Neill H. Stacey

Inhibition of Taurocholate and Ouabain Transport in Isolated Rat Hepatocytes by Cyclosporin A

The use of cyclosporin A in transplantation procedures has been reported to cause hepatotoxicity as evidenced by elevated serum bilirubin and bile salt levels. However, these biochemical abnormalities could also result from interference with hepatic transport processes. This possibility was investigated in the present study in which the effect of cyclosporin A on transport processes was examined in isolated rat liver cells. Taurocholate, ouabain, and alpha-aminoisobutyric acid were selected as compounds known to enter liver cells by distinct active transport systems and cadmium was selected as a substance taken up by a combination of simple and facilitated diffusion. Cyclosporin A was found to cause a dose-related inhibition of both taurocholate and ouabain uptake. On the other hand, the uptake of alpha-aminoisobutyric acid and of cadmium were unaffected by cyclosporin A. These findings indicate a substrate-specific effect of cyclosporin A rather than a general effect on cellular transport. Efflux of taurocholate from preloaded hepatocytes was also inhibited by cyclosporin A. Cyclosporin A caused a decrease in maximum velocity for ouabain uptake with no change in Km. Kinetic analysis for both uptake and efflux of taurocholate showed an unchanged maximum velocity and an increased Km. The data indicate that the ability of liver cells to take up and release bile acids is impaired in the presence of cyclosporin A. These findings provide a possible explanation for the finding of increased serum bile acids during cyclosporin A therapy and suggest that hepatic clearance of other compounds could also be impaired.

Comparison of methods of assessment of metal‐induced lipid peroxidation in isolated rat hepatocytes

There is some controversy about which method of assessment of lipid peroxidation in isolated hepatocytes is most appropriate. The present study was undertaken primarily to compare measurement of concentrations of thiobarbituric acid (TBA) reacting substances with measurement of ethane concentrations in the gas phase of the incubation flask as indicators of lipid peroxidation. Four metal salts, FeCl2, NaVO3, CdCl2, and MnCl2, were selected as agents that interact with the lipid peroxidation process. Furthermore, reduced glutathione (GSH) concentrations and enzyme leakage were assayed to determine whether there was a consistent pattern of interaction between lipid peroxidation and change in GSH concentrations and enzyme leakage. The effects of the metal ions on the concentration of TBA reactants estimated in the whole cell suspension and on the gaseous ethane concentration were similar. However, the assessment of TBA reactants was a little more sensitive, and ethane concentrations continued to climb with incubation time rather than leveling off as observed for TBA reactants. In general, the results of both assays were in good agreement. No consistent pattern of interaction between lipid peroxidation, GSH, and enzyme leakage was discernible in the results of the present study. It is suggested that perhaps the best approach to an experimental situation where lipid peroxidation is thought to be of central importance is measurement of both parameters, TBA reactants and ethane concentrations.

Differential effects of cyclosporin a on the transport of bile acids by human hepatocytes

Cyclosporin A (CsA) treatment has been reported to cause rises in serum bile acids both in humans and rats. It has also been shown to suppress bile flow in situ in rats and inhibit the transport of bile salts by rat hepatocytes. The purpose of this study was to examine the influence of CsA on uptake of radiolabelled cholate (CA), glycocholate (GC) and taurocholate (TC) by isolated human hepatocytes. CsA did not significantly change Vmax for CA uptake [0.23 +/- 0.01 vs 0.25 +/- 0.02 nmol/mg protein/min for control and CsA (10 microM), respectively], but significantly increased Km (37 +/- 2 vs 86 +/- 8 microM). Similarly, Vmax for TC uptake was not affected (0.51 +/- 0.02 vs 0.67 +/- 0.05 nmol/mg protein/min) while Km was significantly increased [46 +/- 3 vs 109 +/- 11 microM for control and CsA (10 microM), respectively]. On the other hand, neither Vmax nor Km for GC uptake was affected by CsA. The data indicate a competitive pattern of inhibition induced by CsA on CA and TC uptake. Furthermore, CsA was found to cause a dose-related inhibition of accumulation of both cholate and taurocholate, but not GC accumulation. None of the concentrations of CsA showed a significant effect on the integrity of the human hepatocytes as assessed by ALT (alanine aminotransferase), AST (aspartate aminotransferase) and LDH (lactate dehydrogenase) release. The findings, in human hepatocytes, are generally consistent with the observations reported from rodent studies. They strongly support the contention that serum bile acid increases in CsA-treated patients are due to interference with the hepatocellular transport and accumulation of particular bile acids.

Inhibition of antibody-dependent cell-mediated cytotoxicity by ethanol

Ethanol has been shown to inhibit spontaneous cell-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity in vitro in a dose-dependent manner. The effect was observed under differing assay conditions such as incubation time and effector: target cell ratio. Acetaldehyde, the primary metabolite of ethanol, did not inhibit these functions of the immune system. A mixture of ethanol and acetaldehyde showed no interaction with respect to spontaneous and antibody-dependent cell-mediated cytotoxicity. These observations may help to explain the apparently increased incidence of infection for individuals who chronically ingest large amounts of alcoholic beverages.

The amelioration of cadmium-induced injury in isolated hepatocytes by reduced glutathione

Although various theories have been proposed to account for the mechanism of cadmium-induced cellular injury, none have received strong, direct support from experimental data. An interference with sulfhydryl groups is one of the proposed mechanisms. The ability of reduced glutathione (GSH) to protect hepatocytes from the toxic effects of cadmium has been investigated. When added just prior to cadmium, GSH has a pronounced protective effect, while an additional 15 min after cadmium resulted in partial protection against the ensuing loss of cellular potassium ion. The protection against loss of aspartate aminotransferase into the medium is pronounced, regardless of whether addition of GSH is at zero time or 15 min after cadmium. Addition of the GSH at either time resulted in reduced levels of cadmium associated with the hepatocytes, and may well account for these protective properties of GSH. Protein-bound sulfhydryl groups showed a small decrease in response to cadmium but only after cell injury had been initiated. These data allow the conclusion that cadmium does not exert its cytotoxic effects by simple, single irreversible interaction with cellular thiol groups. Furthermore, as the added GSH remains extracellular, these data indicate that protection can be afforded at an extracellular level even after intracellular exposure to toxic concentrations of cadmium.

Concentrations of individual serum or plasma bile acids in workers exposed to chlorinated aliphatic hydrocarbons.

Individual serum or plasma bile acid concentrations were measured by high performance liquid chromatography in two groups of workers with differing exposures: to hexachlorobutadiene (HCBD) and a mixture of other chlorinated solvents (SOLVENT) in study A; and trichloroethylene (TCE) in study B. Exposures to HCBD and TCE were associated with highly significant increases in a number of individual and summed bile acid measures, with a dose effect relation shown for HCBD. Exposure to SOLVENT was associated with significant decreases in three bile acid measures but this may have been due to misclassification of exposure. No association was found between any of the exposures and any of the standard tests of liver function. This preliminary study suggests that some chlorinated hydrocarbons are associated with raised bile acid concentrations in the blood of exposed workers. It may be that the changes in such concentrations reflect early and small disturbances of liver function. The significance and mechanism of the changes are yet to be determined.

Individual serum bile acids as early indicators of carbon tetrachloride- and chloroform-induced liver injury☆

Individual serum bile acids (SBA) are emerging as potentially useful early indicators of liver injury. This study was undertaken to compare the usefulness of individual SBA with the routinely used assays for detecting the effects of the hepatotoxicants carbon tetrachloride (CCl4) and chloroform (CHCl3). Serum samples were assayed for liver injury by determination of alanine aminotransferase (ALT), aspartate amino-transferase (AST), alkaline phosphatase (ALP), bilirubin and total bile acid (by enzymatic kit). These results were compared with levels of individual SBA measured by high performance liquid chromatography (HPLC). Liver samples from CCl4-treated rats were taken for light and electron microscopic examination. The highest dose for each chemical caused increases in serum ALT and AST but not ALP. Chloroform at the highest dose increased bilirubin. Total SBA levels as assayed by the kit were elevated in response to CCl4 and CHCl3 at doses below which serum enzymes and bilirubin were increased. Some individual SBA were increased at a still lower dose for each of these two chlorinated solvents. At the lowest dose of CCl4 tested no consistent light microscopic or ultrastructural changes were found. At all the higher doses periacinar cells displayed typical accumulation of lipid droplets and degranulation and dilation of rough endoplasmic reticulum. The extent of the ultrastructural changes were dose-dependent. Thus individual SBA assayed by HPLC may be considered as a very sensitive indicator of liver injury induced by the classical hepatotoxicants carbon tetrachloride and chloroform.

Elevation of individual serum bile acids on exposure to trichloroethylene or α-naphthylisothiocyanate

Rats were treated with trichloroethylene via intraperitoneal (ip) injection or inhalation, or with ip alpha-naphthylisothiocyanate (ANIT). Serum samples were assayed for indices of liver injury including alanine aminotransferase (ALT), sorbitol dehydrogenase (SDH), alkaline phosphatase (AP), and bilirubin. Liver from some rats was examined for histological appearance. These data were compared to levels of individual serum bile acids (SBA) determined by high-performance liquid chromatography. Trichloroethylene and ANIT, each at their highest dose only, caused elevations in ALT, but not SDH or AP. The highest dose of ANIT also caused elevated serum bilirubin and cholangitis in the liver. SBA were also elevated in response to both trichloroethylene and ANIT, but at doses below those at which other parameters of liver function were increased. For both chemicals, taurocholic acid was the most sensitive of the bile acids assayed, being elevated at the lowest doses tested of 10 mumols/kg for trichloroethylene and 5 mumols/kg for ANIT. As the doses were raised more of the individual bile acids showed increases. On exposure to trichloroethylene via inhalation taurocholic acid was one of two SBA to show elevation. Thus, both trichloroethylene and ANIT cause elevation in SBA at doses well below those which cause an increase in standard indicators of liver dysfunction. This suggests that SBA and perhaps taurocholic acid, in particular, may provide a sensitive tool for studying hepatobiliary effects of chemicals.

Interference with hepatocellular substrate uptake by 1,1,1-trichloroethane and tetrachloroethylene☆

The effects of chlorinated aliphatic hydrocarbon solvent exposure on hepatocellular transport of some model substrates have been investigated. Exposure of isolated hepatocytes to 1,1,1-trichloroethane or tetrachloroethylene resulted in suppression of uptake of taurocholate, ouabain, and 2-aminoisobutyric acid but not CdCl2 or 3-O-methyl-D-glucose. The effect was clearly evident at noncytotoxic concentrations, as indicated by the lack of intracellular enzyme leakage and unaltered intracellular K+ ion concentration. Moreover, the ultrastructure of solvent-exposed hepatocytes was similar to that of control cells, except for a reduction in membrane microvilli. The suppression of uptake was reversible provided that sufficient time was allowed for the cells to recover. The mechanism of this inhibition may be associated with energy-linked processes, as uptake of taurocholate, ouabain, and 2-aminoisobutyric acid is energy requiring while uptake of CdCl2 and 3-O-methyl-D-glucose is not. Cellular ATP was reduced in a dose-dependent manner, but a marked depletion occurred only at cytotoxic concentrations. Na(+)-K(+)- and Mg2(+)-ATPase activities in hepatocyte plasma membrane preparations were also inhibited by solvent exposure. The data suggest that 1,1,1-trichloroethane and tetrachloroethylene interfere specifically with energy-dependent hepatic transport functions and that a decrease in ATP levels and/or inhibition of cell membrane ATPases may be the mechanism.

Protective effects of dithiothreitol on cadmium-induced injury in isolated rat hepatocytes.

The biochemical mechanism involved in cadmium-induced cellular injury remains to be elucidated. Various theories have been proposed to account for this action, one of which is an interaction with reduced sulfhydryl (SH) groups of membranes. The ability of a known SH group reducing agent, dithiothreitol (DTT), to interact with cadmium-induced damage to isolated hepatocytes was investigated. Cadmium chloride produced a cytotoxic response similar to that previously demonstrated. When added just prior to cadmium chloride, DTT was found to protect against the metal-induced injury. If the cells were first exposed to cadmium for 15 min before DTT was added, it was found that the cells could be rescued from the ensuing expression of toxicity due to cadmium. This result was regardless of the fact that by 15 min the cells had accumulated a quantity of cadmium that was associated with cell injury. Furthermore, DTT did not exert its action by decreasing the amount of cadmium accumulation by the cells. Under some experimental conditions, an increased cadmium uptake in the presence of DTT was observed. While it is not clear as to whether the chelating or thiol reductive properties of DTT are of prime importance in the demonstrated protection, the data show that toxicity is not due to a simple, single irreversible interaction between cadmium and membrane SH groups.