Microcystin-LR and okadaic acid belong to a family of natural toxins which localize to the liver, inhibit the serine-threonine protein phosphatases PP1 and PP2A, inducing severe detrimental effects on the liver. These compounds are considered acute hepatotoxins and carcinogens. Little information with respect to the metabolism of these two toxins is available. While glutathione and cysteine conjugates of microcystin-LR have been identified in vertebrates and invertebrates, no phase I metabolites have been identified. Recent reports suggest that okadaic acid is metabolically activated to genotoxic products, yet no metabolites of okadaic acid have been identified in vertebrates. The long-term goal of this research is to develop a thorough understanding of the metabolic fate of the two hepatotoxins and to understand how and if xenobiotic metabolizing enzymes are regulated by these environmental toxins and how factors such as age, gender cytochrome P450 (CYP) polymorphisms, tobacco use, diet, and other environmental factors will influence toxicity by affecting the expression and activity of xenobiotic metabolizing enzymes and hence the profile metabolites formed. The objective of this proposal is to generate human metabolites of microcystin-LR and okadaic acid synthetically and enzymatically, using human recombinant xenobiotic metabolizing enzymes, and to create an """"""""average"""""""" metabolite profile for each toxin derived from human primary hepatocytes. We will also examine the effect of parent toxins and individual metabolites on a human liver cell line, HepG2. We will further evaluate the influence of the parent toxins and selected metabolites on gene expression in BALB/c mice. The central hypothesis of the proposed research is that microcystin-LR and okadaic acid may be metabolized by xenobiotic metabolizing enzymes, and that metabolites diversify the effects of exposure to the parent toxins by exhibiting different biological activities in their respective target organs. Preliminary data indicates that microcystin-LR is metabolized by CYP1A2 and that exposure to microcystin-LR induces expression of CYP1A2. This hypothesis will be tested by pursuing four specific aims: 1) synthesize, isolate and characterize metabolites of MC-LR and OA using a combination of enzymatic and chemical synthesis;2) incubate MC-LR and OA with pooled human primary hepatocytes to generate an """"""""average"""""""" metabolite profile;3) Perform gene expression/ microarray experiments in mice with MC-LR, OA and selected MC-LR and OA metabolites;4) Evaluate cytotoxicity and genotoxicity of individual metabolites using human liver HepG2 cell line. This research is innovative because a systematic analysis of MC-LR and OA metabolites and their toxicities has not been undertaken. This work is significant because diet, environment, gender, age, health, prior exposure to xenobiotics and genetic polymorphisms in liver xenobiotic metabolizing enzymes in humans will influence metabolism and susceptibility in different populations. This work will immediately link a xenobiotic metabolizing enzyme, such as a cytochrome P450 (CYP) with a specific metabolite and will evaluate the toxicity of individual metabolites.
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