Mechanisms of Diphenylamine Xenobiotic Reactivity and Toxicity

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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.