Effects of carbon tetrachloride on calcium homeostasis. A critical reconsideration

The incubation of isolated rat hepatocytes with 0.172 mM carbon tetrachloride caused a rapid decrease in the calcium content of both mitochondrial and extramitochondrial compartments. However, the release of Ca2+ from the intracellular stores was not associated with an increase in the cytosolic Ca2+ levels as measured by activation of phosphorylase alpha or by Quin-2 fluorescence. A rapid rise in hepatocyte free calcium was only observed with concentrations of CCl4 higher than 0.172 mM. The lack of activation of phosphorylase alpha was not due to the inhibition of the enzyme by CCl4, since in CCl4-treated hepatocytes the phosphorylase activity could be stimulated by glucagon, butyryl--cAMP or by the increase of cell calcium induced by the addition of A23187. Ca2+-dependent ATPase of plasma membranes was only slightly affected in the early phases of poisoning with CCl4 when both mitochondrial and extramitochondrial calcium pools were already lowered. This led to the conclusion that calcium released from intracellular organelles could be extruded from the cells in sufficient amounts to prevent the increase of the cytosolic levels. A rise in hepatocyte free calcium was observed during the second hour of incubation with CCl4, concomitantly with the appearance of both LDH leakage and plasma membrane blebbing. The addition of EGTA to the medium prevented both the increase in cytosolic Ca2+ and the blebbing suggesting that they were a consequence of an influx of calcium into the cells. However, neither EGTA nor the addition of inhibitors of calcium-dependent phospholipase A2 or non-lysosomal proteases were able to protect against cell death. These latter results suggested that the alterations of calcium distribution induced by CCl4 in isolated hepatocytes were not a primary cause of the toxic effects, although they did not exclude that a sustained rise in cytosolic Ca2+ could contribute in the progression of cell injury.     

Evidence against involvement of calcium in carbon tetrachloride-dependent inhibition of lipid secretion by isolated hepatocytes

Carbon tetrachloride (CCl4)-induced inhibition of very low density lipoprotein (VLDL) secretion was studied in isolated hepatocytes. The hypothesis that inhibition of secretion is due to altered calcium homeostasis following CCl4-dependent inhibition of endoplasmic reticulum calcium sequestration was investigated. Inhibition of VLDL secretion by CCl4 was not dependent on extracellular calcium, since inhibition occurred when extracellular calcium was reduced to 0.1 microM. CCl4 inhibited hepatocyte VLDL secretion more rapidly than it inhibited microsomal calcium sequestration. Further, the concentration of CCl4 that produced half-maximal inhibition of VLDL secretion was about one-half the concentration required to produce half-maximal inhibition of microsomal calcium sequestration. The calcium ionophore A23187 did not mimic the action of CCl4 in inhibiting VLDL secretion under conditions in which A23187 altered cellular calcium homeostasis. The results that an alteration of calcium homeostasis is not involved in inhibition of VLDL secretion by carbon tetrachloride.     

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.     

Comparative studies on rat primary cultured and isolated hepatocytes in the evaluation of a therapeutic agents for liver disease

We have investigated the effect of a therapeutic agent for liver disease, Laennec, on the GOT leakage from freshly isolated and primary cultured rat hepatocytes which were treated with CCl4. By treatment with Laennec together with CCl4, the GOT leakage from isolated hepatocytes increased and that from cultured hepatocytes decreased, compared to those incubated only with CCl4. The results suggest that it is better to use primary cultured hepatocytes than to use freshly isolated hepatocytes to evaluate therapeutic agents for liver disease.     

Calpain released from dying hepatocytes mediates progression of acute liver injury induced by model hepatotoxicants

Liver injury is known to progress even after the hepatotoxicant is long gone and the mechanisms of progressive injury are not understood. We tested the hypothesis that hydrolytic enzymes such as calpain, released from dying hepatocytes, destroy the surrounding cells causing progression of injury. Calpain inhibitor, N-CBZ-VAL-PHE-methyl ester (CBZ), administered 1 h after a toxic but nonlethal dose of CCl(4) (2 ml/kg, ip) to male Sprague Dawley rats substantially mitigated the progression of liver injury (6 to 48 h) and also led to 75% protection against CCl(4)-induced lethality following a lethal dose (LD75) of CCl(4) (3 ml/kg). Calpain leakage in plasma and in the perinecrotic areas increased until 48 h and decreased from 72 h onward paralleling progression and regression of liver injury, respectively, after CCl(4) treatment. Mitigation of progressive injury was accompanied by substantially low calpain in perinecrotic areas and in plasma after CBZ treatment. Normal hepatocytes incubated with the plasma collected from CCl(4)-treated rats (collected at 12 h when most of the CCl(4) is eliminated) resulted in extensive cell death prevented by CBZ. Cell-impermeable calpain inhibitor E64 also protected against progression of CCl(4)-induced liver injury, thereby confirming the role of released calpain in progression of liver injury. Following CCl(4) treatment, calpain-specific breakdown of alpha-fodrin increased, while it was negligible in rats receiving CBZ after CCl(4). Hepatocyte cell death in incubations containing calpain was completely prevented by CBZ. Eighty percent of Swiss Webster mice receiving a lethal dose (LD80) of acetaminophen (600 mg/kg, ip) survived if CBZ was administered 1 h after acetaminophen, suggesting that calpain-mediated progression of liver injury is neither species nor chemical specific. These findings suggest the role of calpain in progression of liver injury.     

The uptake mechanism of gallium-67 into hepatocytes treated with carbon tetrachloride

We have recently reported that transferrin (Tf)-unbound gallium-67 (67Ga) may be taken up into the liver of carbon tetrachloride (CCl4)-treated rats. In the present study, we attempted to clarify detailed mechanism of Tf-unbound 67Ga uptake by hepatocytes treated with CCl4 using in vitro experimental system. Hepatotoxic damages by CCl4 are mostly attributed to radical formed by an action of cytochrome P450. P450 isozymes have a higher expression in the perivenous hepatocytes (PVH) more than periportal hepatocytes (PPH). Therefore, we thought that the uptake of 67Ga which had been used for the detection of liver damage might have a zonal difference. The results of ALT activities showed that the CCl4 exposure for 4 h strongly impaired PVH more than PPH. The uptake of 67Ga by PVH treated with CCl4 was also higher than that by PPH. Moreover, the uptake of 45Ca by PVH was higher than that by PPH. In order to investigate whether 67Ga passed through calcium channel of hepatocytes, we made use of calcium channel blocker and activator. The Ca2+-channel blocker, verapamil, significantly decreased the uptakes of 45Ca and 67Ga by PPH and PVH pretreated with CCl4. The addition of the Ca2+-channel activator, Bay K8644, significantly increased the uptake both of 45Ca and 67Ga by PPH pretreated with CCl4. In the present study, it was demonstrated that the uptake of Tf-unbound 67Ga preferentially occurred in CCl4-damaged PVH and 67Ga was taken up into the hepatocytes in part through calcium channel.     

Muramyl dipeptide and carbon tetrachloride hepatotoxicity in rats: involvement of plasma membrane and calcium homeostasis in protective effect

The present study was carried out to investigate the effect of single and multiple doses of N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) on CCl4-induced hepatotoxicity, and hence some of the mechanisms involved. MDP (8.26 mumol/kg i.v.) was administered to rats according to different protocols followed by a single dose of CCl4 (5.2 mmol/kg i.p.), and either the hepatocytes were subsequently isolated and tested for viability and lipid peroxides formation or the level of serum aminotransferases and lactate dehydrogenase (LD) was measured. The results clearly indicate that MDP pretreatment in a single dose reduced CCl4 hepatotoxicity as judged from viability tests as well as reduction of elevated lipid peroxides induced by CCl4 administration. The level of lactate dehydrogenase was also brought to normal value by single MDP administration. MDP also decreased significantly the CCl4-elevated Ca2+ content of isolated hepatocytes and postmicrosomal supernatant Ca2+. 14C-palmitic acid incorporation was increased significantly for neutral lipids and/or phospholipids in hepatocytes and certain subcellular fractions under MDP treatment in vivo. A different effect was seen after multiple MDP administration which further increased CCl4-induced elevation of aminotransferases. Even the repeated administration of MDP without CCl4 increased the level of the latter enzymes. It may be concluded that a single administration of MDP can protect liver cells from CCl4 injury by a mechanism affecting the plasma membrane.     

Inhibition of liver endoplasmic reticulum calcium pump by CCl4 and release of a sequestered calcium pool

One of the earliest effects observed in rat liver after CCl4 administration is inhibition of an ATP-dependent calcium pump found at the endoplasmic reticulum. This report confirms that the amount of calcium associated with the microsomal fraction is reduced after CCl4 administration and, for the first time, demonstrates time-, dose-, and metabolism-dependent relationships between inhibition of the liver microsomal calcium pump and the amount of calcium found in the microsomal fraction. Furthermore, release of calcium from the endoplasmic reticulum is shown to cause activation of a cytoplasmic enzyme that responds to increases of ionized calcium, glycogen phosphorylase. This suggests that the endoplasmic reticulum calcium pump sequesters an intracellular pool of calcium within the endoplasmic reticulum. This pool of calcium may be released into the cytoplasm as a consequence of inhibition of the calcium pump by CCl4.     

Protection of rat hepatocytes exposed to CCl4 in-vitro by cynandione A, a biacetophenone from Cynanchum wilfordii

To identify hepatoprotective agents from plant sources we use primary cultures of rat hepatocytes injured by CCl4. The hepatoprotective agents are the compounds that mitigate the injury caused by CCl4. Using this system we have investigated the biochemical mechanisms involved in the hepatoprotective activity of cynandione A, a biacetopherone, isolated from the roots of Cynanchum wilfordii Hemsley (Asclepiadaceae). Cynandione A (50 microM) significantly reduced (approximately 50%) the release into the culture medium of glutamic pyruvic transaminase and sorbitol dehydrogenase from the primary cultures of rat hepatocytes exposed to CCl4. Glutathione, superoxide dismutase, catalase and glutathione reductase play important roles in the cellular defence against oxidative stress. Cynandione A appeared to protect primary cultured rat hepatocytes exposed to CCl4 from significant drops in the levels of each of these four specific markers. Cynandione A also ameliorated lipid peroxidation by up to 50% as demonstrated by a reduction in the production of malondialdehyde. These results suggest that cynandione A protected the hepatocytes from CCl4-injury by maintaining the level of glutathione and by inhibiting the production of malondialdehyde, due to its radical scavenging properties.     

Carbon tetrachloride-dependent inhibition of lipid secretion by isolated hepatocytes. Characterization and requirement for bioactivation

Secretion of lipid as very low density lipoprotein (VLDL) by isolated hepatocytes was studied in a system in which the cells were exposed to a constant concentration of carbon tetrachloride (CCl4) throughout the duration of the incubation. Inhibition of secretion was characterized in terms of CCl4 concentration and duration of incubation. Half-maximum inhibition of VLDL secretion occurred at 80 microM CCl4. At 390 microM CCl4, VLDL secretion was inhibited 40% in 2 min and was suppressed completely in 5 min. No CCl4-dependent release of cellular glutamic oxaloacetic transaminase occurred under the conditions studied. Evidence is presented indicating that the metabolism of CCl4 is required for the expression of CCl4-dependent inhibition of lipid secretion. With respect to the parameters studied, the isolated hepatocyte system closely mimics the hepatic response of the intact animal to CCl4.     

In vitro and in vivo hepatoprotective effects of the total alkaloid fraction of Hygrophila auriculata leaves

Objective:

To investigate the total alkaloid fraction of the methanol extract of leaves of Hygrophila auriculata for its hepatoprotective activity against CCl4-induced toxicity in freshly isolated rat hepatocytes, HepG2 cells, and animal models.

Materials and Methods:

Mature leaves of H. auriculata were collected, authenticated, and subjected to methanolic extraction followed by isolation of total alkaloid fraction. Freshly isolated rat hepatocytes were exposed to CCl4 (1%) along with/without various concentrations of the total alkaloid fraction (80–40 µg/ml). Protection of human liver-derived HepG2 cells against CCl4-induced damage was determined by the MTT assay. Twenty-four healthy Wistar albino rats (150–200 g) of either sex were used for the in vivo investigations. Liver damage was induced by administration of 30% CCl4 suspended in olive oil (1 ml/kg body weight, i.p).

Results:

The antihepatotoxic effect of the total alkaloid fraction was observed in freshly isolated rat hepatocytes at very low concentrations (80–40 µg/ml). A dose-dependent increase in the percentage viability was observed when CCl4-exposed HepG2 cells were treated with different concentrations of the total alkaloid fraction. Its in vivo hepatoprotective effect at 80 mg/kg body weight was comparable with that of the standard Silymarin at 250 mg/kg body weight.

Conclusion:

The total alkaloid fraction was able to normalize the biochemical levels which were altered due to CCl4 intoxication.     

Hepatoprotective Effect of the Total Alkaloid Fraction of Solanum pseudocapsicum Leaves

The total alkaloid fraction of the methanol extract of leaves of Solanum pseudocapsicum was tested for its hepatoprotective activity against CCl 4 induced toxicity in freshly isolated rat hepatocytes, HepG2 cells and animal models. The total alkaloid fraction was able to normalise the levels of aspartate amino transferase (ASAT), alanine aminotransferase (ALAT), alkaline phosphatase (ALP), triglycerides (TGL), total proteins, albumin, total bilirubin and direct bilirubin, which were altered due to CCl 4 intoxication in freshly isolated rat hepatocytes and also in animal models. The antihepatotoxic effect of the total alkaloid fraction was observed at very low concentrations (6–10µg/ml) and was found to be superior to that of the standard used. A dose dependent increase in the percentage viability was observed when CCl 4 exposed HepG2 cells were treated with different concentrations of the total alkaloid fraction. The highest percentage viability of HepG2 was observed at a concentration of 10µg/ml. Its in vivo hepatoprotective effect at 20 mg/kg body weight was comparable with that of the standard at 250mg/kg body weight. The total alkaloid fraction merits further investigation to identify the active principles responsible for the hepatoprotective properties. The results from the present investigation also indicate well correlation between the in vivo and in vitro studies.     

The mechanism of adenosine release from hypoxic rat liver cells.

Uptake of [14C]-adenosine into freshly dispersed rat hepatocytes was inhibited 44% by dipyridamole (50 microM) and 60% by nitrobenzylthioinosine (NBTI, 20 microM). The results are consistent with the known ability of these drugs to inhibit adenosine transport in other cell types. The nucleotide analogue, alpha, beta-methylene adenosine diphosphate (AOPCP, 50 microM), inhibited by 84% the degradation of exogenous 5' AMP that occurred rapidly when this substrate alone was presented to isolated hepatocytes. This confirms the ecto-5'-nucleotidase inhibitory properties of this analogue in isolated hepatocytes. During hypoxic incubation, isolated hepatocytes released adenosine, which accumulated in the extracellular volume. Dipyridamole and NBTI each markedly attenuated this extracellular adenosine accumulation. In contrast, AOPCP had no inhibitory effect on net hypoxic adenosine release. It is concluded that hypoxic rat hepatocytes produce adenosine intracellularly and that this adenosine is released via facilitated diffusion to the extracellular space, based on the inhibition observed with the transport inhibitors. The plasma membrane enzyme ecto-5'-nucleotidase does not appear to participate in hypoxic adenosine release from these cells as indicated by the lack of effect of the nucleotidase inhibitor, AOPCP.     

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.     

Amelioration of carbon tetrachloride-induced hepatic necrosis by post-toxicant treatment with cystamine

A quantitative animal model was developed to study amelioration of carbon tetrachloride-induced hepatic injury by post-toxicant administration of cystamine. Amelioration of CCl4-induced injury by post-toxicant cystamine treatment was compared to prevention of injury by cystamine pretreatment and possible mechanisms of the post-toxicant cytoprotective effect were investigated. Pretreatment of rats with cystamine dihydrochloride (300 mg/kg, p.o.) 30 min prior to CCl4 (0.25 ml/kg, i.p.) prevented CCl4-induced hepatic necrosis, plasma enzyme elevations, and hepatic calcium accumulation. When administered up to 12 h after CCl4, a single oral dose of cystamine inhibited necrosis in a dose-dependent manner, but did not reduce CCl4-induced plasma enzyme elevation or hepatic calcium accumulation. Cystamine post-treatment, therefore, does not appear to inhibit toxicant-induced influx of extracellular calcium into toxicant-damaged cells. This also suggests that the influx of extracellular calcium does not necessarily constitute an irreversible event leading to cell death. The mild hypothermia induced by post-toxicant treatment with cystamine did not delay the appearance of the lesion. Evidence for a slightly earlier regeneration of hepatic tissue was noted when cystamine was administered 12 h after CCl4. However, this effect was observed too long after exposure to the toxicant to account for the protection from necrosis observed 24 h after CCl4.     

Sauchinone, a lignan from Saururus chinensis, attenuates CCl4-induced toxicity in primary cultures of rat hepatocytes

We used primary cultures of rat hepatocytes injured by the hepatotoxin, carbon tetrachloride (CCl4), as a test system to screen for hepatoprotective compounds from natural products. Sauchinone was isolated from the aerial parts of Saururus chinensis (Saururaceae) by this method. At a concentration of 50 microM, sauchinone significantly reduced the release into the culture medium of glutamic pyruvic transaminase from CCl4-damaged cultures of rat hepatocytes. It has been determined that glutathione, superoxide dismutase and glutathione peroxidase all play important roles in the cellular defense against oxidative stress. Sauchinone appeared to protect primary cultured rat hepatocytes exposed to CCl4 from significant drops in the levels of each of these three specific markers, respectively. Sauchinone also seemed to ameliorate lipid peroxidation as demonstrated by a reduction in the production of the oxidized lipid byproduct, malondialdehyde. These results suggest that sauchinone may exert hepatoprotective activity through antioxidant activity.     

Hepatoprotective dibenzylbutyrolactone lignans of Torreya nucifera against CCl4-induced toxicity in primary cultured rat hepatocytes

Three dibenzylbutyrolactone lignans, (-)-arctigenin, (-)-traxillagenin, and (-)-4'-demethyltraxillagenin, isolated from the bark of Torreya nucifera SIEB. et ZUCC. (Taxaceae) showed significant hepatoprotective activity in primary cultures of rat hepatocytes injured by carbon tetrachloride (CCl(4)). These lignans reduced the release of glutamic pyruvic transaminase into the culture medium from the CCl(4)-injured primary cultures of rat hepatocytes. Further investigation revealed that the three lignans significantly preserved the level of glutathione (GSH) and activities of superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase in the CCl(4)-injured rat hepatocytes. The lignans also ameliorated lipid peroxidation as demonstrated by a reduction in malondialdehyde-related products. Moreover, these lignans significantly attenuated the GSH reduction caused by diethylmaleate which depletes GSH through the formation of stable conjugates. However, these lignans showed no effect on the GSH synthesis inhibited by buthionine sulfoximine. From these results, it can be concluded that arctigenin, traxillagenin, and 4'-demethyltraxillagenin may protect hepatocytes from CCl(4) injury by maintaining the GSH level.     

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。     

Hepatoprotective phenylpropanoids from Scrophularia buergeriana roots against CCl(4)-induced toxicity: action mechanism and structure-activity relationship

Phenylpropanoids isolated from the roots of Scrophularia buergeriana MIQ. (Scrophulariaceae) protected primary cultures of rat hepatocytes from toxicity induced by carbon tetrachloride (CCl(4) ). In this report, we show that two of these phenylpropanoids, 4-O-E- p-methoxycinnamoyl-alpha-L-rhamnopyranoside ester ( 1) and p-methoxycinnamic acid ( 3) have significant hepatoprotective activity; another phenylpropanoid used for comparison, isoferulic acid ( 11), was equally active. To determine the mechanism(s) by which these three phenylpropanoids exerted their hepatoprotective activity, we measured activities of enzymes involved in the glutathione (GSH) redox system and assayed the level of hepatic mitochondrial GSH. The GSH levels in primary cultures of rat hepatocytes were significantly reduced with CCl(4) insult, but were significantly preserved by the treatment with these three phenylpropanoids. The activities of glutathione disulfide reductase and glutathione-S-transferase which normally decrease in CCl(4) -injured rat hepatocytes were significantly preserved by the treatment with these three phenylpropanoids. In addition, in CCl(4) -injured rat hepatocytes, the increased formation of malondialdehyde, a byproduct of lipid peroxidation, was reduced by the treatment with these phenylpropanoids. We determined the essential structural moiety within these three phenylpropanoids needed to exert hepatoprotective activity. The alpha,beta-unsaturated ester moiety seemed to be essential for exerting hepatoprotective activity.