Cryopreservation of Isolated Rat Hepatocytes: Effects of Iron-Mediated Oxidative Stress on Metabolic Activity

Abstract

In an attempt to quantitatively evaluate the destructive effects of free radicals on metabolism, freshly prepared and cryopreserved isolated rat hepatocytes were exposed to and incubated with Fe2+compounds, reputedly inducing oxygen-derived free radicals (OFR) capable of attacking the lipid structures of cellular membranes. Malondialdehyde (MDA) formation was interpreted as an expression of free radical interaction with polyunsaturated lipids, andin vitroincubations were carried out during the period of constant MDA formation. Protein synthesizing activity was evaluated by incubating control hepatocytes and cells previously exposed to 100 μMof Fe2+, to 100 μMof Fe2+, and 100 μMof desferrioxamine and to 100 μMof desferrioxamine alone with 0.1 μCi ofl-[U-14C]isoleucine and in the presence of these compounds. Membrane transport activity was similarly evaluated by following the cellular uptake of α-amino-[1-14C]isobutyric acid. Protein-synthesizing 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 μMof Fe2+, but was effectively neutralized by the simultaneous addition of 100 μMof desferrioxamine. Cryopreserved hepatocytes equally presented a significantly inhibited amino acid transport activity over the incubation period. The results suggest that the metabolic depression 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.