1. Analyses of (1-chloroethenyl)oxirane headspace and hemoglobin N-valine adducts in erythrocytes indicate selective detoxification of (1-chloroethenyl)oxirane enantiomers
Harrell E Hurst, Md Yeakub Ali Chem Biol Interact. 2007 Mar 20;166(1-3):332-40. doi: 10.1016/j.cbi.2006.04.016. Epub 2006 May 2.
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.
2. Identification and quantitation of N-(carboxymethyl)valine adduct in hemoglobin by gas chromatography/mass spectrometry
J Cai, H E Hurst J Mass Spectrom. 1999 May;34(5):537-43. doi: 10.1002/(SICI)1096-9888(199905)34:53.0.CO;2-H.
A sensitive, specific and reproducible method was developed for the quantitation of the hemoglobin (Hb) adduct N-(carboxymethyl)valine (CMV). This adduct is one of various products from the Maillard reaction, involving reducing sugars and amino acids, proteins or other molecules with a free amino group. Such adducts, including N epsilon-(carboxymethyl)lysine (CML), are called advanced glycation end products (AGE) and have been correlated with aging and severity of diabetes in human tissues. This method was developed to examine the CMV-Hb adduct as a possible AGE formed by reaction of Hb with glucose or other oxidation products. CMV was cleaved selectively from isolated globin using pentafluorophenyl isothiocyanate (PFPITC) in a modified Edman degradation at pH 9.5. The carboxyl group of the adduct was derivatized to its methyl ester with diazomethane. The resulting derivative, 5-isopropyl-1-(methyl acetate)-3-pentafluorophenyl-2-thiohydantoin, was detected by gas chromatography/mass spectrometry with selected ion monitoring (GC/SIM/MS). Quantitation was based on the response factor of the derivative molecular ion (m/z 396) from synthesized CMV and N-(2-carboxyethyl)valine (molecular ion m/z 410) as internal standard. This method exhibits reproducibility and linearity in the range 0.2-100 ng CMV. The limit of quantitation (0.2 ng CMV) gave a signal-to-noise ratio greater than 5:1 using a 1:30 sample aliquot. The GC/SIM/MS method can detect CMV adduct in 5 mg globin samples with relative standard deviations less than 5%. This approach avoids tedious acid hydrolysis and interference from other amino acids. The molecular ion and other CMV derivative ion assignments from samples were confirmed by accurate mass determinations using GC/high resolution SIM/MS. Measurements from random mouse, rat and human globin samples gave mean CMV levels of about 6, 5 and 14 nmol g-1 Hb in these species, respectively.
3. 1,3-butadiene: cancer, mutations, and adducts. Part V: Hemoglobin adducts as biomarkers of 1,3-butadiene exposure and metabolism
J A Swenberg, N I Christova-Gueorguieva, P B Upton, A Ranasinghe, N Scheller, K Y Wu, T Y Yen, R Hayes Res Rep Health Eff Inst. 2000 Mar;(92):191-210; discussion 211-9.
1,3-Butadiene (BD) is an important chemical used largely in the manufacture of synthetic rubber and thermoplastic resins. In addition, it has been identified in cigarette smoke, automobile exhaust, and gasoline vapor. The objective of this research was to develop highly sensitive and specific assays for the detection and quantitation of hemoglobin adducts of three BD metabolites: 1,2-epoxy-3-butene (BDO), 1,2,3,4-diepoxybutane (BDO2), and 1,2-dihydroxy-3,4-epoxybutane (BDO-diol). We have successfully developed an assay for both N-(2-hydroxy-3-butenyl)valine (HBVal) and N-(2,3,4-trihydroxybutyl)valine (THBVal) in hemoglobin. The six adducts measured were the two diastereomers (isomers I and II) of HBVal and the four diastereomers of THBVal (isomers I through IV, which were eluted as three peaks, 1, 2, and 3). HBVal and THBVal were measured in control and exposed B6C3F1 mice and Sprague-Dawley rats (1,000 ppm BD for 13 weeks at 6 hours/day, 5 days/week).
