Role of oxidative stress and glutathione in busulfan toxicity in cultured murine hepatocytes.

UNLABELLED: This study examines busulfan metabolism. Busulfan given in vivo or in vitro decreased hepatocyte glutathione (GSH) by 60 and 50%, respectively. In vitro, busulfan toxicity was prevented by glutathione S-transferase inhibitors or by antioxidants and led to increased production of oxidized GSH and thiobarbituric acid reactive substances. 'Rescue' from toxicity by GSH precursors was prevented by N,N-bis(2-chloroethyl)-N-nitrosourea (BCNU). Depletion of GSH exacerbated toxicity. In GSH-depleted hepatocytes, busulfan decreased GSH by 95% and BCNU did not prevent rescue by GSH precursors. CONCLUSIONS: (1) In hepatocytes with normal GSH: busulfan toxicity requires GSH conjugation, does not cause profound GSH depletion and is mediated by oxidative stress. We postulate that a GSH conjugate promotes oxidative stress. (2) In GSH-depleted hepatocytes: busulfan profoundly depletes GSH; toxicity is mediated by oxidative stress and is prevented by restoring GSH levels; cell death may be due to unopposed endogenous oxidative stress.

Support of sinusoidal endothelial cell glutathione prevents hepatic veno-occlusive disease in the rat.

Depletion of sinusoidal endothelial cell glutathione (GSH) has been proposed as a common mechanism leading to hepatic veno-occlusive disease (HVOD). This study examines whether intraportal infusion of GSH can prevent HVOD in the monocrotaline rat model. HVOD was induced in rats with monocrotaline 160 mg/kg i.g. on day 0. GSH was infused intraportally by mini-osmotic pump. Monocrotaline decreased GSH in sinusoidal endothelial cells, but not in liver homogenate. Infusion of GSH, 2 micromol/hr starting day - 1, prevented the decrease in sinusoidal endothelial cell GSH and protected against histological and clinical evidence of HVOD. Protection by GSH was dose-dependent (0.5-2 micromol/hr). In rats receiving continuous GSH infusion, treatment with buthionine sulfoximine starting day - 2 decreased sinusoidal endothelial cell GSH and attenuated the protective effect of GSH against monocrotaline. GSH infusion starting 24 hours after monocrotaline ("glutathione rescue") offered substantial protection to most rats. N-acetyl-L-cysteine conferred protection, but N-acetyl-D-cysteine (an antioxidant that is not a precursor for GSH) had little or no protective effect, and 4-hydroxy TEMPO, a free radical scavenger, was not protective. Discontinuation of the GSH infusion 5 days after monocrotaline administration led to severe hepatic veno-occlusive disease on day 6. In conclusion, monocrotaline selectively depletes sinusoidal endothelial cell GSH. Intraportal infusion of GSH protects against monocrotaline toxicity, at least partially by maintaining sinusoidal endothelial cell GSH levels. Glutathione infusion started after monocrotaline is partially protective. Monocrotaline induces prolonged changes in the liver that remain suppressed as long as GSH is infused.

Characterization of a reproducible rat model of hepatic veno-occlusive disease.

Lack of a reproducible animal model has hampered progress in understanding hepatic veno-occlusive disease (HVOD). This article characterizes a reproducible model of HVOD. Rats gavaged with monocrotaline, 160 mg/kg, were killed between days 1 and 10. Sections were evaluated by light microscopy with a standardized scoring system, by immunoperoxidase staining with ED-1 (monocytes, macrophages) and ED-2 (Kupffer cells) antibodies, and by transmission (TEM) and scanning electron microscopy (SEM). On days 1 and 2, the earliest manifestations were progressive injury to the sinusoidal wall with loss of sinusoidal lining cells, sinusoidal hemorrhage, and mild damage to central vein (CV) endothelium. On days 3 through 5 ("early HVOD"), there was centrilobular coagulative necrosis, severe injury to sinusoids, severe sinusoidal hemorrhage, and severe CV endothelial damage; inflammation with ED-1-positive cells was most marked on these days. Days 6 and 7 ("late HVOD") were characterized by subendothelial and advential fibrosis of CVs, damage of the CV endothelium with subendothelial hemorrhage, and some restoration of the sinusoidal wall. Between days 8 and 10, sections showed interindividual variation ranging from mild, residual fibrosis to severe, late HVOD. From days 1 through 10, ED-2-positive cells were decreased in number, and the number of ED-1-positive cells was increased. Sinusoidal damage is the earliest change in HVOD. Coagulative necrosis follows sinusoidal injury and resolves with improvement in sinusoidal endothelial cell (SEC) morphology. Moderate-to-severe CV fibrosis occurs after reappearance of sinusoidal lining cells and resolution of hepatocyte necrosis. The inflammatory response within the lobule and CVs is a result of recruitment of monocytes, whereas Kupffer cells are decreased in number.

Glutathione defense in non-parenchymal cells.

Toxicity to nonparenchymal cells can result in disruption of the hepatic microcirculation, altered production of cytokines, and hepatic fibrosis. Many of the relevant insults produce oxidative stress or toxic metabolites that require glutathione detoxification. This article reviews the role of sinusoidal endothelial cell glutathione (GSH) in reperfusion injury, cytomegalovirus infection, and hepatic venoocclusive disease. The effects of oxidative stress and antioxidants on Kupffer cell production of cytokines and, in particular the potential benefit of antioxidants in the setting of reperfusion injury, are discussed. Oxidative stress upregulates collagen gene expression by stellate cells, and this is modulated by antioxidants. Current thinking on intrahepatic GSH and cysteine homeostasis is discussed. Finally, I review the published data on nonparenchymal GSH levels, glutathione S-transferase activity and isoenzyme pattern, and glutathione peroxidase activity.

Sinusoidal endothelial cells as a target for acetaminophen toxicity. Direct action versus requirement for hepatocyte activation in different mouse strains.

Hepatic congestion occurs early in acetaminophen poisoning. This study examines whether acetaminophen is toxic to sinusoidal endothelial cells (SEC), which might lead to microcirculatory disruption. Acetaminophen toxicity was examined in vivo and in vitro in SEC and hepatocytes from C3H-HEN and Swiss Webster mice. In both strains, there was significantly more toxicity to SEC than to hepatocytes; in SEC from C3H-HEN mice, acetaminophen was directly toxic, but the presence of hepatocytes was required for toxicity to Swiss SEC. Acetaminophen, 750 mg/kg, by gavage caused toxicity with variability within and between strains, but all animals died between 3.5 and 6 hr with zone 3 hemorrhagic necrosis. Pretreatment of C3H-HEN SEC with aminobenzotriazole, a suicide inhibitor of P450, abolished toxicity. Baseline glutathione (GSH) levels were comparable, but a 12-hr incubation with acetaminophen decreased GSH by 60 and 8%, respectively, in C3H-HEN and Swiss SEC in single cell type culture. In co-culture, under conditions where Swiss SEC viability declined by 73%, hepatocyte viability and GSH only decreased by 21 and 20%, respectively. In conclusion, acetaminophen was toxic to SEC. It was directly toxic to SEC in one mouse strain and required hepatocyte activation in another strain. The lack of direct toxicity to Swiss SEC may be due to the lack of an activating P450 isozyme. Zone 3 hemorrhagic necrosis in vivo was comparable in both strains, despite differences in the pathways leading to SEC toxicity in vitro. We propose that toxicity to SEC may contribute to hepatic congestion in acetaminophen intoxication.

Effect of decreased glutathione levels in hereditary glutathione synthetase deficiency on dibromoethane-induced genotoxicity in human fibroblasts.

The genotoxic effect of dibromoethane is thought to be due to glutathione S-transferase mediated metabolism. The purpose of this study was to determine whether variations in endogenous glutathione in human cells could modify the genotoxicity of dibromoethane. Genotoxicity of dibromoethane, assessed by sister chromatid exchange, was examined in normal human skin fibroblasts and fibroblasts obtained from individuals with hereditary generalized glutathione synthetase deficiency. Cell proliferation was examined as a measure of dibromoethane toxicity. The number of sister chromatid exchanges induced by dibromoethane was significantly lower in the fibroblasts with glutathione synthetase deficiency compared to control cells. Inhibition of cell proliferation was similar in the glutathione-deficient and normal fibroblasts. In conclusion, low endogenous glutathione levels are protective against dibromoethane-induced genotoxicity in human fibroblasts.

Dinitrochlorobenzene is genotoxic by sister chromatid exchange in human skin fibroblasts.

Dinitrochlorobenzene (DNCB) is clinically efficacious in the therapy of alopecia areata, but its use was limited when it was found to be mutagenic in the Ames test. However, there has been renewed interest in the immunomodulatory benefits of topically applied dinitrochlorobenzene in patients with human immunodeficiency virus and systemic lupus erythematosus. The current study examines the genotoxicity of dinitrochlorobenzene in human skin fibroblasts using sister chromatid exchange. Dinitrochlorobenzene caused a significant increase in sister chromatid exchange at concentrations ranging from 2.5 to 10 microM. Thus, dinitrochlorobenzene is genotoxic in human skin fibroblasts at concentrations well below those used clinically. The potential for long-term toxicity from dinitrochlorobenzene will have to be weighed against the severity and prognosis of the diseases for which it is used.

Cellular target of cyclophosphamide toxicity in the murine liver: role of glutathione and site of metabolic activation.

Hepatic venoocclusive disease (HVOD) is caused by the disruption of the microcirculation by an as-yet unknown mechanism. Previous in vitro studies with azathioprine, monocrotaline, and dacarbazine suggested that toxins that cause HVOD initially causing HVOD target sinusoidal endothelial cells (SEC) perhaps via profound glutathione (GSH) depletion. The current study examines cyclophosphamide toxicity in SEC and hepatocytes, as well as the interplay between the two cell types. Cyclophosphamide was not directly toxic to SEC, but in coculture of SEC and hepatocytes, cyclophosphamide was significantly more toxic to SEC. Two cyclophosphamide metabolites, 4-hydroperoxycyclophosphamide and acrolein, were equally toxic to SEC, and toxicity occurred at 20-fold-lower concentrations than in hepatocytes. 4-Hydroperoxycyclophosphamide depleted GSH by greater than 95% before inducing cell death in SEC. When hepatocyte-GSH levels were sustained with supplemental methionine and serine in coculture, toxicity in both cell types was diminished. In coculture, SEC are significantly more susceptible than hepatocytes to cyclophosphamide toxicity, and this is likely caused by acrolein generated by the hepatocyte. As seen with other toxins implicated in HVOD, the profound depletion of SEC GSH precedes the onset of toxicity. The degree of cyclophosphamide toxicity induced in SEC is determined by both metabolic activation and GSH detoxification in the hepatocytes.

Toxicity of azathioprine and monocrotaline in murine sinusoidal endothelial cells and hepatocytes: the role of glutathione and relevance to hepatic venoocclusive disease.

The mechanisms leading to hepatic venoocclusive disease (HVOD) remain largely unknown. Azathioprine and monocrotaline were studied as part of a series of studies looking at a variety of toxins that induce HVOD to find common features that might be of pathogenic significance. In a previous study, dacarbazine showed selective in vitro toxicity to sinusoidal endothelial cells (SEC) compared with hepatocytes and a key role for SEC glutathione (GSH) was demonstrated. Murine SEC and hepatocytes were isolated and studied in culture. Azathioprine and monocrotaline were found to be selectively more toxic to SEC than to hepatocytes. The relative resistance of hepatocytes to azathioprine was due to enhanced GSH defense: hepatocytes exposed to azathioprine maintained intracellular GSH levels better than SEC, particularly when supplemental GSH precursors were added, and hepatocyte resistance was completely overcome by depletion of intracellular GSH. In contrast, monocrotaline toxicity in hepatocytes was largely unaffected by depletion of GSH, which suggests that selectivity of monocrotaline for SEC may be attributable to differences in metabolic activation. Both compounds are detoxified by GSH in SEC, as demonstrated by enhanced toxicity in the presence of buthionine sulfoximine (BSO) and attenuation of toxicity with exogenous GSH. SEC GSH levels were more than 70% to 80% depleted by monocrotaline and azathioprine, respectively, before cell death. Azathioprine and monocrotaline are selectively toxic to SEC; the mechanism of toxicity in the SEC may be caused by profound GSH depletion.

Mechanisms of drug-induced liver disease.

The liver is the main metabolizing organ in the body for drugs and toxins. The liver is therefore exposed to relatively high levels of electrophilic metabolites and free radicals that may induce toxicity. Furthermore, the liver performs many vital functions that may be disrupted by toxic metabolites. Despite the high exposure to reactive metabolites, drug-induced toxicity is relatively uncommon because of the redundancy of the detoxification systems present in the liver.

Differential toxicity of the protein phosphatase inhibitors microcystin and calyculin A.

Microcystin (Mcyst) and calyculin A (CalA) in vitro inhibit protein phosphatases (PP)1 and 2A activity (IC50 0.1-2.0 nM). This study was aimed at determining the contribution of PP inhibition to Mcyst hepatotoxicity by comparing the effect of these two chemically different inhibitors in perfused rat livers. Both compounds (60 micrograms Mcyst and 6 micrograms CalA/150 ml perfusate) caused cessation of bile flow and inhibition of PP activity after 20 min of perfusion to 8% and 37% of control activity for Mcyst and CalA treatments, respectively. Histopathological findings included loss of cord sinusoidal pattern and of normal liver architecture. There also was hepatocyte swelling, pyknotic changes and necrosis. Mcyst caused a modest increase in perfusion pressure of 1.2 cm of water, whereas CalA caused a 3-fold increase. The most likely explanation for this hemodynamic effect is direct action of CalA on the vascular endothelium and/or sinusoidal and perisinusoidal cells. This possibility was explored with hepatocytes and sinusoidal endothelial cells. PP activity of both cell types was inhibited by 10 to 100 nM CalA followed later by cell lysis, whereas Mcyst (500 nM-2 microM) had no effect on sinusoidal endothelial cells, but inhibited PP activity and caused later lysis in hepatocytes (Mcyst 20-160 nM). Mcyst hepatotoxicity is therefore a direct consequence of PP inhibition in hepatocytes, the loss of sinusoidal integrity following from the primary toxic insult to the hepatocyte. Inhibition of PP activity of the cells of the presinusoidal vasculature and/or nonparenchymal cells results in hepatic hypertension.

Dacarbazine toxicity in murine liver cells: a model of hepatic endothelial injury and glutathione defense.

The pathophysiology of hepatic veno-occlusive disease is poorly understood. These studies were undertaken to determine the initial cellular target and the role of glutathione detoxification of dacarbazine, a toxin implicated in hepatic veno-occlusive disease. Sinusoidal endothelial cells (SECs) and hepatocytes were isolated and plated in culture dishes. Dacarbazine (5-(3,3-dimethyl-triazeno) imidazole-4-carboxamide), 3 and 6 mM, was toxic to SECs but not to hepatocytes. Onset of toxicity occurred between 11 and 12 hr as determined by serial MTT assays and ethidium homodimer dye exclusion. Glutathione detoxification of dacarbazine in SECs was suggested by: (1) depletion of glutathione before onset of toxicity; (2) exacerbation of toxicity by buthionine sulfoximine (BSO) depletion of glutathione; and (3) protection by exogenous glutathione. Protection by exogenous glutathione may be by uptake of intact tripeptide rather than by extracellular hydrolysis: neither acivicin (inhibitor of gamma-glutamyltranspeptidase) nor BSO (inhibitor of gamma-glutamylcysteine synthetase) blocked the protective effect, and glutathione disulfide did not protect. The relative resistance to dacarbazine toxicity seen in hepatocytes is not due to more efficient GSH detoxification, because toxicity was not unmasked in hepatocytes cultures in medium lacking sulfur amino acid precursors of GSH. In conclusion, glutathione status may play an important role in the susceptibility to toxicity. Furthermore, the findings suggest that the SEC is the initial in vivo target of dacarbazine due to a relatively higher level of metabolic activation that more readily overcomes the available detoxification.

Glutathione metabolism and its role in hepatotoxicity.

Glutathione (GSH) fulfills several essential functions: Detoxification of free radicals and toxic oxygen radicals, thiol-disulfide exchange and storage and transfer of cysteine. GSH is present in all mammalian cells, but may be especially important for organs with intense exposure to exogenous toxins such as the liver, kidney, lung and intestine. Within the cell mitochondrial GSH is the main defense against physiological oxidant stress generated by cellular respiration and may be a critical target for toxic oxygen and electrophilic metabolites. Glutathione homeostasis is a highly complex process, which is predominantly regulated by the liver, lung and kidney.

Plasma concentration-response relationships of two formulations of propranolol.

The time-course of beta blockade induced by two formulations of propranolol was compared to their plasma concentration-time curves. Graded infusions of isoproterenol were used to assess the degree of beta blockade at different times after oral administration of 80 mg of propranolol to 11 healthy volunteers. The time-course of drug effect was measured as the decline of the systolic pressor dose 20 (SPD 20) and the chronotropic dose 20 (CD 20). Variability of plasma propranolol concentration was small, varying within subjects from 27% to 36% and between subjects from 19% to 28% at the various sampling times. Pharmacodynamic effects showed a similar reproducibility: intra-individual variation was 15% to 28% for CD 20 and 17% to 32% for SPD 20; interindividual variation was 10% to 24% for CD 20 and 13% to 23% for SPD 20. Pooling of the data of all subjects indicated a parallel decline of drug concentration and effect. However, three of the 11 subjects showed drug effects declining at a faster rate than drug levels. This dissociation between serum concentrations and effects points out the clinical relevance of complementing kinetic studies of propranolol with pharmacodynamic studies. The good reproducibility within subjects and the small interindividual variation suggests that isoproterenol dose-response curves may be a useful tool for such studies.