Hypothermia injury/cold‐induced apoptosis—evidence of an increase in chelatable iron causing oxidative injury in spite of low O ₂ ⁻ /H ₂ O ₂ formation

Abstract

When incubated at 4 degrees C, cultured rat hepatocytes or liver endothelial cells exhibit pronounced injury and, during earlier rewarming, marked apoptosis. 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 classical Fenton chemistry, are involved. However, when we measured the Fenton chemistry educt hydrogen peroxide and its precursor, the superoxide anion radical, formation of both had markedly decreased and steady-state levels of hydrogen peroxide did not alter during cold incubation of either liver endothelial cells or hepatocytes. Similarly, there was no evidence of an increase in O2-/H2O2 release contributing to cold-induced apoptosis occurring on rewarming. In contrast to the release/level of O2- and H2O2, cellular homeostasis of the transition metal iron is likely to play a key role during cold incubation of cultured hepatocytes: the hepatocellular pool of chelatable iron, measured on a single-cell level using laser scanning microscopy and the fluorescent indicator phen green, increased from 3.1 +/- 2.3 microM (before cold incubation) to 7.7 +/- 2.4 microM within 90 min after initiation of cold incubation. This increase in the cellular chelatable iron pool was reversible on rewarming after short periods of cold incubation. The cold-induced increase in the hepatocellular chelatable iron pool was confirmed using the calcein method. These data suggest that free radical-mediated hypothermia 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 O2-/H2O2.

Cold ischemic injury of transplanted kidneys: new insights from experimental studies

“…Free iron at higher levels can catalyze the Haber-Weiss reaction, resulting in the generation of toxic hydroxyl radicals. Earlier studies have shown that iron can be toxic in many forms of renal injury (50,68), including cold storage injury (18,32,57,78). Consistent with a role for iron toxicity in cold storage injury, we have recently found the copious release of free iron during cold storage, mainly of microsomal origin (26).…”

Deregulation of iron homeostasis and cold-preservation injury to rat liver stored in University of Wisconsin solution

“…[7][8][9][10] Existing data also show that during cold storage (CS), the LIP expands in isolated hepatocytes and whole kidney and liver. [9][10][11][12] It has been shown that in isolated organs (liver and kidney), an increase in LIP iron may contribute to I/R injury. [12][13][14][15] However, earlier studies that investigated iron-mediated injury to organs during CS mainly focused on the LIP and the attending lipid peroxidation.…”

Low temperature cell pausing: an alternative short-term preservation method for use in cell therapies including stem cell applications

“…Extreme temperature shifts may also induce apoptosis and necrosis (Mathew et al 2004). Viability evaluation, either immediately after hypothermic exposure or following rewarming to normothermic conditions, may give varied results as cells may be programmed for apoptosis upon their return to 37°C or following continuous culture (Rauen et al 2000). A full examination of these properties during and shortly after hypothermic storage has not been executed for a variety of cell types.…”