Heme oxygenase and heme degradation

The microsomal heme oxygenase system consists of heme oxygenase (HO) and NADPH-cytochrome P450 reductase, and plays a key role in the physiological catabolism of heme which yields biliverdin, carbon monoxide, and iron as the final products. Heme degradation proceeds essentially as a series of autocatalytic oxidation reactions involving heme bound to HO. Large amounts of HO proteins from human and rat can now be prepared in truncated soluble form, and the crystal structures of some HO proteins have been determined. These advances have greatly facilitated the understanding of the mechanisms of individual steps of the HO reaction. HO can be induced in animals by the administration of heme or several other substances; the induction is shown to involve Bach1, a translational repressor. The induced HO is assumed to have cytoprotective effects. An uninducible HO isozyme, HO-2, has been identified, so the authentic HO is now called HO-1. HOs are also widely distributed in invertebrates, higher plants, algae, and bacteria, and function in various ways according to the needs of individual species.     

Involvement of heme oxygenase as antioxidant defense in soybean nodules

Objective: We have previously demonstrated that the inducible form of heme oxygenase plays a critical role in protecting against oxidative stress in mammals. To gain further insight into the functions of this enzyme in plants, we have tested its activity and expression in soybean nodules subjected to cadmium (Cd) stress.

Materials and methods: Four-weeks-old soybean nodulated plants were treated with different cadmium chloride concentrations (0, 50 and 200 μM) during 48 h. Oxidative stress parameters such as TBARS content, GSH levels and antioxidant enzyme activities were measured as well as heme oxygenase activity and expression. Besides, the effect of biliverdin and Zn-protophorphyrin IX were analized.

Results: Treatment with 200 μM Cd during 48 h caused a 67% increase in TBARS content, whereas GSH decreased 44%, and total superoxide dismutase, gluthatione reductase and guaiacol peroxidase were also inhibited 54, 20 and 60%, respectively. A total of 200 μM Cd produced the overexpression of heme oxygenase-1, as well as a 10-fold enhancement of its activity. Co-administration of biliverdin (10 μM) completely prevented the effects caused by Cd. Treatment with Zn protoporphyrin IX, a strong inhibitor of heme oxygenase, expectedly decreased heme oxygenase-1 activity to half. When the inhibitor was given together with Cd, completely prevented the enzyme induction and oxidative stress parameters were significantly enhanced.

Conclusion: Taking together, these results are indicating that heme oxygenase plays a protective role against oxidative cell damage in soybean nodules.     

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.     

Heme oxygenase expression in human central nervous system disorders

In the normal mammalian CNS, heme oxygenase-2 (HO-2) is constitutively, abundantly, and fairly ubiquitously expressed, whereas heme oxygenase-1 (HO-1) mRNA and protein are confined to small populations of scattered neurons and neuroglia. Unlike ho-2, the ho-1 gene in neural (and many systemic) tissues is exquisitely sensitive to upregulation by a host of pro-oxidant and other noxious stimuli. In Alzheimer disease, HO-1 immunoreactivity is significantly augmented in neurons and astrocytes of the hippocampus and cerebral cortex relative to age-matched, nondemented controls and colocalizes to senile plaques, neurofibrillary tangles, and corpora amylacea. In Parkinson disease, HO-1 decorates Lewy bodies of affected dopaminergic neurons and is highly overexpressed in astrocytes residing within the substantia nigra. The ho-1 gene is also upregulated in glial cells within multiple sclerosis plaques; in the vicinity of human cerebral infarcts, hemorrhages, and contusions; and in various other degenerative and nondegenerative human CNS disorders. The products of the heme oxygenase reaction, free ferrous iron, carbon monoxide, and biliverdin/bilirubin, are all biologically active molecules that may profoundly influence tissue redox homeostasis under a wide range of pathophysiological conditions. Evidence adduced from whole animal and in vitro studies indicates that enhanced HO-1 activity may either ameliorate or exacerbate neural injury, effects likely contingent upon the specific model employed, the duration and intensity of HO-1 induction, and the chemistry of the local redox microenvironment. HO-1 hyperactivity also promotes mitochondrial sequestration of nontransferrin iron in oxidatively challenged astroglia and may thereby contribute to the pathological iron deposition and bioenergetic failure amply documented in aging and degenerating human neural tissues.     

Ethylene perception inhibitor 1-MCP decreases oxidative damage of leaves through enhanced antioxidant defense mechanisms in soybean plants grown under high temperature stress

Ethylene is a stress hormone involved in early senescence and abscission of vegetative and reproductive organs under stress conditions. Ethylene perception inhibitors can minimize the impact of ethylene-mediated stress. The effects of high temperature (HT) stress during flowering on ethylene production rate in leaf, flower and pod and the effects of ethylene inhibitor on ethylene production rate, oxidative damage and physiology of soybean are not understood. We hypothesize that HT stress induces ethylene production, which causes premature leaf senescence and flower and pod abscission, and that application of the ethylene perception inhibitor 1-Methyl cyclopropene (1-MCP) can minimize HT stress induced ethylene response in soybean. The objectives of this study were to (1) determine whether ethylene is produced in HT stress; (2) quantify the effects of HT stress and 1-MCP application on oxidative injury; and (3) evaluate the efficacy of 1-MCP at minimizing HT-stress-induced leaf senescence and flower abscission. Soybean plants were exposed to HT (38/28 °C) or optimum temperature (OT; 28/18 °C) for 14 d at flowering stage (R2). Plants at each temperature were treated with 1-MCP (1 μg L⁻¹) gas for 5 h or left untreated (control). High temperature stress increased rate of ethylene production in leaves, flowers and pods, production of reactive oxygen species (ROS), membrane damage, and total soluble carbohydrate content in leaves and decreased photosynthetic rate, sucrose content, F_v/F_m ratio and antioxidant enzyme activities compared with OT. Foliar spray of 1-MCP decreased rate of ethylene production and ROS and leaf senescence traits but enhanced antioxidant enzyme activities (e.g. superoxide dismutase and catalase). In conclusion, HT stress increased ethylene production rates, caused oxidative damage, decreased antioxidant enzyme activity, caused premature leaf senescence, increased flower abscission and decreased pod set percentage. Application of 1-MCP lowered ethylene and ROS production, enhanced antioxidant enzyme activity, increased membrane stability, delayed leaf senescence, decreased flower abscission and increased pod set percentage. The beneficial effects of 1-MCP were greater under HT stress compared to OT in terms of decreased ethylene production, decreased ROS production, increased antioxidant protection, decreased flower abscission and increased pod set percentage.     

Heme oxygenase induction by menadione bisulfite adduct-generated oxidative stress in rat liver

The in vivo effect of menadione bisulfite adduct on both hepatic oxidative stress and heme oxygenase induction was studied. A marked increase in lipid peroxidation was observed 1 h after menadione bisulfite adduct administration. To evaluate liver antioxidant enzymatic defenses, superoxide dismutase, catalase and glutathione peroxidase activities were determined. Antioxidant enzymes significantly decreased 3 h after menadione bisulfite adduct injection. Heme oxygenase activity appeared 6 h after treatment, peaking 9 h after menadione bisulfite adduct administration. Such induction was preceded by a decrease in the intrahepatic GSH pool and an increase in hydrogen peroxide steady-state concentration, both effects taking place some hours before induction of heme oxygenase. Iron ferritin levels and ferritin content began to increase 6 h after heme oxygenase induction, and these increases were significantly higher 15 h after treatment and remained high for at least 24 h after menadione bisulfite adduct injection. Administration of bilirubin entirely prevented heme oxygenase induction as well as the decrease in hepatic GSH and the increase in lipid peroxidation when administered 2 h before menadione bisulfite adduct treatment. These results indicate that the induction of heme oxygenase by menadione bisulfite adduct may be a general response to oxidant stress, by increasing bilirubin and ferritin levels and could therefore provide a major cellular defense mechanism against oxidative damage.     

脑血红素加氧酶系统的作用研究

血红素加氧酶（HO）是降解血红素的微粒体酶系统,催化降解血红素生成一氧化碳（CO）、胆绿素和铁离子,同工酶HO-1和HO-2广泛分布于脑中,具有不同的调控机制,可能发挥着调节NO水平,抗氧化应激,参与神经元退行性变等重要作用。     

Brain microvascular and intracranial artery resistance to atherosclerosis is associated with heme oxygenase and ferritin in Japanese quail

Oxidative stress and increased oxidation of low-density lipoprotein (oxLDL) through free radical-mediated tissue injury may be important factors in the development of extracranial atherosclerotic lesions. However, the roles of oxidative stress and hypercholesterolemia in intracranial atherosclerosis is less established. The induction of heme oxygenase (HO) is a cellular response to oxidative stress, and inducible HO (HO-1) may protect against oxidized lipids such as those produced by oxidative stress. We investigated the effects of oxLDL on cell and tissue viability, HO-1 and ferritin expression in extracranial and intracranial endothelial cells, and the arteries of cholesterol-induced atherosclerosis (CIA) Japanese quail. We report that cultured microvascular endothelial cells from the brain (QBMEC) and carotid (QCEC) differ in their response to oxidative stress. The QCECs are less responsive than QBMECs to oxidative stress induced by oxLDL, as evident by lower expression of HO-1 mRNA, HO activity, and ferritin levels. Furthermore, the higher levels of catalytic iron, thiobarbituric acid reactive substances, and lactate dehydrogenase released in QCECs indicated that these cells are more susceptible to oxidative stress than QBMECs. We also investigated the relationship between extent of atherosclerotic plaque deposition and the extracranial and intracranial arterial expression of HO-1 in quail. The common carotid and vertebral (extracranial) arteries had higher tissue cholesterol levels (starting at 2 weeks of cholesterol-supplementation) and a greater atherosclerotic plaque score (starting at 4 weeks of cholesterol-supplementation) compared with middle cerebral and basilar (intracranial) arteries, and this may be relevant to the effect of aging on the process of atherogenesis. The extracranial arteries also had early and greater levels of lipid peroxidation and catalytic iron coupled with lower expression of HO-1 protein, HO activity, and ferritin compared to the intracranial vessels. These observations suggest that the extracranial and intracranial arterial walls respond differently to oxidation of lipoproteins, and support the feasibility of increased HO-1 expression as a means of protection against oxidant injury.     

S-Allylcysteine, a garlic compound, protects against oxidative stress in 1-methyl-4-phenylpyridinium-induced parkinsonism in mice

S-Allylcysteine (SAC), the most abundant organosulfur compound in aged garlic extract, has multifunctional activity via different mechanisms and neuroprotective effects that are exerted probably via its antioxidant or free radical scavenger action. The 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mouse has been the most widely used model for assessing neuroprotective agents for Parkinson's disease. 1-Methyl-4-phenylpyridinium (MPP⁺) is the stable metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, and it causes nigrostriatal dopaminergic neurotoxicity. Previous studies suggest that oxidative stress, via free radical production, is involved in MPP⁺-induced neurotoxicity. Here, we report on the neuroprotective effect of SAC against oxidative stress induced by MPP⁺ in the striatum of C57BL/6J mice. Mice were pretreated with SAC (125 mg/kg ip) daily for 17 days, followed by administration of MPP⁺ (0.72 mg/kg icv), and were sacrificed 24 h later to evaluate lipid peroxidation, different antioxidant enzyme activities, spontaneous locomotor activity and dopamine (DA) content. MPP⁺ administration resulted in a significant decrease in DA levels in the striatum. Mice receiving SAC (125 mg/kg ip) had significantly attenuated MPP⁺-induced loss of striatal DA levels (32%). The neuroprotective effect of SAC against MPP⁺ neurotoxicity was associated with blocked (100% of protection) of lipid peroxidation and reduction of superoxide radical production — indicated by an up-regulation of Cu-Zn-superoxide dismutase activity — both of which are indices of oxidative stress. Behavioral analyses showed that SAC improved MPP⁺-induced impairment of locomotion (35%). These findings suggest that in mice, SAC attenuates MPP⁺-induced neurotoxicity in the striatum and that an antioxidant effect against oxidative stress may be partly responsible for its observed neuroprotective effects.     

beta-carbolines that accumulate in human tissues may serve a protective role against oxidative stress

beta-Carbolines are tricyclic nitrogen heterocycles formed in plants and animals as Maillard reaction products between amino acids and reducing sugars or aldehydes. They are being detected increasingly in human tissues, and their physiological roles need to be understood. Two beta-carboline carboxylates have been reported to accumulate in the human eye lens. We report here on the identification of another beta-carboline, namely 1-methyl-1-vinyl -2, 3,4-trihydro-beta-carboline-3-carboxylic acid, in the lenses of some cataract patients from India. Analysis of these three lenticular beta-carbolines using photodynamic and antioxidant assays shows all of them to be inert as sensitizers and effective as antioxidants; they quench singlet oxygen, superoxide and hydroxyl radicals and inhibit the oxidative formation of higher molecular weight aggregates of the test protein, eye lens gamma-crystallin. Such antioxidative ability of beta-carbolines is of particular relevance to the lens, which faces continual photic and oxidative stress. The beta-carboline diacid IV is also seen to display an unexpected ability of inhibiting the thermal coagulation of gamma-crystallin and the dithiothreitol-induced precipitation of insulin. These results offer experimental support to earlier suggestions that one of the roles that the beta-carbolines have is to offer protection against oxidative stress to the human tissues where they accumulate.     

Leptospira interrogans requires a functional heme oxygenase to scavenge iron from hemoglobin

The transposon TnSC189 was used to construct a mutant in the putative heme oxygenase gene hemO (LB186) of Leptospira interrogans. Unlike its parent strain, the mutant grew poorly in medium in which hemoglobin was the sole iron source. The putative heme oxygenase was over expressed in a His-tagged form, purified and was demonstrated to degrade heme in vitro. Unexpectedly, it was also found that the L. interrogans growth rate was significantly increased when medium was supplemented with hemoglobin, but only if ferrous iron sources were absent. This result was mirrored in the expression of some iron-related genes and suggests the presence of regulatory mechanisms detecting Fe²⁺ and hemoglobin. This is the first demonstration of a functional heme oxygenase from a spirochete.     

Heme oxygenase-1 prevents superoxide anion-associated endothelial cell sloughing in diabetic rats

Heme oxygenase-1 (HO-1) represents a key defense mechanism against oxidative injury. Hyperglycemia has been linked to increased oxidative stress, leading to endothelial dysfunction, delayed cell replication, and enhanced apoptosis. The effect of streptozotocin (STZ)-induced diabetes on HO activity, HO-1 promoter activity, superoxide anion (O*-2, and the number of circulating endothelial cells was measured. The expression of HO-1/HO-2 protein was unchanged, but HO activity was decreased in aortas of diabetic rats compared with control (p < 0.05). High glucose decreased HO-1 promoter activity (p < 0.05). Hyperglycemia increased O*-2 and this increase was augmented with HO-1 inhibition and diminished with HO-1 upregulation (p < 0.05). Circulating endothelial cells were significantly higher in diabetic rats and were decreased or increased with administration of the HO-1 inducer (CoPP) or inhibitor (SnMP), respectively (p<0.05). In conclusion, HO-1 upregulation in diabetic rats brings about an increase in serum bilirubin, a reduction in O*-2 production, and a decrease in endothelial cell sloughing.     

Glyphosate-induced oxidative stress in rice leaves revealed by proteomic approach

Glyphosate is one of the most widely used herbicides in cereal-growing regions worldwide. In the present work, the protein expression profile of rice leaves exposed to glyphosate was analyzed in order to investigate the alternative effects of glyphosate on plants. Two-week-old rice leaves were subjected to glyphosate or a reactive oxygen species (ROS) inducing herbicide paraquat, and total soluble proteins were extracted and analyzed by two-dimensional gel electrophoresis (2-DE) coupled with matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) analysis. A total of 25 differentially expressed proteins were identified from the glyphosate treated sample, wherein 18 proteins were up-regulated and 7 proteins were down-regulated. These proteins had shown a parallel expression pattern in response to paraquat. Results from the 2-DE analysis, combined with immunoblotting, clearly revealed that ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit was significantly decreased by the treatment of both herbicides. An increased accumulation of antioxidant enzymes including ascorbate peroxidase, glutathione S-transferase, thioredoxin h-type, nucleoside diphosphate kinase 1, peroxiredoxin and a superoxide dismutase [Cu–Zn] chloroplast precursor in the glyphosate-treated sample suggests that a glyphosate treatment possibly generates oxidative stress in plants. Moreover, a gene expression analysis of five antioxidant enzymes by Northern blot confirmed their mRNA levels in the rice leaves. A histo-cytochemical investigation with DAB (3,3-diaminobenzidine) to localize H₂O₂ and increases of the thiobarbituric acid reactive substances (TBARS) concentration revealed that the glyphosate application generates ROS, which resulted in the peroxidation and destruction of lipids in the rice leaves.     

A High Expression of Heme Oxygenase-1 in the Liver of LEC Rats at the Stage of Hepatoma: The Possible Implication of Induction in Uninvolved Tissue

We have examined changes in the expression of heme oxygenase-1 (HO-1), an inducible isoform and HO-2, a constitutive isoform, in the liver of Long-Evans with a Cinnamon-like color (LEC) rat, a mutant strain which spontaneously develops acute hepatitis and hepatoma. HO-1 expression was highly enhanced in the LEC rat livers with jaundice, and then decreased slightly, but overall remained at a higher level than in the Long-Evans with Agouti color (LEA) control rats, as judged by Northern blotting analysis of the whole liver extract. The high expression of HO-1 in the LEC rat liver was, however, not due to the actual cancer lesion but, rather, due to the surrounding uninvolved tissues including hepatocytes. Immunohistochemical analysis also supported this conclusion. Among normal tissues, the expression of HO-1 but not HO-2 was high in only the spleen of both LEC and LEA rats.

The high expression observed in the stage of acute hepatitis and hepatoma stages in the LEC rat is probably due to the oxidative stress caused by the accumulation of free copper and free iron levels which has been reported earlier by our group (Suzuki et al., Carcinogenesis, 1993,14, 1881-1884 and Koizumi et al., Free Radical Research, in press) as well as by free heme levels. The inflammatory cytokines produced by the surrounding tissue at the hepatoma stage would also be expected to play a role in the induction mechanism. The physiological relevance of HO-1 induction might be an adaptive response to oxidative stress and vasodilatory effect of carbon monoxide on sinusoidal circulation.