A.S. Bernacchi

Treatment of carbon tetrachloride-induced liver necrosis with chemical compounds.

Several chemical compounds previously reported by our laboratory as effective in preventing CCl 4 -induced liver necrosis were ineffective when administered after CCl 4 , e.g., pyrazole; 3-amino-1,2,4-triazole; ethyl 2-diethylaminoethyl-2-phenyl-2-ethylmalonate (Sch 5705); and ethyl N -(2-diethylaminoethyl)-2-phenyl-2-ethylmalonate (Sch 5706). In contrast, cystamine, cysteine, and 2-diethylaminoethyl 2,2-diphenylvalerate (SKF 525-A) protected against CCl 4 -induced liver necrosis when given as late as 12 hr after CCl 4 . Liver damage was assessed at 24 hr either histologically or by measurement of plasma isocitric dehydrogenase. Some degree of protection by cystamine and cysteine was also apparent when liver damage was assessed 72 hr after CCl 4 administration.

Therapeutic effectiveness of cystamine and cysteine to reduce liver cell necrosis induced by several hepatotoxins.

Abstract Cystamine or cysteine administration to rats receiving dimethylnitrosamine, thioacetamide, or bromobenzene 3, 6, or 12 hr before, partially prevented the liver necrosis produced by these substances at 24 hr. In contrast, both chemicals were unable to prevent the necrosis induced by allyl alcohol and aflatoxin B 1 . Since at either 6 hr (dimethylnitrosamine) or 12 hr (bromobenzene and thioacetamide), most of activation of the agents and the subsequent interaction with cell constituents has already occurred, these results suggest that cystamine or cysteine protect because they alter cellular response to the hepatotoxins. The results also suggest the possible application of these chemicals in therapeutics.

Disruption of mitochondrial function as the basis of the trypanocidal effect of trifluoperazine onTrypanosoma cruzi

The tricyclic anti-calmodulin drug trifluoperazine (TFP) inhibited growth and motility of epimastigotes ofTrypanosoma cruzi, at concentrations lower than 100 μM, and motility and infectivity of the bloodstream trypomastigote form at 200 μM. Electron microscopy of TFP-treated epimastigotes showed that the major effect was at the mitochondrial level, with gross swelling and disorganization. The oligomycin-sensitive, mitochondrial ATPase was completely inhibited by 20 μM TFP, and the same drug concentration caused a 60% decrease in intracellular ATP content. The results suggest that the trypanocidal effect of TFP may be related more to mitochondrial damage than to the well-known anticalmodulin effect of the drug.

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.

Carbon tetrachloride activation by highly purified liver mitochondrial preparations

Highly purifled rat liver mitochondrial preparations were found to be able to activate CCl4 to reactive metabolites that bind covalently to lipids. Part of the process is of an enzymatic nature, but most of it is non-enzymatic, The enzymatic mitochondrial CCl4 activation operates more efficiently under anaerobic conditions; it requires NADPH, is CO sensitive, is inducible by phenobarbital pretreatment and is only weakly inhibited by high concentrations of cyanide or azide. The non-enzymatic mitochondrial CCl4 activation is not inhibited by CO and proceeds equally well under air or nitrogen.

Effects of repeated administration of rat 2-diethylaminoethyl-2-2-diphenylvalerate-HCl (SKF 525 A) on liver

Repetitive administration to male rats of 2-diethylaminoethyl-2-2-diphenyl-valerate-HCI (SKF 525 A) (50 mg/kg, i.p.), decreases the intensity of [14C]-orotic acid incorporation/mg of RNA but not the 14C-incorporation/g liver. The RNA content/g liver is significantly higher in SKF-treated animals than in controls. Decay of label in liver RNA from [14C] orotic acid pretreated animals, is not significantly different in SKF 525 A treated animals than in controls. SKF 525 A repetitive administration, does not modify the rate of incorporation of 32P in liver microsomal lipid when results are expressed per microgram of inorganic phosphorus but it does when expressed in terms of per gram liver. There is a significant decrease in the decay rate of label from 32P-prelabeled liver microsomal phospholipids when animals are treated with SKF 525 A. There is a significant increase in the protein and phospholipid content in the smooth endoplasmic reticulum fraction. The electron microscopy of liver from SKF 525 A-treated animals, shows the presence of large areas of round vesicles of swollen endoplasmic reticulum, partly due to smooth component and part due to rough component, having detached the ribosomes from their membranes. Results suggest an inhibitory effect of SKF 525 A on RNA and phospholipid degradative processes.

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 gluthathione 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 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.

Carbon tetrachloride induced ultrastructural alterations in pancreatic acinar cells and in the hepatocytes. Similarities and differences

We found that 14C from 14CCl4 gets covalently bound to rat pancreas and liver microsomal lipids. The intensity of this process in 320 times smaller in the former than in the latter organ. We did not detect any cytochrome P-450 (P-450) or ethylmorphine N-demethylase activity in pancreatic microsomes in contrast to the high content of those components occurring in liver. Similarly, CCl4 induced lipid peroxidation is observed in liver preparations but not in pancreas. There was no evidence for any histological alterations in pancreas 24 h after administration of a toxic dose causing marked cnetrolobular necrosis in liver. CCl4 causes profound ultrastructural alterations in the hepatocytes involving early the endoplasmic reticulum, the Golgi complex and the nuclear envelope and later the entire cell. In selected clones of pancreatic acinar cells we observed an intense dilatation in the rough endoplasmic reticulum and polysome detachment from their membranes, the appearance of myelin figures and occasionally of large autophagic vacuoles. No alterations in other organelles were detected but a slight increase in the number of lipid droplets was observed. CH2Cl2 did not behave similarly.