William J. Brattin

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.

Estimation of relative bioavailability of lead in soil and soil-like materials using young swine

In this article we summarize the results of a series of studies that measured the relative bioavailability (RBA) of lead in a variety of soil and soil-like test materials. Reference material (Pb acetate) or Pb-contaminated soils were administered orally to juvenile swine twice a day for 15 days. Blood samples were collected from each animal at multiple times during the course of the study, and samples of liver, kidney, and bone were collected at sacrifice. All samples were analyzed for Pb. We estimated the RBA of a test material by fitting mathematical models to the dose–response curves for each measurement end point and finding the ratio of doses that gave equal responses. The final RBA for a test material is the simple average of the four end point–specific RBA values. Results from 19 different test materials reveal a wide range of RBA values across different exposure materials, ranging from 6 to 105%. This variability in RBA between different samples highlights the importance of reliable RBA data to help improve risk assessments for Pb in soil. Although the RBA value for a sample depends on the relative amounts of the different chemical and physical forms of Pb present, data are not yet adequate to allow reliable quantitative predictions of RBA from chemical speciation data alone.

Evaluation of Small Arms Range Soils for Metal Contamination and Lead Bioavailability

Although small arms ranges are known to be contaminated with lead, the full extent of metal contamination has not been described, nor has the oral bioavailability of lead in these soils. In this work, soil samples from ranges with diverse geochemical backgrounds were sieved to <250 microm and analyzed for total metal content. Soils had consistently high levels of lead and copper, ranging from 4549 to 24 484 microg/g and 223 to 2936 microg/g, respectively, while arsenic, antimony, nickel, and zinc concentrations were 100-fold lower. For lead bioavailability measurements, two widely accepted methods were used: an in vivo juvenile swine relative bioavailability method measuring lead absorption from ingested soils relative to equivalent lead acetate concentrations and an in vitro bioaccessibility procedure which measured acid-extractable lead as a percent of total lead in the soil. For eight samples, the mean relative bioavailability and bioaccessibility of lead for the eight soils was about 100% (108 +/- 18% and 95 +/- 6%, respectively) showing good agreement between both methods. Risk assessment and/or remediation of small arms ranges should therefore assume high bioavailability of lead.

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+.

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 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 intra-hepatocellular 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.