M.I. Díaz Gómez

Species differences in carbon tetrachloride-induced hepatotoxicity: The role of CCl4 activation and of lipid peroxidation

Abstract Carbon tetrachloride-induced liver necrosis was more intense in the mouse than in the guinea pig or the hamster, which are more susceptible than the rat. The chicken was resistant to CCl 4 . The extent of the irreversible binding of 14 C from 14 CCl 4 to cellular components decreases in the following order: mouse = hamster > guinea pig > rat > chicken, while the intensity of the CCl 4 -induced lipid peroxidation decreased in the order rat > hamster = guinea pig > chicken = mouse. The results roughly suggest a better parallelism between intensity of the process of irreversible binding to cellular components and necrosis than between lipid peroxidation and necrosis. In the case of the mouse the necrotic process was observed in absence of lipid peroxidation during the entire 24-hr period of intoxication. It was not possible to absolutely correlate the extent of the irreversible binding to microsomal lipids in the different species with either the characteristics of the spectral changes produced by CCl 4 by interaction with cytochrome P-450 (P-450) or with the content of P-450 or that of the P-450 reductase activity.

Irreversible binding of 14C from 14CCl4 to liver microsomal lipids and proteins from rats pretreated with compounds altering microsomal mixed-function oxygenase activity☆

Abstract 14 C from 14 CCl 4 irreversibly binds to lipid and protein components of different liver subcellular fractions. Microsomal lipids bind more 14 C than does either mitochondria or 105,000 g supernatant. The proteins from the 105,000 g fraction bind more 14 C than those from the other two fractions. The extent of the irreversible binding of 14 C to microsomal lipids and proteins is decreased by the prior treatment of the rats with cystamine, pyrazole, 3-methylcholanthrene or metopirone while it is increased by pretreatment with phenobarbital. The prior treatment with 2-diethylaminoethyl 2,2-diphenyl valerate hydrochloride (SKF 525A) increased the irreversible binding of 14 C to microsomal lipids, but not to microsomal proteins. The results suggest that the irreversible binding of 14 C to microsomal lipids is a more reliable expression of the CCl 4 -activation step than the one to microsomal proteins. The 14 CCl 3 free radicals responsible for the binding of 14 C to lipid would arise during the reduction of the CCl 4 /cytochrome P-450 complex mediated by cytochrome P-450 reductase.

Studies on the mechanism of cystamine prevention of several liver structural and biochemical alterations caused by carbon tetrachloride

Cystamine and cysteamine give type II spectral changes by interaction with microsomal suspensions while CCl4 gives a type I spectral change. Neither cystamine nor cysteamine at saturating concentrations prevent the spectral binding of small amounts of CCl4. Cystamine and cysteamine decelerate the reduction of cytochrome P-450 by cytochrome P-450 reductase. Cystamine administration partially prevents the irreversible binding of 14CCl4 to microsomal lipids. This preventive effect does not significantly change when the 14CCl4 dose is increased about 4-fold. In spite of the fact that this irreversible binding is decreased by about 60% at 6 hr by the previous cystamine administration, lipid peroxidation is only slightly prevented at 6 and 10 hr after CCl4 and prevented to about 30% at 24 hr. Cystamine partially prevented the depression of glucose-6-phosphatase activity and the destruction of cytochrome P-450 caused by CCl4. Cystamine pronouncedly lowers body temperature of the rats, and it is as effective in preventing CCl4-induced necrosis and fatty liver at 24 hr as it is at 48 or 72 hr after CCl4 administration. Cystamine stabilizes lysosomes in vitro but labilizes them when administered to rats. Cysteamine labilizes lysosomes in vitro. These results may suggest that cystamine administration partially prevents several structural and biochemical alterations caused by CCl4 because it inhibits the CCl4-activation step to ·CCl3 free radical, which would take place during the reduction of the cytochrome P-450CCl4 complex by cytochrome P-450 reductase.

Prevention and treatment of carbon tetrachloride hepatotoxicity by cysteine: Studies about its mechanism

Cysteine administration to rats (1.9 g/kg po) prevented the development of the necrosis and fatty liver induced by CCl4. This protective effect was observed when cysteine was given either 30 min before or 1 hr after the administration of CCl4. Cysteine administration did not prevent the irreversible binding of 14C from 14CCl4 to microsomal lipids, but it partially reduced the binding to microsomal proteins at 6 hr after 14CCl4. Cysteine pretreatment did not modify the intensity of the CCl4-induced lipid peroxidation process or cytochrome P-450 destruction, but it partially prevented depression of glucose-6-phosphatase activity by CCl4. The results are compatible with the possibility of a protective action of cysteine at a site in the chain of events leading to necrosis, but not the activation step.

The nature of the in vitro irreversible binding of carbon tetrachloride to microsomal lipids.

Abstract The in vitro irreversible binding of 14 C from 14 CCl 4 to liver microsomal lipids heeds NADPH as a cofactor and is considerably greater in anaerobic mixtures than in air. Sodium dithionite is not able to replace NADPH as a reducing agent. The irreversible binding process is almost completely inhibited by carbon monoxide. The tlc analysis of the in vitro labeled lipids showed that most of the label is associated with phospholipids and oleic acid is the favored target fatty acid. This latter observation was confirmed by glc analysis of the resulting fatty acid methyl esters with simultaneous determination of radioactivity. The products of the interaction between microsomal lipids and enzymatically produced ·CCl 3 reactive intermediate were also found to be formed when different standard fatty acid methyl esters react with CCl 4 in the catalytic presence of benzoyl peroxide, a reaction known to be mediated by ·CCl 3 and ·Cl free radicals. Results are compatible with the hypothesis that CCl 4 is activated to ·CCl 3 and ·Cl free radicals during the reduction of the CCl 4 /cytochrome P-450 complex by NADPH in the catalytic presence of the cytochrome P-450 reductase but not when CCl 4 interacts with reduced cytochrome P-450.

Mechanistic studies on carbon tetrachloride hepatotoxicity in fasted and fed rats

Abstract In agreement with previous reports from other laboratories, the fed rats are more resistant to the toxic action of CCl4 than rats starved overnight. This preventive effect of feeding does not merely reflect a delay in the time for damage to appear, since the protection is even more pronounced at 72 than at 24 hr. The similarity in CCl4 concentrations in the livers of fed and fasted rats excludes an effect due to a decreased absorption of the hepatotoxin. Irreversible binding of 14CCl4 to liver microsomal lipids and lipid peroxidation are only slightly decreased in the livers of fed rats when compared to that of fasted rats. These findings correlate with the similarity of the sleeping time, cytochrome P-450 content and cytochrome P-650 reductase activity in both groups. Preventive effects are apparently due to an interference in the events occurring between CCl4 activation or lipid peroxidation and the resulting damage.

Studies on thioacetamide-induced liver necrosis☆

Even at concentration as high as 20 mm, thioacetamide neither results in any type of spectral change by interaction with liver microsomal suspensions nor modifies the in vitro NADPH cytochrome P-450 reductase activity. Thioacetamide administration to rats does not induce a microsomal lipid peroxidation process. The ability of thioacetamide to induce liver necrosis in rats is age dependent, appearing at about the age of 20 days; the 30-day-old rats are already fully responsive to thioacetamide action. Thioacetamide-induced necrosis is as intense in control males as is in females or in castrated males. The previous administration of inhibitors of cytochrome P-450-mediated transformations does not prevent thioacetamide-induced necrosis (2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride, SKF 525A, or ethyl N-2-diethylaminoethyl-2-phenyl-2-ethylmalonate, Sch 5706). The effect of thioacetamide on livers from phenobarbital-preinduced rats is as intense as it is in control rats. Previous treatment with either cystamine or disulfiram partially prevented thioacetamide-induced liver necrosis. The results suggest that cytochrome P-450 or NADPH cytochrome P-450 reductase does not control the process of activation of thioacetamide to the ultimate necrogen.

Mechanism of in vivo carbon tetrachloride-induced liver microsomal cytochrome P-450 destruction.

Abstract Prior administration of aminotriazole (3-amino-1,2,4-triazole) or pyrazole to rats resulted in a significant prevention of the CCl4-induced decrease in the liver microsomal P-450 content. In A/J mice the CCl4 activation and P-450 destruction occurred in absolute absence of lipid peroxidation as determined by uv absorption. The data suggest that P-450 destruction is mainly mediated by direct attack of CCl4 metabolites rather than by CCl4-induced lipid peroxidation.

Carbon tetrachloride activation in liver microsomes from rats induced with 3-methylcholantrene

Abstract The irreversible binding of 14 C from 14 CCl 4 to microsomal lipids is decreased in animals treated with 3-methylcholantrene (3-MC), while it is increased in animals induced with phenobarbital (PB). CCl 4 -induced lipid peroxidation in 3-MC treated rats is as intense as in controls. Destruction of glucose 6-phosphatase (G6P-ase) by CCl 4 is smaller in 3-MC treated rats than in controls. Destruction of total cytochrome P-450 (P-450 + P 1 -450) by CCl 4 is smaller in 3-MC treated than in PB treated rats but similar to that obtained in controls. Results would indicate that P-450 would participate in CCl 4 activation much more effectively than P 1 -450.

Studies on the role of protein synthesis in cell injury by toxic agents: I. Effect of cycloheximide administration on several factors modulating carbon tetrachloride-induced liver necrosis☆

Abstract Cycloheximide pretreatment (1 mg/kg, ip) slightly attenuated the hepatic necrosis 24 hr after CCl 4 administration and this attenuation was more obvious at 72 hr. A dose of cycloheximide, 2 mg/kg, evoked a marked protective effect even at 24 hr. Cycloheximide administration (1 mg/kg) significantly reduced the body temperature in CCl 4 -treated rats but did not change the CCl 4 concentrations in the liver. Prior treatment of the rats with cycloheximide decreased the amount of binding of 14 CCl 4 to liver microsomal lipids and reduced CCl 4 -induced lipid peroxidation at 1 hr but not at 6 hr after administration of the hepatotoxin. Cycloheximide did not alter the pentobarbital sleeping time of the rats nor did it interact with microsomes to give spectral changes. Cycloheximide did not alter microsomal cytochrome P -450 reductase activity. Administration of cycloheximide prior to CCl 4 did not prevent the CCl 4 -induced decrease in cytochrome P -450 or glucose 6-phosphatase activity caused by the hepatotoxin. However it did prevent the CCl 4 -induced polysome breakdown and it did attenuate the severity of the lesions caused by CCl 4 at the ultrastructural level.