Effects of Sporidesmin on Isolated Liver Cells

Abstract

The mycotoxin sporidesmin has been responsible for major losses in production in New Zealand livestock as the causative agent of the disease facial eczema. In previous studies sporidesmin has been found to be a potent hepatotoxic and inflammatory agent which in livestock and laboratory animals produced hepatocyte and bile duct damage with cessation of bile flow. Isolated rat hepatocytes were prepared by a collagenase perfusion procedure. Yields of approximately 5 x 107 cells/g liver were obtained. Trypan Blue exclusion, respiration, leakage of cytosolic glutathione S-transferase and hormone sensitivity were criteria used to select high viability preparations. Bile acid uptake and efflux in isolated hepatocyte suspensions were monitored by estimating radioactivity in cell free supernatants of aliquots removed at intervals. Uptake of cholate was dependent on hepatocyte numbers and cholate concentration and was blocked by uncouplers. Hepatocytes accumulated taurocholate more rapidly than cholate. Taurocholate efflux was apparently stimulated by uncouplers. Viability of hepatocytes was not significantly altered by incubation of approximately 2 x 107 - 5 x 107 cells/10 ml incubation medium with 0.5 mg sporidesmin for 30-60 min at 37ºC. Under these conditions sporidesmin inhibited cholate uptake and taurocholate efflux. Inhibition of cholate uptake was maximal after 1 min and was not reversed by washing cells free of extracellular sporidesmin. Inhibition was dependent on the cellular sporidesmin concentration. Taurocholate efflux from hepatocytes was apparently stimulated by 0.005-0.05 mg sporidesmin indicating a selective inhibition of uptake. Cholate conjugation was not significantly effected by 0.5 mg sporidesmin. Ultrastructural changes in hepatocytes exposed to 0.5 mg sporidesmin were; loss of surface membrane microvilli, an increase in the zone of organelle free cytoplasm immediately beneath the plasma membrane and some endoplasmic reticulum reorganisation. Lipid droplets with glycogen or similar electron dense particles at the margins were seen in hepatocytes isolated from rats dosed chronically with sporidesmin. An attempt was made to isolate biliary epithelial cells from the white strands of bile duct tissue remaining after mechanical disruption of collagenase perfused liver. Yields of isolated bile duct cells were low therefore it was not possible to observe sporidesmin - bile duct cell interactions in vitro. N-ethylmaleimide (0.1 mM) or dithiothreitol (1 mM) partially protected hepatocytes from sporidesmin (0.5 mg) inhibition of bile acid uptake. Significant protection was not given by other thiols, zinc sulphate, cholesterol, ascorbate or α-tocopherol acetate. Gliotoxin, a dioxodithiapiperazine structurally similar to sporidesmin, had less effect on bile acid uptake than sporidesmin when compared at equal concentrations or equal amounts. Ethynyl estradiol produced an inhibition equivalent to that observed with sporidesmin. When [14C]-sporidesmin was exposed to isolated hepatocytes radioactivity was distributed among the soluble and membrane fractions including a fraction containing the bulk of plasmalemma derived membranes. When total and monomeric actin were measured by inhibition of DNAase-1 sporidesmin did not alter the total actin or G-actin content of hepatocytes. Sporidesmin did not modify the composition of hepatocyte membrane or soluble proteins as analysed by SDS/polyacrylamide gel electrophoresis. The distribution of [14C]-sporidesmin among sheep hepatocyte macromolecules indicated little binding to DNA or RNA and few sporidesmin - macromolecule disulphide linkages. The results are discussed in terms of sporidesmin action on cell membranes and the toxin's effects on bile flow.