Inhibition of human prenatal biosynthesis of all-trans-retinoic acid by ethanol, ethanol metabolites, and products of lipid peroxidation reactions - A possible role for CYP2E1

Biotransformation of all-trans-retinol (t-ROH) and all-trans-retinal (t-RAL ) to all-hans-retinoic acid (t-RA) in human prenatal hepatic tissues (53-84 gestational days) was investigated with HPLC using human adult hepatic tis sues as positive controls. Catalysis of the biotransformation of t-ROH by p renatal human cytosolic fractions resulted in accumulation of t-RAL with mi nimal t-RA. Oxidations of t-ROH catalyzed by prenatal cytosol were supporte d by both NAD(+) and NADP(+), although NAD(+) was a much better cofactor. I n contrast, catalysis of the oxidation of t-RAL to t-RA appeared to be sole ly NAD(+) dependent. Substrate K-m values for conversions of t-ROH to t-RAL and of t-RAL to t-RA were 82.4 and 65.8 mu M, respectively. At concentrati ons of 10 and 90 mM, ethanol inhibited the conversion of t-ROH to t-RAL by 25 and 43%, respectively, but did not inhibit the conversion of t-RAL to t- RA significantly. In contrast, acetaldehyde reduced the conversion of t-RAL to t-RA by 25 and 87% at 0.1 and 10 mM respective concentrations. Several alcohols and aldehydes known to be generated from lipid peroxides also exhi bited significant inhibition of t-RA biosynthesis in human prenatal hepatic tissues. Among the compounds tested, 4-hydroxy-2-nonenal (4-HNE) was highl y effective in inhibiting the conversion of t-RAL to t-RA. A 20% inhibition was observed at a concentration of only 0.001 mM, and nearly complete inhi bition was produced at 0.1 mM. Human fetal and embryonic hepatic tissues ea ch exhibited significant CYP2E1 expression as assessed with chlorzoxazone 6 -hydroxylation, a highly sensitive western blotting technique, and reverse transcriptase-polymerase chain reaction (PCR) (RT-PCR), suggesting that lip id peroxidation can be initiated via CYP2E1-catalyzed ethanol oxidation in human embryonic hepatic tissues. In summary, these studies suggest that eth anol may affect the biosynthesis of t-RA in human prenatal hepatic tissues directly and indirectly. Ethanol and its major oxidative metabolite, acetal dehyde, both inhibit the generation of t-RA. Concurrently, the CYP2E1-catal yzed oxidation of ethanol can initiate lipid peroxidation via generation of a variety of free radicals. The lipid peroxides thereby generated could th en be further converted via CYP2E1-catalyzed reactions to alcohols and alde hydes, including 4-HNE, that act as potent inhibitors of t-RA synthesis. (C ) 1999 Elsevier Science Inc.