β-Caryophyllene Attenuates Focal Cerebral Ischemia-Reperfusion Injury by Nrf2/HO-1 Pathway in Rats

This study aimed to identify the effect of β-caryophyllene (BCP) pretreatment and elucidate the Nrf2/HO-1 signaling mechanism after focal cerebral ischemia-reperfusion (I-R) injury in rats. Adult male Sprague–Dawley rats were randomly assigned to the sham-operated group, I-R group and BCP pretreated I-R group. At 24 h after reperfusion, neurological deficits and infarct volume were evaluated. Pathological changes of neuron in hippocampuses were observed by Nissil staining and transmission electron microscopy (TEM). Oxidative stress was assessed by malondialdehyde (MDA) level, lipid peroxidation (LPO), nitric oxide (NO), superoxide dismutase (SOD) and Catalase (CAT) activity. The expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) were analysed by Western blotting and real-time quantitative polymerase chain reaction (Q-PCR). The protein expression of Bcl-2 and Bax was determined by immunohistochemistry. Apoptotic cells were detected using TUNEL staining. In I-R group, neurological deficit scores, cerebral infarct volume, MDA levels, LPO content, NO level, expression of Bax and TUNEL-positive cells were found to be increased at 24 h after I-R injury, while SOD activity, CAT activity and expression of Bcl-2 were decreased. However, results in the BCP pretreatment groups were reversed. And the protein and mRNA expressions of Nrf2 and HO-1 were significantly up-regulated in the BCP pretreated I-R group. Results of Nissil staining and TEM scan manifested that BCP remarkablely improved neuronal injury after I-R in rats. All the above suggested that BCP pretreatment played a neuroprotective role in cerebral I-R injury, which might be exerted by upregulating the expression of Nrf2 and HO-1 to ameliorate oxidative damage and neuronal apoptosis.     

Impact of silencing HO-2 on EC-SOD and the mitochondrial signaling pathway

The contribution of heme oxygenase HO-2, the primary source of bilirubin and carbon monoxide (CO) under physiological conditions, to the regulation of vascular function has remained largely unexplored. Using siRNA HO-2, we examined the effect of suppressed levels of HO-2 on vascular antioxidant and survival proteins. In vivo HO-2 siRNA treatment decreased the basal levels of EC-SOD, pAKT proteins (serine-473 and threonine-308), without changing Akt protein expression. HO-2 siRNA treatment increased 3-nitrotyrosine (3-NT) and apoptotic signaling kinase-1 (ASK-1) (P < 0.01). HO activity was decreased by the use of siRNA HO-2. We extended these studies to the mitochondria, examining for the presence of HO-1 and its role in the regulation of pro- and anti-apoptotic proteins. HO activity was increased by the administration of CoPP resulting in the translocation of HO-1 into the mitochondria, mainly to the inner face of the mitochondrial inner membrane. These findings suggest that HO-2 is critical in the maintenance of heme homeostasis and also the regulation of apoptosis by controlling levels of EC-SOD, Akt, 3-NT, and ASK-1. In addition, localization of HO-1 in the mitochondrial compartment plays a critical role in mitochondria-mediated apoptosis.     

Genetic overexpression of eNOS attenuates hepatic ischemia-reperfusion injury

Previous studies have shown that endothelial nitric oxide (NO) synthase (eNOS)-derived NO is an important signaling molecule in ischemia-reperfusion (I-R) injury. Deficiency of eNOS-derived NO has been shown to exacerbate injury in hepatic and myocardial models of I-R. We hypothesized that transgenic overexpression of eNOS (eNOS-TG) would reduce hepatic I-R injury. We subjected two strains of eNOS-TG mice to 45 min of hepatic ischemia and 5 h of reperfusion. Both strains were protected from hepatic I-R injury compared with wild-type littermates. Because the mechanism for this protection is still unclear, additional studies were performed by using inhibitors and activators of both soluble guanylyl cyclase (sGC) and heme oxygenase-1 (HO-1) enzymes. Blocking sGC with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and HO-1 with zinc (III) deuteroporphyrin IX-2,4-bisethyleneglycol (ZnDPBG) in wild-type mice increased hepatic I-R injury, whereas pharmacologically activating these enzymes significantly attenuated I-R injury in wild-type mice. Interestingly, ODQ abolished the protective effects of eNOS overexpression, whereas ZnDPBG had no effect. These results suggest that hepatic protection in eNOS-TG mice may be mediated in part by NO signaling via the sGC-cGMP pathway and is independent of HO-1 signal transduction pathways.     

Interleukin-19 (IL-19) induces heme oxygenase-1 (HO-1) expression and decreases reactive oxygen species in human vascular smooth muscle cells

Heme oxygenase-1 (HO-1) has potent anti-inflammatory activity and recognized vascular protective effects. We have recently described the expression and vascular protective effects of an anti-inflammatory interleukin (IL-19), in vascular smooth muscle cells (VSMC) and injured arteries. The objective of this study was to link the anti-inflammatory effects of IL-19 with HO-1 expression in resident vascular cells. IL-19 induced HO-1 mRNA and protein in cultured human VSMC, as assayed by quantitative RT-PCR, immunoblot, and ELISA. IL-19 does not induce HO-1 mRNA or protein in human endothelial cells. IL-19 activates STAT3 in VSMC, and IL-19-induced HO-1 expression is significantly reduced by transfection of VSMC with STAT3 siRNA or mutation of the consensus STAT binding site in the HO-1 promoter. IL-19 treatment can significantly reduce ROS-induced apoptosis, as assayed by Annexin V flow cytometry. IL-19 significantly reduced ROS concentrations in cultured VSMC. The IL-19-induced reduction in ROS concentration is attenuated when HO-1 is reduced by siRNA, indicating that the IL-19-driven decrease in ROS is mediated by HO-1 expression. IL-19 reduces vascular ROS in vivo in mice treated with TNFα. This points to IL-19 as a potential therapeutic for vascular inflammatory diseases and a link for two previously unassociated protective processes: Th2 cytokine-induced anti-inflammation and ROS reduction.     

Isovitexin Exerts Anti-Inflammatory and Anti-Oxidant Activities on Lipopolysaccharide-Induced Acute Lung Injury by Inhibiting MAPK and NF-κB and Activating HO-1/Nrf2 Pathways

Oxidative damage and inflammation are closely associated with the pathogenesis of acute lung injury (ALI). Thus, we explored the protective effect of isovitexin (IV), a glycosylflavonoid, in the context of ALI. To accomplish this, we created in vitro and in vivo models by respectively exposing macrophages to lipopolysaccharide (LPS) and using LPS to induce ALI in mice. In vitro, our results showed that IV treatment reduced LPS-induced pro-inflammatory cytokine secretion, iNOS and COX-2 expression and decreased the generation of ROS. Consistent findings were obtained in vivo. Additionally, IV inhibited H₂O₂-induced cytotoxicity and apoptosis. However, these effects were partially reversed following the use of an HO-1 inhibitor in vitro. Further studies revealed that IV significantly inhibited MAPK phosphorylation, reduced NF-κB nuclear translocation, and upregulated nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) expression in RAW 264.7 cells. In vivo, pretreatment with IV attenuated histopathological changes, infiltration of polymorphonuclear granulocytes and endothelial activation, decreased the expression of ICAM-1 and VCAM-1, reduced the levels of MPO and MDA, and increased the content of GSH and SOD in ALI. Furthermore, IV treatment effectively increased Nrf2 and HO-1 expression in lung tissues. Therefore, IV may offer a protective role against LPS-induced ALI by inhibiting MAPK and NF-κB and activating HO-1/Nrf2 pathways.     

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

Somatic cell gene transfer is a potentially useful strategy to alter lung function. However, achieving efficient transfer to the alveolar epithelium, especially in smaller animals, has not been demonstrated. In this study, the rat heme oxygenase-1 (HO-1) gene was delivered to the lungs of neonatal mice via transpulmonary injection. A bidirectional promoter construct coexpressing both HO-1 and a luciferase reporter gene was used so that in vivo gene expression patterns could be monitored in real time. HO-1 expression levels were also modulated with doxycycline and assessed in vivo with bioluminescent light transmitted through the tissues from the coregulated luciferase reporter. As a model of oxidative stress and HO-1-mediated protection, groups of animals were exposed to hyperoxia. After gene transfer, elevated levels of HO-1 were detected predominantly in alveolar type II cells by immunocytochemistry. With overexpression of HO-1, increased oxidative injury was observed. Furthermore, this model demonstrated a cell-specific effect of lung HO-1 overexpression in oxidative stress. Specific control of expression for therapeutic genes is possible in vivo. The transpulmonary approach may prove useful in targeting gene expression to cells of the alveolar epithelium or to circumscribed areas of the lung.     

Erythropoietin induces heme oxygenase-1 expression and attenuates oxidative stress

Recent studies have established that erythropoietin (EPO) is a pleiotropic cytokine. In this study we investigated whether pleiotropic effects of EPO may involve regulation of heme oxygenase (HO)-1, an anti-oxidative stress protein. A stimulatory effect of EPO on HO-1 expression was demonstrated in cultured renal endothelial cells, in which EPO decreased intracellular oxidative stress and provided cytoprotection against H(2)O(2). These beneficial effects were partially reversed by a HO-1 inhibitor. We then evaluated whether EPO induces HO-1 and ameliorates renal injury in vivo. Administration of EPO to Dahl salt-sensitive (DS) rats with low salt diet, a model of chronic tubulointerstitial injury, reduced proteinuria, and renal injury including peritubular capillaries rarefaction as compared to vehicle-treated DS rats. This renoprotection was associated with up-regulation of HO-1 in the kidney. In conclusion, EPO-induced HO-1 expression is likely to provide cytoprotection against oxidative stress.     

Carbon monoxide blocks oxidative stress-induced hepatocyte apoptosis via inhibition of the p54 JNK isoform

Most chronic liver diseases are accompanied by oxidative stress, which may induce apoptosis in hepatocytes and liver injury. Oxidative stress induces heme oxygenase-1 (HO-1) expression. This stress-responsive cytoprotective protein is responsible for heme degradation into carbon monoxide (CO), free iron, and biliverdin. CO is an important intracellular messenger; however, the exact mechanisms responsible for its cytoprotective effect are not yet elucidated. Thus, we investigated whether HO-1 and CO protect primary hepatocytes against oxidative-stress-induced apoptosis. In vivo, bile duct ligation was used as model of chronic liver disease. In vitro, primary hepatocytes were exposed to the superoxide anion donor menadione in a normal and in a CO-- containing atmosphere. Apoptosis was determined by measuring caspase-9, -6, -3 activity and poly(ADP-ribose) polymerase cleavage, and necrosis was determined by Sytox green staining. The results showed that (1) HO-1 is induced in chronic cholestatic liver disease, (2) superoxide anions time- and dose-dependently induce HO-1 activity, (3) HO-1 overexpression inhibits superoxide-anions-induced apoptosis, and (4) CO blocks superoxide-anions-induced JNK phosphorylation and caspase-9, -6, -3 activation and abolishes apoptosis but does not increase necrosis. We conclude that HO-1 and CO protect primary hepatocytes against superoxide-anions-induced apoptosis partially via inhibition of JNK activity. CO could represent an important candidate for the treatment of liver diseases.     

Translocation of heme oxygenase-1 to mitochondria is a novel cytoprotective mechanism against non-steroidal anti-inflammatory drug-induced mitochondrial oxidative stress, apoptosis, and gastric mucosal injury

The mechanism of action of heme oxygenase-1 (HO-1) in mitochondrial oxidative stress (MOS)-mediated apoptotic tissue injury was investigated. MOS-mediated gastric mucosal apoptosis and injury were introduced in rat by indomethacin, a non-steroidal anti-inflammatory drug. Here, we report that HO-1 was not only induced but also translocated to mitochondria during gastric mucosal injury to favor repair mechanisms. Furthermore, mitochondrial translocation of HO-1 resulted in the prevention of MOS and mitochondrial pathology as evident from the restoration of the complex I-driven mitochondrial respiratory control ratio and transmembrane potential. Mitochondrial translocation of HO-1 also resulted in time-dependent inhibition of apoptosis. We searched for the plausible mechanisms responsible for HO-1 induction and mitochondrial localization. Free heme, the substrate for HO-1, was increased inside mitochondria during gastric injury, and mitochondrial entry of HO-1 decreased intramitochondrial free heme content, suggesting that a purpose of mitochondrial translocation of HO-1 is to detoxify accumulated heme. Heme may activate nuclear translocation of NF-E2-related factor 2 to induce HO-1 through reactive oxygen species generation. Electrophoretic mobility shift assay and chromatin immunoprecipitation studies indicated nuclear translocation of NF-E2-related factor 2 and its binding to HO-1 promoter to induce HO-1 expression during gastric injury. Inhibition of HO-1 by zinc protoporphyrin aggravated the mucosal injury and delayed healing. Zinc protoporphyrin further reduced the respiratory control ratio and transmembrane potential and enhanced MOS and apoptosis. In contrast, induction of HO-1 by cobalt protoporphyrin reduced MOS, corrected mitochondrial dysfunctions, and prevented apoptosis and gastric injury. Thus, induction and mitochondrial localization of HO-1 are a novel cytoprotective mechanism against MOS-mediated apoptotic tissue injury.     

Down-regulation of heme oxygenase-2 is associated with the increased expression of heme oxygenase-1 in human cell lines

Intracellular heme concentrations are maintained in part by heme degradation, which is catalyzed by heme oxygenase. Heme oxygenase consists of two structurally related isozymes, HO-1 and HO-2. Recent studies have identified HO-2 as a potential oxygen sensor. To gain further insights into the regulatory role of HO-2 in heme homeostasis, we analyzed the expression profiles of HO-2 and the biochemical consequences of HO-2 knockdown with specific short interfering RNA (siRNA) in human cells. Both HO-2 mRNA and protein are expressed in the eight human cancer cell lines examined, and HO-1 expression is detectable in five of the cell lines, including HeLa cervical cancer and HepG2 hepatoma. Down-regulation of HO-2 expression with siRNA against HO-2 (siHO-2) caused induction of HO-1 expression at both mRNA and protein levels in HeLa and HepG2 cells. In contrast, knockdown of HO-1 expression did not noticeably influence HO-2 expression. HO-2 knockdown prolonged the half-life of HO-1 mRNA twofold in HeLa cells. Transient transfection assays in HeLa cells revealed that the 4.5-kb human HO-1 gene promoter was activated with selective knockdown of HO-2 in a sequence-dependent manner. Moreover, HO-2 knockdown caused heme accumulation in HeLa and HepG2 cells only when exposed to exogenous hemin. HO-2 knockdown may mimic a certain physiological change that is important in the maintenance of cellular heme homeostasis. These results suggest that HO-2 may down-regulate the expression of HO-1, thereby directing the co-ordinated expression of HO-1 and HO-2.     

A novel cardioprotective agent in cardiac transplantation: metformin activation of AMP-activated protein kinase decreases acute ischemia-reperfusion injury and chronic rejection

The main cause of mortality after the first year from cardiac transplantation is cardiac allograft vasculopathy (CAV), which leads to chronic rejection of the heart. To improve long-term outcomes in cardiac transplantation, treatments to prevent or diminish CAV are actively being researched. Ischemia-reperfusion (I-R) injury has been shown to be the strongest alloantigen-independent factor in the development of CAV. Here, we investigate the use of metformin in murine cardiac transplantation models as a novel cardioprotective agent to limit acute I-R injury and subsequent chronic rejection. We show that metformin treatment activates AMP-activated kinase (AMPK) in vitro and in vivo. In the acute transplantation model, metformin activation of AMPK resulted in significantly decreased apoptosis in cardiac allografts on postoperative day (POD) 1 and 8. In the chronic transplantation model, metformin pretreatment of allografts led to significantly improved graft function and significantly decreased CAV, as measured on POD 52. Taken together, our results in the acute and chronic rejection studies suggest a potential cardioprotective mechanism for metformin; we demonstrate a correlation between metformin-induced decrease in acute I-R injury and metformin-related decrease in chronic rejection. Thus, one of the ways by which metformin and AMPK activation may protect the transplanted heart from chronic rejection is by decreasing initial I-R injury inherent in donor organ preservation and implantation. Our findings suggest novel therapeutic strategies for minimizing chronic cardiac rejection via the use of metformin- and AMPK-mediated pathways to suppress acute I-R injury.     

Thrombin induces heme oxygenase-1 expression in human synovial fibroblasts through protease-activated receptor signaling pathways

Introduction

Thrombin is a key factor in the stimulation of fibrin deposition, angiogenesis, and proinflammatory processes. Abnormalities in these processes are primary features of osteoarthritis (OA). Heme oxygenase (HO)-1 is a stress-inducible rate-limiting enzyme in heme degradation that confers cytoprotection against oxidative injury. Here, we investigated the intracellular signaling pathways involved in thrombin-induced HO-1 expression in human synovial fibroblasts (SFs).

Methods

Thrombin-mediated HO-1 expression was assessed with quantitative real-time (q)PCR. The mechanisms of action of thrombin in different signaling pathways were studied by using Western blotting. Knockdown of protease-activated receptor (PAR) proteins was achieved by transfection with siRNA. Chromatin immunoprecipitation assays were used to study in vivo binding of Nrf2 to the HO-1 promoter. Transient transfection was used to examine HO-1 activity.

Results

Osteoarthritis synovial fibroblasts (OASFs) showed significant expression of thrombin, and expression was higher than in normal SFs. OASFs stimulation with thrombin induced concentration- and time-dependent increases in HO-1 expression. Pharmacologic inhibitors or activators and genetic inhibition by siRNA of protease-activated receptors (PARs) revealed that the PAR1 and PAR3 receptors, but not the PAR4 receptor, are involved in thrombin-mediated upregulation of HO-1. Thrombin-mediated HO-1 expression was attenuated by thrombin inhibitor (PPACK), PKCδ inhibitor (rottlerin), or c-Src inhibitor (PP2). Stimulation of cells with thrombin increased PKCδ, c-Src, and Nrf2 activation.

Conclusion

Our results suggest that the interaction between thrombin and PAR1/PAR3 increases HO-1 expression in human synovial fibroblasts through the PKCδ, c-Src, and Nrf2 signaling pathways.     

Higenamine reduces HMGB1 during hypoxia-induced brain injury by induction of heme oxygenase-1 through PI3K/Akt/Nrf-2 signal pathways

Growing lines of evidence suggests that high mobility group box-1 (HMGB1) plays an important role for promoting inflammation and apoptosis in brain ischemia. Previously, we demonstrated that inducers of heme oxygenase-1 (HO-1) significantly reduce HMGB1 release in inflammatory conditions in vitro and in vivo. Thus, we tested our hypothesis that higenamine protects brain injury by inhibition of middle cerebral artery occlusion (MCAO)-mediated HMGB1 release in vivo, and glucose/glucose oxidase (GOX)-induced apoptosis in C6 cells in vitro due to HO-1 induction. Higenamine increased HO-1 expression in C6 cells in both hypoxia and normoxia, in which the former was much more significant than the latter. Higenamine increased Nrf-2 luciferase activity, translocated Nrf-2 to nucleus, and increased phosphorylation of Akt in C6 cells. Consistent with this, LY 294002, a PI3K inhibitor, inhibited HO-1 induction by higenamine and apoptosis induced by glucose/GOX in C6 cells was prevented by higenamine, which effect was reversed by LY 294002. Importantly, administration of higenamine (i.p) significantly reduced brain infarct size, mortality rate, MPO activity and tissue expression of HMGB1 in MCAO rats. In addition, recombinant high mobility group box 1 induced apoptosis in C6 cells by increasing ratio of Bax/bcl-2 and cleaved caspase c, which was inhibited by higenamine, and all of these effects were reversed by co-treatment with ZnPPIX. Therefore, we conclude that higenamine, at least in part, protects brain cells against hypoxic damages by up-regulation of HO-1. Thus, higenamine may be beneficial for the use of ischemic injuries such as stroke.     

Intestinal preconditioning prevents inflammatory response by modulating heme oxygenase-1 expression in endotoxic shock model

Gut mucosal injury observed during ischemia-reperfusion is believed to trigger a systemic inflammatory response leading to multiple organ failure. It should be interesting to demonstrate this relationship between gut and multiple organ failure in a sepsis model. Intestinal preconditioning (PC) can be used as a tool to assess the effect of intestinal ischemia in inflammatory response after LPS challenge. The aim of this study was to investigate the protective effect of PC against LPS-induced systemic inflammatory and intestinal heme oxygenase-1 (HO-1) expression. ES was performed with LPS (10 mg/kg iv) with or without PC, which was done before LPS. Rats were first subjected to sham surgery or PC with four cycles of 1 min ischemia and 4 min of reperfusion 24 h before LPS challenge or saline administration. PC significantly reduced fluid requirements, lung edema, intestinal lactate production, and intestinal injury. Inflammatory mRNA expressions for intestine and lung ICAM and TNF were significantly reduced after PC, and these effects were significantly abolished by zinc-protoporphyrin (a specific HO-1 activity inhibitor) and mimicked by bilirubin administration. Intestinal PC selectively increased HO-1 mRNA expression in intestine, but we have observed no expression in lungs. These findings demonstrate that intestinal injury is a important event for inflammatory response and multiple organ injury after LPS challenge. Intestinal HO-1 expression attenuates LPS-induced multiple organ failure by modulating intestine injury and its consequences on inflammatory response. Identification of the exact mechanisms responsible for intestine HO-1 induction may lead to the development of new pharmacological interventions.     

Heme oxygenase-1 protects against neutrophil-mediated intestinal damage by down-regulation of neutrophil p47phox and p67phox activity and O2- production in a two-hit model of alcohol intoxication and burn injury

Heme oxygenase-1 (HO-1) has been demonstrated to protect against tissue injury. Furthermore, HO-1 is also shown to be antioxidant. Our recent findings indicate that acute alcohol (EtOH) intoxication exacerbates postburn intestinal and lung tissue damage, and this was found to be neutrophil dependent. Because neutrophil-mediated tissue injury involves the release of superoxide anions (O(2)(-)), the present study examined the role of HO-1 in neutrophil O(2)(-) production following EtOH and burn injury. Furthermore, we investigated whether HO-1 antioxidant properties are mediated via modulation of p47(phox) and/or p67(phox) proteins. Male rats (approximately 250 g) were gavaged with EtOH to achieve a blood EtOH level of approximately 100 mg/dL before burn or sham injury (approximately 12.5% total body surface area). Some rats were treated with HO-1 activator cobalt protoporphyrin IX chloride (Copp; 25 mg/kg body weight) at the time of injury. On day 1 after injury, we found that EtOH combined with burn injury significantly increased neutrophil O(2)(-) production and p47(phox) and p67(phox) activation and decreased caspase-3 activity and apoptosis. This was accompanied with a decrease in neutrophil HO-1 levels. The treatment of animals with HO-1 activator Copp normalized neutrophil HO-1, O(2)(-), p47(phox), and p67(phox) following EtOH and burn injury. The expression of caspase-3, however, was further decreased in Copp-treated sham and EtOH plus burn groups. Moreover, Copp treatment also prevented the increase in intestinal edema and permeability following EtOH and burn injury. Altogether, these findings provide a new insight into the mechanism by which HO-1 regulates neutrophil O(2)(-) production and protect the intestine from damage following EtOH and burn injury.     

How Heme Oxygenase-1 Prevents Heme-Induced Cell Death

Earlier observations indicate that free heme is selectively toxic to cells lacking heme oxygenase-1 (HO-1) but how this enzyme prevents heme toxicity remains unexplained. Here, using A549 (human lung cancer) and immortalized human bronchial epithelial cells incubated with exogenous heme, we find knock-down of HO-1 using siRNA does promote the accumulation of cell-associated heme and heme-induced cell death. However, it appears that the toxic effects of heme are exerted by “loose” (probably intralysosomal) iron because cytotoxic effects of heme are lessened by pre-incubation of HO-1 deficient cells with desferrioxamine (which localizes preferentially in the lysosomal compartment). Desferrioxamine also decreases lysosomal rupture promoted by intracellularly generated hydrogen peroxide. Supporting the importance of endogenous oxidant production, both chemical and siRNA inhibition of catalase activity predisposes HO-1 deficient cells to heme-mediated killing. Importantly, it appears that HO-1 deficiency somehow blocks the induction of ferritin; control cells exposed to heme show ~10-fold increases in ferritin heavy chain expression whereas in heme-exposed HO-1 deficient cells ferritin expression is unchanged. Finally, overexpression of ferritin H chain in HO-1 deficient cells completely prevents heme-induced cytotoxicity. Although two other products of HO-1 activity–CO and bilirubin–have been invoked to explain HO-1-mediated cytoprotection, we conclude that, at least in this experimental system, HO-1 activity triggers the induction of ferritin and the latter is actually responsible for the cytoprotective effects of HO-1 activity.