Core Research Project: Human Metabolites of Algal Hepatoxins

微囊藻毒素-LR和冈田酸属于天然毒素家族，其定位于肝脏，抑制 丝氨酸-苏氨酸蛋白磷酸酶PP 1和PP 2A，诱导严重的有害作用， 肝脏这些化合物被认为是急性肝毒素和致癌物质。信息不多， 关于这两种毒素的代谢情况。而谷胱甘肽和半胱氨酸结合物 尽管在脊椎动物和无脊椎动物中已经鉴定出微囊藻毒素-LR，但尚未发现I相代谢物。 鉴定最近的报告表明，冈田酸被代谢活化为遗传毒性产物，但 在脊椎动物中没有发现冈田酸的代谢物。这项研究的长期目标是 深入了解这两种肝毒素的代谢命运，并了解它们如何 如果异生素代谢酶受到这些环境毒素的调节， 细胞色素P450（CYP 450）多态性与年龄、性别、吸烟、饮食和其他环境因素有关 这些因素通过影响外源物质代谢酶的表达和活性来影响毒性 并因此形成谱代谢物。该提案的目的是产生人类代谢物 微囊藻毒素-LR和冈田酸的合成和酶促，使用人重组异生物质 代谢酶，并创建每种毒素的“平均”代谢物谱，来源于人类 原代肝细胞我们还将研究母体毒素和单个代谢物对 人肝细胞系HepG 2。我们将进一步评估母体毒素的影响， 代谢物对BALB/c小鼠基因表达的影响。这项研究的中心假设是， 微囊藻毒素-LR和冈田酸可被异生物质代谢酶代谢， 代谢物通过表现出不同的生物活性使暴露于母体毒素的影响多样化 在各自的目标器官中。初步数据表明，微囊藻毒素-LR由CYP 1A 2代谢 暴露于微囊藻毒素-LR诱导CYP 1A 2的表达。这一假设将由以下人员进行检验： 追求四个具体目标：1）合成、分离和表征MC-LR和OA的代谢物， 2）将MC-LR和OA与合并的人血清孵育; 原代肝细胞以产生“平均”代谢物谱; 3）进行基因表达/微阵列 用MC-LR、OA和选定的MC-LR和OA代谢物在小鼠中进行的实验; 4）评价细胞毒性 使用人肝HepG 2细胞系研究单个代谢物的遗传毒性。这项研究具有创新性 因为尚未对MC-LR和OA代谢物及其毒性进行系统分析。 这项工作意义重大，因为饮食，环境，性别，年龄，健康，先前暴露于外源性物质 人类肝脏异生物质代谢酶的遗传多态性将影响代谢 不同人群的易感性。这项工作将立即将异生物质代谢 酶，如具有特定代谢物的细胞色素P450（CYP 1A 1），并将评估 单个代谢物。

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.