Macrophage enhancement of galactosamine hepatotoxicity using a rat hepatocyte culture system

Prior administration of endotoxin to rats is known to aggravate the hepatotoxicity of galactosamine. It has been proposed that this exacerbation occurs as a result of the release of cytokines and other humoral factors by resident macrophages (Kupffer cells). In order to study this phenomenon we have utilized a co-culture system consisting of rat activated peritoneal macrophages and rat hepatocytes. Peritoneal macrophages were isolated and cultured; LPS was added as a macrophage activator 16 hours later. Rat hepatocytes were isolated and plated in Transwell COL inserts, which were placed in wells with and without activated macrophages. Cytotoxicity was determined 24 hours later by measuring lactate dehydrogenase (LDH) leakage into the culture medium. In the presence of activated macrophages an approximate 3-fold increase in galactosamine-induced hepatocyte toxicity was observed, as compared to the toxicity in hepatocytes cultured alone. Using this co-culture system, we examined the role of leukotriene D4 (LTD4) and nitric oxide (NO) as mediators of this enhancement. Addition of either LTD4 or NO to hepatocytes cultured alone did not exacerbate galactosamine toxicity. Furthermore, addition of the LTD4 receptor antagonist SK & F 104353 (50 microM) or the NO synthase inhibitor N-monomethyl-L-arginine (1.0 mM) to macrophage/hepatocyte co-cultures did not attenuate the enhanced galactosamine hepatocyte toxicity in the co-cultures. Collectively, these data indicate that this co-culture system will be useful in examining the mechanism of macrophage enhancement of chemical-induced hepatoxicity and, further, suggest that LTD4 and NO may not be involved in the exacerbation of galactosamine toxicity to hepatocyte cultures.