DIFFERENCES IN BASAL AND HYPEROXIA-ASSOCIATED HO EXPRESSION IN OXIDANT-RESISTANT HAMSTER FIBROBLASTS

Heme oxygenase (HO) is the rate-limiting enzyme in the production of b ilirubin from heme, and HO-1 is its inducible isoenzyme. In the metabo lic pathway of HO a potential oxidant, heme, is degraded, a potential antioxidant, bilirubin: is generated, and a potent sequestering agent of redox active iron, ferritin, is thought to be coinduced. Therefore. the sum of the reactions of HO may be useful in antioxidant defense. To explore the role of HO in protection against oxidative stress, we e xamined HO-1 expression in Chinese hamster fibroblasts (HA-1) as well as stable hydrogen peroxide (H2O2)-resistant (OC-14) and 95% O-2-resis tant (O(2)R95) valiant cell lines derived from HA-1, after exposure to 72 h of hyperoxia (95% O-2-5% CO2). Total HO activity, HO-1. protein, and HO-1 mRNA steady-state levels were assessed before exposure and d aily during exposure to hyperoxia. Controls were exposed to 95% air-5% CO2. Confluent monolayers of O(2)R9.5 and OC-14 cells had increased b asal immunoreactive HO-1 protein levels relative to HA-1 cells when th e cells were grown in normoxia, and O(2)R95 had higher total basal HO activity. When exposed to hyperoxia for up to 3 days, O(2)R95 cells, w hich were resistant to oxygen-induced killing, did not show induction of HO-1 mRNA or increased immunoreactive protein, whereas OC-14 and HA -1, which were relatively more sensitive than O(2)R95 to oxygen-relate d cytotoxicity, demonstrated significant increases in HO-1 expression during exposure to hyperoxia. Basal ferritin protein levels were highe st in the O(2)R95 cells, intermediate in OC-14, and lowest in HA-1, bu t ferritin protein did not increase further, with hyperoxic exposure, in any of the cell lines. We conclude that increased constitutive HO-1 expression is associated with resistance to hyperoxia, whereas induct ion of HO-1 mRNA is an index of oxidative injury, since it only occurs after cells have sustained cytotoxic injury. We also conclude that in creased ferritin expression does not necessarily accompany increased H O-1 expression in oxidant stress. We speculate that HO-1 plays a role in protection against hyperoxic damage.