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Maintenance of Zinc-Dependent Hepatic Functions in Rat Hepatocytes Cultured in Medium without Added Zinc¹

Food Science and Human Nutrition Department, University of Florida, Gainesville, Florida 32611

Hepatocytes were cultured with Waymouth's media containing zinc at concentrations of 1 (the endogenous zinc concentration of basal medium), 16 and 48 µmol 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 extra-cellular conditions. Basal medium had no effect on cell zinc, metallothionein (MT) or MTmRNA for up to 48 h but reduced -aminolevulinic acid dehydratase (-ALA-D) activity to 75% of the initial level by 3 h. The addition of zinc at 16 or 48 µmol Zn/L during the initial 3 h of culture did not prevent the decrease in -ALA-D activity. Reintroducing zinc at concentrations of 16 or 48 µmol 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 -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 µmol Zn/L) medium to maintain cell zinc content and some zinc-dependent functions underscores the difficulty of producing zinc deficiency in primary hepatocyte culture.

KEY WORDS: • rats • hepatocytes • metallothionein • -aminolevulinic acid dehydratase • zinc

¹ This research was supported by National Institutes of Health Grant DK 31651 from the National Institute of Diabetes and Digestive and Kidney Diseases, U.S. Department of Agriculture Predoctoral Fellowship, and Boston Family Endowment Funds.

² To whom correspondence should be addressed.

Manuscript received 13 August 1990. Revision accepted 5 November 1990.

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