CRYOPRESERVATION OF ISOLATED RAT HEPATOCYTES - EFFECTS OF IRON-MEDIATED OXIDATIVE STRESS ON METABOLIC-ACTIVITY

In an attempt to quantitatively evaluate the destructive effects of fr ee radicals on metabolism, freshly prepared and cryopreserved isolated rat hepatocytes were exposed to and incubated with Fe2+ compounds, re putedly inducing oxygen-derived free radicals (OFR) capable of attacki ng the lipid structures of cellular membranes. Malondialdehyde (MDA) f ormation was interpreted as an expression of free radical interaction with polyunsaturated lipids, and in vitro incubations were carried out during the period of constant MDA formation. Protein synthesizing act ivity was evaluated by incubating control hepatocytes and cells previo usly exposed to 100 mu M of Fe2+, to 100 mu M of Fe2+, and 100 mu M of desferrioxamine and to 100 mu M of desferrioxamine alone with 0.1 mu Ci of L-[U-C-14]isoleucine and in the presence of these compounds. Mem brane transport activity was similarly evaluated by following the cell ular uptake of alpha-amino-[1-C-14]isobutyric acid. Protein-synthesizi ng activity of freshly prepared and cryopreserved hepatocytes was not affected by Fe2+ treatment, nor by the additions of the iron chelator desferrioxamine. Amino acid transport, however, was inhibited by 100 m u M of Fe2+, but was effectively neutralized by the simultaneous addit ion of 100 mu M of desferrioxamine. Cryopreserved hepatocytes equally presented a significantly inhibited amino acid transport activity over the incubation period. The results suggest that the metabolic depress ion measured in thawed hepatocytes does not result to any large extent from iron-catalysed OFR effects. When OFR production was deliberately induced, the most significant early change was seen in transmembrane amino acid uptake in both fresh and cryopreserved cells. (C) 1997 Acad emic Press.