A CCl4/CHCl3 interaction study in isolated hepatocytes: non-induced and phenobarbital-pretreated cells

The purpose of this study was to evaluate an isolated hepatocyte model for predicting the in vivo hepatotoxicity of carbon tetrachloride (CCl4) and chloroform (CHCl3), alone and in combination. Response surface methodology (RSM) was used to analyze and describe the data. The interaction was evaluated for % initial K+ (cell injury) and % LDH leakage (cell death) in non-induced (untreated) and phenobarbital-pretreated suspended hepatocytes. CCl4 and CHCl3 were delivered alone and in combination in dimethyl sulfoxide (DMSO) to suspended hepatocytes. The maximum observed no-effect level (MONEL) for CCl4 in non-induced cells was 1.0 mM (LDH and K+). In induced cells, the MONEL was 0.25 mM (K+) and 0.5 mM (LDH). The MONEL for CHCl3 in non-induced cells was 5.0 mM (LDH and K+) and in induced cells was 0.5 mM (K+) and 1.0 mM (LDH). Phenobarbital pretreatment enhanced the toxicity of both CCl4 and CHCl3, alone and in combination. RSM analysis of the % initial K+ and % LDH for CCl4 and CHCl3 in combination in noninduced and induced cells showed a greater than additive interaction. The isolated hepatocyte model appears to be a promising system for evaluating the toxicity of chemical mixtures and predicting their in vivo effects.     

Biochemical evaluation of rat hepatocyte primary cultures as a model for carbon tetrachloride hepatotoxicity: comparative studies in vivo and in vitro

In order to evaluate how well the development of CCl4 hepatotoxicity in vivo can be modeled in primary cultures of rat hepatocytes, biochemical alterations were determined in liver samples from rats given CCl4 and in liver cells cultured for 18 hr then exposed to CCl4. Soluble thiol levels matched closely between tissue and hepatocytes (11 vs 12 micrograms-SH/mg protein) prior to exposure. Comparable concentrations of CCl4 were measured in blood (0.30 mM at 30 min) and in culture medium (0.49 mM at 5 min). Simultaneous inhibition of the endoplasmic reticulum calcium pump and stimulation of phosphorylase a activity occurred at early times in vivo (30 min) and in vitro (5 min). Glucose-6-phosphatase was inhibited next in liver (120 min) and in cells (20 min). 5'-Nucleotidase was not affected at any time points examined in either system. Leakage of glutamic-pyruvic transaminase and depletion of glycogen were maximal at later times in vivo (greater than or equal to 8 hr) and in cells (30 min). Total calcium content was increased severalfold in liver tissue (24 hr), but was not elevated in hepatocytes. This lack of calcium accumulation in cells appeared to result from impaired mitochondrial calcium uptake. Thus CCl4-induced biochemical changes followed nearly the same continuum in both models, although the progression was much more rapid in vitro than in vivo.     

The protection from hepatotoxicity of some compounds by the synthetic immunomodulator muramyl dipeptide (MDP) in rat hepatocytes and in vivo

Muramyl dipeptide (MDP) protection from acrolein, chloroform and carbon tetrachloride toxicity was tested using isolated rat hepatocytes prepared by collagenase perfusion method. Hepatotoxin lethal effects were assessed using trypan blue exclusion and ascertained by LD leakage test. Incubation of hepatocyte suspensions with acrolein (143 mumol/ml), CHCl3 (124 mumol/ml) and CCl4 (103.5 mumol/ml) for 15 min reduced viability to 62%, 13% and 27%, respectively. Pretreatment of hepatocytes in incubation media with MDP (20.6 nmol/ml) increased viability significantly to 83%, 27% and 46%, respectively (P less than 0.01 and P less than 0.05). MDP single treatment in vivo (8.26 mumol/kg) produced a three-fold decrease in the high serum aspartate and alanine transaminases induced by CCl4 (5.2 mmol/kg). MDP modulation of CCl4 hepatotoxicity was not accompanied by reduction of lipid peroxides either in liver homogenates, microsomes or hepatocytes in the present conditions. It is suggested that MDP in certain dosages may produce nonspecific stabilization of cytoplasmic membranes towards the studied cytotoxins.     

Protection from CCl4 toxicity by prestimulation of hepatocellular regeneration in partially hepatectomized gerbils

The present investigation was undertaken to test our hypothesis that the slow responses of hepatocellular regeneration and tissue repair after CCl4-induced liver injury are responsible for the high sensitivity of gerbils to the hepatotoxic and lethal effects of CCl4. These studies were conducted in normal and actively regenerating livers using male gerbils 5 or 15 days after partial (2/3) hepatectomy (PH5 and PH15, respectively), or those undergoing sham operation (SH). An LD50 dose of CCl4 (80 microL/kg, i.p.) resulted in a mortality (21%) significantly (P less than 0.05) less than 50% in PH5 gerbils 48 hr after CCl4 administration, whereas the mortality observed in PH15 or SH gerbils was not significantly different from 50%. The elevations of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were significantly (P less than 0.05) less in PH5 gerbils than in PH15 or SH groups after the administration of either the LD50 dose or a low dose (15 microL/kg) of CCl4. Histopathological and histomorphometric examinations also indicated that CCl4-induced liver injury was less severe in PH5 gerbils than in the PH15 and SH groups. The hepatic microsomal cytochrome P450 content measured before CCl4 administration in the PH5 gerbils was decreased (26%) significantly (P less than 0.05) as compared with the SH group, but was not significantly different from that of PH15 gerbils. In vivo metabolism of 14CCl4 and lipid peroxidation in liver tissue were not significantly different among the various groups. Therefore, the protection against CCl4 toxicity observed in PH5 gerbils is unlikely to be due to decreased bioactivation of CCl4 or lipid peroxidation in that group. [3H]Thymidine incorporation into hepatocellular nuclear DNA was 4- to 5-fold higher in PH5 gerbils than in the PH15 and SH groups, indicating active hepatocellular proliferation in PH5 gerbils. [3H]Thymidine incorporation was further increased significantly (P less than 0.05) 24 hr after challenge with a low dose of CCl4 in PH5 gerbils, whereas it remained low until 48 hr after the CCl4 injection in the PH15 or SH group. The protection against CCl4 toxicity afforded by partial hepatectomy was closely associated with active hepatocellular regeneration. The overall results confirm the concept that the high sensitivity of gerbils to CCl4 is due to very sluggish hepatocellular regeneration and tissue repair response to the CCl4-induced liver injury.     

Further examination of the selective toxicity of CCl4 in rat liver slices.

Lipid peroxidation and loss of enzymes located predominantly in either periportal or centrilobular hepatocytes were investigated in precision-cut liver slices from male Sprague-Dawley rats. Pretreatment of animals with 80 mg/kg phenobarbital for the site-specific enzyme studies enhanced and accelerated CCl4 toxicity in slices resulting from increased radical formation. Liver slices were exposed to 0.57 mM CCl4 by vaporization using a roller incubation system at 37 degrees C for a total of 9 hr. Conjugated diene formation, an index of lipid peroxidation, was detected 15 min following CCl4 administration and increased over time. Loss of cytochrome P450 occurred in a time-dependent manner relative to controls where levels in treated slices were 42% of controls at 9 hr. A 48-hr fast prior to termination increased intracellular K+ leakage relative to that present in slices from fed animals. Significant leakage of glucose-6-phosphate dehydrogenase and beta-glucuronidase from centrilobular hepatocytes occurred 9 hr following CCl4 administration. The content of the periportal enzymes (lactate dehydrogenase and sorbitol dehydrogenase) was unchanged in the same slices over the duration of the experiment. Reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide, a mitochondrial selective dye and indicator of viability, was significantly lower in treated slices from phenobarbital-treated animals at 9 hr relative to controls. These studies demonstrate that precision-cut slices are an ideal in vitro system for mechanistic studies and the investigation of site-specific toxicants since the integral architecture of the liver and cellular identity are maintained.     

Changes in cytochrome P450 molecular species in rat liver in chloroform intoxication

The effect of CHCl3 on the composition of hepatic microsomal cytochrome P450 species was compared with that of CCl4 in rats pretreated with phenobarbital (PB) or 3-methylcholanthrene (3MC). The administration of CHCl3 hardly affected cytochrome P450 content in non-treated rat liver, but caused a similar degree of depletion in the content as observed after CCl4 administration in PB-pretreated rats. In the pretreatment with 3MC, the administration of CHCl3 brought about a marked decrease in the content to 24% of control after 12 hr, while CCl4 reduced the content only to one-half of control. It was demonstrated by SDS-polyacrylamide gel electrophoresis and Whatman DE-52 anion-exchange chromatography that 3MC-induced P450 species decreased with CHCl3, while it was affected little by CCl4 treatment. The activity of benzo[a]pyrene hydroxylase was altered together with the change in the content of cytochrome P450 species. The administration of CHCl3 to PB-pretreated rats caused the depletion in PB-induced P450. These findings indicate that cytochrome P450 species induced with 3MC as well as PB are highly susceptible to CHCl3 intoxication, whereas the administration of CCl4 depletes the PB-induced species without affecting the 3MC-induced species.     

Protective action of diethyldithiocarbamate and carbon disulfide against renal injury induced by chloroform in mice

Oral administration of diethyldithiocarbamate (DTC) and carbon disulfide (CS2) protected mice against CHCl3-induced kidney injury, as evidenced by normalization of delayed plasma phenolsulfonphthalein clearance, suppression of increased kidney calcium content and prevention of renal tubular necrosis. In CCl4-treated mice, in which liver microsomal monooxygenase activities were decreased markedly, and kidney microsomal aniline hydroxylase and p-nitroanisole demethylase activities were increased to about twice those of the untreated mice, renal toxicity of CHCl3 was greatly potentiated, and the latter effect was also blocked by both agents. DTC and CS2 per se markedly decreased kidney microsomal aniline hydroxylase and p-nitroanisole demethylase activities at 1 hr after oral administration, accompanying a moderate loss of cytochrome P-450 content, in both normal and CCl4-treated mice. The protection was not due to hypothermia, because pretreatment with DTC or CS2 (p.o.) also prevented the hypothermia induced by CHCl3. The mechanism of the protection may have involved inhibition of metabolic activation of CHCl3 in the kidney rather than in the liver.     

Potentiation of cocaine hepatotoxicity by ethanol in human hepatocytes

The hepatotoxic effects of cocaine on the human liver and the effect of ethanol on cocaine-induced hepatotoxicity have been examined in adult human hepatocytes cultured in chemically defined conditions. Cultures were exposed to concentrations of cocaine ranging from 10(-2) to 10(-5) M. Cytotoxicity was evaluated after 24 hr of continuous exposure to cocaine by measuring the leakage of intracellular LDH and the ability of cells to reduce MTT. According to these end-point parameters, half-maximal cytotoxic concentrations of cocaine for human hepatocytes (IC50) were 6.8 and 7.8 mM, respectively. Lower concentrations of cocaine, however, impaired basic metabolic functions of human hepatocytes. Exposure of cells to 2 mM cocaine for 24 hr resulted in a 50% decrease in hepatic glycogen, a 40% decrease in cellular glutathione content, and a 40% decrease in urea synthesis with respect to control values. For most of the metabolic parameters assayed, significant alterations were observed at 0.5 mM cocaine. Glycogen reloading of hepatocytes began to be inhibited in the presence of 0.60 mM cocaine (IC10). Ethanol greatly potentiated cocaine-induced hepatotoxicity. After a 48 hr pretreatment of human hepatocytes with 50 mM ethanol, low concentration of cocaine (0.25 mM) that had no effects on hepatocyte metabolism in the absence of ethanol caused a 20% inhibition of the urea synthesis rate, a 40% degradation of glycogen stores, and a 30% reduction in glutathione content. The results of our work show that ethanol increases the effects of cocaine on human hepatocytes by a factor of 10.     

Preventive effect of KZ-1026 on acute liver injury induced by CCl4 in rats: histochemical, enzyme-histochemical and ultrastructural studies

The preventive effect of KZ-1026 on acute liver injury induced by CCl4 in rats was evaluated histochemically, enzyme-histochemically and ultrastructurally. Rats that received 50% CCl4 (2 ml/kg) intraperitoneally were sacrificed at 3, 7 and 24 hr. Remarkable reductions in hepatic glycogen, RNA and G-6-Pase activity were observed in the centrilobular area at 3 hr, and ballooned cells appeared in the mid-zone at 7 hr. At 24 hr, the above histochemical parameters in the hepatocytes of the centrilobular area and mid-zone were extensively reduced, while the number of ballooned cells in the mid-zone was increased. KZ-1026 (200 mg/kg) was given orally at 24 and 4 hr before, simultaneously with or 3 hr after CCl4 treatment, and each rat was sacrificed at 24 hr after CCl4 administration. Pretreatment with KZ-1026 24 hr before CCl4 administration prevented reduction of RNA, glycogen and G-6-Phase activity, as well as disruption of rER and proliferation of sER due to CCL4 toxicity. This preventive effect of KZ-1026 was reduced by posttreatment; however, only the decrease in cytoplasmic RNA was well prevented. These results suggested that KZ-1026 is protective against CCl4-induced acute liver injury.     

Mechanism of chloroform nephrotoxicity : I. Time course of chloroform toxicity in male and female mice

Chloroform (CHCl3) nephrotoxicity in male mice could be detected as early as 2 hr after CHCl3 administration (250 microliter/kg, sc) as decreased ability of renal cortical slices to accumulate p-aminohippurate (PAH) and tetraethylammonium (TEA). The decrease was preceded and paralleled by a reduction of renal cortical nonprotein sulfhydryl (NPSH) concentration, an index of tissue reduced glutathione concentration. Histologic alterations were not observed until NPSH concentrations and PAH and TEA accumulation had reached the nadir, 5 hr after CHCl3 administration. Female mice exhibited no evidence of nephrotoxicity to CHCl3 even when the dose was increased to 1000 microliter/kg or when pretreated with diethyl maleate to reduce renal cortical NPSH concentrations prior to CHCl3 injection. The extent of hepatotoxicity was similar in male and female mice and decreases of hepatic NPSH concentrations also were detected by 1.5 hr after CHCl3 administration. The rapid response of the kidney to CHCl3 toxicity in male mice and the similarity of liver toxicity in both sexes suggests that nephrotoxicity occurs independently of hepatotoxicity. Furthermore, the ability to detect these early changes in vivo following CHCl3 administration may permit the development of an in vitro model to evaluate the mechanism of CHCl3 nephrotoxicity.     

Early biochemical alterations in liver mitochondria from carbon tetrachloride poisoned rats

Covalent binding of reactive metabolites of 14CCl4 were found 1 or 3 h after treatment with the solvent in the lipid and protein fractions of highly purified liver mitochondrial of rats. Most of the label was found in the phospholipid (PL) fraction, much less in cholesterol esters (ChE), and only minor quantities in other lipids. The reactive metabolites of 14CCl4 activated by isolated mitochondria interact mostly with ChE and far less with PL and other fractions. Both in vivo and in vitro covalent binding to PL is decreasing in the following order: phosphatidylethanolamine greater than diphosphatidylglycerol greater than phosphatidylcholine greater than sphingomyelin greater than lysophosphatidyl choline. No evidence of lipid peroxidation was found in liver mitochondrial lipids in the first 6 h and only a slight tendency of decrease in arachidonic acid concentration at 24 h. The incorporation of [14C] leucine in mitochondrial, microsomal or cytosolic proteins decreased as early as 1 h after treatment. These results, in agreement with previous reports suggest the existence of multiple sites in liver cells for the activation of CCl4. The transport of altered phospholipids and proteins and the inhibition of protein synthesis might contribute to the propagation of damage from the endoplasmic reticulum to other organelles.     

A comparative study on the irreversible binding of labeled halothane trichlorofluoromethane,chloroform, and carbon tetrachloride to hepatic protein and lipids in vitro and in vivo

1) After intraperitoneal injection of labeled CCl4, CHCl3, and halothane in mice, 14C is preferentially bound to liver endoplasmic protein and lipid. A considerable activity is also associated with mitochondrial constituents. Maximal protein binding (nmol/mg): CCl4: 2.8 (0.5 hrs); CHCl3: 11.5 (6 hrs); halothane: 5 (6 hrs). Lipid binding: CCl4: 6.4 (5 min); CHCl3: 8 (4 hrs); halothane: 13.5 (2 hrs). The form of the binding curves in microsomal and mitochondrial protein and lipid differed with the individual haloalkanes. 2) The irreversible (covalent) binding of 14C from labeled haloalkanes in anaerobic suspensions of isolated rabbit liver microsomes and NADPH after 30 min was for protein (lipid) (nmol/mg): CCl4: 15 (58); CHCl3: 3.4 (3.2); halothane: 2.3 (10); trichlorofluoromethane: 6.5 (30). Anerobic incubation favored dehalogenation, but CHCl3 metabolism and irreversible binding requires oxygen. The greatest differences in the in vitro "covalent" binding rates were observed with CHCl3 in rat, mouse, and rabbit. 3) Altered microsomal cytochrome P-450 concentrations in newborn animals, or produced by pretreatment of rats with phenobarbital, 3-methylcholanthrene (MC), or CoCl2 effected similar, but not proportional changes in the rates of irreversible protein and lipid binding. Upon addition of CCl4 the difference of light absorption of reduced liver microsomes from MC-pretreated rats containing cytochrome P-448 appeared at 452 nm. The irreversible binding rate in these microsomes was also increased. The small accleration in irreversible binding in liver microsomes from rats pretreated with isopropanol is not proportional to the high increase of CCl4 toxicity. 4) Practically no binding to added, soluble albumin or RNA was observed in microsomal incubates. However, 14C is bound to the nicotine-adenine dinucleotides of the NADPH system. All haloalkanes produced a similar increase of NADPH oxidation in incubates of rabbit liver microsomes and NADPH.     

Protection from chlordecone-amplified carbon tetrachloride toxicity by cyanidanol: regeneration studies.

Previous work has shown that chlordecone (CD)-amplified CCl4 hepatotoxicity and lethality can be mitigated by pretreatment with cyanidanol. These studies also revealed that stimulated hepatocellular regeneration might play an important role in the cyanidanol protection of CD-amplified CCl4 toxicity. The present studies conducted over a time course of 0 to 120 hr after CCl4 challenge describe sequential changes in hepatic [3H]thymidine incorporation into hepatocellular nuclear DNA, polyamines and related enzymes, and histomorphometry of liver sections from variously treated rats. Male Sprague-Dawley rats (125-150 g) were maintained on a control diet or on a diet contaminated with CD (10 ppm) for 15 days and/or pretreated with cyanidanol (250 mg/kg, ip) at 48, 24, and 2 hr before a single ip injection of either a standard protocol dose (100 microliters/kg) or a low dose (50 microliters/kg, L) of CCl4 on Day 16 of the dietary protocol. Cyanidanol pretreatment significantly stimulated the hepatic [3H]thymidine incorporation into hepatocellular nuclear DNA of control rats irrespective of CD pretreatment. Similarly, polyamine metabolism was altered favorably for cell division, although mitotic index (metaphase) was not increased. Cyanidanol-stimulated [3H]thymidine incorporation was highly suppressed in rats receiving the CD + CCl4 standard dose combination treatment up to 36 hr, but after this time point a marked increase was observed. Hepatocellular regeneration, quantified histomorphometrically as volume density of cells in metaphase, was progressively increased in rats protected from CD + CCl4 interaction by cyanidanol, starting at 36 hr and lasting until 72 hr. Favorably altered polyamine metabolism was evident from the stimulated ornithine decarboxylase, as well as from the stimulated interconversion of the higher polyamines to maintain increased concentration of putrescine. Challenge by the same dose of CCl4 (100 microliters/kg) to CD-pretreated rats not protected by cyanidanol failed to cause any increase in [3H]thymidine incorporation up to 36 hr and resulted in animal death starting at 36 hr. In the surviving rats, [3H]thymidine incorporation at 48 hr was increased, but was less than 50% of the increase observed in the cyanidanol group. In these rats, attenuation in the stimulation of cell division and insufficiently increased putrescine levels were observed, which are consistent with the inadequate level of hepatocellular regeneration. With rats receiving CD + CCl4(L) combination, the [3H]thymidine incorporation at 48 hr was less than 50% of the increase of cyanidanol-protected rats. Cyanidanol pretreatment to the CD + CCl4 group of rats prevented the decrease in the hepatic DNA levels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of chlorpromazine and trifluoperazine on choline metabolism and phosphatidylcholine biosynthesis in cultured chick heart cells under normoxic and anoxic conditions

The effects of chlorpromazine and trifluoperazine on phosphatidylcholine biosynthesis in the heart were investigated in isolated cardiac cells under normoxic and anoxic conditions. The cells were obtained from 7-day-old chick embryos and were maintained in culture. After 96 hr, cells were maintained either in an incubator with oxygen at room air concentration (normoxia) or in an incubator containing 95% nitrogen and 5% CO2 (anoxia). Pulse chase experiments with [methyl-3H]choline were conducted using a 2-hr incubation with choline. Chlorpromazine and trifluoperazine at 10(-5) M produced a significant (P less than 0.05) increase in the incorporation of choline into both phosphocholine and phospholipid. High concentrations of chlorpromazine or trifluoperazine i.e. 10(-4) M, damaged myocardial cells as reflected in a significant (P less than 0.05) reduction in cellular protein and a further reduction in labelled choline in phosphocholine or phospholipid after adjusting for the lower protein concentrations. Anoxia altered choline metabolism but 6 hr of anoxia was the minimum time needed for the effect to be observable. Anoxia, for 24 hr, produced a significant (P less than 0.05) reduction in labelled choline in phosphocholine without a significant change in incorporation of label in phospholipid or cellular protein. Both chlorpromazine and trifluoperazine at 10(-5) M prevented anoxic-induced changes in phosphocholine metabolism. Thus, chlorpromazine and trifluoperazine affect phospholipid biosynthesis in cardiac cells and prevent anoxia-induced changes in phosphatidylcholine biosynthesis.     

Protective effects of the fractions extracted from the callus of Acer nikoense Maxim. on carbon tetrachloride induced liver injury]

The decoctions extracted from the bark and leaf of Acer nikoense Maxim. (AN) have been used as a folk medicine for eye-wash and hepatic disease. As previously reported, the methanol extract from the bark of AN and the fractions of the methanol extract have protective effects for liver injury induced by carbon tetrachloride (CCl4) in rats. In this study, protective effects of the fractions extracted from the callus of AN were investigated on CCl4-induced liver injury in rat. The active principles for the protection of CCl4-induced liver injury were recognized in a fraction (EF 3-3) obtained by using silica gel chromatography (solvent: CHCl3-MeOH-H2O). The components were further fractionated by silica gel chromatography followed by gel filtration (solvent: CHCl3-MeOH). In addition, the mechanism of the protective action against liver injury induced by CCl4 was also examined. The fractions with protective effects in vivo showed the inhibitory effects on CCl4-dependent lipid peroxidation in microsomal fraction in vitro.     

二至丸提取物对体外肝细胞损伤的保护作用

 目的：研究二至丸乙酸乙酯提取物(ethyl acetate extractsof Erzhi pill,EAEP)对体外肝细胞损伤的保护作用及其机理。方法：培养L-O2型肝细胞,采用CCl4和H2O2体外分别诱导肝细胞损伤,检测培养上清液中天门冬氨酸转换酶(AST)和丙氨酸氨基转换酶(ALT)水平,测定上清液中丙二醛(MDA)的含量和过氧化物岐化酶(SOD)活力及谷胱甘肽过氧化物酶(GSH-Px)活性,MTT法检测细胞存活和增殖活性。结果：① EAEP(0.32～40 μg?mL-1)剂量组可明显降低由CCl4所致肝细胞培养上清液中AST和ALT水平及MDA含量的升高,还可提高由H2O2所致肝细胞存活率和SOD活力及GSH-Px活性的降低。② EAEP(0.32～40 μg?mL-1)剂量组可使H2O2升高的肝细胞培养上清液中ALT和ALT水平及MDA含量明显降低或恢复,还可提高CCl4降低的肝细胞存活率和SOD活力及GSH-Px活性。结论：提示AEEP对体外肝细胞损伤有直接保护作用,该作用可能与其抗氧化作用有关。     

Protective effects of Indigofera tinctoria L. against D-Galactosamine and carbon tetrachloride challenge on 'in situ' perfused rat liver

The effect of pre-treatment with Indigofera tinctoria (IT) extract against the toxicity of D-Galactosamine (D-GalN) and carbon tetrachloride (CCl4) during 'in situ' perfusion of the liver for 2 hr was studied in rats. Release of LDH and levels of urea in the liver effluent perfusate, was studied and the rate of bile flow was monitored. Perfusion with D-Galactosamine (5 mM) or carbon tetrachloride (0.5 mM) resulted in increased LDH leakage, decreased urea levels in the liver effluent and reduction in bile flow. IT pretreatment (500 mg/kg body weight) in vivo ameliorated D-GalN and CCl4 induced adverse changes towards near normalcy and thereby indicates its hepatoprotective effects in rats.     

Role of cyclic AMP in the inhibition of mouse hepatocyte intercellular communication by liver tumor promoters

The liver tumor promoters phenobarbital (PB) (20-500 micrograms/ml) and 1,1-bis(4-chlorophenyl)-2,2,2-trichloroethane (DDT) (1-10 micrograms/ml) inhibited intercellular communication between primary cultured B6C3F1 mouse hepatocytes after 8 hr of treatment. Intercellular communication was detected autoradiographically as the passage and incorporation of [5-3H]uridine nucleotides from prelabeled donor hepatocytes to recipient hepatocytes. The addition of either dibutyryl cyclic AMP (N6,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate) (0.001-0.1 mM) or caffeine (0.01-1 mM) decreased or completely abolished the inhibitory effects of PB and DDT on intercellular communication. Cyclic AMP (adenosine 3':5'-cyclic monophosphate; cAMP) in primary cultured mouse hepatocytes was measured by radioimmunoassay. Cyclic AMP in nontreated, freshly plated cultures declined from 4.2 +/- 0.7 pmol/mg protein after 1 hr in culture to 2.4 +/- 0.5 pmol/mg protein after 8 hr in culture. Phenobarbital at 250 and 500 micrograms/ml significantly decreased cyclic AMP below control values after 1 hr of treatment. However, no difference in the amount of cyclic AMP was detected between control and PB-treated cultures after 2, 4, and 8 hr in culture or with lower PB concentrations. DDT at 10 micrograms/ml decreased cAMP levels in the hepatocytes after 1, 2, 4, and 8 hr of treatment. No effects were seen after 8 hr of treatment or with lower DDT concentrations. DDT (10 micrograms/ml) also decreased cAMP levels in 24-hr-old cultures while PB (500 micrograms/ml) had no effect. Addition of dibutyryl cAMP (0.1 mM) or caffeine (1.0 mM) to freshly plated cultures elevated cAMP levels 50-fold and twofold, respectively. These data suggest that the inhibition of mouse hepatocyte intercellular communication by PB and DDT at the highest concentrations tested may be mediated by transient decreases in intercellular cAMP levels.     

Uptake of gallium-67 by the hepatocytes during liver regeneration in carbon tetrachloride-treated mice

Gallium-67 (67Ga) has been used as a tumor or inflammation-imaging agent in nuclear medicine. We recently reported that the peak of serum alanine aminotransferase (ALT) level was 1 d after carbon tetrachloride (CCl4) treatment in rats. However, the peak of 67Ga uptake by the liver tissue and hepatocytes was 2 d after the treatment. If 67Ga is taken in the hepatic disorder phase, the pattern of 67Ga uptake by the hepatocytes should be consistent with that of the ALT level. In order to answer why it was not, we carried out a detailed examination. The lipid peroxidation of hepatocytes from CCl4-treated mice was greatly increased 1 d after CCl4 treatment; serum ALT was also increased 1 d after the treatment. The uptake of 67Ga by the liver tissue reached a maximum 1 and 2 d after the treatment, while maximum by the hepatocytes was achieved after 2 d. The incorporation of bromodeoxyuridine into the hepatocytes also reached maximum 2 d after the treatment. These results suggest that the uptake of 67Ga by the hepatocytes is carried out during liver regeneration rather than during hepatic disorder by CCl4 treatment.     

The measurement of platelet aggregation and ATP-release in mice with liver damage induced by carbon tetrachloride (CCl4) using a whole blood aggregometer

Time course change in platelet function on liver damaged mice was studied by a whole blood aggregometer. The liver damaged animals were produced by single and multiple injections (p.o.) of 20% CCl4 in olive oil to ddY mice (6 weeks). Platelet aggregation and ATP-release were induced with collagen (final conc. of 2 micrograms/ml), ADP (final conc. of 20 microM) and arachidonic acid (AA: final conc. of 100 microM). After a single injection of CCl4, platelet counts increased at 5 and 12 hr, and then they decreased from 24 to 120 hr. Multiple injections of CCl4 resulted in a significant increase in platelet counts. A single injection of CCl4 suppressed aggregation by collagen at 24 and 48 hr and diminished the rate of ATP-release from 12 to 48 hr. AA-induced platelet aggregation was depressed at 48 hr, and ATP-release was also diminished from 24 to 72 hr after a single injection. ADP-induced platelet aggregation was decreased 24 and 48 hr after a single injection and at 48 hr after multiple injections, while, the rate of ATP-release by ADP was significantly increased after single and multiple injections. These changes in platelet functions might be consistent with our original report on the alterations in coagulative and fibrinolytic activities with CCl4-induced liver injury.