Late protective effects of the anticalmodulin drug fluphenazine on carbon tetrachloride-induced liver necrosis.

Fluphenazine (FP) treatment (50 mg/kg bw, ip in saline) 30 min before or 6 or 10 h after CCl4 administration (1 ml/kg ip in olive oil) significantly prevented the liver necrosis produced by the hepatotoxin at 24 h. FP had enhancing effects on the covalent binding of CCl4 reactive metabolites to cellular constituents and on CCl4 induced lipid peroxidation. FP lowered body temperature of the CCl4-poisoned animals during the 24 h observation period. The obtained results are compatible but do not prove the hypothesis that calmodulin (CaM) had participation in late occurring events preceding necrosis. FP lowering action on body temperature, however, might also play a role in the effects of this drug on the onset of CCl4 induced liver necrosis. FP levels in liver tissue as determined by gas chromatography-mass spectrometry evidenced the presence of the drug in amounts sufficient to inhibit CaM and that suggests that not all preventive effects of FP are due to its indirect actions on the central nervous system via decreased body temperature.

Trifluopromazine late preventive effects on carbon tetrachloride-induced liver necrosis.

Trifluopromazine (TFPro) administration to rats (50 mg/kg, ip) 30 min before or 6 or 10 hr after CCl4 treatment (1 ml/kg ip in olive oil) partially prevented necrogenic effects of this compound at 24 hr. TFPro has only minor effects on the covalent binding (CB) of CCl4-reactive metabolites to cellular constituents and even an enhancing action on CCl4-promoted lipid peroxidation (LP). Determination of TFPro levels in liver 1 and 3 hr after administration by gas chromatography/mass spectrometry showed its presence in that tissue at concentrations well above those needed for calmodulin (CaM) inhibitory effects of this drug. TFPro lowered body temperature in CCl4-treated animals during the 24-hr observation period. Protective effects of TFPro at 6 or 10 hr, when most of the CB and all of the LP has already occurred, suggest but do not prove a role for CaM in late stages of CCl4-induced necrogenic effects. Decreases in the body temperature of CCl4-poisoned animals provoked by TFPro might also play a role in the preventive actions of this drug.

Nicotinamide late protective effects against carbon tetrachloride-induced liver necrosis.

Nicotinamide (NIC) is known to increase the synthesis of pyridine nucleotides and also to inhibit the hydrolysis of them to ADP-ribose, which in turn is involved in Ca2+ release from mitochondria via the ADP ribosylation of crucial mitochondrial proteins. In this work, we test the potential ability of NIC to be a late protective agent against CCl4-induced liver necrosis. We observed that 1 g/kg po NIC, 30 min before or 6 or 10 hr after CCl4 (1 ml/kg), given ip as a 20% (v/v) solution in olive oil, was able to significantly prevent the necrogenic effect of the hepatotoxin at 24 hr as evidenced by determination of isocitric dehydrogenase activity in plasma or by histological observation. NIC administration 6 hr after CCl4 prevented fatty liver induced by hepatotoxin at 24 hr. NIC did not modify CCl4-induced lipid peroxidation process at 1 hr after CCl4 and decreased the covalent binding of 14CCl4 to lipids. NIC decreased the levels of 14CCl4 reaching the liver when given 30 min before hepatotoxin but not when given 6 hr after it. NIC lowered body temperature of rats at 1, 3, and 6 hr and augmented it at 24 hr after CCl4. NIC concentrations in liver as determined by GC/MS/SIM analysis were 21 micrograms/g liver 1 hr after administration and 53 micrograms/g at 3 hr. Late preventive effects of NIC against CCl4 induced liver necrosis when given at 6 or 10 hr after CCl4 are compatible with the hypothesis that NIC restores mitochondrial ability for Ca2+ uptake. This hypothesis remains to be proved and is being further challenged in our laboratory.

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.

Ultrastructural alterations in testes from rats treated with cysteine.

Cysteine administration in relatively large doses has been repetitively employed as preventive agent against chemically induced cell injury or as radioprotector. In this work we report that administration of a dose standard for those purposes (1.9 gr/kg, po in water) causes significant ultrastructurally evident alterations in testes at 24h. Damage involves Sertoli cells and spermatids. Alterations found in the former include dilatation of nuclear membrane and of the smooth (SER) and rough endoplasmic reticulum (RER) and detachment of ribosomes from RER. Cytoplasm appeared more sparse and electron lucent than in controls and contained more lipid droplets and lysosomes. Mitochondria exhibited alterations in shape and size. Damage in spermatids consisted of the appearance of irregular shape of the nucleus and alterations in their acrosomal caps. There was no histochemical evidence for either calcium accumulation or lipid peroxidation occurrence in testes of cysteine-treated animals. Results indicate that the large doses of cysteine employed in prevention of radiation or chemical effects is able to cause injury to Sertoli cells of the testes. Damage observed does not reach irreversible stages but may be sufficient to lead to production of abnormal spermatids.

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.

Glutathione (GSH) content in livers from control and carbon tetrachloride poisoned rats treated with the calmodulin inhibitors thioridazine, imipramine or chlorpromazine.

Administration to rats of the calmodulin (CaM) inhibitors Thioridazine (TDZ) or Imipramine (IMP) (50 mg/kg ip) or Chlorpromazine (CPZ) at a dose of 25 mg/kg, ip 6 hr after olive oil or CCl4 (1 ml/kg, ip as a 20% olive oil solution) significantly increased the GSH content in liver of CCl4 poisoned animals but not in controls. The analysis of present observations and past results with Trifluoperazine, suggest that increases in GSH content in CCl4 poisoned animals treated with protective anticalmodulins are a consequence of prevention and not the cause of their preventive effects.

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.

Increased glutathione (GSH) content in livers of control and carbon tetrachloride poisoned rats treated with the anticalmodulin drug trifluoperazine (TFP).

TFP administration to rats (50 mg/kg, ip) 6 hr after olive oil or CCl4 (1 mg/kg, ip as a 20% olive oil solution) significantly increases the GSH content in liver at 24 hr. The role of GSH increases in the late preventive effects against CCl4 hepatotoxicity is discussed. A link between the calcium/calmodulin (CaM) system and the GSH status in liver is postulated.

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.