Numerous xenobiotic compounds and drugs in our environment are toxic to the eye as such or become toxic after metabolic potentiation. As a cellular defense against their adverse effects, the eye possesses capabilities of physical as well as metabolic detoxification of xenobiotics. However, our knowledge of those mechanisms and ocular capacities of metabolic detoxification is very limited. Drug metabolism involves enhanced generation of active oxygen species (e.g. superoxide, H202). Hence, detoxification of those species is of equal importance and particular attention is directed in this proposal to peroxisomes, a subcellular organelle with which catalase activity is associated. We have previously shown that uveal tissues, especially ciliary body, are richest in drug metabolizing and detoxifying activities. The focus of this application, therefore, is to delineate the nature of those enzymatic activities in ciliary epithelial cells and determine their capacities to metabolize and detoxify xenobiotics and reactive oxygen species, using cultured ciliary epithelial cells. Cultured epithelial cells are used to determine whether the above-mentioned activities are localized to nonpigmented cells or pigmented cells, or to both. Glutathione conjugates of xenobiotics are metabolized to mercapturates by sequential catalysis of several enzymes. Similar enzymes catalyze conversion of leukotriene A4 to leukotriene E4. It is not known whether the enzymes responsible for the metabolism of xenobiotics and the endogenous substrates are identical or different. Cultured cells are also used to determine the nature of the enzymes involved in those reactions at basal level as well as at induced level, i.e. when the enzymes are induced or enhanced by inducer compounds.
The specific aims i nclude: (1) determination of cytochrome P-450 monooxygenases (Phase 1 enzyme) at basal and induced levels in cell cultures, (2) induction of glutathione S-transferase (Phase 2 enzyme).and other enzymes of the mercapturate pathway in cell cultures, (3) determination of the relative activities of mercapturate pathway enzymes with xenobiotics and leukotrienes at basal and induced levels in cell cultures, {4) induction of peroxisomes and peroxisomal enzymes (e.g. catalase) in cell cultures, and (5) purification and characterization of enzymes of the mercapturate pathway and H202 detoxification.
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