Microcystin-LR and okadaic acid belong to a family of natural toxins which localize to the liver, inhibitthe serine-threonine protein phosphatases PP1 and PP2A, inducing severe detrimental effects on theliver. These compounds are considered acute hepatotoxins and carcinogens. Little information withrespect to the metabolism of these two toxins is available. While glutathione and cysteine conjugates ofmicrocystin-LR have been identified in vertebrates and invertebrates, no phase I metabolites have beenidentified. Recent reports suggest that okadaic acid is metabolically activated to genotoxic products, yetno metabolites of okadaic acid have been identified in vertebrates. The long-term goal of this research isto develop a thorough understanding of the metabolic fate of the two hepatotoxins and to understand howand if xenobiotic metabolizing enzymes are regulated by these environmental toxins and how factors suchas age, gender cytochrome P450 (CYP) polymorphisms, tobacco use, diet, and other environmentalfactors will influence toxicity by affecting the expression and activity of xenobiotic metabolizing enzymesand hence the profile metabolites formed. The objective of this proposal is to generate human metabolitesof microcystin-LR and okadaic acid synthetically and enzymatically, using human recombinant xenobioticmetabolizing enzymes, and to create an 'average' metabolite profile for each toxin derived from humanprimary hepatocytes. We will also examine the effect of parent toxins and individual metabolites on ahuman liver cell line, HepG2. We will further evaluate the influence of the parent toxins and selectedmetabolites on gene expression in BALB/c mice. The central hypothesis of the proposed research is thatmicrocystin-LR and okadaic acid may be metabolized by xenobiotic metabolizing enzymes, and thatmetabolites diversify the effects of exposure to the parent toxins by exhibiting different biological activitiesin their respective target organs. Preliminary data indicates that microcystin-LR is metabolized by CYP1A2and that exposure to microcystin-LR induces expression of CYP1A2. This hypothesis will be tested bypursuing four specific aims: 1) synthesize, isolate and characterize metabolites of MC-LR and OA using acombination of enzymatic and chemical synthesis; 2) incubate MC-LR and OA with pooled humanprimary hepatocytes to generate an 'average' metabolite profile; 3) Perform gene expression/ microarrayexperiments in mice with MC-LR, OA and selected MC-LR and OA metabolites; 4) Evaluate cytotoxicityand genotoxicity of individual metabolites using human liver HepG2 cell line. This research is innovativebecause 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 xenobioticsand genetic polymorphisms in liver xenobiotic metabolizing enzymes in humans will influence metabolismand susceptibility in different populations. This work will immediately link a xenobiotic metabolizingenzyme, such as a cytochrome P450 (CYP) with a specific metabolite and will evaluate the toxicity ofindividual metabolites.
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