Effects of p-Cresol on Oxidative Stress, Glutathione Depletion, and Necrosis in HepaRG Cells: Comparisons to Other Uremic Toxins and the Role of p-Cresol Glucuronide Formation.

Sang Zhu,Yan Rong,Tony K L Kiang

doi:10.3390/pharmaceutics13060857

Abstract

The toxicological effects of p-cresol have primarily been attributed to its metabolism products; however, very little human data are available in the key organ (i.e., liver) responsible for the generation of these metabolites. Experiments were conducted in HepaRG cells utilizing the following markers of cellular toxicity: 2′-7′-dichlorofluorescein (DCF; oxidative stress) formation, total cellular glutathione (GSH) concentration, and lactate dehydrogenase (LDH; cellular necrosis) release. Concentrations of p-cresol, p-cresol sulfate, and p-cresol glucuronide were determined using validated assays. p-Cresol exposure resulted in concentration- and time-dependent changes in DCF (EC50 = 0.64 ± 0.37 mM at 24 h of exposure) formation, GSH (EC50 = 1.00 ± 0.07 mM) concentration, and LDH (EC50 = 0.85 ± 0.14 mM) release at toxicologically relevant conditions. p-Cresol was also relatively more toxic than 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid, indole-3-acetic acid, indoxyl sulfate, kynurenic acid, and hippuric acid on all markers. Although the exogenous administration of p-cresol sulfate and p-cresol glucuronide generated high intracellular concentrations of these metabolites, both metabolites were less toxic compared to p-cresol at equal-molar conditions. Moreover, p-cresol glucuronide was the predominant metabolite generated in situ from p-cresol exposure. Selective attenuation of glucuronidation (without affecting p-cresol sulfate formation, while increasing p-cresol accumulation) using independent chemical inhibitors (i.e., 0.75 mM l-borneol, 75 µM amentoflavone, or 100 µM diclofenac) consistently resulted in further increases in LDH release associated with p-cresol exposure (by 28.3 ± 5.3%, 30.0 ± 8.2% or 27.3 ± 6.8%, respectively, compared to p-cresol treatment). These novel data indicated that p-cresol was a relatively potent toxicant, and that glucuronidation was unlikely to be associated with the manifestation of its toxic effects in HepaRG cells.

Highlights

Introduction pCresol, a part of the protein-bound uremic toxin milieu, is derived from colonic amino acids tyrosine and phenylalanine [1]
The toxicological effects of p-cresol have primarily been attributed to its metabolism end products [2], which are considered relatively significant toxic species amongst a large variety of uremic toxins known to date [2,3]
Positive Control and Concentration/Time-Course Responses of p-Cresol in HepaRG Cells t-BOOH was utilized as an assay-based positive control for each toxicity marker [25,26,27]

Summary

Introduction

A part of the protein-bound uremic toxin milieu, is derived from colonic amino acids tyrosine and phenylalanine [1]. The toxicological effects of p-cresol have primarily been attributed to its metabolism end products [2], which are considered relatively significant toxic species amongst a large variety of uremic toxins known to date [2,3]. Sulfonation is the predominant pathway, evidenced by the relatively higher plasma total concentrations of p-cresol sulfate in various clinical reports [5,6,7,8,9,10]. Due to differences in protein binding, the biologically active unbound concentrations of p-cresol glucuronide and p-cresol sulfate are comparable [6,7,8,9], and higher plasma concentrations of the free glucuronide have been documented [10]. A shift to the production of p-cresol glucuronide from p-cresol sulfate has been observed in patients with advanced kidney disease [5,7], 4.0/).

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