title: Measurement of the hemoglobin N-(2-oxoethyl)valine adduct in ethyl carbamate-treated mice.
abstract: Ethyl carbamate (EC) is bioactivated by CYP2E1 through vinyl carbamate to its epoxide, a reactive electrophile. This carcinogen reacts with macromolecules, including hemoglobin (Hb). This report defines a method to examine levels of N-(2-oxoethyl) adduct on the N-terminal valine of Hb after EC treatment at carcinogenic doses. Concentrations were determined 24 hr following an oral dose of EC (1 mg/g body wt) to strains A/J and C57BL/6 mice. Globin samples were isolated by precipitation in acidified acetone, washed, dried, and stored frozen at -20 degrees C until analyzed. Weighed aliquots were treated with sodium borohydride to reduce the aldehyde of the 2-oxoethyl group to the N-(2-hydroxyethyl) adduct. The adduct valine was cleaved using phenylisothiocyanate to form a substituted phenylthiohydantoin derivative of N-(2-hydroxyethyl)valine in a modified Edman degradation. After reaction with N,O-bis(trimethylsilyl)trifluoroacetamide, the resultant product, 1-(2'-trimethylsilyloxy)ethyl-5-isopropyl-3-phenyl-2-thiohydantoin , was quantified by GC/MS with selected ion monitoring of the molecular ion using synthetic N-(3-hydroxypropyl)valine as an internal standard. No adducts were detected without NaBH4 reduction. Strain A/J mice treated with EC (1 mg/g, N = 10) yielded mean +/- standard deviation (SD) adduct level values of 13.3 +/- 1.03 nmol/g globin; saline-treated A/J controls (N = 7) gave background levels of 4.43 +/- 0.69 nmol/g globin. Strain C57BL/6 mice treated with EC (1 mg/g, N = 6) exhibited mean +/- SD values of 12.0 +/- 1.92 nmol/g globin, while control mice of this strain (N = 4) had adduct levels of 7.23 +/- 1.19 nmol/g globin. These results are consistent with findings of others that bioactivation of EC produces N-(2-oxoethyl)valine hemoglobin adducts. Although the difference between mouse strains in mean total adduct levels following EC treatment was not significant, the differences evident in comparisons within strains due to treatment, between strains in endogenous background levels, and between strains in estimates of mean increases in adduct concentrations resulting from EC treatment were highly significant (p < 0.01). This assay provides a biomarker system for assessment of production from EC of the electrophilic metabolites which are believed to be genotoxic following metabolic activation in vivo.
authors.affiliation: Department of Pharmacology and Toxicology, School of Medicine, University of Louisville, Kentucky 40292.
authors.forename: J
authors.initials: J
authors.lastname: Cai
authors.forename: S R
authors.initials: SR
authors.lastname: Myers
authors.forename: H E
authors.initials: HE
authors.lastname: Hurst
mesh_terms: D000818:Animals; D001711:Biotransformation; D002138:Calibration; D002273:Carcinogens / Q000493:pharmacokinetics / Q000633:toxicity*; D008401:Gas Chromatography-Mass Spectrometry; D006454:Hemoglobins / Q000187:drug effects* / Q000378:metabolism*; D006868:Hydrolysis; D008297:Male; D051379:Mice; D008805:Mice, Inbred A; D008810:Mice, Inbred C57BL; D010084:Oxidation-Reduction; D010669:Phenylthiohydantoin / Q000031:analogs & derivatives / Q000032:analysis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014520:Urethane / Q000493:pharmacokinetics / Q000633:toxicity*; D014633:Valine / Q000032:analysis / Q000378:metabolism
chemical_list: C092376:1-(2'-trimethylsilyloxy)ethyl-5-isopropyl-3-phenyl-2-thiohydantoin; D002273:Carcinogens; D006454:Hemoglobins; D010669:Phenylthiohydantoin; D014520:Urethane; D014633:Valine