JNK interaction with Sab mediates ER stress induced inhibition of mitochondrial respiration and cell death.

Our aim was to better understand the mechanism and importance of sustained c-Jun N-terminal kinase (JNK) activation in endoplasmic reticulum (ER) stress and effects of ER stress on mitochondria by determining the role of mitochondrial JNK binding protein, Sab. Tunicamycin or brefeldin A induced a rapid and marked decline in basal mitochondrial respiration and reserve-capacity followed by delayed mitochondrial-mediated apoptosis. Knockdown of mitochondrial Sab prevented ER stress-induced sustained JNK activation, impaired respiration, and apoptosis, but did not alter the magnitude or time course of activation of ER stress pathways. P-JNK plus adenosine 5'-triphosphate (ATP) added to isolated liver mitochondria promoted superoxide production, which was amplified by addition of calcium and inhibited by a blocking peptide corresponding to the JNK binding site on Sab (KIM1). This peptide also blocked tunicamycin-induced inhibition of cellular respiration. In conclusion, ER stress triggers an interaction of JNK with mitochondrial Sab, which leads to impaired respiration and increased mitochondrial reactive oxygen species, sustaining JNK activation culminating in apoptosis.

Case definition and phenotype standardization in drug-induced liver injury.

Drug-induced liver injury (DILI) is the most frequent reason cited for the withdrawal of approved drugs from the market and accounts for up to 15% of the cases of acute liver failure. Investigators around the globe have begun to identify and study patients with DILI; several large registries and tissue banks are being established. In order to gain the maximum scientific benefit from these efforts, the definitions and terminology related to the clinical phenotypes of DILI must be harmonized. For this purpose, an international DILI Expert Working Group of clinicians and scientists reviewed current DILI terminology and diagnostic criteria so as to develop more uniform criteria that would define and characterize the spectrum of clinical syndromes that constitute DILI. Consensus was established with respect to the threshold criteria for definition of a case as being DILI, the pattern of liver injury, causality assessment, severity, and chronicity. Consensus was also reached on approaches to characterizing DILI in the setting of chronic liver diseases, including autoimmune hepatitis (AIH).

Recurrent drug-induced liver injury (DILI) with different drugs in the Spanish Registry: the dilemma of the relationship to autoimmune hepatitis.

BACKGROUND & AIMS: Multiple instances of DILI in the same patient with drugs of similar structure or function as well as completely unrelated drugs are not well understood and poorly documented. We have sought evidence of the frequency and characteristics of patients who have experienced two DILI episodes due to different drugs. METHODS: All cases of DILI systematically collected in the Spanish DILI Registry between 1994 and 2009 were retrieved. Data on demographics, clinical, laboratory and pathological findings, and outcome were analyzed. RESULTS: Nine patients (mean age 67 years, four women) out of 742, 1.21%, had evidence of two DILI episodes caused by different drugs. In four cases DILI was associated with structurally related drugs and in an additional two cases the drugs had a common target. In another case, unrelated antibiotics were implicated. In only two cases, the two drugs/herbals were not related in structure or function. All but one patient exhibited hepatocellular damage. The type of damage was consistent in both DILI episodes. Four cases presented as autoimmune hepatitis (AIH) in the second episode. CONCLUSIONS: Multiple episodes of DILI in association with different drugs occur infrequently. In each individual, the type of injury was similar during the two DILI episodes, regardless of the causative drug. Second episodes of DILI are more likely to be associated with features of AIH. It remains uncertain if this is drug-induced unmasking of true AIH or DILI with autoimmune features. These cases illustrate the dilemma faced by clinicians in distinguishing these possibilities.

Diagnosis, management and prevention of drug-induced liver injury.

Drug-induced liver injury (DILI) is increasingly being recognised as a significant cause of both acute and chronic liver disease. The most commonly implicated agents are paracetamol, antimicrobials, non-steroidal anti-inflammatory drugs, statins, isoniazid and herbal remedies. Drug-induced hepatotoxicity is generally idiosyncratic in nature. The pathogenesis of DILI remains enigmatic, but involves exposure to the toxic agent, mitochondrial injury, failure of adaptation, and innate and adaptive immune responses. Diagnosis of drug-induced liver diseases can be difficult, but the key to causality is to diligently exclude other causes of liver injury, and to identify a characteristic clinical drug-related signature. Management of drug-induced liver injury is symptomatic, with early referral to a liver transplant unit at the first hint of liver failure, especially in those with non-paracetamol-induced liver injury. Prevention of drug hepatotoxicity includes increased vigilance during pre-clinical drug development and clinical trials, alanine aminotransferase monitoring with certain drugs, better marketing strategies, and the future identification of both diagnostic and prognostic biomarkers.

Tauroursodeoxycholic acid protects hepatocytes from ethanol-fed rats against tumor necrosis factor-induced cell death by replenishing mitochondrial glutathione.

Mitochondrial glutathione (GSH) plays a key role against tumor necrosis factor alpha (TNF)-induced apoptosis because its depletion is known to sensitize hepatocytes to TNF. The present study examined the role of tauroursodeoxycholic acid (TUDCA) administration to chronic ethanol-fed rats on mitochondrial GSH levels and kinetics, mitochondrial membrane physical properties, TNF-induced peroxide formation, and subsequent hepatocyte survival. TUDCA selectively increased the levels of GSH in mitochondria without an effect on cytosolic GSH. This outcome was accompanied by improved initial rate of GSH transport examined at low (1 mmol/L) and high (10 mmol/L) GSH concentrations both in intact mitochondria and mitoplasts prepared from ethanol-fed livers. Assessment of membrane fluidity revealed an increased order parameter in mitochondria and mitoplasts from ethanol-fed rats compared with pair-fed controls, which was prevented by TUDCA administration. Compared with hepatocytes from pair-fed rats, TNF stimulated peroxide generation in hepatocytes from ethanol-fed rats, preceding TNF-induced cell death. Administration of TUDCA to ethanol-fed rats prevented TNF-induced peroxide formation and cell death, an effect that was reversed on depletion of the recovered mitochondrial GSH levels by (R,S)-3-hydroxy-4-pentenoate before TNF treatment. The protective effect of TUDCA against TNF was not because of activation of phosphatidylinositol 3-kinase, discarding a role for a survival-dependent pathway. Thus, these findings reveal a novel role of TUDCA in protecting hepatocytes in long-term ethanol-fed rats through modulation of mitochondrial membrane fluidity and subsequent normalization of mitochondrial GSH levels.

Drug-induced liver disorders: implications for drug development and regulation.

Drug-induced hepatotoxicity is a frequent cause of liver disease. Although often presenting as acute hepatitis and/or cholestasis, virtually any clinical-pathological pattern of acute or chronic liver disease can occur. Most reactions occur in a small proportion of the population using a particular drug. Each drug associated with hepatotoxicity tends to have a characteristic signature regarding latency and pattern of injury. The mechanism can be drug metabolism-dependent or related to the chemical properties of the parent drug. The former are immune mediated or due to metabolic idiosyncrasy. Monitoring serum ALT levels is of unproven effectiveness but should be considered when there is an increased risk of delayed onset serious hepatitis-like reactions. The key for the future is improved identification of toxic potential in preclinical studies, clinical trials and postmarketing experience. The elucidation of the genetic and environmental mechanisms contributing to delayed idiosyncratic reactions is a major barrier to overcome in this field.

How is the liver primed or sensitized for alcoholic liver disease?

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Hidekazu Tsukamoto and Yoshiyuki Takei. The presentations were (1) Tribute to Professor Rajendar K. Chawla, by Craig J. McClain; (2) Dysregulated TNF signaling in alcoholic liver disease, by Craig J. McClain, S. Joshi-Barve, D. Hill, J Schmidt, I. Deaciuc, and S. Barve; (3) The role of mitochondria in ethanol-mediated sensitization of the liver, by Anna Colell, Carmen Garcia-Ruiz, Neil Kaplowitz, and Jose C. Fernandez-Checa; (4) A peroxisome proliferator (bezafibrate) can prevent superoxide anion release into hepatic sinusoid after acute ethanol administration, by Hirokazu Yokoyama, Yukishige Okamura, Yuji Nakamura, and Hiromasa Ishii; (5) S-adenosylmethionine affects tumor necrosis factor-alpha gene expression in macrophages, by Rajendar K. Chawla, S. Barve, S. Joshi-Barve, W. Watson, W. Nelson, and C. McClain; (6) Iron, retinoic acid and hepatic macrophage TNFalpha gene expression in ALD, by Hidekazu Tsukamoto, Min Lin, Mitsuru Ohata, and Kenta Motomura; and (7) Role of Kupffer cells and gut-derived endotoxin in alcoholic liver injury, by N. Enomoto, K. Ikejima, T. Kitamura, H. Oide, Y. Takei, M. Hirose, B. U. Bradford, C. A. Rivera, H. Kono, S. Peter, S. Yamashina, A. Konno, M. Ishikawa, H. Shimizu, N. Sato, and R. Thurman.

Regulation of GSH in alphaA-expressing human lens epithelial cell lines and in alphaA knockout mouse lenses.

PURPOSE: To study the mechanism of regulation of GSH in HLE-B3 cells expressing alphaA-crystallin (alphaA) and in alphaA knockout mouse lenses. METHODS: GSH levels and maximal rates of GSH synthesis were measured in immortalized, alphaA-transfected HLE-B3 cells containing varying amounts of alphaA. The mRNA and protein for the rate-limiting enzyme for GSH synthesis, gamma-glutamylcysteine synthetase (GCS), were also determined in alphaA- and mock-transfected cells by Northern blot analysis and Western blot analysis of heavy (GCS-HS) and light (GCS-LS) subunits. The effect of absence of alphaA and alphaB on lens GSH concentrations was evaluated in whole lenses of alphaA knockout and alphaB knockout mice as a function of age. GCS-HS mRNA and protein were determined in young, precataractous and cataractous alphaA knockout lenses. RESULTS: GSH levels were significantly higher in HLE-B3 cells expressing alphaA- compared with mock-transfected cells and were correlated positively with alphaA content. Mean rate of GSH synthesis was also higher in alphaA-expressing cells than in mock controls (0.84 vs. 0.61 nmol. min(-1) per mg protein, respectively). GCS-HS mRNA and GCS-LS mRNA were approximately twofold higher in alphaA-expressing cells, whereas the heavy and light GCS subunit proteins increased by 80% to 100%. In alphaA(-/-) mouse lenses, GSH level was not different from that of wild type up to 2 months from birth, after which it dropped to approximately 50% of controls. On the other hand, GCS-HS and GCS-LS proteins showed a significant decrease before cataract formation as early as 15 days after birth. GSH level in cataract-free alphaB(-/-) lenses was similar to that of wild type for up to 14 months. CONCLUSIONS: Expression of alphaA caused an increase in cellular GSH, in part, because of an increase in mRNA and protein of both GCS subunits. GSH levels decreased with increasing age in cataractous alphaA(-/-) lenses but not in the noncataractous alphaB(-/-) lenses. It is suggested that neonatal precataractous lenses (with normal GSH and decreased GCS) may maintain their GSH level by other compensatory mechanisms such as increased GSH transport.

Radiation inactivation studies of hepatic sinusoidal reduced glutathione transport system.

Sinusoidal transport of reduced glutathione (GSH) is a carrier-mediated process. Perfused liver and isolated hepatocyte models revealed a low-affinity transporter with sigmoidal kinetics (K(m) approximately 3.2-12 mM), while studies with sinusoidal membrane vesicles (SMV) revealed a high-affinity unit (K(m) approximately 0.34 mM) besides a low-affinity one (K(m) approximately 3.5-7 mM). However, in SMV, both the high- and low-affinity units manifested Michaelis-Menten kinetics of GSH transport. We have now established the sigmoidicity of the low-affinity unit (K(m) approximately 9) in SMV, consistent with other models, while the high-affinity unit has been retained intact with Michaelis-Menten kinetics (K(m) approximately 0.13 mM). We capitalized on the negligible cross-contributions of the two units to total transport at the low and high ends of GSH concentrations and investigated their characteristics separately, using radiation inactivation, as we did in canalicular GSH transport (Am. J. Physiol. 274 (1998) G923-G930). We studied the functional sizes of the proteins that mediate high- and low-affinity GSH transport in SMV by inactivation of transport at low (trace and 0.02 mM) and high (25 and 50 mM) concentrations of GSH. The low-affinity unit in SMV was much less affected by radiation than in canalicular membrane vesicles (CMV). The target size of the low-affinity sinusoidal GSH transporter appeared to be considerably smaller than both the canalicular low- and high-affinity transporters. The high-affinity unit in SMV was markedly inactivated upon irradiation, revealing a single protein structure with a functional size of approximately 70 kDa. This size is indistinguishable from that of the high-affinity GSH transporter in CMV reported earlier.

Mechanisms of liver cell injury.

Liver cell death is triggered by a number of insults arising from the external environment or from within the cell. These insults may engage cell surface receptors with death domaines leading to a proteolytic cascade involving initiator and executioner caspases and an apoptotic demise. Alternatively, the insults may profoundly disrupt mitochondrial function and result in loss of homeostasis accompanied by activation of hydrolases and a necrotic or lytic demise. The distinction between apoptotic and necrotic cell death has become blurred recently by the recognition that the same stimuli can induce either form of cell death as well as caspase independent apoptosis. Mitochondria play a key role in the shape of cell death; selective release of mediators amplifies the apoptosis program and profound loss of mitochondrial function leads to necrosis. Reactive oxygen metabolites and nitric oxide participate as initiating factors and modulators. The extensive knowledge gained in recent years about the mechanisms of cell death will undoubtedly lead to new and exciting advances in the prevention and treatment of liver diseases. Important targets include death receptors, death signaling mechanisms, the mitochondrial permeability transition and approaches which selectively inhibit or activate cell death in parenchymal versus nonparenchymal cells.

GSH transport in human cerebrovascular endothelial cells and human astrocytes: evidence for luminal localization of Na+-dependent GSH transport in HCEC.

The purpose of the present study was to identify and localize glutathione (GSH) transport in an in vitro tissue culture model of blood-brain barrier (BBB). The localization of Na+-dependent GSH transport in an immortalized cell line of human cerebrovascular endothelial cells (HCEC) and asymmetry of transport in Transwell studies were investigated. Initial studies with cultured HCEC established a significant (45%) Na+-dependency for GSH uptake in cultured HCEC pretreated with acivicin, an inhibitor of gamma-glutamyltranspeptidase (GGT). Transendothelial electrical resistance (TEER) and uptake of [35S]GSH from luminal and abluminal fluids of HCEC were measured in Na+-containing and Na+-free (choline chloride) buffers using cells grown on gelatin-coated membrane filters. TEER of HCEC monolayers in regular medium was 40.1 +/- 8.0 ohms cm2. Human astrocyte-conditioned medium (ACM) caused no change in TEER, but increased GGT activity approximately threefold when measured in cell lysates. Luminal and abluminal GSH uptake increased in a time-dependent fashion and were not affected by inhibition of GGT activity with acivicin. Sodium dependency was only observed for luminal uptake (Na+-containing 2.41 +/- 0.15 vs. Na+-free 0.96 +/- 0.03 pmol/30 min/million cells, p < 0.001) but not for abluminal uptake (1.02 +/- 0.13 vs. 1.11 +/- 09, p > 0.05). Apparent efflux via the luminal membrane was lower in the presence of sodium as compared to that without sodium, further suggesting that a Na+-dependent uptake process for GSH is operative at this membrane. GSH uptake and efflux were also demonstrated in neonatal rat and fetal human astrocytes, both exhibiting partial Na+-dependency of uptake. In conclusion, our results show for the first time, that HCEC and astrocytes take up GSH by both Na+-dependent and -independent mechanisms. The Na+-dependent GSH transport process in HCEC appears to be localized to luminal plasma membranes of HCEC.

Colchicine protects mice from the lethal effect of an agonistic anti-Fas antibody.

The aim of this study was to determine whether colchicine, which has been reported to protect against various hepatotoxic insults, influences the susceptibility of mice to the agonistic anti-Fas antibody, Jo2. All mice that were pretreated with colchicine (2 mg/kg) survived the lethal challenge of intraperitoneal administration of 10 microg of Jo2, whereas all control mice pretreated with gamma-lumicolchicine succumbed to the challenge. Twelve micrograms of Jo2 killed less than half of colchicine-pretreated mice and its lethal effects were delayed relative to control mice, which all died within 8 hours. Other microtubule-disrupting agents such as Taxol, vinblastine, and nocodazole also improved the survival of mice treated with the lethal dose of Jo2. Histologic examination showed that colchicine protected against Jo2-induced fulminant liver injury, and TUNEL assay demonstrated that colchicine protected against massive apoptosis of hepatocytes. Hepatocytes isolated from colchicine-pretreated mice exhibited decreased susceptibility to Jo2-induced apoptosis. In addition, colchicine pretreatment reduced surface expression of Fas and decreased Jo2- and TNF-alpha-induced apoptosis of cultured hepatocytes in the presence of actinomycin D, but did not affect the susceptibility of cultured sinusoidal endothelial cells to Jo2-induced apoptosis. Remarkably, Fas and TNF receptor-1 mRNA and intracellular protein levels increased after colchicine treatment, indicating that colchicine protects against death ligand-induced apoptosis in the liver by decreasing death-receptor targeting to the cell surface.

Cell death at the millennium. Implications for liver diseases.

Cell death occurs by apoptosis or necrosis. Although these are morphologically distinct, they share similar initiating events (death receptor ligation, chemicals, drug hypoxia, oxidative stress), and usually involve the participation of mitochondria. The ultimate shape of cell death depends on the extent of functional collapse of mitochondria, which either leads to a rapid loss of ATP, swelling and lysis, or a more selective release of cytochrome c in the presence of sufficient ATP to activate executioner caspases, leading to the development of apoptosis. Apoptosis and necrosis participate in the pathogenesis of most liver diseases. Therapies targeting the death receptors, initiator caspases and mitochondria show potential promise in various liver disease, whereas targeting inhibition of executioner caspases may rapidly or in delayed fashion switch from apoptotic to necrotic cell death.

GSH transport in immortalized mouse brain endothelial cells: evidence for apical localization of a sodium-dependent GSH transporter.

We have previously shown GSH transport across the blood-brain barrier in vivo and expression of transport in Xenopus laevis oocytes injected with bovine brain capillary mRNA. In the present study, we have used MBEC-4, an immortalized mouse brain endothelial cell line, to establish the presence of Na+-dependent and Na+-independent GSH transport and have localized the Na+-dependent transporter using domain-enriched plasma membrane vesicles. In cells depleted of GSH with buthionine sulfoximine, a significant increase of intracellular GSH could be demonstrated only in the presence of Na+. Partial but significant Na+ dependency of [35S]GSH uptake was observed for two GSH concentrations in MBEC-4 cells in which gamma-glutamyltranspeptidase and gamma-glutamylcysteine synthetase were inhibited to ensure absence of breakdown and resynthesis of GSH. Uniqueness of Na+-dependent uptake in MBEC-4 cells was confirmed with parallel uptake studies with Cos-7 cells that did not show this activity. Molecular form of uptake was verified as predominantly GSH, and very little conversion of [35S]cysteine to GSH occurred under the same incubation conditions. Poly(A)+ RNA from MBEC expressed GSH uptake with significant (approximately 40-70%) Na+ dependency, whereas uptake expressed by poly(A)+ RNA from HepG2 and Cos-1 cells was Na+ independent. Plasma membrane vesicles from MBEC were separated into three fractions (30, 34, and 38% sucrose, by wt) by density gradient centrifugation. Na+-dependent glucose transport, reported to be localized to the abluminal membrane, was found to be associated with the 38% fraction (abluminal). Na+-dependent GSH transport was present in the 30% fraction, which was identified as the apical (luminal) membrane by localization of P-glycoprotein 170 by western blot analysis. Localization of Na+-dependent GSH transport to the luminal membrane and its ability to drive up intracellular GSH may find application in the delivery of supplemented GSH to the brain in vivo.

Protection from oxidant injury by sodium-dependent GSH uptake in retinal Müller cells.

Glutathione (GSH) is known to play an important role in regulating oxidative damage to cells. The present study was initiated to examine the effect of exogenous GSH on oxidative injury in a retinal Müller cell line and to characterize GSH transport in these cells. Rat Müller cells (rMC-1) were incubated with varying concentrations of t-butylhydroperoxide (t-BHP) to induce oxidative stress, and cell viability was measured after addition of GSH. In other studies, kinetics of GSH uptake and Na+-dependency were examined by incubating cells with35S-GSH in Na+-containing and Na+-free buffers. GSH uptake was studied with GSH at concentrations varying from 0. 05-10 m m in NaCl buffer. In the presence of sodium, extracellular GSH provided protection against t-BHP-induced oxidant injury to rMC-1 cells; in contrast, the amino acid precursors of GSH did not have any effect on cell viability. GSH was taken up by rMC-1 cells in a concentration- and sodium-dependent manner. Kinetic studies revealed both a high affinity (Km approximately 0.31 m m) and low affinity Km( approximately 4.2 m m) component. Furthermore, GSH depletion had no significant effect on the rate of GSH uptake. The results show that physiological concentrations of GSH can protect Müller cells from oxidative injury. Both Na+-dependent and Na+-independent transport systems for GSH exist in Müller cells, and the Na+-dependent GSH transporter may be involved in the protective role of GSH.