Effects of p-cresol on oxidative stress, glutathione depletion, and necrosis in HepaRG cells: comparative effects to other uremic toxins and role of p-cresol glucuronide formation

• Author / Creator

  Zhu, Sang

• Background: The toxicological effects of p-cresol have primarily been attributed to its metabolism end products; however, very little human toxicological data are available in the key organ (i.e. the liver) responsible for the generation of these metabolites. We hypothesized that p-cresol is relatively more toxic than the other protein-bound uremic toxins, and that the glucuronidation of p-cresol or the glucuronide end product can lead to toxicities in the human liver. Our objectives were: i) to characterize the concentration- and temporal- effects of p-cresol on toxicity markers; ii) to compare the effects of p-cresol and other protein bound uremic toxins on toxicity markers in HepaRG cells; iii) to determine the metabolic activities of HepaRG cells in the production of major p-cresol conjugated metabolites; and iv) to characterize the effects of exogenously-administered and in-situ generated p-cresol glucuronide in the manifestation of toxicities in HepaRG cells. Methods: The HepaRG cell line was utilized as the experimental model. The toxicity markers were 2’-7’-dichlorofluorescein (DCF; marker of oxidative stress) formation, total cellular glutathione (GSH) concentration, and lactate dehydrogenase (LDH; marker of cellular necrosis) release. The concentrations of p-cresol were measured by ultra-high performance liquid chromatography assay (UPLC). The concentrations of p-cresol sulfate and p-cresol glucuronide were quantified using ultra-high performance liquid chromatography-tandem mass spectrometry assay (UPLC-MS/MS). Results: p-Cresol exposure resulted in concentration- and time-dependent changes in DCF formation, GSH concentration, and LDH release in HepaRG cells at potentially 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 tested markers. Although the exogenous administration of p-cresol sulfate and p-cresol glucuronide generated high intracellular concentrations of these metabolites, both metabolites were significantly 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. The selective attenuation of glucuronidation (without affecting p-cresol sulfate formation, while increasing p-cresol accumulation) using independent chemical inhibitors (i.e. L-borneol, amentoflavone, or diclofenac) consistently resulted in further increases in LDH release associated with p-cresol exposure. Conclusion: These novel data indicated that p-cresol was a relatively potent toxicant, and that glucuronidation was unlikely associated with the manifestation of its toxic effects in HepaRG cells.

• Subjects / Keywords

  • p-cresol
  • p-cresol glucuronide
  • p-cresol sulfate
  • uremic toxins
  • HepaRG

• Graduation date

  Fall 2021

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-t7pz-ka66

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.