Evaluation of In Situ Generated Valproyl 1-<i>O</i>-<i>β</i>-Acyl Glucuronide in Valproic Acid Toxicity in Sandwich-Cultured Rat Hepatocytes

Surendradoss, Jayakumar; Chang, Thomas K. H.; Abbott, Frank S.

doi:10.1124/dmd.114.059352

Cited by 10 publications

(6 citation statements)

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“…Because the rates of MPA depletion and MPAG formation were linear between 0 and 12 h, 3 or 6 h (suitable for modulatory experiments) was selected as the optimum incubation times in subsequent experiments. Only concentrations in the culture medium were determined because the majority of analytes were found in the medium (vs. cell lysates) in similar cellular systems (Surendradoss, Chang, & Abbott, ). Additionally, the sum of concentrations of MPA and MPAG detected in the cell culture supernatant in our model (at various incubation times, Figure ) was close to that of the original substrate (i.e.…”

Section: Resultsmentioning

confidence: 99%

Development and validation of a sensitive liquid‐chromatography tandem mass spectrometry assay for mycophenolic acid and metabolites in HepaRG cell culture: Characterization of metabolism interactions between p‐cresol and mycophenolic acid

Rong

Kiang

2019

Biomedical Chromatography

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Mycophenolic acid (MPA), a frequently used immunosuppressant, exhibits large interpatient pharmacokinetic variability. This study (a) developed and validated a sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for MPA and metabolites [MPA glucuronide (MPAG) and acyl-glucuronide (AcMPAG)] in the culture medium of HepaRG cells; and (b) characterized the metabolism interaction between MPA and p-cresol (a common uremic toxin) in this in vitro model as a potential mechanism of pharmacokinetic variability. Chromatographic separation was achieved with a C 18 column (4.6 × 250 mm,5 μm) using a gradient elution with water and methanol (with 0.1% formic acid and 2 mM ammonium acetate). A dual ion source ionization mode with positive multiple reaction monitoring was utilized. Multiple reaction monitoring mass transitions (m/z) were: MPA (320.95 → 207.05), MPAG (514.10 → 303.20) and AcMPAG (514.10 → 207.05). MPA-d 3 (323.95 → 210.15) and MPAG-d 3 (517.00 → 306.10) were utilized as internal standards. The calibration curves were linear from 0.00467 to 3.2 μg/mL for MPA/MPAG and from 0.00467 to 0.1 μg/mL for AcMPAG. The assay was validated based on industry standards. p-Cresol inhibited MPA glucuronidation (IC 50 ≈ 55 μM) and increased MPA concentration (up to >2-fold) at physiologically relevant substrate-inhibitor concentrations (n = 3). Our findings suggested that fluctuations in p-cresol concentrations might be in part responsible for the large pharmacokinetic variability observed for MPA in the clinic.

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Section: Resultsmentioning

confidence: 99%

Development and validation of a sensitive liquid‐chromatography tandem mass spectrometry assay for mycophenolic acid and metabolites in HepaRG cell culture: Characterization of metabolism interactions between p‐cresol and mycophenolic acid

Rong

Kiang

2019

Biomedical Chromatography

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“…Additionally, primary human hepatocytes in sandwich culture treated with chlorpromazine showed changes in the genetic signature related to fibrosis, inflammation, necrosis and steatosis after short-term and long-term chlorpromazine treatment [236]. Primary rat hepatocytes, cultured between two layers of collagen, showed lipid droplets following incubation with VPA, amiodarone, tetracycline or tamoxifen [237].…”

Section: Models In Nafld Researchmentioning

confidence: 99%

Strategies, models and biomarkers in experimental non-alcoholic fatty liver disease research

Willebrords¹,

Pereira

Maes³

et al. 2015

Progress in Lipid Research

142

130

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Non-alcoholic fatty liver disease encompasses a spectrum of liver diseases, including simple steatosis, steatohepatitis, liver fibrosis and cirrhosis and hepatocellular carcinoma. Non-alcoholic fatty liver disease is currently the most dominant chronic liver disease in Western countries due to the fact that hepatic steatosis is associated with insulin resistance, type 2 diabetes mellitus, obesity, metabolic syndrome and drug-induced injury. A variety of chemicals, mainly drugs, and diets is known to cause hepatic steatosis in humans and rodents. Experimental non-alcoholic fatty liver disease models rely on the application of a diet or the administration of drugs to laboratory animals or the exposure of hepatic cell lines to these drugs. More recently, genetically modified rodents or zebrafish have been introduced as non-alcoholic fatty liver disease models. Considerable interest now lies in the discovery and development of novel non-invasive biomarkers of non-alcoholic fatty liver disease, with specific focus on hepatic steatosis. Experimental diagnostic biomarkers of non-alcoholic fatty liver disease, such as (epi)genetic parameters and '-omics'-based read-outs are still in their infancy, but show great promise. In this paper, the array of tools and models for the study of liver steatosis is discussed. Furthermore, the current state-of-art regarding experimental biomarkers such as epigenetic, genetic, transcriptomic, proteomic and metabonomic biomarkers will be reviewed.

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“…Only non-parametric testing with more stringent thresholds was utilized. The Mann-Whitney rank sum test was used to compare two groups, and the Kruskal-Wallis analysis of variance on ranks followed by Student-Newman-Keuls post hoc test were used to compare multiple groups (SigmaStat 3.5, Systat Software, San Jose, CA, USA) [26,27,38]. A p value < 0.05 was deemed a priori as the threshold for significance.…”

Section: Discussionmentioning

confidence: 99%

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

2021

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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.

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Evaluation of In Situ Generated Valproyl 1-O-β-Acyl Glucuronide in Valproic Acid Toxicity in Sandwich-Cultured Rat Hepatocytes

Cited by 10 publications

References 46 publications

Development and validation of a sensitive liquid‐chromatography tandem mass spectrometry assay for mycophenolic acid and metabolites in HepaRG cell culture: Characterization of metabolism interactions between p‐cresol and mycophenolic acid

Development and validation of a sensitive liquid‐chromatography tandem mass spectrometry assay for mycophenolic acid and metabolites in HepaRG cell culture: Characterization of metabolism interactions between p‐cresol and mycophenolic acid

Strategies, models and biomarkers in experimental non-alcoholic fatty liver disease research

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

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