Induction of heme oxygenase 1 in radiation nephropathy: role of angiotensin II

Datta, P. K., Moulder, J. E., Fish, B. L., Cohen, E. P. and Lianos, E. A. Induction of Heme Oxygenase 1 in Radiation Nephropathy: Role of Angiotensin II. Radiat. Res. 155, 734-739 (2001). In a rat model of radiation-induced nephropathy, we investigated changes in expression of heme oxygenase 1 (Hmox1, also known as HO-1), an enzyme that catalyzes conversion of heme into biliverdin, carbon monoxide and iron. The study explored whether radiation induces Hmox1 expression in the irradiated kidney and whether angiotensin II (AII) mediates Hmox1 expression in glomeruli isolated from irradiated kidneys. To assess the effects of radiation on Hmox1 expression, rats received 20 Gy bilateral renal irradiation and were randomized to groups receiving an AII type 1 (AT(1)) receptor antagonist (L-158,809) or no treatment. Drug treatment began 9 days prior to bilateral renal irradiation and continued for the duration of the study. Estimation of Hmox1 levels in glomerular protein lysates assessed by Western blot analysis revealed a significant increase in Hmox1 protein at 50 and 65 days postirradiation. In animals treated with the AT(1) receptor antagonist, there was no induction of Hmox1, suggesting that AII may be a mediator of Hmox1 induction. To confirm that AII stimulates Hmox1 expression, animals were infused with 200, 400 or 800 ng/kg min(-1) of AII for 18-19 days, and Hmox1 protein levels in glomeruli were assessed. There was a significant induction of Hmox1 in glomeruli of animals infused with 800 ng/kg min(-1) of AII. These studies demonstrate that glomerular Hmox1 expression is elevated in the middle phase of radiation nephropathy and that AII can increase glomerular Hmox1 levels.     

Induction of hepatic heme oxygenase activity by bromobenzene

Hepatic heme oxygenase, an enzyme which converts heme to carbon monoxide and bile pigment in vitro, is inducible by heme but also by large “toxic” doses of such nonheme substances as hormones, endotoxin, and heavy metal ions. When we gave rats a single hepatotoxic dose of allyl alcohol, ethionine, acetaminophen, furosemide, or endotoxin, hepatic heme oxygenase activity rose modestly (two- to fivefold) after 20 h. In contrast, administration of bromobenzene (5 mmol/kg) induced heme oxygenase in the liver an average of 15-fold after 20 h but was without effect on the enzyme in the kidney or spleen. The change in heme oxygenase was accompanied by a loss in cytochrome P-450 concentration and, in rats labeled with 5-δ-amino[¹⁴C]levulinic acid, an increased rate of degradation of hepatic [¹⁴C]heme to ¹⁴CO. Induction of heme oxygenase by bromobenzene was blocked by cycloheximide, an inhibitor of protein synthesis, but not by actinomycin D, an inhibitor of RNA synthesis. This suggests that bromobenzene stimulates de novo enzyme synthesis at the step of translation. Subtoxic doses of bromobenzene (less than 1 mmol/kg) gave proportionately greater induction of heme oxygenase. Furthermore, induction of the enzyme remained unaffected when bromobenzene hepatotoxicity was blocked by pretreatment of rats with SKF-525A, 3-methylcholanthrene, or cysteine (which supplements liver sulfhydryl content), or when hepatotoxicity was enhanced by pretreatment with phenobarbital or with diethylmaleate (which depletes hepatic glutathione). These data suggest that with induction of heme oxygenase by bromobenzene, neither liver cell necrosis nor alteration in hepatic sulfhydryl metabolism is indispensible. The latter characteristic differs from induction of the enzyme by metal ions in which depletion of sulfhydryl-containing constituents has been thought to be essential. We conclude that bromobenzene is a novel inducer of heme oxygenase activity in the liver, differing from other nonheme substances in potency and specificity for the liver, and in utilizing mechanism(s) which require neither production of hepatotoxicity, depletion of hepatic glutathione, nor sensitivity to actinomycin D.     

Induction of heme oxygenase 1 in liver of spontaneously diabetic rats

It has been suggested that diabetes induces an increase in oxidative stress; the increased expression of heme-oxygenase 1 (HO-1) in liver is believed to be a sensitive marker of the stress response. The aim of this study was to examine whether diabetes is able to induce HO-1 expression in liver. The specific mRNA was amplified by RT/PCR and calibrated with amplified β-actin mRNA.

The mRNA HO-1 levels in the liver of spontaneously diabetic rats were increased by 1.8 fold compared with non diabetics; this supports the hypothesis of weak but significant oxidative damage due to chronic hyperglycaemia. This work represents the first in vivo study exploring the semi-quantitative expression of HO-1 in the liver of spontaneously diabetic rats.     

Induction in mouse peritoneal macrophages of 34 kDa stress protein and heme oxygenase by sulfhydryl-reactive agents

The synthesis of 34-kDa stress protein was enhanced, with a simultaneous increase in heme oxygenase activity, when mouse macrophages were exposed to diethylmaleate or sodium arsenite. After 7 h of exposure to the sulfhydryl agents, the 34-kDa protein was the most actively synthesized protein. Immunoblot analysis showed that the induced 34-kDa protein reacted with an antibody raised against bovine heme oxygenase. Cadmium ions or 1-chloro-2,4-dinitrobenzene also induced the 34-kDa protein which reacted with the antibody. Treatments of the cells with buthionine sulfoximine or hydrogen peroxide weakly induced the protein, while diamide treatment or heat shock was without effect. These results are consistent with our previous findings that heavy metal ions including arsenite and cadmium ions induce heme oxygenase (32-kDa stress protein) in human cell lines [Taketani, S., Kohno, H., Yoshinaga, T., & Tokunaga, R. (1989) FEBS Lett. 245, 173-176], and also suggest that the formation of glutathione conjugate with sulfhydryl-reactive agents may mediate the induction of the stress protein in mouse peritoneal macrophages.     

Induction of heme oxygenase 1 by Ginkgo biloba in neuronal cultures and potential implications in ischemia.

Ginkgo biloba extract (EGb 761) is a standardized extract originating in traditional Chinese medicine. Ginkgo biloba dried leaves have been used for centuries to treat various neurological conditions. The constituents from the extract are likely to have synergistic effects that have been shown to be protective against oxidative stress injury. However, the cellular mechanisms of protection afforded by Ginkgo biloba are still unclear. The cascade leading to neuronal cell death in acute and chronic neurodegenerative conditions, such as cerebral ischemia and Alzheimer's disease, has been postulated to be mediated by free radical damage. We tested the hypothesis that the neuroprotective action of EGb 761 could be due partially to an induction of heme oxygenase I (HO1). We and others have previously reported that modulation of HO total activity may well have direct physiological implications in stroke and in Alzheimer's disease. Heme oxygenase acts as an antioxidant enzyme by degrading heme into iron, carbon monoxide, and biliverdin which is rapidly converted into bilirubin. Through the use of primary neuronal cultures, we demonstrated that EGb 761 induces HO1 in a dose-dependent manner (0, 10, 50, 100 and 500 microg/ml) and time-dependent manner with a maximal induction at 8 hr. We are proposing that several of the protective effects of EGb 761 in ischemia could be mediated through beneficial actions of heme degradation and its metabolites.     

Induction of heme oxygenase mRNA by cobalt protoporphyrin in rat liver

The effect of cobaltic(III)-protoporphyrin on heme oxygenase activity and mRNA content was examined in vivo in the adult male rat liver. The activity of heme oxygenase, the rate-limiting enzyme in the degradation of heme, was enhanced, as expected, by cobalt protoporphyrin (25 mumol/kg body weight) in a time-dependent manner. Levels of enzyme activity were increased 2-fold by 8-16 h following treatment and were 6-fold higher than baseline values by 48 h. Administration of cobalt protoporphyrin resulted in a marked increase in heme oxygenase mRNA in the liver. Within 2 h of treatment, mRNA levels had increased 7.9-fold. The induction of heme oxygenase mRNA was maximal at 8 h when the levels were 58.5-fold above baseline. At every time point tested, the increase in heme oxygenase mRNA was several fold greater than that of enzyme activity.     

Mechanism of heme degradation by heme oxygenase

Heme oxygenase catalyzes the three step-wise oxidation of hemin to alpha-biliverdin, via alpha-meso-hydroxyhemin, verdoheme, and ferric iron-biliverdin complex. This enzyme is a simple protein which does not have any prosthetic groups. However, heme and its two metabolites, alpha-meso-hydroxyhemin and verdoheme, combine with the enzyme and activate oxygen during the heme oxygenase reaction. In the conversion of hemin to alpha-meso-hydroxyhemin, the active species of oxygen is Fe-OOH, which self-hydroxylates heme to form alpha-meso-hydroxyhemin. This step determines the alpha-specificity of the reaction. For the formation of verdoheme and liberation of CO from alpha-meso-hydroxyhemin, oxygen and one reducing equivalent are both required. However, the ferrous iron of the alpha-meso-hydroxyheme is not involved in the oxygen activation and unactivated oxygen is reacted on the 'activated' heme edge of the porphyrin ring. For the conversion of verdoheme to the ferric iron-biliverdin complex, both oxygen and reducing agents are necessary, although the precise mechanism has not been clear. The reduction of iron is required for the release of iron from the ferric iron-biliverdin complex to complete total heme oxygenase reaction.     

Evidence against a regulatory role for heme oxygenase in hepatic heme synthesis

The role of a microsomal enzyme system, heme oxygenase, in the regulation of intracellular heme concentration and of the heme biosynthetic pathway was investigated. It was determined that alterations in heme oxygenase activity were not consistent with the observed alterations of heme biosynthesis produced by the administration of various drugs. It is concluded that heme oxygenase does not play a role in the regulation of heme biosynthesis under these circumstances.     

Induction of heme oxygenase produces load-independent cardioprotective effects in hypertensive rats.

Although heme oxygenase (HO) has been suggested to be involved in the regulation of cardiovascular function through production of carbon monoxide (CO), the pathophysiological significance of HO in hypertensive organ damage remains unknown. We examined the effects of inducing HO-1 mRNA by stannous chloride (SnCl2) on cardiac hypertrophy in stroke-prone spontaneously hypertensive rats (SHR-SP/Izm). Chronic administration of SnCl2 resulted in a significant decrease in left ventricular (LV) weight/body weight ratio and LV brain natriuretic peptide (BNP) mRNA levels as a marker of cardiac hypertrophy and a significant increase in LV HO-1 mRNA levels and LV cGMP contents in SHR-SP/Izm, while there was no significant change in systemic blood pressure. These results provide the first evidence that induction of HO in the heart attenuates cardiac hypertrophy in load-independent mechanism in genetically hypertensive rats.     

Induction of DNA strand breakage and base oxidation by nitroxyl anion through hydroxyl radical production.

Nitroxyl anion (NO-), the one-electron reduction product of nitric oxide (NO*), has been reported to be formed under various physiological conditions and to be cytotoxic, although the mechanism responsible for the toxic effects has not been identified. We have studied the effects of NO- generated from Angeli's salt (sodium trioxodinitrate) or Piloty's acid (N-hydoxybenzenesulfonamide) on DNA strand breakage and DNA base oxidation in vitro. Induction of strand breakage was dose- and time-dependent upon incubation of plasmid pBR322 with Angeli's salt or Piloty's acid. Similarly, 8-oxo-2'-deoxyguanosine and malondialdehyde were formed when calf-thymus DNA or 2'-deoxyribose, respectively, were incubated with Angeli's salt. Electron acceptors (ferricyanide, 4-hydroxy-TEMPO), that convert NO to NO*, inhibited the reactions, indicating that NO , but not NO*, is responsible for the reactions. Furthermore, the reactions were also inhibited by the presence of hydroxyl radical (HO*) scavengers, antioxidants, metal chelators and superoxide dismutase and catalase, implying involvement of free HO*. These results suggest that NO- is a possible endogenous source of HO*, that may be formed either directly from the reaction product of NO- with NO* (N2O2*-) or indirectly through H2O2 formation. Thus NO may play an important role as a cause of diverse pathophysiological conditions such as inflammation and neurodegenerative diseases.     

A microassay for heme oxygenase activity using thin-layer chromatography.

A sensitive and facile assay for heme oxygenase (HO) has been developed. The basis of the assay is the detection of [14C]bilirubin formation in a coupled enzyme assay involving HO and biliverdin reductase actions, respectively. Separation of substrate from product is accomplished by thin-layer chromatography with subsequent quantitation by liquid scintillation counting of radioactive material present on chromatograms. As little as 20 micrograms of total cellular protein derived from cells growing in a standard 25-cm2 culture flask is sufficient for detection of HO enzyme activity using this assay. The reaction is inhibited by tin-protoporphyrin (10 microM final concentration), a specific inhibitor of HO. The linearity of the enzyme reaction with respect to incubation time and amount of protein used was established. Comparison of the new HO assay with a spectrophotometric assay was made, and good agreement of the results from both methods was found. The assay described here should facilitate measurements of this important heme-degrading enzyme in tissue culture studies and cases where limited amounts of material are available.     

Induction of heme oxygenase by delta 12-prostaglandin J2 in porcine aortic endothelial cells.

delta 12-Prostaglandin (PG)J2 stimulated the synthesis of a 31,000-dalton protein (termed p31) and the induction of cellular heme oxygenase activity in porcine aortic endothelial cells. A good correlation was observed between the time courses and dose dependencies of the induction of p31 synthesis and that of heme oxygenase activity by delta 12-PGJ2. Hemin, a known inducer of heme oxygenase, also induced p31 synthesis as well as heme oxygenase activity in the cells. On two-dimensional gel electrophoresis, p31 induced by delta 12-PGJ2 exhibited an isoelectric point of 5.4, which coincided exactly with that induced by hemin. These results indicate that the p31 induced by delta 12-PGJ2 in porcine aortic endothelial cells is heme oxygenase.     

Regulation of heme oxygenase expression by alcohol, hypoxia and oxidative stress

AIM:To study the effect of both acute and chronic alcohol exposure on heme oxygenases(HOs) in the brain,liver and duodenum.METHODS:Wild-type C57BL/6 mice,heterozygous Sod2 knockout mice,which exhibit attenuated manganese superoxide dismutase activity,and liver-specific ARNT knockout mice were used to investigate the role of alcohol-induced oxidative stress and hypoxia.For acute alcohol exposure,ethanol was administered in the drinking water for 1 wk.Mice were pair-fed with regular or ethanol-containing Lieber De Carli liquid diets for 4 wk for chronic alcohol studies.HO expression was analyzed by real-time quantitative polymerase chain reaction and Western blotting.RESULTS:Chronic alcohol exposure downregulated HO-1 expression in the brain but upregulated it in the duodenum of wild-type mice.It did not alter liver HO-1 expression,nor HO-2 expression in the brain,liver or duodenum.In contrast,acute alcohol exposure decreased both liver HO-1 and HO-2 expression,and HO-2 expression in the duodenum of wild-type mice.The decrease in liver HO-1 expression was abolished in ARNT+/-mice.Sod2+/-mice with acute alcohol exposure did not exhibit any changes in liver HO-1 and HO-2 expression or in brain HO-2 expression.However,alcohol inhibited brain HO-1 and duodenal HO-2 but increased duodenal HO-1 expression in Sod2+/-mice.Collectively,these findings indicate that acute and chronic alcohol exposure regulates HO expression in a tissue-specific manner.Chronic alcohol exposure alters brain and duodenal,but not liver HO expression.However,acute alcohol exposure inhibits liver HO-1 and HO-2,and also duodenal HO-2 expression.CONCLUSION:The inhibition of liver HO expression by acute alcohol-induced hypoxia may play a role in the early phases of alcoholic liver disease progression.     

Induction of heme oxygenase-1 by chamomile protects murine macrophages against oxidative stress

AimsProtection of cells from oxidative insult may be possible through direct scavenging of reactive oxygen species, or through stimulation of intracellular antioxidant defense mechanisms by induction of antioxidant gene expression. In this study we investigated the cytoprotective effect of chamomile and elucidated the underlying mechanisms.Main methodsThe cytoprotective effect of chamomile was examined on H₂O₂-induced cellular stress in RAW 264.7 murine macrophages.Key findingsRAW 264.7 murine macrophages treated with chamomile were protected from cell death caused by H₂O₂. Treatment with 50 μM H₂O₂ for 6 h caused significant increase in cellular stress accompanied by cell death in RAW 264.7 macrophages. Pretreatment with chamomile at 10–20 μg/mL for 16 h followed by H₂O₂ treatment protected the macrophages against cell death. Chamomile exposure significantly increased the expression of antioxidant enzymes viz. heme oxygenase-1 (HO-1), peroxiredoxin-1 (Prx-1), and thioredoxin-1 (Trx-1) in a dose-dependent manner, compared with their respective controls. Chamomile increased nuclear translocation of Nrf2 with increased phosphorylated Nrf2 levels, and binding to the antioxidant response element in the nucleus.SignificanceThese molecular findings for the first time provide insights into the mechanisms underlying the induction of phase 2 enzymes through the Keap1-Nrf2 signaling pathway by chamomile, and provide evidence that chamomile possesses antioxidant and cytoprotective properties.     

The role of heme oxygenase signaling in various disorders

Modern methods of cell and molecular biology, augmented by molecular technology, have great potential for helping to unravel the complex mechanisms of various diseases. They also have the potential to help us try to dissect the events which follow the altered physiological conditions. Thus, there is every reason to believe that some of the potential mechanisms will be translated sooner or later into the clinic. Heme oxygenase (HO)-related mechanisms play an important role in several aspects of different diseases. In the past several years, significant progress has been made in our understanding of the function and regulation of HO. The objective of this article is to review current knowledge relating to the importance of HO mechanism in various diseases including myocardial ischemia/reperfusion, hypertension, cardiomyopathy, organ transplantation, endotoxemia, lung diseases, and immunosuppression. The morbidity and mortality of these diseases remain high even with optimal medical management. Furthermore, in this review, we consider various factors influencing the HO system and finally assess current pharmacological approaches to their control.     

Protective role of heme oxygenase in the blood vessel wall during atherogenesis.

Several lines of evidence suggest that antioxidant processes and (or) endogenous antioxidants inhibit proatherogenic events in the blood vessel wall. Heme oxygenase (HO), which catabolizes heme to biliverdin, carbon monoxide, and catalytic iron, has been shown to have such antioxidative properties. The HO-1 isoform of heme oxygenase is ubiquitous and can be increased several fold by stimuli that induce cellular oxidative stress. Products of the HO reaction have important effects: carbon monoxide is a potent vasodilator, which is thought to play a role in modulation of vascular tone; biliverdin and its by-product bilirubin are potent antioxidants. Although HO induction results in an increase in catalytic free iron release, the enhancement of intracellular ferritin protein through HO-1 has been reported to decrease the cytotoxic effects of iron. Oxidized LDL has been shown to increase HO-1 expression in endothelial and smooth muscle cell cultures, and during atherogenesis. Further evidence of HO-1 expression associated with atherogenesis has been demonstrated in human, murine and rabbit atherosclerotic lesions. Moreover, genetic models of HO deficiency suggest that the actions of HO-1 are important in modulating the severity of atherosclerosis. Recent experiments in gene therapy using the HO gene suggest that interventions aimed at HO in the vessel wall could provide a novel therapeutic approach for the treatment or prevention of atherosclerotic disease.