Halothane-induced cytotoxicity to rat centrilobular hepatocytes in primary culture is not increased under low oxygen concentration

Abstract

Background: Halothane can be metabolized by both oxidative and reductive pathways in the liver. This anesthetic can induce direct liver injury preferentially localized in centrilobular areas, probably in relation with lower oxygen tension. The reductive pathway has been related to liver damage; however, a correlation between lower oxygen concentration in centrilobular areas, the extent of reductive metabolism of halothane, and the degree of liver injury has not yet been demonstrated. This study was designed to better evaluate the toxicity of the reduced metabolites by using centrilobular and periportal rat hepatocyte subpopulations. Methods: Adult rat hepatocytes, either as whole cell preparations or after separation in centrilobular and periportal cell subpopulations, were placed in primary culture and exposed to either 2% or 4% halothane under various oxygen concentrations. The enriched centrilobular hepatocyte subpopulations isolated by the digitonin-collagenase method were characterized by immunolocalization of glutamine synthetase. Three oxygen concentrations were tested: 5%, 20%, and 95%, and the main parameters measured were cell viability and fluoride ion formation. Results: Viability of centrilobular hepatocytes was similar under 5% and 20% O2, but the unpurified hepatocyte population was more susceptible to 5% O2 (P < 0.01). Significantly higher cytochrome P-450 content was found in whole hepatocyte populations under 5% versus 20% oxygen, indicating that centrilobular hepatocytes that contained higher cytochrome P-450 monooxygenase activities were less sensitive to low oxygen concentration. Halothane toxicity to centrilobular hepatocytes was enhanced under 95% versus 20% O2 (P-0.05). By contrast, no significant difference was observed when the cells were maintained under 5% O2, although fluoride ions, indicative of reductive metabolism of halothane, were found in much higher amounts in the culture medium. Moreover, under 20% O2, halothane toxicity was significantly greater in centrilobular versus unpurified hepatocytes (P < 0.05). Conclusions: Isolated centrilobular hepatocytes appear to be more sensitive to halothane than their periportal counterparts in vitro. However, the authors' results support the conclusion that increased reductive metabolism of halothane induced by decreasing oxygen concentration is not a critical parameter for the occurrence of liver damage in these cells.