Determination of p-phenylenediamine and its metabolites by liquid chromatography/electrochemistry
Abstract
The compound p-phenylenediamine (p-PDA) is a suspected carcinogen and mutagen commonly used in hair dyes. Despite its widespread usage, the metabolism of p-PDA is largely unexplored. It is proposed that p-PDA will be metabolized by the liver to benzoquinonediimine, which by analogy with a similar metabolite of aceteminophen, may bind covalently with protein and cause toxicity. Liquid chromatography/electrochemistry (LCEC) was used to investigate the in vitro metabolism of p-PDA by microsomes. Strong evidence for the formation of benzoquinonediimine as a metabolite of p-phenylenediamine was obtained by the detection of the glutathione conjugate of p-phenylenediamine (p-PDA-GSH) in mouse liver microsomal incubations. Hydrodynamic voltammograms support identification of the metabolite. They correlate well with the product of glutathione and oxidized p-phenylenediamine prepared in a novel synthesis using vitreous carbon as a mild oxidizing agent. No evidence was observed for the production of ring-hydroxylated metabolites. The mechanism of the production of p-PDA-GSH was investigated using specific inhibitors and inducers. Catalase and superoxide dismutase showed no true inhibitory effects, thereby eliminating peroxidase, H$\sb2$O$\sb2$, and superoxide ion. Glutathione transferase proved ineffective in producing p-PDA-GSH. Flavin monomine oxidase was eliminated after CO showed definite inhibition. The involvement of cytochrome P-450 is supported by CO, metyrapone, and cysteamine dose-dependent inhibition and the requirement of NADPH and O$\sb2$. However, pretreatment with phenobarbital did not increase p-PDA-GSH production. Apparent kinetic parameters for the production of p-PDA-GSH were determined to be K$\sb{\rm m} = 1.36 \times 10\sp{-4}$ M and V$\sb{\rm max} = 6.87 \times 10\sp{-8}$ M/s for non-induced mouse liver microsomes. In an attempt to investigate the binding of p-PDA to protein, an alternative to techniques utilizing radiolabelling was developed. After hydrolyzing protein containing the bound electroactive xenobiotic of interest, amino acid conjugates of the xenobiotic can be detected by LCEC, thereby bypassing the synthesis of a radiolabelled compound. This technique was applied to determination of protein-bound hydroquinone by detecting the cysteine conjugate of hydroquinone after acid hydrolysis of a microsomal incubation.
Degree
Ph.D.
Advisors
Kissinger, Purdue University.
Subject Area
Analytical chemistry
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