FORMATION AND REACTIVITY OF TOXIC QUINONE METHIDES

有毒醌甲基化物的形成和反应性

• 批准号: 2155070
• 负责人: JOHN A THOMPSON
• 金额: $ 17.17万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-01 至 1997-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2155070
• 关键词: DNA binding protein; adduct; alkylation; binding proteins; chemical binding; chemical structure function; chemical substitution; chemical synthesis; cytochrome P450; cytotoxicity; enzyme activity; flavonoids; free radical scavengers; gas chromatography mass spectrometry; hepatotoxin; high performance liquid chromatography; laboratory rat; mass spectrometry; nuclear magnetic resonance spectroscopy; oxidation reduction reaction; oxidative stress; peroxidases; phenols; quinones; ultraviolet spectrometry

Hydroxylated and polyhydroxylated aromatics are widespread in the environment, especially as constituents of edible plants. Many of these phenolic compounds can be oxidized enzymatically to electrophilic quinone methides, but, with a few exceptions, the involvement of quinone methides in mediating the adverse effects of substituted phenols has not been considered. Both cytochrome P450 and peroxidase activity can catalyze quinone methide formation, so the alkylation of cellular components by this pathway may occur in many different tissues. Preliminary data demonstrate wide variation both in the formation and reactivity of quinone methides due to the structure of the phenolic precursor. The present application addresses relationships between phenol structure and P450- catalyzed quinone methide formation, reactivity, and cytotoxicity. To accomplish these goals, the following specific aims are proposed. (1) Determine the influence of chemical structure on the oxidation of phenolic compounds to quinone methides. (a) The effects of specific ortho oxygen- containing substituents, and unsaturated para alkyl substituents will be examined, as these are commonly found in naturally-occurring phenols, and are expected to affect quinone methide formation and reactivity. These studies will be extended to investigate quinone methide formation from mutagenic and non-mutagenic flavonoids. (b) Influences of substrate structure on cytochrome P450 isozyme selectivities will be studied, as previous results have demonstrated isozymic differences in the catalysis of quinone methide formation. Mechanistic information on this oxidative pathway will be obtained with deuterium-labeled analogs and free radical scavengers. For comparison with P450 activity, quinone methide formation by a model peroxidase system also will be investigated. (2) Investigate the effects of quinone methide structure on electrophilic reactivity. Rates of reactions of quinone methides with water, glutathione, nucleophilic amino acids, and purine deoxynucleosides will be measured. This data will be correlated with quinone methide structures to deduce general principles underlying their reactivity, and gain insight into intracellular binding selectivities. Reactions of selected quinone methides with pure proteins and DNA will also be investigated to generate qualitative and quantitative data concerning the particular residues affected. (3) Investigate the roles of quinone methides in mediating the toxicity of phenolic compounds. Isolated rat hepatocytes will be incubated with substituted phenols which produce quinone methides of varying reactivities with nucleophiles. The effects of quinone methide formation on cell viability, and on biochemical parameters related to toxicity will be determined. The covalent binding of quinone methides to proteins and DNA will be investigated to determine intracellular sites of alkylation. The resulting data will provide substantial insight into the roles of quinone methides in the cytotoxicity of phenolic compounds.

羟基化和多羟基化的芳香剂在 环境，特别是作为食用植物的成分。其中许多 酚类化合物可以用酶氧化为亲电醌 甲基甲基，但除了少数例外，醌 在介导取代酚的不良反应时，尚未是 经过考虑的。细胞色素P450和过氧化物酶活性都可以催化 奎因酮甲基形成，因此细胞成分的烷基化 该途径可能发生在许多不同的组织中。初步数据 证明喹酮的形成和反应性均变化很大 由于酚类前体的结构而引起的甲基化。现在 应用程序解决了苯酚结构与P450-之间的关系 催化的喹酮甲基形成，反应性和细胞毒性。到 实现这些目标，提出了以下特定目标。 （1） 确定化学结构对酚类氧化的影响 化合物对喹酮甲烷。 （a）特定的氧气的影响 包含取代基和不饱和PARA烷基取代基将是 被检查的，因为这些通常在自然出现的苯酚中发现，并且 预计会影响醌甲基的形成和反应性。这些 将扩展研究以研究醌甲基的形成 诱变和非毒素类黄酮。 （b）底物的影响 将研究细胞色素p450同工酶选择性的结构，如 先前的结果表明催化中的同源差异 喹酮甲基的形成。有关此氧化的机械信息 途径将使用氘标记的类似物和自由基获得 清道夫。为了与P450活性进行比较 还将通过模型过氧化物酶系统进行研究。 （2）调查 醌甲基结构对亲电反应性的影响。 喹酮甲基与水，谷胱甘肽的反应速率， 将测量亲核氨基酸和嘌呤脱氧核苷。 这些数据将与喹酮甲基结构相关以推断 反应性的一般原则，并深入了解 细胞内结合选择性。选定奎因酮的反应 还将研究具有纯蛋白质和DNA的甲基 有关特定残基的定性和定量数据 做作的。 （3）研究醌甲基在介导的作用 酚类化合物的毒性。孤立的大鼠肝细胞将孵化 用替代的酚，产生醌甲基的变化 用亲核试剂的重复性。喹酮甲基形成的影响 关于细胞活力，与毒性有关的生化参数 可以确定。醌甲基与蛋白质和蛋白质的共价结合 将研究DNA以确定烷基化的细胞内部位。 由此产生的数据将提供对角色的实质性见解 酚醛化合物的细胞毒性中的醌甲基。