MACROPHAGE ENHANCEMENT OF GALACTOSAMINE HEPATOTOXICITY USING A RAT HEPATOCYTE CULTURE SYSTEM

Prior administration of endotoxin to rats is known to aggravate the he patotoxicity of galactosamine. It has been proposed that this exacerba tion occurs asa result of the release of cytokines and other humoral f actors by resident macrophages (Kupffer cells). In order to study this phenomenon we have utilized a co-culture system consisting of rat act ivated peritoneal macrophages and rat hepatocytes. Peritoneal macropha ges were isolated and cultured; LPS was added as a macrophage activato r 16 hours later. Rat hepatocytes were isolated and plated in Transwel l(R)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 pr esence of activated macrophages an approximate 3-fold increase in gala ctosamine-induced hepatocyte toxicity was observed, as compared to the toxicity in hepatocytes cultured alone. Using this coculture system, we examined the role of leukotriene D-4 (LTD(4)) and nitric oxide (NO) as mediators of this enhancement. Addition of either LTD(4) or NO to hepatocytes cultured alone did not exacerbate galactosamine toxicity. Furthermore, addition of the LTD(4) receptor antagonist SK and F 10435 3 (50 mu M) or the NO synthase inhibitor N-monomethyl-L-arginine (1.0 mM) to macrophage/hepatocyte co-cultures did not attenuate the enhance d galactosamine hepatocyte toxicity in the co-cultures. Collectively, these data indicate that this co-culture system will be useful in exam ining the mechanism of macrophage enhancement of chemical-induced hepa totoxicity and, further, suggest that LTD(4) and NO may not be involve d in the exacerbation of galactosamine toxicity to hepatocyte cultures .