Inhibition of galactosamine cytotoxicity in an in vivo/in vitro hepatocellular toxicity model.

A combined in vivo/in vitro model of galactosamine hepatotoxicity was employed to test whether previously reported cytoprotective actions of cystamine administration on galactosamine-induced hepatic injury in vivo could be attributed to a direct action of cystamine on toxicant-challenged hepatocytes. In this model, male Sprague-Dawley rats received a 400 mg/kg galactosamine challenge via intraperitoneal injection 1 hr prior to portal vein cannulation for hepatocyte isolation. Isolated cells are established in monolayer culture and galactosamine-induced cellular injury is then expressed over the ensuing 24-48 hr in culture. Consistent with the biochemical basis of galactosamine-induced hepatocellular injury in vivo, cytotoxicity could be prevented by in vitro uridine treatments within 3 hr of the in vivo galactosamine challenge, but not when added 12 hr later. Cystamine, in contrast, exhibited a cytoprotective effect even when added to cultures 12 hr after the in vivo toxicant challenge. Post-toxicant cytoprotection by cystamine in vitro was concentration dependent and did not produce an alteration of hepatocyte nonprotein sulfhydryl content. Post-toxicant cytoprotection by uridine and cystamine in this in vivo/in vitro model of toxicity were fully consistent with in vivo protection from galactosamine-induced necrosis by these agents. This model eliminates potential "extrahepatic" mechanisms for cystamine's hepatoprotective effect and demonstrates a direct cytoprotective action on galactosamine-challenged hepatocytes.

Isolation and maintenance of monolayer hepatocytes from the livers of toxin-treated rats.

Hepatocytes were isolated from male Sprague-Dawley rats 30 min after oral challenge with carbon tetrachloride (CCl4), or 1 hr after ip injection of D(+)-galactosamine (GAL). Cell preparations of comparable yield and initial viability were obtained from toxin-treated and respective control animals with equivalent 1-hr attachment efficiencies to tissue culture plastic. Cells from toxin-treated rats exhibited significant degeneration over 48 hr in culture. This degeneration included loss of viable cell density on the monolayer and increased lactate dehydrogenase activity in the culture media compared to controls. Toxicity expressed in culture was dose dependent for both CCl4 (0.1-2.5 ml/kg) and GAL (100-400 mg/kg). Loss of cell viability in vitro and ultrastructural degeneration followed a time course consistent with hepatocellular necrosis produced by these agents in vivo. As a model for studying late occurring events in the progression from initiation of toxic cell injury to cell death, this methodology offers several potential advantages over strict in vivo or in vitro models. The analytical advantages of an in vitro cell model are incorporated with initiation of injury in vivo by physiologically relevant doses of hepatotoxins. Limited bioactivating potential of monolayer hepatocytes, and time course limitations of suspension hepatocytes for toxicity studies, are also circumvented in this model.