The incidence of alcoholism is much higher in Native Americans and Mexican Americans that in Caucasians. The reasons may be sociological, psychological, biochemical, or a combination of these factors. In addition to a higher incidence of alcoholism, there is also an increased susceptibility of alcohol related liver disease in these two populations which may, in part, be related to metabolism of alcohol. P4502E1 is known to metabolize several compounds including ethanol to hepatotoxic metabolites. Moreover, chronic exposure to alcohol causes an increase in the expression of this particular P450 enzyme. This increase in expression may play a role in the development of occult liver disease from alcohol and other hepatotoxins metabolized by this enzyme. A focus of this proposal is to determine whether P4502E1 plays a role in the susceptibility of certain individuals to alcohol-related liver injury. this will be investigated by screening lymphocytes from 3 separate ethnic populations, Native Americans, Mexican Americans and Caucasians both alcoholic and non-alcoholic, for differences in the expression of the enzyme. To determine enzyme expression, lymphocytes will be isolated from whole blood collected from alcoholic and nonalcoholic subjects from all three subpopulations. P4502E1 will be monitored by immunoblot analysis of microsomes isolated from the lymphocytes using a mono- specific antibody prepared to human liver P4502E1. Detection of an elevation in this enzyme may serve as a predictor that chemical or alcohol-induced hepatotoxicity may result. This proposal focuses on different ethnic populations to determine whether this enzyme can be used as a biochemical marker of alcohol-dependent liver disease in subpopulations which are considered to be at high risk. Identification of specific RFLPs in the regulatory region of CYP2E1 is also proposed which will be correlated with phenotypic expression, ethnicity and alcoholism. Further characterization of P4502E1 in lymphocytes will involve examining whether ethanol causes an increase in P4502E1 mRNA with a subsequent increase in the enzyme. This will be accomplished by Northern blot analysis of mRNA isolated from alcoholic and non-alcoholic subjects and results may provide a mechanism in which ethanol increases P4502E1 in lymphocytes. Preliminary evidence from our laboratory suggests that the lymphocyte enzyme exhibits a molecular weight (MW) difference from that of the liver P450. Thus, the structure of the lymphocyte protein will also be examined and compared to liver P4502E1 in order to assess whether the altered structure modifies the activity of the enzyme. Whether the expression and regulation of P4502E1 in lymphocytes parallels that which is in liver will be determined in a clinical study using the drug chlorozoxazone as a marker of liver P4502E1 function. If the enzyme is inducible by alcohol in lymphocytes and its regulation and expression mirrors that of the liver, human blood may be a readily accessible tissue to phenotype and study regulation of liver P4502E1 in humans.
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