the mutational spectra observed in molecular analyses of BD-associated mutagenicity, in both humans and mice, show a shift towards deletion mutants when compared to the spontaneous spectrum (Ma et al., 2000; Meng et al., 2000; Recio et al., 2001). Finally, the correlation between acutely induced genomic damage and the induction of Hprt mutants might indicate that a substantial number of lesions occurring within the Hprt gene itself are improperly repaired. This evidence should be further evaluated in controlled exposures to genotoxicants to validate the possible relationship between genomic DNA damage and the incorporation of mutations into DNA. The primary goal of our research program is to evaluate variation in human sensitivity following exposure to environmentally relevant levels of BD (Ward et al., 1996, 2001; Abdel-Rahman et al., 2001, 2003; Ammenheuser et al., 2001). To address individual susceptibility to BD and develop an accurate assessment of risk, however, variation in responses attributable to genetic and physiological factors must be considered. Therefore, to adequately assess the genetic impacts of BD exposure, models of human sensitivity need to be used. Mice deficient in mEH activity are significantly more sensitive to both the subchronic genotoxicity of BD and the acute genotoxicity of the reactive epoxide, BDO2. The mEH knockout mouse used in these experiments, applied in the context of simulated yet environmentally relevant exposures (i.e., low, chronic doses), should provide a system uniquely suited to model the ultimate in human mEH sensitivity. REFERENCES Abdel-Rahman SZ, Ammenheuser MM, Ward JB Jr. 2001. Human sensitivity to 1,3-butadiene: role of microsomal epoxide hydrolase polymorphisms. Carcinogenesis 22:415– 423. Abdel-Rahman SZ, El-Zein RA, Ammenheuser MM, Yang Z, Stock TH,
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Morandi M, Ward JB Jr. 2003. Variability in human sensitivity to 1,3-butadiene: influence of the allelic variants of the microsomal epoxide hydrolase gene. Environ Mol Mutagen 41:140 –146. Ammenheuser MM, Bechtold WE, Abdel-Rahman SZ, Rosenblatt JI, Hastings-Smith DA, Ward JB Jr. 2001. Assessment of 1,3-butadiene exposure in polymer production workers using hprt mutation in lymphocytes as a biomarker. Environ Health Perspect 109:1249 – 1255. Anderson D, Dobrzynka MM, Jackson LI, Yu TW, Brinkworth MH. 1997. Somatic and germ cell effects in rats and mice after treatment with 1,3-butadiene and its metabolites, 1,2-epoxybutene and 1,2,3,4diepoxybutane. Mutat Res 391:233–242. Cochrane JE, Skopek TR. 1994. Mutagenicity of butadiene and its epoxide metabolites. II. Mutational spectra of butadiene, 1,2-epoxybutene and diepoxybutane at the hprt locus in splenic T cells from exposed B6C3F1 mice. Carcinogenesis 15:719 –723. Hassett C, Aicher L, Sidhu JS, Omiecinski CJ. 1994. Human microsomal epoxide hydrolase: genetic polymorphism and functional expression in vitro of amino acid variants. Hum Mol Genet 3:421– 428. Hassett C, Lin J, Carty CL, Laurenzana EM, Omiecinski CJ. 1997. Human hepatic microsomal epoxide hydrolase: comparative analysis of polymorphic expression. Arch Biochem Biophys 337:275–283. Huff JE, Melnick RL, Solleveld HA, Haseman JK, Powers M, Miller RA. Multiple organ carcinogenicity of 1,3-butadiene in B6C3F1 mice after 60 weeks of inhalation exposure. Science 227:548 –549. IARC. 1999. IARC monographs on the evaluation of carcinogenic risks to humans, vol. 71. Re-evaluation of some organic chemicals, hydrazine and hydrogen peroxide. Lyon, France: IARC. p 109. Jackson MA, Stack HF, Rice JM, Waters MD. 2000. A review of the genetic and related effects of 1,3-butadiene in rodents and humans. Mutat Res 463:181–123. Ma H, Wood TG, Ammenheuser MM, Rosenblatt JI, Ward JB Jr. 2000. Molecular analysis of hprt mutant lymphocytes from 1,3-butadiene-exposed workers. Environ Mol Mutagen 36:59 –71. Meng Q, Recio L, Reilly AA, Wong B, Bauer B, Walker VE. 1998a. Comparison of the mutagenic potency of 1,3-butadiene at the Hprt locus of T-lymphocytes following inhalation exposure of female B6C3F1 mice and F344 rats. Carcinogenesis 19:1019 –1027. Meng Q, Skopek TR, Walker DM, Hurley-Leslie S, Chen T, Zimmer DM, Walker VE. 1998b. Culture and propagation of Hprt mutant T-
lymphocytes isolated from mouse spleen. Environ Mol Mutagen 32:236 –243. Meng Q, Henderson RF, Chen T, Heflich RH, Walker DM, Bauer MJ, Reilly AA, Walker VE. 1999a. Mutagenicity of 1,3-butadiene at the Hprt locus of T-lymphocytes following inhalation exposure of female mice and rats. Mutat Res 429:107–125. Meng Q, Henderson RF, Walker DM, Bauer MJ, Reilly AA, Walker VE. 1999b. Mutagenicity of the racemic mixtures of butadiene monoepoxide and butadiene diepoxide at the Hprt locus of T-lymphocytes following inhalation exposures of female mice and rats. Mutat Res 429:127–140. Meng Q, Singh N, Heflich RH, Bauer MJ, Walker VE. 2000. Comparison of the mutations at Hprt exon 3 of T-lymphocytes from B6C3F1 mice and F344 rats exposed by inhalation to 1,3-butadiene or the racemic mixture of 1,2:3,4-diepoxybutane. Mutat Res 464:169 – 184. Miyata M, Kudo G, Lee Y-H, Yang TJ, Gelboin HV, Fernandez-Salguero P, Kimura S, Gonzalez FJ. 1999. Targeted disruption of the microsomal epoxide hydrolase gene: microsomal epoxide hydrolase is required for the carcinogenic activity of 7,12-dimethylbenz[a]anthracene. J Biol Chem 274:23963–23968. Sinal CJ, Miyata M, Tohkin M, Nagata K, Bend JR, Gonzalez FJ. 2000. Targeted disruption of soluble epoxide hydrolase reveals a role in blood pressure regulation. J Biol Chem 275:40504 – 40510. Skopek TR, Walker VE, Cochrane JE, Craft TR, Cariello NF. 1982. Mutational spectrum at the Hprt locus in splenic T cells of B6C3F1 mice exposed to n-ethyl-n-nitrosourea. Proc Natl Acad Sci USA 89:7866 –7870. Recio L, Steen A-M, Pluta LJ, Meyer KG, Saranko CJ. 2001. Mutational spectrum of 1,3-butadiene and metabolites 1,2-epoxybutene and 1,2,3,4-diepoxybutane to assess mutagenic mechanisms. Chem Biol Interact 136-136:325–341. Ward JB Jr, Ammenheuser MM, Whorton EB Jr, Bechtold WE, Kelsey KT, Legator MS. 1996. Biological monitoring for mutagenic effects of occupational exposure to butadiene. Toxicology 113:84 –90. Ward JB Jr, Abdel-Rahman SZ, Henderson RF, Stock TH, Morandi M, Rosenblatt JI, Ammenheuser MM. 2001. Assessment of butadiene exposure in synthetic rubber manufacturing workers in Texas using frequencies of hprt mutant lymphocytes as a biomarker. Chem Biol Interact 135-136:465– 483.
