Management of Oxidative Stress by Heme Oxygenase-1 in Cisplatin-induced Toxicity in Renal Tubular Cells

Induction of heme oxygenase-1 (HO-1) may serve as an immediate protective response during treatment with the cytostatic drug cisplatin (CDDP). Oxidative pathways participate in the characteristic nephrotoxicity of CDDP. In the present study, cultured tubular cells (LLC-PK1) were used to investigate whether induction of HO provided protection against CDDP by maintaining the cellular redox balance. The antioxidants, &#102 -tocopherol (TOCO) and N -acetylcysteine (NAC), were used to demonstrate that elevation of ROS levels contribute to the development of CDDP-induced cytotoxicity. Chemical modulators of HO activity were used to investigate the role of HO herein. Hemin was used to specifically induce HO-1, while exposure of the cells to tin-protoporphyrin (SnPP) was shown to inhibit HO activity. Hemin treatment prior to CDDP-exposure significantly decreased the generation of ROS to control levels, while inhibition of HO increased the ROS levels beyond the levels measured in cells treated with CDDP alone. Furthermore, HO induction protected significantly against the cytotoxicity of CDDP, although this protection was limited. Similar results were obtained when the cells were preincubated with TOCO, suggesting that mechanisms other than impairment of the redox ratio are important in CDDP-induced loss of cell viability in vitro. In addition, SnPP treatment exacerbated the oxidative response and cytotoxicity of CDDP, especially at low CDDP concentrations. We therefore conclude that HO is able to directly limit the CDDP-induced oxidative stress response and thus serves as safeguard of the cellular redox balance.     

Inhibition of vascular smooth muscle cell proliferation by chronic hemin treatment

Hemin, an oxidized form of heme, is an essential regulator of gene expression and cell cycle progression. Our laboratory previously reported (34) that chronic hemin treatment of spontaneously hypertensive rats reversed the eutrophic inward remodeling of small peripheral arteries. Whether long-term treatment of cultured vascular smooth muscle cells (VSMCs) with hemin alters the proliferation status of these cells has been unknown. In the present study, hemin treatment at 5 muM for 4, 7, 14, and 21 days significantly inhibited the proliferation of cultured rat aortic VSMCs (A-10 cells) by arresting cells at G0/G1 phases so as to decelerate cell cycle progression. Heme oxygenase (HO) activity and inducible HO-1 protein expression were significantly increased by hemin treatment. HO inhibitor tin protoporphyrin IX (SnPP) abolished the effects of hemin on cell proliferation and HO activity. Interestingly, hemin-induced HO-1 expression was further increased in the presence of SnPP. Hemin treatment had no significant effect on the expression of constitutive HO-2. Expression of p21 protein and the level of reactive oxygen species (ROS) were decreased by hemin treatment, which was reversed by application of SnPP. After removal of hemin from culture medium, inhibited cell proliferation and increased HO-1 expression in VSMCs were returned to control level within 1 wk. Transfection with HO-1 small interfering RNA significantly knocked down HO-1 expression and decreased HO activity, but had no effect on HO-2 expression, in cells treated with or without hemin for 7 days. The inhibitory effect of hemin on cell proliferation was abolished in HO-1 silenced cells. It is concluded that induction of HO-1 and, consequently, increased HO activity are responsible for the chronic inhibitory effect of hemin on VSMC proliferation. Changes in the levels of p21 and ROS might also participate in the cellular effects of hemin.     

Heme oxygenase modulates oxidant-signaled airway smooth muscle contractility: role of bilirubin

Reactive oxygen species (ROS) increase the contractile response of airway smooth muscle (ASM). Heme oxygenase (HO) catabolizes heme to the powerful antioxidant bilirubin. Because HO is expressed in the airways, we investigated its effects on ASM contractility and ROS production in guinea pig trachea. HO expression was higher in the epithelium than in tracheal smooth muscle. Incubation of tracheal rings (TR) with the HO inhibitor tin protoporphyrin (SnPP IX) or the HO substrate hemin increased and decreased, respectively, ASM contractile response to carbamylcholine. The effect of hemin was reversed by SnPP and mimicked by the antioxidants superoxide dismutase (SOD) and catalase. Hemin significantly reduced the effect of carbamylcholine in rings treated with the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), compared with ODQ-treated rings without hemin incubation, suggesting that the CO-guanosine 3',5'-cyclic monophosphate pathway was not involved in the control of tracheal reactivity. SnPP and hemin increased and decreased ROS production by TR by 18 and 38%, respectively. Bilirubin (100 pM) significantly decreased TR contractility and ROS production. Hemin, bilirubin, and SOD/catalase decreased phosphorylation of the contractile protein myosin light chain, whereas SnPP significantly augmented it. These data suggest that modulation of the redox status by HO and, moreover, by bilirubin modulates ASM contractility by modulating levels of phosphorylated myosin light chain.     

Tin-protoporphyrin potentiates arsenite-induced DNA strand breaks, chromatid breaks and kinetochore-negative micronuclei in human fibroblasts

Numerous reports have shown that oxidative stress is involved in arsenite-induced genetic damage. Arsenite is also a potent inducer of heme oxygenase (HO)-1. To understand whether HO-1 could function as a cellular antioxidant and protect cells from arsenite injury, the effects of tin-protoporphyrin (SnPP), a competitive inhibitor of HO-1, on arsenite-induced genetic damage were examined in human skin fibroblasts (HFW). In the present study, we found that SnPP at 100 microM significantly potentiated arsenite-induced cytotoxicity, DNA strand breaks (assayed by alkaline single cell gel electrophoresis(SCGE)), and chromatid breaks. Although arsenite alone mainly induced kinetochore-plus micronuclei (K(+)-MN), SnPP only synergistically enhanced kinetochore-negative micronuclei (K(-)-MN). The increase in K(-)-MN by SnPP cotreatment was consistent with the increase in DNA strand breaks and chromatid breaks caused by SnPP. However, at higher arsenite doses, K(+)-MN was significantly reduced by SnPP. Pretreatment of HFW cells with hemin, an inducer of HO-1, significantly attenuated the cytotoxicity of arsenite. Therefore, the present results suggest that HO-1 induction by arsenite plays certain roles in protecting cells from arsenite-induced injury.     

Heme-oxygenase-mediated iron accumulation in the liver

Heme oxygenase (HO) isozymes, HO-1 and HO-2, catalyze the conversion of heme to iron, carbon monoxide, and biliverdin. The present study was aimed at elucidating the role of the HO system in iron accumulation and oxidative stress in the liver. We have also studied the regulation of an iron exporter, ferroportin-1 (FPN-1), as an adaptive response mechanism to increased iron levels. Sprague-Dawley rats were treated with HO inducer hemin or HO inhibitor tin-protoporphyrin IX (SnPPIX) for 1 month. A portion of liver tissues was subjected to RT-PCR for HO-1, HO-2, and FPN-1 gene expression as well as an HO activity assay. Paraffin-embedded tissues were stained for iron with Prussian blue. Hepatic iron concentration was measured by High Resolution-Inductively Coupled Plasma-Mass Spectrometry. 8-hydroxy-2'-deoxyguanosine (8-OHdG) stain, a sensitive and specific marker of oxidative DNA damage, was performed to assess oxidative stress. Hemin treatment led to augmented HO expression and activity in association with increased iron accumulation and oxidative stress. FPN-1 expression was also found to be upregulated. SnPPIX treatment reduced HO activity, intracellular iron levels, and oxidative stress as compared to controls. Our data provides evidence of increased HO activity as an important pro-oxidant mechanism leading to iron accumulation in the liver.     

Scavenging activity of indole compounds against cisplatin-induced reactive oxygen species

The antioxidant effects of indole compounds such as melatonin (MLT), tryptophan, and serotonin, on cisplatin (cis-diaminedichloroplatinum, or CDDP)-induced reactive oxygen species (ROS) generation were examined by electron spin resonance (ESR). In addition, DNA fragmentation by CDDP-induced ROS and the effect of MLT on it were analyzed in primary cultures of rat renal tubular epithelial cells. MLT and serotonin had scavenging effects on CDDP-induced hydroxy radicals (*OH), and the scavenging activity of MLT was higher than that of serotonin. The exposure of primary-cultured renal tubular cells to CDDP caused severe cytotoxicity. Tryptophan, serotonin, and 6-OH-MLT did not reduce the CDDP-induced cytotoxicity, whereas MLT did. CDDP exposure induced DNA fragmentation in primary-cultured renal tubular cells, but the simultaneous administration of MLT inhibited the DNA fragmentation. These results indicate that MLT inhibits CDDP-induced cytotoxicity by directly scavenging *OH, and that MLT markedly reduces renal cytotoxicity and DNA fragmentation caused by CDDP-induced ROS in vitro.     

5-Aminolevulinic Acid Protects against Cisplatin-Induced Nephrotoxicity without Compromising the Anticancer Efficiency of Cisplatin in Rats In Vitro and In Vivo

Background/Aims

Nephrotoxicity is a frequent and major limitation in cisplatin (CDDP)-based chemotherapy. 5-Aminolevulinic acid (ALA) is widely distributed in animal cells, and it is a precursor of tetrapyrole compounds such as heme that is fundamentally important in aerobic energy metabolism. The aim of this study is to evaluate the protective role of ALA in CDDP-induced acute kidney injury (AKI).

Method

We used CDDP-induced AKI rat model and cultured renal tubular cells (NRK-52E). We divided four groups of rats: control, CDDP only, CDDP + ALA(post);(ALA 10 mg/kg + Fe in drinking water) after CDDP, CDDP + ALA(pre & post).

Result

CDDP increased Cr up to 6.5 mg/dl, BUN up to 230 mg/dl, and ALA significantly reduced these changes. ALA ameliorates CDDP-induced morphological renal damages, and reduced tubular apoptosis evaluated by TUNEL staining and cleaved caspase 3. Protein and mRNA levels of ATP5α, complex(COX) IV, UCP2, PGC-1α in renal tissue were significantly decreased by CDDP, and ALA ameliorates reduction of these enzymes. In contrast, Heme Oxigenase (HO)-1 level is induced by CDDP treatment, and ALA treatment further up-regulates HO-1 levels. In NRK-52E cells, the CDDP-induced reduction of protein and mRNA levels of mitochondrial enzymes was significantly recovered by ALA + Fe. CDDP-induced apoptosis were ameliorated by ALA + Fe treatment. Furthermore, we evaluated the size of transplantated bladder carcinoma to the rat skin, and ALA did not change the anti cancer effects of CDDP.

Conclusion

These data suggested that the protective role of ALA in cisplatin-induced AKI is via protection of mitochondrial viability and prevents tubular apoptosis. Also there are no significant effects of ALA on anticancer efficiency of CDDP in rats. Thus, ALA has the potential to prevent CDDP nephrotoxicity without compromising its anticancer efficacy.     

Interaction between endothelial heme oxygenase-2 and endothelin-1 in altered aortic reactivity after hypoxia in rats

The aim of this study was to determine whether increased expression of heme oxygenase (HO) contributes to impairment of aortic contractile responses after hypoxia through effects on reactivity to endothelin-1 (ET-1). Thoracic aortas from normoxic rats and rats exposed to hypoxia (10% O2) for 16 or 48 h were mounted in organ bath myographs for contractile studies, fixed in paraformaldehyde, or frozen in liquid nitrogen for protein extraction. In rings from normoxic rats, the HO inhibitor tin protoporphyrin IX (SnPP IX, 10 microM) did not alter the response to phenylephrine or ET-1. In rings from rats exposed to 16-h hypoxia, maximum tension generated in response to these agonists was higher in endothelium-intact but not -denuded rings in the presence of SnPP IX. In rings from rats exposed to 48-h hypoxia SnPP IX increased contraction in endothelium-intact but not -denuded rings. In endothelium-intact aortic rings from rats exposed to 16-h hypoxia incubated with endothelin A receptor-specific antagonist BQ-123 (10(-7) M), SnPP IX did not alter phenylephrine-induced contraction. Aortic ET-1 protein levels, measured by radioimmunoassay, were increased in rats exposed to hypoxia for 16 and 48 h. Western blotting showed that HO-1 and HO-2 protein were increased after 16 h of hypoxia and returned to near-control levels after 48 h. Increase in HO-1 protein was detected in endothelium-intact and -denuded rings. Removal of endothelium abolished the increase in HO-2 immunoreactivity. Immunohistochemistry localized expression of HO-1 protein to vascular smooth muscle, whereas HO-2 was only detected in endothelium. HO-2 is expressed by aortic endothelial cells early during hypoxic exposure and impairs ET-1-mediated potentiation of contraction to alpha-adrenoceptor stimulation.     

The role of oxidative stress in the ochratoxin A-mediated toxicity in proximal tubular cells

Balkan endemic nephropathy (BEN), a disease characterized by progressive renal fibrosis in human patients, has been associated with exposure to ochratoxin A (OTA). This mycotoxin is a frequent contaminant of human and animal food products, and is toxic to all animal species tested. OTA predominantly affects the kidney and is known to accumulate in the proximal tubule (PT). The induction of oxidative stress is implicated in the toxicity of this mycotoxin.In the present study, primary rat PT cells and LLC-PK(1) cells, which express characteristics of the PT, were used to investigate the OTA-mediated oxidative stress response. OTA exposure of these cells resulted in a concentration-dependent elevation of reactive oxygen species (ROS) levels, depletion of cellular glutathione (GSH) levels and an increase in the formation of 8-oxoguanine.The OTA-induced ROS response was significantly reduced following treatment with alpha-tocopherol (TOCO). However, this chain-braking anti-oxidant did not reduce the cytotoxicity of OTA and was unable to prevent the depletion of total GSH levels in OTA-exposed cells. In contrast, pre-incubation of the cell with N-acetyl-L-cysteine (NAC) completely prevented the OTA-induced increase in ROS levels as well as the formation of 8-oxoguanine and completely protected against the cytotoxicity of OTA. In addition, NAC treatment also limited the GSH depletion in OTA-exposed PT- and LLC-PK(1) cells.From these data, we conclude that oxidative stress contributes to the tubular toxicity of OTA. Subsequently, cellular GSH levels play a pivotal role in limiting the short-term toxicity of this mycotoxin in renal tubular cells.     

Catalytic inactive heme oxygenase-1 protein regulates its own expression in oxidative stress

Heme oxygenase-1 (HO-1) catalyzes the degradation of heme and forms antioxidant bile pigments as well as the signaling molecule carbon monoxide. HO-1 is inducible in response to a variety of chemical and physical stress conditions to function as a cytoprotective molecule. Therefore, it is important to maintain the basal level of HO-1 expression even when substrate availability is limited. We hypothesized that the HO-1 protein itself could regulate its own expression in a positive feedback manner, and that this positive feedback was important in the HO-1 gene induction in response to oxidative stress. In cultured NIH 3T3 cells, transfection of HO-1 cDNA or intracellular delivery of pure HO-1 protein resulted in activation of a 15-kb HO-1 promoter upstream of luciferase as visualized by bioluminescent technology and increased HO-1 mRNA and protein levels. These effects were independent of HO activity because an enzymatically inactive mutant form of HO-1 similarly activated the HO-1 promoter and incubation with HO inhibitor metalloporphyrin SnPP did not affect the promoter activation. In addition, HO-1-specific siRNA significantly reduced hemin and cadmium chloride-mediated HO-1 induction. Furthermore, deletion analyses demonstrated that the E1 and E2 distal enhancers of the HO-1 promoter are required for this HO-1 autoregulation. These experiments document feed-forward autoregulation of HO-1 in oxidative stress and suggest that HO-1 protein has a role in the induction process. We speculate that this mechanism may be useful for maintaining HO-1 expression when substrate is limited and may also serve to up-regulate other genes to promote cytoprotection and to modulate cell proliferation.     

Atrial natriuretic peptide reduces cyclosporin toxicity in renal cells: role of cGMP and heme oxygenase-1.

Using cultured proximal renal tubular epithelial cells (LLC-PK1), the present study investigates the effect of atrial natriuretic peptide (ANP) on cytotoxicity induced by cyclosporin A (CsA). Preincubation with ANP (1-100 nM) protected LLC-PK1 cells from CsA-induced toxicity in a concentration-dependent manner. A cytoprotective effect comparable to ANP was observed when preincubating the cells with 8-bromo cGMP (1-100 microM) or the antioxidant heme oxygenase (HO) metabolite bilirubin (0.1-10 microM). ANP or cGMP produced increases in HO-1 protein levels at concentrations that were also effective in cellular protection. Moreover, incubation with ANP or 8-bromo cGMP led to increased HO activity, i.e., formation of bilirubin in the cell lysate (up to 3-fold over basal). Tin protoporphyrin-IX (SnPP; 19 microM), an inhibitor of HO activity, completely abolished ANP-induced cytoprotection. Our results demonstrate that HO-1 is a cellular target of ANP and cGMP in renal cells. HO-1 induction and ensuing formation of antioxidant metabolites may be a novel pathway by which ANP protects from CsA-dependent nephrotoxicity and preserves renal function.     

The Ganglioside GM3 Is Associated with Cisplatin-Induced Apoptosis in Human Colon Cancer Cells

Cisplatin (cis-diamminedichloroplatinum, CDDP) is a well-known chemotherapeutic agent for the treatment of several cancers. However, the precise mechanism underlying apoptosis of cancer cells induced by CDDP remains unclear. In this study, we show mechanistically that CDDP induces GM3-mediated apoptosis of HCT116 cells by inhibiting cell proliferation, and increasing DNA fragmentation and mitochondria-dependent apoptosis signals. CDDP induced apoptosis within cells through the generation of reactive oxygen species (ROS), regulated the ROS-mediated expression of Bax, Bcl-2, and p53, and induced the degradation of the poly (ADP-ribosyl) polymerase (PARP). We also checked expression levels of different gangliosides in HCT116 cells in the presence or absence of CDDP. Interestingly, among the gangliosides, CDDP augmented the expression of only GM3 synthase and its product GM3. Reduction of the GM3 synthase level through ectopic expression of GM3 small interfering RNA (siRNA) rescued HCT116 cells from CDDP-induced apoptosis. This was evidenced by inhibition of apoptotic signals by reducing ROS production through the regulation of 12-lipoxigenase activity. Furthermore, the apoptotic sensitivity to CDDP was remarkably increased in GM3 synthase-transfected HCT116 cells compared to that in controls. In addition, GM3 synthase-transfected cells treated with CDDP exhibited an increased accumulation of intracellular ROS. These results suggest the CDDP-induced oxidative apoptosis of HCT116 cells is mediated by GM3.     

Heme Oxygenase-1 Protects Retinal Endothelial Cells against High Glucose- and Oxidative/Nitrosative Stress-Induced Toxicity

Diabetic retinopathy is a leading cause of visual loss and blindness, characterized by microvascular dysfunction. Hyperglycemia is considered the major pathogenic factor for the development of diabetic retinopathy and is associated with increased oxidative/nitrosative stress in the retina. Since heme oxygenase-1 (HO-1) is an enzyme with antioxidant and protective properties, we investigated the potential protective role of HO-1 in retinal endothelial cells exposed to high glucose and oxidative/nitrosative stress conditions. Retinal endothelial cells were exposed to elevated glucose, nitric oxide (NO) and hydrogen peroxide (H(2)O(2)). Cell viability and apoptosis were assessed by MTT assay, Hoechst staining, TUNEL assay and Annexin V labeling. The production of reactive oxygen species (ROS) was detected by the oxidation of 2',7'-dichlorodihydrofluorescein diacetate. The content of HO-1 was assessed by immunobloting and immunofluorescence. HO activity was determined by bilirubin production. Long-term exposure (7 days) of retinal endothelial cells to elevated glucose decreased cell viability and had no effect on HO-1 content. However, a short-time exposure (24 h) to elevated glucose did not alter cell viability, but increased both the levels of intracellular ROS and HO-1 content. Moreover, the inhibition of HO with SnPPIX unmasked the toxic effect of high glucose and revealed the protection conferred by HO-1. Oxidative/nitrosative stress conditions increased cell death and HO-1 protein levels. These effects of elevated glucose and HO inhibition on cell death were confirmed in primary endothelial cells (HUVECs). When cells were exposed to oxidative/nitrosative stress conditions there was also an increase in retinal endothelial cell death and HO-1 content. The inhibition of HO enhanced ROS production and the toxic effect induced by exposure to H(2)O(2) and NOC-18 (NO donor). Overexpression of HO-1 prevented the toxic effect induced by H(2)O(2) and NOC-18. In conclusion, HO-1 exerts a protective effect in retinal endothelial cells exposed to hyperglycemic and oxidative/nitrosative stress conditions.     

Heme oxygenase-1 is an important modulator in limiting glucose-induced apoptosis in human umbilical vein endothelial cells

Hyperglycaemia is associated with oxidative stress. The inducible isoform of heme oxygenase (HO-1) is an effective system to counteract oxidative stress, yet it is unclear how hyperglycaemia affects HO-1. In this study, we explored: 1) the HO-1 protein content and HO activity in human umbilical vein endothelial cells (HUVECs) exposed to different glucose concentrations, and 2) the mechanisms which account for the high glucose-induced effects on HO-1. We evaluated HO-1 protein expression, HO activity, apoptosis and reactive oxygen species (ROS) in HUVECs treated for 48 h with 5.5, 10 and 20 mM glucose. A dose-dependent production of reactive oxygen species was observed. At 10 mM glucose, an increase of HO-1 protein expression and HO activity was observed, whereas at 20 mM, there was no change in protein content and activity relative to at 5.5 mM glucose. HO-1 protein expression in HUVECs exposed to 20 mM of glucose was increased in the presence of 20 U/ml superoxide dismutase (SOD). HO-1 gene silencing augments ROS production both at 5.5 and 10 mM glucose, leading to an increased apoptosis. We conclude that, in endothelial cells, the regulation of HO-1 by glucose is dependent upon levels of glucose itself. Lack of homeostatic HO-1 upregulation fails to protect from oxidative damage and results in a higher rate of apoptotic cell death.     

Carbon monoxide and bilirubin from heme oxygenase-1 suppresses reactive oxygen species generation and plasminogen activator inhibitor-1 induction

Heme oxygenase-1 (HO-1) responds to a variety of oxidative stresses. We examined whether HO-1 expression influences pro-thrombotic processes, in which the involvement of oxidative stress has been reported. Since HO-1 knockout mice with a C57/BL6J background were not viable, embryonic cells from HO-1 deficient mice (E11.5) were used. Cell viability, the level of plasminogen activator inhibitor-1 (PAI-1) expression and reactive oxygen species (ROS) generation of HO-1 deficient cells in response to the exposures to hydrogen peroxide and oxidized LDL were compared to those with wild-type cells. We also examined the effects of glutathione (GSH), desferrioxamine (DFO) and diphenyleneiodonium (DPI: an NADPH oxidase inhibitor) as well as of the HO reaction products, bilirubin (BR) and carbon monoxide (CO) on PAI-1 expression and ROS generation. PAI-1 expression and ROS generation were markedly elevated in HO-1 deficient cells compared to wild-type cells. Exposure to oxidized LDL significantly elevated PAI-1 expression and ROS production in HO-1 deficient cells. Interestingly, these increases in HO-1 deficient cells were significantly lowered by BR, CO, GSH and DPI while DFO had little effect. Furthermore, BR and CO were effective to improve viabilities of HO-1 deficient cells. These results suggest that HO-1 may be required to suppress ROS generation and the production of pro-thrombotic molecules such as PAI-1.     

Catalytically inactive heme oxygenase-2 mutant is cytoprotective

Heme oxygenase (HO) catalyzes the rate-limiting step in heme degradation, producing iron, carbon monoxide, and bilirubin/biliverdin. HO consists of two isozymes: HO-1, which is an oxidative stress-response protein, and HO-2, which is constitutively expressed. HO-2 accounts for most HO activity within the nervous system. Its posttranslational modifications and/or interactions with other proteins make HO-2 a unique regulator of cellular homeostasis. Our previous results revealed that brain infarct volume was enlarged in HO-2 knockout mice. A similar neuroprotective role of HO-2 was shown using primary cortical neurons. To better understand the neuroprotective mechanism of HO-2, we used a catalytically inactive mutant, HO-2H45A, and investigated its cellular effects in response to hemin and hydrogen peroxide-induced cytotoxicity. We observed that HO-2WT overexpression in the HEK293 cell lines became less sensitive to hemin, whereas the inactive mutant HO-2H45A was more sensitive to hemin as compared to control. Interestingly, HO-2WT- and HO-2H45A-overexpressing cells were both protected against H2O2-induced oxidative stress and had less oxidatively modified proteins as compared to control cells. These data indicate that when HO-2 cannot metabolize the prooxidant heme, more cytotoxicity is found, whereas, interestingly, the catalytically inactive HO-2H45A was also able to protect cells against oxidative stress injury. These results suggest the multiplicity of action of the HO-2 protein itself.     

Ferritin in Parkinsonian and control brains

Heme oxygenase-1 (HO-1) is a stress protein expressed in various pathological conditions associated with oxidative stress. Brain HO-1 expression and activity in response to LPS treatment showed regional variability with the highest levels in the substantia nigra (SN) and hippocampus. HO-1 induction by LPS was redox-sensitive and associated with increased levels of NO synthase and arginase, two proteins involved in the regulation of cellular redox state. Brain HO-2 and HO-3 expression, studied by quantitative RT-PCR, did not show significant changes. Our data suggest an interaction between NO and the HO system in the brain after LPS treatment. As SN and hippocampus are involved in Parkinson's and Alzheimer's diseases, understanding interaction of these proteins in the brain will help to elucidate the mechanisms involved in neurodegeneration.     

Heme oxygenase-1 gene expression attenuates angiotensin II-mediated DNA damage in endothelial cells

Heme oxygenase (HO) catalyzes the conversion of heme to biliverdin with the release of iron and carbon monoxide. HO-1 is inducible by inflammatory conditions, which cause oxidative stress in endothelial cells. Overexpression of human HO-1 in endothelial cells may have the potential to provide protection against a variety of agents that cause oxidative stress. We investigated the physiological significance of human HO-1 overexpression using a retroviral vector on attenuation of angiotensin II (Ang II)-mediated oxidative stress. Comet and glutathione (GSH) levels were used as indicators of the levels of oxidative stress. Comet assay was performed to evaluate damage on DNA, whereas GSH levels were measured to determine the unbalance of redox potential. Pretreatments with inducers, such as heme 10 microM, SnCl(2) 10 microM, and inhibitors, such as tin-mesoporphyrin 10 microM was followed by treatment with Ang II 200 ng/ml. Pretreatment with heme or SnCl(2) provoked significant reductions (P < 0.01) of tail moment in the comet assay. Opposite effects were evident by pretreatment for 16 hr with tin-mesoporphyrin. A decrease in tail moment levels was found in human endothelial cells transduced with the human HO-1 gene. The addition of Ang II (200 ng/ml) to human dermal microvessel endothelial cell-1 for 16 hr resulted in a significant (P < 0.05) reduction of GSH contents control endothelial cells but not in endothelial cells transduced with HO-1 gene. The results presented indicated that stimulation or overexpression of HO-1 attenuated DNA damages caused by exposures of Ang II.     

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

It is often postulated that the cytoprotective nature of heme oxygenase (HO-1) explains the inducible nature of this enzyme. However, the mechanisms by which protection occurs are not verified by systematic evaluation of the physiological effects of HO. To explain how induction of HO-1 results in protection against oxygen toxicity, hamster fibroblasts (HA-1) were stably transfected with a tetracycline response plasmid containing the full-length rat HO-1 cDNA construct to allow for regulation of gene expression by varying concentrations of doxycycline (Dox). Transfected cells were exposed to hyperoxia (95% O(2)/5% CO2) for 24 h and several markers of oxidative injury were measured. With varying concentrations of Dox, HO activity was regulated between 3- and 17-fold. Despite cytoprotection with low (less than fivefold) HO activity, high levels of HO-1 expression (greater than 15-fold) were associated with significant oxygen cytotoxicity. Levels of non-heme reactive iron correlated with cellular injury in hyperoxia whereas lower levels of heme were associated with cytoprotection. Cellular levels of cyclic GMP and bilirubin were not significantly altered by modification of HO activity, precluding a substantial role for activation of guanylate cyclase by carbon monoxide or for accumulation of bile pigments in the physiological consequences of HO-1 overexpression. Inhibition of HO activity or chelation of cellular iron prior to hyperoxic exposure decreased reactive iron levels in the samples and significantly reduced oxygen toxicity. We conclude that there is a beneficial threshold of HO-1 overexpression related to the accumulation of reactive iron released in the degradation of heme. Therefore, despite the ready induction of HO-1 in oxidant stress, accumulation of reactive iron formed makes it unlikely that exaggerated expression of HO-1 is a cytoprotective response.