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. 1999 Nov 15;344(Pt 1):153–158.

Haem oxygenase shows pro-oxidant activity in microsomal and cellular systems: implications for the release of low-molecular-mass iron.

N J Lamb 1, G J Quinlan 1, S Mumby 1, T W Evans 1, J M Gutteridge 1
PMCID: PMC1220625  PMID: 10548545

Abstract

Haem oxygenase-1 (HO-1) is a highly inducible stress protein that removes haem from cells with the release of biliverdin, carbon monoxide and low-molecular-mass iron (LMrFe). Several antioxidant functions have been ascribed to HO; its induction is considered to be a protective event. However, LMrFe produced during haem catabolism might elicit a pro-oxidant response, with deleterious consequences. We therefore investigated the delicate balance between pro-oxidant and antioxidant events with the use of a microsomal lipid peroxidation (LPO) system. By using microsomal-bound HO in an NADPH-dependent LPO system, we assessed the pro-oxidant nature of the released LMrFe and the antioxidant effect of the released bilirubin. Hb, a biologically relevant substrate for HO, was included with the microsomes to supplement the source of haem iron and to promote LPO. We found significant increases in microsomal LPO, by using the thiobarbituric acid (TBA) test, after incubation with Hb. This Hb-stimulated peroxidation was inhibited by HO inhibitors and by iron chelators, suggesting a HO-driven, iron-dependent mechanism. GLC-MS was employed to measure the specific LPO product 4-hydroxy-2-nonenal and to confirm our TBA test results. A HO inhibitor attenuated an increase in intracellular LMrFe that occurred after treatment of rat pulmonary artery smooth-muscle cells with Hb. Additionally, exogenously added bilirubin at an equimolar concentration to the LMrFe present in both microsomal and liposomal systems was unable to prevent the pro-oxidant effect of the iron. Under certain circumstances HO can act as a pro-oxidant and seems to have a role in stimulating microsomal LPO.

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Selected References

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  1. Alam J., Shibahara S., Smith A. Transcriptional activation of the heme oxygenase gene by heme and cadmium in mouse hepatoma cells. J Biol Chem. 1989 Apr 15;264(11):6371–6375. [PubMed] [Google Scholar]
  2. Applegate L. A., Luscher P., Tyrrell R. M. Induction of heme oxygenase: a general response to oxidant stress in cultured mammalian cells. Cancer Res. 1991 Feb 1;51(3):974–978. [PubMed] [Google Scholar]
  3. Balla G., Jacob H. S., Balla J., Rosenberg M., Nath K., Apple F., Eaton J. W., Vercellotti G. M. Ferritin: a cytoprotective antioxidant strategem of endothelium. J Biol Chem. 1992 Sep 5;267(25):18148–18153. [PubMed] [Google Scholar]
  4. Camhi S. L., Alam J., Otterbein L., Sylvester S. L., Choi A. M. Induction of heme oxygenase-1 gene expression by lipopolysaccharide is mediated by AP-1 activation. Am J Respir Cell Mol Biol. 1995 Oct;13(4):387–398. doi: 10.1165/ajrcmb.13.4.7546768. [DOI] [PubMed] [Google Scholar]
  5. Carraway M. S., Ghio A. J., Taylor J. L., Piantadosi C. A. Induction of ferritin and heme oxygenase-1 by endotoxin in the lung. Am J Physiol. 1998 Sep;275(3 Pt 1):L583–L592. doi: 10.1152/ajplung.1998.275.3.L583. [DOI] [PubMed] [Google Scholar]
  6. Choi A. M., Alam J. Heme oxygenase-1: function, regulation, and implication of a novel stress-inducible protein in oxidant-induced lung injury. Am J Respir Cell Mol Biol. 1996 Jul;15(1):9–19. doi: 10.1165/ajrcmb.15.1.8679227. [DOI] [PubMed] [Google Scholar]
  7. Dennery P. A., Spitz D. R., Yang G., Tatarov A., Lee C. S., Shegog M. L., Poss K. D. Oxygen toxicity and iron accumulation in the lungs of mice lacking heme oxygenase-2. J Clin Invest. 1998 Mar 1;101(5):1001–1011. doi: 10.1172/JCI448. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dennery P. A., Sridhar K. J., Lee C. S., Wong H. E., Shokoohi V., Rodgers P. A., Spitz D. R. Heme oxygenase-mediated resistance to oxygen toxicity in hamster fibroblasts. J Biol Chem. 1997 Jun 6;272(23):14937–14942. doi: 10.1074/jbc.272.23.14937. [DOI] [PubMed] [Google Scholar]
  9. Esterbauer H., Schaur R. J., Zollner H. Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radic Biol Med. 1991;11(1):81–128. doi: 10.1016/0891-5849(91)90192-6. [DOI] [PubMed] [Google Scholar]
  10. Faivre-Fiorina B., Caron A., Fassot C., Fries I., Menu P., Labrude P., Vigneron C. Presence of hemoglobin inside aortic endothelial cells after cell-free hemoglobin administration in guinea pigs. Am J Physiol. 1999 Feb;276(2 Pt 2):H766–H770. doi: 10.1152/ajpheart.1999.276.2.H766. [DOI] [PubMed] [Google Scholar]
  11. Gutteridge J. M. A method for removal of trace iron contamination from biological buffers. FEBS Lett. 1987 Apr 20;214(2):362–364. doi: 10.1016/0014-5793(87)80088-1. [DOI] [PubMed] [Google Scholar]
  12. Gutteridge J. M. Free radicals in disease processes: a compilation of cause and consequence. Free Radic Res Commun. 1993;19(3):141–158. doi: 10.3109/10715769309111598. [DOI] [PubMed] [Google Scholar]
  13. Gutteridge J. M., Hou Y. Y. Iron complexes and their reactivity in the bleomycin assay for radical-promoting loosely-bound iron. Free Radic Res Commun. 1986;2(3):143–151. doi: 10.3109/10715768609088066. [DOI] [PubMed] [Google Scholar]
  14. Gutteridge J. M. Iron promoters of the Fenton reaction and lipid peroxidation can be released from haemoglobin by peroxides. FEBS Lett. 1986 Jun 9;201(2):291–295. doi: 10.1016/0014-5793(86)80626-3. [DOI] [PubMed] [Google Scholar]
  15. Gutteridge J. M., Quinlan G. J. Malondialdehyde formation from lipid peroxides in the thiobarbituric acid test: the role of lipid radicals, iron salts, and metal chelators. J Appl Biochem. 1983 Aug-Oct;5(4-5):293–299. [PubMed] [Google Scholar]
  16. Gutteridge J. M. Superoxide-dependent formation of hydroxyl radicals from ferric-complexes and hydrogen peroxide: an evaluation of fourteen iron chelators. Free Radic Res Commun. 1990;9(2):119–125. doi: 10.3109/10715769009148579. [DOI] [PubMed] [Google Scholar]
  17. Halliwell B., Gutteridge J. M. Formation of thiobarbituric-acid-reactive substance from deoxyribose in the presence of iron salts: the role of superoxide and hydroxyl radicals. FEBS Lett. 1981 Jun 15;128(2):347–352. doi: 10.1016/0014-5793(81)80114-7. [DOI] [PubMed] [Google Scholar]
  18. Hur T., Squibb K., Cosma G., Horowitz S., Piedboeuf B., Bowser D., Gordon T. Induction of metallothionein and heme oxygenase in rats after inhalation of endotoxin. J Toxicol Environ Health A. 1999 Feb 12;56(3):183–203. doi: 10.1080/009841099158141. [DOI] [PubMed] [Google Scholar]
  19. Immenschuh S., Iwahara S., Satoh H., Nell C., Katz N., Muller-Eberhard U. Expression of the mRNA of heme-binding protein 23 is coordinated with that of heme oxygenase-1 by heme and heavy metals in primary rat hepatocytes and hepatoma cells. Biochemistry. 1995 Oct 17;34(41):13407–13411. doi: 10.1021/bi00041a018. [DOI] [PubMed] [Google Scholar]
  20. Keyse S. M., Tyrrell R. M. Heme oxygenase is the major 32-kDa stress protein induced in human skin fibroblasts by UVA radiation, hydrogen peroxide, and sodium arsenite. Proc Natl Acad Sci U S A. 1989 Jan;86(1):99–103. doi: 10.1073/pnas.86.1.99. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lee P. J., Alam J., Sylvester S. L., Inamdar N., Otterbein L., Choi A. M. Regulation of heme oxygenase-1 expression in vivo and in vitro in hyperoxic lung injury. Am J Respir Cell Mol Biol. 1996 Jun;14(6):556–568. doi: 10.1165/ajrcmb.14.6.8652184. [DOI] [PubMed] [Google Scholar]
  22. Lee P. J., Alam J., Wiegand G. W., Choi A. M. Overexpression of heme oxygenase-1 in human pulmonary epithelial cells results in cell growth arrest and increased resistance to hyperoxia. Proc Natl Acad Sci U S A. 1996 Sep 17;93(19):10393–10398. doi: 10.1073/pnas.93.19.10393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Maines M. D., Kappas A. Metals as regulators of heme metabolism. Science. 1977 Dec 23;198(4323):1215–1221. doi: 10.1126/science.337492. [DOI] [PubMed] [Google Scholar]
  24. Maines M. D. The heme oxygenase system: a regulator of second messenger gases. Annu Rev Pharmacol Toxicol. 1997;37:517–554. doi: 10.1146/annurev.pharmtox.37.1.517. [DOI] [PubMed] [Google Scholar]
  25. McCoubrey W. K., Jr, Huang T. J., Maines M. D. Isolation and characterization of a cDNA from the rat brain that encodes hemoprotein heme oxygenase-3. Eur J Biochem. 1997 Jul 15;247(2):725–732. doi: 10.1111/j.1432-1033.1997.00725.x. [DOI] [PubMed] [Google Scholar]
  26. Nakai K., Sakuma I., Ohta T., Ando J., Kitabatake A., Nakazato Y., Takahashi T. A. Permeability characteristics of hemoglobin derivatives across cultured endothelial cell monolayers. J Lab Clin Med. 1998 Oct;132(4):313–319. doi: 10.1016/s0022-2143(98)90045-2. [DOI] [PubMed] [Google Scholar]
  27. Nath K. A., Balla G., Vercellotti G. M., Balla J., Jacob H. S., Levitt M. D., Rosenberg M. E. Induction of heme oxygenase is a rapid, protective response in rhabdomyolysis in the rat. J Clin Invest. 1992 Jul;90(1):267–270. doi: 10.1172/JCI115847. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Nath K. A. The functional significance of induction of heme oxygenase by oxidant stress. J Lab Clin Med. 1994 Apr;123(4):461–463. [PubMed] [Google Scholar]
  29. Nutter L. M., Sierra E. E., Ngo E. O. Heme oxygenase does not protect human cells against oxidant stress. J Lab Clin Med. 1994 Apr;123(4):506–514. [PubMed] [Google Scholar]
  30. Otterbein L., Sylvester S. L., Choi A. M. Hemoglobin provides protection against lethal endotoxemia in rats: the role of heme oxygenase-1. Am J Respir Cell Mol Biol. 1995 Nov;13(5):595–601. doi: 10.1165/ajrcmb.13.5.7576696. [DOI] [PubMed] [Google Scholar]
  31. Pederson T. C., Aust S. D. The mechanism of liver microsomal lipid peroxidation. Biochim Biophys Acta. 1975 Apr 7;385(2):232–241. doi: 10.1016/0304-4165(75)90351-7. [DOI] [PubMed] [Google Scholar]
  32. Quinlan G. J., Evans T. W., Gutteridge J. M. 4-hydroxy-2-nonenal levels increase in the plasma of patients with adult respiratory distress syndrome as linoleic acid appears to fall. Free Radic Res. 1994 Aug;21(2):95–106. doi: 10.3109/10715769409056561. [DOI] [PubMed] [Google Scholar]
  33. Rizzardini M., Carelli M., Cabello Porras M. R., Cantoni L. Mechanisms of endotoxin-induced haem oxygenase mRNA accumulation in mouse liver: synergism by glutathione depletion and protection by N-acetylcysteine. Biochem J. 1994 Dec 1;304(Pt 2):477–483. doi: 10.1042/bj3040477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Ryter S., Kvam E., Richman L., Hartmann F., Tyrrell R. M. A chromatographic assay for heme oxygenase activity in cultured human cells: application to artificial heme oxygenase overexpression. Free Radic Biol Med. 1998 Apr;24(6):959–971. doi: 10.1016/s0891-5849(97)00380-8. [DOI] [PubMed] [Google Scholar]
  35. Shibahara S., Yoshida T., Kikuchi G. Mechanism of increase of heme oxygenase activity induced by hemin in cultured pig alveolar macrophages. Arch Biochem Biophys. 1979 Oct 15;197(2):607–617. doi: 10.1016/0003-9861(79)90285-6. [DOI] [PubMed] [Google Scholar]
  36. Stocker R., Ames B. N. Potential role of conjugated bilirubin and copper in the metabolism of lipid peroxides in bile. Proc Natl Acad Sci U S A. 1987 Nov;84(22):8130–8134. doi: 10.1073/pnas.84.22.8130. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Stocker R. Induction of haem oxygenase as a defence against oxidative stress. Free Radic Res Commun. 1990;9(2):101–112. doi: 10.3109/10715769009148577. [DOI] [PubMed] [Google Scholar]
  38. Stocker R., Yamamoto Y., McDonagh A. F., Glazer A. N., Ames B. N. Bilirubin is an antioxidant of possible physiological importance. Science. 1987 Feb 27;235(4792):1043–1046. doi: 10.1126/science.3029864. [DOI] [PubMed] [Google Scholar]
  39. Svingen B. A., Buege J. A., O'Neal F. O., Aust S. D. The mechanism of NADPH-dependent lipid peroxidation. The propagation of lipid peroxidation. J Biol Chem. 1979 Jul 10;254(13):5892–5899. [PubMed] [Google Scholar]
  40. Tampo Y., Yonaha M. A microsomal membrane component associated with iron reduction in NADPH-supported lipid peroxidation. Lipids. 1995 Jan;30(1):55–62. doi: 10.1007/BF02537042. [DOI] [PubMed] [Google Scholar]
  41. Taylor J. L., Carraway M. S., Piantadosi C. A. Lung-specific induction of heme oxygenase-1 and hyperoxic lung injury. Am J Physiol. 1998 Apr;274(4 Pt 1):L582–L590. doi: 10.1152/ajplung.1998.274.4.L582. [DOI] [PubMed] [Google Scholar]
  42. Tenhunen R., Marver H. S., Schmid R. The enzymatic catabolism of hemoglobin: stimulation of microsomal heme oxygenase by hemin. J Lab Clin Med. 1970 Mar;75(3):410–421. [PubMed] [Google Scholar]
  43. Trakshel G. M., Kutty R. K., Maines M. D. Purification and characterization of the major constitutive form of testicular heme oxygenase. The noninducible isoform. J Biol Chem. 1986 Aug 25;261(24):11131–11137. [PubMed] [Google Scholar]
  44. Vile G. F., Basu-Modak S., Waltner C., Tyrrell R. M. Heme oxygenase 1 mediates an adaptive response to oxidative stress in human skin fibroblasts. Proc Natl Acad Sci U S A. 1994 Mar 29;91(7):2607–2610. doi: 10.1073/pnas.91.7.2607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Wills E. D. Lipid peroxide formation in microsomes. General considerations. Biochem J. 1969 Jun;113(2):315–324. doi: 10.1042/bj1130315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Wills E. D. Lipid peroxide formation in microsomes. The role of non-haem iron. Biochem J. 1969 Jun;113(2):325–332. doi: 10.1042/bj1130325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Yoshida M., Egawa K., Kasai N. Effect of endotoxin and its degradation products on hepatic mixed-function oxidase and heme enzyme systems in mice. Toxicol Lett. 1982 Jul;12(2-3):185–190. doi: 10.1016/0378-4274(82)90184-9. [DOI] [PubMed] [Google Scholar]
  48. da Silva J. L., Morishita T., Escalante B., Staudinger R., Drummond G., Goligorsky M. S., Lutton J. D., Abraham N. G. Dual role of heme oxygenase in epithelial cell injury: contrasting effects of short-term and long-term exposure to oxidant stress. J Lab Clin Med. 1996 Sep;128(3):290–296. doi: 10.1016/s0022-2143(96)90030-x. [DOI] [PubMed] [Google Scholar]

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