Prelytic damage of red cells in filtrates from peroxidizing microsomes.

When liver microsomes are incubated in the presence of reduced nicotinamide adenine dinucleotide phosphate (NADPH), their constituent lipids undergo peroxidative degeneration. If erythrocytes are present in such a peroxidizing system, they hemolyze. Filtrates obtained by ultrafiltration of peroxidizing microsomal systems were found to have the capacity to produce prelytic damage in red cells. Filtrates obtained from microsomes that had not undergone peroxidative lipid decomposition were inert. The toxic activity in the active filtrates was not due to continuing oxidation of NADPH nor to continuing liver microsomal lipid peroxidation. Neither the chemical identity of the toxic product or products in active filtrates nor the mechanisms involved in the erythrocyte damage are known at this time.

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.

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.

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.

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.

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).

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.

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.

Determination of lipid conjugated dienes with tetracyanoethylene-14C: significance for study of the pathology of lipid peroxidation.

A method for quantitative analysis of conjugated diene unsaturation has been developed utilizing tetracyanoethylene-14C (TCNE-14C) in a Diels-Alder condensation. The amount of C14 found in the Diels-Alder adduct has been shown to be a measure of conjugated diene content. The method has proven successful in analysis of a variety of triglycerides, phospholipids, and peroxidized tissue lipids. In the course of this work, a method for removing the fatty acid substituents from phospholipids using lithium aluminum hydride was developed. TCNE-14C analysis for conjugated dienes in rat liver microsomal lipids after dosing with CCl4 or BrCCl3 has provided conclusive evidence that the increase in ultraviolet absorption at 233 nm of these lipids is due to conjugated dienes.

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.