Detection of cells resistant to the cytotoxic action of CCl4 in rat hepatocyte populations following a single exposure to diethylnitrosamine and subsequent injections of phenobarbital

A single intraperitoneal injection of hepatocarcinogen diethylnitrosamine induced emergence of a new subpopulation of "small" hepatocytes (64-128 mkm2), disappearing by the 30th day after carcinogen injection. But 5 injections of the tumor promotor phenobarbital 7 days after carcinogen treatment prolonged the existence of such "small" hepatocytes up to 3 months. The quantitative cytochemical measurement of succinic dehydrogenase activity (respiratory enzyme of mitochondria) showed these cells to be resistant to cytotoxic action of CCl4. These data are consistent with the earlier reported results obtained in analogous experiments with 4-dimethylaminoazobenzene and phenobarbital.     

CCl4-induced increase of hepatocyte free arachidonate level: pathogenesis and contribution to cell death

A significant increase of the intracellular level of free arachidonic acid was observed in intact rat hepatocytes after poisoning with very low concentrations of CCl4 (0.129-0.172 mM), shown not to exert direct solvent effect. It seems likely that activation of phospholipase A2 (PLA2) is the mechanism mainly responsible for the rise of cytosolic arachidonate, since the latter is prevented by the PLA2 inhibitors indomethacin and mepacrine. The CCl4-induced delay of arachidonic acid incorporation within the cell membrane phospholipids partly contributes to its intracellular accumulation in the early phases of the poisoning. The lack of any significant protection by metabolic inhibitors (SKF 525A, metyrapone), antioxidant compounds (promethazine, diphenylphenylenediamine DPPD) or antioxidant procedures (rat pretreatment with vitamin E) leads to exclude an involvement of CCl4 biotransformation in the increase of intracellular free arachidonate. Finally, the PLA2 inhibitors employed in this study did not afford protection against the enzymic leakage of CCl4-treated hepatocytes.     

LC-MS/MS methods for the detection of isoprostanes (iPF2alpha-III and 8,12-iso-iPF2alpha-VI) as biomarkers of CCl4-induced oxidative damage to hepatic tissue

Isoprostanes are formed after peroxidation of arachidonic acid and are promising biomarkers for reactive oxygen species. A LC-MS/MS based method was developed for the quantitation of two isoprostanes (iPF2alpha-III and 8,12-iso-iPF2alpha-VI) in hepatocytes, tissue and urine samples of rats. A column switching method was used to reduce sample preparation to a minimum. Precision was 9.4% and accuracy was between 96 and 114% for free iPF2alpha-III in tissue at concentrations from 1.9 to 6.1 ng/g. Treatment of rats with CCl4 to induce oxidative stress resulted in a dose-dependent increase (two- to three-fold) of iPF2alpha-III and 8,12-iso-iPF2alpha-VI in liver and kidney. For both isoprostanes an increase of four- to five-fold was observed in CCl4 treated hepatocytes and six- to eight-fold in CCl4 treated and glutathione depleted hepatocytes. In conclusion, the presented method is sensitive, specific and precise to be applied for the quantitation of iPF2alpha-III and 8,12-iso-iPF2alpha-VI which are shown to increase by CCl4 treatment in vitro and in vivo.     

The role of lipid peroxidation in CCl4-induced damage to liver microsomal enzymes: comparative studies in vitro using microsomes and isolated liver cells

The question as to whether CCl4 decreases the activities of glucose-6-phosphatase and cytochrome P-450 in liver endoplasmic reticulum mainly through its action in stimulating lipid peroxidation has been investigated using Promethazine to block lipid peroxidation. The investigation, moreover, has compared the effects of CCl4, with and without Promethazine, on isolated rat hepatocytes with corresponding effects on rat liver microsomal suspensions. Our data give no support for the view that products of lipid peroxidation are the main cause of the decrease in cytochrome P-450 observed in CCl4-intoxication. However, our present results are consistent with lipid peroxidation being a major contributory factor to the decrease in glucose-6-phosphatase activity observed in CCl4-induced liver injury.     

Mechanisms responsible for carbon tetrachloride-induced perturbation of mitochondrial calcium homeostasis

Incubation of isolated hepatocytes with CCl4 results in early reduction of the intracellular calcium content, mostly due to loss from the mitochondrial compartment. CCl4 treatment directly affects mitochondrial functions as indicated by the inhibition of Ca2+ uptake in cells permeabilized to the ion by digitonin exposure and by the reduction of intracellular ATP content in hepatocytes incubated in a glucose-free medium. Such mitochondrial damage is not caused by CCl4-induced stimulation of lipid peroxidation since it is not prevented by alpha-tocopherol, used at a concentration able to inhibit completely peroxidative reactions without interfering with CCl4 activation. All data together are in favour of a direct action of CCl4-reactive metabolites on liver cell calcium homeostasis.     

Pathogenesis of carbon tetrachloride-induced hepatocyte injury bioactivation of CCI4 by cytochrome P450 and effects on lipid homeostasis

The CCl4-induced development of liver damage was studied in monolayer cultures of primary rat hepatocytes: (1) CCl4 caused accumulation of triglycerides in hepatocytes following cytochrome P450 induction with beta-naphthoflavone or metyrapone. Ethanol or a high dose of insulin plus triiodothyronine had the same effect. (2) CCl4 increased the synthesis of fatty acids and triglycerides and the rate of lipid esterification. Cholesterol and phospholipid synthesis from acetate was also increased. (3) CCl4 reduced beta-oxidation of fatty acids as assessed by CO2-release and ketone body formation. Hydrolysis of triglycerides was also reduced. (4) The content of unsaturated fatty acids in microsomal lipids was decreased by almost 50% after incubation with CCl4, while saturated fatty acids increased slightly. (5) CCl4 exerted a pronounced inhibitory effect on the exocytosis of macromolecules (albumin), but did not affect secretion of bile acids from hepatocytes.     

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.     

Uncoupling protein-2 induction in rat hepatocytes after acute carbon tetrachloride liver injury

This study is focused on the role of UCP-2 in hepatic oxidative metabolism following acute CCl(4) administration to rats. UCP-2 mRNA, almost undetectable in the liver of controls, was significantly increased 24 h after CCl(4) administration, peaked at 72 h and then tended to disappear. UCP-2 protein, undetectable in controls, increased 48-72 h after CCl(4) treatment. Experiments with isolated liver cells indicated that in control rats UCP-2 was expressed in non-parenchymal cells and not in hepatocytes, whereas in CCl(4)-treated rats UCP-2 expression was induced in hepatocytes and was not affected in non-parenchymal cells. Addition of CCl(4) to the culture medium of hepatocytes from control rats failed to induce UCP-2 expression. Liver mitochondria from CCl(4)-treated rats showed an increase of H(2)O(2) release at 12-24 h, followed by a rise of TBARS. Vitamin E protected liver from CCl(4) injury and reduced the expression of UCP-2. Treatment with GdCl(3) prior to CCl(4), in order to inhibit Kupffer cells, reduced TBARS and UCP-2 mRNA increase in hepatic mitochondria. Our data indicate that CCl(4) induces the expression of UCP-2 in hepatocytes with a redox-dependent mechanism involving Kupffer cells. A role of UCP-2 in moderating CCl(4)-induced oxidative stress during tissue regeneration after injury is suggested.     

15—Mt—PGF2α对CCL4所致大鼠离体肝细胞损伤的保护作用

采用大鼠离体肝细胞原代培养２４ｈ,并利用四氯化碳ＣＣｌ４造成急性肝细胞损伤模型,检定１５－甲基－前列腺素Ｆ２α（１５－Ｍｔ－ＰＧＦ２α）对肝细胞损伤的影响。结果表明：（１）１５－Ｍｔ－ＰＧＦ２α可显著降低中毒肝细胞脂质过氧化物水平,抑制肝细胞脂质过氧化,并降低谷丙转氨酶（ＧＰＴ）和谷草转氨酶（ＧＯＴ）水平,稳定脂质膜。（２）显著促进中毒肝细胞ＲＮＡ和ＤＮＡ的合成。（３）超微结构证实１５－Ｍｔ－ＰＧＦ２α能减轻ＣＣｌ４对肝细胞脂质膜,染色质,线粒体,内质网和核蛋白体的损害。     

Reestablishment of the heterogeneous distribution of hepatic glutamine synthetase during regeneration after CCl4-intoxication

Intoxication of rats with CCl4 (1 ml/kg) resulted in the almost complete loss of glutamine synthetase (GS) specific activity and immunologically detectable enzyme protein known to be expressed exclusively in some hepatocytes of the perivenous zone of the liver acinus. During regeneration the specific activity as well as the original number of GS-positive (GS+) hepatocytes were reestablished. However, while the GS+ hepatocytes in control livers were arranged in up to 3 cell layers surrounding the central veins the same number of GS+ hepatocytes in regenerated livers formed a single cell layer only, most likely because the central veins were enlarged in diameter. Investigation of the nuclear pattern of GS+ and GS- hepatocytes of control animals in primary cultures revealed striking differences characterized by significantly more mononuclear diploid, binuclear diploid, and binuclear tetraploid cells among the GS+ hepatocytes and predominantly mononuclear tetraploid cells (70%) among the GS- hepatocytes. Immediately after liver damage by CCl4 and during regeneration small but significant changes in the nuclear pattern were noted for GS- hepatocytes. However, the first GS+ cells appearing during early regeneration showed a pattern of ploidy classes close to the original one found for GS- hepatocytes. These results indicate that new GS+ hepatocytes may be derived from formerly GS- cells which are induced to express GS if they have reached the border of the central veins.     

Cyclic nucleotides levels in the liver of rats treated with CCl4.

Treatment of rats with two different doses of CCl4 (respectively 2.5 and 0.5 ml/kg body wt. intragastrically) is followed by a rapid increase in the cAMP content of the liver. With 0.5 ml of CCl4, the increase occurs as early as 30 min after poisoning, namely about 4-5 h before the onset of triglyceride accumulation in the liver. The maximum increase has been at 6 h after administration of the hepatotoxin. In both experimental conditions, normal values are recovered only after 36-48 h. cGMP level appears unmodified during the whole observation period. Therefore the ratio cGMP/CAMP decreases consistently. The ATP level decreases significantly between 2 and 12 h. The increase in liver triglycerides level after CCl4 can be also a consequence of an impairment of microtubule function, leading to a decreased release of lipoprotein micelles from hepatocytes into the blood stream.     

Hepatocyte cytotoxicity induced by various hepatotoxins mediated by cytochrome P-450IIE1: protection with diethyldithiocarbamate administration.

The objective of this study was to determine whether the thiol drug, diethyldithiocarbamate (DEDC) and its two metabolites, disulfiram (DS) and carbon disulfide (CS2) could be used as inhibitors of cytochrome P-450IIE1 to protect hepatocytes from cytotoxic xenobiotics. (1) Hepatocytes isolated from rats following pyrazole administration to induce cytochrome P-450IIE1 were much more susceptible to carbon tetrachloride (CCl4) and dimethylnitrosamine (DMN) than hepatocytes from untreated rats. Microsomes isolated from P-450IIE1-induced liver were also much more effective at catalysing a NADPH-dependent metabolism of CCl4 and DMN. The activities of aniline hydroxylase and p-nitroanisole-O-demethylase increased whereas ethoxyresorufin-O-dealkylase activity was much less induced and pentoxyresorufin-O-dealkylase activity was decreased. The P-450IIE1 antibody markedly inhibited the NADPH-dependent metabolism of these compounds indicating that IIE1 is a major catalyst of the microsomal metabolism of CCl4 and DMN. (2) Hepatocytes isolated from rats treated with DEDC or its metabolites, DS and CS2, on the other hand, were resistant to CCl4 and DMN. Microsomes isolated from the liver of animals treated with DEDC or DS or CS2 were also much less effective at catalysing the NADPH-dependent metabolism of the above compounds. DEDC markedly decreased the activities of aniline hydroxylase, p-nitroanisole-O-demethylase and pentoxyresorufin-O-dealkylase but had no effect on ethoxyresorufin-O-dealkylase activity. (3) Hepatocytes isolated from pyrazole-treated rats were also more susceptible to bromobenzene (BB) and naphthalene-induced cytotoxicity than hepatocytes from untreated rats. Furthermore, DEDC or CS2 administration beforehand significantly protected hepatocytes against both xenobiotics. (4) By contrast, hepatocytes isolated from P-450IIE1 induced rats were not more susceptible to lactonitrile or cyclophosphamide. Instead, cyclophosphamide was activated by phenobarbital-induced P-450 isozymes whereas lactonitrile was activated by alcohol dehydrogenase. Hepatocytes isolated from DEDC-treated rats were also resistant to cyclophosphamide but not lactonitrile. (5) The above results suggest that P-450IIE1 catalyses the cytotoxic activation of CCl4, DMN, BB and naphthalene but not of lactonitrile or cyclophosphamide. Furthermore, the administration of DEDC and its metabolites, disulfiram or CS2, inactivates P-450IIE1 so that the hepatocytes become resistant to these hepatotoxins.     

Stimulation of arachidonic acid metabolism by CCl4 in isolated rat hepatocytes

Arachidonic acid metabolism was evaluated in isolated rat hepatocytes after CCl4 exposure. CCl4 induced dose-dependently the synthesis and release of prostacyclin (PGI2) and thromboxane (TXB2). Treatment with prostaglandin E2 (PGE2) 30 min after exposure to CCl4, significantly reduced the cell damage as well as the release of TXB2 from the cells.     

Changes in the activity of matrix metalloproteinases in regenerating rat liver after CCl4-induced injury

Molecular mechanisms involved in mediating alteration in cell matrix interaction have been examined by studying the changes in the activity of matrix metalloproteinases (MMPs) in CCl4-induced regenerating liver, using zymography and ELISA. Activity of MMPs (72 kD, 92 kD and 130 kD gelatinases) in the rat liver increased progressively during acute injury till the 4th day and then decreased to near normal level after CCl4 administration (0.5 ml/100 g body wt.) on the 6th day. Hepatocyte lysate of injured liver on the 4th day showed significantly higher levels of MMP2 and MMP9 compared to the control. In the culture medium of hepatocytes, the levels of MMP2 and MMP9 increased progressively with the duration of culture, indicating that hepatocytes are the major source of these MMPs in regenerating liver. These results suggest an involvement of MMPs in matrix degradation and remodeling during regeneration after acute liver injury induced by CCl4.     

Ultrastructural changes in isolated rat hepatocytes exposed to different CCl4 concentrations

Ultrastructural data are presented on time-course changes in isolated rat hepatocyte suspensions exposed either to 1.2 or 1.8 mM CCl4 for up to 1 h. The subcellular changes at the lower concentration, but not the higher, are shown to closely parallel those reported to occur in rat hepatocytes following ingestion of CCl4.     

The in vitro NADPH-dependent inhibition by CCl4 of the ATP-dependent calcium uptake of hepatic microsomes from male rats. Studies on the mechanism of the inactivation of the hepatic microsomal calcium pump by the CCl3.radical

The hepatotoxicity of CCl4 is mediated through its initial reduction by cytochrome P-450 to the CCl3.radical. This radical then damages important metabolic systems such as the ATP-dependent microsomal Ca2+ pump. Previous studies from our laboratory on isolated microsomes have shown that NADPH in the absence of toxic agents inhibits this pump. We have now found in in vitro incubations that CCl4 (0.5-2.5 mM) enhanced the NADPH-dependent inhibition of Ca2+ uptake from 28% without CCl4 to a maximum of 68%. These concentrations are in the range found in the livers and blood of lethally intoxicated animals (Dambrauskas, T., and Cornish, H. H. (1970) Toxicol. Appl. Pharmacol. 17, 83-97; Long, R.M., and Moore, L. (1988) Toxicol. Appl. Pharmacol. 92, 295-306) and are toxic to cultured hepatocytes (Long, R. M., and Moore, L. (1988) Toxicol. Appl. Pharmacol. 92, 295-306). The inhibition of Ca2+ uptake was due both to a decrease in the Ca2(+)-dependent ATPase and to an enhanced release of Ca2+ from the microsomes. The NADPH-dependent CCl4 inhibition was greater under N2 and was totally prevented by CO. GSH (1-10 mM) added during the incubation with CCl4 prevented the inhibition. This protection was also seen when the incubations were performed under nitrogen. When samples were preincubated with CCl4, the CCl4 metabolism was stopped, and then the Ca2+ uptake was determined; GSH reversed the CCl4 inhibition of Ca2+ uptake. This reversal showed saturation kinetics for GSH with two Km values of 0.315 and 93 microM when both the preincubation and the Ca2+ uptake were performed under air, and 0.512 and 31 microM when both were performed under nitrogen. Cysteine did not prevent the NADPH-dependent CCl4 inhibition of Ca2+ uptake. CCl4 increased lipid peroxidation in air, but no lipid peroxidation was seen under nitrogen. Lipid peroxidation was only modestly reversed by GSH. GSH did not remove 14C bound to samples preincubated with the 14CCl4. Although EDTA (100 microM) decreased the CCl4 inhibition, the metal-complexing agents deferoxamine (100 microM) and diethyldithiocarbamate (100 microM) had no effect on the inhibition of the pump. Similarly, the reactive oxygen scavengers catalase (65 micrograms/ml), superoxide dismutase (15 micrograms/ml), mannitol (10 mM), and dimethyl sulfoxide (50 mM) also had no effect. Our results suggest that the initial toxicity of CCl4 for the Ca2+ pump results from the metabolism of CCl4 to the CCl3. radical. This radical then directly oxidizes the Ca2+ pump, leading to decreased Ca2+ uptake.(ABSTRACT TRUNCATED AT 400 WORDS)     

Protective role of fructose 1,6-bisphosphate during CCl4 hepatotoxicity in rats.

Rats were injected intraperitoneally with CCl4 (2.5 ml/kg body wt.) and the hepatotoxicity was compared with that of rats receiving the same dose of CCl4 and an intraperitoneal injection of fructose 1,6-bisphosphate (2 g/kg body wt.). A 50-70% decrease in plasma aspartate aminotransferase and alanine aminotransferase activities was observed in the latter treatment, indicating a protective role of the sugar bisphosphate in CCl4 hepatotoxicity. The protection was accompanied by elevated hepatic activities of ornithine decarboxylase at 2, 6 and 24 h, S-adenosylmethionine decarboxylase at 6 h, and spermidine N1-acetyltransferase at 2 h. The increase in the enzymes involved in polyamine metabolism was shown in our previous work [Rao, Young & Mehendale (1989) J. Biochem. Toxicol. 4, 55-63] to correlate with increased polyamine synthesis or interconversion, which was related to the extent of hepatocellular regeneration. The hepatic contents of fructose 1,6-bisphosphate and ATP significantly decreased after CCl4 treatment, and administration of the sugar bisphosphate increased hepatic ATP. Fructose 1,6-bisphosphate, an intermediary metabolite of the glycolytic pathway, may decrease CCl4 toxicity by increasing the ATP in the hepatocytes. The ATP generated is useful for hepatocellular regeneration and tissue repair, events which enable the liver to overcome CCl4 injury.     

A genome-wide screen identifies genes required for formation of the wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine in Saccharomyces cerevisiae

We recently showed that the gamma-subunit of Kluyveromyces lactis killer toxin (gamma-toxin) is a tRNA endonuclease that cleaves tRNA(mcm5s2UUC Glu), tRNA(mcm5s2UUU Lys), and tRNA(mcm5s2UUG Gln) 3' of the wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U). The 5-methoxycarbonylmethyl (mcm(5)) side chain was important for efficient cleavage by gamma-toxin, and defects in mcm(5) side-chain synthesis correlated with resistance to gamma-toxin. Based on this correlation, a genome-wide screen was performed to identify gene products involved in the formation of the mcm(5) side chain. From a collection of 4826 homozygous diploid Saccharomyces cerevisiae strains, each with one nonessential gene deleted, 63 mutants resistant to Kluyveromyces lactis killer toxin were identified. Among these, eight were earlier identified to have a defect in formation of the mcm(5) side chain. Analysis of the remaining mutants and other known gamma-toxin resistant mutants revealed that sit4, kti14, and KTI5 mutants also have a defect in the formation of mcm(5). A mutant lacking two of the Sit4-associated proteins, Sap185 and Sap190, displays the same modification defect as a sit4-null mutant. Interestingly, several mutants were found to be defective in the synthesis of the 2-thio (s(2)) group of the mcm(5)s(2)U nucleoside. In addition to earlier described mutants, formation of the s(2) group was also abolished in urm1, uba4, and ncs2 mutants and decreased in the yor251c mutant. Like the absence of the mcm(5) side chain, the lack of the s(2) group renders tRNA(mcm5s2UUC Glu) less sensitive to gamma-toxin, reinforcing the importance of the wobble nucleoside mcm(5)s(2)U for tRNA cleavage by gamma-toxin.     

Promethazine inhibits the formation of aldehydic products of lipid peroxidation but not covalent binding resulting from the exposure of rat liver fractions to CCl4.

Promethazine is known to have protective activity in relation to CCl4-induced liver necrosis. This hepatoprotective property has been investigated with regard to the free radical scavenging and antioxidant properties of promethazine using isolated hepatocytes and microsomal suspensions. CCl4 is activated in both systems to free radical metabolites that bind covalently to lipid and protein, and initiate lipid peroxidation. A large number of carbonyl products is produced during CCl4-induced lipid peroxidation; promethazine strongly inhibits the production of all classes of carbonyl compounds in both microsomal suspensions and isolated hepatocytes. In contrast, promethazine is a very weak inhibitor of the covalent binding of metabolites of CCl4. We conclude that promethazine acts by scavenging the trichloromethylperoxyl radical and lipid peroxyl radicals, and is a weak scavenger of the trichloromethyl radical. These data, when considered together with the hepatoprotective effects of promethazine, suggest that lipid peroxidation is of relatively more importance than covalent binding in the pathogenesis of CCl4-induced liver necrosis.     

Proliferation and differentiation of ductular progenitor cells and littoral cells during the regeneration of the rat liver to CCl4/2-AAF injury

Restoration of centrolobular injury induced by carbon tetrachloride (CCl4), when hepatocyte proliferation is inhibited by treatment with N-2-acetylaminofluorene (AAF), is accomplished by proliferation of ductular progenitor cells, that arise intraportally and extend into the liver lobule. This pattern contrasts to the restitutive proliferation of hepatocytes when AAF is not administered, and the proliferation of non-ductular periportal oval cells follows periportal necrosis induced by allyl alcohol. The expanding ducts stain for alphafetoprotein (AFP), OV-6, pan-cytokeratin (CKPan), and laminin. The neoductular proliferation is accompanied by fibronectin-positive Kupffer cells and desmin-positive stellate (Ito) cells, which may play critical roles not only in controlling proliferation and differentiation of ductular progenitor cells, but also in reestablishing hepatic cord structure. When AAF is discontinued 7 days after injury, clusters of small hepatocytes appear next to the neoductules. Some of these small hepatocytes, as well as some larger hepatocytes adjacent to the ducts, stain for AFP and for carbamoylphosphate synthetase I (CPS-I), suggesting that the ductular progenitor cells may differentiate into hepatocytes when AAF is withdrawn. The restitutive process is facilitated by clearing of the central necrotic zone by infiltrating macrophages and co-migration of mature hepatocytes, with Kupffer cells and stellate cells, into the necrotic zone.