Maintenance of zinc-dependent hepatic functions in rat hepatocytes cultured in medium without added zinc.

Hepatocytes were cultured with Waymouth's media containing zinc at concentrations of 1 (the endogenous zinc concentration of basal medium), 16 and 48 mumols Zn/L to examine the effects of extracellular zinc on a variety of zinc-related functions. The zinc concentrations were chosen with the intention of simulating zinc-deficient, adequate and excess extracellular conditions. Basal medium had no effect on cell zinc, metallothionein (MT) or MTmRNA for up to 48 h but reduced delta-aminolevulinic acid dehydratase (delta-ALA-D) activity to 75% of the initial level by 3 h. The addition of zinc at 16 or 48 mumols Zn/L during the initial 3 h of culture did not prevent the decrease in delta-ALA-D activity. Reintroducing zinc at concentrations of 16 or 48 mumols Zn/L to hepatocytes after the initial 3 h of culture in basal medium significantly increased cell zinc, MT and MTmRNA levels and fully restored delta-ALA-D activity by 24 h. Medium zinc had no apparent effect on membrane integrity assessed as leakage of lactate dehydrogenase activity into culture media or de novo protein synthesis as examined by two-dimensional gel electrophoresis of 35S-labeled proteins. Hepatocytes cultured in basal medium resisted losses in cell zinc concentration even when EDTA and bovine serum albumin were present in culture medium. Kinetic experiments using 65Zn suggest hepatocytes maintain zinc concentrations by reducing zinc efflux. The ability of hepatocytes cultured in basal (1 mumol Zn/L) medium to maintain cell zinc content and some zinc-dependent functions underscores the difficulty of producing zinc deficiency in primary hepatocyte culture.