Heme Oxygenase Activity and Heme Binding in a Neonatal Mouse Model

<b><i>Background:</i></b> Severe hemolytic disease of the newborn leads to the release of pro-oxidative free heme (FH). Heme oxygenase (HO) is primarily responsible for detoxifying FH. <b><i>Objective:</i></b> To investigate the protective effects of HO in a model of heme overload. <b><i>Methods:</i></b> For in vitro studies, NIH3T3 HO-1-<i>luc</i> cells were incubated with 10, 30, or 60 µ<smlcap>M</smlcap> FH or methemalbumin (MHA). HO-1 promoter activity was assessed 3, 6, and 24 h after treatment. Cell survival was indexed by viability assays. For in vivo studies, 1- and 5-week-old wild-type (Wt) or HO-1-heterozygous (Het, HO-1^+/-) mice were given 60 µmol FH or MHA/kg intraperitoneally. After 24 h, plasma aspartate aminotransferease (AST)/alanine transaminase (ALT) and hemopexin, liver HO activity, and lipid peroxidation (LP) were determined. <b><i>Results: </i></b>In HO-1-<i>luc</i> cells, HO-1 promoter activity peaked 6 h after incubation with 30 µ<smlcap>M</smlcap> FH (1.6-fold) or 60 µ<smlcap>M</smlcap> MHA (2.1-fold) over baseline. Twenty-four hours after exposure to 60 µ<smlcap>M</smlcap> FH, a decrease in viability of 80% was found, compared with no decrease after exposure to 60 µ<smlcap>M</smlcap> MHA. In 1-week-old Wt and HO-1 Het pups given 60 µmol FH/kg, HO activity significantly increased 3.5- and 3.1-fold, respectively. No changes in LP or AST/ALT levels were observed. In adult Wt and HO-1 Het mice, HO activity increased (3.0- and 2.6-fold, respectively). LP and AST levels significantly increased 28.4- and 2.7-fold, respectively, in adult HO-1 Het mice.<b> </b>Hemopexin levels at baseline were higher in adults compared with newborns for both Wt and Het mice. In addition, FH induced hemopexin levels in both adults and newborns, but to a lesser degree in newborns. <b><i>Conclusions:</i></b> FH is highly toxic in vitro, but its toxicity is abolished when bound to albumin. Newborns appear to be protected from the pro-oxidative effects of FH, which may be mediated by heme binding and a higher absolute HO activity at baseline and after FH-mediated induction.