In vivo induction of hepatic P4502E1 by ethanol: role of increased enzyme synthesis.
P4502E1 (2E1), an ethanol-inducible P450 enzyme, plays an important role in the bioactivation of certain hepatotoxins and chemical carcinogens. Different mechanisms of 2E1 induction by ethanol and other agents (e.g., acetone) have been proposed, ranging from enhanced de novo enzyme synthesis caused by an increase in 2E1 mRNA and/or the efficiency with which it is translated to decreased enzyme degradation stemming from substrate stabilization. To evaluate these mechanisms, we first examined the time course of hepatic 2E1 protein induction in rats pair-fed liquid diets containing 36% of total calories as either ethanol or dextrin-maltose (controls) for 28 days. Western blot analysis with anti-2E1 immunoglobulins revealed that 2E1 reached a new steady-state level (eightfold greater than that found with controls) after ethanol feeding for 10 days and remained elevated for the duration of treatment. Microsomal p-nitrophenol hydroxylation, a 2E1-catalyzed reaction, exhibited a similar induction time course, with the maximal increase in enzyme activity also observed on Day 10 of ethanol administration. We then determined steady-state 2E1 protein turnover in ethanol-fed and control animals that were given [35S]methionine plus[3H]aminolevulinate to radiolabel 2E1 apoprotein and the prosthetic heme group, respectively. Monophasic exponential decay curves showed that hepatic 2E1 protein and heme half-lives (27-28 h and 17 h, respectively) did not differ between the treatment groups. However, rates of 2E1 synthesis, assessed by measuring initial rates of incorporation of [35S]methionine and [3H]aminolevulinate into 2E1 apoprotein and heme, were increased in animals fed ethanol. Our results indicate that the in vivo induction of hepatic 2E1 protein by ethanol involves increased enzyme synthesis rather than decreased enzyme degradation. This enhancement of de novo 2E1 synthesis most likely entails the ethanol-mediated increase of steady-state levels of 2E1 mRNA and/or the stimulation of its translational efficiency.
Tsutsumi
M
M
Alcohol Research and Treatment Center, Bronx VA Medical Center, New York 10468.
Lasker
J M
JM
Takahashi
T
T
Lieber
C S
CS
D000818:Animals; D001835:Body Weight / Q000187:drug effects; D019392:Cytochrome P-450 CYP2E1; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis*; D004790:Enzyme Induction / Q000187:drug effects; D000431:Ethanol / Q000494:pharmacology*; D015870:Gene Expression / Q000187:drug effects; D017403:In Situ Hybridization; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D010089:Oxidoreductases, N-Demethylating / Q000096:biosynthesis*; D011233:Precipitin Tests; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D013311:Streptozocin / Q000494:pharmacology
D012333:RNA, Messenger; D000431:Ethanol; D013311:Streptozocin; D003577:Cytochrome P-450 Enzyme System; D019392:Cytochrome P-450 CYP2E1; D010089:Oxidoreductases, N-Demethylating