Hypothermia injury/cold-induced apoptosis - evidence of an increase in chelatable iron causing oxidative injury in spite of low O-2(-)/H2O2 formation

When incubated at 4 degrees C, cultured rat hepatocytes or liver endothelia l cells exhibit pronounced injury and, during earlier rewarming, marked apo ptosis. Both processes are mediated by reactive oxygen species, and marked protective effects of iron chelators as well as the protection provided by various other antioxidants suggest that hydroxyl radicals, formed by classi cal Fenton chemistry, are involved. However, when we measured the Fenton ch emistry educt hydrogen peroxide and its precursor, the superoxide anion rad ical, formation of both had markedly decreased and steady-state levels of h ydrogen peroxide did not alter during cold incubation of either liver endot helial cells or hepatocytes. Similarly, there was no evidence of an increas e in O-2(-)/H2O2 release contributing to cold-induced apoptosis occurring o n rewarming. In contrast to the release/ level of O-2(-) and H2O2, cellular homeostasis of the transition metal iron is likely to play a key role duri ng cold incubation of cultured hepatocytes: the hepatocellular pool of chel atable iron, measured on a single-cell level using laser scanning microscop y and the fluorescent indicator phen green, increased from 3.1 +/- 2.3 mu M (before cold incubation) to 7.7 +/- 2.4 mu M within 90 min after initiatio n of cold incubation. This increase in the cellular chelatable iron pool wa s reversible on rewarming after short periods of cold incubation. The cold- induced increase in the hepatocellular chelatable iron pool was confirmed u sing the calcein method. These data suggest that free radical-mediated hypo thermia injury/cold-induced apoptosis is primarily evoked by alterations in the cellular iron homeostasis/a rapid increase in the cellular chelatable iron pool and not by increased formation of O-2(-)/H2O2.