Hepatotoxicity due to clofibrate is oxygen-dependent in the perfused rat liver

Toxicity of clofibrate, a hypolipidemic drug, was assessed in livers from fasted rats perfused in both the anterograde and the retrograde directions. Oxygen uptake decreased steadily following infusion of clofibrate (15 mM) and was diminished by about 40% in 15 min. Cell damage, assessed by the appearance of lactate dehydrogenase (LDH) in the effluent perfusate, began within 20 min. Maximal values for LDH release into perfusate were around 250 U/g/hr after perfusion with clofibrate for 40 min. Inhibition of oxygen uptake and release of LDH into the perfusate was dose-dependent (half-maximal effect = ca. 12 mM clofibrate). Nearly 90% of hepatocytes in oxygen-rich, periportal regions but only about 30% in oxygen-poor, pericentral areas took up trypan blue, an indicator of irreversible cell death, following perfusion with clofibrate in the anterograde direction. In contrast, when livers were perfused in the retrograde direction, 85% of cells in upstream, oxygen-rich pericentral regions were damaged whereas only about 30% in downstream areas were stained. When local oxygen tension was lowered by reducing the flow rate to one-quarter of normal, trypan blue uptake in periportal areas was diminished nearly completely (ca. 5% of cells were stained). Incubation in vitro of isolated cylinders of periportal and pericentral tissue with clofibrate at 800 or 200 microM oxygen led to about three times greater LDH release in incubations carried out at high than at low oxygen tension. This experiment led us to rule out the involvement of clofibrate delivery in the mechanism of zone-specific toxicity. Subsequently, local rates of oxygen uptake were measured using miniature oxygen electrodes placed on the liver surface. Clofibrate decreased oxygen uptake about 30% in oxygen-rich, periportal regions of the liver lobule, yet had no effect on respiration in downstream, pericentral areas. These phenomena can best be explained by a direct effect of clofibrate on active mitochondria in periportal regions of the liver lobule where oxygen uptake predominates, since state 3 but not state 4 rates of respiration were inhibited by clofibrate in isolated mitochondria (half-maximal effect = ca. 1.8 mM clofibrate). Thus, toxicity of clofibrate in upstream, periportal areas of the liver lobule is dependent on local oxygen tension and affects actively respiring mitochondria. This may lead to local cell death and be responsible for initiating a sequence of events leading to the well-known carcinogenic effects of this compound.