Mechanisms of Diphenylamine Xenobiotic Reactivity and Toxicity

二苯胺异生物质反应性和毒性的机制

• 批准号: RGPIN-2020-06305
• 负责人: Siraki, Arno
• 金额: $ 2.33万
• 依托单位: University of Alberta
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=741151
• 关键词: Mechanisms; Diphenylamine; Xenobiotic; Reactivity; Toxicity

Background: Diphenylamine (DPA) occurs both naturally and synthetically, and DPA derivatives (DPAs) have widespread industrial use, including in the manufacturing of dyes, rubbers, pesticides, pharmaceuticals, and are found in munition sites. As a result of their widespread use, DPAs also occur in wastewater treatment plants in Canada, and in fish. Xenobiotics like DPAs are not typically reactive but may be oxidized to produce reactive metabolites. The latter are of toxicological concern since they can weaken cells and cause damage to their proteins and DNA. This proposal focuses on unexplored pathways of DPAs metabolism and the ensuing biological consequences. The ubiquitous use and environmental occurrence of DPA derivatives necessitate these studies. Rationale: Preliminary studies have shown that some DPAs can form two types of reactive metabolites: free radical metabolites and hydroxylated metabolites. These metabolites were formed from the heme protein, myeloperoxidase (MPO), which is found in specific blood cells. However, MPO does not usually produce hydroxylated metabolites, and the latter can be further oxidized to quinone- like reactive metabolites. As such, there are fundamental questions about how MPO produces two different types of reactive metabolites. Moreover, metabolism studies using industrial antioxidant DPAs, which are massively produced and imported, have not been reported. Therefore, the potential of DPAs toxicity has not been thoroughly evaluated, nor is it known how relatively harmful different DPAs metabolites are compared to their unmetabolized precursors. Hypotheses: 1) MPO can produce both free radical and hydroxylated metabolites of DPAs. 2) MPO enzymatic activity is a requirement for the toxicity of DPAs. Aims: 1) To characterize the reactive metabolites formed from DPAs by isolated MPO and MPO-positive vs MPO-negative cells. 2) To determine the cellular effects and responses from DPAs in MPO-positive vs MPO-negative cells. 3) To determine damage of specific macromolecule (protein, DNA) intracellular targets by DPAs, and to carry out proteomic and metabolomic studies. 4) To understand the basis of metabolism and toxicity using structure-activity relationships. Significance: This proposal will reveal significant advances in our understanding of how MPO produces different reactive metabolites of DPAs, and evaluate their cellular toxicity pathways. As thousands of tonnes of DPAs being used in certain industrial processes, it is vital to study the effects of DPAs to appreciate their potential hazards. These research findings can contribute to government policymakers regarding the use of DPAs. The application of diverse techniques that involve analytical techniques, mechanistic toxicology, omics technologies, and computational analyses are proposed, which will benefit the training of highly qualified personnel in Canada.

背景资料：二苯胺（DPA）既存在于天然物质中，也存在于合成物质中，DPA衍生物（DPA）具有广泛的工业用途，包括制造染料、橡胶、杀虫剂、药品，并存在于弹药场。由于其广泛使用，DPA也出现在加拿大的废水处理厂和鱼类中。像DPA这样的异生物质通常不具有反应性，但可以被氧化以产生反应性代谢物。后者具有毒理学意义，因为它们会削弱细胞并对其蛋白质和DNA造成损害。该提案的重点是未探索的DPAs代谢途径和随之而来的生物后果。DPA衍生物的普遍使用和环境发生需要这些研究。基本原理：初步研究表明，一些DPA可以形成两种类型的反应性代谢产物：自由基代谢产物和羟基化代谢产物。这些代谢物由血红素蛋白、髓过氧化物酶（MPO）形成，髓过氧化物酶存在于特定的血细胞中。但MPO通常不产生羟基化代谢物，后者可进一步氧化为醌样反应性代谢物。因此，关于MPO如何产生两种不同类型的反应性代谢物存在根本问题。此外，还没有报道使用大量生产和进口的工业抗氧化剂DPAs进行代谢研究。因此，DPAs的潜在毒性尚未得到彻底评价，也不知道不同DPAs代谢物与其未代谢前体相比相对有害。 假设：1）MPO既能产生自由基，又能产生DPAs的羟基化代谢产物。 2)MPO酶活性是DPA毒性的必要条件。目的：1）研究MPO阳性和阴性细胞对DPAs反应性代谢产物的影响。 2)确定MPO阳性与MPO阴性细胞中DPA的细胞效应和反应。 3)确定DPA对特定大分子（蛋白质、DNA）细胞内靶点的损伤，并进行蛋白质组学和代谢组学研究。 4)利用构效关系了解代谢和毒性的基础。 重要性：这一提议将揭示我们对MPO如何产生不同的DPAs反应性代谢物的理解的重大进展，并评估其细胞毒性途径。由于在某些工业过程中使用了数千吨DPA，因此研究DPA的影响以了解其潜在危害至关重要。这些研究结果有助于政府决策者使用DPA。提出了涉及分析技术、机械毒理学、组学技术和计算分析的多种技术的应用，这将有利于加拿大高素质人才的培养。