Adduct Formation in the Toxicity of Dithiocarbamates

描述(申请人提供)：人类接触二硫代氨基甲酸盐的情况发生在农业、工业和医药行业。虽然对降解和代谢途径的了解正在发展，但目前对二硫代氨基甲酸酯生物影响的分子靶点和机制知之甚少。该项目的长期目标是描绘二硫代氨基甲酸酯及其代谢物在生物系统内的相互作用，并确定这些相互作用作为毒性机制和生物标记物的相关性。以前的研究表明，某些二硫代氨基甲酸酯能够产生选择性雪旺细胞毒性，并伴随着神经中铜和脂质过氧化产物水平的增加。在这一应用中的研究是在工作假设的指导下进行的，即这些二硫代氨基甲酸酯与内源性铜结合，形成亲脂络合物，并在髓鞘内蓄积，导致脂质过氧化、氧化损伤和脱髓鞘增加。这一假说将通过两个特定的目的来验证：1)确定二硫代氨基甲酸盐介导的节段性脱髓鞘是否需要铜的积累和氧化应激；2)确定二硫代氨基甲酸酯介导的周围神经脱髓鞘的分子靶点和细胞反应。目的1)合成对铜的亲和力不同的二硫代氨基甲酸酯及其铜络合物的溶解性，并评估它们在体内对脂质过氧化和脱髓鞘的相对效力，b)确定非二硫代氨基甲酸铜螯合剂铜必利酮提高神经中铜和脂质过氧化的能力，c)通过确定这些影响的剂量反应和时间关系来确定这些影响是否为二硫代氨基甲酸酯对髓鞘损伤的直接影响，以及d)确定对氧化损伤和铜毒性的妥协的转基因模型是否对二硫代氨基甲酸酯介导的脱髓鞘更敏感。目标2将通过a)确定蛋白质表达的变化，b)识别受损的蛋白质，c)测量铜酶活性和d)表征二硫代氨基甲酸盐在神经中产生过量铜的位置和化学形态，从而实现目标2。这些研究的意义在于建立结构-活性关系，用于预测可能通过类似机制发挥作用的制剂，识别易感人群，制定基于机制的暴露建议，并预测长期低水平暴露的影响。

DESCRIPTION (provided by applicant): Human exposure to dithiocarbamates occurs in agriculture, industry and medicine. Although an understanding of degradative and metabolic pathways is evolving, there is currently little knowledge on the molecular targets and mechanisms underlying the biological affects of dithiocarbamates. The long-range objectives of this project are to delineate the interactions of dithiocarbamates and their metabolites within biological systems and to determine the relevance of these interactions as mechanisms of toxicity and biomarkers. Previous studies revealed the ability of certain dithiocarbamates to produce selective Schwann cell toxicity accompanied by increased levels of copper and lipid peroxidation products in nerve. Investigations in this application are guided by the working hypothesis that these dithiocarbamates bind endogenous copper, form lipophilic complexes and accumulate within myelin resulting in increased lipid peroxidation, oxidative injury and demyelination. This hypothesis will be tested through two specific aims: 1) To determine if copper accumulation and oxidative stress are required for dithiocarbamate-mediated segmental demyelination and 2) To determine the molecular targets and cellular responses in dithiocarbamate-mediated peripheral nerve demyelination. Aim 1 will be achieved through: a) synthesis of dithiocarbamates differing in their affinity for copper and their copper complex solubility and evaluating their relative potency for lipid peroxidation and demyelination in vivo, b) determining the ability of the non-dithiocarbamate copper chelating agent, cuprizone, to elevate copper and lipid peroxidation in nerve, c) determining if lipid peroxidation and copper accumulation are direct affects of dithiocarbamates through defining the dose response and temporal relationship of these affects to the onset of myelin injury and d) determining if transgenic models with compromised defense to oxidative injury and copper toxicity are more sensitive to dithiocarbamate-mediated demyelination. Aim 2 will be achieved through a) determining changes in protein expression, b) identifying damaged proteins, c) measuring cuproenzyme activities and d) characterizing the location and chemical species of excess copper produced in nerve by dithiocarbamates. The significance of these studies lies in establishing structure-activity relationships useful for predicting agents that may act through similar mechanisms, identifying susceptible populations, developing mechanistically based exposure recommendations and predicting the affects of long term low level exposures.