The objectives of the proposed research are 1) to determine the degree to which changes in cytochrome P450IIE1 alter the pulmonary metabolism of xenobiotics and 2) to elucidate the role of the lung in the metabolism of ethanol and other alcohols.
One aim of the first objective is to determine the degree to which pyridine as a model inducer modulates the metabolism of substrates of the ethanol-inducible cytochrome P450IIE1 isozyme and thereby alters their toxic effects on the lung. These include benzene, urethane and styrene. The use of selective inducers will allow for the analysis of the importance of P450IIE1 and other isozymes in metabolizing and altering the toxicity of the pneumotoxicant O,O,S-trimethyl phosphorothioate. Related to these studies is the identification of cell types, with emphasis on Clara cells and type II alveolar cells, which contain P450IIE1 using immunohistochemistry in lung sections and isolated cell preparations and immunoblotting.
The second aim i s to examine the regulation of pulmonary cytochrome P450IIE1 to understand the mechanistic basis for the selective induction of metabolism of some substrates but not others. Protein synthesis will be measured to determine if inducers of the apoprotein are affecting translation rather then transcription. Translational efficiency of P450IIE1 mRNA will be measured, and apoprotein stabilization will be evaluated.
One aim of the second objective is to examine the synthesis of pulmonary esterified fatty acids in response to ethanol and longer chain alcohols. Rates of in formation will be measured, and the types and amounts of these esters synthesized in vivo will be determined.
A second aim i s to evaluate the role of the lung in the metabolism of long chain alcohols via alcohol dehydrogenase (ADH) and the microsomal ethanol oxidizing system (MEOS). Of particular interest are propanol, isopropanol, butanol, pentanol and octanol. For ADH, apparent Km and Vmax values will be determined as well as pH dependency. For MEOS, the rate of metabolism and susceptibility to methylpyrazole inhibition will be examined. Throughout, several approaches will be taken including: 1) comparing tissue specificities of the lung with the liver, a well characterized organ, 2) integrating findings by examining these pathways at levels ranging from subcellular fractions to intact isolated cells, 3) comparing pyridine with inducers of the phenobarbital and polycyclic aromatic hydrocarbon types, and 4) employing biochemical measurements, such as bronchoalveolar lavage fluid analysis, and histopathology to evaluate pulmonary injury. These studies will elucidate the role of the lung in the biotransformation of xenobiotics including alcohols and the ability of pyridine as a model inducer of P450IIE1 and other inducers to alter these metabolic pathways and the potential resultant toxicities associated with these chemicals.
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