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Freshly isolated rat hepatocytes were used to study the mechanism of cell death induced by N-hydroxy-2-acetylaminofluorene (N-OH-AAF). Exposure to 1.0 mM N-OH-AAF resulted in more than 90% cell death (as measured by LDH leakage) of hepatocytes isolated from male rats within 6 hr. Only 36% of the hepatocytes isolated from female rats died within this period. When inorganic sulfate was omitted from the incubation medium, a 6 hr exposure to 1.0 mM N-OH-AAF resulted in only 40% cell death of male hepatocytes. These findings are in accordance with the sex difference and sulfation dependence of N-OH-AAF hepatotoxicity observed in the rat in vivo. N-OH-AAF decreased glutathione (GSH) in male hepatocytes in a concentration-dependent manner. This GSH consumption was only partly dependent on the presence of inorganic sulfate. No lipid peroxidation was observed during N-OH-AAF exposure; N-OH-AAF even prevented endogenous and diethyl maleate (DEM)-induced lipid peroxidation. No reduction of free protein thiol groups was found after exposure to N-OH-AAF, even after 75% cell death had occurred. A reduction of protein thiols after N-OH-AAF exposure was observed in GSH depleted hepatocytes (obtained by DEM plus vitamin E pretreatment). Under these conditions N-OH-AAF-induced cell death occurred earlier. Therefore, GSH protects against protein thiol depletion by N-OH-AAF in control cells. N-OH-AAF-induced cell death was preceded by a loss of intracellular ATP. It is concluded, therefore, that neither lipid peroxidation nor depletion of protein thiols, but possibly loss of intracellular ATP, is involved in the sulfation-dependent cytotoxic mechanism of N-OH-AAF in isolated rat hepatocytes.

In order to investigate rat hepatocyte heterogeneity in asialoglycoprotein transport, rats were pretreated with N-hydroxy-2-acetylaminofluorene (N-OH-AAF, 90 mumol/kg, i.v.) to damage zone 1 hepatocytes, or with carbon tetrachloride (CCl4, 2.1 mmole/kg, p.o.) to damage zone 3 hepatocytes. Twenty-four hours after pretreatment, the asialoglycoprotein dog intestinal alkaline phosphatase (As-ALPase) was injected and plasma disappearance and biliary excretion were measured. In addition, the acinar distribution of the hepatic binding of As-ALPase 10 min after injection in vivo, or after incubation of fixed liver sections with As-ALPase in vitro, was determined by enzyme histochemistry. In control rats, a rapid biexponential plasma disappearance was observed and 6.4 +/- 1.5% of the dose was excreted into bile after 60 min. Hepatocyte binding occurred predominantly in zone 3, both after administration in vivo and after incubation with liver sections in vitro. In rats with zone 1 liver damage, both the half-lives of the first and of the second phase were strongly increased, but biliary excretion did not change significantly. Both in vivo and in vitro the relatively weak binding of As-ALPase in zone 1 of the liver was abolished, whereas binding to zone 3 cells was normal or only slightly decreased. After CCl4-pretreatment histochemically detectable binding to zone 3 cells was completely abolished, leaving only the relatively weak binding in zone 1. Unexpectedly, a normal plasma disappearance and biliary excretion rate were found in these rats. The discrepancy between the pharmacokinetic results, which point to a predominant involvement of zone 1 cells in As-ALPase transport, and the enzyme histochemical studies, which show preferential binding of As-ALPase in zone 3, is discussed.