Mechanisms of Diphenylamine Xenobiotic Reactivity and Toxicity

背景：二苯胺(DPA)既有天然的，也有人工合成的，DPA的衍生物在工业上有广泛的用途，包括在染料、橡胶、杀虫剂、药品的制造中，并在弹药现场发现。由于DPA的广泛使用，加拿大的废水处理厂和鱼类中也存在DPA。像DPA这样的异生物质通常不是反应性的，但可能被氧化产生反应性代谢物。后者引起毒理学关注，因为它们会削弱细胞，并对细胞的蛋白质和DNA造成损害。这项建议侧重于尚未探索的DPA代谢途径和随之而来的生物学后果。DPA衍生物的无处不在的使用和环境中的存在使这些研究成为必要。理论基础：初步研究表明，一些DPA可以形成两种类型的反应性代谢物：自由基代谢物和羟基代谢物。这些代谢物是由特定血细胞中发现的血红素蛋白髓过氧化物酶(MPO)形成的。然而，MPO通常不会产生羟基化的代谢物，后者可以进一步被氧化成类苯醌的反应性代谢物。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性的问题。此外，大量生产和进口的工业抗氧化剂DPA的代谢研究尚未见报道。因此，DPA的潜在毒性还没有得到彻底的评估，也不知道相对有害的不同DPA代谢物与其未代谢前体的比较情况。假设：1)MPO可产生DPAs的自由基代谢产物和羟化代谢产物。2)MPO酶活性是DPA毒性的必要条件。目的：1)用分离的MPO细胞和MPO阳性细胞与MPO阴性细胞比较DPA形成的反应性代谢产物。2)研究DPA对MPO阳性细胞和MPO阴性细胞的细胞效应和反应。3)确定DPAs对细胞内特定大分子(蛋白质、DNA)的损伤作用，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。意义：这项提议将揭示我们在理解MPO如何产生DPA的不同反应性代谢物方面取得的重大进展，并评估它们的细胞毒性途径。由于在某些工业过程中使用了数以千计的DPA，因此研究DPA的影响以认识其潜在的危害是至关重要的。这些研究结果可以为政府政策制定者在使用DPA方面做出贡献。提出了分析技术、机械毒理学、组学技术和计算分析等多种技术的应用，这将有助于加拿大高素质人才的培养。

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.