Effect of ruthenium red on the induction by Ca2+ ions of the beta- and gamma states of comuton regulation of mitochondrial respiration and oxidative phosphorylation

Preincubation of liver mitochondria (Mch) with Ca2+ ions at inorganic phosphate concentration less than I mM in the presence of liver cell soluble phase (CSP) induced rotenone-independent tissue-specific uncoupling of oxidative phosphorylation (beta state of comuton regulation) and rotenone-stimulated tissue-specific uncoupling (gamma state of comuton regulation). The reduction in K+ ion concentration in the incubation medium entirely inhibited the induction of beta state. Tissue-specific stimulation of the rat liver Mch respiration in substrate-containing medium was increased after rotenone addition. Ruthenium red was added to the medium before and after the end of Mch preincubation with Ca2+ in the presence of CSP. The results suggest that limited Ca2+ transport in Mch is necessary for the induction of beta and gamma states of comuton regulation. Ca2+ ejected from Mch also participates in the induction of beta state of comuton regulation. Comuton receptor on the mitochondrial membrane surface is devoid of glyco- and mucoprotein components bound by ruthenium red.     

The induction of the beta state of the comuton regulation of mitochondrial oxidative phosphorylation by 2,4-DNP and malonate

The effect of 2,4-DNP and malonate on tissue-specific uncoupling of oxidative phosphorylation (OP) of rat liver and kidney mitochondria by homologous comutons has been studied. The addition of 2,4-DNP in the presence of comuton induced beta state of comuton regulation. Transfer of liver mitochondria from alpha to beta state also resulted from partial inhibition of succinate dehydrogenase activity of addition of 0.25-0.35 mM malonate. This suggests that the transfer to beta state may be caused by de-energization of mitochondria.     

Involvement of endogenous CRF in carbon tetrachloride-induced acute liver injury in rats

Central neuropeptides play important roles in many physiological and pathophysiological regulation mediated through the autonomic nervous system. In regard to the hepatobiliary system, several neuropeptides act in the brain to regulate bile secretion, hepatic blood flow, and hepatic proliferation. Central injection of corticotropin-releasing factor (CRF) aggravates carbon tetrachloride (CCl4)-induced acute liver injury through the sympathetic nervous pathway in rats. However, still nothing is known about a role of endogenous neuropeptides in the brain in hepatic pathophysiological regulations. Involvement of endogenous CRF in the brain in CCl4-induced acute liver injury was investigated by centrally injecting a CRF receptor antagonist in rats. Male fasted Wistar rats were injected with CRF receptor antagonist alpha-helical CRF-(9-41) (0.125-5 microg) intracisternally just before and 6 h after CCl4 (2 ml/kg) administration, and blood samples were obtained before and 24 h after CCl4 injection for measurement of hepatic enzymes. The liver sample was removed 24 h after CCl4 injection, and histological changes were examined. Intracisternal alpha-helical CRF-(9-41) dose dependently (0.25-2 microg) reduced the elevation of alanine aminotransferase and aspartate aminotransferase levels induced by CCl4. Intracisternal alpha-helical CRF-(9-41) reduced CCl4-induced liver histological changes, such as centrilobular necrosis. The effect of central CRF receptor antagonist on CCl4-induced liver injury was abolished by sympathectomy and 6-hydroxydopamine pretreatment but not by hepatic branch vagotomy or atropine pretreatment. These findings suggest the regulatory role of endogenous CRF in the brain in experimental liver injury in rats.     

CCl4致小鼠肝损伤中几种免疫介质含量变化的研究

本文通过研究CCl4致小鼠肝损伤组织匀浆和血浆一些免疫介质含量的变化以探讨这些免疫介质在CCl4诱发肝损伤过程中作用机制。分别选用30只健康成年小鼠,雌雄各半,随机分成对照组和CCl4负荷组,每组15只。通过腹腔注射CCl4诱发肝损伤后,分别在第2、4、6周检测肝组织匀浆cAMP、cGMP和MDA及血浆IL-2、TNF-α水平的变化。结果显示,在整个实验期内,CCl4组肝组织匀浆cAMP水平均低于或明显低于对照组;cGMP在实验第2周后,高于或显著高于对照组;cAMP／cGMP比值呈现下降趋势,并低于或明显低于对照组;MDA含量明显高于对照组。在整个实验期内,CCl4组血浆IL-2水平下降或显著下降;TNF-α水平则均高于或显著高于对照组。结果提示,CCl4负荷诱发免疫介质cAMP、cGMP、TNF-α和IL-2发生剧烈变化,在介导肝损伤过程中可能起重要作用。     

Aqueous extract of Anoectochilus formosanus attenuate hepatic fibrosis induced by carbon tetrachloride in rats.

The aim of this study was to investigate the effects of aqueous extract of Anoectochilus formosanus (AFE) on liver fibrogenesis in carbon tetrachloride (CCl4)-induced cirrhosis. Fibrosis was induced in rats by oral administration of CCl4 (20%, 0.5 ml/rat, p.o.) twice a week for 8 weeks. AFE (0.5 and 2.0 g/kg, p.o., daily for 8 weeks) was administered to rats simultaneously. AFE showed reducing actions on the elevated levels of GOT and GPT caused by CCl4. Liver fibrosis in rats induced by CCl4 led to the drop of serum albumin concentration; the AFE increased the albumin concentration. The CCl4-induced liver fibrosis markedly caused liver atrophy and splenomegalia, while AFE increased the liver weight, and decreased the spleen weight. The CCl4-induced liver fibrosis decreased the protein content, and increased collagen contents in rat's liver. AFE significantly increased the contents of protein and reduced the amount of collagen in the liver. In CCl4-treated rats, glutathione concentrations of liver were not affected. AFE significantly increased liver glutathione concentrations. All these results clearly demonstrate that AFE can reduce the liver fibrogensis in rats induced by CCl4.     

Chromium (III) decreases carbon tetrachloride-originated trichloromethyl radical in mice.

Effects of the administration of trivalent chromium (Cr(III)) to mice and the activation of carbon tetrachloride (CCl4) to form trichloromethyl radicals (.CCl3) in the liver were studied. The lipid peroxidation in liver microsomes induced in vitro by CCl4 in the presence of NADPH was decreased by the preadministration of Cr(III) to mice. The activity of NADPH-cytochrome C reductase, which presumably catalyzes the formation of .CCl3 from CCl4 in liver microsomes, was depressed by Cr(III) administration and kept at a level lower than that of the control group for at least 2 hr after CCl4 dosing. Furthermore, the frequency of appearances of ESR signals of .CCl3 in the liver homogenate of mice 1 min after CCl4 administration was markedly decreased by Cr(III) preadministration, similarly to DL-alpha-tocopherol. These results suggest that Cr(III) preadministered to mice decreases the formation of .CCl3 from CCl4, an activating process of CCl4, in the liver, presumably by scavenging the radical.     

Mitochondrial phospholipid bilayer structure is ruined after liver oxidative injury in vivo

The purpose of this study was to investigate whether, after oxidative injury in vivo, liver mitochondrial phospholipids suffered from structural defects similar to those we have previously observed after either chemical oxidation or respiration state IV incubation of isolated mitochondria in vitro. Oxidative injury of the liver was simulated by endogastric administration of CCl4 to rats in variable amounts for different times, under various conditions. Measurements of the phospholipid bilayer packing order were carried out by electron paramagnetic resonance (EPR) spectrometry of oriented planar samples of phospholipids extracted from liver mitochondria, spin labeled with 5-doxylstearoyl-lecithin. Disordering of the bilayer was revealed by the anisotropy loss of EPR spectra and reached a maximum value 4.5 h after CCl4 administration, vanishing thereafter. The observed disorder also increased with the amount of CCl4 administered, showing distinct dose-dependence, while administration of resveratrol soon after carbon tetrachloride decreased bilayer disordering by 50%. On the contrary, the order parameter S of spin labeled lecithin in isolated mitochondrial membranes from intoxicated rats revealed no change in membrane fluidity after oxidative stress. It is concluded that the phospholipid damage leading to disturbed bilayer geometry after oxidative attack already observed in model membranes and in isolated mitochondria in vitro also occurs in a simulated pathological state in vivo, indicating its possible occurrence also in real oxidative stress-linked pathologies as a contribution to the onset/sustaining of related diseases.     

L-tryptophan administration promotes the reversion of pre-established chronic liver injury in rats treated with carbon tetrachloride

We examined the effect of L-tryptophan (Trp) administration on the reversion of CCl(4)-induced chronic liver injury after hepatotoxicant withdrawal in rats. When rats treated with CCl(4) twice a week for 6 weeks were released from CCl(4) treatment for 2 weeks, there was an incomplete reversion of liver injury. The reversion was enhanced by 2 weeks of daily intraperitoneal administration of Trp (50 mg/kg body weight), starting just after CCl(4) withdrawal. There were increases in the levels of thiobarbituric acid reactive substances, an index of lipid peroxidation, Ca(2+), triglycerides, and Trp, and decreases in tryptophan 2,3-dioxygenase activity and serum triglyceride concentrations in the liver of rats treated with CCl(4) for 6 weeks. Serum albumin concentrations and in vitro hepatic protein synthesis activity did not change in the CCl(4)-treated rats. The changes in the CCl(4)-treated rats were partially attenuated 2 weeks after CCl(4) withdrawal. The attenuation was enhanced by 2 weeks of daily Trp administration. The increases in hepatic thiobarbituric acid reactive substances and triglycerides and the decreases in hepatic tryptophan 2,3-dioxygenase activity and serum triglyceride concentrations observed 2 weeks after CCl(4) withdrawal were almost completely attenuated by Trp administration. In vitro hepatic protein synthesis in CCl(4)-treated and untreated rats was increased by 2 weeks of daily Trp administration. These results indicate that Trp administration promotes the reversion of pre-established chronic liver injury in rats treated with CCl(4,) and suggest that Trp exerts this effect by enhancing the improvement of several parameters of liver dysfunction associated with chronic liver injury and by stimulating hepatic protein synthesis.     

Hepatoprotective activity of ellagic acid against carbon tetrachloride induced hepatotoxicity in rats

Administration of CCl4 to normal rats and consequent oral feeding with ellagic acid (50 mg/kg) provided a significant protection against the biochemical alterations in serum and liver produced by CCl4. In vitro experiments showed that liver microsomes from animals treated with ellagic acid and CCl4, decreased lipid peroxidation compared to microsome prepared from rats exposed to CCl4 alone.     

Role of the functional state of the liver RES in the pathogenesis of toxic liver injury

In rats to which E. coli endotoxin (250 micrograms/kg i.p.) was administered 24 h before they were given tetrachlormethane (CCl4) (1.5 ml/kg intragastrically), stimulation of liver DNA synthesis was observed during the first 48 h after administration of the hepatatoxin. In experimental rats to which prodigiosan (a Serratia marcescens polysaccharide, 250 micrograms/kg i.p.) was administered 24 h before CCl4 (1.5 ml/kg i.p.), liver damage 24 h after CCl4 poisoning was expressed less--judging from the size of liver necrosis and the size of glycogen-free zones in the liver lobules than in the controls. To elucidate the role of activated macrophages in the induction of liver resistance to CCl4, liver injury caused by this hepatotoxin was compared after the pre-administration of protein extract from the Kupffer cells or hepatocytes of prodigiosan-stimulated rats. In rats given the larger dose of Kupffer cell extract (6 mg/ml i.p.), the necrotic foci formed after the administration of CCl4 were significantly smaller. The results confirm the conception that liver macrophages participate in the development of resistance to CCl4.     

Hepatocyte heterogeneity in ammonia metabolism: impairment of glutamine synthesis in CCl4 induced liver cell necrosis with no effect on urea synthesis

After induction of a perivenous liver cell necrosis by CCl4 pretreatment of the rat, ammonia uptake by perfused liver is decreased. This was due to an inhibition of glutamine synthesis from added ammonia, whereas urea synthesis was not affected by CCl4 pretreatment. The data confirm recent findings on hepatocyte heterogeneity in ammonia metabolism and are explained by an impairment of perivenous glutamine synthetase, but not of periportal urea synthesis, by the perivenous liver cell necrosis induced by CCl4. Regarding the pathogenesis of hyperammonemia in acute severe liver disease like CCl4 poisoning, the data point to a role of an impaired glutamine synthesis, but not to an impairment of urea synthesis.     

Hepatocyte mitochondrion respiratory chain in rats with experimental toxic hepatitis

The purpose of this study was to examine hepatocyte mitochondrion respiratory chain in rats subjected to ethanol and CCl4 administration within 4 weeks to induce an experimental hepatitis. Oxygen consumption was determined as a measure of mitochondrion respiration chain function. The development of liver pathology was accompanied by fat accumulation, fibrosis, triglycerides and lipid peroxidation increase. Respiratory chain characteristics damage was found. Endogenous oxygen consumption by hepatocytes isolated from pathological liver was found 34% higher compared to control. Exogenous malate and pyruvate substrates delivery didn't stimulate cell respiration. Rotenone (the inhibitor of the I complex) decreased 27% oxygen consumption by pathological hepatocytes while dinitrophenol produced 37% cell respiration increase. States 3 (V3) and 4 (V4) mitochondrial respiration with malate + glutamate as substrates were found to be 70 and 56% higher accordingly compared to control level. V3 and Vd (dinitrophenol respiration) for mitochondria from pathological liver didn't differ from control when being tested with malate + glutamate or succinate as substrates. Cytochrome c oxidase activity increased (+ 80%) as compared to control. Administration of hypolipidemic agent simvastatin simultaneously with ethanol and CC14 resulted in decrease liver fat accumulation, fibrosis and peroxidation products. Simvastatin administration caused hepatocyte endogenous respiration decrease while malate + pyruvate, dinitrophenol or rotenone delivery produced oxygen consumption alterations similar to control. However, when isolated mitochondria from liver of simvastatin treated animals being tested the decrease of oxidative phosphorylation coupling for substrates malate + glutamate was found. While simvastatin did not cause changes in cytochrome c oxidase activity. We propose the hypothesis that the NCCR complex in rat mitochondria with experimental toxic hepatitis works extensively on superoxydanion production. Alterations of SCCR, Coenzyme Q-cytochrome c-reductase, cytochrome c oxidase and ATP-synthase activities have an adaptive nature to compensate for impaired NCCR function.     

Oxygen- and carbon-centered free radical formation during carbon tetrachloride metabolism. Observation of lipid radicals in vivo and in vitro

Free radical reactions involved in the metabolism of carbon tetrachloride by rat liver have been considered to be a cause of at least part of the injury resulting from exposure to this halocarbon. In an earlier study employing electron spin resonance and spin-trapping techniques, we demonstrated that trichloromethyl (13.CCl3) radicals are readily observed in rat liver microsomes metabolizing 13CCl4, and that the same radical could be shown to form in vivo in the liver of intact rats given a single dose of 13CCl4. This report describes the production of lipid dienyl (L.) and oxygen-centered lipid radicals (LO. or LOO., or both) in in vitro systems metabolizing 13CCl4, and also the formation of lipid dienyl radicals (L.) in liver of intact animals exposed to CCl4. The radicals appear to be produced in a sequence of reactions governed among other things by the oxygen tension in the system. The lipid radicals (L.) which form in intact liver of CCl4-treated rats are apparently the result of an attack on lipids of the endoplasmic reticulum by 13.CCl3 radicals formed by reductive cleavage to CCl4 and are the initial intermediates in the process of lipid peroxidation. These investigations demonstrate that while the events occurring in liver microsomes in vitro appear to parallel those which take place in intact liver in vivo, the conditions in vivo make the spin-trapping studies of radicals in intact animals much more selective than it is in vitro for a given spin trap, and requires the use of more than one type of spin-trapping agent to detect different radical species in vivo.     

Early growth response (EGR)-1 is required for timely cell-cycle entry and progression in hepatocytes after acute carbon tetrachloride exposure in mice

Cell-cycle induction in hepatocytes protects from prolonged tissue damage after toxic liver injury. Early growth response (Egr)-1(-/-) mice exhibit increased liver injury after carbon tetrachloride (CCl(4)) exposure and reduced TNF-α production. Because TNF-α is required for prompt cell-cycle induction after liver injury, here, we tested the hypothesis that Egr-1 is required for timely hepatocyte entry into the cell cycle after CCl(4)-induced liver injury. Acute liver injury was induced by a single injection of CCl(4). Assays were employed to assess indices of the cell cycle in liver after CCl(4) exposure. Bromodeoxyuridine incorporation peaked in wild-type mice at 48 h after CCl(4) but was reduced by 80% in Egr-1(-/-) mice. Proliferating-cell nuclear-antigen immunohistochemistry revealed blocks in cell-cycle entry and progression to DNA synthesis in Egr-1-deficient mice 48 h after CCl(4). Cyclin D, important for G0/G1 progression, was reduced at baseline and 36 h after CCl(4). Cyclin E1, required for G1/S-phase transition, was reduced in Egr-1(-/-) mice 24 and 48 h after CCl(4) exposure and was associated with reduced phosphorylation of the retinoblastoma protein. Proliferation in Egr-1(-/-) mice was delayed, rather than blocked, because indices of cell-cycle progression were restored 72 h after CCl(4) exposure. We concluded that Egr-1 was required for prompt cell-cycle entry (G0- to G1-phase) and G1/S-phase transition after toxic liver injury. These data support the hypothesis that Egr-1 provides hepatoprotection in the CCl(4)-injured liver, attributable, in part, to timely cell-cycle induction and progression.     

Effects of retinoic acid on the development of liver fibrosis produced by carbon tetrachloride in mice

The role of retinoic acid (RA) in liver fibrogenesis was previously studied in cultured hepatic stellate cells (HSCs). RA suppresses the expression of alpha2(I) collagen by means of the activities of specific nuclear receptors RARalpha, RXRbeta and their coregulators. In this study, the effects of RA in fibrogenesis were examined in carbon tetrachloride (CCl4) induced liver fibrosis in mice. Mice were treated with CCl4 or RA and CCl4, along side control groups, for 12weeks. RA reduced the amount of histologically detectable fibrosis produced by CCl4. This was accompanied by a attenuation of the CCl4 induced increase in alpha2(I) collagen mRNA and a lower (2-fold versus 3-fold) increase in liver hydroxyproline. Furthermore, RA reduced the levels of 3-nitrotyrosine (3-NT) protein adducts and thiobarbituric acid (TBA) reactive substance (TBARS) in the liver, which are formed as results of oxidative stress induced by CCl4 treatment. These in vivo findings support our previous in vitro studies in cultured HSC of the inhibitory effect of RA on type I collagen expression. The data also provide evidence that RA reduces CCl4 induced oxidative stress in liver, suggesting that the anti-fibrotic role of RA is not limited to the inhibition of type I collagen expression.     

Kupffer cells inhibition prevents hepatic lipid peroxidation and damage induced by carbon tetrachloride

The aim of this work was to determine if the action mechanism of gadolinium on CCl(4)-induced liver damage is by preventing lipid peroxidation (that may be induced by Kupffer cells) and its effects on liver carbohydrate metabolism. Four groups of rats were treated with CCl(4), CCl(4)+GdCl(3), GdCl(3), and vehicles. CCl(4) was given orally (0.4 g 100 g(-1) body wt.) and GdCl(3) (0.20 g 100 g(-1) body wt.) was administered i.p. All the animals were killed 24 h after treatment with CCl(4) or vehicle. Glycogen and lipid peroxidation were measured in liver. Alkaline phosphatase, gamma-glutamyl transpeptidase, alanine amino transferase activities and bilirubins were measured in rat serum. A liver histological analysis was performed. CCl(4) induced significant elevations on enzyme activities and bilirubins; GdCl(3) completely prevented this effect. Liver lipid peroxidation increased 2.5-fold by CCl(4) treatment; this effect was also prevented by GdCl(3). Glycogen stores were depleted by acute intoxication with CCl(4). However, GdCl(3) did not prevent this effect. The present study shows that Kupffer cells may be responsible for liver damage induced by carbon tetrachloride and that lipid peroxidation is produced or stimulated by Kupffer cells, since their inhibition with GdCl(3) prevented both lipid peroxidation and CCl(4)-induced liver injury.     

Protective effects of melatonin against carbon tetrachloride hepatotoxicity in rats

Melatonin is an indolamine, mainly secreted by the pineal gland into the blood of mammalian species. The potential for protective effects of melatonin on carbon tetrachloride (CCl(4))-induced acute liver injury in rats was investigated in this work. CCl(4) exerts its toxic effects by generation of free radicals; it was intragastrically administered to male Wistar rats (4 g kg(-1) body weight) at 20 h before the animals were decapitated. Melatonin (15 mg kg(-1) body weight) was administered intraperitoneally three times: 30 min before and at 2 and 4 h after CCl(4) injection. Rats injected with CCl(4) alone showed significant lipid and hydropic dystrophy of the liver, massive necrosis of hepatocytes, marked increases in free and conjugated bilirubin levels, elevation of hepatic enzymes (alanine aminotransferase and aspartate aminotransferase) in plasma, as well as NO accumulation in liver and in blood. Melatonin administered at a pharmacological dose diminished the toxic effects of CCl(4). Thus it decreased both the structural and functional injury of hepatocytes and clearly exerted hepatoprotective effects. Melatonin administration also reduced CCl(4)-induced NO generation. These findings suggest that the effect of melatonin on CCl(4)-induced acute liver injury depends on the antioxidant action of melatonin.     

The effects of dietary flaxseed on the Fischer 344 rat. III. Protection against CCl(4)-induced liver injury

The hepatotoxic effect of carbon tetrachloride (CCl(4)) administered by gavage at 0.25 ml CCl(4) (1:1 in olive oil) per 100 g body weight was examined 24 h later in regular chow fed (RC) and 10% flax chow fed (FC) male and female Fischer 344 rats. CCl(4)-treated RC rats were subdued, lethargic and unkempt. CCl(4)-treated FC rats were much less affected. CCl(4) treatment resulted in loss of weight in RC and FC rats. In males, the weight loss was 6.7% body mass in RC rats compared to 5.6% body mass in FC rats. In females, the weight loss was 7.5% body mass in both RC and FC rats. While CCl(4) treatment increased the level of the liver injury marker plasma alanine aminotransferase (ALT) in RC rats, this CCl(4) effect was significantly attenuated in FC rats. In male rats, the ALT increase was 435-fold in RC rats and 119-fold in FC rats, over that of their respective controls. In female rats, the ALT increase was 454-fold in RC rats and 381-fold in FC rats, over that of their respective controls. These results provide evidence that flax consumption protects the liver against injury and that the extent of the protection is sex dependent. CCl(4) had no effect on the plasma level of gamma-glutamyltranspeptidase (gammaGT) in RC and FC rats supporting the contention that plasma gammaGT is not a useful marker for acute liver injury which is seen in this model. The activity of gammaGT was increased in the livers of FC rats compared to RC rats: 2.7-fold in males and 1.5-fold in females. In RC rats, the activity of liver gammaGT was decreased by CCl(4) treatment: 70% in the male and 25% in the female. However, this CCl(4) effect was reversed or abolished by flax consumption. Compared to RC rats: in male FC rats, CCl(4) actually increased the activity of liver gammaGT 1.28-fold; while in female FC rats, the depressing effect of CCl(4) treatment was abolished. The flax-induced preservation of gammaGT in the liver in response to injury may be involved in the observed hepatoprotection through generation of GSH. In RC male rats, CCl(4) treatment effected a 25% reduction in plasma glucose levels. There was no decrease in CCl(4)-treated FC male rats. In female rats, CCl(4) treatment effected a 21% decrease in plasma glucose levels in both RC and FC rats. In conclusion, multiple parameters for acute CCl(4)-induced injury were attenuated in the FC compared to the RC rat. That flaxseed consumption conferred greater protection against liver injury in the male than in the female suggests an involvement of the estrogenic lignan component of flaxseed. We discuss the possibility that this hepatoprotection is through a flax lignan-induced increase in reduced glutathione related to a flax effect on the activity of liver gammaGT in the resting state and the maintenance of its activity in response to injury.     

Influence of parathyroidectomy on liver glycogen in rats treated with carbon tetrachloride

Carbon tetrachloride (CCl4) brings about a rise in cytosolic free calcium which may lead to glycogen mobilization. Therefore, glycogen and glucose-6-phosphatase (G-6-pase) levels in the liver of parathyroidectomized (PTX) rats following CCl4 treatment have been estimated. CCl4 depletes both glycogen and G-6-pase levels in the liver. PTX followed by CCl4 administration, however, fails to restore liver glycogen and G-6-pase levels. The results suggest that neither cytosolic Ca2+ nor phospholipase A2 mediation is needed for glycogen mobilization, however, glucocorticoid intervention might have a role in such mechanisms.