Effect of CYP2E1 genotype on acrylonitrile hemoglobin adducts

Abstract

Acrylonitrile (ACN) is metabolized by two pathways: conjugation with glutathione, and oxidation to cyanoethylene oxide catalyzed by CYP2El. Measurement of ACN metabolites indicated an absence of oxidation to cyanoethylene oxide in CYP2El-null mice [Sumner et al., Chem. Res. Toxicol. 12: 1110-1116, (1999)]. Hemoglobin adducts provide a long-term measure of the internal dose of a reactive chemical. ACN reacts with hemoglobin to form a number of adducts, including N-(2-cyanoethyl)valine (CEVal). We have developed a new method for analysis of CEVal using LC-MS/MS. After a modified Edman degradation with phenylisothiocyanate as derivatizing agent, the resulting adducted valine phenylrhiohydantoin derivative was isolated by solid phase extraction, and chromatographed by reverse phase HPLC. Elution was monitored by selected reaction monitoring on a PE Sciex API 3000 triple quadrupole mass spectrometer. The method can measure adducts from natural abundance and isotope labeled ACN. To investigate the impact of the CYP2El genotype on ACN internal dose, [l, 2, 3-uC] ACN (20 mg/kg) was administered to wild type and CYP2El-null mice, and blood was collected for CEVal analysis. In the wild type mice, CEVal levels were approximately 3 fold lower than in the CYP2El-null mice (1487 ± 728 fmol/mg globin vs. 4863 ± 911 fmol/mg globin). These data indicate that, in the absence of CYP2El and the capability to oxidize ACN, the internal dose of acrylonitrile in the CYP2El-null mice increased to greater than 3 times the levels in wild type mice.