Mortality Patterns Among Industrial Workers Exposed to Chloroprene and Other Substances: Extended Follow-Up.

OBJECTIVES: To update the U.S. portion of an historical cohort mortality study of workers with potential exposure to chloroprene (CD) and vinyl chloride (VC) with focus on lung and liver cancer. METHODS: Subjects were 6864 workers from two sites with vital status determined through 2017 for 99% of subjects and cause of death for 97.2% of deaths. Historical exposures to CD and VC were estimated quantitatively. We performed external and internal mortality comparisons. RESULTS: External comparisons revealed mostly deficits in deaths; internal comparisons revealed no consistent evidence of exposure-response relationships with CD or VC. CONCLUSIONS: Our update continues to support the conclusion that the risk of death from lung or liver cancer is unrelated to exposure to CD or VC at levels experienced by workers in the two U.S. sites.

Incorporation of in vitro metabolism data and physiologically based pharmacokinetic modeling in a risk assessment for chloroprene.

Objective: To develop a physiologically based pharmacokinetic (PBPK) model for chloroprene in the mouse, rat and human, relying only on in vitro data to estimate tissue metabolism rates and partitioning, and to apply the model to calculate an inhalation unit risk (IUR) for chloroprene.Materials and methods: Female B6C3F1 mice were the most sensitive species/gender for lung tumors in the 2-year bioassay conducted with chloroprene. The PBPK model included tissue metabolism rate constants for chloroprene estimated from results of in vitro gas uptake studies using liver and lung microsomes. To assess the validity of the PBPK model, a 6-hr, nose-only chloroprene inhalation study was conducted with female B6C3F1 mice in which both chloroprene blood concentrations and ventilation rates were measured. The PBPK model was then used to predict dose measures - amounts of chloroprene metabolized in lungs per unit time - in mice and humans.Results: The mouse PBPK model accurately predicted in vivo pharmacokinetic data from the 6-hr, nose-only chloroprene inhalation study. The PBPK model was used to conduct a cancer risk assessment based on metabolism of chloroprene to reactive epoxides in the lung, the target tissue in mice. The IUR was over100-fold lower than the IUR from the EPA Integrated Risk Information System (IRIS), which was based on inhaled chloroprene concentration. The different result from the PBPK model risk assessment arises from use of the more relevant tissue dose metric, amount metabolized, rather than inhaled concentrationDiscussion and conclusions: The revised chloroprene PBPK model is based on the best available science, including new test animal in vivo validation, updated literature review and a Markov-Chain Monte Carlo analysis to assess parameter uncertainty. Relying on both mouse and human metabolism data also provides an important advancement in the use of quantitative in vitro to in vivo extrapolation (QIVIVE). Inclusion of the best available science is especially important when deriving a toxicity value based on species extrapolation for the potential carcinogenicity of a reactive metabolite.

Regression analysis of current status data in the presence of a cured subgroup and dependent censoring.

This paper discusses regression analysis of current status data, a type of failure time data where each study subject is observed only once, in the presence of dependent censoring. Furthermore, there may exist a cured subgroup, meaning that a proportion of study subjects are not susceptible to the failure event of interest. For the problem, we develop a sieve maximum likelihood estimation approach with the use of latent variables and Bernstein polynomials. For the determination of the proposed estimators, an EM algorithm is developed and the asymptotic properties of the estimators are established. Extensive simulation studies are conducted and indicate that the proposed method works well for practical situations. A motivating application from a tumorigenicity experiment is also provided.

Weighted gene co-expression network analysis of pneumocytes under exposure to a carcinogenic dose of chloroprene.

AIMS: Occupational exposure to chloroprene via inhalation may lead to acute toxicity and chronic pulmonary diseases, including lung cancer. Currently, most research is focused on epidemiological studies of chloroprene production workers. The specific molecular mechanism of carcinogenesis by chloroprene in lung tissues still remains obscure, and specific candidate therapeutic targets for lung cancer are lacking. The present study identifies specific gene modules and valuable hubs associated with lung cancer. MAIN METHODS: We downloaded the dataset GSE40795 from the Gene Expression Omnibus (GEO) and divided the dataset into the non-carcinogenic dose chloroprene exposed mice group and the carcinogenic dose chloroprene exposed mice group. With a systemic biological view, we discovered significantly altered gene modules between the two groups and identified hub genes in the carcinogenic dose exposed group using weighted co-expression network analysis (WGCNA). KEY FINDINGS: A total of 2434 differentially expressed genes were identified. Twelve gene modules with multiple biological activities were related to the carcinogenesis of chloroprene in lung tissue. Seven hub genes that were critical for the carcinogenesis of chloroprene in lung tissue were ultimately identified, including Cftr, Hip1, Tbl1x, Ephx1, Cbr3, Antxr2 and Ccnd2. They were implicated in inflammatory response, cell transformation, gene transcription regulation, phase II detoxification, angiogenesis, cell adhesion, motility and the cell cycle. SIGNIFICANCE: The seven hub genes may become valuable candidates for risk assessment biomarkers and therapeutic targets in lung cancer.

A constrained maximum likelihood approach to evaluate the impact of dose metric on cancer risk assessment: application to ß-chloroprene.

ß-Chloroprene (2-chloro-1,3-butadiene, CD) is used in the manufacture of polychloroprene rubber. Chronic inhalation studies have demonstrated that CD is carcinogenic in B6C3F1 mice and Fischer 344 rats. However, epidemiological studies do not provide compelling evidence for an increased risk of mortality from total cancers of the lung. Differences between the responses observed in animals and humans may be related to differences in toxicokinetics, the metabolism and detoxification of potentially active metabolites, as well as species differences in sensitivity. The purpose of this study was to develop and apply a novel method that combines the results from available physiologically based kinetic (PBK) models for chloroprene with a statistical maximum likelihood approach to test commonality of low-dose risk across species. This method allows for the combined evaluation of human and animal cancer study results to evaluate the difference between predicted risks using both external and internal dose metrics. The method applied to mouse and human CD data supports the hypothesis that a PBK-based metric reconciles the differences in mouse and human low-dose risk estimates and further suggests that, after PBK metric exposure adjustment, humans are equally or less sensitive than mice to low levels of CD exposure.

Cross-species transcriptomic analysis of mouse and rat lung exposed to chloroprene.

ß-Chloroprene (2-chloro-1,3-butadiene), a monomer used in the production of neoprene elastomers, is of regulatory interest due to the production of multiorgan tumors in mouse and rat cancer bioassays. A significant increase in female mouse lung tumors was observed at the lowest exposure concentration of 12.8 ppm, whereas a small, but not statistically significant increase was observed in female rats only at the highest exposure concentration of 80 ppm. The metabolism of chloroprene results in the generation of reactive epoxides, and the rate of overall chloroprene metabolism is highly species dependent. To identify potential key events in the mode of action of chloroprene lung tumorigenesis, dose-response and time-course gene expression microarray measurements were made in the lungs of female mice and female rats. The gene expression changes were analyzed using both a traditional ANOVA approach followed by pathway enrichment analysis and a pathway-based benchmark dose (BMD) analysis approach. Pathways related to glutathione biosynthesis and metabolism were the primary pathways consistent with cross-species differences in tumor incidence. Transcriptional BMD values for the pathway were more similar to differences in tumor response than were estimated target tissue dose surrogates based on the total amount of chloroprene metabolized per unit mass of lung tissue per day. The closer correspondence of the transcriptional changes with the tumor response is likely due to their reflection of the overall balance between metabolic activation and detoxication reactions, whereas the current tissue dose surrogate reflects only oxidative metabolism.

A frailty model approach for regression analysis of multivariate current status data.

This paper discusses regression analysis of multivariate current status failure time data (The Statistical Analysis of Interval-censoring Failure Time Data. Springer: New York, 2006), which occur quite often in, for example, tumorigenicity experiments and epidemiologic investigations of the natural history of a disease. For the problem, several marginal approaches have been proposed that model each failure time of interest individually (Biometrics 2000; 56:940-943; Statist. Med. 2002; 21:3715-3726). In this paper, we present a full likelihood approach based on the proportional hazards frailty model. For estimation, an Expectation Maximization (EM) algorithm is developed and simulation studies suggest that the presented approach performs well for practical situations. The approach is applied to a set of bivariate current status data arising from a tumorigenicity experiment.

Epidemiologic evidence for chloroprene carcinogenicity: review of study quality and its application to risk assessment.

This article evaluates the quality and weight of evidence associated with epidemiologic studies of cancer among occupational cohorts exposed to chloroprene. The focus is on liver, lung, and lymphohematopoietic cancers, which had been increased in early studies. Literature searches identified eight morbidity/mortality studies covering seven chloroprene-exposed cohorts from six countries. These studies were summarized and their quality was assessed using the 10 criteria suggested by the U.S. Environmental Protection Agency. The limitations within this literature (primarily the early studies) included crude exposure assessment, incomplete follow-up, uncertain baseline rates, and uncontrolled confounding by factors such as smoking, drinking, and co-exposure to benzene and vinyl chloride. Four cohorts were studied by the same group of investigators, who reported no overall increased associations for any cancers. This four-cohort study was by far the most rigorous, having the most comprehensive exposure assessment and follow-up and the most detailed documentation. This study also contained the two largest cohorts, including an American cohort from Louisville, Kentucky, that ranked at or near the top for each of the 10 quality criteria. There was evidence of a strong healthy worker effect in the four-cohort study, which could have hidden small excess risks. Small increased risks were suggested by internal or company-specific analyses, but these were most likely caused by uncontrolled confounding and low baseline rates. Overall, the weight of evidence does not support any substantial link between chloroprene exposure and cancer, but inconsistencies and a lack of control for major confounders preclude drawing firmer conclusions.

Regression analysis of multivariate interval-censored failure time data with application to tumorigenicity experiments.

This paper discusses multivariate interval-censored failure time data that occur when there exist several correlated survival times of interest and only interval-censored data are available for each survival time. Such data occur in many fields. One is tumorigenicity experiments, which usually concern different types of tumors, tumors occurring in different locations of animals, or together. For regression analysis of such data, we develop a marginal inference approach using the additive hazards model and apply it to a set of bivariate interval-censored data arising from a tumorigenicity experiment. Simulation studies are conducted for the evaluation of the presented approach and suggest that the approach performs well for practical situations.

Efficient estimation for the proportional hazards model with bivariate current status data.

We consider efficient estimation of regression and association parameters jointly for bivariate current status data with the marginal proportional hazards model. Current status data occur in many fields including demographical studies and tumorigenicity experiments and several approaches have been proposed for regression analysis of univariate current status data. We discuss bivariate current status data and propose an efficient score estimation approach for the problem. In the approach, the copula model is used for joint survival function with the survival times assumed to follow the proportional hazards model marginally. Simulation studies are performed to evaluate the proposed estimates and suggest that the approach works well in practical situations. A real life data application is provided for illustration.

International Symposium on the Evaluation of Butadiene and Chloroprene Health Risks.

These proceedings represent nearly all the platform and poster presentations given during the International Symposium on Evaluation of Butadiene and Chloroprene Health Risks, held in Charleston, South Carolina, USA, on September 20-22, 2005. The Symposium was attended by 78 participants representing private industry (37), academia (21), government (11), not-for-profit organizations (5), and consulting (4). The program followed the format of previous symposia on butadiene, chloroprene, and isoprene in London UK (2000) and butadiene and isoprene in Blaine, Washington USA (1995). This format enabled the exchange of significant new scientific results and discussion of future research needs. Isoprene was not evaluated during the 2005 Symposium because of lack of new data. For background information, the reader is referred to the proceedings of the London 2000 meeting for a thorough historical perspective and overview of scientific and regulatory issues concerning butadiene, chloroprene, and isoprene [Chem.-Biol. Interact. (2001) 135-136:1-7]. The Symposium consisted of seven sessions: (1) Introduction and Opening Remarks, (2) Butadiene/styrene-butadiene rubber (SBR)--Process Overview, Exposure and Health Effects/Human Studies; (3) Chloroprene--Process Overview, Exposure and Health Effects/Human Studies; (4) Mode of Action/Key Events; (5) Risk Assessment; (6) Poster Presentations; and (7) Panel Discussion and Future Directions. The Symposium concluded with a discussion by all participants of issues that arose throughout the course of the Symposium. The Proceedings of the Symposium published in this Special Issue are organized according to the Sessions outlined above. The purpose of this foreword is to summarize the presentations and their key findings and recommend future research directions for each chemical.

Chloroprene: overview of studies under consideration for the development of an IRIS assessment.

Beta-chloroprene (C(4)H(5)Cl, chloroprene, 2-chloro-1,3-butadiene, CASRN 126-99-8) is a volatile, flammable liquid monomer utilized primarily in the manufacture of neoprene (polychloroprene) elastomer used in belts, hoses, gloves, wire coatings, and tubing. Absorption into the body occurs primarily via the respiratory system and may occur via the gastrointestinal tract or the skin. Once absorbed, chloroprene is widely distributed as evidenced by effects in several target organs including nose and lung, liver, and skin. Chloroprene metabolism is believed to include cytochrome P450 oxidation to a monoepoxide, hydrolysis by epoxide hydrolases, and glutathione conjugation. Similar to 1,3-butadiene, the epoxide is considered to be the toxic moiety, and species differences in metabolic capacity may influence the severity of effects as well as what tissues are affected. EPA has not previously developed an assessment of chloroprene's potential for human health effects. Existing human epidemiological studies offer little data on noncancer effects, and the associations of exposure with increased cancer (liver and lung) mortality reported are inconclusive. Recent epidemiological studies (submitted for publication) could offer information that may impact chloroprene's health assessment. Multiple-site tumors have been reported in rats and mice exposed to chloroprene by inhalation; nevertheless, there are marked differences in strain sensitivities (i.e., tumors in F344 rats versus no tumors in Wistar rats). Recently developed physiologically based toxicokinetic models may allow for the resolution of species and tissue differences and sensitivities as well as exposure-dose-response relationships relevant to humans. (This presentation does not necessarily reflect EPA policy.).

Evaluation of genetic alterations in cancer-related genes in lung and brain tumors from B6C3F1 mice exposed to 1,3-butadiene or chloroprene.

1,3-Butadiene and chloroprene are multisite carcinogens in B6C3F1 mice with the strongest tumor response being the induction of lung neoplasms in females. Incidence of brain tumors in mice exposed to 1,3-butadiene was equivocal. This article reviews the efforts of our laboratory and others to uncover the mechanisms of butadiene and chloroprene induced lung and brain tumor responses in the B6C3F1 mouse. The formation of lung tumors by these chemicals involved mutations in the K-ras cancer gene and loss of heterozygosity in the region of K-ras on distal chromosome 6, while alterations in p53 and p16 were implicated in brain tumorigenesis.

The metabolism and molecular toxicology of chloroprene.

Chloroprene (2-chloro-1,3-butadiene, 1) is oxidised by cytochrome P450 enzymes in mammalian liver microsomes to several metabolites, some of which are reactive towards DNA and are mutagenic. Much less of the metabolite (1-chloroethenyl)oxirane (2a/2b) was formed by human liver microsomes compared with microsomes from Sprague-Dawley rats and B6C3F1 mice. Epoxide (2a/2b) was a substrate for mammalian microsomal epoxide hydrolases, which showed preferential hydrolysis of the (S)-enantiomer (2b). The metabolite 2-chloro-2-ethenyloxirane (3a/3b) was rapidly hydrolysed to 1-hydroxybut-3-en-2-one (4) and in competing processes rearranged to 1-chlorobut-3-en-2-one (5) and 2-chlorobut-3-en-1-al (6). The latter compound isomerised to (Z)-2-chlorobut-2-en-1-al (7). In microsomal preparations from human, rat and mouse liver, compounds 4, 5 and 7 were conjugated by glutathione both in the absence and presence of glutathione transferases. There was no evidence for the formation of a chloroprene diepoxide metabolite in any of the microsomal systems. The major adducts from the reaction of (1-chloroethenyl)oxirane (2a/2b) with calf thymus DNA were identified as N7-(3-chloro-2-hydroxy-3-buten-1-yl)-guanine (20) and N3-(3-chloro-2-hydroxy-3-buten-1-yl)-2'-deoxyuridine (23), with the latter being derived by alkylation at N-3 of 2'-deoxycytidine, followed by deamination. Adducts in DNA were identified by comparison with those derived from individual deoxyribonucleosides. The metabolite (Z)-2-chlorobut-2-en-1-al (7) formed principally two adducts with 2'-deoxyadenosine which were identified as a pair of diastereoisomers of 3-(2'-deoxy-beta-d-ribofuranosyl)-7-(1-hydroxyethyl)-3H-imidazo[2,1-i]purine (25). The chlorine atom of chloroprene thus leads to different intoxication and detoxication profiles compared with those for butadiene and isoprene. The results infer that in vivo oxidations of chloroprene catalysed by cytochrome P450 are more important in rodents, whereas hydrolytic processes catalysed by epoxide hydrolases are more pronounced in humans. The reactivity of chloroprene metabolites towards DNA is important for the toxicology of chloroprene, especially when detoxication is incomplete.

Toxicology of 1,3-butadiene, chloroprene, and isoprene.

The diene monomers, 1,3-butadiene, chloroprene, and isoprene, respectively, differ only in substitution of a hydrogen, a chlorine, or a methyl group at the second of the four unsaturated carbon atoms in these linear molecules. Literature reviewed in the preceding sections indicates that these chemicals have important uses in synthesis of polymers, which offer significant benefits within modern society. Additionally, studies document that these monomers can increase the tumor formation rate in various organs of rats and mice during chronic cancer bioassays. The extent of tumor formation versus animal exposure to these monomers varies significantly across species, as well among strains within species. These studies approach, but do not resolve, important questions of human risk from inhalation exposure. Each of these diene monomers can be activated to electrophilic epoxide metabolites through microsomal oxidation reactions in mammals. These epoxide metabolites are genotoxic through reactions with nucleic acids. Some of these reactions cause mutations and subsequent cancers, as noted in animal experiments. Significant differences exist among the compounds, particularly in the extent of formation of highly mutagenic diepoxide metabolites, when animals are exposed. These metabolites are detoxified through hydrolysis by epoxide hydrolase enzymes and through conjugation with glutathione with the aid of glutathione S-transferase. Different strains and species perform these reactions with varying efficacy. Mice produce these electrophilic epoxides more rapidly and appear to have less adequate detoxification mechanisms than rats or humans. The weight of evidence from many studies suggests that the balance of activation versus detoxification offers explanation of differing sensitivities of animals to these carcinogenic actions. Other aspects, including molecular biology of the many processes that lead through specific mutations to cancer, are yet to be understood. Melnick and Sills (2001) compared the carcinogenic potentials of these three dienes, along with that of ethylene oxide, which also acts through an epoxide intermediate. From the number of tissue sites where experimental animal tumors were detected, butadiene offers greatest potential for carcinogenicity of these dienes. Chloroprene and then isoprene appear to follow in this order. Comparisons among these chemicals based on responses to external exposures are complicated by differences among studies and of species and tissue susceptibilities. Physiologically based pharmacokinetic models offer promise to overcome these impediments to interpretation. Mechanistic studies at the molecular level offer promise for understanding the relationships among electrophilic metabolites and vital genetic components. Significant improvements in minimization of industrial worker exposures to carcinogenic chemicals have been accomplished after realization that vinyl chloride caused hepatic angiosarcoma in polymer production workers (Creech and Johnson 1974; Falk et al. 1974). Efforts continue to minimize disease, particularly cancer, from exposures to chemicals such as these dienes. Industry has responded to significant challenges that affect the health of workers through efforts that minimize plant exposures and by sponsorship of research, including animal and epidemiological studies. Governmental agencies provide oversight and have developed facilities that accomplish studies of continuing scientific excellence. These entities grapple with differences in perspective, objectives, and interpretation as synthesis of knowledge develops through mutual work. A major challenge remains, however, in assessment of significance of environmental human exposures to these dienes. Such exposure levels are orders of magnitude less than exposures studied in experimental or epidemiological settings, but exposures may persist much longer and may involve unknown but potentially significant sensitivities in the general population. New paradigms likely will be needed for toxicological evaluation of these human exposures, which are ongoing but as yet are not interpreted.

Spatial and temporal trend evaluation of ambient concentrations of 1,3-butadiene and chloroprene in Texas.

This paper provides information on 1,3-butadiene (BD) and chloroprene as atmospheric pollutants in Texas and reviews available emission estimates and monitoring data. Ambient BD concentrations in most areas of Texas are predominantly influenced by on-road and off-road vehicular emissions or biomass burning, since BD is a product of combustion. However, large industrial point sources of BD emissions in Texas locally influence ambient concentrations. Total industrial BD emissions to the atmosphere in Texas for 2003 were estimated at 695 tonnes per year (TPY), approximately 70% of the total reported national industrial BD air emissions. Since 1998, there have not been any large industrial sources of chloroprene emissions in Texas, and total industrial chloroprene emissions for 2003 was estimated at only 0.09 TPY. Chloroprene was never detected at air monitoring sites. In 2003, the Texas Commission on Environmental Quality (TCEQ) monitored BD ambient air concentrations at 57 sites, some of which have been operational since 1992. These air monitors provide information on ambient BD concentrations in Texas and allow spatial and temporal trend evaluation. In 2003, annual average concentrations at monitoring sites in Texas ranged from less than the reporting limit of 0.01 to 3.2 parts per billion by volume (ppbv) with an overall average of 0.2 ppbv. This overall average is reduced to 0.1 ppbv if BD data from monitoring sites in Port Neches and Milby Park in Houston, which are located downwind of significant point sources of BD, are excluded. Ambient air monitoring has been conducted in Port Neches and in Milby Park in Houston since 1996 and 1999, respectively. At the Port Neches monitor, trend evaluation indicates that ambient concentrations of BD have declined since 1996 due to cooperative agreements with industries emitting BD. Annual average BD concentrations at the Port Neches monitor decreased from 8.3ppbv in 1996 to 1.3 ppbv in 2003, giving an 8-year average of 3.8 ppbv. Annual average BD concentrations at the Milby Park monitor varied between 2.1 and 4.4 ppbv from 1999 through 2003, giving a 5-year average of 3.1 ppbv. The results of cancer cluster studies based on Cancer Registry 1995-2001 incidence data and 1993-2002 mortality data conducted by the Texas Department of State Health Services for zip codes 77017/77012 (Houston) and 77651 (Port Neches) will be presented.

Reproductive and developmental toxicity of inhaled 2,3-dichloro-1,3-butadiene in rats.

Inhalation developmental and reproductive toxicity studies were conducted with 2,3-dichloro-1,3-butadiene (DCBD), a monomer used in the production of synthetic rubber. In the reproductive toxicity study, Crl:CD(SD)IGS BR rats (24/sex/group) were exposed whole body by inhalation to 0, 1, 5, or 50 ppm DCBD (6 h/day) for approximately 10-11 weeks total, through premating (8 weeks; 5 days/week), cohabitation of mating pairs (up to 2 weeks, 7 days/week), post-cohabitation for males (approximately 7 days) and from conception to implantation (gestation days 0-7 [GD 0-7]), followed by a recovery period (GD 8-21) for presumed pregnant females. Estrous cyclicity was evaluated during premating (last 3 weeks) and cohabitation. Reproductive organs and potential target organs, sperm parameters, and GD 21 fetuses (viability, weight, external alterations) were evaluated. In the developmental study, pregnant Crl:CD(SD)IGS BR rats (22/group) were exposed whole body by inhalation to 0, 1, 10, or 50 ppm DCBD (6 h/day) on GD 6-20; dams were necropsied on GD 21 (gross post-mortem only) and fetuses were evaluated (viability, weight, and external, visceral and skeletal exams). During the in-life portion of the studies, body weight, food consumption, and clinical observation data were collected. At 50 ppm, gasping and labored breathing occurred in both studies during the first few exposures; body weight and food consumption parameters were affected in parental animals from both studies, but were more severely affected in the developmental study. Fetal weight was decreased in the developmental study at 50 ppm. Degeneration of the nasal olfactory epithelium was observed in the reproduction study at 50 ppm. There were no effects on reproductive function, embryo-fetal viability, or increases in fetal structural alterations in either study. The no-observed-adverse-effect level (NOAEL) for reproductive toxicity was 50 ppm. The NOAEL for systemic toxicity in the reproduction study was 5 ppm based on adverse effects on body weight and food consumption parameters and nasal olfactory epithelial toxicity at 50 ppm in parental rats. The NOAEL for maternal and developmental toxicity was 10 ppm based on reduced maternal weight gain and food consumption and reduced fetal weight at 50 ppm in the developmental toxicity study.

A new role for glutathione: protection of vitamin B12 from depletion by xenobiotics.

NADPH in microsomes reduces the hydroxocob(III)alamin form of vitamin B12 to cob(II)alamin and the supernucleophilic cob(I)alamin, which are both highly reactive toward xenobiotic epoxides formed by mammalian metabolism of dienes such as the industrially important chemicals chloroprene and 1,3-butadiene. With styrene, the metabolically formed styrene oxide is reactive toward cob(I)alamin but not cob(II)alamin. Such reactions in humans could lead to vitamin B12 deficiency, which is implicated in pernicious anemia, cancer, and degenerative diseases. However, glutathione inhibits the reduction of hydroxocob(III)alamin by formation of the 1:1 complex glutathionylcobalamin. This blocks reactions of the cobalamins with metabolically formed epoxides. The interaction between glutathione and vitamin B12 could protect against diseases related to vitamin B12 depletion.