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，定位于肝脏，抑制 丝氨酸-苏氨酸蛋白磷酸酶PP1和PP2A，对 肝脏。这些化合物被认为是急性肝毒素和致癌物。提供的信息很少 关于这两种毒素的新陈代谢是可用的。而谷胱甘肽和半胱氨酸的结合物 已经在脊椎动物和无脊椎动物中发现了微囊藻毒素-LR，还没有发现I相代谢产物 已确认身份。最近的报告表明冈田酸被代谢激活为遗传毒性产物，但 在脊椎动物中还没有发现冈田酸的代谢物。这项研究的长期目标是 对这两种肝毒素的代谢去向有深入的了解，并了解它们是如何 如果异源代谢酶受这些环境毒素的调节，以及这些因素是如何 由于年龄、性别、细胞色素P450(CYP)基因多态性、吸烟、饮食等环境因素的影响 影响外源代谢酶表达和活性的因素会影响毒性 因此形成了轮廓代谢物。这项提议的目标是产生人类代谢物 利用人重组异种生物合成和酶促合成微囊藻毒素-LR和冈田酸 代谢酶，并为来自人类的每一种毒素创建一个“平均”代谢物图谱 原代肝细胞。我们还将研究母体毒素和个体代谢物对 人肝细胞系HepG2。我们将进一步评估母体毒素的影响，并选择 代谢产物对BALB/c小鼠基因表达的影响。拟议研究的中心假设是 微囊藻毒素-LR和冈田酸可被异源代谢酶代谢， 代谢产物通过表现不同的生物活性使暴露于母体毒素的影响多样化 在它们各自的目标器官中。初步数据表明微囊藻毒素-LR是由细胞色素P1A2代谢的 暴露于微囊藻毒素-LR可诱导细胞色素P1A2的表达。这一假设将通过以下方式检验 追求四个具体目标：1)合成、分离和鉴定MC-LR和OA的代谢物 酶法与化学合成法相结合；2)MC-LR和OA与混合人孵育 原代肝细胞产生“平均”代谢物；3)进行基因表达/微阵列 MC-LR、OA及精选MC-LR和OA代谢物的小鼠实验；4)评价细胞毒性 以及利用人肝细胞株HepG2对单个代谢物的遗传毒性。本研究具有创新性。 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、以前接触过外源生物 人类肝脏异源代谢酶基因多态性将影响新陈代谢 以及在不同人群中的易感性。这项工作将立即将一种异生代谢联系起来 酶，如具有特定代谢物的细胞色素P450(CYP)，并将评估 个体代谢物。

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.