Patricia B. Upton

1,3-Butadiene: Biomarkers and application to risk assessment

1,3-Butadiene (BD) is a known rodent and human carcinogen that is metabolized mainly by P450 2E1 to three epoxides, 1,2-epoxy-3-butene (EB), 1,2:3,4-diepoxybutane (DEB) and 1,2-epoxy-3,4-butanediol (EB-diol). The individual epoxides vary up to 200-fold in their mutagenic potency, with DEB being the most mutagenic metabolite. It is important to understand the internal formation of the individual epoxides to assign the relative risk for each metabolite and to understand the molecular mechanisms responsible for major species differences in carcinogenicity. We have conducted extensive exposure-biomarker studies on mice, rats and humans. Using low exposures that range from current occupational levels to human exposures from tobacco smoke has provided evidence that mice are very different from humans, with mice forming ∼200 times more DEB than humans at exposures of 0.1-1.5ppm BD. While no gender differences have been noted in mice and rats for globin adducts or N-7 guanine adducts, female rats and mice had 2-3-fold higher Hprt mutations and DNA-DNA cross-links, suggesting a gender difference in DNA repair. Numerous molecular epidemiology studies have evaluated globin adducts and Hprt mutations, SCEs and chromosomal abnormalities. None of the blinded studies have shown evidence of human genotoxicity at current occupational exposures and studies of globin adducts have shown similar or lower formation of adducts in females than males. If one calculates the EB dose-equivalents for the three species, mice clearly differ from rats and humans, being ∼44 and 174 times greater than rats and humans, respectively. These data provide a scientific basis for improved risk assessment of BD.

Analysis of Diepoxide-Specific Cyclic N-Terminal Globin Adducts in Mice and Rats after Inhalation Exposure to 1,3-Butadiene

1,3-Butadiene is an important industrial chemical used in the production of synthetic rubber and is also found in gasoline and combustion products. It is a multispecies, multisite carcinogen in rodents, with mice being the most sensitive species. 1,3-Butadiene is metabolized to several epoxides that form DNA and protein adducts. Previous analysis of 1,2,3-trihydroxybutyl-valine globin adducts suggested that most adducts resulted from 3-butene-1,2-diol metabolism to 3,4-epoxy-1,2-butanediol, rather than from 1,2;3,4-diepoxybutane. To specifically examine metabolism of 1,3-butadiene to 1,2;3,4-diepoxybutane, the formation of the 1,2;3,4-diepoxybutane–specific adduct N,N-(2,3-dihydroxy-1,4-butadiyl)-valine was evaluated in mice treated with 3, 62.5, or 1250 ppm 1,3-butadiene for 10 days and rats exposed to 3 or 62.5 ppm 1,3-butadiene for 10 days, or to 1000 ppm 1,3-butadiene for 90 days, using a newly developed immunoaffinity liquid chromatography tandem mass spectrometry assay. In addition, 2-hydroxy-3-butenyl-valine and 1,2,3-trihydroxybutyl-valine adducts were determined. The analyses of several adducts derived from 1,3-butadiene metabolites provided new insight into species and exposure differences in 1,3-butadiene metabolism. Mice formed much higher amounts of N,N-(2,3-dihydroxy-1,4-butadiyl)–valine than rats. The formation of 2-hydroxy-3-butenyl-valine and N,N-(2,3-dihydroxy-1,4-butadiyl)–valine was similar in mice exposed to 3 or 62.5 ppm 1,3-butadiene, whereas 2-hydroxy-3-butenyl-valine was 3-fold higher at 1250 ppm. In both species, 1,2,3-trihydroxybutyl-valine adducts were much higher than 2-hydroxy-3-butenyl-valine and N,N-(2,3-dihydroxy-1,4-butadiyl)–valine. Together, these data show that 1,3-butadiene is primarily metabolized via the 3-butene-1,2-diol pathway, but that mice are much more efficient at forming 1,2;3,4-diepoxybutane than rats, particularly at low exposures. This assay should also be readily adaptable to molecular epidemiology studies on 1,3-butadiene-exposed workers

Using DNA and hemoglobin adducts to improve the risk assessment of butadiene.

The purpose of this paper is to review what we know about various biomarkers of butadiene in animal, human and in vitro studies, and to draw inferences from these data that impact on the accurate assessment of human risks for cancer. Studies comparing the DNA and hemoglobin adducts of butadiene with exposure, metabolism and genotoxicity have provided a great deal of insight that is applicable to biologically based risk assessment. First, the DNA and hemoglobin adduct data strongly support the conclusion that 3,4-epoxy-1,2-butanediol is the major electrophile available for binding to these macromolecules. Biomarker studies have also provided insight into the possibility of a sensitive population associated with the GSTT1 null genotype. While it is clear that lymphocytes from GSTT1 null individuals are more sensitive for the induction of sister chromatid exchanges (SCE) following in vitro exposure to 1,2,3,4-diepoxybutane, there was no such increase in SCE or other biomarkers of genotoxicity in workers exposed to 1-3 p.p.m. butadiene, regardless of GST genotype. The globin adduct data also demonstrate that there is roughly a tenfold range for interindividual differences in the metabolism of butadiene. This type of analysis represents an excellent means for providing scientific data for this critical determinant. Another useful application of hemoglobin adducts in risk assessment was demonstrated by regressing data for various endpoints for genotoxicity against that individuals biologically effective dose, thereby providing an independent mechanism for evaluation that excludes any possible confounding by inappropriate controls. Finally, biomarker studies have identified critical gaps in our knowledge that are needed for the accurate assessment of butadiene. Most notable of these is the lack of diepoxide-specific biomarkers in mice, rats and humans.

DNA adducts: Effects of low exposure to ethylene oxide, vinyl chloride and butadiene

Dose-response relationships of genotoxic agents differ greatly depending on the agent and the endpoint being evaluated. Simple conclusions that genotoxic effects are linear cannot be applied universally. The shape of the molecular dose of DNA adducts varies from linear, to supralinear, to sublinear depending on metabolic activation and detoxication, and repair of individual types of DNA adducts. For mutagenesis and other genotoxicity endpoints, the dose-response reflects the molecular dose of each type of DNA adduct, cell proliferation, as well as endogenous factors that lead to mutagenesis such as the formation and repair of endogenous DNA adducts. These same factors are important when interpreting the shape of dose-response data for carcinogenesis of genotoxic agents, however, tumor background variability adds additional complexity. Endogenously formed DNA adducts may be identical to those formed by chemicals, as in the case of vinyl chloride and ethylene oxide, or they may be those associated with oxidative stress. Data presented in this paper demonstrate that the exogenous number of adducts induced by 5 days of exposure to 10 ppm vinyl chloride is only 2. 2-fold greater than that present as a steady-state amount in unexposed control rats. Similar data are shown for ethylene oxide. Extremely sensitive methods have been developed for measuring the molecular dose of genotoxins. These methods can detect DNA adducts as low as 1 per 10(9) to 10(10). However, in view of the high number of endogenous DNA adducts that are present in all cells, it is unlikely that causal relationships can be attributed to very low numbers of such DNA adducts. Effects of both exogenous and endogenous DNA adducts need to be factored into the interpretation of chemical exposures.

In vivo mutagenicity of ethylene oxide at the hprt locus in T-lymphocytes of B6C3F1 lacI transgenic mice following inhalation exposure

Ethylene oxide (EO) is a direct-acting alkylating agent with the potential to induce cytogenetic alterations, mutations, and cancer. In the present study, the in vivo mutagenicity of EO at the hypoxanthine guanine phosphoribosyltransferase (hprt) locus of T-lymphocytes was evaluated following inhalation exposure of male B6C3F1 lacI transgenic mice. For this purpose, groups of male Big Blue mice at 6-8 (n = 4/group) and 8-10 (n = 5/group) weeks of age were exposed to 0, 50, 100, or 200 ppm EO for 4 weeks (6 h/day, 5 days/week). At necropsy, T-cells were isolated from thymus and/or spleen and cultured in the presence of concanavalin A, IL-2, and 6-thioguanine [Skopek, T.R., V.E. Walker, J.E. Cochrane et al. (1992) Proc. Natl. Acad. Sci. USA, 89, 7866-7870]. The time course for expression of hprt-negative lymphocytes in thymus was determined in mice necropsied 2 h, 2 weeks, and 8 weeks after exposure to 200 ppm EO. The dose-response for hprt mutant T-cells in thymus and spleen was defined in mice necropsied 2 and 8 weeks post-exposure, respectively. The hprt mutant frequency (Mf) in thymus of exposed mice was increased 2 h after exposure and reached a maximum of 7.5 +/- 0.9 x 10(-6) (average Mf +/- SE) at 2 weeks post-exposure, compared with 2.3 +/- 0.8 x 10(-6) in thymus of control mice. Dose-related increases in hprt Mfs were found in thymus from mice exposed to 100 and 200 ppm EO. In addition, a nonlinear dose-dependent increase in hprt Mfs was observed in splenic T-cells, with greater mutagenic efficiency (mutations per unit dose) found at higher concentrations than at lower concentrations of EO. Average induced Mfs (i.e. induced Mf = treatment Mf - background Mf) in splenic T-cells were 1.6, 4.6, and 11.9 x 10(-6) following exposures to 50, 100, or 200 ppm EO, respectively, while the average control Mf value was 2.2 +/- 0.3 x 10(-6). In aliquots of lymphocytes (both B- and T-cells) isolated from spleen for analysis of lacI mutations in the same animals, only two of three EO-exposed mice at the 200 ppm exposure level demonstrated an elevated lacI Mf and these elevations were apparently due to the in vivo replication of preexisting mutants and not due to the induction of new mutations associated with EO exposure [Sisk, S., L.J. Pluta, K.G. Meyer and L. Recio (1996) Mutation Res., submitted]. These data demonstrate that repeated inhalation exposures to high concentrations of EO produce dose-related increases in mutations at the hprt locus of T-lymphocytes in male lacI transgenic mice of B6C3F1 origin.

Detection of cyclophosphamide-induced mutations at the Hprt but not the lacI locus in splenic lymphocytes of exposed mice.

The relative sensitivities and specificities of the endogenous Hprt gene and the lacI transgene as mutational targets were evaluated in splenic lymphocytes from male standard B6C3F1 mice (only Hprt assayed) and from lacI transgenic B6C3F1 mice treated at 6–7 weeks‐ of‐age with the indirect‐acting agent, cyclophosphamide (CP). To define the effects of the time elapsed since CP treatment on Hprt mutant frequencies (Mfs), nontransgenic mice were given single i.p. injections of 25 mg CP/kg or vehicle (PBS) alone and then necropsied 2, 4, 6, 8, or 10 weeks after treatment. Peak Mfs were found at 6 weeks postexposure, with mean Mf values ranging from 2.27 to 3.27 × 10–5 using two different lots of CP in standard packaging (compared with mean control Mf values of 0.14 to 0.26 × 10–5 in various experiments). To determine the dose response for Hprt Mfs, nontransgenic mice were given single doses of 0, 12.5, 25, 50, or 100 mg CP/kg and necropsied 4 weeks postexposure. These treatments produced a supralinear dose response curve for CP‐induced Hprt Mfs. Based on these experiments, CP mutagenicities at Hprt and lacI were compared in transgenic mice treated with 0, 25, or 100 mg CP/kg (using another lot of CP in ISOPAC® bottles; Sigma) and necropsied 6 weeks later. There was a significant increase in Hprt Mfs in treated transgenic mice (100 mg CP/kg: 0.75 ± 0.09 × 10–5; 25 mg CP/kg: 0.39 ± 0.05 × 10–5) versus controls (0.10 ± 0.01 × 10–5); however, the Mfs in lacI of lymphocytes from the same CP‐treated animals were not significantly different from controls (100 mg CP/kg: 9.4 ± 1.1 × 10–5; 25 mg CP/kg: 6.7 ± 0.8 × 10–5; control: 7.7 ± 0.7 × 10–5). Hprt mutational spectra data in CP‐treated transgenic and nontransgenic mice were different from those of control mice, whereas the spectra of mutations in lacI of lymphocytes from Big Blue® transgenic mice were not significantly changed after CP treatment. These data indicate that, under these treatment conditions, CP‐induced mutations in splenic lymphocytes were detectable in the Hprt gene but not the lacI transgene of this nontarget tissue for CP‐induced cancer. Environ. Mol. Mutagen. 34:167–181, 1999

Pyrimido[1,2-a]-purin-10(3H)-one, M1G, is less prone to artifact than base oxidation

Pyrimido[1,2-a]-purin-10(3H)-one (M1G) is a secondary DNA damage product arising from primary reactive oxygen species (ROS) damage to membrane lipids or deoxyribose. The present study investigated conditions that might lead to artifactual formation or loss of M1G during DNA isolation. The addition of antioxidants, DNA isolation at low temperature or non-phenol extraction methods had no statistically significant effect on the number of M1G adducts measured in either control or positive control tissue samples. The number of M1G adducts in nuclear DNA isolated from brain, liver, kidney, pancreas, lung and heart of control male rats were 0.8, 1.1, 1.1, 1.1, 1.8 and 4.2 M1G/108 nt, respectively. In rat liver tissue, the mitochondrial DNA contained a 2-fold greater number of M1G adducts compared with nuclear DNA. Overall, the results from this study demonstrated that measuring M1G is a reliable way to assess oxidative DNA damage because the number of M1G adducts is significantly affected by the amount of ROS production, but not by DNA isolation procedures. In addition, this study confirmed that the background number of M1G adducts reported in genomic DNA could have been overestimated by one to three orders of magnitude in previous reports.

Hemoglobin adducts in 1,3-butadiene exposed Czech workers: Female-male comparisons

We previously reported results of a molecular epidemiological study of female and male 1,3-butadiene (BD) exposed Czech workers showing that females appeared to absorb or metabolize less BD per unit exposure concentration than did males, based on metabolite concentrations in urine (Chem. Biol. Interact. 166 (2007) 63-77). However, that unexpected observation could not be verified at the time because the only additional BD metabolite measurement attempted was for 1,2,3,4-diepoxybutane (DEB) as reflected in specific N,N[2,3-dihydroxy-1,4-butyl]valine (pyr-Val) hemoglobin adduct concentrations, which were not quantifiable in any subject with the method then employed. Neither somatic gene mutations nor chromosome aberrations were associated with BD exposure levels in that study, consistent with findings in an earlier Czech study of males only. We have since measured production and accumulation of the 1,2-dihydroxy-3,4-epoxybutane (EBD) metabolite as reflected in N-[2,3,4-trihydroxy-butyl]valine (THB-Val) hemoglobin adduct concentrations. The mean THB-Val concentration was significantly higher in exposed males than in control males (922.3pmol/g and 275.5pmol/g, respectively), but exposed and control females did not differ significantly (224.5pmol/g and 181.1pmol/g, respectively). In both the control and exposed groups mean THB-Val concentrations were significantly higher for males than females. THB-Val concentrations were significantly correlated with mean 8-h TWA exposures for both males and females, but the rate of increase with increasing BD exposure was significantly lower for females. THB-Val concentrations also increased with increasing urine M2 metabolite [isomeric mixture of 1-hydroxy-2-{N-actylcysteinyl}-3-butene and 2-hydroxy-1-{N-acetylcysteinyl}-3-butene] concentrations in both sexes but the rate of increase was also lower in females than in males. There were no significant correlations between THB-Val concentrations and either somatic gene mutations or chromosome aberrations in either males or females. These results using another biomarker to measure a second metabolite of BD support the original conclusion that females absorb or metabolize less BD than males per unit exposure and indicate that the size of the difference increases with exposure. This observation in humans differs from findings in rodents where at prolonged exposures to high BD levels the females form higher amounts of hemoglobin adducts than do males, a difference that disappears at shorter duration lower exposure levels, while female susceptibility to BD induced mutations and tumorgenesis in rodents appears to persist at all BD exposure levels.

Mutagenicity at the Hprt locus in T cells of female mice following inhalation exposures to low levels of 1,3-butadiene.

A study was conducted to test the hypothesis that repeated low level exposures to 1,3-butadiene (BD), approaching the OSHA occupational threshold for this chemical, produce a significant mutagenic response in mice. Female B6C3F1 mice (4-5 weeks of age) were exposed by inhalation for 2 weeks (6 h/day, 5 days/week) to 0 or 3 ppm BD, and then necropsied at 4 weeks after the cessation of exposures to measure the frequency of mutations (MF) at the Hprt locus using the T-lymphocyte clonal assay. At necropsy, T cells were isolated from spleen and cultured in the presence of mitogen, growth factors, and a selection agent. Cells were scored for growth on days 8-9 after plating to determine cloning efficiencies (CEs) and Hprt MFs. There was a marginal but significant reduction in the growth of splenic T cells from mice exposed to 3 ppm (n=27) compared with control mice (n=24) (P=0.004), suggesting the occurrence of BD-induced cytotoxicity at this low exposure concentration. In addition, the average Hprt MF in mice exposed to 3 ppm BD [1.54+/-0.82 (S.D.)x10(-6)] was significantly increased by 1.6-fold over the average control value of 0.96+/-0.51 (S.D.)x10(-6) (P=0.004). Comparisons of these data to earlier Hprt mutagenicity studies of mice exposed to high concentrations of BD (where significant mutagenic but not cytotoxic effects were observed) indicate that the ability to detect the cytotoxic and mutagenic responses of T cells to low levels of BD was enhanced by using a much larger sample size than usual for both the control and treatment groups. Additional analyses of the quantitative relationships between CE and MF demonstrated that CE had no significant effect upon MF values in sham-exposed control mice or mice exposed to low-level BD. Furthermore, the approaches for assessing the impact of CE and clonality on Hprt MFs in these control and BD-exposed mice were applied with the same rigor as in in vivo Hprt mutagenicity studies in human children. The overall study results support the conclusion that short-term low-level BD exposure is mutagenic in the mouse.

Formation of Hydroxymethyl DNA Adducts in Rats Orally Exposed to Stable Isotope Labeled Methanol

Methanol is a large volume industrial chemical and widely used solvent and fuel additive. Methanols well known toxicity and use in a wide spectrum of applications has raised long-standing environmental issues over its safety, including its carcinogenicity. Methanol has not been listed as a carcinogen by any regulatory agency; however, there are debates about its carcinogenic potential. Formaldehyde, a metabolite of methanol, has been proposed to be responsible for the carcinogenesis of methanol. Formaldehyde is a known carcinogen and actively targets DNA and protein, causing diverse DNA and protein damage. However, formaldehyde-induced DNA adducts arising from the metabolism of methanol have not been reported previously, largely due to the absence of suitable DNA biomarkers and the inability to differentiate what was due to methanol compared with the substantial background of endogenous formaldehyde. Recently, we developed a unique approach combining highly sensitive liquid chromatography-mass spectrometry methods and exposure to stable isotope labeled chemicals to simultaneously quantify formaldehyde-specific endogenous and exogenous DNA adducts. In this study, rats were exposed daily to 500 or 2000 mg/kg [¹³CD₄]-methanol by gavage for 5 days. Our data demonstrate that labeled formaldehyde arising from [¹³CD₄]-methanol induced hydroxymethyl DNA adducts in multiple tissues in a dose-dependent manner. The results also demonstrated that the number of exogenous DNA adducts was lower than the number of endogenous hydroxymethyl DNA adducts in all tissues of rats administered 500 mg/kg per day for 5 days, a lethal dose to humans, even after incorporating an average factor of 4 for reduced metabolism due to isotope effects of deuterium-labeled methanol into account.