Robert L. Waller

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.

Determination of lipid conjugated dienes with tetracyano-ethylene-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.

Effect of halomethanes on intracellular calcium distribution in hepatocytes

Exposure of isolated rat hepatocytes to hepatotoxic halomethanes results in a 40-60% decrease in intracellular Ca2+ content. The order of halomethane potency (CBrCl3 CCl4 CHCl3) suggests that this effect requires halomethane metabolism by the hepatic mixed function oxidase system. Although the Ca2+ sequestering ability of the endoplasmic reticulum is destroyed by CBrCl3 and CCl4, it appears that much of the Ca2+ lost from the cell is mitochondrial in origin. Paradoxically, saturating concentrations of CCl4 cause a marked increase in cell Ca2+. CCl4 also causes an acute increase in cytoplasmic free Ca2+ (from about 60 nM to about 90 nM), but this effect does not appear to require CCl4 metabolism and is probably a result of direct action of CCl4 on the plasma membrane.

Cytosolic free calcium concentration and intracellular calcium distribution in lymphocytes from cystic fibrosis patients

Lymphocytes prepared from normal individuals and patients with cystic fibrosis (CF) were compared with regard to intracellular Ca2+ concentration, distribution, and handling. No difference between control and CF was found in the concentration of cytosolic free Ca2+ (98 +/- 5 vs 102 +/- 7 nM), and no difference was observed in the kinetics with which control and CF cells restored cytoplasmic Ca2+ toward normal following a perturbation induced by cold-exposure. However, total intracellular Ca2+ is about 25% higher in CF lymphocytes than in control. Of this excess Ca2+, about 50% appears to be sequestered in mitochondria. This suggests that some difference in Ca2+ handling does exist, but the significance of this in cystic fibrosis remains to be determined.

Erythrocyte cytosolic free Ca2+ and plasma membrane Ca2+-ATPase activity in cystic fibrosis

The properties of the Ca2+, Mg2+-ATPase of erythrocyte membranes from patients with cystic fibrosis (CF) were extensively compared to that of healthy controls. Following removal of an endogenous membrane inhibitor of the ATPase, activation of the enzyme by Ca2+, calmodulin, limited tryptic digestion or oleic acid, as well as inhibition by trifluoperazine, were studied. The only properties found to be significantly different (CF cells vs controls) were calmodulin-stimulated peak activity (90 vs 101, P less than 0.02) and trypsin-activated peak activity (92 vs 102, P less than 0.02). No significant difference could be measured in the steady-state Ca2+-dependent phosphorylation of CF and control erythrocyte membranes indicating similar numbers of enzyme molecules per cell. The functional state of Ca2+ homeostasis in intact erythrocytes was investigated by measuring the resting cytosolic free Ca2+ levels using quin-2. Both CF and control erythrocytes maintained cytosolic free Ca2+ between 20 to 30 nM. Addition of 50 uM trifluoperazine resulted in an increase in erythrocyte cytosolic free Ca2+ to about 50 nM in both CF and control cells. Estimates of erythrocyte membrane permeability using the steady-state uptake of 45Ca into intact erythrocytes revealed no differences between CF and control cells. These results confirm that there is a small decrease in the calmodulin-stimulated activity of the erythrocyte Ca2+, Mg2+-ATPase in CF. However, this deficit is apparently not large enough to impair the ability of the CF erythrocyte to maintain normal resting levels of cytosolic free Ca2+.

Methanethiol inhibition of mitochondrial respiration.

Abstract Methanethiol and other alkylthiols appear to be at least partly responsible for fetor hepaticus, the unpleasant breath-odor of comatose patients with severe liver disease. We have found that methanethiol strongly inhibits rat liver mitochondrial respiration. At 1 μ m methanethiol, glutamate oxidation by rat liver mitochondria was inhibited 10%, and at 224 μ m methanethiol, glutamate oxidation was completely inhibited. This inhibition was shown to be at least partly reversible. Oxidative phosphorylation is not uncoupled from mitochondrial respiration by methanethiol nor is it specifically blocked. Methanethiol inhibits mitochondrial respiration apparently by reacting with cytochrome c oxidase.

Evaluation of a role for phosgene production in the hepatotoxic mechanism of action of carbon tetrachloride and bromotrichloromethane

During aerobic incubations of rat liver microsomes containing a NADPH generating system, 1.54% of added carbon tetrachloride (14CCl4), and 3.05% of added Bromotrichloromethane (CBrCl3) could be recovered as the 2-oxothiazolidene-4-carboxylic acid derivative of phosgene. Actual nanomolar quantities of phosgene formed were very small in comparison to input concentrations of phosgene necessary to depress microsomal cytochrome P-450 and glucose-6-phosphatase. Cysteine had no statistically significant effect on covalent binding of 14CCl4 metabolites to either microsomal lipids or proteins. Furthermore, presence of cysteine had no protective effect against loss of cytochrome P-450, glucose-6-phosphatase, or the capacity of microsomes to sequester calcium ions, all of which losses occur in vitro as a result of the metabolism of either CCl4 or CBrCl3. The low level of phosgene production, the lack of any effect of cysteine on the degree of covalent binding of CCl4 metabolites, the failure of cysteine to afford any protection against CCl4- or CBrCl3-dependent loss of microsomal enzyme activity, and the relative ineffectiveness of phosgene itself as a microsomal poison argue against the possibility that formation of phosgene plays a significant role in the liver injury resulting from CCl4 or CBrCl3 intoxication. Our experiments, however, do not rule out the possibility that some phosgene production and subsequent toxicological action may occur in a hydrophobic microenvironment of the endoplasmic reticulum.

Infection and immunity to Pseudomonas

Cystic fibrosis (CF) is a genetic disease that is inherited in an auto-somally-recessive fashion. The CF gene has been recently discovered. Its protein product, cystic fibrosis transmembrane regulator protein, plays an important role in the transport of substances across the cell membrane (1). The phenotypic defects in CF involve functional alterations of ion transport across affected exocrine glands and epithelial tissues, however, most of the morbidity and mortality in this disease is a result of chronic progressive pulmonary disease associated with infection. A consistent primary abnormality affecting the function of cells involved in host immunity has not been identified. The characteristic susceptibility of CF patients to chronic Pseudomonas aeruginosa lung infection may be indicative of a colonization or growth advantage in the CF environment for this bacterium or of exaggeration of CF abnormalities in the lung or immune cells caused by certain P. aeruginosa components or exoproducts.

Destruction of Microsomal Calcium Pump Activity: A Possible Secondary Mechanism in BrCCl3 and CCl4 Liver Cell Injury

In vitro rat liver microsomes free of Fe2+ ions peroxidize minimally at 37° when NADPH and either CC14 or BrCC13 are added. Although the lipid peroxidation dependent on toxigenic haloalkanes in these Fe2+-free microsome systems is very low, it is considerably more efficient in causing loss of cytochrome P-450 and glucose6-phosphatase than is the far more vigorous lipid peroxidation dependent on presence of Fe2+ ions. In particular, the Ca2+-pump activity of isolated microsomes was almost completely destroyed when malonic dialdehyde (MDA) production was as little as 8 pg per gram equivalent microsomes. Moore et al. (1976) had shown previously that the capacity of liver microsomes to sequester Ca2+ was severely depressed 30 min after CC14 administration to rats. We have shown that this effect is already manifested within 3 min after CC14 administration, by which time peroxidative decomposition of microsomal lipids can be detected. The time course of the destruction of the liver microsomal Ca2+ pump after CC14 administration to rats was essentially identical to the time course of microsomal lipid peroxidation, as revealed by the appearance of conjugated diene configurations in microsomal lipids.

Use of vitamin e deficient red cells to detect a dialyzable hemolytic factor produced by peroxidizing rat liver microsomes

Abstract The tendency of rat red blood cells to hemolyze in the presence of peroxidizing rat liver microsomes is greatly increased if the red cells are obtained from vitamin E deficient rats. Adequate dietary vitamin E supplementation imparts resistance against hemolysis. Dietary butylated hydroxytoluene or the level of erythrocyte glutathione or total thiols are relatively unimportant factors in determining red cell sensitivity to hemolysis induced by perixiziding microsomes. When separated from peroxidizing microsomes by a dialysis membrane, vitamin E deficient cells are completely hemolyzed. Hemolytically active material can be separated from peroxidized microsomes by dialysis at 0°C.