Induction of rat liver microsomal epoxide hydrolase by thiazole and pyrazine: hydrolysis of 2-cyanoethylene oxide

Liver microsomal epoxide hydrolase (mEH) is active in the detoxificationof epoxide-containing carcinogens. The effects of thiazole andpyrazine, constituents of tobacco and tobacco smoke as wellas of a variety of foods, on the expression and regulation ofmEH were examined in rats (200 mg/kg body wt/day, i.p., 1/emdash3 days). Immunoblot analyses using rabbit anti-rat mEH antibodyrevealed a significant increase in mEH levels in hepatic microsomesisolated from either thiazole- or pyrazine-treated animals.Another protein (43 kd) cross-reacting with polyclonal mEH antibodywas found to be increased concomitantly following pyrazine treatment.Northern and slot blot analyses showed substantial increasesin mEH mRNA following either thiazole or pyrazine treatment.The level of mEH mRNA increased 17-fold at 24 h following thiazoletreatment, relative to control. Approximately 20- and 16-foldincreases in mEH mRNA were also observed at 48 and 72 h respectivelyfollowing treatment with pyrazine. The level of polymerase chainreaction (PCR)-amplified mEH DNA derived from poly(A)+ RNA wasclearly elevated following either thiazole or pyrazine treatmentrelative to that from untreated animals. Both sense and antisensestrands of PCR-amplified mEH DNA were cloned into an M13mpl9phage vector in order to examine the nucleotide sequences ofPCR-amplified mEH DNA derived from the poly(A)+ RNA isolatedfrom thiazole- or pyrazine-treated animals. Sequence analysesrevealed that the sequence of PCR-amplified DNA from the inducedmRNA was identical to that published for mEH cDNA. Epoxide hydrolaseactivity toward the hydrolysis of 2-cyanoethylene oxide (CEO),the epoxide metabolite of the rat carcinogen acrylonitrile,was not significant in hepatic microsomes from untreated rats,but was substantially induced by treatment with thiazole orpyrazine. Microsomal hydrolysis activity was heat-sensitiveand potently inhibited by l, l, l-trichloropropene-2, 3-oxide,indicating that mEH was the catalyst. The V_max for the hydrolysisof CEO by hepatic microsomes from thiazole-treated rats (13.4nmol/min/mg protein) was 1.5-fold greater than that with microsomesfrom pyrazine-treated rats, whereas similar K_m values ( 1 mM)were observed for both microsomal preparations. These kineticdata correlate well with the increases in mEH mRNA observedafter administration of thiazole or pyrazine to rats. Theseresults provide evidence that administration of thiazole orpyrazine induces mEH with a large increase in mEH mRNA, andthat the induced mEH catalyzes the hydrolysis of CEO.