title: Analyses of (1-chloroethenyl)oxirane headspace and hemoglobin N-valine adducts in erythrocytes indicate selective detoxification of (1-chloroethenyl)oxirane enantiomers.
abstract: Chloroprene (2-chloro-1,3-butadiene, CAS 126-99-8, CP) is a colorless volatile liquid used in manufacture of polychloroprene, a synthetic rubber polymer. National Toxicology Program inhalation studies of CP in rats and mice gave clear evidence of carcinogenic activity. CP is metabolized by CYP2E1 to electrophilic epoxides, including R- and S-(1-chloroethenyl)oxirane (CEO), which form adducts with nucleic acids and other nucleophiles including glutathione and hemoglobin. As detection of these epoxide metabolites in vivo is technically challenging, measurements of CEO-Hb adducts may provide biomarkers of exposure to bioactivated metabolites of CP. The present studies involved exposure of C57BL/6 mouse erythrocytes (RBC) in vitro to pure enantiomers of CEO. Headspace analysis of CEO using Cyclodex-B capillary GC/MS with selected ion monitoring enabled separation, specific detection, and quantification of CEO enantiomers as reactions proceeded in vitro with RBC. These analyses indicated that R-CEO was much more persistent when incubated in vitro with RBC, while S-CEO disappeared rapidly. After periods of exposure of RBC to various concentrations of R- or S-CEO, erythrocytes were lysed and globin isolated. Covalent adducts, formed by reaction of CEO with N-terminal valine in Hb, were analyzed following Edman cleavage and trimethylsilylation. SIM-GC/MS analyses using a 5%-phenyl-dimethylsiloxane capillary column enabled quantification of CEO-Hb adducts. These analyses produced two chromatographic peaks of CEO-valine adduct derivatives, which were tentatively identified from mass spectra, reaction, and abundance data to be 1-(3-chloro-2-trimethylsilyloxybut-3-en-1-yl)-5-isopropyl-3-phenyl-2-thiohydantoin and 1-[2-chloro-1-(trimethylsilyloxymethyl)prop-2-en-1-yl]-5-isopropyl-3-phenyl-2-thiohydantoin. Analyses quantified significantly greater levels of adducts formed from R-CEO than from S-CEO. Studies involving pretreatment of RBC with glutathione-depleting diethyl maleate diminished the selective detoxification of S-CEO, and suggest enantiomeric selectivity of mouse glutathione-S-transferase as a mechanism of differential detoxification of CEO enantiomers. These results indicate more rapid detoxification of S-CEO by mouse RBC in vitro, while R-CEO may persist to react with cellular nucleophiles.
authors.affiliation: Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, KY 40292, USA. h.hurst@louisville.edu
authors.forename: Harrell E
authors.initials: HE
authors.lastname: Hurst
authors.forename: Md Yeakub
authors.initials: MY
authors.lastname: Ali
mesh_terms: D000818:Animals; D002849:Chromatography, Gas; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism*; D005027:Ethylene Oxide / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification / Q000633:toxicity; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006454:Hemoglobins / Q000032:analysis*; D008658:Inactivation, Metabolic; D007700:Kinetics; D013058:Mass Spectrometry; D051379:Mice; D008810:Mice, Inbred C57BL; D010084:Oxidation-Reduction; D013237:Stereoisomerism; D014633:Valine / Q000032:analysis*; D014835:Volatilization / Q000187:drug effects
chemical_list: C439367:(1-chloroethenyl)oxirane; D006454:Hemoglobins; D005982:Glutathione Transferase; D005978:Glutathione; D014633:Valine; D005027:Ethylene Oxide