Phase 1 and 2 metabolism in freshly isolated hepatocytes and subcellular fractions from rat, mouse, chicken and ox livers

By Swales, N.J. - Caldwell, J.

In toxicological studies hepatocytes offer an excellent alternative to whole-animal experiments, provided their metabolic competence has been established. We have compared Phase 1 and 2 metabolism in rat, mouse, chicken and ox liver microsomes and cytosol with freshly isolated hepatocytes. The relative amounts of total cytochrome P450 in microsomes and hepatocytes were equivalent. Rat liver had the highest P450 content while chicken liver had the lowest content (148.2(+/-75.7) and 20.6(+/-11.5) pmol mg-1 hepatocellular protein, respectively). The metabolism of testosterone was assessed to determine selective cytochrome P450 isoenzyme activities. Only two metabolite products were common to all four species, namely 6 beta-hydroxytestosterone (6 beta-OHT) and androstenedione (ASD), which co-eluted with 6-dehydrotestosterone (6DHT). 16 alpha-OHT was present in all incubations except for ox microsomes. The rate of metabolism of testosterone was generally lower in microsomes than hepatocytes, with the exception of the ox, but the pattern and quantity of metabolite formation was similar. The quantity of total products formed was 15- to 27-fold higher in rat and mouse livers than in chicken or ox. The major product formed in freshly isolated hepatocytes from mice and chickens was ASD/6DHT which accounted for 60% and 76% of the total metabolites, respectively. ASD/6DHT formation accounted for only 33% and 17% of the total metabolites formed by rat and ox hepatocytes, respectively. 2 alpha-OHT production occurred in rat and mouse hepatocytes (14% of the total metabolites in rat and 7% in mouse hepatocytes) but was lacking in chicken or ox cells. The stability of P450 isoforms in culture was species-dependent. Rat and mouse hepatocyte cultures lost 54% and 31% of their initial P450 content after 72 h, while there was no loss in chicken hepatocytes over the same period. There was a good correlation between the relative glutathione S-transferase (GST) activities in cytosol and freshly isolated hepatocytes. Mouse liver exhibited highest GST activity (664.2(+/-203.5)) compared with rat, chicken or ox (320.4(+/- 64.0), 341.5(+/-13.9) and 256.3(+/-109.9) nmol min-1 mg-1 cytosolic protein, respectively).