Effects of chlorinated hydrocarbons on cellular volume regulation in slices of rat liver.

The effects of carbon tetrachloride and 1,2-dichloroethane (1,2DCE) on the recovery of slices of rat liver from cellular swelling in vitro were studied. Slices took up water during pre-incubation at 1 degrees C, then cellular volume and ultrastructure were rapidly restored during subsequent incubation at 38 degrees C. Ouabain (2 mm) inhibited water extrusion by less than 50%, while inducing formation of peri-canalicular vesicles, apparently derived from the Golgi apparatus. Neither CCl(4) nor 1,2DCE (up to 10 mm) affected the initial extrusion of water at 38 degrees C in the absence of ouabain, but renewed swelling occurred after 60 min with either agent; this was associated with loss of membrane selectivity and some histological damage. By contrast, 1,2DCE inhibited water extrusion in the presence of ouabain after less intensive exposure, for example with 5 mm-1,2DCE for 60 min or 10 mm for 15-30 min. With ouabain present, 1,2DCE (10 mm) caused marked swelling of the endoplasmic reticulum, reduced the peri-canalicular vesicles seen with ouabain alone and reduced the formation of canalicular microvilli. Both CCl(4) and 1,2DCE inhibited the ATP-dependent accumulation of Cl(-) by isolated vesicles of the Golgi apparatus, The delayed swelling of hepatocytes at high concentrations of 1,2DCE and CCl(4) in the absence of ouabain is probably a non-specific consequence of membrane damage. By contrast, 1,2DCE specifically inhibits the ouabain-resistant extrusion of water, possibly by interfering with a postulated mechanism for the exocytotic expulsion of water.

Effects of monensin on ATP levels and cell functions in rat liver and lung in vitro.

Effects of the proton-alkali cation-exchanging ionophore, monensin, on aspects of cellular metabolism and ionic exchanges have been studied in rat tissues in vitro. Incubation of liver slices at 38 degrees C with 0.1 microM monensin induced time-dependent vesiculation, initially in the Golgi region, reduction of ATP content and of protein synthesis. At 1 microM, monensin also reduced net, active movements of K+, Na+, Cl- and water in liver slices and inhibited state 3 respiration in isolated mitochondria. The respiratory inhibitor, amytal, similarly reduced ATP content and protein synthesis at concentrations lower than those inhibiting ion transport in slices. Low concentrations of monensin (0.1-1.0 microM) had similar effects on ATP and ion transport in slices of adult lung. By contrast, late-fetal liver and lung were much less sensitive to monensin; in these tissues, glycolysis sustained substantial levels of ATP. Monensin also induced vesiculation of the Golgi apparatus in fetal lung cells. It is concluded that by lowering ATP levels, monensin can markedly alter various metabolic activities in those cells which depend primarily on oxidative phosphorylation for their metabolic energy.

Comparison of metabolic effects of carbon tetrachloride and 1,2-dichloroethane added in vitro to slices of rat liver.

Carbon tetrachloride and 1,2-dichloroethane (1,2DCE) were added in vitro to freshly prepared slices of rat liver and the time- and concentration-dependence of their toxic effects on several metabolic parameters determined. With each agent, the most sensitive effect was an increase of malondialdehyde production by a microsomal preparation isolated from the treated slices. The next most sensitive parameter was the inhibition of amino acid incorporation into slice proteins, followed by inhibition of net K(+) accumulation and the induction of early necrotic changes, as indicated by loss of histological staining with azure II. Substantially greater exposures were required to reduce cellular ATP and to initiate entry of Ca(2+). This sequence was similar with both agents, but CCl(4) was the more potent in each case. When added in combinations of submaximally effective concentrations, the two agents produced at least additive inhibitions of protein synthesis and K(+) accumulation. We conclude that metabolic effects in liver slices can be a useful in vitro test for potential toxicity of chlorinated hydrocarbons. Amino acid incorporation and K(+) transport are the most convenient indicator systems, combining considerable sensitivity to relatively low levels of exposure with convenience of measurement.