Resistance to hyperoxia with heme oxygenase-1 disruption: role of iron

doi:10.1016/S0891-5849(02)01295-9

Free Radical Biology and Medicine

Volume 34, Issue 1, 1 January 2003, Pages 124-133

https://doi.org/10.1016/S0891-5849(02)01295-9 Get rights and content

Abstract

In many models, a protective role for heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme degradation, has been demonstrated. Also, HO-1 null mice (KO) are more susceptible to inflammation and hypoxia and transplant rejection. Nonetheless, their response to hyperoxia (> 95% O₂) has not yet been evaluated. Surprisingly, after acute hyperoxic exposure, KO had significantly decreased markers of lung oxidative injury and survived chronic hyperoxia as well as wild-type (WT) controls. Disrupted HO-1 expression was associated with decreased lung reactive iron and iron-associated proteins, decreased NADPH cytochrome cP450 reductase activity, and decreased lung peroxidase activity compared to WT. Injection of tin protoporphyrin, an inhibitor of HO, in the WT decreased acute hyperoxic lung injury, whereas transduction of human HO-1 in the KO reversed the relative protection of the KO to acute injury and worsened hyperoxic survival. This suggests that disruption of HO-1 protects against hyperoxia by diminishing the generation of toxic reactive intermediates in the lung via iron and H₂O₂.

Introduction

Heme oxygenase (HO) degrades heme and forms bilirubin and carbon monoxide (CO). This requires the action of the microsomal enzyme NADPH cytochrome c P450 reductase (cP450), which serves to donate electrons to the reaction. The HO-1 isoenzyme is also a stress protein that is readily induced in oxidative stress [1]. We have shown that moderate overexpression of HO-1 improves resistance to heme-mediated injury [2] and to oxygen toxicity [3]. It is felt that this protective effect is likely due to pro-oxidant (heme) degradation and antioxidant (bilirubin) formation. Nonetheless, iron is released from the reaction and this could obviate these benefits because of the known toxicities of this metal [4]. In fact, in cultured fibroblasts, lower levels (< 5-fold increase) of HO activity were cytoprotective but higher levels (> 15-fold) were toxic because of the reactive iron released [5].

So far, the effects of HO-1 disruption on hyperoxic lung injury are unknown, but HO-2 knockouts (KO) exposed to hyperoxia sustain more injury than wild-type mice (WT). These animals also had increased HO-1 expression compared to the WT [6]. Did the disruption of HO-2 or the increased HO-1 result in the injury? Preliminary observations indicate that mice overexpressing HO-1 in alveolar type II cells have increased oxygen toxicity compared to WT [7]. Worsened oxygen toxicity is also seen in neonatal mice with increased HO-1 in alveolar type II cells after transpulmonary transduction [8], suggesting that increased HO-1 confers susceptibility to hyperoxia. Nonetheless, aging HO-1 KO are more susceptible to inflammation [9] and a HO-1-deficient human had increased endothelial injury [10]. Furthermore, rats given HO-1 cDNA intratracheally had improved resistance to oxygen toxicity [11] and HO-1 KO mice have increased hypoxic pulmonary vasoconstriction and inflammation, illustrating contradictory effects of HO-1 in the lung.

Perhaps increased lung HO-1 and iron release is not beneficial in hyperoxia because iron worsens this injury paradigm 12, 13. Furthermore, cP450, found in the HO complex could generate H₂O₂ in the presence of oxygen, leading to worsened oxidative stress as shown with other NADPH oxidoreductases [14].

To better understand the mechanism by which HO-1 affects lung hyperoxic defense, mice with targeted disruption of HO-1 [15] and wild-type littermates (WT) were exposed to hyperoxia and HO expression, indices of oxidative injury and iron metabolism were examined, as was the activity of cP450.

Section snippets

Materials and methods

Homozygote C57Bl/6 × 129/Sv KO were obtained by targeted disruption of the HO-1 gene as previously described [15]. Heterozygotes were mated and the pups genotyped so as to recover KO and WT. The KO mice were smaller than their WT littermates but were healthy at the time of experiments and had no obvious deformities. Animal protocols were reviewed and approved by the Animal Care Institutional Review Panel of Stanford University.

HO-1 KO have lowered lung HO activity in hyperoxia

Prior to hyperoxic exposure, total lung HO activity was similar in the HO-1 KO mice compared to WT controls (Fig. 1A) as was exhaled CO (VeCO) (Fig. 1B). This was associated with increased lung HO-2 protein levels in the KO as compared to WT (Fig. 1C). After acute hyperoxic exposure, lung HO activity was increased in the WT but unchanged in the KO (Fig. 1A). In this case, HO-2 protein was increased only in KO lungs (Fig. 1C), whereas HO-1 protein was only increased in the WT lungs (Fig. 1C).

Discussion

Increased HO may favor antioxidant defense by removal of a potential pro-oxidant heme, and formation of antioxidant pigments but iron released from heme can participate in the Fenton reaction, leading to increased toxic radicals [4]. In fact, early induction of HO-1 results in subsequent mitochondrial iron sequestration in astrocytes, leading to increased oxidative stress [34]. The HO-1 KO have a targeted disruption of HO-1 but they can degrade heme via HO-2 and are not devoid of HO activity.

Acknowledgements

We thank Douglas R. Spitz and Julia E. Sim for their expert assistance in antioxidant enzyme assays and Ron Wong and Hedrik J. Vreman, Ph.D., for their assistance with the VeCO measurements. We acknowledge Dr. Susumu Tonegawa and Ken D. Poss for the development of the mutant strains. This work was funded by the National Institutes of Health Grant HL-52701 and HL-58752, the Mary L. Johnson and Hess funds of Stanford University (P.A.D.), the American Lung Association of Florida Career

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Original contributionResistance to hyperoxia with heme oxygenase-1 disruption: role of iron

Abstract

Introduction

Section snippets

Materials and methods

HO-1 KO have lowered lung HO activity in hyperoxia

Discussion

Acknowledgements

J. Biol. Chem

Free Radic. Biol. Med

Regul. Pept

Neuron

Lab. Invest

J. Biol. Chem

Arch. Biochem. Biophys

Biochem. Biophys. Res. Commun

J. Biol. Chem

Methods Enzymol

Anal. Biochem

Methods Enzymol

J. Biol. Chem

Arch. Biochem. Biophys

J. Biol. Chem

Transfection of the human heme oxygenase gene into rabbit coronary microvessel endothelial cellsprotective effect against heme and hemoglobin toxicity

Proc. Natl. Acad. Sci. USA

Superoxide-dependent formation of hydroxyl radicals in the presence of iron salts. Detection of ‘free’ iron in biological systems by using bleomycin-dependent degradation of DNA

Biochem. J

Reversal of HO-1 related cytoprotection with increased expression is due to reactive iron

FASEB J

Lung oxidative injury in the absence of heme oxygenase-2 in mice

J. Clin. Invest

HO-1 expression in type II pneumocytes after transpulmonary gene delivery

Am. J. Physiol. Lung Cell. Mol. Physiol

Heme oxygenase 1 is required for mammalian iron reutilization

Proc. Natl. Acad. Sci. USA

Original contribution
Resistance to hyperoxia with heme oxygenase-1 disruption: role of iron