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Title: Bile acid-induced necrosis in primary human hepatocytes and in patients with obstructive cholestasis

Abstract

Accumulation of bile acids is a major mediator of cholestatic liver injury. Recent studies indicate bile acid composition between humans and rodents is dramatically different, as humans have a higher percent of glycine conjugated bile acids and increased chenodeoxycholate content, which increases the hydrophobicity index of bile acids. This increase may lead to direct toxicity that kills hepatocytes, and promotes inflammation. To address this issue, this study assessed how pathophysiological concentrations of bile acids measured in cholestatic patients affected primary human hepatocytes. Individual bile acid levels were determined in serum and bile by UPLC/QTOFMS in patients with extrahepatic cholestasis with, or without, concurrent increases in serum transaminases. Bile acid levels increased in serum of patients with liver injury, while biliary levels decreased, implicating infarction of the biliary tracts. To assess bile acid-induced toxicity in man, primary human hepatocytes were treated with relevant concentrations, derived from patient data, of the model bile acid glycochenodeoxycholic acid (GCDC). Treatment with GCDC resulted in necrosis with no increase in apoptotic parameters. This was recapitulated by treatment with biliary bile acid concentrations, but not serum concentrations. Marked elevations in serum full-length cytokeratin-18, high mobility group box 1 protein (HMGB1), and acetylated HMGB1 confirmed inflammatory necrosismore » in injured patients; only modest elevations in caspase-cleaved cytokeratin-18 were observed. These data suggest human hepatocytes are more resistant to human-relevant bile acids than rodent hepatocytes, and die through necrosis when exposed to bile acids. These mechanisms of cholestasis in humans are fundamentally different to mechanisms observed in rodent models. - Highlights: • Cholestatic liver injury is due to cytoplasmic bile acid accumulation in hepatocytes. • Primary human hepatocytes are resistant to BA-induced injury compared to rodents. • Primary human hepatocytes largely undergo necrosis in response to BA toxicity. • Cholestatic liver injury in vivo is predominantly necrotic with minor apoptosis. • Rodent models of bile acid toxicity may not recapitulate the injury in man.« less

Authors:
;  [1]; ;  [2];  [3];  [1]; ; ;  [4];  [5]; ;  [2];  [1];  [3];  [1]
  1. Department of Pharmacology, Toxicology & Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States)
  2. MRC Centre for Drug Safety Science, Department of Molecular and Clinical Pharmacology, Institute of Translational Medicine, University of Liverpool, Liverpool (United Kingdom)
  3. Department of Internal Medicine, University of Kansas Medical Center, Kansas City, KS (United States)
  4. Department of Surgery, University of Kansas Medical Center, Kansas City, KS (United States)
  5. Department of Pathology, University of Kansas Medical Center, Kansas City, KS (United States)
Publication Date:
OSTI Identifier:
22465712
Resource Type:
Journal Article
Resource Relation:
Journal Name: Toxicology and Applied Pharmacology; Journal Volume: 283; Journal Issue: 3; Other Information: Copyright (c) 2015 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; AMINOTRANSFERASES; APOPTOSIS; BILE; BILIARY TRACT; CHOLIC ACID; CONCENTRATION RATIO; GLYCINE; HUMAN POPULATIONS; INFLAMMATION; INJURIES; LEVELS; LIVER; LIVER CELLS; NECROSIS; PATIENTS; POLYPEPTIDES; RODENTS; TOXICITY

Citation Formats

Woolbright, Benjamin L., Dorko, Kenneth, Antoine, Daniel J., Clarke, Joanna I., Gholami, Parviz, Li, Feng, Kumer, Sean C., Schmitt, Timothy M., Forster, Jameson, Fan, Fang, Jenkins, Rosalind E., Park, B. Kevin, Hagenbuch, Bruno, Olyaee, Mojtaba, and Jaeschke, Hartmut, E-mail: hjaeschke@kumc.edu. Bile acid-induced necrosis in primary human hepatocytes and in patients with obstructive cholestasis. United States: N. p., 2015. Web. doi:10.1016/J.TAAP.2015.01.015.
Woolbright, Benjamin L., Dorko, Kenneth, Antoine, Daniel J., Clarke, Joanna I., Gholami, Parviz, Li, Feng, Kumer, Sean C., Schmitt, Timothy M., Forster, Jameson, Fan, Fang, Jenkins, Rosalind E., Park, B. Kevin, Hagenbuch, Bruno, Olyaee, Mojtaba, & Jaeschke, Hartmut, E-mail: hjaeschke@kumc.edu. Bile acid-induced necrosis in primary human hepatocytes and in patients with obstructive cholestasis. United States. doi:10.1016/J.TAAP.2015.01.015.
Woolbright, Benjamin L., Dorko, Kenneth, Antoine, Daniel J., Clarke, Joanna I., Gholami, Parviz, Li, Feng, Kumer, Sean C., Schmitt, Timothy M., Forster, Jameson, Fan, Fang, Jenkins, Rosalind E., Park, B. Kevin, Hagenbuch, Bruno, Olyaee, Mojtaba, and Jaeschke, Hartmut, E-mail: hjaeschke@kumc.edu. Sun . "Bile acid-induced necrosis in primary human hepatocytes and in patients with obstructive cholestasis". United States. doi:10.1016/J.TAAP.2015.01.015.
@article{osti_22465712,
title = {Bile acid-induced necrosis in primary human hepatocytes and in patients with obstructive cholestasis},
author = {Woolbright, Benjamin L. and Dorko, Kenneth and Antoine, Daniel J. and Clarke, Joanna I. and Gholami, Parviz and Li, Feng and Kumer, Sean C. and Schmitt, Timothy M. and Forster, Jameson and Fan, Fang and Jenkins, Rosalind E. and Park, B. Kevin and Hagenbuch, Bruno and Olyaee, Mojtaba and Jaeschke, Hartmut, E-mail: hjaeschke@kumc.edu},
abstractNote = {Accumulation of bile acids is a major mediator of cholestatic liver injury. Recent studies indicate bile acid composition between humans and rodents is dramatically different, as humans have a higher percent of glycine conjugated bile acids and increased chenodeoxycholate content, which increases the hydrophobicity index of bile acids. This increase may lead to direct toxicity that kills hepatocytes, and promotes inflammation. To address this issue, this study assessed how pathophysiological concentrations of bile acids measured in cholestatic patients affected primary human hepatocytes. Individual bile acid levels were determined in serum and bile by UPLC/QTOFMS in patients with extrahepatic cholestasis with, or without, concurrent increases in serum transaminases. Bile acid levels increased in serum of patients with liver injury, while biliary levels decreased, implicating infarction of the biliary tracts. To assess bile acid-induced toxicity in man, primary human hepatocytes were treated with relevant concentrations, derived from patient data, of the model bile acid glycochenodeoxycholic acid (GCDC). Treatment with GCDC resulted in necrosis with no increase in apoptotic parameters. This was recapitulated by treatment with biliary bile acid concentrations, but not serum concentrations. Marked elevations in serum full-length cytokeratin-18, high mobility group box 1 protein (HMGB1), and acetylated HMGB1 confirmed inflammatory necrosis in injured patients; only modest elevations in caspase-cleaved cytokeratin-18 were observed. These data suggest human hepatocytes are more resistant to human-relevant bile acids than rodent hepatocytes, and die through necrosis when exposed to bile acids. These mechanisms of cholestasis in humans are fundamentally different to mechanisms observed in rodent models. - Highlights: • Cholestatic liver injury is due to cytoplasmic bile acid accumulation in hepatocytes. • Primary human hepatocytes are resistant to BA-induced injury compared to rodents. • Primary human hepatocytes largely undergo necrosis in response to BA toxicity. • Cholestatic liver injury in vivo is predominantly necrotic with minor apoptosis. • Rodent models of bile acid toxicity may not recapitulate the injury in man.},
doi = {10.1016/J.TAAP.2015.01.015},
journal = {Toxicology and Applied Pharmacology},
number = 3,
volume = 283,
place = {United States},
year = {Sun Mar 15 00:00:00 EDT 2015},
month = {Sun Mar 15 00:00:00 EDT 2015}
}
  • 3{alpha}-6{alpha}-Dihydroxy-7{alpha}-fluoro-5{beta}-cholanoate (UPF-680), the 7{alpha}-fluorine analog of hyodeoxycholic acid (HDCA), was synthesized to improve bioavailability and stability of ursodeoxycholic acid (UDCA). Acute rat biliary fistula and chronic cholestasis induced by 17{alpha}-ethynyl-estradiol (17EE) models were used to study and compare the effects of UPF-680 (dose range 0.6-6.0 {mu}mol/kg min) with UDCA on bile flow, biliary bicarbonate (HCO{sub 3} {sup -}), lipid output, biliary bile acid composition, hepatic enzymes and organic anion pumps. In acute infusion, UPF-680 increased bile flow in a dose-related manner, by up to 40.9%. Biliary HCO{sub 3} {sup -} output was similarly increased. Changes were observed in phospholipid secretionmore » only at the highest doses. Treatment with UDCA and UPF-680 reversed chronic cholestasis induced by 17EE; in this model, UDCA had no effect on bile flow in contrast to UPF-680, which significantly increased bile flow. With acute administration of UPF-680, the biliary bile acid pool became enriched with unconjugated and conjugated UPF-680 (71.7%) at the expense of endogenous cholic acid and muricholic isomers. With chronic administration of UPF-680 or UDCA, the main biliary bile acids were tauro conjugates, but modification of biliary bile acid pool was greater with UPF-680. UPF-680 increased the mRNA for cytochrome P450 7A1 (CYP7A1) and cytochrome P450 8B (CYP8B). Both UDCA and UPF-680 increased the mRNA for Na{sup +} taurocholate co-transporting polypeptide (NCTP). In conclusion, UPF-680 prevented 17EE-induced cholestasis and enriched the biliary bile acid pool with less detergent and cytotoxic bile acids. This novel fluorinated bile acid may have potential in the treatment of cholestatic liver disease.« less
  • Intrahepatic cholestasis is a clinical syndrome with systemic and intrahepatic accumulation of excessive toxic bile acids that ultimately cause hepatobiliary injury. Appropriate regulation of bile acids in hepatocytes is critically important for protection against liver injury. In the present study, we characterized the protective effect of alisol B 23-acetate (AB23A), a natural triterpenoid, on alpha-naphthylisothiocyanate (ANIT)-induced liver injury and intrahepatic cholestasis in mice and further elucidated the mechanisms in vivo and in vitro. AB23A treatment dose-dependently protected against liver injury induced by ANIT through reducing hepatic uptake and increasing efflux of bile acid via down-regulation of hepatic uptake transporters (Ntcp)more » and up-regulation of efflux transporter (Bsep, Mrp2 and Mdr2) expression. Furthermore, AB23A reduced bile acid synthesis through repressing Cyp7a1 and Cyp8b1, increased bile acid conjugation through inducing Bal, Baat and bile acid metabolism through an induction in gene expression of Sult2a1. We further demonstrate the involvement of farnesoid X receptor (FXR) in the hepatoprotective effect of AB23A. The changes in transporters and enzymes, as well as ameliorative liver histology in AB23A-treated mice were abrogated by FXR antagonist guggulsterone in vivo. In vitro evidences also directly demonstrated the effect of AB23A on FXR activation in a dose-dependent manner using luciferase reporter assay in HepG2 cells. In conclusion, AB23A produces protective effect against ANIT-induced hepatotoxity and cholestasis, due to FXR-mediated regulation of transporters and enzymes. - Highlights: • AB23A has at least three roles in protection against ANIT-induced liver injury. • AB23A decreases Ntcp, and increases Bsep, Mrp2 and Mdr2 expression. • AB23A represses Cyp7a1 and Cyp8b1 through inducing Shp and Fgf15 expression. • AB23A increases bile acid metabolism through inducing Sult2a1 expression. • FXR activation is involved in the hepatoprotective effect of AB23A.« less
  • Sandwich-cultured hepatocytes (SCH) are used commonly to investigate hepatic transport protein-mediated uptake and biliary excretion of substrates. However, little is known about the disposition of endogenous bile acids (BAs) in SCH. In this study, four endogenous conjugated BAs common to rats and humans [taurocholic acid (TCA), glycocholic acid (GCA), taurochenodeoxycholic acid (TCDCA), and glycochenodeoxycholic acid (GCDCA)], as well as two BA species specific to rodents (α- and β-tauromuricholic acid; α/β TMCA), were profiled in primary rat and human SCH. Using B-CLEAR{sup ®} technology, BAs were measured in cells + bile canaliculi, cells, and medium of SCH by LC-MS/MS. Results indicatedmore » that, just as in vivo, taurine-conjugated BA species were predominant in rat SCH, while glycine-conjugated BAs were predominant in human SCH. Total intracellular BAs remained relatively constant over days in culture in rat SCH. Total BAs in control (CTL) cells + bile, cells, and medium were approximately 3.4, 2.9, and 8.3-fold greater in human than in rat. The estimated intracellular concentrations of the measured total BAs were 64.3 ± 5.9 μM in CTL rat and 183 ± 56 μM in CTL human SCH, while medium concentrations of the total BAs measured were 1.16 ± 0.21 μM in CTL rat SCH and 9.61 ± 6.36 μM in CTL human SCH. Treatment of cells for 24 h with 10 μM troglitazone (TRO), an inhibitor of the bile salt export pump (BSEP) and the Na{sup +}-taurocholate cotransporting polypeptide (NTCP), had no significant effect on endogenous BAs measured at the end of the 24-h culture period, potentially due to compensatory mechanisms that maintain BA homeostasis. These data demonstrate that BAs in SCH are similar to in vivo, and that SCH may be a useful in vitro model to study alterations in BA disposition if species differences are taken into account. -- Highlights: ► Bile acids (BAs) were measured in rat and human sandwich-cultured hepatocytes (SCH). ► Cell and medium BA concentrations were estimated using B-CLEAR{sup ®} technology. ► Endogenous BA profiles in SCH were similar to those reported in vivo for each species. ► Species differences were evident in endogenous BA profiles of rat vs human SCH. ► 10 µM troglitazone had no effect on endogenous BA profiles in rat or human SCH at 24 h.« less
  • An alkaline unwinding assay was used to quantitate the induction of DNA strand breaks (DNA SB) in the livers of rats and mice treated in vivo, in rodent hepatocytes in primary culture, and in CCRF-CEM cells, a human lymphoblastic leukemia cell line, following treatment with tri-(TCA), di-(CA), and mono-(MCA) chloroacetic acid and their corresponding aldehydes, tri-(chloralhydrate, CH), di(DCAA) and mono-(CAA) chloroacetaldehyde. None of the chloracetic acids induced DNA SB in the livers of rats at 4 hr following a single administration of 1-10 mmole/kg. TCA (10 mmole/kg) and DCA (5 and 10 mmole/kg) did produce a small amount of strandmore » breakage in mice (7% at 4hr) but not at 1 hr. N-nitrosodiethylamine (DENA), an established alkylating agent and a rodent hepatocarcinogen, produced DNA SB in the livers of both species. TCA, DCA, and MCA also failed to induce DNA strand breaks in splenocytes and epithelial cells derived from the stomach and duodenum of mice treated in vivo. None of the three chloroacetaldehydes induced DNA SB in either mouse or rat liver. These studies provide further evidence that the chloroacetic acids lack genotoxic activity not only in rodent liver, a tissue in that they induce tumors, but in a variety of other rodent tissues and cultured cell types. Two of the chloroacetaldehydes, DCAA and CAA, are direct acting DNA damaging agents in CCRF-CEM cells, but not in liver or splenocytes in vivo or in cultured hepatocytes. CH showed no activity in any system investigated. 58 refs., 6 figs., 2 tabs.« less
  • Acetaminophen (APAP) overdose is the most prevalent cause of drug-induced liver injury in western countries. Numerous studies have been conducted to investigate the mechanisms of injury after APAP overdose in various animal models; however, the importance of these mechanisms for humans remains unclear. Here we investigated APAP hepatotoxicity using freshly isolated primary human hepatocytes (PHH) from either donor livers or liver resections. PHH were exposed to 5 mM, 10 mM or 20 mM APAP over a period of 48 h and multiple parameters were assessed. APAP dose-dependently induced significant hepatocyte necrosis starting from 24 h, which correlated with the clinicalmore » onset of human liver injury after APAP overdose. Interestingly, cellular glutathione was depleted rapidly during the first 3 h. APAP also resulted in early formation of APAP-protein adducts (measured in whole cell lysate and in mitochondria) and mitochondrial dysfunction, indicated by the loss of mitochondrial membrane potential after 12 h. Furthermore, APAP time-dependently triggered c-Jun N-terminal kinase (JNK) activation in the cytosol and translocation of phospho-JNK to the mitochondria. Both co-treatment and post-treatment (3 h) with the JNK inhibitor SP600125 reduced JNK activation and significantly attenuated cell death at 24 h and 48 h after APAP. The clinical antidote N-acetylcysteine offered almost complete protection even if administered 6 h after APAP and a partial protection when given at 15 h. Conclusion: These data highlight important mechanistic events in APAP toxicity in PHH and indicate a critical role of JNK in the progression of injury after APAP in humans. The JNK pathway may represent a therapeutic target in the clinic. - Highlights: • APAP reproducibly causes cell death in freshly isolated primary human hepatocytes. • APAP induces adduct formation, JNK activation and mitochondrial dysfunction in PHH. • Mitochondrial adducts and JNK translocation are delayed in PHH compared to mice. • JNK inhibitor SP600125 partially protects against APAP-induced cell death in PHH. • N-acetylcysteine provides significant protection even if administered 6 h after APAP.« less