Carbon tetrachloride toxicity as a model for studying free-radical mediated liver injury

A single dose of CCl4 when administered to a rat produces centrilobular necrosis and fatty degeneration of the liver. These hepatotoxic effects of CCl4 are dependent upon its metabolic activation in the liver endoplasmic reticulum to reactive intermediates, including the trichloromethyl free radical. Positive identification of the formation of this free radical in vivo, in isolated liver cells and in microsomal suspensions in vitro has been achieved by e.s.r. spin-trapping techniques. The trichloromethyl radical has been found to be relatively unreactive in comparison with the secondarily derived peroxy radical CCl3O2., although each free radical species contributes significantly to the biological disturbances that occur. Major early perturbations produced to liver endoplasmic reticulum by exposure in vivo or in vitro to CCl4 include covalent binding and lipid peroxidation; studies of these processes occurring during CCl4 intoxication have uncovered a number of concepts of general relevance to free-radical mediated tissue injury. Lipid peroxidation produces a variety of substances that have high biological activities, including effects on cell division; many liver tumours have a much reduced rate of lipid peroxidation compared with normal liver. A discussion of this rather general feature of liver tumours is given in relation to the liver cell division that follows partial hepatectomy.     

Mechanism of carbon tetrachloride-induced hepatotoxicity. Hepatocellular damage by reactive carbon tetrachloride metabolites

CCl4-induced liver damage was modeled in monolayer cultures of rat primary hepatocytes with a focus on involvement of covalent binding of CCl4 metabolites to cell components and/or peroxidative damage as the cause of injury. (1) Covalent binding of 14C-labeled metabolites was detected in hepatocytes immediately after exposure to CCl4. (2) Low oxygen partial pressure increased the reductive metabolism of CCl4 and thus covalent binding. (3) [14C]-CCl4 was bound to lipids and to proteins throughout subcellular fractions. Binding occurred preferentially to triacylglycerols and phospholipids, with phosphatidylcholine containing the highest amount of label. (4) The lipid peroxidation potency of CCl4 revealed subtle differences compared to other peroxidative substances, viz., ADP-Fe3+ and cumol hydroperoxide, respectively. (5) CCl4, but not the other peroxidative substances, decreased the rate of triacylglycerol secretion as very low density lipoproteins. (6) The anti-oxidant vitamin E (alpha-tocopherol) blocked lipid peroxidation, but not covalent binding, and secretion of lipoproteins remained inhibited. (7) The radical scavenger piperonyl butoxide prevented CCl4-induced lipid peroxidation as well as covalent binding of CCl4 metabolites to cell components, and also restored lipoprotein metabolism. The results confirm that covalent binding of the CCl3* radical to cell components initiates the inhibition of lipoprotein secretion and thus steatosis, whereas reaction with oxygen, to form CCl3-OO*, initiates lipid peroxidation. The two processes are independent of each other, and the extent to which either process occurs depends on partial oxygen pressure. The former process may result in adduct formation and, ultimately, cancer initiation, whereas the latter results in loss of calcium homeostasis and, ultimately, apoptosis and cell death.     

Administration of the tris salt of alpha-tocopheryl hemisuccinate inactivates CYP2E1, enhances microsomal alpha-tocopherol levels and protects against carbon tetrachloride-induced hepatotoxicity

A series of tocopherol compounds were examined for their capacity to protect against carbon tetrachloride (CCl4)-induced hepatotoxicity in rats. Of the tocopherol compounds tested in our study, only the tris salt of d-alpha-tocopheryl hemisuccinate (TS-tris) protected against CCl4-induced hepatotoxicity. The administration of d-alpha-tocopherol (alpha-T) and the nonhydrolyzable tocopherol ether, d-alpha-tocopheryloxybutyrate tris salt (TSE-tris), failed to protect against CCl4-induced hepatotoxicity. TS-tris was the only tocopherol which significantly decreased CYP2E1 activity after 18 h. This decrease in CYP2E1 activity is likely to limit the activation of CCl4 and protect against CCl4-induced hepatotoxicity. Our results also suggest that TS-tris protection against CCl4-induced hepatotoxicity correlates with the enhanced capacity of TS-tris to deliver alpha-T and increase the antioxidant status of hepatocytes. TSE-tris did not increase cellular alpha-T levels, while administration of TS-tris produced large increases in alpha-T levels in liver homogenates as well as in liver nuclei, microsomes, mitochondria and plasma membranes. This enhanced ability to deliver tocopherol equivalents to parenchymal liver cells may be related in part to the ability of TS-tris to form liposomes in aqueous solutions. TS-tris administration protected against CCl4-induced microsomal lipid peroxide formation and inactivation of the microsomal enzyme glucose-6-phosphatase (G6Pase). Supplementation of animals with alpha-T protected against microsomal lipid peroxide formation but not against the inactivation of G6Pase. Based on our findings, we propose that high cellular levels of alpha-T protect against CCl4-induced hepatotoxicity by scavenging CCl4 radicals as well as protecting against lipid peroxidation. Our results do not support the importance of microsomal lipid peroxidation as an early event in acute CCl4-induced hepatic necrosis.     

The role of lipid peroxidation in CCl4-induced damage to liver microsomal enzymes: comparative studies in vitro using microsomes and isolated liver cells

The question as to whether CCl4 decreases the activities of glucose-6-phosphatase and cytochrome P-450 in liver endoplasmic reticulum mainly through its action in stimulating lipid peroxidation has been investigated using Promethazine to block lipid peroxidation. The investigation, moreover, has compared the effects of CCl4, with and without Promethazine, on isolated rat hepatocytes with corresponding effects on rat liver microsomal suspensions. Our data give no support for the view that products of lipid peroxidation are the main cause of the decrease in cytochrome P-450 observed in CCl4-intoxication. However, our present results are consistent with lipid peroxidation being a major contributory factor to the decrease in glucose-6-phosphatase activity observed in CCl4-induced liver injury.     

Lipid peroxidation in purified plasma membrane fractions of rat liver in relation to the hepatoxicity of carbon tetrachloride

Preparations of rat liver sinusoidal plasma membrane have been tested for their ability to metabolize the hepatotoxin carbon tetrachloride (CCl4) to reactive free radicals in vitro and compared in this respect with standard preparations of rat liver microsomes. The sinusoidal plasma membranes were relatively free of endoplasmic reticulum-associated activities such as the enzymes of the cytochrome P450 system and glucose-6-phosphatase. CCl4 metabolism was measured as (i) covalent binding of [14C]-CCl4 to membrane protein, (ii) electron spin resonance spin-trapping of CCl3. radicals and (iii) CCl4-induced lipid peroxidation. By all of these tests, purified sinusoidal plasma membranes were found unable to metabolize CCl4. The fatty acid composition of the plasma membranes was almost identical to that of the microsomal preparation and both membrane fractions exhibited similar rates of the lipid peroxidation that was stimulated non-enzymically by gamma-radiation or incubation with ascorbate and iron. The absence of CCl4-induced lipid peroxidation in the plasma membranes seems to be due, therefore, to an absence of CCl4 activation rather than an inherent resistance to lipid peroxidation. We conclude that damage to the hepatocyte plasma membrane during CCl4 intoxication is not due to a significant local activation of CCl4 to CCl3. within that membrane.     

Correlations between common tests for assessment of liver damage: indices of the hepatoprotective activity of promethazine in carbon tetrachloride hepatotoxicity

The effects of promethazine (PM) on different aspects of the hepatotoxic action of CCl4 in the rat were investigated with the objective of finding rapid and reliable indicators of hepatoprotective effects. The study was based on definitive histological assessment of liver damage caused by CCl4 in the presence and absence of PM: PM (78 mumol kg-1, i.p.) protected against CCl4-induced hepatic necrosis 24 h after a low dose of CCl4 (1.3 mmol kg-1) but not against a higher dose (13.0 mmol kg-1). The large increases in plasma activities of GOT, GPT and LDH produced by dosing with CCl4 were partially inhibited by the administration of PM. PM and CCl4 caused a synergistic and long-lasting decrease in body temperature (2-3 degrees C for 8-10 h). Modifying the toxicity with PM, together with a low dose of CCl4, helped to minimize secondary effects of CCl4, to clarify the sequence of toxic events, and to assess the sensitivity of some standard tests of hepatotoxicity. Simultaneous measurement of over 20 commonly used biochemical screening tests in individual animals 3 or 6 h after treatment permitted direct correlation of a wide variety of concentrations, activities and effects. For example, liver CHCl3 concentrations (as a measure of CCl4 metabolism) correlate strongly with increases in diene conjugation of microsomal lipids (as a measure of CCl4-induced lipid peroxidation); malonaldehyde production appears to be less sensitive as a measure of lipid peroxidation in vivo than diene conjugation. The changes induced in each parameter and the correlations between them are discussed with reference to the overall nature of the hepatotoxic reaction and its modification by PM.     

Separation and characterization of the aldehydic products of lipid peroxidation stimulated by carbon tetrachloride or ADP-iron in isolated rat hepatocytes and rat liver microsomal suspensions.

Carbonyl products were separated and identified in suspensions of rat liver microsomal fractions and in isolated hepatocytes, after stimulation of lipid peroxidation by incubation with the pro-oxidants CCl4 and ADP-iron. The carbonyl products were allowed to react with 2,4-dinitrophenylhydrazine, and the derivatives were extracted and separated by t.l.c. into three zones of non-polar materials, and one fraction of polar derivatives that remained at the origin. Separation of the individual non-polar hydrazones in each zone by h.p.l.c. demonstrated that zone I prepared from microsomal fraction or hepatocytes incubated with CCl4 or ADP-iron contained mainly 4-hydroxyhex-2-enal, 4-hydroxynon-2-enal and 4-hydroxynona-2,5-dienal. Zone III consisted mainly of the alkanals propanal, pentanal and hexanal, the 2-alkenals propenal, pent-2-enal, hex-2-enal, hept-2-enal, oct-2-enal and non-2-enal, the ketones butanone, pentan-2-one and pentan-3-one, and deca-2,4-dienal. Incubation of a microsomal fraction with ADP-iron was much more effective in producing malonaldehyde and other carbonyl products than an incubation with CCl4. Despite such quantitative differences, there were no obvious qualitative differences in the h.p.l.c. spectra obtained from zones I and III. However, the stoichiometric evaluation of fatty acid loss and the production of malonaldehyde and other carbonyls suggests that the pathways of lipid peroxidation triggered by CCl4 and ADP-iron are different. The accumulation of carbonyl products of lipid peroxidation in isolated hepatocytes is strongly affected by their metabolism; in particular, 4-hydroxyalkenals were found to be metabolized very rapidly. Nonetheless, both CCl4 and ADP-iron produced stimulation in the production of malonaldehyde and non-polar carbonyl production. After incubation of rat hepatocytes with CCl4 or ADP-iron it was found that approx. 50% of the total amount of non-polar carbonyls produced during incubation escaped into the external medium. This was not leakage from dead cells, as 90-95% of the hepatocytes had retained their integrity at the end of the incubation. Release of carbonyl products from cells stimulated to undergo lipid peroxidation may be a mechanism for spreading an initial intracellular disturbance to affect critical targets outside the parent cell.     

Lipid peroxidation and covalent binding in the early functional impairment of liver Golgi apparatus by carbon tetrachloride

The onset of the lipoprotein secretory block provoked by CCl4 in the whole animal was monitored after purification of liver Golgi membranes. Both lipid transit through the apparatus and hexosylation of the lipoprotein are markedly inhibited 5-15 min after poisoning. Pre-treating the animal with alpha-tocopherol, shown to prevent lipid peroxidation without modifying the covalent binding due to CCl4 metabolites, affords little protection against lipid accumulation in the Golgi, but total preservation of galactosyl transferase activity. While haloalkylation therefore appears to be the major mechanism of damage in the early phases of CCl4-induced derangement of lipid secretion, lipid peroxidation is probably more involved later; this is indicated by the marked, though never complete, protection against fatty liver afforded at 24 h after CCl4 poisoning by supplementation of the membrane with alpha-tocopherol.     

Hepatoprotective and antioxidant effects of Morus bombycis Koidzumi on CCl4-induced liver damage

The antioxidant activity and liver protective effect of Morus bombycis Koidzumi were investigated. Aqueous extracts of M. bombycis Koidzumi had higher superoxide radical scavenging activity than other types of extracts. The aqueous extract at a dose of 100 mg/kg showed significant hepatoprotective activity when compared with that of a standard agent. The biochemical results were confirmed by histological observations indicating that M. bombycis Koidzumi extract together with CCl(4) treatment decreased ballooning degeneration. The water extract recovered the CCl(4)-induced liver injury and showed antioxidant effects in assays of FeCl(2)-ascorbic acid-induced lipid peroxidation in rats. Based on these results, we suggest that the hepatoprotective effect of the M. bombycis Koidzumi extract is related to its antioxidative activity.     

Free radical scavenging action of the medicinal herb Limonium wrightii from the Okinawa islands

Free radical scavenging action of Limonium wrightii O. kunthe was examined in vitro and in vivo by using electron spin resonance spectrometer and chemiluminescence analyzer. A water extract of L. wrightii showed a strong scavenging action for the 1,1-diphenyl-2-picrylhydrazyl, or superoxide anion and moderate for hydroxyl radical. The extract also depressed production of reactive oxygen species from polymorphonuclear leukocytes stimulated by phorbor-12-mysistate acetate and inhibited lipid peroxidation in rat liver microsomes. When the extract was given intraperitoneally to mice prior to carbon tetrachloride (CCl4) treatment, CCl4-induced liver toxicity, as seen by an elevation of serum aspartate aminotransferase and alanine aminotransferase activities, was significantly reduced. Gallic acid was identified as the active component of L. wrightii with a strong free radical scavenging action. Our results demonstrate the free radical scavenging action of L. wrightii and that gallic acid contributes to these actions.     

Inhibition of carbon tetrachloride-induced lipid peroxidation by novel antioxidants in rat hepatic microsomes: dissociation from hepatoprotective effects in vivo

The ability of two novel antioxidants, U-74,006F and U-78,517G, as well as the known antioxidant N,N'-diphenyl-p-phenylenediamine to inhibit lipid peroxidation induced by carbon tetrachloride (CCl4) was investigated in Aroclor 1254-induced rat hepatic microsomes. All three compounds completely inhibited lipid peroxidation in microsomes as measured by the formation of thiobarbituric acid reactive substances (TBARS). Inhibition of lipid peroxidation was not a function of decreased bioactivation of CCl4, as the compounds did not substantially inhibit benzphetamine N-demethylase activity or covalent binding of [14-C]CCl4 to lipid or protein. Parallel studies examined the hepatoprotective effects of the compounds in vivo. Rats were pretreated with antioxidant or vehicle prior to administration of CCl4 (300 or 600 microL/kg i.p.). Sera were collected 24 h postadministration of CCl4 and analyzed for alanine aminotransferase (ALT) and alkaline phosphatase (ALP) activities and total bilirubin. Administration of CCl4 produced elevations in ALT, moderate changes in bilirubin, and no change in ALP activities. Histological examination of CCl4-treated livers revealed lipidosis and centrilobular necrosis. The antioxidants partially improved the clinical chemistry parameters, but had minimal effects on the histological lesion. In contrast to the complete inhibition of lipid peroxidation observed in the in vitro studies, none of the antioxidants markedly protected against CCl4-induced toxicity in vivo.     

Protection against lipid peroxidation by a microsomal glutathione-dependent labile factor

Glutathione (GSH) protects rat liver microsomes against ascorbic acid (0.2 mM)/ferrous iron (10 microM)-induced lipid peroxidation for some time. The inhibitory effect of GSH is concentration-dependent (0.1-1.0 mM). Our data suggest that GSH acts by preventing initial radical formation rather than via radical scavenging or GSH--peroxidase activity. A labile GSH-dependent factor is involved in the inhibition of microsomal lipid peroxidation by GSH, inasmuch as heating the microsomes abolishes the GSH effect. We found that besides heating, lipid peroxidation also destroys the GSH-dependent factor. Consequently, continuous radical stress will produce lipid peroxidation, despite the presence of GSH. Moreover, a detrimental effect of in vivo-induced lipid peroxidation (CCl4-treatment) on the GSH-dependent factor was observed. The implications of the present data for the genesis of and the protection against peroxidative damage are discussed.     

Protective effects of melatonin against carbon tetrachloride hepatotoxicity in rats

Melatonin is an indolamine, mainly secreted by the pineal gland into the blood of mammalian species. The potential for protective effects of melatonin on carbon tetrachloride (CCl(4))-induced acute liver injury in rats was investigated in this work. CCl(4) exerts its toxic effects by generation of free radicals; it was intragastrically administered to male Wistar rats (4 g kg(-1) body weight) at 20 h before the animals were decapitated. Melatonin (15 mg kg(-1) body weight) was administered intraperitoneally three times: 30 min before and at 2 and 4 h after CCl(4) injection. Rats injected with CCl(4) alone showed significant lipid and hydropic dystrophy of the liver, massive necrosis of hepatocytes, marked increases in free and conjugated bilirubin levels, elevation of hepatic enzymes (alanine aminotransferase and aspartate aminotransferase) in plasma, as well as NO accumulation in liver and in blood. Melatonin administered at a pharmacological dose diminished the toxic effects of CCl(4). Thus it decreased both the structural and functional injury of hepatocytes and clearly exerted hepatoprotective effects. Melatonin administration also reduced CCl(4)-induced NO generation. These findings suggest that the effect of melatonin on CCl(4)-induced acute liver injury depends on the antioxidant action of melatonin.     

Dimerumic acid as an antioxidant of the mold, Monascus anka

We previously reported that the mold Monascus anka, traditionally used for fermentation of food, showed antioxidant and hepatoprotective actions against chemically induced liver injuries. In the present study, the antioxidant component of M. anka was isolated and identified. The antioxidant was elucidated to be dimerumic acid. DPPH (1,1-diphenyl-2-picrylhydrazyl) radical was significantly scavenged by the antioxidant whereas hydroxyl radical and superoxide anion were moderately scavenged. When the antioxidant (12 mg/kg) was given to mice prior to carbon tetrachloride (CCl(4), 20 microl/kg, ip) treatment, the CCl(4)-induced liver toxicity in mice seen in an elevation of serum aspartate aminotransferase and alanine aminotransferase activities was depressed, suggesting the hepatoprotective action of the antioxidant. The liver microsomal glutathione S-transferase activity, which is known to be activated by oxidative stress or active metabolites, was increased by CCl(4) treatment and the increase was also depressed by pretreatment with the mold antioxidant. Thus these data confirmed that the dimerumic acid isolated from M. anka is the potential antioxidant and protective against CCl(4)-induced liver injury.     

Protective effect of gossypitrin on carbon tetrachloride-induced in vivo hepatotoxicity

Carbon tetrachloride (CCl4) is a known environmental biohazard, which induces lipid peroxidation (LPO) and oxidative damage in rat liver. In this study, the hepatoprotective effect of Gossypitrin, a flavonoid extracted from Hibiscus elatus S.W, was investigated against the CCl4-induced in vivo hepatotoxicity. The levels of malondialdehyde (MDA) were assayed as an index of LPO and the levels of catalase (CAT) activity as a biomarker of oxidative damage. Leakage of aspartate aminotransferase (ALT) and lactate dehydrogenase (LDH), liver weight/body weight ratio as well as morphological parameters were used as signs of hepatotoxicity. CCl4 (1 ml/kg), intraperitoneally injected into rats, caused increased MDA production and CAT activity, and also a significant ALT and LDH leakage as compared to levels of these constituents in the control group. Changes in morphology, including steatosis, cells forming balloon cells and necrosis were evaluated in the hepatotoxin-induced damage. Treatment of rats with Gossypitrin (3.98, 5.97 and 8.95 mg/kg) 2 h before and 2 h after CCl4 injection, protected hepatocytes against cell injury induced by CCl4 and its efficacy as an antioxidant was similar to vitamin E (used as a reference antioxidant). These results are consistent with the conclusion that the toxicity of CCl4 is due to LPO and the generation of reactive oxygen species (ROS), and that Gossypitrin's protective effects relate to its direct radical scavenging ability and other antioxidative processes induced by its structure.     

Hepatoprotective and free radical scavenging effects of Nelumbo nucifera.

Ethanol extracts from Nelumbo nucifera (ENN) seeds were studied for possible antioxidative and hepatoprotective effects. Antioxidative effects were measured spectrophotometrically by reduction of 2,2'-Diphenyl-1-picrylhydrazyl (DPPH) radicals. Hepatoprotective effects were tested using carbon tetrachloride (CCl4) and aflatoxin B1 (AFB1)-induced hepatocyte toxicity models. ENN showed potent free radical scavenging effects with a median inhibition concentration of 6.49 microg/ml. Treatment of hepatocytes with ENN inhibited both the production of serum enzymes and cytotoxicity by CCl4. The genotoxic and cytotoxic effects of AFB1 were also inhibited by ENN in dose-dependent manners. These hepatoprotective effects of ENN against CCl4 and AFB1 might result from its potent antioxidative properties.     

The effect of the administration of cobaltic protoporphyrin IX on drug metabolism, carbon tetrachloride activation and lipid peroxidation in rat liver microsomes

The effects of cobaltic protoporphyrin IX (CPP) administration on hepatic microsomal drug metabolism, carbon tetrachloride activation and lipid peroxidation have been investigated using male Wistar rats. CPP (125 mumol/kg, 72 h before sacrifice) profoundly decreased the levels of hepatic microsomal heme, particularly cytochrome P-450. Consequently, the associated mixed-function oxidase systems were equally strongly depressed. An unexpected finding was that CPP administration also greatly decreased the activity of NADPH/cytochrome c reductase, a result not generally found with the administration of the more widely used cytochrome P-450 depleting agents, cobaltous chloride. Activation of carbon tetrachloride, measured as covalent binding of [14C] CCl4, spin-trapping of CCl3 and CCl4-stimulated lipid peroxidation, was much lower in liver microsomes from CPP-treated rats. Other microsomal lipid peroxidation systems, utilising cumene hydroperoxide or NADPH/ADP-Fe2+, were also depressed in parallel with the decrease in microsomal enzyme activities.     

Two Mechanisms of Cc14-Induced Fatty Liver: Lipid Peroxidation Or Covalent Binding Studied in Cultured Rat Hepatocytes

With cultured hepatocytes it was studied whether CCl₄-induced inhibition of secretion of VLDL and HDL from liver cells is a consequence of covalent binding of CC1₄ metabolites (i.e. CO,; CC1,00) to cell constituents or of membrane damage by lipid peroxidation. Comparing the kinetics of inhibition of lipoprotein secretion with that of CCl₄-bioactivation it was found, that covalent binding of (^HC)-CC1₄ occurred at early time points (5 min) after CC1₄ administration and inhibited the lipoprotein secretion. At 100μM CC1₄ it was depressed by 53% within 60min. Incubations of CC1₄-treated cells with increasing concentrations of vitamin E blocked lipid peroxidation, but lipoprotein secretion was still inhibited. Piperonyl butoxid, a radical scavenger, protected against CCl₄-induced inhibition of lipoprotein section, lipid peroxidation and covalent binding.

These results show that during the early phases of CC1₄ poisoning fat accumulation is the consequence of covalent binding of CC1₄ metabolities to cell structures.     

Prophylactic effect of aqueous propolis extract against acute experimental hepatotoxicity in vivo

Propolis has been extensively used in folk medicine for the management of a wide spectrum of disorders. In a previous study, we demonstrated the protective effect of the aqueous propolis extract (APE) against the injurious effects of carbon tetrachloride (CCl4) on hepatocytes in vitro. The present investigation was carried out to show whether the hepatoprotective effect of the extract could also be manifested in vivo. Rats were given APE orally for 14 consecutive days, before being subjected to a single intraperitoneal injection of CCl4. One day after the CCl4 injection, the animals were sacrificed, hepatocytes were isolated and liver homogenates were prepared for the assessment of liver injury. In isolated hepatocytes, APE afforded protection against CCl4-induced injury as manifested by a decrease in the leakage of the cytosolic enzyme lactate dehydrogenase (LDH), decreased generation of lipid peroxide and maintenance of cellular reduced glutathione (GSH) content. In principle, similar findings were observed in liver homogenates. The present findings show that APE has in vivo hepatoprotective potential which could be attributed at least in part to the maintenance of cellular GSH content. The latter effect seems to play an important role in conserving the integrity of biomembranes as it was associated with a decrease in lipid peroxidation and reduced leakage of cytosolic LDH.     

A note on the stability of conjugated diene absorption of rat liver microsomal lipids after carbon tetrachloride poisoning

Liver microsomal lipid peroxidation has been observed in fatal human CCl(4) poisoning, in rats with fatty livers induced by CCl(4) or by yellow phosphorus, and in mice poisoned with 1,1,2,2-tetrachloroethane. These observations suggest the possibility that other instances of toxic liver injury may involve lipid peroxidation. Cases of acute, fatal, toxic liver injury (e.g., from halothane anesthesia) are not likely to occur at or near laboratories equipped to determine whether any lipid peroxidation might have taken place. The data presented indicate that rat livers may be stored frozen for at least 7 days with no demonstrable diminution in CCl(4)-induced conjugated diene absorption of liver microsomal lipids.