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Restoring HOmeostasis: is heme oxygenase-1 ready for the clinic?
Opinion
TRENDS in Pharmacological Sciences
Vol.28 No.5
Restoring HOmeostasis: is heme oxygenase-1 ready for the clinic? Jeffrey R. Scott*, Beek Y. Chin*, Martin H. Bilban and Leo E. Otterbein Transplant Research Center, Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA
Inflammation and immunity result in a wide range of disease processes, including atherosclerosis, vascular thrombosis and sepsis. Heme oxygenase-1 (HO-1) is a key enzyme that is integral to the temporal and spatial regulation of the host response and, together with its products carbon monoxide (CO) and bilirubin, is crucial for maintaining homeostasis and the preservation of function and life. An increasing number of reports demonstrates that HO-1, CO and bilirubin regulate the immune response. As CO and bilirubin enter clinical trials, there are obstacles to be addressed before their full therapeutic potential can be achieved. In this article, we delineate the challenges that lie ahead regarding toxicity, pharmacokinetics and mechanisms of action to be able to take full advantage of the powerful cytoprotective properties of these agents for clinical benefit. Introduction Two products of the heme degradation pathway [bilirubin and carbon monoxide (CO)] have long been viewed by the medical and scientific community as metabolic waste products and/or toxic substances (Figure 1). These widely held views could have skewed the objectivity of researchers and masked beneficial properties associated with these compounds at physiological concentrations. For example, severe hyperbilirubinemia results in neonatal kernicterus or bilirubin encephalopathy, and prolonged exposure to atmospheric CO at high concentrations can result in chemical asphyxiation and neurological impairment [1,2]. However, key insights into the homeostatic function of the heme oxygenase (HO) pathway – and growing evidence of cytoprotective properties associated with administering exogenous CO, biliverdin or bilirubin at relatively low concentrations – have highlighted several approaches that might hold great therapeutic potential (Figure 2). It is becoming clear that further advancement and public acceptance of this rapidly emerging field will require a paradigm shift that identifies these compounds as being ‘conditionally therapeutic’: CO at appropriately low concentrations and bilirubin at high-normal or slightly supernormal concentrations [2]. Such a shift in perspective will not only challenge widely held beliefs about the function and physiological relevance of these compounds but also stimulate research from an unsullied perspective in the greater medical and scientific community. We hope that this shift Corresponding author: Otterbein, L.E. ([email protected]). Authors contributed equally. Available online 9 April 2007.
*
www.sciencedirect.com
will lead to a broader understanding of the properties associated with these compounds and provide a basis for the rapid development of cytoprotective therapies that mimic the endogenous HO pathway. There are two, welldescribed isoforms of HO: HO-1, the stress-inducible isoform, and HO-2, which is constitutively expressed [3,4]. The expression of HO-1 and the production of CO, biliverdin and bilirubin in response to stress [2] enable the use of these agents in conditions in which they normally would not provide salutary actions because they are produced too late and at levels too low to avoid serious pathology in many instances. Therefore, the ability to induce HO-1 or administer CO and/or biliverdin and bilirubin before a given insult at levels above those that might be produced physiologically has great potential for therapy. Fe2+, which is also a product of heme degradation, and ferritin have not been tested sufficiently for such a statement to be made, but ferritin is also an effective protective molecule in various disease states [5]. We focus here on CO and biliverdin, which both modulate the inflammatory and immune responses. The statement by Paracelsus (1493–1541) that ‘all substances are poisons; there is none which is not a poison. The right dose differentiates a poison from a remedy’ perhaps summarizes the concept that every agent is dangerous when given at the appropriate dose. Thoroughly evaluating the properties of CO from a fresh perspective invokes the concept of ‘hormesis’, which might better describe the physiological response to this gas molecule. The term hormesis, which was coined by Schultz more than a century ago and has been used by others more recently [6,7], describes chemicals that are stimulatory at low doses but that become inhibitory at higher concentrations: a nonlinear toxicological perspective that contradicts traditional toxicological models (Figure 3). In fact, compounds such as dioxins that are traditionally considered to be toxic might have beneficial properties at lower concentrations that have been ignored for decades [6,7]. It is an error to assume that, once a compound is labeled as being toxic at high concentrations, all exposure must be limited because it provides an allusion of negative physiological outcome even at lower concentrations. In 1952, Sjo¨strand identified that the decomposition of hemoglobin led to CO production in vivo [8], almost two decades before HO was identified as the enzyme responsible for this reaction. One wonders why the finding of a gaseous molecule produced endogenously was not further
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Figure 1. Heme degradation pathway driven by HO-1. Shown is the generation of CO, biliverdin and iron. Biliverdin is converted to bilirubin by biliverdin reductase.
investigated with regard to a potential role in biological processes until decades later. Novel findings regarding CO since the discovery of nitric oxide might have been viewed differently because these gases share similar vasoactive and immunological properties [9]. In hindsight, if interpreted in this context, the data obtained for NO might have provided the rationale for
research into the potential therapeutic value of CO more than half a century ago. Recent clinical evidence confirms that carboxyhemoglobin and end-tidal CO levels are significantly elevated during systemic inflammation in both trauma and septic patients [10–12]. These data strongly indicate that endogenous CO production is increased through the HO pathway, although this seems paradoxical
Figure 2. The cytoprotective effects of CO and the bile pigments. Shown are the effects in different cell types and the pathologies that are ameliorated in their presence. www.sciencedirect.com
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Figure 3. The concept of hormesis. In this concept, compounds exhibit a ’J-shaped’ dose–response profile. At low concentrations, there exist potent beneficial or stimulatory effects that shift to a classic linear dose–response toxicity at higher concentrations. Although CO and bilirubin have been labeled as having toxic properties at high concentrations, recent evidence indicates that each is consistent with fitting a hormetic profile whereby, at low concentrations, CO and bilirubin modulate inflammatory, apoptotic and proliferative processes to re-establish and maintain homeostasis and to exert therapeutic benefits.
at first glance in view of conventional attitudes vis-a`-vis CO toxicity; further investigation invites a paradigm shift that would include a characterization of CO as conditionally therapeutic. The term conditionally therapeutic implies that one would expect to extrapolate the efficacy data from animals to humans under the correct and appropriate conditions. A clinical investigator would appreciate the fact that the available data from humans are sparse, necessitating clinical testing. Optimizing the concentration of exogenous CO and taking advantage of the innate affinity of this gas for hemoglobin could represent an efficient in vivo delivery system if an extravascular gradient were to exist for CO removal. Explosion of HO research At >3300, the number of recent publications directly related to evaluating the homeostatic or cytoprotective properties associated with the HO pathway is remarkable [13,14]. Findings from Hmox1-knockout mice and the first reported case of human HO-1 deficiency [15–17] indicate that the activity of this enzyme is essential for modulating the inflammatory response [18,17], apoptosis [19] and cellular proliferation [20]. Furthermore, HO inhibitors attenuate HO-induced cytoprotection and exacerbate cellular injury [21,22]. It is also clear that humans differ quantitatively in their ability to upregulate HO-1 in response to various stressful stimuli because of variability in two distinct HO-1 promoter polymorphisms [23]. Thus, the administration of CO, biliverdin or bilirubin at appropriate doses is an alternative to HO-1-mediated protection, and these agents could function as novel therapeutics in www.sciencedirect.com
multiple disease states [24–28]. CO and biliverdin can both influence diverse cellular processes depending on cell type and model (Figure 2). CO CO is a colorless, odorless gas that has traditionally been considered an environmental pollutant and a lethal chemical asphyxiant at high concentrations. This is primarily because of the high affinity of CO for hemoglobin, which is 245 times that of oxygen in humans, with a half-life of 3–5 h under normal atmospheric conditions [29]. Additionally, CO binds to other heme-containing moieties such as nitric oxide synthase and guanylate cyclase [30,31]. Cytochrome c oxidase is also a subcellular target of CO, binding to which leads to alterations in the electron transport chain [10]. However, it is important to note that CO, like all compounds, has both a toxic and a therapeutic range. Indeed, despite CO having negative effects on hemoprotein function at high levels, it might be beneficial at low concentrations – it is the dosing differences that define the functional consequences (i.e. toxicity versus benefit). Before the discovery that low concentrations of exogenous CO [<250 parts per million (ppm)] have therapeutic effects, animal reports relied heavily on classical CO toxicological studies, in which the hypotheses and endpoints were grave and severe. These toxicological studies provided data and conclusions, such as the LC50 in rats (1807 ppm for 4 h), in addition to observations of detrimental effects of CO on motor ability [32,33]. Although there were no comparative studies in humans at the time,
Opinion
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most of the reports were collected serendipitously from individuals exposed to mixtures of CO and other toxic products (e.g. exhaust fumes or leaky furnaces). By contrast, a study in 1986 determined that individuals exposed to CO at 40 ppm for 60 days were not negatively affected [1]. Currently, the permissible exposure limit for CO in the USA is 35 ppm for 8 h/day [33]. Against the vast toxicological data in the literature, experimental findings from the past five years have highlighted the therapeutic potential of CO as a novel gaseous therapy at concentrations <200 ppm, which seems to alter several key components of the inflammatory cascade [34,35]. More recently, the emergence of CO-releasing molecules (CORMs) is providing excellent alternatives to exogenous gas exposure with similar biological effects [36]. These findings lead to the conundrum of how a substance with known toxic properties can exert beneficial effects. Biliverdin or bilirubin As with CO, the history of bilirubin dates back centuries. Hippocrates viewed the body as consisting of four humors, one of which was black and one yellow, existing in a state of dynamic equilibrium and, if they were not in balance, disease would result. In 1916, van den Bergh developed a definitive test for the measurement of bilirubin that continues to dominate all clinical and investigative thinking in the field [37]. Schmorl labeled the lesion ‘kernicterus’ in 1904 to describe a pathology of the brain in neonates [38]. Even in adults, elevated levels of bilirubin are considered with alarm and are usually associated with liver failure. Hence, the conventional wisdom of the 20th century was – and, until recently, continued to be – that bilirubin was a potentially toxic waste-product with limited biological use. In 1987, bilirubin was defined as the most potent antioxidant molecule in serum [39], making it a potential cytoprotective agent in, for example, the brain, where it could scavenge free radicals and protect against oxidative damage [40]; biliverdin continued to be labeled as an intermediary in the breakdown of heme [41]. Like CO, the administration of biliverdin and/or bilirubin has recently proven to be potently cytoprotective in ameliorating the pathology associated with several disease processes such as ischemia–reperfusion injury, transplant rejection and inflammatory bowel disease [25,31,42,43]. Several reports show that individuals with high-normal or slightly elevated levels of bilirubin (including individuals with Gilbert’s syndrome, who have bilirubin levels two or three times those of normal individuals) have a markedly reduced incidence of atherosclerotic disease and a low prevalence of ischemic heart disease [44]. Experimental and clinical observations continue to shed light on the way in which bilirubin should be viewed by the greater medical and scientific community in both newborns and adults. In addition to its antioxidant capabilities, it is possible that biliverdin reductase regulates protective gene expression, including that of the gene encoding HO-1 [45]. One can envisage a therapeutic cycle wherein biliverdin and bilirubin induce the expression of HO-1, which then produces additional endogenous biliverdin and CO. A redox www.sciencedirect.com
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cycle proposed by Sedlak and Snyder communicates with a separate bile pigment cycle in which biliverdin and bilirubin are interconverted, with the reciprocity increasing the overall antioxidant power [45]. The increase in the number of publications demonstrating that exogenous delivery of biliverdin or bilirubin leads to protective effects supports the use of these metabolites as powerful therapeutics, yet generates questions that demand further inquiry, investigation and evaluation (Box 1). Future therapeutic strategies It is becoming clear that the next major challenge surrounding the clinical applicability of the HO-1 system is to identify accurately the particular disease states that will respond positively to altered HO-1 activity and/or exogenous administration of one or more of its products. Most of the preclinical data support translational studies with HO-1. Three therapeutic strategies exist that hold the strongest potential for efficacy, depending on the disease or the mode of injury: (i) pre-induction of HO-1 before a known injurious insult; (ii) pre-treatment; and/or (iii) post-treatment with exogenous byproducts of the HO pathway (i.e. CO, and biliverdin or bilirubin). A key concern when translating this knowledge into a treatment strategy with clinical use is the concept of achieving sufficient HO-1 induction and localization independently of injury. To achieve elevated HO-1 levels, such systems would include the use of a viral vector system or a trans activator of transcription (TAT) protein. However, the innate immune response initiated by local leukocytes after virus or foreign-protein administration arguably represents a potential injurious or inflammatory insult
Box 1. The evolution of HO-1 as a homeostatic mediator Are the diagnoses in newborns of kernicterus or encephalopathy, in addition to the negative correlation of bilirubin levels with atherosclerosis and ischemic heart disease, associative or are biliverdin and bilirubin directly involved in these diseases? Have these compounds been overlooked as potential therapeutics? Have biliverdin or bilirubin, and CO been inaccurately classified as ‘vestigial biological products’ that are akin to ammonia and urea? Because it has been demonstrated that the expression of HO-1 is greatest during embryonic development [46] – when CO and bilirubin levels are typically elevated – are such coincidental phenomena dose or concentration gradient effects, or simply hormetic effects? Does either have a crucial role in development? In addition, the gene encoding HO-1 is clearly a stress response gene and, thus, the systemic elevation of levels of the gene product is expected. Perhaps this explains, in part, why the phenotype of HO-1-deficient mice and humans exhibits heightened pathophysiological qualities. Indeed, the first reported HO-1-deficient patient was severely mentally retarded. This raises important issues that demand inquiry: Were these effects consequences of insufficient biliverdin or bilirubin and/or CO levels in utero? Are there threshold levels at which biliverdin or bilirubin and/or CO operates to ensure the production of ideal, beneficial or necessary elements that promote survival of the organism? Does the presence of these products re-establish homeostasis following a stress response, thereby indicating that HO-1 induction provides the products necessary for this process to occur?
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that limits applicability in the clinical setting. In these conditions, HO-1 itself might emerge as a therapeutic, assuming that there are no substrate limitations. The exogenous administration of CO and/or biliverdin and bilirubin is promising, akin to most novel pharmaceuticals; as such, these agents require standard clinical phase trials to determine efficacy, toxicological profiling, tissue distribution and half-life. Using the field of transplantation as an example of a large population of patients with an unmet clinical need, how would one take advantage of the HO-1 system? By using the three therapeutic approaches mentioned, it would be possible to pre-induce HO-1 in the donor, in the organ ex vivo or in the recipient before and, perhaps, after transplantation. Alternatively, CO and/or biliverdin or bilirubin could also be used to mimic HO-1 and attenuate both acute and chronic transplant rejection. Assuming that one or all of these approaches were efficacious, would they require a lifelong commitment, as with traditional immunosuppression, supplanted entirely as a new therapeutic option or would they override completely the current standards of care regarding immunosuppressive therapies? If an individual could inhale CO for 10–15 min or take a tablet containing a CORM and/or biliverdin or bilirubin daily, might these be acceptable, if novel, therapies? The use of CORMs has the advantage of enabling additional routes of administration compared with gas delivery, including targeted delivery to different organs and varying kinetics of delivery. CORMS, and biliverdin or bilirubin would also enable conjugation to other molecules such as nonsteroidal anti-inflammatory drugs, statins and immunosuppressive drugs and would not only combat the side effects of these compounds but also improve their efficacy. Perhaps these therapies would need to be administered only during the first week post-transplantation to induce tolerance, or alternatively be administered to only the donor and not the recipient. Obviously, the complexity of such a regimen requires standard testing and evaluation according to US Food and Drug Administration (http:// www.fda.gov/) regulations. Concluding remarks The potential for therapy would be enormous if one could harness the beneficial properties of the heme degradation pathway. For now, there are only questions for which research must continue to provide much-needed answers. It is up to the medical and scientific community to consider a paradigm shift, to take advantage of these conditionally therapeutic modalities and, ultimately, to use this intricate host defense and homeostasis-restoring system by applying its properties appropriately to promote health and to alleviate disease. Clearly, evolution has succeeded with HO-1. Disclosure statement L.E.O. is a paid consultant of Linde Healthcare. Acknowledgements This work was supported by NIH grants HL-071797 and HL-076167 (to L.E.O.). We thank the Julie Henry Fund at the Transplant Center of the Beth Israel Deaconess Medical Center for their support. www.sciencedirect.com
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References 1 ACGIH (1986) Documentation of the Threshold Limit Values and Biological Exposure Indices (5th edn), ACGIH 2 Ollinger, R. et al. (2007) Bilirubin and biliverdin treatment of atherosclerotic diseases. Cell Cycle 6, 39–43 3 Tenhunen, R. et al. (1969) Microsomal heme oxygenase. Characterization of the enzyme. J. Biol. Chem. 244, 6388–6394 4 Maines, M.D. (2004) The heme oxygenase system: past, present, and future. Antioxid. Redox Signal. 6, 797–801 5 Berberat, P.O. et al. (2003) Heavy chain ferritin acts as an antiapoptotic gene that protects livers from ischemia reperfusion injury. FASEB J. 17, 1724–1726 6 Schulz, H. (1888) Ueber Hefegifte. Pfluegers Arch. Gesamte Physiol. Menschen Tiere. 42, 517 7 Calabrese, E.J. and Baldwin, L.A. (2001) Hormesis: U-shaped dose responses and their centrality in toxicology. Trends Pharmacol. Sci. 22, 285–291 8 Sjo¨strand, T. (1952) The formation of carbon monoxide by the decomposition of haemoglobin in vivo. Acta Physiol. Scand. 26, 338–344 9 Ryter, S.W. and Otterbein, L.E. (2004) Carbon monoxide in biology and medicine. Bioessays 26, 270–280 10 Moncure, M. et al. (1999) Carboxyhemoglobin elevation in trauma victims. J. Trauma 46, 424–427 11 Zegdi, R. et al. (2002) Increased endogenous carbon monoxide production in severe sepsis. Intensive Care Med. 28, 793–796 12 Kawashima, A. et al. (2002) Heme oxygenase-1 deficiency: the first autopsy case. Hum. Pathol. 33, 125–130 13 Ryter, S.W. et al. (2006) Heme oxygenase-1/carbon monoxide: from basic science to therapeutic applications. Physiol. Rev. 86, 583–650 14 Otterbein, L.E. et al. (2003) Heme oxygenase-1: unleashing the protective properties of heme. Trends Immunol. 24, 449–455 15 Wiesel, P. et al. (2000) Endotoxin-induced mortality is related to increased oxidative stress and end-organ dysfunction, not refractory hypotension, in heme oxygenase-1-deficient mice. Circulation 2, 3015– 3022 16 Kapturczak, M.H. et al. (2004) Heme oxygenase-1 modulates early inflammatory responses: evidence from the heme oxygenase-1deficient mouse. Am. J. Pathol. 165, 1045–1053 17 Yachie, A. et al. (1999) Oxidative stress causes enhanced endothelial cell injury in human heme oxygenase-1 deficiency. J. Clin. Invest. 103, 129–135 18 Fujita, T. et al. (2001) Paradoxical rescue from ischemic lung injury by inhaled carbon monoxide driven by derepression of fibrinolysis. Nat. Med. 7, 598–604 19 Brouard, S. et al. (2000) Carbon monoxide generated by heme oxygenase 1 suppresses endothelial cell apoptosis. J. Exp. Med. 192, 1015–1026 20 Taille, C. et al. (2003) Heme oxygenase inhibits human airway smooth muscle proliferation via a bilirubin-dependent modulation of ERK1/2 phosphorylation. J. Biol. Chem. 278, 27160–27168 21 Sass, G. et al. (2003) Heme oxygenase-1 and its reaction product, carbon monoxide, prevent inflammation-related apoptotic liver damage in mice. Hepatology 38, 909–918 22 Yang, Y. et al. (2003) Selective protection of renal tubular epithelial cells by heme oxygenase (HO)-1 during stress-induced injury. Kidney Int. 64, 1302–1309 23 Exner, M. et al. (2004) The role of heme oxygenase-1 promoter polymorphisms in human disease. Free Radic. Biol. Med. 37, 1097– 1104 24 Otterbein, L.E. et al. (1999) Carbon monoxide provides protection against hyperoxic lung injury. Am. J. Physiol. 276, L688–L694 25 Fondevila, C. et al. (2004) Biliverdin therapy protects rat livers from ischemia and reperfusion injury. Hepatology 40, 1333–1341 26 Sato, K. et al. (2001) Carbon monoxide generated by heme oxygenase-1 suppresses the rejection of mouse-to-rat cardiac transplants. J. Immunol. 166, 4185–4194 27 Moore, B.A. et al. (2003) Inhaled carbon monoxide suppresses the development of post-operative ileus in the murine small intestine. Gastroenterology 124, 377–391 28 Kruger, A.L. et al. (2006) Up-regulation of heme oxygenase provides vascular protection in an animal model of diabetes through its antioxidant and antiapoptotic effects. J. Pharmacol. Exp. Ther. 319, 1144–1152
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29 Smith, R.P. (1986) Toxic responses of the blood. In Casarett and Doull’s Toxicology, the Basic Science of Poisons (3rd edn) (Klaassen, C.D. et al., eds), pp. 223–244, MacMillan Publishing 30 White, K.A. and Marletta, M.A. (1992) Nitric oxide synthase is a cytochrome P-450 type hemoprotein. Biochemistry 31, 6627–6631 31 Brune, B. and Ullrich, V. (1987) Inhibition of platelet aggregation by carbon monoxide is mediated by activation of guanylate cyclase. Mol. Pharmacol. 32, 497–504 32 Gosselin, R.E. et al. (1984) Clinical Toxicology of Commercial Products (5th edn), Williams & Wilkins 33 NIOSH (1986) Registry of toxic effects of chemical substances: carbon monoxide. US Department of Health and Human Services, Public Health Service, Centers for Disease Control, National Institute for Occupational Safety and Health 34 Otterbein, L.E. et al. (2003) Carbon monoxide suppresses arteriosclerotic lesions associated with chronic graft rejection and with balloon injury. Nat. Med. 9, 183–190 35 Nakao, A. et al. (2003) Carbon monoxide inhalation protects rat intestinal grafts from ischemia/reperfusion injury. Am. J. Pathol. 163, 1587–1598 36 Boczkowski, J. et al. (2006) CO–metal interaction: vital signaling from a lethal gas. Trends Biochem. Sci. 31, 614–621 37 Hymans van den Bergh, A.A. and Mueller, P. (1916) Determination of direct and indirect bilirubin. Biochem. 77, 90–92
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38 Schmorl, C.G. (1904) Zur kenntnis des ikterus neonatorum, insbesondere der dabei auftretenden gehirnveranderungen. Dent. Path. Gesell. 6, 109–112 39 Stocker, R. et al. (1987) Bilirubin is an antioxidant of possible physiological importance. Science 235, 1043–1046 40 Dore, S. and Snyder, S.H. (1999) Neuroprotective action of bilirubin against oxidative stress in primary hippocampal cultures. Ann. N. Y. Acad. Sci. 890, 167–172 41 Novotny, L. and Vitek, L. (2003) Inverse relationship between serum bilirubin and atherosclerosis in men: a meta-analysis of published studies. Exp. Biol. Med. (Maywood) 228, 568–571 42 Clark, J.E. et al. (2000) Heme oxygenase-1-derived bilirubin ameliorates postischemic myocardial dysfunction. Am. J. Physiol. Heart Circ. Physiol. 278, H643–H651 43 Nakao, A. et al. (2004) Biliverdin protects the functional integrity of a transplanted syngeneic small bowel. Gastroenterology 127, 595–606 44 Vitek, L. et al. (2002) Gilbert syndrome and ischemic heart disease: a protective effect of elevated bilirubin levels. Atherosclerosis 160, 449– 456 45 Sedlak, T.W. and Snyder, S.H. (2004) Bilirubin benefits: cellular protection by a biliverdin reductase antioxidant cycle. Pediatrics 113, 1776–1782 46 Kreiser, D. et al. (2003) Gestational pattern of heme oxygenase expression in the rat. Pediatr. Res. 54, 172–178
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Restoring HOmeostasis: is heme oxygenase-1 ready for the clinic? Jeffrey R. Scott*, Beek Y. Chin*, Martin H. Bilban and Leo E. Otterbein Transplant Research Center, Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA
Inflammation and immunity result in a wide range of disease processes, including atherosclerosis, vascular thrombosis and sepsis. Heme oxygenase-1 (HO-1) is a key enzyme that is integral to the temporal and spatial regulation of the host response and, together with its products carbon monoxide (CO) and bilirubin, is crucial for maintaining homeostasis and the preservation of function and life. An increasing number of reports demonstrates that HO-1, CO and bilirubin regulate the immune response. As CO and bilirubin enter clinical trials, there are obstacles to be addressed before their full therapeutic potential can be achieved. In this article, we delineate the challenges that lie ahead regarding toxicity, pharmacokinetics and mechanisms of action to be able to take full advantage of the powerful cytoprotective properties of these agents for clinical benefit. Introduction Two products of the heme degradation pathway [bilirubin and carbon monoxide (CO)] have long been viewed by the medical and scientific community as metabolic waste products and/or toxic substances (Figure 1). These widely held views could have skewed the objectivity of researchers and masked beneficial properties associated with these compounds at physiological concentrations. For example, severe hyperbilirubinemia results in neonatal kernicterus or bilirubin encephalopathy, and prolonged exposure to atmospheric CO at high concentrations can result in chemical asphyxiation and neurological impairment [1,2]. However, key insights into the homeostatic function of the heme oxygenase (HO) pathway – and growing evidence of cytoprotective properties associated with administering exogenous CO, biliverdin or bilirubin at relatively low concentrations – have highlighted several approaches that might hold great therapeutic potential (Figure 2). It is becoming clear that further advancement and public acceptance of this rapidly emerging field will require a paradigm shift that identifies these compounds as being ‘conditionally therapeutic’: CO at appropriately low concentrations and bilirubin at high-normal or slightly supernormal concentrations [2]. Such a shift in perspective will not only challenge widely held beliefs about the function and physiological relevance of these compounds but also stimulate research from an unsullied perspective in the greater medical and scientific community. We hope that this shift Corresponding author: Otterbein, L.E. ([email protected]). Authors contributed equally. Available online 9 April 2007.
*
www.sciencedirect.com
will lead to a broader understanding of the properties associated with these compounds and provide a basis for the rapid development of cytoprotective therapies that mimic the endogenous HO pathway. There are two, welldescribed isoforms of HO: HO-1, the stress-inducible isoform, and HO-2, which is constitutively expressed [3,4]. The expression of HO-1 and the production of CO, biliverdin and bilirubin in response to stress [2] enable the use of these agents in conditions in which they normally would not provide salutary actions because they are produced too late and at levels too low to avoid serious pathology in many instances. Therefore, the ability to induce HO-1 or administer CO and/or biliverdin and bilirubin before a given insult at levels above those that might be produced physiologically has great potential for therapy. Fe2+, which is also a product of heme degradation, and ferritin have not been tested sufficiently for such a statement to be made, but ferritin is also an effective protective molecule in various disease states [5]. We focus here on CO and biliverdin, which both modulate the inflammatory and immune responses. The statement by Paracelsus (1493–1541) that ‘all substances are poisons; there is none which is not a poison. The right dose differentiates a poison from a remedy’ perhaps summarizes the concept that every agent is dangerous when given at the appropriate dose. Thoroughly evaluating the properties of CO from a fresh perspective invokes the concept of ‘hormesis’, which might better describe the physiological response to this gas molecule. The term hormesis, which was coined by Schultz more than a century ago and has been used by others more recently [6,7], describes chemicals that are stimulatory at low doses but that become inhibitory at higher concentrations: a nonlinear toxicological perspective that contradicts traditional toxicological models (Figure 3). In fact, compounds such as dioxins that are traditionally considered to be toxic might have beneficial properties at lower concentrations that have been ignored for decades [6,7]. It is an error to assume that, once a compound is labeled as being toxic at high concentrations, all exposure must be limited because it provides an allusion of negative physiological outcome even at lower concentrations. In 1952, Sjo¨strand identified that the decomposition of hemoglobin led to CO production in vivo [8], almost two decades before HO was identified as the enzyme responsible for this reaction. One wonders why the finding of a gaseous molecule produced endogenously was not further
0165-6147/$ – see front matter ß 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tips.2007.03.006
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Vol.28 No.5
201
Figure 1. Heme degradation pathway driven by HO-1. Shown is the generation of CO, biliverdin and iron. Biliverdin is converted to bilirubin by biliverdin reductase.
investigated with regard to a potential role in biological processes until decades later. Novel findings regarding CO since the discovery of nitric oxide might have been viewed differently because these gases share similar vasoactive and immunological properties [9]. In hindsight, if interpreted in this context, the data obtained for NO might have provided the rationale for
research into the potential therapeutic value of CO more than half a century ago. Recent clinical evidence confirms that carboxyhemoglobin and end-tidal CO levels are significantly elevated during systemic inflammation in both trauma and septic patients [10–12]. These data strongly indicate that endogenous CO production is increased through the HO pathway, although this seems paradoxical
Figure 2. The cytoprotective effects of CO and the bile pigments. Shown are the effects in different cell types and the pathologies that are ameliorated in their presence. www.sciencedirect.com
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Figure 3. The concept of hormesis. In this concept, compounds exhibit a ’J-shaped’ dose–response profile. At low concentrations, there exist potent beneficial or stimulatory effects that shift to a classic linear dose–response toxicity at higher concentrations. Although CO and bilirubin have been labeled as having toxic properties at high concentrations, recent evidence indicates that each is consistent with fitting a hormetic profile whereby, at low concentrations, CO and bilirubin modulate inflammatory, apoptotic and proliferative processes to re-establish and maintain homeostasis and to exert therapeutic benefits.
at first glance in view of conventional attitudes vis-a`-vis CO toxicity; further investigation invites a paradigm shift that would include a characterization of CO as conditionally therapeutic. The term conditionally therapeutic implies that one would expect to extrapolate the efficacy data from animals to humans under the correct and appropriate conditions. A clinical investigator would appreciate the fact that the available data from humans are sparse, necessitating clinical testing. Optimizing the concentration of exogenous CO and taking advantage of the innate affinity of this gas for hemoglobin could represent an efficient in vivo delivery system if an extravascular gradient were to exist for CO removal. Explosion of HO research At >3300, the number of recent publications directly related to evaluating the homeostatic or cytoprotective properties associated with the HO pathway is remarkable [13,14]. Findings from Hmox1-knockout mice and the first reported case of human HO-1 deficiency [15–17] indicate that the activity of this enzyme is essential for modulating the inflammatory response [18,17], apoptosis [19] and cellular proliferation [20]. Furthermore, HO inhibitors attenuate HO-induced cytoprotection and exacerbate cellular injury [21,22]. It is also clear that humans differ quantitatively in their ability to upregulate HO-1 in response to various stressful stimuli because of variability in two distinct HO-1 promoter polymorphisms [23]. Thus, the administration of CO, biliverdin or bilirubin at appropriate doses is an alternative to HO-1-mediated protection, and these agents could function as novel therapeutics in www.sciencedirect.com
multiple disease states [24–28]. CO and biliverdin can both influence diverse cellular processes depending on cell type and model (Figure 2). CO CO is a colorless, odorless gas that has traditionally been considered an environmental pollutant and a lethal chemical asphyxiant at high concentrations. This is primarily because of the high affinity of CO for hemoglobin, which is 245 times that of oxygen in humans, with a half-life of 3–5 h under normal atmospheric conditions [29]. Additionally, CO binds to other heme-containing moieties such as nitric oxide synthase and guanylate cyclase [30,31]. Cytochrome c oxidase is also a subcellular target of CO, binding to which leads to alterations in the electron transport chain [10]. However, it is important to note that CO, like all compounds, has both a toxic and a therapeutic range. Indeed, despite CO having negative effects on hemoprotein function at high levels, it might be beneficial at low concentrations – it is the dosing differences that define the functional consequences (i.e. toxicity versus benefit). Before the discovery that low concentrations of exogenous CO [<250 parts per million (ppm)] have therapeutic effects, animal reports relied heavily on classical CO toxicological studies, in which the hypotheses and endpoints were grave and severe. These toxicological studies provided data and conclusions, such as the LC50 in rats (1807 ppm for 4 h), in addition to observations of detrimental effects of CO on motor ability [32,33]. Although there were no comparative studies in humans at the time,
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most of the reports were collected serendipitously from individuals exposed to mixtures of CO and other toxic products (e.g. exhaust fumes or leaky furnaces). By contrast, a study in 1986 determined that individuals exposed to CO at 40 ppm for 60 days were not negatively affected [1]. Currently, the permissible exposure limit for CO in the USA is 35 ppm for 8 h/day [33]. Against the vast toxicological data in the literature, experimental findings from the past five years have highlighted the therapeutic potential of CO as a novel gaseous therapy at concentrations <200 ppm, which seems to alter several key components of the inflammatory cascade [34,35]. More recently, the emergence of CO-releasing molecules (CORMs) is providing excellent alternatives to exogenous gas exposure with similar biological effects [36]. These findings lead to the conundrum of how a substance with known toxic properties can exert beneficial effects. Biliverdin or bilirubin As with CO, the history of bilirubin dates back centuries. Hippocrates viewed the body as consisting of four humors, one of which was black and one yellow, existing in a state of dynamic equilibrium and, if they were not in balance, disease would result. In 1916, van den Bergh developed a definitive test for the measurement of bilirubin that continues to dominate all clinical and investigative thinking in the field [37]. Schmorl labeled the lesion ‘kernicterus’ in 1904 to describe a pathology of the brain in neonates [38]. Even in adults, elevated levels of bilirubin are considered with alarm and are usually associated with liver failure. Hence, the conventional wisdom of the 20th century was – and, until recently, continued to be – that bilirubin was a potentially toxic waste-product with limited biological use. In 1987, bilirubin was defined as the most potent antioxidant molecule in serum [39], making it a potential cytoprotective agent in, for example, the brain, where it could scavenge free radicals and protect against oxidative damage [40]; biliverdin continued to be labeled as an intermediary in the breakdown of heme [41]. Like CO, the administration of biliverdin and/or bilirubin has recently proven to be potently cytoprotective in ameliorating the pathology associated with several disease processes such as ischemia–reperfusion injury, transplant rejection and inflammatory bowel disease [25,31,42,43]. Several reports show that individuals with high-normal or slightly elevated levels of bilirubin (including individuals with Gilbert’s syndrome, who have bilirubin levels two or three times those of normal individuals) have a markedly reduced incidence of atherosclerotic disease and a low prevalence of ischemic heart disease [44]. Experimental and clinical observations continue to shed light on the way in which bilirubin should be viewed by the greater medical and scientific community in both newborns and adults. In addition to its antioxidant capabilities, it is possible that biliverdin reductase regulates protective gene expression, including that of the gene encoding HO-1 [45]. One can envisage a therapeutic cycle wherein biliverdin and bilirubin induce the expression of HO-1, which then produces additional endogenous biliverdin and CO. A redox www.sciencedirect.com
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cycle proposed by Sedlak and Snyder communicates with a separate bile pigment cycle in which biliverdin and bilirubin are interconverted, with the reciprocity increasing the overall antioxidant power [45]. The increase in the number of publications demonstrating that exogenous delivery of biliverdin or bilirubin leads to protective effects supports the use of these metabolites as powerful therapeutics, yet generates questions that demand further inquiry, investigation and evaluation (Box 1). Future therapeutic strategies It is becoming clear that the next major challenge surrounding the clinical applicability of the HO-1 system is to identify accurately the particular disease states that will respond positively to altered HO-1 activity and/or exogenous administration of one or more of its products. Most of the preclinical data support translational studies with HO-1. Three therapeutic strategies exist that hold the strongest potential for efficacy, depending on the disease or the mode of injury: (i) pre-induction of HO-1 before a known injurious insult; (ii) pre-treatment; and/or (iii) post-treatment with exogenous byproducts of the HO pathway (i.e. CO, and biliverdin or bilirubin). A key concern when translating this knowledge into a treatment strategy with clinical use is the concept of achieving sufficient HO-1 induction and localization independently of injury. To achieve elevated HO-1 levels, such systems would include the use of a viral vector system or a trans activator of transcription (TAT) protein. However, the innate immune response initiated by local leukocytes after virus or foreign-protein administration arguably represents a potential injurious or inflammatory insult
Box 1. The evolution of HO-1 as a homeostatic mediator Are the diagnoses in newborns of kernicterus or encephalopathy, in addition to the negative correlation of bilirubin levels with atherosclerosis and ischemic heart disease, associative or are biliverdin and bilirubin directly involved in these diseases? Have these compounds been overlooked as potential therapeutics? Have biliverdin or bilirubin, and CO been inaccurately classified as ‘vestigial biological products’ that are akin to ammonia and urea? Because it has been demonstrated that the expression of HO-1 is greatest during embryonic development [46] – when CO and bilirubin levels are typically elevated – are such coincidental phenomena dose or concentration gradient effects, or simply hormetic effects? Does either have a crucial role in development? In addition, the gene encoding HO-1 is clearly a stress response gene and, thus, the systemic elevation of levels of the gene product is expected. Perhaps this explains, in part, why the phenotype of HO-1-deficient mice and humans exhibits heightened pathophysiological qualities. Indeed, the first reported HO-1-deficient patient was severely mentally retarded. This raises important issues that demand inquiry: Were these effects consequences of insufficient biliverdin or bilirubin and/or CO levels in utero? Are there threshold levels at which biliverdin or bilirubin and/or CO operates to ensure the production of ideal, beneficial or necessary elements that promote survival of the organism? Does the presence of these products re-establish homeostasis following a stress response, thereby indicating that HO-1 induction provides the products necessary for this process to occur?
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that limits applicability in the clinical setting. In these conditions, HO-1 itself might emerge as a therapeutic, assuming that there are no substrate limitations. The exogenous administration of CO and/or biliverdin and bilirubin is promising, akin to most novel pharmaceuticals; as such, these agents require standard clinical phase trials to determine efficacy, toxicological profiling, tissue distribution and half-life. Using the field of transplantation as an example of a large population of patients with an unmet clinical need, how would one take advantage of the HO-1 system? By using the three therapeutic approaches mentioned, it would be possible to pre-induce HO-1 in the donor, in the organ ex vivo or in the recipient before and, perhaps, after transplantation. Alternatively, CO and/or biliverdin or bilirubin could also be used to mimic HO-1 and attenuate both acute and chronic transplant rejection. Assuming that one or all of these approaches were efficacious, would they require a lifelong commitment, as with traditional immunosuppression, supplanted entirely as a new therapeutic option or would they override completely the current standards of care regarding immunosuppressive therapies? If an individual could inhale CO for 10–15 min or take a tablet containing a CORM and/or biliverdin or bilirubin daily, might these be acceptable, if novel, therapies? The use of CORMs has the advantage of enabling additional routes of administration compared with gas delivery, including targeted delivery to different organs and varying kinetics of delivery. CORMS, and biliverdin or bilirubin would also enable conjugation to other molecules such as nonsteroidal anti-inflammatory drugs, statins and immunosuppressive drugs and would not only combat the side effects of these compounds but also improve their efficacy. Perhaps these therapies would need to be administered only during the first week post-transplantation to induce tolerance, or alternatively be administered to only the donor and not the recipient. Obviously, the complexity of such a regimen requires standard testing and evaluation according to US Food and Drug Administration (http:// www.fda.gov/) regulations. Concluding remarks The potential for therapy would be enormous if one could harness the beneficial properties of the heme degradation pathway. For now, there are only questions for which research must continue to provide much-needed answers. It is up to the medical and scientific community to consider a paradigm shift, to take advantage of these conditionally therapeutic modalities and, ultimately, to use this intricate host defense and homeostasis-restoring system by applying its properties appropriately to promote health and to alleviate disease. Clearly, evolution has succeeded with HO-1. Disclosure statement L.E.O. is a paid consultant of Linde Healthcare. Acknowledgements This work was supported by NIH grants HL-071797 and HL-076167 (to L.E.O.). We thank the Julie Henry Fund at the Transplant Center of the Beth Israel Deaconess Medical Center for their support. www.sciencedirect.com
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References 1 ACGIH (1986) Documentation of the Threshold Limit Values and Biological Exposure Indices (5th edn), ACGIH 2 Ollinger, R. et al. (2007) Bilirubin and biliverdin treatment of atherosclerotic diseases. Cell Cycle 6, 39–43 3 Tenhunen, R. et al. (1969) Microsomal heme oxygenase. Characterization of the enzyme. J. Biol. Chem. 244, 6388–6394 4 Maines, M.D. (2004) The heme oxygenase system: past, present, and future. Antioxid. Redox Signal. 6, 797–801 5 Berberat, P.O. et al. (2003) Heavy chain ferritin acts as an antiapoptotic gene that protects livers from ischemia reperfusion injury. FASEB J. 17, 1724–1726 6 Schulz, H. (1888) Ueber Hefegifte. Pfluegers Arch. Gesamte Physiol. Menschen Tiere. 42, 517 7 Calabrese, E.J. and Baldwin, L.A. (2001) Hormesis: U-shaped dose responses and their centrality in toxicology. Trends Pharmacol. Sci. 22, 285–291 8 Sjo¨strand, T. (1952) The formation of carbon monoxide by the decomposition of haemoglobin in vivo. Acta Physiol. Scand. 26, 338–344 9 Ryter, S.W. and Otterbein, L.E. (2004) Carbon monoxide in biology and medicine. Bioessays 26, 270–280 10 Moncure, M. et al. (1999) Carboxyhemoglobin elevation in trauma victims. J. Trauma 46, 424–427 11 Zegdi, R. et al. (2002) Increased endogenous carbon monoxide production in severe sepsis. Intensive Care Med. 28, 793–796 12 Kawashima, A. et al. (2002) Heme oxygenase-1 deficiency: the first autopsy case. Hum. Pathol. 33, 125–130 13 Ryter, S.W. et al. (2006) Heme oxygenase-1/carbon monoxide: from basic science to therapeutic applications. Physiol. Rev. 86, 583–650 14 Otterbein, L.E. et al. (2003) Heme oxygenase-1: unleashing the protective properties of heme. Trends Immunol. 24, 449–455 15 Wiesel, P. et al. (2000) Endotoxin-induced mortality is related to increased oxidative stress and end-organ dysfunction, not refractory hypotension, in heme oxygenase-1-deficient mice. Circulation 2, 3015– 3022 16 Kapturczak, M.H. et al. (2004) Heme oxygenase-1 modulates early inflammatory responses: evidence from the heme oxygenase-1deficient mouse. Am. J. Pathol. 165, 1045–1053 17 Yachie, A. et al. (1999) Oxidative stress causes enhanced endothelial cell injury in human heme oxygenase-1 deficiency. J. Clin. Invest. 103, 129–135 18 Fujita, T. et al. (2001) Paradoxical rescue from ischemic lung injury by inhaled carbon monoxide driven by derepression of fibrinolysis. Nat. Med. 7, 598–604 19 Brouard, S. et al. (2000) Carbon monoxide generated by heme oxygenase 1 suppresses endothelial cell apoptosis. J. Exp. Med. 192, 1015–1026 20 Taille, C. et al. (2003) Heme oxygenase inhibits human airway smooth muscle proliferation via a bilirubin-dependent modulation of ERK1/2 phosphorylation. J. Biol. Chem. 278, 27160–27168 21 Sass, G. et al. (2003) Heme oxygenase-1 and its reaction product, carbon monoxide, prevent inflammation-related apoptotic liver damage in mice. Hepatology 38, 909–918 22 Yang, Y. et al. (2003) Selective protection of renal tubular epithelial cells by heme oxygenase (HO)-1 during stress-induced injury. Kidney Int. 64, 1302–1309 23 Exner, M. et al. (2004) The role of heme oxygenase-1 promoter polymorphisms in human disease. Free Radic. Biol. Med. 37, 1097– 1104 24 Otterbein, L.E. et al. (1999) Carbon monoxide provides protection against hyperoxic lung injury. Am. J. Physiol. 276, L688–L694 25 Fondevila, C. et al. (2004) Biliverdin therapy protects rat livers from ischemia and reperfusion injury. Hepatology 40, 1333–1341 26 Sato, K. et al. (2001) Carbon monoxide generated by heme oxygenase-1 suppresses the rejection of mouse-to-rat cardiac transplants. J. Immunol. 166, 4185–4194 27 Moore, B.A. et al. (2003) Inhaled carbon monoxide suppresses the development of post-operative ileus in the murine small intestine. Gastroenterology 124, 377–391 28 Kruger, A.L. et al. (2006) Up-regulation of heme oxygenase provides vascular protection in an animal model of diabetes through its antioxidant and antiapoptotic effects. J. Pharmacol. Exp. Ther. 319, 1144–1152
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29 Smith, R.P. (1986) Toxic responses of the blood. In Casarett and Doull’s Toxicology, the Basic Science of Poisons (3rd edn) (Klaassen, C.D. et al., eds), pp. 223–244, MacMillan Publishing 30 White, K.A. and Marletta, M.A. (1992) Nitric oxide synthase is a cytochrome P-450 type hemoprotein. Biochemistry 31, 6627–6631 31 Brune, B. and Ullrich, V. (1987) Inhibition of platelet aggregation by carbon monoxide is mediated by activation of guanylate cyclase. Mol. Pharmacol. 32, 497–504 32 Gosselin, R.E. et al. (1984) Clinical Toxicology of Commercial Products (5th edn), Williams & Wilkins 33 NIOSH (1986) Registry of toxic effects of chemical substances: carbon monoxide. US Department of Health and Human Services, Public Health Service, Centers for Disease Control, National Institute for Occupational Safety and Health 34 Otterbein, L.E. et al. (2003) Carbon monoxide suppresses arteriosclerotic lesions associated with chronic graft rejection and with balloon injury. Nat. Med. 9, 183–190 35 Nakao, A. et al. (2003) Carbon monoxide inhalation protects rat intestinal grafts from ischemia/reperfusion injury. Am. J. Pathol. 163, 1587–1598 36 Boczkowski, J. et al. (2006) CO–metal interaction: vital signaling from a lethal gas. Trends Biochem. Sci. 31, 614–621 37 Hymans van den Bergh, A.A. and Mueller, P. (1916) Determination of direct and indirect bilirubin. Biochem. 77, 90–92
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38 Schmorl, C.G. (1904) Zur kenntnis des ikterus neonatorum, insbesondere der dabei auftretenden gehirnveranderungen. Dent. Path. Gesell. 6, 109–112 39 Stocker, R. et al. (1987) Bilirubin is an antioxidant of possible physiological importance. Science 235, 1043–1046 40 Dore, S. and Snyder, S.H. (1999) Neuroprotective action of bilirubin against oxidative stress in primary hippocampal cultures. Ann. N. Y. Acad. Sci. 890, 167–172 41 Novotny, L. and Vitek, L. (2003) Inverse relationship between serum bilirubin and atherosclerosis in men: a meta-analysis of published studies. Exp. Biol. Med. (Maywood) 228, 568–571 42 Clark, J.E. et al. (2000) Heme oxygenase-1-derived bilirubin ameliorates postischemic myocardial dysfunction. Am. J. Physiol. Heart Circ. Physiol. 278, H643–H651 43 Nakao, A. et al. (2004) Biliverdin protects the functional integrity of a transplanted syngeneic small bowel. Gastroenterology 127, 595–606 44 Vitek, L. et al. (2002) Gilbert syndrome and ischemic heart disease: a protective effect of elevated bilirubin levels. Atherosclerosis 160, 449– 456 45 Sedlak, T.W. and Snyder, S.H. (2004) Bilirubin benefits: cellular protection by a biliverdin reductase antioxidant cycle. Pediatrics 113, 1776–1782 46 Kreiser, D. et al. (2003) Gestational pattern of heme oxygenase expression in the rat. Pediatr. Res. 54, 172–178
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