Metabolic activation of haloalkanes and tests in vitro for mutagenicity.

1. During incubation of 14CCl4, 14CHCl3, [14C]halothane, or 14CCl3F with liver microsomes and NADPH, considerable radioactivity is bound irreversibly to endoplasmic protein and lipid. However, no 14C was detected in the ribosomal RNA. 2. None of the four haloalkanes studied induced mutations after incubation with liver microsomes and the bacterial tester strains S. typhimurium TA 1535 and TA 1538. 3. Very low or no activity was associated with soluble protein or RNA added to incubation mixtures of the four haloalkanes with liver microsomes.     

Potentiation of carbon tetrachloride induced hepatotoxicity by thinner inhalation

The interaction of thinner and carbon tetrachloride (CCl4) induced hepatotoxicity was studied in the rats using the activity of plasma GOT and GPT, liver triglyceride and histopathologic changes of liver necrosis as indices. The animals were housed in a chamber with the continuous flow of thinner vapour (1.11 g/litre/hr) for 2 hrs prior to i.p. administration of CCl4 (0.1 ml/kg BW) at 18 hrs after thinner inhalation. Thinner inhalation potentiated CCl4 induced hepatotoxicity in a dose-dependent manner. The maximal enhanced effect was observed at 24 hrs after CCl4 administration by which the activities of PGOT and PGPT were significantly increased (3 folds). Thinner itself caused an additive effect on CCl4 induced liver triglyceride accumulation. At 18 hrs after thinner inhalation, the activity of NADPH cytochrome C reductase was markedly increased (2.2 folds) but no change in the activity of aminopyrine N-demethylase which was able to increase the 14.CCl3 free radicals and binding to both the hepatic microsomal proteins (1.8 folds) and lipids (1.4 folds). In addition, thinner pretreatment somehow increased hepatic lipid peroxidation by 1.4 folds. These results suggest that thinner pretreatment causes an increase in mixed function oxidases to activate the formation of .CCl3 free radicals and binding to the microsomal proteins and lipids, which in turn stimulate hepatic damage via lipid peroxidation in the membrane.     

Irreversible binding of 3-14C-antipyrine to hepatic protein in vivo and in metabolizing liver microsomes

Summary After i.p. injection of 3-¹⁴C-antipyrine (10 mole=1.9 mg with 10 Ci per 10 g of body weight) to mice radioactivity was irreversibly bound to liver proteins. The irreversible binding reached maximal values of 0.15 nmole/mg protein in liver microsomes after 30–60 min.During 60 min incubation with liver microsomes of mice and rabbits (phenobarbital pretreated) and a NADPH-regenerating system 3-¹⁴C-antipyrine was irreversibly bound to microsomal protein at a rate of 1.5 nmole/mg protein (mouse) and 3 nmole/mg protein (rabbit).In identical incubates with rabbit liver microsomes the 4-hydroxylation of antipyrine was 24 nmole/mg protein in 60 min and formaldehyde production from antipyrine 3 nmole/mg protein in 60 min.In incubates with rabbit liver microsomes the binding rate was 80–90% inhibited by 1mM metyrapone, SKF 525-A and trichloropropene epoxide respectively; 4-hydroxylation was 70–80% inhibited by the same substances. In the presence of 1 mM GSH, cysteine or ethylene diamine binding was 30–40% inhibited, whereas 4-hydroxylation showed no inhibition.Some of the results were presented at the 17th Spring Meeting of the Deutsche Pharmakologische Gesellschaft, Mainz, March 1976     

Mechanism of chloroform nephrotoxicity. III. Renal and hepatic microsomal metabolism of chloroform in mice

In vitro studies with male ICR mouse renal cortical slices have indicated that chloroform (CHCl3) is metabolized by the kidney to a nephrotoxic intermediate, possibly by a cytochrome P-450-dependent mechanism similar to that occurring in the liver. In this investigation, metabolism of 14CHCl3 by microsomes prepared from renal cortex and liver provided definitive evidence for a role of cytochrome P-450 in the renal metabolism and toxicity of CHCl3. 14CHCl3 was metabolized to 14CO2 and covalently bound radioactivity by male renal cortical microsomes; metabolism required oxygen, a NADPH regenerating system, was dependent on incubation time, microsomal protein concentration, and substrate concentration, and was inhibited by carbon monoxide. Consistent with the absence of CHCl3 nephrotoxicity in female mice, little or no metabolism of 14CHCl3 by female renal cortical microsomes was detected. CHCl3 produced a type I binding spectrum with oxidized male renal cortical and hepatic microsomes. Incubation of glutathione with microsomes and 14CHCl3 increased the amount of aqueous soluble metabolites detected with a concomitant decrease of metabolism to 14CO2 and covalently bound radioactivity, suggesting the formation of a phosgene conjugate as has been described for hepatic CHCl3 metabolism. These data support the hypothesis that renal cytochrome P-450 metabolizes CHCl3 to a nephrotoxic intermediate.     

Metabolic activation of the tricyclic antidepressant amineptine--I. Cytochrome P-450-mediated in vitro covalent binding

Incubation of [14C]amineptine (1 mM) with hamster liver microsomes resulted in the irreversible binding of an amineptine metabolite to microsomal proteins. Covalent binding measured in the presence of various concentrations of amineptine (0.0625-1 mM) followed Michaelis-Menten kinetics. Pretreatment with phenobarbital increased not only the Vmax, but also the Km, for this binding. Covalent binding required NADPH and molecular oxygen and was decreased when the incubation was made in the presence of inhibitors of cytochrome P-450 such as piperonyl butoxide (4 mM), SKF 525-A (4 mM) or carbon monoxide (80:20 CO-O2 atmosphere). In contrast, binding was increased when microsomes from untreated hamsters were incubated in the presence of 0.5 mM 1,1,1-trichloropropene 2,3-oxide, an inhibitor of epoxide hydrolase. Metabolic activation also occurred in kidney microsomes. In vitro covalent binding to kidney microsomal proteins required NADPH and was decreased by piperonyl butoxide (4 mM) but was not increased by pretreatment with phenobarbital. We conclude that amineptine is activated by hamster liver and kidney microsomes into a chemically reactive metabolite that covalently binds to microsomal proteins.     

Protective effects of the fractions extracted from the callus of Acer nikoense Maxim. on carbon tetrachloride induced liver injury]

The decoctions extracted from the bark and leaf of Acer nikoense Maxim. (AN) have been used as a folk medicine for eye-wash and hepatic disease. As previously reported, the methanol extract from the bark of AN and the fractions of the methanol extract have protective effects for liver injury induced by carbon tetrachloride (CCl4) in rats. In this study, protective effects of the fractions extracted from the callus of AN were investigated on CCl4-induced liver injury in rat. The active principles for the protection of CCl4-induced liver injury were recognized in a fraction (EF 3-3) obtained by using silica gel chromatography (solvent: CHCl3-MeOH-H2O). The components were further fractionated by silica gel chromatography followed by gel filtration (solvent: CHCl3-MeOH). In addition, the mechanism of the protective action against liver injury induced by CCl4 was also examined. The fractions with protective effects in vivo showed the inhibitory effects on CCl4-dependent lipid peroxidation in microsomal fraction in vitro.     

Biochemical mechanism of metallothionein--carbon tetrachloride interaction in vitro

To elucidate the mechanism underlying the protective effect of metallothionein (MT) against carbon tetrachloride (CCl4) toxicity, in vitro experiments were carried out to study the interaction of metallothionein and CCl4. Results from this study showed that incubation of Cd,Zn-MT with CCl4 in the presence of hepatic microsomes and NADPH resulted in a time-dependent depletion of MT thiols with a concurrent reduction in the metal-binding sites of the protein. Moreover, this reaction also released Zn and Cd from MT. Results from experiments conducted to determine whether or not the CCl4-induced decrease in MT-thiol content was due to the scavenging of CCl4 metabolite(s) showed that the trichloromethyl radical, chloroform and phosgene as well as the products of CCl4-induced microsomal lipid peroxidation were not directly involved. Although covalent binding of 14CCl4 to MT was detected following incubation in the presence of a microsomal bioactivation system, it did not account for the CCl4-induced loss of MT thiol groups for the following reasons: (i) prior oxidation of sulfhydryl groups of MT by hydrogen peroxide did not alter the binding; and (ii) anaerobiosis did not alter the extent of covalent binding but obliterated the inhibitory effect of CCl4 on MT thiol content. Measurement of the thiol content of CCl4-treated MT after treatment with 1,4-dithiothreitol revealed that all the thiol groups that were lost subsequent to CCl4 treatment could be regenerated. These data suggest that CCl4-linked oxidation of MT, rather than the covalent binding of 14CCl4 metabolite(s), may be responsible for the CCl4-induced loss of metal binding sites of MT with the concurrent release of Zn and Cd. However, the precise role of the metal released during the oxidation of MT in CCl4 toxicity remains to be defined.     

Irreversible binding of 14C-labelled trichloroethylene to mice liver constituents in vivo and in vitro

1. ¹⁴C-labelled trichloroethylene was injected i.p. into male mice (10 μmole/g of b.w.). The radioactivity irreversibly bound to hepatic protein reached highest levels after 6 h: 2 nmole/mg in cytosol protein, 4.4 nmole/mg in mitochondrial protein, and 7.6 nmole/mg in microsomal protein.
2. The commercial trichloroethylene contained radioactive impurities binding to proteins without metabolic activation. Purification by various extractions removed 60–70% of those materials. In aerobic incubates of mice hepatic microsomes and NADPH the covalent binding rate of the purified trichloroethylene was 1.4 nmole/mg protein in 60 min. The activity of rat liver microsomes was approximately 40% less. Covalent binding increased 2-fold with microsomes of mice pretreated with phenobarbital.

     

Irreversible protein binding of acrylonitrile

1. After i.p. injection of [2,3-14C]acrylonitrile to rats, a significant portion of radioactivity becomes irreversibly attached to proteins of liver, lung, spleen and other tissues. 2. When rat liver microsomes were incubated with [2,3-14C]acrylonitrile, a time-dependent irreversible binding of radioactivity occurred to microsomal proteins. This binding was not dependent on NADPH. A high extent of binding to heat-inactivated microsomes indicated that no enzymic metabolic step was involved. 3. The irreversible binding of [2,3-14C]acrylonitrile to rat liver microsomal protein in vitro was inhibited by thiols (cysteine, glutathione, mercaptoethanol). The greatest inhibitory potency was displayed by dithiocarb (diethyl dithiocarbamate).     

The mechanism of the suicidal, reductive inactivation of microsomal cytochrome P-450 by carbon tetrachloride

1. Stoichiometric losses of microsomal haem and cytochrome P-450 were observed when carbon tetrachloride (CCl4) was incubated anaerobically with rat liver microsomes using NADPH or sodium dithionite as a reducing agent. A rapid destruction of haem was also observed during the non-enzymatic reductive incubation of CCl4 with soluble haem preparations (methaemalbumin) in presence of sodium dithionite. The results indicate that haem is both the site and the target of the suicidal activation of CCl4 by cytochrome P-450. 2. When an additional, fluorimetric assay for haem determination was used, an equimolar loss of protoporphyrin IX fluorescence was also observed in both the enzymatic and non-enzymatic system, indicating that the haem moiety of cytochrome P-450 has undergone a structural change, involving either loss or labilization of the porphyrin tetrapyrrolic structure. In both systems the loss of porphyrin was prevented by carbon monoxide (CO). 3. A dichlorocarbene-cytochrome P-450 ligand complex is partially responsible for the difference spectrum obtained on addition of CCl4 to anaerobically reduced rat liver microsomes. A molar extinction coefficient for this complex has been calculated. The carbene trapping agent 2,3-dimethyl-2-butene (DMB) strongly inhibited (greater than 95%) the formation of this spectrum but did not modify the loss of haem in reduced CCl4-supplemented microsomal incubations. The results suggest that dichlorocarbene (:CCl2) is not significantly involved in CCl4-dependent haem destruction. 4. Pretreatment of rats with different microsomal enzyme inducers was responsible for similar but not identical patterns of :CCl2 and CO formation and haem loss during incubation of CCl4 with reduced microsomes. This indicates a critical role of CCl4 metabolism in the suicidal destruction of cytochrome P-450 haem and suggests that the apoprotein of cytochrome P-450 is capable of modulating not only the metabolism of CCl4 to :CCl2 but also the hydrolysis of :CCl2 to CO. 5. Inactivation of cytochrome P-450 by CCl4 with reduced microsomes from Aroclor-pretreated rats was saturable and followed pseudo first-order kinetics. This provides further evidence to conclude that CCl4 activation is a suicidal process where the reactive metabolite(s) formed bind to haem, we predict, in a one to one stoichiometry. 6. The partition ratio between loss of cytochrome P-450 haem and CCl4 metabolism by liver microsomes from Aroclor pretreated rats has been investigated using limiting concentrations of CCl4. It was calculated that approximately 26 molecules of CCl4 had to be metabolised to achieve the loss of one molecule of haem.     

Metabolic activation of 2,4-diaminoanisole, a hair-dye component--II. Role of cytochrome P-450 metabolism in irreversible binding in vitro.

Incubation of rat liver microsomes with radiolabeled 2,4-diaminoanisole (2,4-DAA) in the presence of NADPH and oxygen led to the formation of irreversibly bound products to microsomal protein. The binding was inhibited by a CO:O₂ atmosphere and by an antibody against NADPH cytochrome c reductase. In vivo and in vitro inhibitors of cytochrome P-450 decreased the binding and phenobarbital-pretreatment increased binding, whereas β-napthoflavone-pretreatment was without effect. Binding of ring-labeled 2,4-DAA was much higher than with methyl-labeled-2,4-DAA. Experiments with [³H]-ring-and [¹⁴C]-ring-labeled-2,4-DAA indicated some loss of tritium; this was confirmed by isolation of labile tritium. Substitution of the hydrogens in the methyl group with deuterium led to increases in both binding and mutagenicity of 2,4-DAA. Formation of formaldehyde and a small amount of methanol could be demonstrated during the oxidative metabolism of methyl-labeled-2,4-DAA. Addition of superoxide dismutase and ascorbic acid inhibited binding, and a small amount of irreversible binding could be demonstrated when NADPH was replaced by a xanthine-xanthine oxidase system. Microsomes from rat kidneys also activated 2.4-DAA in the presence of NADPH. Thin-layer chromatography revealed that 30–40 per cent of 2.4-DAA was oxidized during 10 min of incubation with liver microsomes. And a tentative scheme involving aromatic hydroxylation, oxidative demethylation and N-hydroxylation for the microsomal metabolism of 2,4-DAA is presented. Irreversible binding could also be shown with liver microsomal RNA in vitro, whereas no binding to exogenously added DNA could be found.     

Liver microsomal uptake of (14C)vinyl chloride and transformation to protein alkylating metabolites in vitro.

[1,2-¹⁴C]Vinyl chloride gas was incubated with rat liver microsomes in an all-glass vacuum system. Microsomal uptake and irreversible protein binding of vinyl chloride radioactivity was determined. Both uptake of vinyl chloride by microsomes and alkylation of proteins by vinyl chloride metabolites were dependent on incubation time, enzymatically active microsomes, NADPH, oxygen, and the partial pressure of vinyl chloride in the atmosphere, and could be inhibited by carbon monoxide. During incubation in presence of NADPH, 10 times more vinyl chloride was taken up by microsomes than in absence of NADPH. Uptake of vinyl chloride by albumin solutions and liposomal suspensions was in a similar range compared to the microsomal uptake without NADPH. Addition of glutathione and cytoplasmic fractions to microsomal incubations with NADPH led to an increase in microsomal uptake of vinyl chloride and to a decrease in protein alkylation by vinyl chloride metabolites. If trichloropropene oxide was present in the microsomal incubation, the protein alkylation reaction by vinyl chloride metabolites was increased twofold, while the microsomal uptake of vinyl chloride was not influenced. Our results are consistent with the view that the microsomal uptake of vinyl chloride radioactivity is due to transformation of vinyl chloride gas to nonvolatile metabolites by microsomal enzymes and that chloroethylene oxide might be the primary microsomal metabolite of vinyl chloride capable of reacting with proteins.     

Metabolism-dependent covalent binding of (S)-[5-3H]nicotine to liver and lung microsomal macromolecules

Incubation of (S)-[5-3H]nicotine with rabbit liver microsomes in the presence of dioxygen and NADPH results in the formation of metabolites that bind covalently to microsomal macromolecules (250-550 pmol/mg of protein/hr). The partition ratio [(S)-nicotine metabolized/(S)-nicotine equivalents covalently bound] ranged between 250:1 and 500:1. The addition of SKF 525-A, cytochrome c, or n-octylamine inhibited both (S)-nicotine metabolism and covalent binding whereas phenobarbital pretreatment increased the rates of metabolism and covalent binding. Sodium cyanide, which forms stable adducts with the cytochrome P-450-generated iminium ion metabolites of (S)-nicotine and a variety of other tertiary amines, inhibited covalent binding but also decreased the rate of (S)-nicotine metabolism. The metabolism-dependent covalent binding of (S)-nicotine and its conversion to the delta 1',5'-iminium species were observed also in microsomal incubations prepared from rabbit lung and human liver tissues.     

Comparison of covalent binding from halothane metabolism in hepatic microsomes from phenobarbital-induced and hyperthyroid rats

1. Hepatic microsomal suspensions from rats pretreated with saline, phenobarbital or triiodothyronine were incubated with 14C-halothane under aerobic and anerobic conditions. 2. Metabolism of halothane by microsomes from phenobarbital-induced rats under anaerobic conditions resulted in covalent binding of 14C to microsomal lipids, and to a lesser extent, microsomal proteins, as seen in previous studies. Covalent binding was decreased with incubation under aerobic conditions. 3. Metabolism of halothane by microsomal suspensions from hyperthyroid rats produced much less covalent binding to microsomal lipids and proteins, with binding similar to, or less than, that observed with microsomes from saline-treated rats. The covalent binding of halothane to protein of microsomes from hyperthyroid rats was dependent upon metabolism, and was inhibited by SKF 525A, reduced glutathione, or cytosol. 4. The in vitro observations with respect to covalent binding are inconsistent with previous reports on halothane hepatotoxicity in hyperthyroid rats in vivo. This inconsistency and the relatively small extent of covalent binding with microsomes from hyperthyroid rats observed, suggests that covalent binding is not an important mechanism of halothane hepatotoxicity in the hyperthyroid rat model.     

Protective action of diethyldithiocarbamate and carbon disulfide against renal injury induced by chloroform in mice

Oral administration of diethyldithiocarbamate (DTC) and carbon disulfide (CS2) protected mice against CHCl3-induced kidney injury, as evidenced by normalization of delayed plasma phenolsulfonphthalein clearance, suppression of increased kidney calcium content and prevention of renal tubular necrosis. In CCl4-treated mice, in which liver microsomal monooxygenase activities were decreased markedly, and kidney microsomal aniline hydroxylase and p-nitroanisole demethylase activities were increased to about twice those of the untreated mice, renal toxicity of CHCl3 was greatly potentiated, and the latter effect was also blocked by both agents. DTC and CS2 per se markedly decreased kidney microsomal aniline hydroxylase and p-nitroanisole demethylase activities at 1 hr after oral administration, accompanying a moderate loss of cytochrome P-450 content, in both normal and CCl4-treated mice. The protection was not due to hypothermia, because pretreatment with DTC or CS2 (p.o.) also prevented the hypothermia induced by CHCl3. The mechanism of the protection may have involved inhibition of metabolic activation of CHCl3 in the kidney rather than in the liver.     

Phenobarbital pretreatment alters the localization of CCl4-induced changes in rat liver microsomal fatty acids

Phenobarbital treatment induces an isozyme(s) of liver microsomal cytochrome P450 susceptible to CCl4 and enhances the latter's lethality. We have now studied phenobarbital's effect on the specificity of phosphatidyl fatty acid changes in rat liver microsomes. Male Sprague-Dawley rats were pretreated with three daily ip doses of phenobarbital (50 mg/kg) or saline and then orally dosed with CCl4 (2.5 ml/kg). Liver microsomes were prepared 7.5 to 180 min after CCl4 treatment, the lipid fraction was extracted, diene conjugate content was determined, and phospholipids were separated by HPLC for fatty acid content determination. Protein, phospholipid, and phosphatidyl fatty acid residue loss occurred early (7.5 to 30 min) and in some cases later (60 to 180 min) in both pretreated groups, suggesting that two phases of CCl4-mediated injury occurred. Phenobarbital pretreatment accelerated the CCl4-induced formation of diene conjugates in the microsomal lipids. In studies on the separated phospholipids, phenobarbital alone altered microsomal fatty acid content, primarily decreasing arachidonic acid in favor of linoleate, particularly in phosphatidylserine. During the early phase of CCl4 injury, phenobarbital pretreatment shifted the major loss of arachidonic acid from phosphatidylserine to phosphatidylethanolamine. During the later phase, arachidonic acid loss was still prominent, but the most extensive CCl4-induced changes in fatty acids occurred in the neutral lipid fraction, regardless of pretreatment. These changes included loss of neutral lipid linoleic and docosahexanoic acids associated with an increase in palmitic acid. These data demonstrate that phenobarbital pretreatment is associated with a shift in the predominant phospholipid locus from phosphatidylserine to phosphatidylethanolamine for the early CCl4-induced fatty acid changes in rat liver microsomes.     

Metabolism of 3-methylindole in human tissues.

Bioactivation of 3-methylindole (3MI), a highly selective pneumotoxin in goats, was investigated in human lung and liver tissues in order to provide information about the susceptibility of humans to 3MI toxicity. Human lung microsomes were prepared from eight organ transplantation donors and liver microsomes from one of the donors were utilized. The 3MI turnover rate with human lung microsomes was 0.23 +/- 0.06 nmol/mg/min, which was lower than the rate with the human liver microsomes (7.40 nmol/mg/min). The activities were NADPH dependent and inhibited by 1-aminobenzotriazole, a potent cytochrome P-450 suicide substrate inhibitor. Covalent binding of 3MI reactive intermediates to human tissues was determined by incubation of 14C-3MI and NADPH with human lung and liver microsomal proteins. Although human lung microsomes displayed measurable covalent binding activity (2.74 +/- 2.57 pmol/mg/min), the magnitude of this reaction was only 4% as large as that seen with human liver microsomes (62.02 pmol/mg/min). However, the covalent binding was protein dependent and also was inhibited by 1-aminobenzotriazole. Therefore, the bioactivation of 3MI to covalently binding intermediates is catalyzed by cytochrome P-450 in human pulmonary tissues. These activities were compared to those activities measured with tissues from goats. Proteins from goat and human pulmonary and hepatic microsomal incubations were incubated with radioactive 3MI, and radioactive proteins were analyzed by SDS-PAGE and HPLC and visualized by autoradiography and radiochromatography, respectively. The results showed that a 57-kDa protein was clearly the most prominently alkylated target associated with 3MI reactive intermediates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Suicidal inactivation of human cytochrome P-450 by carbon tetrachloride and halothane in vitro.

A significant loss of human cytochrome P-450 was observed during the anaerobic incubation of NADPH-reduced human liver microsomes obtained from surgical samples, in presence of carbon tetrachloride or halothane. In order to prevent any interference in the classical spectrum of cytochrome P-450 with CO, the method of Johannesen & DePierre (1978) was modified to obtain cytochrome P-450 determination. The enzyme inactivation reaction showed pseudo-first order kinetics and was accompanied by destruction of the haem tetrapyrrolic structure, as indicated by a significant loss of its porphyrin fluorescence. Values of about 200 and 700 were calculated for the partition ratio between metabolic turnover of the substrate and enzyme inactivation during reductive incubation of one of these microsomal preparations with limiting concentrations of CCl4 and halothane, respectively. The results indicate that human liver cytochrome P-450 can be inactivated reductively in vitro by CCl4 and halothane reactive metabolites and suggest that a suicide type of mechanism, similar to that which was recently demonstrated to occur, for both substrates, with rat liver microsomes (Manno et al. 1988a & 1991), may also be involved in the inactivation of the human enzyme(s).     

Mechanism of antihepatotoxic activity of atractylon, I: effect on free radical generation and lipid peroxidation1.

The mechanism of antihepatotoxic action of atractylon, a main sesquiterpenic constituent of ATRACTYLODES rhizomes, was studied. Atractylon inhibited carbon tetrachloride (CCl (4))-induced cytotoxicity in primary cultured rat hepatocytes and CCl (4)-induced lipid peroxidation by rat liver microsomes. However, atractylon increased the free radical generation by CCl (4) with rat liver microsomes in the presence of a radical trapping agent, phenyl T-butyl nitrone (PBN). In addition, atractylon generated free radical PER SE. Experiments using (13)CCl (4) instead of CCl (4) indicated that the increased free radicals consisted of those from (13)CCl (4) and from atractylon. Accumulated data support that although both CCl (4) and atractylon generate free radicals respectively by rat liver microsomes, free radical from CCl (4) conducts lipid peroxidation and produces liver lesion, while atractylon forms free radical which scavenges CCl (3) radical in the absence of PBN, inhibits lipid peroxidation by CCl (4) and suppresses CCl (4)-induced liver lesion.