Disruption of NaS1 sulfate transport function in mice leads to enhanced acetaminophen-induced hepatotoxicity (original) (raw)
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Drug Metabolism and Disposition
Cytosolic sulfotransferases (SULTs), including SULT1A, SULT1B, SULT1E and SULT2A isoforms, play noteworthy roles in xenobiotic and endobiotic metabolism. We quantified the protein abundance of SULT1A1, SULT1A3, SULT1B1 and SULT2A1 in human liver cytosol samples (n=194) by LC-MS/MS proteomics. The data were analyzed for their association with age, sex, genotype, and ethnicity of the donors. SULT1A1, SULT1B1, and SULT2A1 showed significant age-dependent protein abundance, whereas SULT1A3 was invariable across 0-70 years. The respective mean abundance of SULT1A1, SULT1B1, and SULT2A1 in neonatal samples was 24, 19 and 38% of the adult levels. Interestingly, unlike UDPglucuronosyltransferases (UGTs) and cytochrome P450 enzymes (CYPs), SULT1A1 and SULT2A1 showed the highest abundance during early childhood (1 to <6 years), which gradually decreased by ~40% in adolescents and adults. SULT1A3 and SULT1B1 abundances were significantly lower in African Americans as compared to Caucasians. Multiple linear regression analysis further confirmed the association of abundance of SULTs with age, ethnicity, and genotype. To demonstrate clinical application of the characteristic SULT ontogeny profiles, we developed and validated a proteomics-informed physiologically based pharmacokinetic (PBPK) model. The latter confirmed the higher fractional contribution of sulfation over glucuronidation in the metabolism of acetaminophen in children. The study thus highlights that ontogeny-based age-dependent fractional contribution (fm) of individual drug metabolizing enzymes has better potential in prediction of drug-drug interactions and the effect of genetic polymorphisms in the pediatric population.
Pharmacological Reports, 2018
Background-Non-opioid and opioid analgesics, as over-the-counter or prescribed medications, are widely used for the management of a diverse array of pathophysiological conditions. Previous studies have demonstrated the involvement of human cytosolic sulfotransferase (SULT) SULT1A1 in the sulfation of acetaminophen, O-desmethylnaproxen (O-DMN), and tapentadol. The current study was designed to investigate the impact of single nucleotide polymorphisms (SNPs) of the human SULT1A1 gene on the sulfation of these analgesic compounds by SULT1A1 allozymes. Methods-Human SULT1A1 genotypes were identified by database search. cDNAs corresponding to nine SULT1A1 nonsynonymous missense coding SNPs (cSNPs) were generated by site-directed mutagenesis. Recombinant wild-type and SULT1A1 allozymes were bacterially expressed and affinity-purified. Purified SULT1A1 allozymes were analyzed for sulfation activity using an established assay procedure. Results-Compared with the wild-type enzyme, SULT1A1 allozymes were shown to display differential sulfating activities toward three analgesic compounds, acetaminophen, Odesmethylnaproxen (O-DMN), and tapentadol, as well as the prototype substrate 4NP. Conclusion-Results obtained indicated clearly the impact of genetic polymorphisms on the drug-sulfation activity of SULT1A1 allozymes. Such information may contribute to a better understanding about the differential metabolism of acetaminophen, O-DMN, and tapentadol in individuals with different SULT1A1 genotypes.
Metabolomic markers predictive of hepatic adaptation to therapeutic dosing of acetaminophen
Background: Drug induced liver injury (DILI) remains a prominent global issue and acetaminophen (APAP) overdose represents a common cause of hepatic injury and DILI. Transient alanine aminotransferase (ALT) elevations have been documented while adhering to recommended daily dosing. However, no metabolites have been identified in pre-treatment samples predicting which patients will develop these transient increases. Methods: This was a secondary analysis of samples collected from a parent study describing the course of ALT levels in subjects receiving therapeutic APAP dosing. Two hundred and four subjects recruited from Denver, Colorado received 4 g APAP/daily for at least 16 days. Subjects were grouped by ALT at any monitored time point above 60 units/L (n ¼ 25) vs. no increase (n ¼ 179). Serum samples from days 0, 7, 16, and 31 were run on ultra-high performance liquid chromatography mass spectrometry. We report the metabolomic results of samples analyzed prior to APAP administration and over time. Significant changes in metabolite and demographic variable expressions were explored using t-tests with false discovery rate correction, chi square, and partial least squares discriminant analyses. Results: Within pre-treatment day 0 samples, allantoate and ornithine were significantly elevated in subjects of the ALT elevation group (p ¼ .032). Baseline ALT (p ¼ .011) and alkaline phosphatase (p ¼ .006) were also significant. These metabolites were significant independent of race, ethnicity, gender, or BMI. Conclusions: Allantoate and ornithine are directly involved in pathways related to nitrogen release and urea production. Further investigation into alterations in the glutathione metabolism and urea cycle pathways may lead to a greater understanding of the mechanisms associated with hepatic adaptation for a variety of pharmaceuticals.
Increased resistance to acetaminophen hepatotoxicity in mice lacking glutathione S -transferase Pi
Proceedings of the National Academy of Sciences, 2000
Overdose of acetaminophen, a widely used analgesic drug, can result in severe hepatotoxicity and is often fatal. This toxic reaction is associated with metabolic activation by the P450 system to form a quinoneimine metabolite, N -acetyl- p -benzoquinoneimine (NAPQI), which covalently binds to proteins and other macromolecules to cause cellular damage. At low doses, NAPQI is efficiently detoxified, principally by conjugation with glutathione, a reaction catalyzed in part by the glutathione S -transferases (GST), such as GST Pi. To assess the role of GST in acetaminophen hepatotoxicity, we examined acetaminophen metabolism and liver damage in mice nulled for GstP ( GstP1/P2 (−/−) ). Contrary to our expectations, instead of being more sensitive, GstP null mice were highly resistant to the hepatotoxic effects of this compound. No significant differences between wild-type ( GstP1/P2 (+/+) ) mice and GstP1/P2 (−/−) nulls in either the rate or route of metabolism, particularly to glutathio...
Archives of Biochemistry and Biophysics, 2018
Sulfoconjugation has been shown to be critically involved in the metabolism of acetaminophen (APAP), morphine, tapentadol and O-desmethyl tramadol (O-DMT). The objective of this study was to investigate the effects of single nucleotide polymorphisms (SNPs) of human SULT1A3 and SULT1A4 genes on the sulfating activity of SULT1A3 allozymes toward these analgesic compounds. Twelve non-synonymous coding SNPs (cSNPs) of SULT1A3/SULT1A4 were investigated, and the corresponding cDNAs were generated by site-directed mutagenesis. SULT1A3 allozymes, bacterially expressed and purified, exhibited differential sulfating activity toward each of the four analgesic compounds tested as substrates. Kinetic analyses of SULT1A3 allozymes further revealed significant differences in binding affinity and catalytic activity toward the four analgesic compounds. Collectively, the results derived from the current study showed clearly the impact of cSNPs of the coding genes, SULT1A3 and SULT1A4, on the sulfating activity of the coded SULT1A3 allozymes toward the tested analgesic compounds. These findings may have implications in the pharmacokinetics as well as the toxicity profiles of these analgesics administered in individuals with distinct SULT1A3 and/or SULT1A4 genotypes.
Inflammopharmacology, 1998
A1-AIi AK, A1-Mustafa ZH, Qaw FS, Fayz M. Paracetamol-induced hepatotoxicity: lack of enhancement of the hepatoprotective effect of N-acetylcysteine by sodium sulphate. Inflammopharmacology. 1998;6:235-241 The potential role of sodium sulphate in possible enhancement of the hepatoprotective action of Nacetylcysteine (NAC) in paracetamol (PCM) overdose was examined. The effects of sodium sulphate (200 mg/kg) in combination with NAC (400 mg/kg) admininstered intraperitoneally 2 h post-PCM dose, on mortality rate and plasma activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were investigated in mice 24 h after receiving a single oral dose of 400 mg/kg PCM. In addition, the effect on the mortality rate of PCM-treated animals of co-administering 400 mg/ kg sodium sulphate with NAC (200 or 400 mg/kg) was also studied. NAC alone caused a marked reduction in the mortality rate of PCM-treated mice and a sharp drop in their plasma AST and ALT activities to near normal values. However, no additional reduction in plasma levels of AST and ALT was observed when sodium sulphate was co-administered with NAC. Similarly, sodium sulphate (200 mg/kg) administered alone to PCM-treated mice had no effect on the high mortality rate or the elevation in plasma AST and ALT activities observed in these animals. Furthermore, increasing the dose of sodium sulphate to 400 mg/kg did not influence the mortality rate. It is therefore concluded that sodium sulphate neither protects against paracetamol-induced hepatotoxicity nor enhances the hepatoprotective action of N-acetylcysteine.
PLoS ONE, 2011
Background: Glutathione S-transferases (GSTs) metabolize drugs and xenobiotics. Yet despite high protein sequence homology, expression of p-class GSTs, the most abundant of the enzymes, varies significantly between species. In mouse liver, hepatocytes exhibit high mGstp expression, while in human liver, hepatocytes contain little or no hGSTP1 mRNA or hGSTP1 protein. p-class GSTs are known to be critical determinants of liver responses to drugs and toxins: when treated with high doses of acetaminophen, mGstp1/2+/+ mice suffer marked liver damage, while mGstp1/22/2 mice escape liver injury. Methodology/Principal Findings: To more faithfully model the contribution of p-class GSTs to human liver toxicology, we introduced hGSTP1, with its exons, introns, and flanking sequences, into the germline of mice carrying disrupted mGstp genes. In the resultant hGSTP1+mGstp1/22/2 strain, p-class GSTs were regulated differently than in wild-type mice. In the liver, enzyme expression was restricted to bile duct cells, Kupffer cells, macrophages, and endothelial cells, reminiscent of human liver, while in the prostate, enzyme production was limited to basal epithelial cells, reminiscent of human prostate. The human patterns of hGSTP1 transgene regulation were accompanied by human patterns of DNA methylation, with bisulfite genomic sequencing revealing establishment of an unmethylated CpG island sequence encompassing the gene promoter. Unlike wild-type or mGstp1/22/2 mice, when hGSTP1+mGstp1/22/2 mice were overdosed with acetaminophen, liver tissues showed limited centrilobular necrosis, suggesting that p-class GSTs may be critical determinants of toxininduced hepatocyte injury even when not expressed by hepatocytes. Conclusions: By recapitulating human p-class GST expression, hGSTP1+mGstp1/22/2 mice may better model human drug and xenobiotic toxicology.
Altered Disposition of Acetaminophen in Nrf2-null and Keap1-knockdown Mice
Toxicological Sciences, 2009
Acetaminophen (AA) is a widely used antipyretic drug that causes hepatotoxicity at high doses. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that mitigates electrophilic stress from AA by inducing genes, such as NAD(P)H:quinone oxidoreductase 1 (Nqo1), multidrug resistance-associated proteins (Mrps), and glutathione (GSH) synthesis enzymes. To determine whether Nrf2 activation alters the biotransformation and excretion of AA, male wild-type, Nrf2-null, and Keap1 (Kelch-like ECH-associated protein 1)-knockdown (Keap1-kd) mice (which have increased activation of Nrf2) were administered a single subtoxic dose of AA (50 mg/kg, iv), after which, AA and its metabolites (AA-glucuronide [AA-GLUC]; AAsulfate [AA-SULF]; AA-glutathione [AA-GSH]) were quantified in plasma, bile, and liver. AA-GLUC concentrations were reduced in plasma and elevated in livers of Nrf2-null mice due to decreased glucuronidation activity and lower expression of the basolateral efflux transporter Mrp3. In contrast, Keap1-kd mice had higher plasma and lower hepatic AA-GLUC concentrations, due to higher Mrp3 expression. Lower glucuronidation activity of Nrf2-null mice increased the proportion of AA available for sulfation, resulting in elevated AA-SULF concentrations in plasma, bile, and liver. Decreased AA-sulfation activity in Keap1-kd mice resulted in lower AA-SULF concentrations. AA-GSH conjugates were increased in Nrf2-null mice and tended to be lower in Keap1-kd mice. Furthermore, Nqo1, an enzyme capable of detoxifying the reactive intermediate of AA metabolism, N-acetyl-p-benzoquinone imine (NAPQI), had 85% lower activity in Nrf2-null mice and 415% higher activity in Keap1-kd mice relative to wild-type. In conclusion, lack of Nrf2 results in decreased AA glucuronidation, leading to increased AA available for NAPQI formation and decreased efflux of AA-GLUC via Mrp3; however, activation of Nrf2, as in Keap1-kd mice, results in decreased sulfotransferase activity, decreased AA-SULF formation, and enhanced elimination of AA-GLUC due to increased expression of Mrp3.
PROTEOMICS, 2003
GST pi (GSTP) is a member of the glutathione S-transferase (EC 2.5.1.18; GST) family of enzymes that catalyse the conjugation of electrophilic species with reduced glutathione and thus play an important role in the detoxification of electrophilic metabolites. Deletion of GSTP in mice has previously been shown to lead to enhanced susceptibility to chemical-induced skin carcinoma, consistent with its known metabolic functions. A decreased susceptibility to paracetamol hepatotoxicity has also been observed, which has not been fully explained. One possibility is that deletion of the GSTP gene locus results in compensatory changes in other proteins involved in defence against chemical stress. We have therefore used complementary protein expression profiling techniques to perform a systematic comparison of the protein expression profiles of livers from GSTP null and wild-type mice. Analysis of liver proteins by two-dimensional electrophoresis confirmed the absence of GSTP in null mice whereas GSTP represented 3-5% of soluble protein in livers from wild-type animals. There was a high degree of quantitative and qualitative similarity in other liver proteins between GSTP null and wild-type mice. There was no evidence that the absence of GSTP in null animals resulted in enhanced expression of other GST isoforms in the null mice (GST alpha, 1.48%, GST mu, 1.68% of resolved proteins) compared with the wild-type animals (GST alpha, 1.50%, GST mu, 1.40%). In contrast, some members of the thiol specific antioxidant family of proteins, notably antioxidant protein 2 and thioredoxin peroxidases, were expressed at a higher level in the GSTP null mouse livers. These changes presumably reflect the recently described role of GSTP in cell signalling and may underlie the protection against paracetamol toxicity seen in these animals.