The long-term objective of this proposal for extension of the research grant is to fully understand the role of the alcohol-inducible forms of cytochrome P-450, P-450IIE1 and P-450IIE2, in alcohol metabolism and in the chemical toxicities, mutagenesis, and carcinogenesis associated with alcohol abuse.
The specific aims are (a) to purify alcohol-inducible cytochrome P-450 IIE2 to homogeneity after heterologous expression and to determine whether P-450 IIE1 in kidney, nasal mucosa, and lung, and P-450 IIE2 in lung are different from their counterparts in liver, (b) to determine whether P-450 IIE1 and IIE2 and any other variants differ in their activities toward biologically occurring substrates such as glycerol and other alcohol, acetone and other carbonyl compounds, retinoids, and lipid hydroperoxides; (c) to compare the activities of P- 450 IIE1 and IIE2 toward a variety of xenobiotics and in the reduction of O2 to H2O2 and oxygen radicals in the absence of added substrates; and (d) to study differences in the developmental expression of the P- 450 IIE1 and IIE2 genes. Although we have purified the unstable P-450 IIE2 in small amounts from neonatal rabbit liver, the number of animals required to obtain enough of the enzyme for thorough characterization is prohibitive. A full-length CDNA for this cytochrome will be cloned and expressed in yeast and E. coli and the cytochrome will be isolated from large scale cultures. Catalytic activities will be measured in the reconstituted system toward various added substrates. Our recent finding that P-450 IIE1 is the most active of the cytochromes tested in the reductive Beta-scission of fatty acid hydroperoxides to oxoacids and hydrocarbons raises the important question of whether the chronic exposure of animals and humans to alcohol leads to a loss of membrane integrity because of lipid alterations. As time permits, we will explore the pathological effects of liquid peroxidation related to alcohol intake as well as the possibility that the products of lipid peroxidation may serve some useful biological functions. Although P-450 IIE1 is known to metabolize a large variety of foreign compounds, including ethanol and other alcohols, ketones, nitrosamines, and halogenated alkanes, alkanes, and ethers, as well as aromatic compounds, almost no information is yet available with P-450 IIE2. The substrates to be tested will include the prohepatotoxin acetaminophen, procarcinogens such as N-nitrosodimethylamine and N-nitrodiethylamine, and the drug chlorzoxazone. Preliminary results indicate that P-450 IIE2 MRNA and protein are detectable immediately after birth, whereas IIE1 MRNA (and,therefore, protein) are not detectable until day 14. The nature of this differential regulation will be studied, including the effects on the progeny of alcohol administration to pregnant or nursing rabbits, and attempts will be made to determine whether the two cytochromes have functional differences that are important during the first few weeks of life. The results may help elucidate some of the biochemical effects in the fetal alcohol syndrome. In addition, the mechanism by which IIE1 and IIE2 mRNA are elevated in the Watanabe hyperlipidemic rabbits will be determined.
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