The role of physiological oxygen partial pressures in lipid peroxidation. Theoretical considerations and experimental evidence

The oxidative breakdown of membrane lipids, i.e. lipid peroxidation, is considered to be decisively involved in a number of toxicological and pathological processes including liver injury caused by iron overload and halogenated alkanes such as CCl4. Within the physiological oxygen partial pressure (PO2) range, i.e. at PO2 between 1 and 100 mmHg, lipid peroxidation depends on O2 in a complex manner. For instance, CCl4-induced lipid peroxidation exhibits a distinct maximum at PO2 of around 7 mmHg, and iron-induced lipid peroxidation shows marked differences in its O2 dependence between an early lag phase and a later phase of self-accelerating propagation. The O2 dependence of lipid peroxidation is either determined by the O2 dependence of initiation or the O2 dependence of propagation. Factors decisive for this are presented and the underlying alterations in the pattern of the peroxidation-related reactions delineated.     

Lipid peroxidation and cell viability in isolated hepatocytes in a redesigned oxystat system: evaluation of the hypothesis that lipid peroxidation, preferentially induced at low oxygen partial pressures, is decisive for CCl4 liver cell injury

An oxystat system is described which is capable of maintaining steady-state oxygen partial pressures (PO2) at levels between 0.1 and 300 mm Hg for hours or even days in incubations of respiring cells. The system was used to study effects of the hepatotoxin carbon tetrachloride (CCl4) on lipid peroxidation and cell viability in isolated hepatocytes from phenobarbital-pretreated rats at various steady-state PO2. At PO2 below 35 mm Hg, with a maximum effect at 7 mm Hg, CCl4 induced an immediate lipid peroxidation, the rate of which slowed down during further incubation. AT PO2 between 35 and 70 mm Hg, CCl4 initially induced only slight lipid peroxidation, while there was a significant increase in lipid peroxidation after approximately 30 min. At PO2 above 100 mm Hg, no lipid peroxidation was induced by CCl4. At PO2 of 70 mm Hg and below, with the maximum effect at 3 mm Hg, CCl4 also induced marked losses of cell viability. Under anaerobic conditions and at PO2 greater than 70 mm Hg, CCl4 was without effect on the viability of the liver cells. Cells isolated from the pericentral area of the liver lobule showed more lipid peroxidation and loss of cell viability than cells from the periportal area of the lobule. These results provide further evidence for the decisive role of lipid peroxidation, preferentially induced at low PO2, in CCl4 liver injury.     

Alterations of the microsomal glucose-6-phosphatase system evoked by ferrous iron- and haloalkane free-radical-mediated lipid peroxidation

Alterations of catalytic activities of the microsomal glucose-6-phosphatase system were examined following either ferrous iron- or halothane (CF3CHBrCl) and carbon tetrachloride (CCl4) free-radical-mediated peroxidation of the microsomal membrane. Enzyme assays were performed in native and solubilized microsomes using either glucose 6-phosphate or mannose 6-phosphate as substrate. Lipid peroxidation was assessed by the amounts of malondialdehyde equivalents formed. Regardless of whether the experiments were performed in the presence of NADPH/Fe3+, NADPH/CF3CHBrCl, or NADPH/CCl4, with the onset of lipid peroxidation, mannose-6-phosphatase activity of the native microsomes increased immediately, while further alterations in catalytic activities were only detectable when lipid peroxidation had passed characteristic threshold values: above 2 nmol malondialdehyde/mg microsomal protein, glucose-6-phosphatase activity of the native microsomes was lost, and at 10 nmol malondialdehyde/mg microsomal protein, glucose-6-phosphatase and mannose-6-phosphatase activity of the solubilized microsomes started to decline. It is concluded that the latter alterations are due to an irreversible damage of the phosphohydrolase active site of the glucose-6-phosphatase system, while the changes observed at earlier stages of microsomal lipid peroxidation may also reflect alterations of the transporter components of the glucose-6-phosphatase system. Virtually no changes in the catalytic activities of the glucose-6-phosphatase system occurred under anaerobic conditions, indicating that CF3CHCl and CCl3 radicals are without direct damaging effect on the glucose-6-phosphatase system. Further, maximum effects of carbon tetrachloride and halothane on lipid peroxidation and enzyme activities were observed at an oxygen partial pressure (PO2) of 2 mmHg, providing additional evidence for the crucial role of low PO2 in the hepatotoxicity of both haloalkanes.     

Protective effects of N-acetyl-L-cysteine against acute carbon tetrachloride hepatotoxicity in rats

In recent years, N-acetyl-L-cysteine (NAC) has been widely investigated as a potentially useful protective and antioxidative agent to be applied in many pathological states. The aim of the present work was further evaluation of the mechanisms of the NAC protective effect under carbon tetrachloride-induced acute liver injuries in rats. The rat treatment with CCl4 (4 g/kg, intragastrically) caused pronounced hepatolysis observed as an increase in blood plasma bilirubin levels and hepatic enzyme activities, which agreed with numerous previous observations. The rat intoxication was accompanied by an enhancement of membrane lipid peroxidation (1.4-fold) and protein oxidative damage (protein carbonyl group and mixed protein-glutathione disulphide formations) in the rat liver. The levels of nitric oxide in blood plasma and liver tissue significantly increased (5.3- and 1.5-fold, respectively) as blood plasma triacylglycerols decreased (1.6-fold). The NAC administration to control and intoxicated animals (three times at doses of 150 mg/kg) elevated low-molecular-weight thiols in the liver. The NAC administration under CCl4-induced intoxication prevented oxidative damage of liver cells, decreased membrane lipid peroxidation, protein carbonyls and mixed protein-glutathione disulphides formation, and partially normalized plasma triacylglycerols. At the same time the NAC treatment of intoxicated animals did not produce a marked decrease of the elevated levels of blood plasma ALT and AST activities and bilirubin. The in vitro exposure of human red blood cells to NAC increased the cellular low-molecular-weight thiol levels and retarded tert-butylhydroperoxide-induced cellular thiol depletion and membrane lipid peroxidation as well as effectively inhibited hypochlorous acid-induced erythrocyte lysis. Thus, NAC can replenish non-protein cellular thiols and protect membrane lipids and proteins due to its direct radical-scavenging properties, but it did not attenuate hepatotoxicity in the acute rat CCl4-intoxication model.     

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.     

Kupffer cells inhibition prevents hepatic lipid peroxidation and damage induced by carbon tetrachloride

The aim of this work was to determine if the action mechanism of gadolinium on CCl(4)-induced liver damage is by preventing lipid peroxidation (that may be induced by Kupffer cells) and its effects on liver carbohydrate metabolism. Four groups of rats were treated with CCl(4), CCl(4)+GdCl(3), GdCl(3), and vehicles. CCl(4) was given orally (0.4 g 100 g(-1) body wt.) and GdCl(3) (0.20 g 100 g(-1) body wt.) was administered i.p. All the animals were killed 24 h after treatment with CCl(4) or vehicle. Glycogen and lipid peroxidation were measured in liver. Alkaline phosphatase, gamma-glutamyl transpeptidase, alanine amino transferase activities and bilirubins were measured in rat serum. A liver histological analysis was performed. CCl(4) induced significant elevations on enzyme activities and bilirubins; GdCl(3) completely prevented this effect. Liver lipid peroxidation increased 2.5-fold by CCl(4) treatment; this effect was also prevented by GdCl(3). Glycogen stores were depleted by acute intoxication with CCl(4). However, GdCl(3) did not prevent this effect. The present study shows that Kupffer cells may be responsible for liver damage induced by carbon tetrachloride and that lipid peroxidation is produced or stimulated by Kupffer cells, since their inhibition with GdCl(3) prevented both lipid peroxidation and CCl(4)-induced liver injury.     

Protective effects of an extract of young radish (Raphanus sativus L) cultivated with sulfur (sulfur-radish extract) and of sulforaphane on carbon tetrachloride-induced hepatotoxicity

The protective effects of an extract of young radish (Raphanus sativus L) cultivated with sulfur (sulfur-radish extract) and of sulforaphane, an isothiocyanate, on carbon tetrachloride (CCl(4))-induced liver injury were observed in mice. CCl(4) produced a marked increase in the serum level of alanine aminotransferase (ALT), primed lipid peroxidation, and resulted in intense necrosis due to oxidative stress. Oral administration of the sulfur-radish extract and of sulforaphane after CCl(4)-induced liver injury both decreased the serum level of ALT, reduced the necrotic zones, inhibited lipid peroxidation, and induced phase 2 enzymes without affecting cytochrome P450-2E1 (CYP2E1). These results suggest that the administration of the sulfur-radish extract and of sulforaphane may partially prevent CCl(4)-induced hepatotoxicity, possibly by indirectly acting as an antioxidant by improving the detoxification system.     

Criteria for determination of lipid peroxidation in tissues: estimation in liver of mice intoxicated with carbon tetrachloride

The profiles of lipid peroxidation products in liver homogenates or microsomal membranes prepared from CCl4-intoxicated mice were determined by several commonly employed methods. The level of conjugated dienes peaked within 30 min and then decreased, suggesting the transitory nature of lipid peroxides in vivo. Values for thiobarbituric acid positive material peaked 30 min after CCl4 treatment, diminished thereafter for a time, and gradually rose to a new maximum at 24 h; the first peak appears to represent lipid peroxides and the second represents further degradation products including malondialdehyde. Fluorescence intensity (excitation, 360 nm; emission, 430 nm) was closely correlated with the second peak. Our findings support the involvement of lipid peroxidation in CCl4-induced hepatotoxicity in mice and emphasize the necessity for several analytical indices of lipid peroxidation performed at different time intervals.     

Protective effect of colchiceine against acute liver damage

Pretreatment of rats with colchiceine (10 micrograms/day/rat) for seven days protected against CCl4-induced liver damage. CCl4 intoxication was demonstrated histologically and by increased serum activities of alanine amino transferase (ALT), alkaline phosphatase (Alk. Phosph.) gamma glutamyl transpeptidase (GGTP), bilirubins and decreased activity of glucose-6-phosphatase (G-6Pase). Furthermore, an increase in liver lipid peroxidation and a decrease in plasma membrane GGTP and Alk. Phosph. activities were found. Colchiceine increased 1.5-fold the LD50 of CCl4 and prevented the release of intracellular enzymes as well as the decrease in GGTP and Alk. Phosph. activities in plasma membranes. It also completely prevented the lipid peroxidation induced by CCl4 and limited the extent of the histological changes.     

Cytochrome P-450 and oxygen toxicity. Oxygen-dependent induction of ethanol-inducible cytochrome P-450 (IIE1) in rat liver and lung

The ethanol-inducible form of cytochrome P-450 (P-450IIE1) has previously been shown to exhibit an unusually high rate of oxidase activity with the subsequent formation of reactive oxygen species, e.g., hydrogen peroxide, and to be the main contributor of microsomal oxidase activity in liver microsomes from acetone-treated rats [Ekstr?m & Ingelman-Sundberg (1989) Biochem. Pharmacol. (in press)]. The results here presented indicate that oxygen exposure of rats causes an about 4-fold induction of P-450IIE1 in rat liver and lung microsomes. The induction in liver was not accompanied by any measurable increase in the P-450IIE1 mRNA levels, but the enhanced amount of P-450IIE1 accounted for 60% of the net 50% increase in the level of hepatic P-450 as determined spectrophotometrically. The induction of P-450IIE1 was maximal after 60 h of O2 exposure, and concomitant increases in the rates of liver microsomal CCl4-dependent lipid peroxidation, O2 consumption, NADPH oxidation, O2- formation, H2O2 production, and NADPH-dependent microsomal lipid peroxidation were seen. Liver microsomes from oxygen-treated rats had very similar properties to those of microsomes isolated from acetone-treated rats with respect to the P-450IIE1 content and catalytic properties, but different from those of thyroxine-treated animals. Treatment of rats with the P-450IIE1 inducer acetone in combination with oxygen exposure caused a potentiation of the NADPH-dependent liver and lung microsomal lipid peroxidation and decreased the survival time of the rats. The results reached indicate a role for cytochrome P-450 and, in particular, for cytochrome P-450IIE1 in oxygen-mediated tissue toxicity.     

The roles played by crucial free radicals like lipid free radicals, nitric oxide, and enzymes NOS and NADPH in CCl(4)-induced acute liver injury of mice

Mice were administered a single dose of carbon tetrachloride (CCl(4)) to induce acute liver injury. We found that lactate dehydrogenase (LDH) and glutamic pyruvic transaminase (GPT) levels in serum, as well as the level of thiobarbituric acid reaction substances (TBARS) in liver homogenate increased significantly in a manner both dose dependent and time dependent after CCl(4) administration. Such results suggest that the liver is susceptible to CCl(4) treatment and that lipid peroxidation is associated with CCl(4)-induced liver injury. The spin-trapping electron paramagnetic resonance (EPR) method was used to detect nitric oxide (NO) level in liver. The chemiluminescence method was also employed to measure the NO(2)(-)/NO(3)(-) concentration in serum. The NO levels in liver tissues and NO(2)(-)/NO(3)(-) concentration in serum were found to decrease significantly both in a dose-dependent manner and in time course after CCl(4) treatment. The nitric oxide synthase (NOS) II activity in the liver, in contrast, was found to increase significantly. Our study suggests that not only should the expression of NOS be analyzed but NO organ and blood concentration must be measured in the study of diseases involving nitric oxide. L-arginine treatment had no significant effect on the liver function of CCl(4)-treated mice. It was found that NO donor sodium nitroprusside (SNP; 50 or 100 microg/kg) treatment resulted in decreases of LDH, GPT, and TBARS levels, leading to a protective effect on CCl(4)-treated mice. On the other hand, N(G)-nitro-L-arginine methyl ester (L-NAME, 100 or 300 mg/kg) treatment caused more severe liver damage. Moreover, we have found in an in vitro EPR study that SNP could scavenge lipid peroxyl radical LOO&z.rad;. The above results together suggest that NO may protect CCl(4)-induced liver injury through scavenging lipid radical, inhibiting the lipid peroxidation chain reaction. On the basis of our analysis, we put forth two explanations for the stated discrepancy between NOS II and NO production: (i) NO was used up gradually in terminating lipid peroxidation and (ii) NADPH was depleted (on the basis of correlation evidence only).     

Carbon tetrachloride-induced lipid peroxidation dependent on an ethanol-inducible form of rabbit liver microsomal cytochrome P-450

Treatment of rats with ethanol or rabbits with either imidazole or pyrazole, agents known to induce the ethanol-inducible form of liver microsomal cytochrome P-450 (P-450 LMeb), caused, compared to controls, 3-25-fold enhanced rates of CCl4-dependent lipid peroxidation or chloroform production in isolated liver microsomes. No significant differences were seen when the rate of CCl4-dependent lipid peroxidation was expressed relative to the amount of P-450 LMeb in the various types of microsomal preparations. In reconstituted membranous systems, this type of P-450 was a 100-fold more effective catalyst of CCl4 metabolism than either of the cytochromes P-450 LM2 or P-450 LM4. It is proposed that the induction of this isozyme provides the explanation on a molecular level for the synergism seen of ethanol on CCl4-dependent hepatotoxicity.     

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.     

Protective effect of aminoguanidine, a nitric oxide synthase inhibitor, against carbon tetrachloride induced hepatotoxicity in mice

The present study was undertaken to evaluate the effect of aminoguanidine (AG) on carbon tetrachloride (CCl4)-induced hepatotoxicity. Treatment of mice with CCl4 (20 microl/kg, i.p.) resulted in damage to centrilobular regions of the liver, increase in serum aminotransferase and rise in lipid peroxides level 24 hours after CCl4 administration. Pretreatment of mice with AG (50 mg/kg, i.p.) 30 minutes before CCl4 was found to protect mice from the CCl4-induced hepatic toxicity. This protection was evident from the significant reduction in serum aminotransferase, inhibition of lipid peroxidation and prevention of CCl4-induced hepatic necrosis revealed by histopathology. Aminoguanidine, a relatively specific inhibitor of inducible nitric oxide synthase, did not inhibit the in vitro lipid peroxidation. Taken together, these data suggest a potential role of nitric oxide as an important mediator of CCl4-induced hepatotoxicity.     

Effective protection of Terminalia catappa L. leaves from damage induced by carbon tetrachloride in liver mitochondria

The protective effects of chloroform extracts of Terminalia catappa L. leaves (TCCE) on carbon tetrachloride (CCl4)-induced liver damage and the possible mechanisms involved in the protection were investigated in mice. We found that increases in the activity of serum aspartate aminotransferase and alanine aminotransferase and the level of liver lipid peroxidation (2.0-fold, 5.7-fold and 2.8-fold) induced by CCl4 were significantly inhibited by oral pretreatment with 20, 50 or 100 mg/kg of TCCE. Morphological observation further confirmed the hepatoprotective effects of TCCE. In addition, the disruption of mitochondrial membrane potential (14.8%), intramitochondrial Ca2+ overload (2.1-fold) and suppression of mitochondrial Ca2+-ATPase activity (42.0%) in the liver of CCl4-insulted mice were effectively prevented by pretreatment with TCCE. It can be concluded that TCCE have protective activities against liver mitochondrial damage induced by CCl4, which suggests a new mechanism of the hepatoprotective effects of TCCE.     

Effect of bacterial lipopolysaccharide on the content of lipid peroxidation products in lungs and other organs of mice

The influence of lipopolysaccharide fromEscherichia coli (LPS, 17 mg/kg body weight) on the lipid peroxidation process in organs of mice was studied. The content of conjugated dienes (CD), lipid peroxides (LP), malondialdehyde (MDA) (all three lipid peroxidation by-products), peroxidase (PO) activity and wet-to-dry weight ratio in lungs, heart, spleen, kidneys and liver were determined 1.5 h after intravenous injection of LPS. Animals observed at this time-point had reduced activity and decreased body temperature by about 2°C, however, all analysed organs did not reveal any changes of wet-to-dry weight ratio comparing to organs from mice injected with sterile, pyrogen free 0,9% NaCl. Only extracts from heart and lungs showed significant increase in the tissue level of at least two lipid peroxidation products. The heart content of CD, MDA, and LP was about 1.5-, 1.3-, and 2.4-fold higher than in control group. In lungs CD and MDA increased 3.3- and 1.3-times but in spleen only content of LP was elevated. In these organs the suppression of PO activity was also observed. Liver and kidneys did not reveal any convincing enhancement of lipid peroxidation process and alterations of PO activity. Since free radical reactions are involved in lipid peroxidation process and inactivation of PO these results suggest that heart, lungs and spleen are the organs mostly exposed to oxidative stress during the first 1.5 h after single injection of LPS in mice.Abbreviations CD conjugated dienes - LP lipid peroxides - LPS lipopolysaccharide - MDA malondialdehyde - PMNL polymorphonuclear leukocytes - PO peroxidase - TBA thiobarbituric acid     

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.     

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.