Prevention of CCl4-induced liver necrosis by the calcium chelator arsenazo III.

Arsenazo III (AIII) (100 mg/kg ip in saline) administration to Sprague-Dawley male rats 30 min before or 6 or 10 hr after CCl4 [1 ml/kg ip as a 20% (v/v) solution in olive oil] significantly prevented liver necrosis but not fatty liver caused by the hepatotoxin at 24 hr as demonstrated either by histology or by determination of isocitric acid dehydrogenase in plasma. AIII did not modify the CCl4 concentrations reaching the liver, the intensity of the covalent binding of CCl4-reactive metabolites to hepatic microsomal lipids, or the CCl4-promoted lipid peroxidation process at either 1 or 3 hr of poisoning. AIII administration enhanced glutathione (GSH) levels in liver and significantly prevented the CCl4-induced minor decreases in GSH content and the CCl4-induced increases in calcium content at 24 hr of intoxication. AIII treatment further enhanced the CCl4-induced decreases in body temperature of the poisoned rats. Results suggest that AIII's preventive effects might be related to its very well-known calcium-chelating properties, but that additional factors related to AIII's ability to increase GSH content in liver or to decrease body temperature of CCl4-intoxicated animals may also play a role.

Prevention of carbon tetrachloride-induced liver necrosis by the chelator alizarin sodium sulfonate.

The administration of the calcium chelator alizarin sodium sulfonate (ASR) (100 mg/kg ip in saline) 30 min before or 6 or 10 hr after CCl4 (1 ml/kg ip as a 20% v/v solution in olive oil) partially prevents the necrogenic effect of the hepatotoxin at 24 hr, but prevention of CCl4 fat accumulation was not observed. Protective action cannot be attributed to potential decreasing effects of ASR on CCl4 levels reaching the liver, on the covalent binding of CCl4-reactive metabolites to cellular components, or on CCl4-induced lipid peroxidation because ASR does not modify these parameters significantly. ASR administration increases GSH levels in livers of both control and CCl4-poisoned animals and decreases the calcium content of intoxicated animals at 24 hr of poisoning. ASR significantly lowers the body temperature of CCl4-treated animals at different times of the intoxication process. Present and previous results from our laboratory on the preventive effects of another very specific calcium chelator, calcion, and several anticalmodulins suggest that the beneficial effects of ASR might be associated with its calcium chelating ability. Other protective effects of ASR, such as lowering body temperature or increasing GSH content in liver, cannot be excluded.

Modulation of the course of CCl4-induced liver injury by the anti-calmodulin drug thioridazine.

Thioridazine (TDZ) administration to rats (50 mg/kg i.p.) 6 or 10 h after CCl4 treatment (1 ml/kg in olive oil i.p.) partially prevented necrogenic effects of this compound at 24 h but not at 72 h. TDZ did not have inhibitory effects on CCl4 activation, covalent binding (CB) of reactive metabolites to cellular constituents or CCl4-induced lipid peroxidation (LP). Moreover, TDZ had enhancing effects on both LP and CB. TDZ was able to increase protein and phospholipid synthesis and slightly but significantly enhanced protein but not phospholipid degradation in livers from control rats. TDZ administration decreased calcium liver content in CCl4-poisoned animals but did not change the intensity of CCl4-induced fatty liver. TDZ lowered body temperature in CCl4-treated animals during the 24 h observation period. These results and previous studies from our laboratory suggest calcium and calmodulin (CaM) participation in the CCl4 necrogenic effects on the liver but not in the hepatotoxin-induced fatty liver. TDZ-lowering effects on body temperature might also be a determinant in the delaying effects of this drug on the onset of CCl4-induced necrosis. Present experiments did allow discrimination between these two or other possible mechanisms for TDZ modulation effects.

Further studies on the mechanism of the late protective effects of phenylmethylsulfonyl fluoride on carbon tetrachloride-induced liver necrosis.

We previously reported that phenylmethylsulfonyl fluoride (PMSF) administration to rats (100 mg/kg, ip in olive oil) as late as 6 or 10 hr after CCl4 (1 ml/kg, ip as a 20% v/v solution in olive oil) can partially prevent the necrogenic response to the hepatotoxin at 24 hr. Here we confirm that observation by electron microscopy and provide further evidence that only in these circumstances were nuclear clumping of chromatin, slight dilatation of the endoplasmic reticulum, myelin figures and lipid droplets in the cytoplasm, large numbers of lysosomes and peroxisomes, glycogen, and slightly swollen mitochondria observable in the protected animals. A very minor part of the late protective effects of PMSF might be due to the effects of this drug on decreasing the intensity of covalent binding of CCl4-reactive metabolites or the intensity of CCl4-induced lipid peroxidation still occurring 6 or 10 hr after CCl4. PMSF administration did not prevent CCl4-induced decreases in cytochrome P450 content or glucose-6-phosphatase activity but partially prevented CCl4-induced calcium accumulation in liver. PMSF treatment increased glutathione and glycogen content in CCl4-poisoned animals, but did not markedly modify protein/phospholipid synthesis or degradation processes. Results suggest that the late protective effects of PMSF administration in CCl4-induced liver necrosis might be due to a favorable modulation of the calcium-calmodulin system similar to that previously described for other drugs.

Carbon tetrachloride-induced liver cell injury in the Mongolian gerbil (Meriones unguiculatus).

1. Male Mongolian gerbils (Meriones unguiculatus) liver activates CCl4 to free radicals that bind covalently to cellular components (CB) and stimulate a lipid peroxidation (LP) process to a larger extent than the rat liver. 2. CCl4 administration results in a less intense necrogenic effect in gerbils than in rats and does not cause fatty liver. 3. CCl4 causes less intense effects on liver ultrastructure or calcium metabolism but more marked depression of glucose 6 phosphatase activity (G6P-ase) in gerbils than in rats. 4. Results suggest that a better ability of gerbil liver to keep calcium homeostasis than rat liver might be the cause of their relative resistance to necrosis. Higher intensity of CB and LP in gerbils than in rats might explain more intense effects on G6P-ase.

Late preventive effects against carbon tetrachloride-induced liver necrosis of the calcium chelating agent Calcion.

In agreement with the hypothesis that changes in calcium homeostasis might be significant in late stages of chemically-induced liver cell injury, a calcium chelating agent, Calcion, was able to partially prevent CCl4-induced liver necrosis observed at 24 h, when treatment was given as late as 6 or 10 h after the hepatotoxin. Calcion had minor or no effects on covalent binding of reactive metabolites to cellular components, or on lipid peroxidation or on CCl4 levels reaching the liver. Calcion treatment of CCl4-poisoned animals decreased CCl4-induced calcium increases in liver and increased glutathione levels decreased by hepatotoxin at 24 h. Calcion treatment was not able to prevent CCl4-induced fatty liver. Calcion protective effects were body temperature dependent but they were cancelled when Calcion-treated poisoned animals were kept normothermic. Results suggest that Calcion protective effects might be linked to calcium chelation or alternatively that they might derive from decreases in body temperature.

Further studies on the late preventive effects of the anticalmodulin trifluoperazine on carbon tetrachloride-induced liver necrosis.

Trifluoperazine (TFP) (50 mg/kg ip) administration to rats 6 or 10 hr after CCl4 (1 ml/kg ip in olive oil) significantly prevented liver necrosis but not fatty liver caused by the hepatotoxin at 24 hr as evidenced by either histology or electron microscopy. TFP given 6 hr after CCl4 significantly decreased the CCl4-induced increases in liver calcium content. TFP raised four to five times the liver glycogen content in control rats but was unable to modify decreased glycogen content of CCl4 poisoned animals. TFP administration increased phospholipid and protein synthesis as evidenced by studies on 32P incorporation into microsomal phospholipid and by experiments on [14C]leucine incorporation in microsomal protein fractions from control rat livers. No significant changes were observed in microsomal phospholipid degradation as studied by decay of label from 32P-prelabeled microsomal lipids or in increased protein degradation as evidenced by decay of label from [14C-guanidino]arginine-prelabeled microsomal proteins found in livers of control rats after TFP treatment. Electron microscopy observations of liver from control animals treated with TFP evidenced accumulation of glycogen in areas close to smooth endoplasmic reticulum (SER); large Golgi areas with an abundant number of lysosomes, and minor dilatation effects on the rough endoplasmic reticulum (RER) and nuclear membrane. Results suggest that TFP preventive effects might be due to the anticalmodulin actions of this drug.

Effects of carbon tetrachloride on the liver of chickens. Early biochemical and ultrastructural alterations in the absence of detectable lipid peroxidation.

Administration of CCl4 i.p. to Leghorn chickens did not promote lipid peroxidation of liver microsomal lipids, as evidenced by either increased diene conjugation or by decreased arachidonic acid content. The hepatotoxin did not produce liver necrosis 24 h after dosing, but decreased the cytochrome P-450 content, and aminopyrine N-demethylase and glucose 6 phosphatase activities at 1, 3, 6 and 24 h. CCl4 administration produced dilation of the rough endoplasmic reticulum and detachment of ribosomes from their membranes. These observations suggest that lipid peroxidation is not the key event in the production of these biochemical and ultrastructural alterations, elicited by CCl4.

Imipramine prevention of carbon tetrachloride-induced liver necrosis at late states of the intoxication process.

Imipramine administration (50 mg kg-1, i.p.) to Sprague-Dawley male rats (240-290 g) 6 or 10 h after CCl4 (1 ml kg-1, i.p.) partially prevents liver necrosis induced by the hepatotoxin. When imipramine is given 30 min before CCl4, it inhibits in part the CCl4-induced lipid peroxidation and the covalent interactions of reactive metabolites with microsomal lipids or proteins and partially prevents CCl4-induced cytochrome P-450 destruction, but not glucose 6 phosphatase activity depression. Imipramine administration prior to CCl4 does not modify levels of the hepatotoxin reaching the liver or the body temperature of CCl4 treated animals. Early preventive effects of imipramine on cytochrome P-450, might be attributed to inhibition of covalent interactions of reactive metabolites. The hypothesis that imipramine exerted late preventive effects by interfering with calcium deleterious effects or by modulation of protein and phospholipid synthesis or degradation is analyzed.

Late preventive effects of trifluoperazine on carbon tetrachloride-induced hepatic necrosis.

As a very preliminary test for a possible role of calmodulin in CCl4-induced hepatic injury, we studied the effects of the anticalmodulin drug trifluoperazine (TFP) on several deleterious actions of CCl4 on the liver. TFP administrated 30 min before or 6 or 10 hr after CCl4 significantly prevented hepatic necrosis induced by the hepatotoxin at 24 hr but not at 72 hr. TFP did not modify the CCl4 concentrations reaching the liver, or the intensity of the covalent binding of CCl4-reactive metabolites to hepatic microsomal proteins or lipids or the CCl4-induced cytochrome P-450 and glucose 6 phosphatase destruction. TFP administration decreased body temperature between 0 and 1 degree C in controls and between 1.2 and 3.5 degrees C in CCl4-treated animals during the 24-hr observation period. When TFP-treated CCl4-poisoned animals were kept normothermic, protective effects were eliminated. One possibility is that the protective effect of TFP might be due to a nonspecific action related to decreased body temperature. Alternatively, prevention might result from TFP inhibition of a late-occurring process critical for CCl4-induced cell necrosis requiring calmodulin participation. If this alternative were in operation, protective consequences of this inhibitory effect of TFP should be either canceled or counteracted in the normothermic TFP + CCl4-treated animal.

Late preventive effects of several anticalmodulin drugs on galactosamine-induced liver necrosis.

Four anticalmodulin drugs: trifluoperazine (TFP), pimozide (PMZ), thioridazine (TDZ) and imipramine (IMP) (50 mg/kg, ip) were able to partially prevent Galactosamine (GAL) (600 mg/kg, ip) induced liver necrosis when given 6 h after the hepatotoxin. IMP was also effective 10 h after GAL. The possibility of calmodulin participation in late stages of GAL-induced liver injury is analyzed.

Late preventive effects on carbon tetrachloride-induced necrosis of the inhibitor of proteases 1-1-chloro-3-tosyl amido-7-amino-2-heptanone (TLCK).

The administration to rats of the inhibitor of proteases 1-1-chloro-3-tosyl-amido-7-amino-2-heptanone (TLCK) 6 or 10 h after carbon tetrachloride (CCl4), significantly prevented liver necrosis induced by this hepatotoxin at 24 h. The present and previous results from our laboratory suggest that degradative processes mediated by proteases and esterases might play a role in late stages of CCl4-induced liver cell injury.

Carbon tetrachloride-induced early biochemical alterations but not necrosis in pigeon's liver.

In contrast to what is well known to occur in rats, pigeons receiving CCl4 (1 ml/kg i.p.) were not susceptible to necrogenic effects of the hepatotoxin at 24 h. There were, however, other early biochemical alterations observable, such as depression of glucose 6 phosphatase activity, decrease in the cytochrome P-450 content and in aminopyrine-N-demethylase activity in pigeon liver microsomes at 3 and 6 h after CCl4 administration. Pigeon liver was able to activate CCl4 to reactive metabolites that bind covalently to lipids, but no CCl4-induced lipid peroxidation was proved by the diene hyperconjugation technique in pigeon liver microsomes at 1, 3 or 6 h after administration. Results suggest that covalent binding of CCl4-reactive metabolites are more relevant to early biochemical alterations induced by CCl4 than is lipid peroxidation. Absence of CCl4-induced necrosis in pigeon liver could be attributable to a smaller intensity of covalent binding interactions observed, when compared to susceptible species, and to absence of lipid peroxidation.

Late preventive effects of phenylmethylsulfonyl-fluoride on carbon tetrachloride-induced liver necrosis.

The administration to rats of phenylmethylsulfonylfluoride (PMSF) 6 or 10 h after tetrachloride (CCl4) significantly prevented liver necrosis induced by this hepatotoxin at 24 h but not at 72 h. Preventive effects of PMSF were not due to interference with CCl4 absorption from the peritoneum since CCl4 levels in livers of treated and untreated animals were not significantly different. The present and previous results from our laboratory suggest that degradative processes mediated by proteases and esterases might play a role in late reversible stages of CCl4-induced liver cell injury.

Late preventive effects on dimethylnitrosamine, thioacetamide or galactosamine-induced liver necrosis of the inhibitor of proteases, phenylmethylsulfonyl fluoride.

Phenylmethylsulfonyl fluoride (PMSF) administration to rats, was effective in partially preventing liver necrosis induced by thioacetamide, dimethylnitrosamine or galactosamine, when given 6 hr after the hepatotoxins. In the case of galactosamine but not of the other necrogenic chemicals, protection was also observed when PMSF was given 10 hr after this compound. These results and previous studies from our laboratory suggest participation of protein degradation at late stages of liver injury by these chemicals.

Studies on the mechanism of glutathione prevention of carbon tetrachloride-induced liver injury.

The prior administration of reduced glutathione (GSH) partially prevents carbon tetrachloride (CCl4)-induced liver necrosis observed at 24 h after administration of the hepatotoxin. No prevention occurs when observations are made at 72 h. GSH pretreatment does not significantly modify the intensity of the covalent binding of CCl4 reactive metabolites to microsomal lipids or the intensity of the CCl4-induced lipid peroxidation process at either 1, 3 or 6 h after poisoning. GSH administration does not significantly prevent CCl4-induced cytochrome P-450 destruction or glucose 6 phosphatase activity depression. Pretreatment with GSH does not significantly modify the levels of CCl4 or i.p. administered CCl4 reaching the liver at 1, 3 or 6 h after intoxication. Pretreatment with GSH significantly prevents CCl4-induced decreases in body temperature. Results are interpreted as suggesting that GSH prevents CCl4-induced liver necrosis by changing the liver cell's response to injury rather than by modification of early events of the process such as lipid peroxidation or covalent binding of reactive metabolites.

Prevention of thioacetamide-induced liver necrosis by prior administration of substrates of microsomal flavin-containing monooxygenase.

Prior administration of chlorpromazine (CPZ), imipramine (IMP), mercaptoethylamine (MEA), 1-(1-naphthyl)2-thiourea (ANTU) or phenyl-thiocarbamide (PTC) but not 1,4-dithio-1-threitol (DTT), was able effectively to prevent most of thioacetamide (TAC) -induced liver necrosis. These and previous observations suggest that liver microsomal flavin-containing monooxygenase critically controls the process of activation of TAC to the ultimate necrogen.

Tryptophan potentiation of the late cysteine preventive effects in carbon tetrachloride-induced necrosis.

Carbon tetrachloride (CCl4) (1 ml/kg/ip) induces a very intense necrotic effect on rat liver at 24 hr after administration. Cysteine (950 mg/kg/po) given 6 h after CCl4 exerted a very weak preventive effect on CCl4-induced necrosis, while tryptophan (300 mg/kg/po) did not. When both aminoacids are given together a very marked protective effect is observed. A possible participation of protein synthesis stimulation in the late protective effects of cysteine on CCl4-induced liver necrosis is discussed.

Carbon tetrachloride-induced liver injury in the rabbit.

CCl4 administration to rabbits leads to early destruction of liver microsomal cytochrome P-450, to depression of glucose 6 phosphatase, to ultrastructurally revealable alterations and to an intense necrosis and fat accumulation in liver. Despite the known resistance of rabbit liver microsomes to lipid peroxidation, CCl4 administration to rabbits promoted lipid peroxidation of their liver microsomal lipids as revealable by the diene hyperconjugation technique, at periods of time from 1 to 12 h. Nevertheless, the intensity of this process is not equivalent to that occurring in rat liver microsomes, since the arachidonic acid content of rabbit liver microsomal lipids does not decrease at either 6 or 24 h after CCl4 administration. Rabbit liver is able to activate CCl4 to reactive metabolites that bind covalently to lipids. Relevance of covalent binding of CCl4 reactive metabolites and CCl4-promoted lipid peroxidation to CCl4-induced rabbit liver injury is analysed.

Prevention of carbon tetrachloride-induced liver necrosis by several amino acids.

Aspartic acid, cystine, methionine and tyrosine were protective against carbon tetrachloride (CCl4)-induced liver necrosis 24 h after its administration, when given 30 min before the hepatotoxin. Aspartic acid, cystine and tyrosine were also effective when given as late as 6 h after CCl4. The protective effects of these amino acids, however, were no longer evident when observations of CCl4-induced necrosis were made at 72 h, except for cystine, which retained its protective potential. Protective amino acid administration did not modify the concentration of CCl4 in liver, nor did it decrease the intensity of the covalent binding of CCl4 reactive metabolites to cellular constituents or the CCl4-induced lipid peroxidation. Consequently, protection cannot be attributed to modulation of these parameters. Cystine, tyrosine and aspartic acid significantly lowered body temperature of the CCl4-treated rats, while methionine did not. Combined, these results suggest that the protective effect is not attributable to lowering of body temperature in CCl4-treated animals. Protection probably results from changes in the cell response to injury promoted by amino acid administration.