Rolf Dahl

Role of Oxidant Stress in the Permeability Transition Induced in Rat Hepatic Mitochondria by Hydrophobic Bile Acids

Hydrophobic bile acids may cause hepatocellular necrosis and apoptosis during cholestatic liver diseases. The mechanism for this injury may involve mitochondrial dysfunction and the generation of oxidant stress. The purpose of this study was to determine the relationship of oxidant stress and the mitochondrial membrane permeability transition (MMPT) in hepatocyte necrosis induced by bile acids. The MMPT was measured spectrophotometrically and morphologically in rat liver mitochondria exposed to glycochenodeoxycholic acid (GCDC). Freshly isolated rat hepatocytes were exposed to GCDC and hepatocellular necrosis was assessed by lactate dehydrogenase release, hydroperoxide generation by dichlorofluorescein fluorescence, and the MMPT in cells by JC1 and tetramethylrhodamine methylester fluorescence on flow cytometry. GCDC induced the MMPT in a dose- and Ca2+-dependent manner. Antioxidants significantly inhibited the GCDC-induced MMPT and the generation of hydroperoxides in isolated mitochondria. Other detergents failed to induce the MMPT and a calpain-like protease inhibitor had no effect on the GCDC-induced MMPT. In isolated rat hepatocytes, GCDC induced the MMPT, which was inhibited by antioxidants. Blocking the MMPT in hepatocytes reduced hepatocyte necrosis and oxidant stress caused by GCDC. Oxidant stress, and not detergent effects or the stimulation of calpain-like proteases, mediates the GCDC-induced MMPT in hepatocytes. We propose that reducing mitochondrial generation of reactive oxygen species or preventing increases in mitochondrial Ca2+ may protect the hepatocyte against bile acid-induced necrosis.

Licorice Compounds Glycyrrhizin and 18β-Glycyrrhetinic Acid Are Potent Modulators of Bile Acid-induced Cytotoxicity in Rat Hepatocytes

The accumulation of hydrophobic bile acids results in cholestatic liver injury by increasing oxidative stress, mitochondrial dysfunction, and activation of cell signaling pathways. Licorice root and its constituents have been utilized as antihepatotoxic agents. The purpose of this study was to evaluate the potential modulation by a primary component of licorice root, glycyrrhizin (GL), and its metabolite, 18β-glycyrrhetinic acid (GA), in a hepatocyte model of cholestatic liver injury. Preincubation of fresh rat hepatocyte suspensions with GL or GA reduced glycochenodeoxycholic acid (GCDC)-dependent reactive oxygen species generation, with GA more potent than GL. Interestingly, GL and GA had opposing effects toward GCDC-induced cytotoxicity; GA prevented both necrosis and apoptosis, whereas GL enhanced apoptosis. GCDC promoted activation of caspase 10, caspase 3, and PARP; all were inhibited by GA but not GL. Induction of apoptosis by GCDC was also associated with activation of JNK, which was prevented by GA. Activation of caspase 9 and dissipation of mitochondrial membrane potential were prevented by GA but not GL. In liver mitochondrial studies, GL and GA were both potent inhibitors of the mitochondrial permeability transition, reactive oxygen species generation, and cytochrome c release at submicromolar concentrations. Results from this study suggest that GL exhibits pro-apoptotic properties, whereas GA is a potent inhibitor of bile acid-induced apoptosis and necrosis in a manner consistent with its antioxidative effect.

Secretion of cytokines and growth factors into autosomal dominant polycystic kidney disease liver cyst fluid

The principal extrarenal manifestation of autosomal dominant polycystic kidney disease (ADPKD) involves formation of liver cysts derived from intrahepatic bile ducts. Autocrine and paracrine factors secreted into the cyst would be positioned to modulate the rate of hepatic cyst growth. The aim of this study was to identify potential growth factors present in human ADPKD liver cyst fluid. Cytokine array and enzyme‐linked immunosorbent assay analysis of human ADPKD liver cyst fluid detected epithelial neutrophil attractant 78, interleukin (IL)‐6 (503 ± 121 pg/mL); and IL‐8 (4,488 ± 355 pg/mL); and elevated levels of vascular endothelial growth factor compared with non‐ADPKD bile (849 ± 144 pg/mL vs. 270 pg/mL maximum concentration). ADPKD liver cyst cell cultures also released IL‐8 and vascular endothelial growth factor, suggesting that cystic epithelial cells themselves are capable of secreting these factors. Western blotting of cultured cyst cells and immunostaining of intact cysts demonstrate that cysteine‐X‐cysteine receptor 2, an epithelial neutrophil attractant 78 and IL‐8 receptor, is expressed at the apical domain of cyst lining epithelial cells. Suggesting the cystic epithelial cells may exist in hypoxic conditions, electron microscopy of the ADPKD liver cyst epithelium revealed morphological features similar to those observed in ischemic bile ducts. These features include elongation, altered structure, and diminished abundance of apical microvilli. In conclusion, IL‐8, epithelial neutrophil attractant 78, IL‐6, and vascular endothelial growth factor may serve as autocrine and paracrine factors to direct errant growth of ADPKD liver cyst epithelia. Interruption of these signaling pathways may provide therapeutic targets for inhibiting liver cyst expansion. (Hepatology 2004;40:836–846).

Subcutaneous vitamin E ameliorates liver injury in an in vivo model of steatocholestasis

Several genetic metabolic liver diseases share the pathological features of combined steatosis and cholestasis, or steatocholestasis. The aims of this study were to develop and characterize an in vivo model for steatocholestasis and to evaluate the effects of an antioxidant treatment on liver injury, oxidative stress, and mitochondrial perturbations in this model. Obese and lean Zucker rats received intravenous (IV) injections of glycochenodeoxycholic acid (GCDC) and were killed 4 hours later. Liver enzymes were measured; the liver histology was assessed, and hepatic mitochondria were analyzed for mitochondrial lipid peroxidation. In separate experiments, rats received daily injections of subcutaneous (SQ) vitamin E before GCDC infusion. Bile acid–induced injury (serum AST and ALT and liver histology) was more severe in the obese rats than in the lean rats, characterized predominantly by extensive cell necrosis with minimal evidence of apoptosis. SQ vitamin E provided significant protection against IV GCDC‐induced hepatic injury, in vitro GCDC‐induced permeability transition, and cytochrome C and apoptosis‐inducing factor release from isolated mitochondria. Conclusion: Steatosis sensitizes the liver to bile acid–induced necrotic hepatocyte injury, which is responsive to vitamin E therapy. (HEPATOLOGY 2007.)

β-Carotene Prevents Bile Acid-Induced Cytotoxicity in the Rat Hepatocyte: Evidence for an Antioxidant and Anti-Apoptotic Role of β-Carotene In Vitro

Hydrophobic bile acids are implicated in the pathogenesis of cholestatic liver disorders through mechanisms involving oxidative stress and mitochondrial dysfunction. Antioxidants ameliorate bile acid–induced cytotoxicity in rat hepatocyte suspensions. The purpose of the current study was to evaluate the potential protective role of β-carotene (βC), a putative fat-soluble antioxidant that is reduced in patients with cholestasis, against bile acid–induced hepatotoxicity. In freshly isolated rat hepatocyte suspensions that were exposed to the toxic hydrophobic bile acid glycochenodeoxycholic acid (100 or 500 μM), βC (100 μM) decreased generation of reactive oxygen species by >50%, similar to the inhibition afforded by β-tocopherol. Commensurate with this antioxidant effect, 100 μM βC also protected hepatocytes against both glycochenodeoxycholic acid–induced cellular necrosis and apoptosis, which was associated with reduction in caspase 3 activation, inhibition of mitochondrial cytochrome c release in rat hepatocytes, and prevention of the mitochondrial permeability transition in both liver mitochondria and rat hepatocytes. A lower concentration of βC (50 μM) produced similar antioxidant and anti-apoptotic protection but with less inhibition against cell necrosis, suggesting that the higher concentration of βC may have conferred additional cytoprotection not directly related to its antioxidant function. These results demonstrate that the antioxidant effects of βC may provide hepatoprotection against cholestatic liver injury by preventing bile acid–induced oxidative stress and mitochondrial perturbations.

Increased Hepatic Na,K-ATPase Activity during Hepatic Regeneration Is Associated with Induction of the β1-Subunit and Expression on the Bile Canalicular Domain

Cellular and molecular mechanisms regulating the activity of the sodium pump or Na,K-ATPase during proliferation of hepatocytes following 70% liver resection have not been defined. Na,K-ATPase may be regulated by synthesis of its α- and β-subunits, by sorting to either the sinusoidal or apical plasma membrane domains, or by increasing membrane lipid fluidity. This study investigated the time course of changes during hepatic regeneration for Na,K-ATPase activity, lipid composition and fluidity, and protein content of liver plasma membrane subfractions. As early as 4 h after hepatic resection, Na,K-ATPase activity was increased selectively in the bile canalicular fraction. It reached a new steady state at 12 h and remained elevated for 2 days. Although hepatic regeneration was associated with a reduced cholesterol/phospholipid molar ratio and increased fluidity, measured with two different probes, these changes in lipid metabolism were in the sinusoidal membrane domain. The Na,K-ATPase β1-subunit, but not the α1-subunit, was increased selectively at the bile canalicular surface as shown by immunoblotting of liver plasma membrane subfractions and the morphological demonstration at both the light and electron microscopic levels. Furthermore, cycloheximide blocked the rise in β1-subunit mRNA levels. Since the time course for β1-subunit accumulation was similar to that for activation of Na,K-ATPase activity, this change implicated the β1-subunit in activating sodium pump activity.

Ectopic expression of MHC class II genes (RT1.B(I) β/α) in rat hepatocytes in vivo and in culture can be elicited by treatment with the pregnane X receptor agonists pregnenolone 16α-carbonitrile and dexamethasone

Abstract The synthetic steroid, pregnenolone-16-α-carbonitrile (PCN), has served for decades as a probe for a postulated series of hepatic defenses activated under situations of environmental “stress”. PCN, an antiglucocorticoid, and also such glucocorticoids as dexamethasone (Dex) appear to stimulate hepatic metabolism and elimination of xenobiotics by binding to the nuclear pregnane X receptor (PXR) which then interacts with a distinct DNA response element associated with induction of cytochrome P450 3A genes. To explore the full domain of genes controlled by PCN/PXR, we used differential display to detect rat liver mRNA species selectively induced by PCN or by Dex. Sequence analysis identified one of many PCN induced cDNA fragments as RT1.B(I)β, a member of the major histocompatability class II (MHC) gene family usually found only in antigen presenting cells. Northern blot analysis of RNA from rat liver or from cultured hepatocytes confirmed that amounts of RT1.B(I)β mRNA and also of its companion gene, RT1.B(I)α mRNA, became readily detectable within 3–6 hours following treatment with PCN or Dex, whereas no induction was observed in spleen RNA. Induction by PCN of RT1.B(I)β immunoreactive protein was localized to the hepatocytes as judged by immunofluorescence. We conclude that ectopic expression of MHC II genes, an unprecedented effect of steroids or drugs, is rapidly evoked by PCN acting on the liver, directly. The concept of a set of genes coordinately controlled to maintain homeostasis in parenchymal tissues during toxic stress must now be extended to include the immune system.

Resistance of Young Rat Hepatic Mitochondria to Bile Acid-Induced Permeability Transition: Potential Role of Alpha Tocopherol

Retention of bile acids within the liver is a primary factor in the pathogenesis of cholestatic liver disorders, which are more common in human infants. The objective of this study was to evaluate developmental changes in mitochondrial factors involved in bile acid-induced hepatocyte injury. Hepatic mitochondria from adult rats (aged 9 wk) underwent a mitochondrial permeability transition (MPT) and release of cytochrome c upon exposure to glycochenodeoxycholic acid. In contrast, mitochondria from young rats (age 6–36 d) were resistant to MPT induction and cytochrome c release. Neither mitochondrial levels of MPT-associated proteins (voltage-dependent anion channel, cyclophilin D, or adenine nucleotide translocase), Bcl-2 family proteins, nor antioxidant enzymes explained this resistance. Mitochondria from young rats contained 2- to 3-fold higher α-tocopherol (α-TH). In vivo α-TH enrichment of adult hepatic mitochondria increased their MPT resistance. Tetra-linoleoyl cardiolipin (TL-CL), the primary molecular species of CL, was reduced in mitochondria of the young rat; however, enrichment with CL and TL-CL only modestly increased their MPT susceptibility. In conclusion, we observed an unexpected resistance in young rats to bile acid induction of mitochondrial cell death pathways, which may be related to developmental differences in membrane composition.