Phospholipase A2 activation and cell injury in isolated rat hepatocytes exposed to bromotrichloromethane, chloroform, and 1,1-dichloroethylene as compared to effects of carbon tetrachloride.

It was found that the four toxigenic agents, CCl4, CHCl3, CBrCl3, and 1,1-dichloroethylene (vinylidene chloride) all share the property of activating phospholipase A2 (PLA2) of isolated hepatocytes in suspension, as determined over a 60- or 120-min time period. In all cases, PLA2 activation, measured as the appearance of lysophosphatidylethanolamine, preceded the release of lactic dehydrogenase during incubation of the cells at 37 degrees C. It is concluded that for these halogenated hydrocarbons phospholipase A2 activation may be part of the chain of causality leading from initial bioactivation to ultimate cell death.

An indirect method demonstrating that CCl4-dependent hepatocyte injury is linked to a rise in intracellular calcium ion concentration.

The capacity of CCl4 to activate phospholipase A2 (PLA2) of isolated hepatocytes in suspension, and the toxicity of CCl4 against such hepatocytes, was confirmed. It was shown that these actions of CCl4 are independent of the concentration of Ca2+ ions in the suspension medium over a 25,000-fold range. In particular, toxicity of CCl4 does not require high Ca2+ in the suspension medium. The Ca(2+)-ionophore A23187 can also activate PLA2, and it also is toxic. However, in this case activation of PLA2 and toxicity require a high medium Ca2+ ion concentration. These radically different modes of action of CCl4 and A23187 permitted an indirect confirmation of the hypothesis that the activation of PLA2 by CCl4, and the toxicity of CCl4 depend on CCl4-dependent mobilization of intracellular stores of Ca2+, and a rise in the concentration of Ca2+ ions in the hepatocyte cytosol.

Increased prooxidant action of hepatic cytosolic low-molecular-weight iron in experimental iron overload.

In the iron-loaded liver there may be an increase in the putative intracellular transit pool of iron, components of which could be catalytically active in stimulating lipid peroxidation. To study the levels of low-molecular-weight, catalytically active iron in the liver, cytosolic ultrafiltrates were tested in an assay containing rat liver microsomes and NADPH. Malondialdehyde production was used as an index of lipid peroxidation. This assay system was sensitive enough to detect 0.25 mumol/L ferrous iron; progressive but non-linear increases in malondialdehyde were produced as the iron concentration was increased to 5 mumol/L. Ultrafiltrates from hepatic cytosol of iron-loaded rats had greater prooxidant action than did those from controls. When added to the assay, deferoxamine, an iron chelator, completely suppressed the prooxidant action of hepatic ultrafiltrates, showing that this activity is iron-dependent. Deferoxamine administered intraperitoneally to control animals at a dose of 1 gm/kg completely inhibited the prooxidant effect of hepatic ultrafiltrates prepared from rats killed after 1, 2 and 3 hr. Partial inhibition was observed at 4 hr; by 6 hr the inhibitory effect of deferoxamine was completely lost. Administration of deferoxamine (1 gm/kg intraperitoneally, 1 hr before killing) completely inhibited the prooxidant action of hepatic ultrafiltrates in moderately iron-loaded rats and controls but had no protective effect in heavily iron-loaded rats. These results support the concept that iron overload results in an increase in a hepatic cytosolic pool of low-molecular-weight iron that is catalytically active in stimulating lipid peroxidation. This pool can be chelated transiently in vivo by deferoxamine in moderate, but not heavy, iron overload.

Liver cell calcium homeostasis in carbon tetrachloride liver cell injury: new data with fura2.

The calcium fluorescent probe fura2 was used to measure concentration of free calcium in the cytosol of isolated rat hepatocytes in suspension. The resting level in untreated hepatocytes was 121 nM. On addition of CCl4 at a concentration of 0.5 mM, cytosolic free calcium rose sharply and reached a statistically significant (P less than 0.05) steady plateau level of about 190 nM within five minutes. With a concentration of 1.0 mM CCl4, cytosolic free calcium rose within ten minutes to a plateau level of about 200 nM. Use of fura2, along with the capacity of Mn2+ ions to effectively quench fura2 fluorescence, provided the basis for a simple and decisive method to determine whether the added CCl4 was permeabilizing the hepatocyte plasma membrane by direct solvent action. It was found that up to a concentration of 1.0 mM, CCl4 did not permeabilize the plasma membrane, but direct attack on the plasma membrane was unequivocally demonstrated for concentrations of 2 mM CCl4 and above. Finally, an hypothesis is presented for resolution of the puzzling dilemma that emerged from the observation, reported from two laboratories, that CCl4 can rapidly mobilize liver mitochondrial calcium despite the well-known relative resistance of these organelles to the damaging effects of this toxic agent.

Lipid peroxidation and associated hepatic organelle dysfunction in iron overload.

Iron overload can have serious health consequences. Since humans lack an effective means to excrete excess iron, overload can result from an increased absorption of dietary iron or from parenteral administration of iron. When the iron burden exceeds the body's capacity for safe storage, the result is widespread damage to the liver, heart and joints, and the pancreas and other endocrine organs. Clear evidence is now available that iron overload leads to lipid peroxidation in experimental animals, if sufficiently high levels of iron are achieved. In contrast, there is a paucity of data regarding lipid peroxidation in patients with iron overload. Data from experiments using an animal model of dietary iron overload support the concept that iron overload results in an increase in an hepatic cytosolic pool of low molecular weight iron which is catalytically active in stimulating lipid peroxidation. Lipid peroxidation is associated with hepatic mitochondrial and microsomal dysfunction in experimental iron overload, and lipid peroxidation may underlie the increased lysosomal fragility that has been detected in homogenates of liver samples from both iron-loaded human subjects and experimental animals. Some current hypotheses focus on the possibility that the demonstrated functional abnormalities in organelles of the iron-loaded liver may play a pathogenic role in hepatocellular injury and eventual fibrosis. The recent demonstration that hepatic fibrosis is produced in animals with long-term dietary iron overload will allow this model to be used to further investigate the relationship between lipid peroxidation and hepatic injury in iron overload.

Potentiation of carbon tetrachloride hepatotoxicity by chlordecone: dose-response relationships and increased covalent binding in vivo.

Chlordecone greatly potentiates carbon tetrachloride (CCl4) hepatotoxicity. In order to quantitate the degree of this potentiation, the effects of a range of doses of CCl4 on two microsomal enzymatic functions and liver enzyme release were examined in chlordecone-treated and control rats. Male Sprague-Dawley rats were pretreated with 15 mg chlordecone per kilogram body weight (BW) intragastrically or with vehicle. After 48 hours, 0 to 250 microliters CCl4 per 100 g body weight were given intraperitoneally (IP), and the rats were killed 24 hours later. Chlordecone treatment produced approximately a 17-fold potentiation of the CCl4-dependent loss of cytochrome P-450 and glucose-6-phosphatase activity, so that a dose of 6 microliters CCl4 per 100 g body weight in the chlordecone-treated animals resulted in a similar amount of damage as observed with 100 microliters CCl4 per 100 g body weight in controls. A similar potentiation by chlordecone was seen with CCl4 induced increases in serum glutamic-oxaloacetic transaminase (SGOT) levels. Chlordecone treatment also increased hepatic cytochrome P-450 levels by 67% and resulted in an increase in the covalent binding of [14-C]-CCl4-derived metabolites to microsomal protein and lipid in vivo.

Chlordecone does not interfere with hepatic repair after carbon tetrachloride or partial hepatectomy.

The effect of chlordecone (CD) on hepatic repair, measured either as recovery of microsomal enzymatic functions or as the induction of cytosolic thymidine kinase (TK) activity, was evaluated in rats given carbon tetrachloride (CCl4). Carbon tetrachloride was administered to CD-potentiated and control animals using doses of this hepatotoxin which produce similar degrees of damage at 24 hours in both groups of animals (6 and 100 microliters CCl4 per 100 g body weight, respectively). Chlordecone had no significant effect on the time course of recovery of microsomal cytochrome P-450 content or glucose-6-phosphatase activity following CCl4 administration. Hepatic TK activity was measured 48 hours after CCl4 administration as a biochemical index of the hepatic regenerative response. Thymidine kinase activity was increased eightfold in CD-treated rats receiving 6 microliters CCl4 per 100 g body weight, whereas in controls a similar induction of TK activity was produced by 100 microliters CCl4 per 100 g body weight. Therefore, the TK response in CD-treated rats receiving CCl4 is appropriate for the amount of damage produced, suggesting that CD does not inhibit the hepatic regenerative response to CCl4-induced injury. The effect of CD on hepatic repair was also examined in rats receiving a two-thirds partial hepatectomy. Pretreatment of animals with CD had no significant effect on the increase in TK activity produced 24 hours after partial hepatectomy. These results offer no support for the idea that CD impairs hepatic repair after either partial hepatectomy or CCl4 administration.

Potentiation by chlordecone of the defect in hepatic microsomal calcium sequestration induced by carbon tetrachloride.

The effect of carbon tetrachloride (CCl4) on the capacity of hepatic microsomes to sequester calcium was studied following pretreatment of rats with chlordecone. Chlordecone pretreatment alone had no effect on the kinetics of calcium uptake by hepatic microsomes. It was found, however, that chlordecone pretreatment of rats potentiated by sixfold the potency of CCl4 to suppress microsomal calcium sequestration capacity when measured one hour after CCl4 administration.

Pathological mechanisms in carbon tetrachloride hepatotoxicity.

Liver cell injury induced by carbon tetrachloride involves initially the metabolism of carbon tetrachloride to trichloromethyl free-radical by the mixed function oxidase system of the endoplasmic reticulum. It is postulated that secondary mechanisms link carbon tetrachloride metabolism to the widespread disturbances in hepatocyte function. These secondary mechanisms could involve the generation of toxic products arising directly from carbon tetrachloride metabolism or from peroxidative degeneration of membrane lipids. The possible involvement of radical species such as trichloromethyl (.CCl3), trichloromethylperoxy (.OOCCl3), and chlorine (.Cl) free radicals, as well as phosgene and aldehydic products of lipid peroxidation, as toxic intermediates is discussed. Data do not support the view that an increase in cytosolic free calcium is important in the toxic action of carbon tetrachloride or bromotrichloromethane. In addition, carbon tetrachloride-induced inhibition of very low density lipoprotein secretion by hepatocytes is not a result of elevated levels of cytosolic free calcium.

Halomethane-induced inhibition of protein synthesis in isolated hepatocytes.

The effect of several halomethanes on protein synthesis has been studied in isolated hepatocytes. When cells are added to medium preequilibrated with CCl4 or CBrCl3, protein synthesis is inhibited after a lag period of 4 to 10 min. The concentrations of CBrCl3, CCl4, and CHCl3 which cause a 50% inhibition of protein synthesis are about 6 microM, 400 microM, and 4 mM, respectively. This order of potency parallels the rate at which these compounds are metabolized by the hepatic mixed function oxidase, suggesting that metabolism is required for toxicity. The inhibitory effect caused by 18 min of exposure to CBrCl3 is not reversed when the toxin is removed, indicating that inhibition involves some irreversible modification of cellular material. Unexpectedly, the inhibitory effect caused by 18 min of exposure of CCl4 is about 30-40% reversed when the toxin is removed. This suggests that CCl4 causes inhibition not only by a metabolism-dependent (irreversible) pathway, but by a metabolism-independent (reversible) mechanism as well. Extracellular Ca2+ is not required for CCl4 inhibition of protein synthesis.

Evidence against involvement of calcium in carbon tetrachloride-dependent inhibition of lipid secretion by isolated hepatocytes.

Carbon tetrachloride (CCl4)-induced inhibition of very low density lipoprotein (VLDL) secretion was studied in isolated hepatocytes. The hypothesis that inhibition of secretion is due to altered calcium homeostasis following CCl4-dependent inhibition of endoplasmic reticulum calcium sequestration was investigated. Inhibition of VLDL secretion by CCl4 was not dependent on extracellular calcium, since inhibition occurred when extracellular calcium was reduced to 0.1 microM. CCl4 inhibited hepatocyte VLDL secretion more rapidly than it inhibited microsomal calcium sequestration. Further, the concentration of CCl4 that produced half-maximal inhibition of VLDL secretion was about one-half the concentration required to produce half-maximal inhibition of microsomal calcium sequestration. The calcium ionophore A23187 did not mimic the action of CCl4 in inhibiting VLDL secretion under conditions in which A23187 altered cellular calcium homeostasis. The results that an alteration of calcium homeostasis is not involved in inhibition of VLDL secretion by carbon tetrachloride.

Assessment of the role of calcium ion in halocarbon hepatotoxicity.

Halogenated hydrocarbons (CCl4, BrCCl3, 1,1-dichloroethylene, bromobenzene) cause a wide spectrum of dysfunction and injury in liver cells. An early effect of CCl4, BrCCl3, and 1,1-dichloroethylene is destruction of the Ca2+-sequestering ability of the endoplasmic reticulum, and it has been suggested that this lesion leads to subsequent disruption of other cell functions. Work to test this hypothesis has begun in this and other laboratories. While it appears that redistribution of intracellular Ca2+ does occur following these agents, the importance of this in cell injury is not fully resolved. Current results suggest Ca2+ redistribution may be involved in some cases (e.g., surface blebbing caused by bromobenzene), but not in others (e.g., inhibition of lipid secretion by CCl4).

Carbon tetrachloride-dependent inhibition of lipid secretion by isolated hepatocytes. Characterization and requirement for bioactivation.

Secretion of lipid as very low density lipoprotein (VLDL) by isolated hepatocytes was studied in a system in which the cells were exposed to a constant concentration of carbon tetrachloride (CCl4) throughout the duration of the incubation. Inhibition of secretion was characterized in terms of CCl4 concentration and duration of incubation. Half-maximum inhibition of VLDL secretion occurred at 80 microM CCl4. At 390 microM CCl4, VLDL secretion was inhibited 40% in 2 min and was suppressed completely in 5 min. No CCl4-dependent release of cellular glutamic oxaloacetic transaminase occurred under the conditions studied. Evidence is presented indicating that the metabolism of CCl4 is required for the expression of CCl4-dependent inhibition of lipid secretion. With respect to the parameters studied, the isolated hepatocyte system closely mimics the hepatic response of the intact animal to CCl4.

Evidence against a role for disturbed hepatocellular calcium homeostasis in the fatty liver of carbon tetrachloride hepatotoxicity.

A consensus does not exist regarding the nature of mechanisms linking the initial events of CCl4 metabolism to emergence of the classical indices of CCl4 liver cell injury. The possibility that a CCl4-dependent disturbance of intrahepatocellular calcium homeostasis might be a linking mechanism was investigated with isolated hepatocytes in suspension. CCl4-dependent inhibition of very low density lipoprotein secretion was studied. On the basis of kinetic data, dose-response data, and failure of elevated cytosolic calcium levels to inhibit lipid secretion, it was concluded that disturbed intracellular calcium homeostasis probably is not important in CCl4-dependent inhibition of secretion of very low density lipoproteins.

A new direction in the study of carbon tetrachloride hepatotoxicity.

An unknown chain of causality links the early events of CCl4 metabolism to emergence of the classical spectrum of pathological changes elicited by this hepatotoxin. Recent developments suggest that an early disturbance in hepatocellular Ca2+ homeostasis may be involved. The possibility that generation of toxic products of lipid peroxidation may act as toxicological "second messengers" linking events near cytochrome P-450 to distant parts of the cell seems unlikely. This mini-review attempts to highlight major recent developments underlying the current situation.

Carbon tetrachloride and bromotrichloromethane toxicity. Dual role of covalent binding of metabolic cleavage products and lipid peroxidation in depression of microsomal calcium sequestration.

We have investigated the importance of covalent binding and lipid peroxidation on the depression of microsomal calcium sequestration associated with in vitro metabolism of 14CCl4. Studies with CBrCl3 are also reported. In aerobic systems, promethazine was used to block lipid peroxidation, measured as malondialdehyde (MDA) generation. Effects of low levels of lipid peroxidation were tested in Fe2+-supplemented systems free of halogenated hydrocarbons. The results indicate that microsomal calcium sequestration can be depressed significantly by metabolism of either CCl4 or CBrCl3 in the absence of MDA generation, or by lipid peroxidation occurring in the absence of halogenated hydrocarbons.

Hepatic lipid peroxidation in vivo in rats with chronic iron overload.

Peroxidative decomposition of cellular membrane lipids is a postulated mechanism of hepatocellular injury in parenchymal iron overload. In the present study, we looked for direct evidence of lipid peroxidation in vivo (as measured by lipid-conjugated diene formation in hepatic organelle membranes) from rats with experimental chronic iron overload. Both parenteral ferric nitrilotriacetate (FeNTA) administration and dietary supplementation with carbonyl iron were used to produce chronic iron overload. Biochemical and histologic evaluation of liver tissue confirmed moderate increases in hepatic storage iron. FeNTA administration produced excessive iron deposition throughout the hepatic lobule in both hepatocytes and Kupffer cells, whereas dietary carbonyl iron supplementation produced greater hepatic iron overload in a periportal distribution with iron deposition predominantly in hepatocytes. Evidence for mitochondrial lipid peroxidation in vivo was demonstrated at all three mean hepatic iron concentrations studied (1,197, 3,231, and 4,216 micrograms Fe/g) in both models of experimental chronic iron overload. In contrast, increased conjugated diene formation was detected in microsomal lipids only at the higher liver iron concentration (4,161 micrograms Fe/g) achieved by dietary carbonyl iron supplementation. When iron as either FeNTA or ferritin was added in vitro to normal liver homogenates before lipid extraction, no conjugated diene formation was observed. We conclude that the presence of conjugated dienes in the subcellular fractions of rat liver provide direct evidence of iron-induced hepatic mitochondrial and microsomal lipid peroxidation in vivo in two models of experimental chronic iron overload.