Causality assessment versus guilt-by-association in drug hepatotoxicity.
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Chemical and Drug Induced Liver Injury↗
Biomedical subjects
N Kaplowitz
Publications and source records attributed to N Kaplowitz.
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.
Animals↗
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.
Animals↗
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.
Chemical and Drug Induced Liver Injury↗
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.
Animals↗
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.
Animals↗
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.
Animals↗
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.
Animals↗
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.
Animals↗
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.
Animals↗
Hepatotoxicity of psychotropic drugs.
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Antipsychotic Agents↗
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.
Animals↗
Avoiding hepatic injury from drugs.
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Anti-Inflammatory Agents, Non-Steroidal↗
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.
Animals↗
Changes in glutathione homeostasis during liver regeneration in the rat.
We have shown previously that plating primary cultures of rat hepatocytes under low density, which stimulates hepatocytes to shift from the G0 to the G1 phase of the cell cycle, resulted in increased levels of glutathione (GSH) and cysteine, and increased activity of gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in GSH synthesis (Lu et al., Am. J. Physiol. 1992;263:C1181-C1189). In the current work we examined changes in GSH homeostasis after two-thirds partial hepatectomy (PH). Male Sprague-Dawley rats underwent two-thirds PH or sham operation. GSH, oxidized glutathione (GSSG), cysteine, GSH efflux, DNA synthesis, changes in GCS subunit messenger RNA (mRNA), and protein levels were measured 12 and 24 hours after PH. Both liver GSH and cysteine levels were doubled at 12 hours and remained elevated at 24 hours after PH. GSSG levels also increased, but the ratio of GSH to GSSG levels remained unchanged. The increase in GSH and cysteine levels preceded the increase in DNA synthesis. Sinusoidal GSH efflux was unchanged after two-thirds PH, but biliary GSH efflux decreased. However, total GSH efflux was minimally altered after two-thirds PH. The increase in GSH can be largely accounted for by the increase in both cysteine availability and the activity of GCS. The steady-state mRNA and protein levels of the GCS heavy subunit were increased at 12 hours after PH. The mRNA level of the GCS light subunit was unchanged. In summary, early in the course of liver regeneration the steady-state hepatic GSH levels double because of an increase in the biosynthesis of GSH.
Animals↗
Selective glutathione depletion of mitochondria by ethanol sensitizes hepatocytes to tumor necrosis factor.
BACKGROUND & AIMS: Tumor necrosis factor (TNF)-alpha induces cell injury by generating oxidative stress from mitochondria. The purpose of this study was to determine the effect of ethanol on the sensitization of hepatocytes to TNF-alpha. METHODS: Cultured hepatocytes from ethanol-fed (ethanol hepatocytes) or pair-fed (control hepatocytes) rats were exposed to TNF-alpha, and the extent of oxidative stress, gene expression, and viability were evaluated. RESULTS: Ethanol hepatocytes, which develop a selective deficiency of mitochondrial glutathione (mGSH), showed marked susceptibility to TNF-alpha. The susceptibility to TNF-alpha, manifested as necrosis rather than apoptosis, was accompanied by a progressive increase in hydrogen peroxide that correlated inversely with cell survival. Nuclear factor kappaB activation by TNF-alpha was significantly greater in ethanol hepatocytes than in control hepatocytes, an effect paralleled by the expression of cytokine-induced neutrophil chemoattractant. Similar sensitization of normal hepatocytes to TNF-alpha was obtained by depleting the mitochondrial pool of GSH with 3-hydroxyl-4-pentenoate. Restoration of mGSH by S-adenosyl-L-methionine or by GSH-ethyl ester prevented the increased susceptibility of ethanol hepatocytes to TNF-alpha. CONCLUSIONS: These results indicate that mGSH controls the fate of hepatocytes in response to TNF-alpha. Its depletion caused by alcohol consumption amplifies the power of TNF-alpha to generate reactive oxygen species, compromising mitochondrial and cellular functions that culminate in cell death.
Animals↗
Role of the liver in interorgan homeostasis of glutathione and cyst(e)ine.
The most widely recognized function of reduced glutathione (GSH) is its defense against toxic compounds, whether exogenous, such as electophilic xenobiotics, or endogenous, such as reactive oxygen species, generated during normal oxidative metabolism and/or stress. However another no less significant role of GSH-namely its function as a reservoir and vehicle for packaging and transport of cyst(e)ine-has been receiving increasing attention. Because GSH is relatively more auto-oxidation resistant and stable than cyst(e)ine (CYSH), it serves as the preferred form for storage and transport of the latter especially in the extracellular and relatively much less reduced (than intracellular) milieu, where CYSH oxidizes to cystine (CYSS) rapidly. Over the past two decades, significant work has been going on to delineate the intra- and extrahepatic (interorgan) turnover, transport, and disposal of GSH and define the quantitative role of these processes in interorgan homeostasis of GSH, CYSH, and CYSS. These studies have identified the liver as the central organ of interorgan GSH homeostasis, with sinusoidal GSH efflux as the major determinant of plasma GSH, CYSH, CYSS, and thiol-disulfide status of plasma. This article focuses on the principal components and determinants of interorgan homeostasis of GSH and its breakdown products. It also presents the current state of knowledge under both normal and diseased states.
Animals↗
Mitochondrial glutathione: importance and transport.
Accumulating evidence pointing to mitochondria as critical participants in the control of apoptotic and necrotic cell death and in the development of specific disease states has led to a renaissance on the study of these organelles. Because mitochondria are the major consumers of molecular oxygen within cells, they stand as one of the most important generators of reactive oxygen species and therefore constitute potential targets of therapeutic intervention in pathologic states in which oxidative stress originates from these organelles. In this regard, mitochondria are specific targets of ethanol intoxication, thereby leading to reported morphologic and functional alterations of mitochondria. Because mitochondria are also indispensable for the maintenance of cell functions, their dysfunction induced by ethanol may be a key event in the development of alcoholic liver disease. Indeed, chronic ethanol feeding in experimental animals has been reported to cause a selective deficiency in the availability of reduced glutathione (GSH) in mitochondria due to the impaired functioning of the specific mitochondrial carrier that translocates GSH from cytosol into the mitochondrial matrix. Such a selective depletion sensitizes hepatocytes from chronic ethanol-fed animals to the oxidative effects of cytokines, e.g., tumor necrosis factor (TNF). Restoration of mitochondrial GSH by the in vivo administration of S-adenosyl-L-methionine or the in vitro use of GSH ethyl ester prevents the susceptibility of hepatocytes to TNF. Although the nature of this specific carrier has not yet been uncovered, the elucidation of the mechanisms whereby ethanol leads to its impaired activity may provide important clues as to its function and mechanism of action, which in turn may be useful toward the definitive characterization and identification of this important carrier.
Animals↗