A cytochrome P450-independent monooxygenase activity in porcine liver was first identified in 1964 and subsequently purified and characterized. This enzyme is a unique flavin-containing monooxygenase (FMO) which plays an equal, if not more important role that cytochrome P450 in the metabolic activation of a wide variety of nitrogen-, sulfur-, and phosphorous-containing xenobiotics, including aryl- and alkylamines found in cigarette smoke condensates and phosphonate type insecticides. As such, this enzyme is thought to play an important role in the early events of chemical carcinogenesis and toxicity. The FMO is an integral membrane protein localized both in the endoplasmic reticulum and nucleus. Two research groups have reported independently that two, and perhaps as many as four related isozymes of FMO exist. One of the major isozymes is found predominantly in the lung while the other predominantly in the liver. Both enzymes appear to be under hormonal and ontogenic control. Recently, two rabbit cDNA clones have been isolated representing the major hepatic and pulmonary forms of FMO. The two mRNAs share 56% sequence identity and exhibit tissue-specific expressions patterns. Employing reverse transcriptase/PCR, both of these cDNAs have also been isolated and cloned in the PI's laboratory. The research objectives of the current proposal are as follows: first, the expression of the lung and liver isozymes will be examined as a function of tissue, sex, fetal development and time of gestation in the doe. Second, the question of transcriptional or post-transcriptional control will be answered by examining the rate of transcription by run-off assays in isolated nuclei. Third, the expression of the FMO isozymes, particularly in response to steroid treatment, will be examined both in established human cell lines. Fourth, the genes for the human cell lines. Fourth, the genes for the human and rabbit FMO isozymes will be isolated and characterized by restriction endonuclease mapping and DNA sequence analysis. Fifth, specific cis-regulatory sequences on the FMO genes will be identified by fusion with a heterologous reporter gene and transient expression assays. Finally, mammalian and yeast heterologous expression systems will be developed. The yeast system will be used to answer questions regarding structure/activity relationships for the FMO isozymes using chimeric constructions and site-directed mutagenesis. Although beyond the scope of this proposal, the mammalian system will be useful in determining the ability of the FMO system to activate specific procarcinogens and/or protoxins, as well as testing the consequences of that activation.
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