Glutathione Depletion In Liver Research Articles

S-adenosylmethionine treatment prevents carbon tetrachloride-induced S-adenosylmethionine synthetase inactivation and attenuates liver injury.

Administration of carbon tetrachloride to rats resulted in induction of hepatic fibrosis and a 60% reduction of hepatic S-adenosylmethionine synthetase activity without producing any significant modification of hepatic levels of S-adenosylmethionine synthetase messenger RNA. The reduction of S-adenosylmethionine synthetase activity was corrected by treatment with S-adenosylmethionine (3 mg/kg/day, intramuscularly). Administration of carbon tetrachloride also produced a 45% depletion of liver glutathione (reduced form) that was corrected by S-adenosylmethionine treatment. After the rats received carbon tetrachloride, a 2.3-fold increase in liver collagen was observed; prolyl hydroxylase activity was 2.5 times greater than that seen in controls. These increases were attenuated in animals treated with carbon tetrachloride and S-adenosylmethionine. The attenuation by S-adenosylmethionine treatment of the fibrogenic effect of carbon tetrachloride was associated with a decrease in the number of rats in which cirrhosis developed.

Effects of endurance training and exercise on tissue antioxidative capacity and acetaminophen detoxification

Both acute acetaminophen toxicity and physical exercise are accompanied by structural and functional damage to tissues. For acute acetaminophen toxicity, this damage occurs mainly in the liver. This damage, which is believed to be initially caused by oxidation and/or arylation, occurs only after depletion of liver glutathione (GSH). GSH normally protects against oxidation and/or arylation. Prolonged physical exercise also depletes GSH in the body. We hypothesized that with endurance training (repeated oxidant stress) tissues will develop mechanisms to prevent GSH depletion. Our results show that, for the same amount of submaximal exercise, trained rats are able to maintain their levels of GSH or their GSH redox status (in the liver, heart, skeletal muscle and plasma) in contrast to their untrained counterparts. Also, upon administration of acetaminophen, trained rats show a less pronounced depletion in liver GSH than untrained rats. We also hypothesized that training may lead to improved maintenance of tissue GSH homeostasis because of induction in the enzyme pathways of protection. We observe that training significantly increases (50-70%) glutathione peroxidase and reductase, glucose-6-phosphate dehydrogenase, and catalase activity in heart and skeletal muscle. Since GSH, in addition to providing cellular protection, also functions in other physiological processes including transport and metabolism, the training-induced benefits seen here may have more far-reaching consequences than ever before realized.

Changes in the biliary excretion of organic anions following exhaustive exercise in rats

The effect of exhaustive exercise on the hepatobiliary transport of organic anions was investigated in rats. Animals were run on a rodent treadmill at 24 m/min up a 12% grade (152 ± 15 min). Exercise resulted in significant hypoglycaemia (−46%) and increased plasma levels of lactate (+12%), together with a marked reduction of glycogen concentration in the liver (−72%). When bromosulphthalein was administered i.v., its maximal biliary excretion ( Tm) was significantly reduced (−30%), and plasma and liver concentrations of the dye were increased (+31% and +56%, respectively). The decrease corresponded both to the excretion of the conjugated and unconjugated dye (−30% and −33%, respectively). Cytosolic glutathione S-transferase activity in the liver was not affected by exercise, but there was a significant reduction in the hepatic concentration of glutathione (−50%). The Tm of dibromosulphthalein was also significantly reduced (−36%) and its plasma and liver concentrations increased (+67% and +33%, respectively) in exercised rats. The results suggest that, in addition to the direct effect of liver glutathione depletion, other factors must be involved in the impairment of the biliary excretion of organic anions caused by exercise.

Silybin dihemisuccinate protects against glutathione depletion and lipid peroxidation induced by acetaminophen on rat liver.

Acetaminophen hepatotoxicity is characterized by glutathione depletion, cellular necrosis, and, in some instances, by the induction of lipid peroxidation. Silybin dihemisuccinate, a soluble form of the flavonoid silymarin, protects rats against liver glutathione depletion and lipid peroxidation induced by acute acetaminophen intoxication. Other biochemical parameters such as serum transaminases did not show the drastic increase observed under acetaminophen intoxication when animals were treated with the flavonoid. Preliminary results suggest that silybin dihemisuccinate may be another antidote against acetaminophen hepatotoxicity.

Prevention of acetaminophen-induced hepatotoxicity by dimethyl sulfoxide

Dimethyl sulfoxide (DMSO) has previously been shown to protect against acetaminophen (APAP)-induced hepatotoxicity, but the mechanism of this effect was not clear. Treatment of mice with 1 mg/kg DMSO 4 h before 250 mg/kg APAP resulted in significantly less hepatotoxicity than with APAP alone, as measured by serum glutamic pyruvic transaminase (SGPT) content 24 h after APAP. Protection was also evident when 1 ml/kg DMSO was given 4, but not 8 h after 250 mg/kg APAP. The APAP-induced depletion of liver glutathione was prevented in mice pretreated with DMSO, although DMSO alone had no effect on liver glutathione levels. The hepatic concentration of cytochrome P-450 ( P450) 4 h after treatment of mice with 1 ml/kg DMSO, was significantly decreased compared to saline-treated animals. However, while this DMSO pretreatment significantly decreased the activity of cytochrome P-450-linked aminopyrine- N-demethylase, it increased the activity of aniline hydroxylase. Covalent binding of [ 14C]APAP to hepatic protein in vivo was significantly decreased in mice pretreated with DMSO. Covalent binding of [ 14C]AaAP to hepatic microsomal protein in vitro was not significantly altered after in vivo treatment with DMSO. However, the presence of DMSO in the in vitro incubation mixture significantly decreased covalent binding of [ 14C]APAP in a dose-dependent manner compared to microsomal fractions from untreated, saline-treated or DMSO pretreated animals. These data suggest that the DMSO-induced alterations in cytochrome P-450 content and activity may not be the cause of the observed protective action of this chemical. The ability to competitively inhibit APAP bioactivation or to directly scavenge free radicals produced during APAP metabolism, including the

4-Hydroxynonenal and other lipid peroxidation products are formed in mouse liver following intoxication with allyl alcohol

Some recent reports indicate that lipid peroxidation might play a crucial role in the production of allyl alcohol hepatotoxicity. Previous work from our laboratory has suggested that in the case of bromobenzene, a hepatotoxin sharing the ability of allyl alcohol to induce a marked depletion of liver glutathione, liver injury is likely to be mediated by lipid peroxidation. In particular, we demonstrated that 4-hydroxynonenal and other aldehydes derived from lipid peroxidation can be detected in the liver of bromobenzene-poisoned mice. In the present study, we report also the in vivo formation of 4-hydroxynonenal and other aldehydes after allyl alcohol poisoning. 24-h-fasted mice were intoxicated with allyl alcohol (1.5 mmol/kg body wt., i.p.) and killed 1–3 h later. 4-Hydroxynonenal and other carbonyls were looked for in liver extracts in the form of 2,4-dinitrophenylhydrazone derivatives. After fractionation of liver extracts by means of thin-layer chromatography (TLC), a well-resolved peak corresponding to standard 4-hydroxynonenal was obtained in the high-pressure liquid chromatography analysis. Total carbonyls (as 2,4-dinitrophenylhydrazones) were separated by TLC into three fractions, according to their different polarity. The amounts of carbonyls present in each fraction were determined by ultraviolet-visible spectroscopy. In addition, several products were identified in the fraction of the ‘non-polar carbonyls’ corresponding to alkanals and alk-2-enals.

Potential use of cimetidine for treatment of acetaminophen overdose.

Acetaminophen, a drug frequently taken in intentional and accidental overdose, causes liver toxicity when concentration of the cytochrome P-450-derived metabolite exceeds the metabolic capacity of available glutathione. Present treatment of acetaminophen overdose involves oral N-acetylcysteine (NAC), which enhances liver glutathione synthesis. An alternative or additive approach to therapy would be to inhibit the formation of the toxic metabolite by inhibiting the cytochrome P-450 system. The H2-receptor antagonist cimetidine inhibits the cytochrome P-450 system, does not interfere with the administration or function of NAC, and therefore affords additive protection. Also, it has little effect on the nontoxic routes of elimination of acetaminophen and is itself quite nontoxic. That cimetidine protects against acetaminophen toxicity in animal models has been demonstrated on the basis of improved survival, as well as decreases in several critical elements used to monitor acetaminophen toxicity: classic histologic changes, aminotransferase activity, metabolite covalent binding, and liver glutathione depletion. Administration of cimetidine well after the overdose is also protective. In contrast, animal models of acetaminophen toxicity demonstrate that ranitidine does not afford protection from acetaminophen hepatotoxicity. Clinical data in well-done trials in humans will be needed to support the experimental animal data.

Hepatotoxicity of precocene I in rats: Role of metabolic activation in vivo

The mechanism of the hepatotoxicity of precocene I has been investigated in male, Sprague-Dawley rats. Administration of a single dose of precocene I caused a large depletion of liver glutathione (GSH) levels that was both time and dose dependent. Concomitant with the decrease of liver GSH, there was an increase in serum glutamic pyruvic transaminase (GPT) levels which was also time and dose dependent. Administration of a single dose of [4- 3H]precocene I resulted in extensive covalent binding of the radiolabel to liver proteins and DNA in the liver; the extent of binding increased with increasing dose. Treatment of the rats with the mixed-function oxidase inhibitor piperonyl butoxide, before administration of precocene I, significantly decreased the proportion of the radiolabel bound covalently to proteins and DNA, although the total radioactivity (bound and unbound) in the liver remained the same. Piperonyl butoxide pretreatment limited both the liver GSH depletion and the hepatic necrosis normally caused by precocene I. These results are consistent with the view that the hepatotoxicity of precocene I is due to reactive metabolites formed through cytochrome P-450 mediated metabolism of precocene I.

Severe depletion in liver glutathione during physical exercise

Physical exercise at submaximal levels in rats results in a progressive depletion of liver glutathione to about 20% of the levels found in sedentary controls which persists for several hours following the cessation of exercise. Skeletal muscle appears to be spared this severe depletion phenomenon. The levels of plasma glutathione show a transient increase at the beginning of the exercise bout followed by a linear decrease with increased running time of the animals. These results may be particularly relevant when attempting to understand the effects of physical exercise on a large number of cellular and organismal functions that are known or suspected to depend critically on the glutathione status of the liver.

Glutathione depletion by phorone organ specificity and effect on hepatic microsomal mixed-function oxidase system.

Phorone (diisopropylidene acetone) led to a strong depletion of cellular glutathione in liver, kidney and heart but not in lung or brain upon administration to mice or rats. Its efficacy in lowering hepatic glutathione levels was comparable to that of diethylmaleate. Unlike this agent, however, phorone did not affect the microsomal mixed-function oxidase system at all. Our findings favor the use of phorone when studying the effect of a decreased glutathione content on detoxication, bioactivation or drug metabolism mechanisms, as it merely interacts with glutathione itself.

The Effects of Diethyldithiocarbamate on the Hepatotoxic Action and Antitumor Activity of N-Methylformamide in Mice

The oral administration of diethyldithiocarbamate (DTC) prevented hepatic necrosis induced by N-methylformamide (NMF) in ddY-strain mice, in more susceptible BALB/c mice and in diethylmaleate-treated mice in which NMF-hepatotoxicity was potentiated, as evidenced by suppression of increases of plasma glutamic pyruvic transaminase activity and liver calcium content or by histological observations. Early depletion of liver glutathione following NMF administration was also prevented by DTC. DTC markedly delayed the in vivo metabolism of NMF as indicated by a prolonged retention of plasma and liver NMF levels and an enhancement of urinary excretion of NMF. These observations support a bioactivation mechanism for NMF hepatotoxicity, and the hepatoprotective action of DTC may be due to an inhibition of the metabolic activation of NMF. Hepatotoxic manifestations after repeated administration of NMF also tended to be ameliorated by simultaneous treatment with DTC. Cotreatment with DTC, however, decreased the antitumor activity of NMF against Ehrlich ascites tumors, and Sarcoma 180. This also implies the involvement of a bioactivation mechanism in the antitumor action of NMF, but further studies are necessary to confirm this point. The possible therapeutic value of DTC as a hepatoprotector may be diminished by the suppression of the antitumor activity of NMF.

Enhanced inhibition of hepatic microsomal calcium pump activity by CCl 4 treatment of isopropanol-pretreated rats

Pretreatment of rats with isopropanol enhanced both hepatotoxicity and calcium pump inhibition after CCl 4 exposure in vivo or in vitro. Animals were given isopropanol (1.25 ml/kg) 18 hr before CCl 4 (0.01 to 1.0 ml/kg). CCl 4 hepatotoxicity, judged as increased appearance of glutamic-pyruvate transaminase in serum, was enhanced by isopropanol pretreatment. Pretreatment of rats with isopropanol made CCl 4 as much as 20- to 30-fold more potent as an inhibitor of the calcium pump. Inhibition of another endoplasmic reticulum enzyme, glucose 6-phosphatase, was also enhanced by isopropanol pretreatment. In contrast to the effect of CCl 4 in control animals, in isopropanol-pretreated rats given CCl 4, depletion of liver glutathione was observed. Altered CCl 4 metabolism in isopropanol-pretreated animals may result in production of increased amounts of phosgene (or other metabolites) responsible for inhibition of the liver microsome calcium pump and glutathione depletion.

Increased biliary secretion and loss of hepatic glutathione in rat liver after nifurtimox treatment

Treatment of rats with nifurtimox, a nitrofuran derivative widely used for the treatment of Chagas' disease, induced a time- and dose-dependent depletion of liver glutathione, maximal effects being obtained with 200 mg nifurtimox/kg body weight. Extra release of both oxidized (GSSG) and reduced (GSH) glutathione into bile contributed to this depletion. Glutathione excretion into bile accounted for only part of liver glutathione loss, thus indicating that, in addition to the GSH-peroxidase reaction (resulting in GSSG generation), other glutathione-related processes were involved in nifurtimox detoxification. Bile flow, bile salt excretion, liver lipid conjugated diene content, liver glutathione reductase and glutathione peroxidase activities, and serum alanine aminotransferase (ALAT) activity were not affected by the nifurtimox treatment, thus ruling out widespread damage of the liver cell by nifurtimox. Nevertheless, the extra GSH release in the nifurtimox-treated rats may indicate an alteration of the hepatocyte membrane.

Effect of chronic ethanol administration on bromobenzene liver toxicity in the rat

Female Sprague-Dawley rats were pair-fed a nutritionally adequate liquid diet containing either ethanol or isocaloric carbohydrate for 3 weeks. In vitro studies showed that chronic ethanol pretreatment preferentially increased the liver microsomal biotransformation of bromobenzene to p-bromophenol (via the toxic 3,4-epoxide) rather than to o-bromophenol (via the nontoxic 2,3-epoxide) and could thus potentiate bromobenzene hepatotoxicity. Bromobenzene administration (500 mg/kg body weight, ip), after an overnight fast, was associated in ethanol-pretreated rats with greater and accelerated liver glutathione depletion and subsequent decrease in liver cytochrome P-450 content than in controls. As assessed histologically and by determination of the rise in activities of serum enzyme markers of liver necrosis, chronic ethanol pretreatment, however, mainly resulted in earlier onset and resolution of bromobenzene-induced liver necrosis, with only a mild increase in the maximal severity of liver lesions. These results suggest that the twofold increase in liver microsomal bromobenzene 3,4-epoxidation by ethanol, being much less than that seen after phenobarbital pretreatment in our animal model and in that of others, is apparently not sufficient to markedly affect the severity of bromobenzene-induced liver toxicity.

Combined effects of polychlorinated biphenyls and methylmercury on hepatic microsomal monooxygenases and the hepatotoxic action of bromobenzene.

The combined effects of polychlorinated biphenyls (PCB) and methylmercury were investigated by assaying the activities of hepatic enzymes and by measuring the binding of bromobenzene to microsomal protein. Rats were fed normal or PCB-diet (KC-400-KC-500, 1:1, 50 ppm) for 14 days and methylmercuric chloride (10 mg Hg/kg, s.c.) was given once daily for the last 2 days. The inducing effects of PCB on microsomal cytochrome P-450, cytochrome b5, aminopyrine N-demethylase, aniline hydroxylase, and p-nitroanisole O-demethylase were counteracted by methylmercury. Glucose 6-phosphatase activity was additively decreased by the combination of PCB and methylmercury. The activity of glucose 6-phosphate dehydrogenase in soluble fraction was increased by PCB but reduced by methylmercury. The toxicity of bromobenzene was enhanced by PCB but the effect of PCB was counteracted by methylmercury. The depletion of liver glutathione and the elevation of serum transaminases by bromobenzene were remarkably potentiated by PCB. Methylmercury counteracted the effect of PCB on serum transaminases but not that on liver glutathione. The amount of bromine covalently bound with liver microsomal protein after an injection of bromobenzene and the radioactivity bound with microsomal protein after in vitro incubation of 14C-bromobenzene with microsomes were fortified by PCB pretreatment but depressed by the combining administration of methylmercury.

Effectiveness of metyrapone in the treatment of acetaminophen toxicity in mice

Metyrapone tartrate, 400 mg/kg i.p. raised the LD 50 for acetaminophen from 340 mg/kg i.p. to 540 mg/kg i.p. in fasting male Swiss white mice. The minimum protective dose of metyrapone was 200 mg/kg. Metyrapone was effective in preventing death when given up to 2 h after acetaminophen administration. The LD 50 for metyrapone tartrate was 760 mg/kg i.p. Metyrapone decreased or prevented acetaminophen induced hepatic damage measured either by histology or plasma glutamate pyruvate transaminase activity. Metyrapone tartrate, 400 mg/kg i.p., inbibited the severe liver glutathione depletion seen with acetaminophen alone. It is proposed that metyrapone protects mice from acetaminophen induced liver toxicity and death by inhibiting the oxidation of acetaminophen to a toxic intermediate.

Acute paracetamol intoxication of starved mice leads to lipid peroxidation in vivo

Lipid peroxidation was monitored in female mice in vivo by the measurement of exhalated hydrocarbons. In liver homogenates in vitro lipid peroxidation as determined by malondialdehyde formation, and hepatic total glutathione levels were measured. After a dose of 500 mg/kg i.p. of paracetamol, the hepatic glutathione of fed mice decreased from 61 nmoles/mg liver protein to 30 nmoles/mg, while the animals expired 5 nmoles of ethane/kga · hr. The same dose in starved mice led to a glutathione level of 6 nmoles/mg and an exhalation rate of 125–150 nmoles ethane/kg · hr. In vivo determined and post-mortem in vitro determined lipid peroxidation correlated with a coefficient of 0.66. If hepatic glutathione was depleted to the same extent by administration of diethylmaleate, no significant lipid peroxidation was found. Our findings demonstrate that the drug-induced depletion of liver glutathione leads in vivo to lipid peroxidation, provided that the glutathione level has been diminished by starvation. The data indicate that glutathione depletion alone by other mechanisms does not account for lipid peroxidation. Hence the hepatoprotective role of liver glutathione against drug-induced liver injury has to be reconsidered in detail. This investigation shows a suitable model for such studies.

The glutathione-linked metabolism of 2-allyl-2-isopropy-lacetamide in rats. Further evidence for the formation of a reactive metabolite

2-Allyl-2-isopropylacetamide (AIA) causes a depletion of liver glutathione in rats only if the animals have been pretreated with phenobarbitone. Phenobarbitone stimulates the excretion in bile of a component derived from AIA and glutathione which is apparently not the same as the conjugate formed by reaction of the two components in simple solutions. The significance of these findings are considered in relation to the suggestion that AIA is metabolised to an epoxide by the microsomal enzyme system; in addition several differences between AIA and the non-porphyrogenic compound, acrylamide, are discussed.

Biochemical studies on the mechanism of action of liver poisons III. Depletion of liver glutathione in ethionine poisoning

Abstract Administration of ethionine to female rats was followed by rapid fall of the hepatic reduced glutathione content, concomitant with a progressive impairment of the cell-free protein-synthesizing capacity and a decline of the ATP level. In male rats and in adrenalectomized female rats, the onset of the first two alterations was considerably delayed. In contrast, the time course and the severity of the ethionine-induced ATP depletion were essentially the same in all instances. Treatment with CCl 4 , despite the similarity of its hepatotoxic effects to those of ethionine, resulted in no appreciable decrease of the liver reduced glutathione level. Administration of methionine counteracted both the reduced glutathione decline and the other biochemical lesions induced by ethionine, whereas injection of adenine, though preventing the ATP depletion, failed to reverse the depression of reduced glutathione. The available evidence suggests that the decrease in the reduced glutathione level reflects a diminished capacity of the liver for oxidized glutathione reduction and its resultant inability to compensate the normal process of continuous reduced glutathione oxidation. The possible role of the altered reduced glutathione metabolism in the pathogenesis of ethionine hepatotoxicity is discussed.