Effects of copper on rat hepatoma HTC cells and primary cultured rat hepatocytes

Cellular responses to copper, applied in concentrations varying from 0.5 to 200 microM Cu2+, were investigated in two different cell types: rat hepatoma cells (HTC) and primary cultured rat hepatocytes. Accumulation of 64Cu, copper (AAS) levels, cellular viability parameters (cell growth and proliferation, LDH leakage, total cell protein, K+ uptake, and ATP levels), and cell toxicity parameters (metallothionein (MT), glutathione (GSH) and superoxide dismutase (SOD)) were examined over 24 hr incubation periods. Accumulation of radiolabeled copper (applied copper concentrations: 15-200 microM Cu2+) showed a four-fold increase in HTC cells (0.88-3.45 nmol Cu/mg cell protein) and a three-fold increase in hepatocytes (4.94-14.66 nmol Cu/mg cell protein), although quantitative uptake in HTC cells was five times lower. Most of the copper accumulated in the hepatoma cells and hepatocytes was found predominantly in the particulate fraction (i.e., cell membranes and organelles), while only a small quantity was present in the soluble fraction (cell cytosol). Metallothionein concentrations in HTC cells were increased from 43 pmol/mg cell protein (0.5 microM Cu2+ application) up to 223 pmol/mg cell protein (200 microM Cu2+ application), whereas MT in rat hepatocytes were elevated from 139 pmol/mg cell protein to 546 pmol/mg cell protein over the same range of administered Cu2+. Metallothionein synthesis rendered both cell types well equipped to deal with increasing intracellular copper levels. In hepatocytes however, MT synthesis resulted in decreasing non-MT-associated copper levels in the cytosol for Cu administrations up to 100 microM. Above that point however, MT failed to stay in line with increasing cytosolic Cu levels, resulting in cytotoxic effects shown by changes in cell viability and GSH/SOD levels. In HTC cells MT synthesis suppressed the free Cu levels in the cytosol to below 0.1 nmol Cu/mg cell protein over the total range of copper concentrations applied. The results presented indicate that hepatoma HTC cells are more capable of dealing with high accumulated Cu levels than the better established rat hepatocytes. Furthermore, it is clear that comparison of these two cell types regarding their ability to respond on (sub)toxic Cu should be discussed with full consideration for the copper applications involved.