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.

羟基化和多羟基化芳族化合物广泛存在于 环境，特别是作为可食用植物的成分。许多这些 酚类化合物可被酶促氧化成亲电子醌 甲基化物，但是，除了少数例外，醌甲基化物的参与 在介导取代酚的不利影响方面， 考虑了细胞色素P450和过氧化物酶活性都可以催化 醌甲基化物的形成，所以细胞成分的烷基化， 该途径可发生在许多不同的组织中。初步数据 证明了醌的形成和反应性的广泛变化 由于酚类前体的结构，本 本申请涉及苯酚结构和P450之间的关系。 催化的醌甲基化物形成、反应性和细胞毒性。到 为了实现这些目标，提出了以下具体目标。（一） 确定化学结构对酚类氧化的影响 化合物到醌甲基化物。(a)特定邻位氧的影响- 不饱和帕拉烷基取代基， 检查，因为这些是常见的天然存在的酚，和 预期会影响醌甲基化物的形成和反应性。这些 研究将扩展到调查醌甲基化物的形成， 致突变和非致突变类黄酮。(b)基质的影响 将研究细胞色素P450同工酶选择性的结构， 以前的结果已经证明了同工酶的差异，在催化 醌甲基化物的形成。关于这种氧化的机制信息 途径将获得与氘标记的类似物和自由基 食腐动物为了与P450活性比较，醌甲基化物形成 通过模型过氧化物酶系统也将被研究。(2)探讨 醌甲基化物结构对亲电反应活性的影响。 醌甲基化物与水，谷胱甘肽， 将测量亲核氨基酸和嘌呤脱氧核苷。 该数据将与醌甲基化物结构相关，以推断 其反应性的一般原则，并深入了解 细胞内结合选择性。选择醌的反应 甲基化物与纯蛋白质和DNA也将被研究，以产生 关于特定残留物的定性和定量数据 影响。(3)研究醌甲基化物在介导 酚类化合物的毒性。将孵育分离的大鼠肝细胞 用取代的酚产生不同的醌甲基化物， 与亲核试剂反应。醌甲基化物形成的影响 对细胞活力和与毒性有关的生化参数的影响， 被确定。醌甲基化物与蛋白质的共价结合， 将对DNA进行研究，以确定细胞内烷基化位点。 由此产生的数据将提供实质性的洞察力的作用， 醌甲基化物的酚类化合物的细胞毒性。