The long term objective is to define ocular drug metabolizing and detoxifying enzymes and their genetic aspects, and to develop preventive measures against ocular drug toxicities. A survey of 250 drugs revealed that 70% are cataractogenic and 30% produce glaucoma. Only 6% cause retinopathy/neuropathy. Toxic effects of drugs (and their metabolites) on anterior tissues lead to cataracts and glaucoma. The higher susceptibility of anterior tissues to drug toxicity than posterior tissues is not well understood but aqueous humor production and drug metabolizing activities associated with ciliary body appear to be important factors. Mice pretreated with cytochrome P450 (CYP) inducers developed cataracts when overdosed with acetaminophen (APAP) but no changes occur in posterior tissues. APAP cataract, therefore, is a useful model to investigate ocular toxicities of drugs that preferentially affect anterior tissues. Drug toxicity is often accompanied by oxidative stress because superoxide anion, H2O2 and other reactive oxygen species are generated by drug metabolism. The ciliary body contains a novel selenium - independent glutathione peroxidase (GPX). The PI will determine its primary structure by molecular cloning. GPX may play an important role in the detoxification of peroxide in ciliary body. There are 2 Specific Aims. (1) Acetaminophen Cataract. The PI hypothesizes that the APAP metabolite N-acetyl-p-benzoquinone (NAPQI) produced mainly by hepatic CYP enzymes and secreted with aqueous humor targets mitochondria of anterior tissues and causes cataract. Questions addressed include: (i) Can NAPQI mimic APAP ocular toxicity affecting primarily anterior tissues? (ii) Do compounds that stimulate NAPQI detoxification, but not CYP inhibitors, prevent NAPQI toxicity? (2) Glutathione Peroxidase. Questions addressed to characterize GPX and its role in ocular tissues are: (i) Where is the cellular location of GPX in eye and other organs? (ii) GPX belongs to the family of antioxidant enzymes distinct from SeGSH peroxidases. Cysteine was suggested to be at the catalytic center of the family of enzymes but not tested. Is the sole cysteine of GPX important for activity? (iii) A gene essentially identically to GPX gene is activated in cultured keratinocytes in response to keratinocyte growth factor (FGF-7). Therefore the PI will investigate whether FGF-7 activates GPX gene in cultured ciliary epithelial cells.
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