G. Ugazio

Initiation of free radical reactions and hepatotoxicity in rats poisoned with carbon tetrachloride or bromotrichloromethane

Morphological studies [1, 2] have shown that bromotrichloromethane behaves as a more dangerous hepatotoxin than carbon tetrachloride.The present investigation extends those preliminary findings and offers new biochemical evidences for the hypothesis suggested by the relationship between the toxicity and the level of the bond dissociation energy. The electron paramagnetic resonance signal and the UV-spectrum of double bond shifting in microsomal lipids show that CBrCl3 administration induces a more rapid and extensive free radical reaction in liver than CCl4. More severe pathological phenomena are coincident with a more profound pro-oxidant effect of bromotrichloromethane. In fact, 0.26 mmol of CBrCl3 prove lethal after having caused massive fatty liver and necrosis, while an equimolecular amount of CCl4 induces a lower and transient hepatic triglyceride accumulation and necrosis. Under the same experimental conditions, the lethal dose of carbon tetrachloride is twenty times higher (5.16 mmol per 100 g body weight). CCl4-poisoned rats develop a remarkable dissociation of liver polysomes. In animals given an equivalent dose of CBrCl3 these damages occur within a shorter lag period and are more severe. Moreover, the recovery takes place much later than when carbon tetrachloride is used.A clear inverse relationship exists between the level of bond dissociation energy of these two halogenoalkanes (respectively, 49 and 68 kcal mol−1, for CBrCl3 and CCl4) and their potency to stimulate free radical reactions and to produce liver injury.

Lipoperoxidation after carbon tetrachloride poisoning in rats previously treated with antioxidants.

Abstract Propyl gallate (PG), reduced glutathione (GSH) and N , N ′-diphenyl- p -phenylenediamine (DPPD), administered to rats prior to carbon tetrachloride, protect against hepatic fat infiltration until the fourth hour after poisoning. This effect does not seem to be mediated by a block in lipid mobilization from depot fat. A preliminary treatment with DPPD succeeds in inhibiting the double bond shifting in liver microsomal lipids within 30 min after dosing with CCl 4 . The early peroxidative alteration occurs at the normal rate after the administration of either GSH or PG. The amount of lipid-bound radiocarbon and of 14 CO 2 exhaled within 2 h after intragastric 14 C-labelled carbon tetrachloride is not affected by the preliminary protection with the antioxidants. CCl 4 metabolites and/or lipoperoxides impair the in vitro combination of serum apoprotein with lipid. No changes are observed when lipoperoxidation is inhibited by antioxidants. These findings are interpreted as a support for the hypothesis that the possible contribution given by the enhancement of lipid peroxidation to the pathogenesis of CCl 4 -induced fatty liver could depend on further structural and functional alterations occurring in the cytoplasmic environment of hepatocytes rather than the early radical attack onto the unsaturated lipids of liver microsomes. The functional integrity and the supply of the protein carrier for the triglyceride secretion mechanisms could be considered a target of the hepatotoxic action of CCl 4 , at the molecular level.

Protection by glutathione and propyl gallate on the impaired in vitro amino acid incorporation into liver microsomal protein of CCl4-poisoned rats

Abstract The endogenous mRNA directed amino acid incorporation into rat liver microsomes in vitro is lowered as soon as 30 min after CCl4 poisoning; at the same time a strong decrease in rat liver polysomes appears. Previous treatment with the watersoluble antioxidants glutathione and propyl gallate completely prevents these changes in polysome activity and structure. These results seem to indicate a direct relationship between lipoperoxidation and alteration in protein synthesis.

Effect of drug pretreatment on CBrCl3-induced liver injury☆

Abstract This study deals with the interaction of administered drugs with CBrCl3- induced toxicity. In vivo lipoperoxidation, fatty infiltration and necrosis of the liver were followed after poisoning with CBrCl3 in animals pretreated with phenobarbital, 2-diethylaminoethyl-2,2-diphenylvalerate (SKF-525A) or N, N′-diphenyl-p-phenylanediamine (DPPD). Double-bond shifting in liver microsomal lipids appeared to be enhanced by administration of barbiturate, whereas SKF-525A and DPPD lowered the extent of the peroxidative attack. Both hepatic triglyceride accumulation and leakage of liver enzymes (alanine aminotransferase [GPT] and aspartate aminotransferase [GOT]) into the plasma behaved similarly in the animals dosed orally with CBrCl3 (10 μl per 100 g body wt.). Lethality by CBrCl3 was reduced in rats pretreated with a small dose of CCl4 (25 μl per 100 g body wt.) which protects against an otherwise lethal dose of CCl4. In our opinion, this phenomenon indicates that the lethal effect of CBrCl3 also depends on the actual efficiency of the drug-metabolizing enzyme systems in the liver.

Mechanism of Protection Against Carbon Tetrachloride Toxicity. I. Prevention of Lethal Effects by Partial Surgical Hepatectomy

Both partial surgical hepatectomy and a challenge with a small dose of CCl4 depress the metabolism of xenobiotics in the liver. In fact, hepatocytes become provided with metabolic activity rates which are peculiar of either embryo or newborn rat liver. These experiments have shown that partial surgical hepatectomy prevents rats from death caused by otherwise lethal doses of CCl4. At the same time, sham-operated animals survive to a limited extent after a large dose of the halogen compound. Investigations carried out on the metabolic efficiency of liver microsomes, both in vito and in vivo, clearly demonstrate that the preventive effect against CCl4 depends mainly on the impaired metabolic activity of endoplasmic reticulum.

Toxicity of halogenated hydrocarbons in pretreated rats — an experimental model for the study of integrated permissible limits of environmental poisons

SummaryThe heavy use and the severe toxicity of several halogenocompounds render these pollutants very important agents in occupational and environmental pathology. Both the biological conditions and the environment by itself might influence their metabolism and then the degree of the resulting toxic effects.The present investigation extends the most significant parameters in toxicity studies to a number of largely diffused halogenocompounds, with special regard to a possible potentiation through the coexistence of other pollutants or by inducing treatments, such as isopropanol or phenobarbital administration.Chloroform by itself elicits toxic effects which are influenced by isopropanol challenge. Halothane and trichloroethylene appear devoid of any intrinsic poisonous action on the liver, and cause hepatic injury only in phenobarbital treated rats. In our experimental conditions, bromobenzene, fluobrene® and dichloromethane do not produce any detectable alteration in the liver. On the contrary, both isopropanol and phenobarbital render the rat particularly susceptible to the toxic effects of CCl4. In fact, treatment with alcohol reduces by two orders of magnitude the vapour concentration of the poison which causes liver necrosis and fatty infiltration, while dosing with barbiturate reduces to one tenth the effective dose of CCl4. which leads to fatty liver.These data underline the importance of the possible interaction between the halogenocompounds and the environmental factors or individual habits. Further, they clearly suggest the necessity of taking into account these experimental conditions in setting the maximum permissible limits (MAC or TLV) of halogenocompounds in the environment.

Interference of DPPD with hepatic drug metabolizing enzyme system

Summary The direct effects by in vivo administered DPPD on hepatic microsomal enzyme activities have been investigated with the aim to elucidate the mechanism of protection by this compound against toxic liver injuries. The efficiency of hepatic drug metabolizing system has been evaluated both in vitro and in vivo . Furthermore, liver necrosis (serum GPT activity) was estimated in rats poisoned with carbon tetrachloride after DPPD treatment. The administration of DPPD lowers down the activity of liver aminopyrine demethylase and enlarges the hexobarbital sleeping time. CCl 4 alone inhibits the microsomal enzymes, while a preliminary treatment with DPPD fails in preventing but renders more remarkable the block induced by the halogenoalkane. However, the antioxidant completely protects against liver necrosis in CCl 4 -poisoned rats. These findings support the hypothesis that the biological activities of DPPD can be mediated also by inhibition of drug metabolizing enzymes bound to the endoplasmic reticulum.

Involvement of capsaicin-sensitive nerves in paraquat-induced mortality

Paraquat (PQ), a broad spectrum herbicide, produces severe lung inflammation and necrosis resulting in pulmonary fibrosis and respiratory failure. Tachykinins are peptides released by sensory C fibers and have the ability of influencing respiratory functions and cellular proliferation. To examine whether the damage caused by PQ involves tachykinins, rats were depleted in their content of tachykinins by systemic treatment with capsaicin prior to PQ exposure. The animal subjected to this treatment showed a 3-fold higher viability compared to those treated with PQ alone (75 vs 27%). Depletion of reduced glutathione (GSH) is associated with oxidative stress produced by reactive oxygen intermediates during PQ metabolism. This is considered to be critical in the pathogenesis of lung damage by PQ. PQ treatment induced a significant depletion of GSH during the first days and a similar effect was also observed in the group of capsaicin-pretreated rats. Four weeks after PQ treatment the levels of GSH were similar to controls in rat pretreated or not with capsaicin plus PQ. This may indicate that the reduced levels of GSH may be associated to the toxicity observed in the acute phase, but not of importance in the final PQ-induced mortality. Neutral endopeptidase (NEP) is an enzyme considered to be critical in controlling the levels of tachykinins. Exposure of crude membrane preparations of rat lung to PQ resulted in a dose-dependent inhibition of NEP activity. Since NEP inactivation may occur in lung following a PQ exposure in vivo, the results indicate that during PQ intoxication a more sustained activity of tachykinins may be present, producing effects such as cell proliferation, fluid extravasation and bronchoconstriction. In conclusion, this finding supports the hypothesis that neuropeptides released from capsaicin-sensitive nerves could be involved in the modulation of PQ-induced lung damage.

Bromsulphalein (BSP) kinetics in the rat: a new approach in evaluating experimental hepatotoxicity.

SummaryAn animal model for the identification and definition of toxic liver damage, based on the investigation of the BSP metabolism in the rat is proposed.Different hepatotoxins can induce specific functional alteration on the different steps of the BSP hepatobiliary transport, mainly the uptake by hepatocytes and the biliary excretion.Removal curves of BSP from the plasma compartment as well as the biliary secretion were evaluated in rats treated with either α-naphthylisothiocyanate (ANIT) for metabolic cholestasis, or with carbon tetrachloride (CCl4) for fatty infiltration and necrosis of the liver.The data were compared with those obtained with untreated rats and with animals submitted either to complete or incomplete mechanically induced cholestasis.Our results lead to the conclusion that a satisfactory discrimination among different types of liver damage may be obtained when only two plasma parameters of BSP metabolism are considered: the disappearance rate for the early 5 min (K), and 15-min plasma BSP retention (R15).The model is proposed as a suitable tool for the evaluation of experimental hepatotoxicity in living rats giving a characterisation of the functional alteration and a measure of liver impairment.

Mechanism of Protection against Carbon Tetrachloride Toxicity Ii. Lethality in Rats Fed a Polyunsaturated Fatty Acid Deficient Diet

A PUFA-deficient diet causes deficiency symptoms and alters the fatty acid pattern in liver microsomal lipids. However, CCl4 lethality and sleeping time remain unchanged while the hepatic level of cytochrome P450 is only slightly lowered by the dietary regimen. In accordance, the amplitude of double bond shifting in microsomal lipids is far from being depressed in animals deprived of the peroxidative substrate. In fact, the experimental treatment does not impair intestinal absorption, liver uptake and metabolism of CCl4 given orally. Finally, both in vitro and in vivo peroxidative challenge of arachidonic acid content in hepatic microsomes causes comparable alterations of this parameter, whatever the initial fatty acid pattern following the dietary regimen. These findings provide evidence excluding an influence of the fatty acid composition of the diet on the severity of damages due to halogen-alkane exposure.