Abstract 1151 Neonatal Disease Oriented Research: Steroids & Oxygen: Perinatal Effects Poster Symposium, Sunday, 5/2

It is hypothesized that heme oxygenase (HO), the rate limiting enzyme in bilirubin production, functions to protect against lung hyperoxic injury because it metabolizes a pro-oxidant heme and forms an antioxidant, bilirubin. Previous studies have demonstrated that adult mice with absent HO-2 (HO-2 KO), the constitutive enzyme, have increased oxidative stress but have increased HO-1, the inducible form. In order to eliminate the compensatory induction of HO-1 and to determine the effect of diminished total HO activity on oxidative injury in neonatal animals, tin mesoporphyrin (SnMP, 40 nmol/g body weight), a competitive inhibitor of HO was injected intraperitoneally in both wild type (WT) and HO-2 KO pups prior to exposure to hyperoxia (>95% oxygen). Pups from the same litter served as controls and were injected with the vehicle (10% ethanolamine) alone. We measured total HO activity by gas chromatography, HO-1 immunoreactive protein by Western blot analysis, and HO-1 mRNA by Northern blot analysis, and measured indices of oxidative injury (lipid peroxidation, protein oxidation) as well as lung microsomal heme and reactive iron content. After 72 hours of hyperoxia, HO-2 KO pups showed an increase in lung HO activity (2.2 fold) and an increase in lung HO-1 immunoreactive protein and mRNA as compared to WT. Paradoxically, injection with SnMP in both HO-2 KO and WT pups resulted in further increased lung HO activity particularly in the WT pups (1.8 fold) and to a lesser extent in the HO-2 KO (1.2 fold). Lung HO-1 immunoreactive protein was also increased with SnMP injection in both WT and KO (1.6 fold in KO and 1.5 fold in WT) compared to sham-injected controls. Microsomal heme content was relatively unchanged in all groups. Lung protein oxidation was highest in the HO-2 KO and lowest in the WT exposed to hyperoxia. Furthermore, SnMP injection prior to hyperoxic exposure was associated with a lowering of protein carbonyls in the HO-2 KO whereas increased lung protein carbonyls were seen in the similarly treated WT compared to sham-injected controls. These findings demonstrate that SnMP induces lung HO-1 expression in vivo. Also, it appears that there is a narrow optimal range of lung HO-1 expression since the more pronounced increase in lung HO activity in the SnMP treated WT was not protective in hyperoxia whereas the more modest increase in lung HO activity in the SnMP treated HO-2 KO appeared to be. Furthermore, since HO-2 KO had increased lung HO activity and HO-1 immunoreactive protein compared to WT, we speculate that HO-2 may serve as a regulator of HO-1 expression. It will be critical to evaluate the effects of metalloporphyrin administration on HO-1 induction in the clinical setting where increased lung oxidative stress is likely to occur. Funded by NIH grant HL-52701, the Hess Funds and a Major Undergraduate Research Opportunity Grant of Stanford University