Translational initiation regulators are hypophosphorylated in rat liver during ethionine-mediated ATP depletion

Administration of ethionine to female rats is known to inhibit hepatic protein synthesis by reducing the level of hepatic ATP. Administration of methionine and/or adenine rapidly restores the ATP levels and protein synthesis. The ethionine administration causes a progressive disaggregation of hepatic polysomes, suggesting that the initiation step of protein synthesis is inhibited. Recent studies indicate that changes in initiation are associated with alterations in the phosphorylation states of translational initiation regulators such as eukaryotic initiation factor (eIF) 4E, eIF4E-binding protein 1 (4E-BP1), and the 70-kDa ribosomal protein S6 kinase (S6K1). We found that these initiation regulators are hypophosphorylated in rat liver during ethionine-mediated ATP depletion (60% of the control value). Furthermore, the restoration of the ATP levels by the administration of methionine and adenine brought about a complete recovery of the phosphorylation states of all these regulators. The present data suggest that hypophosphorylation of various initiation regulators represents the primary event in the ethionine-induced breakdown of polysomes and inhibition of protein synthesis in the liver. Possible involvement of mammalian target of rapamycin (mTOR), as a sensor of intracellular ATP level, was also discussed.     

Effect of ethionine on the rough endoplasmic reticulum from male and female rat liver

Ethionine causes a decrease in the amount of rough endoplasmic reticulum in rat liver, the effect being greater in female than in male rats. Rough endoplasmic reticulum isolated from rat liver 24 hr after ethionine injection and stripped of its ribosomes partially lost itsin vitro ribosome binding capacity. However, no differences were detected between the binding affinities of ribosomes, isolated from either untreated animals or intoxicated rats, to stripped rough membranes derived from normal rats. Structural changes occur in the rough endoplasmic reticulum of the ethionine treated rats, while the ribosomes are still bound to the membrane.     

Early alteration in liver of rats fed 2-fluorenylacetamide investigated by L-ethionine probe

The metabolic activity of liver of rats fed a diet containing 0.03% 2-N-fluorenylacetamide (FAA) was investigated using the probe of L-[ethyl-14C]ethionine (613 mumol L-E/100 g body wt.). Shortly after the onset of the carcinogenic regimen, the capacity of liver to accumulate S-adenosylethionine (SAE) began to decline, reaching its minimum (30% of the concentration in control rats) within 3 weeks. This decreased capacity to accumulate SAE results from the FAA-induced decrease in activity of ATP-L-methionine adenosyltransferase. The concentration of hepatic ATP assayed without L-ethionine (L-E) probe also declined during the first 2 weeks of the carcinogenic regimen, but then increased, attaining the normal values within 2 more weeks. Administering the L-E probe to the FAA-fed rats produced an even greater drop in hepatic ATP concentration during the first 2 weeks; however from the third week on, the L-E dose produced no depressing effect, despite the SAE accumulation remaining at its same depressed levels and, therefore, trapping the same amount of ATP as in the previous weeks. The results show that the modification of L-E metabolism and of ATP turnover, observed previously in DL-E fed rats, need not be specific for the carcinogen fed and can occur even when the carcinogens are metabolized by different enzymatic systems.     

Hepatic glutathione concentrations linked to ethionine toxicity in rats

1. The effect of injected ethionine on liver GSH concentrations was studied in male and female rats. 2. Liver GSH concentrations were markedly and consistently decreased in 5hr. and elevated within 24-44hr. By 72hr. the amount of liver GSH of ethionine-poisoned rats had the same values as saline-injected control rats. At no time was erythrocyte GSH concentration affected by ethionine. 3. The concentrations of non-protein thiol compounds, glycogen and ATP were also significantly decreased when the rats were killed 5hr. after ethionine injection. 4. ATP, adenine or methionine did not prevent the decrease of liver GSH produced by ethionine. From these and other observations we conclude that ethionine is not an antagonist of methionine with respect to GSH metabolism. 5. The metabolic relationship between ethionine toxicity and liver GSH concentration is briefly discussed.     

Hepatoprotective effect of green tea (Camellia sinensis) extract against tamoxifen-induced liver injury in rats

Tamoxifen citrate (TAM), is widely used for treatment of breast cancer. It showed a degree of hepatic carcinogenesis. The purpose of this study was to elucidate the antioxidant capacity of green tea (Camellia sinensis) extract (GTE) against TAM-induced liver injury. A model of liver injury in female rats was done by intraperitoneal injection of TAM in a dose of 45mg Kg(-1) day(-1), i.p. for 7 successive days. GTE in the concentration of 1.5 %, was orally administered 4 days prior and 14 days after TAM-intoxication as a sole source of drinking water. The antioxidant flavonoid; epicatechin (a component of green tea) was not detectable in liver and blood of rats in either normal control or TAM-intoxicated group, however, TAM intoxication resulted in a significant decrease of its level in liver homogenate of tamoxifenintoxicated rats. The model of TAM-intoxication elicited significant declines in the antioxidant enzymes (glutathione-S-transferase,glutathione peroxidase, superoxide dismutase and catalase) and reduced glutathione concomitant with significant elevations in TBARS (thiobarbituric acid reactive substance) and liver transaminases; sGPT (serum glutamate pyruvate transaminase) and sGOT (serum glutamate oxaloacetate transaminase) levels. The oral administration of 1.5 % GTE to TAM-intoxicated rats, produced significant increments in the antioxidant enzymes and reduced glutathione concomitant with significant decrements in TBARS and liver transaminases levels. The data obtained from this study speculated that 1.5 % GTE has the capacity to scavenge free radical and can protect against oxidative stress induced by TAM intoxication. Supplementation of GTE could be useful in alleviating tamoxifen-induced liver injury in rats.     

Effect of picroliv on glutathione metabolism in liver and brain of Mastomys natalensis infected with Plasmodium berghei.

Administration of picroliv, the active principle from Picrorhiza kurrooa, at a dose of 6 mg/kg, po for two weeks showed significant protection against changes in liver and brain glutathione metabolism of Plasmodium berghei infected Mastomys natalensis. The depletion of reduced glutathione level and inhibition of glutathione-S-transferase, glutathione reductase and glutathione peroxidase activities due to P. berghei infection were markedly recovered by picroliv. The increased levels of lipid peroxidation products in damaged tissues were also reduced along with the recovery of glutathione metabolism.     

The effect of dimethyl dimethoxy biphenyl dicarboxylate (DDB) against tamoxifen-induced liver injury in rats: DDB use is curative or protective

Tamoxifen citrate is an anti-estrogenic drug used for the treatment of breast cancer. It showed a degree of hepatic carcinogenesis, when it used for long term as it can decrease the hexose monophosphate shunt and thereby increasing the incidence of oxidative stress in liver rat cells leading to liver injury. In this study, a model of liver injury in female rats was done by intraperitoneal injection of tamoxifen in a dose of 45 mg/kg body weight for 7 successive days. This model produced a state of oxidative stress accompanied with liver injury as noticed by significant declines in the antioxidant enzymes (glutathione-S-transferase, glutathione peroxidase and catalase) and reduced glutathione concomitant with significant elevations in TBARS (thiobarbituric acid reactive substance) and liver transaminases; sGPT (serum glutamate pyruvate transaminase) and sGOT (serum glutamate oxaloacetate transaminase) levels. The oral administration of dimethyl dimethoxy biphenyl dicarboxylate (DDB) in a dose of 200 mg/kg body weight daily for 10 successive days, resulted in alleviation of the oxidative stress status of tamoxifen-intoxicated liver injury in rats as observed by significant increments in the antioxidant enzymes (glutathione-S-transferase, glutathione peroxidase and catalase) and reduced glutathione concomitant with significant decrements in TBARS and liver transaminases; sGPT and sGOT levels. The administration of DDB before tamoxifen intoxication (as protection) is more little effective than its curative effect against tamoxifen-induced liver injury. The data obtained from this study speculated that DDB can mediate its biochemical effects through the enhancement of the antioxidant enzyme activities and reduced glutathione level as well as decreasing lipid peroxides.     

Inhibition of the initiation of hepatic protein synthesis during ethionine mediated ATP depletion in vivo: modification to ribosomal subunits, evidence of impaired ternary complex formation and a subcellular redistribution of eIF-2 alpha

Acute ethionine intoxication is known to induce a reversible hepatic injury in female rats by reducing the level of hepatic ATP. The injury indirectly impairs the initiation of hepatic protein synthesis, with resultant polysome disaggregation. Administration of adenine rapidly restores the ATP levels and protein synthesis. Analysis of liver polysome and ribosomal subunits reveals that polysome disaggregation occurs following 3 h of the intoxication, and reaggregation occurs following the administration of adenine. Inactive hepatic ribosomes accumulate as monomers and disomes when analysed by sucrose gradient sedimentation in low-salt buffers. High-salt buffers dissociate the inactive ribosomes into the component 40 S and 60 S subunits. The level of higher density, 1.48 g/cc, 40 S subunit increases during the inhibition of protein synthesis, while the lower density, 1.41 g/cc, 40 S subunit species does not change significantly. Hepatic microsomal and cytosolic extracts examined for their ability to support the formation of the ternary complex of eIF-2-GTP and [35S]Met-tRNAi demonstrate that during acute ethionine intoxication, ternary complex formation in the two extracts decrease 65% and 85%, respectively. These changes are coincident with polysome disaggregation. Administration of adenine to reverse the intoxication restores the ternary complex forming ability of the cytosolic extract, but does not affect the activity of the microsomal salt wash extracts. Mixing experiments indicate the accumulation of an inhibitor of ternary complex formation in the microsomal salt wash fraction. The application of quantitative western blotting demonstrates that the level of antigenic eIF-2 alpha in the microsomal salt wash extract increases 31% during the inhibition. These observations are consistent with the idea that the inhibition of the initiation of hepatic protein synthesis induced by ethionine is mediated by eIF-2 alpha phosphorylation. The latter results in an inhibition of ternary complex formation, redistribution of eIF-2 to the microsome fraction, polysomal disaggregation, and accumulation of inactive ribosomal subunits.     

Effect of gender differences and voluntary exercise on antioxidant capacity in rats

The effects of gender difference and voluntary exercise on antioxidant capacity in rats were evaluated. The subjects were divided into two groups, physically active and sedentary. In the sedentary group, the level of hydroxyl radical in the liver was higher (P<0.001) in male rats than in female rats, however, in the physically active group, the level in male rats was lower (P<0.05) than in female rats. The levels of reduced glutathione (GSH) in physically active males and females were higher compared to those in the sedentary group. The physically active group also showed an increase in antioxidant enzymes, such as glutathione peroxidase (GPx), glutathione reductase (GR) and superoxide dismutase activities. The level of liver GSH was higher in physically active females than in physically active males. For both groups, GPx and GR activities in females were significantly higher than in males. These results indicate that female rats have an intrinsically higher antioxidant capacity, which resulted in increased levels of GSH via the glutathione redox cycle and gamma-glutamyl cycle enzymes. The adaptation to altered antioxidant capacity, induced by physical activity, appeared to be affected by gender differences.     

Time-dependent enhancement in mitochondrial glutathione status and ATP generation capacity by schisandrin B treatment decreases the susceptibility of rat hearts to ischemia-reperfusion injury

In the present study, we examined the time-dependent changes in the mitochondrial glutathione status and ATP generation capacity in the myocardium as well as the susceptibility of the myocardium to ischemia-reperfusion (IR) injury in female Sprague Dawley rats treated with a single pharmacological dose (1.2 mmol/kg) of schisandrin B (Sch B). Sch B treatment produced a time-dependent enhancement in myocardial mitochondrial glutathione status, as evidenced by increases in myocardial mitochondrial reduced glutathione (GSH) level and activities of glutathione reductase, Se-glutathione peroxidase (GPX) and glutathione S-transferases, with the response reaching maximum at 48 h post-dosing and then declining gradually to the control level at 96 h post-dosing. The enhancement of mitochondrial glutathione status was associated with an increase in myocardial ATP generation capacity, with the value peaking at 72 h post-dosing. These beneficial effects of Sch B on the myocardium was paralleled by a time-dependent decrease in the susceptibility to IR injury, with the maximum protection demonstrable at 48 h post-dosing. The cardioprotection was associated with increases in myocardial GSH level and activities of glutathione antioxidant enzymes (except for GPX whose activity was suppressed) as well as tissue ATP level/ATP generation capacity. The results suggest that Sch B treatment can precondition the myocardium by enhancing the mitochondrial glutathione status and ATP generation capacity, thereby protecting against IR injury.     

Protective effect of glucose-cysteine adduct on thein situ perfused rat liver

Summary Insitu perfusion of rat liver was performed with a medium containing glucose-cysteine adduct [2-(D-gluco-pentahydroxypentyl) thiazolidine-4-carboxylic acid, glc-cys] and its effect on glutathione (GSH) and ATP levels and bile production was examined. The GSH content in the liver was maintained at the original level during perfusion with 1 mM glc-cys for 2h, while it decreased significantly in the absence of glc-cys. After 4h of perfusion without glc-cys, ATP content and bile production decreased significantly besides the decrease in GSH content, but they were maintained at the original levels with glc-cys. When the perfusion was performed with the liver of rats injected with diethyl maleate (DEM), the GSH level, which was decreased to 6.0% of the control by DEM injection, was restored to 22.6% of the original level by perfusion with 2mM glc-cys for 30 min. Data indicate that glccys is a cysteine prodrug with protective action on the liver.     

Effect of allylisopropylacetamide on glutathione metabolism in the rat liver. The possible role of glutathione in the induction of 5-aminolaevulinate synthase

Administration of allylisopropylacetamide to rats caused a marked decline in the concentrations of reduced and oxidized glutathione in the liver. However, this decrease occurred in the presence of uninhibited activities of gamma-glutamylcysteine synthase and glutathione reductase, and unaltered activities of glutathione transferases A, B and C. The administration of cysteine, the rate-limiting precursor of glutathione formation, to rats treated with allylisopropylacetamide potentiated the inductive effects of the agent on 5-aminolaevulinate synthase, and markedly decreased the extent of decrease in glutathione concentrations by the agent. Conversely, the administration of diethyl maleate, which depletes the hepatic glutathione concentrations, to allylisopropylacetamide-pretreated rats (1h) diminished the extent of 5-aminolaevulinate synthase induction and the production of porphyrins by nearly 50%, when measured at 16h. This treatment did not alter the extent of non-enzymic degradation of liver haem by allylisopropylacetamide. When diethyl maleate was administered to the animals possessing high 5-aminolaevulinate synthase activity (at 3, 7 and 15h after allylisopropylacetamide), in 1h the enzyme activity was markedly decreased. Diethyl maleate had no effect on induction of 5-aminolaevulinate synthase by 3,5-diethoxycarbonyl-1,4-dihydrocollidine, also a potent porphyrinogenic agent. Diethyl maleate alone neither inhibited 5-aminolaevulinate synthase activity nor decreased the cellular content of porphyrins and haem. The data suggest that the decreases observed in the glutathione concentrations after allylisopropylacetamide administration are not the result of decreased production of the tripeptide. Rather, they most likely reflect the increased utilization of glutathione. The findings further suggest that the inhibition by diethyl maleate of allylisopropylacetamide-stimulated 5-aminolaevulinate synthase involves the inhibition of induction processes.     

High-fat diet prevents eating response and attenuates liver ATP decline in rats given 2,5-anhydro-D-mannitol

Administration of the fructose analog 2,5-anhydro-D-mannitol (2,5-AM) stimulates eating in rats fed a low-fat diet but not in those fed a high-fat diet that enhances fatty acid oxidation. The eating response to 2,5-AM treatment is apparently triggered by a decrease in liver ATP content. To assess whether feeding a high-fat diet prevents the eating response to 2,5-AM by attenuating the decrease in liver ATP, we examined the effects of the analog on food intake, liver ATP content, and hepatic phosphate metabolism [using in vivo 31P-NMR spectroscopy (NMRS)]. Injection (intraperitoneal) of 300 mg/kg 2,5-AM increased food intake in rats fed a high-carbohydrate/low-fat diet, but not in those fed high-fat/low-carbohydrate (HF/LC) food. Liver ATP content decreased in all rats given 2,5-AM compared with saline, but it decreased about half as much in rats fed the HF/LC diet. NMRS on livers of anesthetized rats indicated that feeding the HF/LC diet attenuates the effects of 2,5-AM on liver ATP by reducing phosphate trapping. These results suggest that rats consuming a high-fat diet do not increase food intake after injection of 2,5-AM, because the analog is not sufficiently phosphorylated and therefore fails to decrease liver energy status below a level that generates a signal to eat.     

Enhancement of lipid peroxidation in rat liver on acute exposure to styrene and acrylamide a consequence of glutathione depletion

Lipid peroxidation, glutathione level and activity of glutathione-S-transferase were studied in liver and brain of rats 4 and 3 h after a single i.p. administration of 0, 25, 75, 100 mg/kg acrylamide or 0, 50, 100, 200, 600 mg/kg styrene, respectively. In liver both acrylamide and styrene caused an increase in lipid peroxidation and decrease in glutathione contents and activity of glutathione-S-transferase in a dose dependent manner, while in brain only acrylamide produced a decrease in glutathione content. The decrease in glutathione content was not always associated with increase of lipid peroxidation. The enhancement of lipid peroxidation occurred only when glutathione contents were depleted to certain critical levels. No effect of acrylamide or styrene was seen on lipid peroxidation under in vitro conditions. The addition of glutathione in the incubation mixture significantly inhibited the rate of lipid peroxidation of liver homogenates of acrylamide and styrene treated animals.The results suggest that enhancement of lipid peroxidation in liver on exposure to acrylamide or styrene is a consequence of depletion of glutathione to certain critical levels. The inhibition of glutathione-S-transferase activity by acrylamide and styrene suggests that detoxication of these neurotoxic compounds could be suppressed following acute exposure.     

Effects of emodin treatment on mitochondrial ATP generation capacity and antioxidant components as well as susceptibility to ischemia-reperfusion injury in rat hearts: single versus multiple doses and gender difference

Effects of emodin (EMD) treatment on mitochondrial ATP generation capacity and antioxidant components as well as susceptibility to ischemia-reperfusion (I-R) injury were examined in male and female rat hearts. Isolated-perfused hearts prepared from female rats were less susceptible to I-R injury than those of male rats. I-R caused significant decreases in ATP generation capacity and reduced glutathione (GSH) and alpha-tocopherol (alpha-TOC) levels as well as glutathione reductase, Se-glutathione peroxidase and Mn-superoxide dismutase (SOD) activities. The lower susceptibility of female hearts to myocardial I-R injury was associated with higher levels of GSH and alpha-TOC as well as activity of SOD than those of male hearts. EMD treatment at 3 daily doses (0.6 or 1.2 mmol/kg) could enhance myocardial mitochondrial ATP generation capacity and antioxidant components in both male and female rat hearts, but it only significantly protected against I-R injury in female hearts. Treatment with a single dose of EMD invariably enhanced mitochondrial antioxidant components and protected against I-R injury in both male and female hearts. The gender-dependent effect of EMD treatment at multiple doses may be related to the differential antioxidant response in the myocardium and/or induction of drug metabolizing enzymes in the liver.     

The effect of melaxen and valdoxan on the activity of the glutathione antioxidant system in rats with experimental hyperthyroidism

The activity of the glutathione system and conjugated diene content (CD) have been investigated in the liver and blood serum of rats with experimental hyperthyroidism treated with melaxen and valdoxan. The study of glutathione reductase (GR), glutathione peroxidase (GP) and glutathione transferase (GST) activities increased under this pathology has shown that administration of these compounds decreased these activities towards control levels. Melaxen and valdoxan administration increased reduced glutathione (GSH) content as compared with untreated hyperthyroid rats. This increase may be associated with its decreased utilization for detoxification of toxic products of free radical oxidation (FRO). Administration of the melatonin correcting drugs also tended to normalize the CD level increased in the liver and blood serum of hyperthyroid rats. Results of this study indicate that melaxen and valdoxan exhibit positive effect on free radical homeostasis. This appears to be accompanied by a decrease in the load of the glutathione antioxidant system in comparison with the examined pathology.     

Effects of ethionine on tRNA methylation in male and female rats

Administration of hepatocarcinogens to rats results in an increase in tRNA methyltransferase activity in the target tissues. Ethionine is active as a carcinogen only in female rats and only in females is this increase in enzyme activity seen. However, ethionine also causes the formation of methyl-deficient tRNA in the liver. Other hepatocarcinogens do not do this. Ethionine is equally effective in this action in males and females. Thus, the two actions of ethionine are completely separable, and the methyl-deficiency of tRNA is caused by an activity not identical with the carcinogenic one.     

Prevention of N-nitrosodiethylamine-induced hepatocarcinogenesis by S-allylcysteine

Chemopreventive effect of S-allylcysteine (constituent of garlic) on N-nitrosodiethylamine (NDEA)-induced hepatocarcinogenesis was evaluated in Wistar rats. Significantly decreased lipid peroxidation products (thiobarbituric acid reactive substances-TBARS and lipid hydroperoxides) with increased level of reduced glutathione, increased activities of glutathione S-transferase, and glutathione peroxidase were observed in liver of NDEA-treated rats when compared with control rats. The activities of superoxide dismutase and catalase were significantly decreased in tumor tissue when compared with control. Administration of S-allylcysteine (SAC) showed the inhibition of tumor incidence, modulated the lipid peroxidation, and increased the reduced glutathione, glutathione-dependent enzymes, superoxide dismutase, and catalase in NDEA-induced carcinogenesis. From our results, we speculate that S-allylcysteine mediates its chemopreventive effects by modulating lipid peroxidation, GST stimulation, and by increasing the antioxidants. Hence SAC prevents cells from loss of oxidative capacity in NDEA-induced hepatocarcinogenesis.     

Ethionine regulates cell motile activity through LPA receptor-3 in liver epithelial WB-F344 cells

Lysophosphatidic acid (LPA) receptors (LPA₁ to LPA₆) indicate a variety of cellular responses, such as cell proliferation, migration, differentiation, and morphogenesis. However, the role of each LPA receptor is not functionally equivalent. Ethionine, an ethyl analog of methionine, is well known to be one of the potent liver carcinogens in rats. In this study, to assess whether ethionine may regulate cell motile activity through LPA receptors, rat liver epithelial (WB-F344) cells were treated with ethionine for 48 h. In cell motility assay with a cell culture insert, the treatment of ethionine at 1.0 and 10 μM enhanced significantly high cell motile activity, compared with untreated cells. The expression levels of LPA receptor genes in cells treated with ethionine were measured by quantitative real time RT-PCR analysis. The expression of the Lpar3 gene in ethionine-treated cells was significantly higher than that in untreated cells. Furthermore, to confirm an involvement of LPA₃ on cell motility increased by ethionine, the Lpar3 knockdown cells were also used. The cell motile activity by ethionine was completely suppressed in the Lpar3 knockdown cells. These results suggest that LPA signaling through LPA₃ may be involved in cell motile activity stimulated by ethionine in WB-F344 cells.     

Ethionine-induced changes in the activities of S-adenosylmethionine synthetase isozymes from rat liver

Isozyme patterns of $\text{S}$-adenosylmethionine synthetase have been measured with and without dimethylsulfoxide in rat liver incuced by ethionine. The activity of the α-form is increased following administration of ethionine plus adenine for 2 consecutive days, and gradually decreased to control level on the 7th day after treatment, whereas the activity of β-form is relatively unaffected. Methyl-deficient transfer RNA and enhanced levels of transfer RNA-methylating enzymes were found in the livers of female rats after the treatment for 2 days, following which they gradually returned to control level.