Heme oxygenase (HO) is the rate limiting enzyme in bilirubin production. The HO-1 isoform is inducible by a variety of oxidant stresses including hyperoxia(1). In its reaction, heme, a prooxidant is degraded, bilirubin, an antioxidant is formed(2). Thus, the overall effect of HO may be to protect against oxidative stress. Previous results from rat fibroblasts transfected with an expression vector containing the rat HO-1 cDNA have led us to speculate that there may be a beneficial threshold of HO-1 overexpression. To further investigate this hypothesis, we have established a variable overexpression model of HO-1 using the Tet-Off™ Gene Expression System (Clontech) which is based on the tetracycline resistance system found in E. coli(3). In this system, gene expression is controlled by the interaction of a tet-responsive transcriptional activator protein and a tet-responsive element where gene expression is tightly regulated by varying concentrations of either tetracycline or doxycycline (Dox) in a dose dependent manner. After insertion of the full length rat HO-1 cDNA into the pBI-L response plasmid, two consecutive stable transfections were performed and >60 stable colonies were screened in order to find a cell line allowing for regulated variable HO-1 overexpression. This cell line was grown in 75cm flasks to >80% confluence. To verify HO-1 overexpression, total HO activity was measured by gas chromatography and HO-1 protein was assessed by Western analysis. By varying the concentration of Dox between 0 and 10ng/ml, we were able to regulate HO-1 expression between 1.5 and 20 fold. Once confluent, cell lines expressing a range of HO-1 protein were exposed to hyperoxia (95% O2/5% CO2) for 72 h and non-specific indicators of cell injury were measured by Trypan blue exclusion and LDH release. Lipid peroxidation, protein oxidation, and total glutathione were also evaluated as specific markers of oxygen toxicity. Lower HO-1 overexpression was associated with increased protection against oxygen damage; however, higher expression was associated with increased markers of cell injury. In this model, we conclude that there is a beneficial threshold of HO-1 overexpression beyond which detrimental effects become apparent. We suggest that measures to enhance protection against oxygen toxicity by HO-1 overexpression be undertaken with a thorough understanding of the potential dual nature of HO-1.