Rauen U, Petrat F, Li T, De Groot H
Institut für Physiologische Chemie, Universitätsklinikum, D-45122 Essen, Germany.
FASEB J. 2000 Oct;14(13):1953-64. doi: 10.1096/fj.00-0071com.
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.
在4℃孵育时，培养的大鼠肝细胞或肝内皮细胞会出现明显损伤，并且在早期复温过程中会发生显著凋亡。这两个过程均由活性氧介导，铁螯合剂的显著保护作用以及其他各种抗氧化剂提供的保护表明，经典芬顿化学产生的羟基自由基参与其中。然而，当我们测量芬顿化学反应物过氧化氢及其前体超氧阴离子自由基时，两者的生成均显著减少，并且在肝内皮细胞或肝细胞的冷孵育过程中，过氧化氢的稳态水平并未改变。同样，没有证据表明复温时O2-/H2O2释放增加导致冷诱导的凋亡。与O2-和H2O2的释放/水平相反，过渡金属铁的细胞内稳态在培养的肝细胞冷孵育过程中可能起关键作用：使用激光扫描显微镜和荧光指示剂酚绿在单细胞水平上测量的可螯合铁的肝细胞池，在冷孵育开始后90分钟内从3.1±2.3微摩尔（冷孵育前）增加到7.7±2.4微摩尔。在短时间冷孵育后复温时，细胞内可螯合铁池的这种增加是可逆的。使用钙黄绿素法证实了冷诱导的肝细胞内可螯合铁池增加。这些数据表明，自由基介导的低温损伤/冷诱导的凋亡主要是由细胞铁稳态的改变/细胞内可螯合铁池的快速增加引起的，而不是由O2-/H2O2生成增加引起的。
