| 7689039 |
Kim SG, Kedderis GL, Batra R, Novak RF: Induction of rat liver microsomal epoxide hydrolase by thiazole and pyrazine: hydrolysis of 2-cyanoethylene oxide. Carcinogenesis. 1993 Aug;14(8):1665-70.
Liver microsomal epoxide hydrolase (mEH) is active in the detoxification of epoxide-containing carcinogens. The effects of thiazole and pyrazine, constituents of tobacco and tobacco smoke as well as of a variety of foods, on the expression and regulation of mEH were examined in rats (200 mg/kg body wt/day, i.p., 1-3 days). Immunoblot analyses using rabbit anti-rat mEH antibody revealed a significant increase in mEH levels in hepatic microsomes isolated from either thiazole- or pyrazine-treated animals. Another protein (approximately 43 kd) cross-reacting with polyclonal mEH antibody was found to be increased concomitantly following pyrazine treatment. Northern and slot blot analyses showed substantial increases in mEH mRNA following either thiazole or pyrazine treatment. The level of mEH mRNA increased 17-fold at 24 h following thiazole treatment, relative to control. Approximately 20- and 16-fold increases in mEH mRNA were also observed at 48 and 72 h respectively following treatment with pyrazine. The level of polymerase chain reaction (PCR)-amplified mEH DNA derived from poly (A)+ RNA was clearly elevated following either thiazole or pyrazine treatment relative to that from untreated animals. Both sense and antisense strands of PCR-amplified mEH DNA were cloned into an M13mp19 phage vector in order to examine the nucleotide sequences of PCR-amplified mEH DNA derived from the poly (A)+ RNA isolated from thiazole- or pyrazine-treated animals. Sequence analyses revealed that the sequence of PCR-amplified DNA from the induced mRNA was identical to that published for mEH cDNA. Epoxide hydrolase activity 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 or pyrazine. Microsomal hydrolysis activity was heat-sensitive and potently inhibited by 1,1,1-trichloropropene-2,3-oxide, indicating that mEH was the catalyst. The Vmax for the hydrolysis of CEO by hepatic microsomes from thiazole-treated rats (13.4 nmol/min/mg protein) was 1.5-fold greater than that with microsomes from pyrazine-treated rats, whereas similar Km values (approximately 1 mM) were observed for both microsomal preparations. These kinetic data correlate well with the increases in mEH mRNA observed after administration of thiazole or pyrazine to rats. These results provide evidence that administration of thiazole or pyrazine induces mEH with a large increase in mEH mRNA, and that the induced mEH catalyzes the hydrolysis of CEO. |
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