Toxicity of myoglobin and haemoglobin: oxidative stress in patients with rhabdomyolysis and subarachnoid haemorrhage

Haemolytic events, such as those following rhabdomyolysis and subarachnoid haemorrhage, often result in pathological complications such as vasoconstriction. Haem-protein cross-linked myoglobin and haemoglobin are generated by ferric-ferryl redox cycling, and thus can be used as markers of oxidative stress. We have found haem-protein cross-linked myoglobin in the urine of patients suffering from rhabdomyolysis and haem-protein cross-linked haemoglobin in the cerebrospinal fluid of patients following subarachnoid haemorrhage. These findings provide strong evidence that these respiratory haem proteins can be involved in powerful oxidation processes in vivo. We have previously proposed that these oxidation processes in rhabdomyolysis include the formation of potent vasoconstrictor molecules, generated by the myoglobin-catalysed oxidation of membranes, inducing nephrotoxicity and renal failure. Haem-protein cross-linked haemoglobin in cerebrospinal fluid suggests that a similar mechanism of lipid oxidation is present and that this may provide a mechanistic basis for the delayed vasospasm that follows subarachnoid haemorrhage.     

Heme oxygenase-1 induction prevents neuronal damage triggered during mitochondrial inhibition: Role of CO and bilirubin

Heme oxygenase (HO) catalyzes the breakdown of heme to iron, carbon monoxide (CO), and biliverdin, the latter being further reduced to bilirubin (BR). A protective role of the inducible isoform, HO-1, has been described in pathological conditions associated with reactive oxygen species (ROS) and oxidative damage. The aim of this study was to investigate the role of HO-1 in the neurotoxicity induced by the mitochondrial toxin 3-nitropropionic acid (3-NP) in primary cultures of cerebellar granule neurons (CGNs). Toxicity of 3-NP is associated with ROS production, and this metabolic toxin has been used to mimic pathological conditions such as Huntington's disease. We found that cell death caused by 3-NP exposure was exacerbated by inhibition of HO with tin mesoporphyrin (SnMP). In addition, HO-1 up-regulation induced by the exposure to cobalt protoporphyrin (CoPP) before the incubation with 3-NP, prevented the cell death and the increase in ROS induced by 3-NP. Interestingly, addition of SnMP to CoPP-pretreated CGNs exposed to 3-NP, abolished the protective effect of CoPP suggesting that HO activity was responsible for this protective effect. This was additionally supported by the fact that CORM-2, a CO-releasing molecule, and BR, were able to protect against cell death and the increase in ROS induced by 3-NP. Our data clearly show that HO-1 elicits in CGNs a neuroprotective action against the neurotoxicity of 3-NP and that CO and BR may be involved, at least in part, in this protective effect. The present results increase our knowledge about the role of HO-1 in neuropathological conditions.     

The presence of an extractable substance in the CSF of humans with cerebral vasospasm after subarachnoid haemorrhage that correlates with phosphatase inhibition

The cellular events leading to cerebral vasospasm after subarachnoid haemorrhage are poorly understood, although an increase in smooth muscle myosin light chain phosphorylation has been observed. This study set out to determine if phosphatase inhibition may be involved in the pathological maintenance of tension observed during vasospasm. We found that 1 nM okadaic acid, a type 2A protein phosphatase inhibitor, elicited an increase in rate of O(2) consumption in the porcine carotid artery similar to that by cerebrospinal fluid (CSF) from vasospastic patients (CSF(V), n=5) (control 0.23+/-0.03, CSF(V) 0.84+/-0.16 and okadaic acid 0.85+/-0.02 micromol min(-1) g dwt(-1)). It was also observed that phosphatase inhibition with 1 nM okadaic acid significantly slowed relaxation after a stretch in a similar fashion to CSF(V) haemorrhage. CSF from vasospastic subarachnoid haemorrhage patients, but not from those without vasospasm, contains an extractable substance which modulates myosin light chain phosphorylation in vitro. A phosphatase preparation obtained from the porcine carotid artery dephosphorylated 63+/-2% of the phosphorylated (MLC(20)) substrate in vitro, and non-vasospastic CSF treated enzyme dephosphorylated 60+/-2.6%. Okadaic acid inhibited phosphatase dephosphorylated only 7.5+/-1% of the substrate where CSF(V) treated enzyme dephosphorylated 22+/-2.8% of the substrate. We conclude that inhibition of smooth muscle phosphatase may be involved in the mechanisms associated with cerebral vasospasm after subarachnoid haemorrhage.     

Curcumin-induced fibroblast apoptosis and in vitro wound contraction are regulated by antioxidants and heme oxygenase: implications for scar formation

Fibroblast apoptosis plays a crucial role in normal and pathological scar formation and therefore we studied whether the putative apoptosis-inducing factor curcumin affects fibroblast apoptosis and may function as a novel therapeutic. We show that 25-μM curcumin causes fibroblast apoptosis and that this could be inhibited by co-administration of antioxidants N -acetyl- l -cysteine (NAC), biliverdin or bilirubin, suggesting that reactive oxygen species (ROS) are involved. This is supported by our observation that 25-μM curcumin caused the generation of ROS, which could be completely blocked by addition of NAC or bilirubin. Since biliverdin and bilirubin are downstream products of heme degradation by heme oxygenase (HO), it has been suggested that HO-activity protects against curcumin-induced apoptosis. Interestingly, exposure to curcumin maximally induced HO-1 protein and HO-activity at 10–15 μM, whereas, at a concentration of >20-μM curcumin HO-1-expression and HO-activity was negligible. NAC-mediated inhibition of 25-μM curcumin-induced apoptosis was demonstrated to act in part via restored HO-1-induction, since the rescuing effect of NAC could be reduced by inhibiting HO-activity. Moreover pre-induction of HO-1 using 5-μM curcumin protected fibroblasts against 25-μM curcumin-induced apoptosis. On a functional level, fibroblast-mediated collagen gel contraction, an in vitro wound contraction model, was completely prevented by 25-μM curcumin, while this could be reversed by co-incubation with NAC, an effect that was also partially HO-mediated. In conclusion, curcumin treatment in high doses (>25 μM) may provide a novel way to modulate pathological scar formation through the induction of fibroblast apoptosis, while antioxidants, HO-activity and its effector molecules act as a possible fine-tuning regulator.     

Photo-isomerization and oxidation of bilirubin in mammals is dependent on albumin binding

The bilirubin (BR) photo-conversion in the human body is a protein-dependent process; an effective photo-isomerization of the potentially neurotoxic Z,Z-BR as well as its oxidation to biliverdin in the antioxidant redox cycle is possible only when BR is bound on serum albumin. We present a novel analytical concept in the study of linear tetrapyrroles metabolic processes based on an in-depth mapping of binding sites in the structure of human serum albumin (HSA). A combination of fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and molecular modeling methods was used for recognition of the binding site for BR, its derivatives (mesobilirubin and bilirubin ditaurate), and the products of the photo-isomerization and oxidation (lumirubin, biliverdin, and xanthobilirubic acid) on HSA. The CD spectra and fluorescent quenching of the Trp–HSA were used to calculate the binding constants. The results of the CD displacement experiments performed with hemin were interpreted together with the findings of molecular docking performed on the pigment–HSA complexes. We estimated that Z,Z-BR and its metabolic products bind on two independent binding sites. Our findings support the existence of a reversible antioxidant redox cycle for BR and explain an additional pathway of the photo-isomerization process (increase of HSA binding capacity; the excess free [unbound] BR can be converted and also bound to HSA).     

Generation of bile pigments by haem oxygenase: a refined cellular strategy in response to stressful insults

The family of haem oxygenase enzymes is unique in nature for its role in haem degradation. Haem is cleaved at the alpha-meso position by haem oxygenase with the support of electrons donated by cytochrome P450 reductase, the first products of this reaction being CO, iron and biliverdin. Biliverdin is then converted to bilirubin by biliverdin reductase. If haem is viewed as a substrate for an anabolic pathway, it becomes evident that haem oxygenases do not break down haem for elimination from the body, but rather use haem to generate crucial molecules that can modulate cellular functions. The facts that biliverdin and bilirubin are potent antioxidants and that CO is both a vasoactive and signalling molecule sustain this idea. The existence of a constitutive haem oxygenase (HO-2), mainly present in the vasculature and nervous system, and an inducible haem oxygenase (HO-1), which is highly expressed during stress conditions in all tissues, also suggests that cells have evolved a fine control of this enzymic pathway to ultimately regulate haem consumption and to ensure production of CO, biliverdin/bilirubin and iron during physiological and pathophysiological situations. This review will focus primarily on the biological actions of biliverdin and bilirubin derived from the haem oxygenase/biliverdin reductase systems and their potential roles in counteracting oxidative and nitrosative stress.     

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.     

Complex-formation and redox reactions of bilirubin and biliverdin with zinc(II), cadmium(II) and copper(II) ions

Transition metal complexes of bilirubin and biliverdin were studied spectrophotometrically, in DMSO and in a boric acid-NaOH buffer mixture at pH 10.5. In the zinc(II) and cadmium(II)- bilirubin systems, 2:1 complexes are formed. Both in aqueous and in DMSO medium, the copper(II) ion oxidizes bilirubin to biliverdin. With all three metal ions, biliverdin forms 1:1 complexes, the stabilities of which are higher than those of the corresponding bilirubin complexes. Accordingly, these metal ions accelerate the oxidative transformations of bilirubin.     

Role of hemin in the inhibition of mutagenic activity of 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and other aminoazaarenes

The mechanism of inhibition by hemin of the mutagenic activities of food pyrolysate aminoazaarenes, particularly that of Trp-P-2 (3-amino-1-methyl-5H-pyrido[4,3-b]indole), was investigated. Hemin efficiently inhibited the metabolic activation by S9 of Trp-P-2, as demonstrated by high-performance liquid chromatographic analysis of the reaction mixtures in which Trp-P-2 had been treated with S9 in the presence or absence of hemin. N-Hydroxy-Trp-P-2, an activated form of Trp-P-2 having direct mutagenicity on Salmonella typhimurium TA98, undergoes spontaneous oxidative degradation in its aqueous solution, and the presence of hemin in the solution accelerated the degradation significantly. The presence of excess hemin with N-hydroxy-Trp-P-2 completely abolished the mutagenic activity of this mutagen towards Salmonella. A UV-visible spectroscopic study has suggested the formation of a complex between hemin and N-hydroxy-Trp-P-2/Trp-P-2. In support of this view, the fluorescence spectrum of a Trp-P-2 solution was quenched efficiently by the addition of hemin. These observations indicate that this complex formation plays a role in the observed multiple actions of hemin. Similar inhibitory actions of hemin on several other direct-acting aminoazaarene mutagens are also described, as well as the inhibition activities of protoporphyrin, chlorophyllin, biliverdin and bilirubin.     

Effect of bilirubin infusion on excretion of bilirubin and biliverdin in rabbits

Intravenous infusion of bilirubin (BR) at 171 micrograms/min/kg into rabbits resulted in biliary concentration of BR increasing from 3.8 (control) to 243 mg/dl and BR excretion increasing from 1.7 to 66 micrograms/min/kg. BR infusion resulted in biliary concentrations of biliverdin (BV) increasing from 9.1 to 30 g/dl and BV excretion increasing from 4.2 to 8.2 micrograms/min/kg. BR infusion produced a progressive decline in bile flow. BV was the predominant bile pigment in control rabbits fed either an alfalfa-based or chlorophyll-free diet. These results imply that rabbits can oxidize BR to BV.     

Neuroprotective role of heme-oxygenase 1 against iodoacetate-induced toxicity in rat cerebellar granule neurons: Role of bilirubin

Heme oxygenase (HO) catalyses the breakdown of heme to iron, carbon monoxide and biliverdin, the latter being further reduced to bilirubin. A protective role of the inducible isoform, HO-1, has been described in pathological conditions associated with the production of reactive oxygen species (ROS). The aim of this study was to investigate the role of HO-1 in the neurotoxicity induced by iodoacetate (IAA) in primary cultures of cerebellar granule neurons (CGNs). IAA, an inhibitor of the glycolysis pathway, reduces cell survival, increases ROS production and enhances HO-1 expression in CGNs. Furthermore, the induction of HO-1 expression by cobalt protoporphyrin (CoPP) prevented cell death and ROS production induced by IAA, whereas the inhibition of HO activity with tin mesoporphyrin exacerbated the IAA-induced neurotoxicity. The protective effect elicited by CoPP was reproduced by bilirubin addition, suggesting that this molecule may be involved in the protective effect of HO-1 induction in this experimental model.     

Immunohistochemical localization of the antioxidant enzymes biliverdin reductase and heme oxygenase-2 in human and pig gastric fundus

The intrinsic antioxidant capacities of the bile pigments biliverdin and bilirubin are increasingly recognized since both heme degradation products can exert beneficial cytoprotective effects due to their scavenging of oxygen free radicals and interaction with antioxidant vitamins. Several studies have been published on the localization of the carbon monoxide producing enzyme heme oxygenase-2 (HO-2), which concomitantly generates biliverdin; histochemical data on the distribution of biliverdin reductase (BVR), converting biliverdin to bilirubin, are still very scarce in large mammals including humans. The present study revealed by means of immunohistochemistry the presence of BVR and HO-2 in mucosal epithelial cells and in the endothelium of intramural vessels of both human and porcine gastric fundus. In addition, co-labeling with the specific neural marker protein-gene product 9.5 (PGP 9.5) demonstrated that both BVR and HO-2 were present in all intrinsic nerve cell bodies of both submucous and myenteric plexuses, while double labeling with c-Kit antibody confirmed their presence in intramuscular interstitial cells of Cajal (ICC). Our results substantiate the hypothesis that BVR, through the production of the potent antioxidant bilirubin, might be an essential component of normal physiologic gastrointestinal defense in man and pig.     

PEP-1-HO-1 prevents MPTP-induced degeneration of dopaminergic neurons in a Parkinson’s disease mouse model

Heme oxygenase-1 (HO-1) degrades heme to carbon dioxide, biliverdin, and Fe²⁺, which play important roles in various biochemical processes. In this study, we examined the protective function of HO-1 against oxidative stress in SH-SY5Y cells and in a Parkinson’s disease mouse model. Western blot and fluorescence microscopy analysis demonstrated that PEP-1-HO-1, fused with a PEP-1 peptide can cross the cellular membranes of human neuroblastoma SH-SY5Y cells. In addition, the transduced PEP-1-HO-1 inhibited generation of reactive oxygen species (ROS) and cell death caused by 1-methyl-4-phenylpyridinium ion (MPP⁺). In contrast, HO-1, which has no ability to transduce into SH-SY5Y cells, failed to reduce MPP⁺-induced cellular toxicity and ROS production. Furthermore, intraperitoneal injected PEP-1-HO-1 crossed the blood-brain barrier in mouse brains. In a PD mouse model, PEP-1-HO-1 significantly protected against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced toxicity and dopaminergic neuronal death. Therefore, PEP-1-HO-1 could be a useful agent in treating oxidative stress induced ailments including PD. [BMB Reports 2014; 47(10): 569-574]     

In vitro permeability and metabolic stability of bile pigments and the effects of hydrophilic and lipophilic modification of biliverdin

Bile pigments, including bilirubin and biliverdin are tetrapyrrolic, dicarboxylic acids capable of forming conjugates at their propionic acid groups via ester or amide bonds. They possess substantial antioxidant and anti-mutagenic activities and therefore their intestinal absorption might influence the development of cardiovascular disease and cancer. The aim of this study was to investigate whether altering the physico-chemical properties of bile pigments would improve their permeability in an in vitro assay of absorption. Native and synthetically modified bile pigments were tested for gastrointestinal permeability and metabolic stability using the Caco-2 cell line. In addition, a gross measure of their toxic effects was tested in a red blood cell co-incubation assay. The apparent permeability of unconjugated bilirubin (1), bilirubin ditaurate (2) and biliverdin (3) through Caco-2 cell monolayers was determined to be 10.4+/-1.2x10(-7), 35.2+/-3.4x10(-7) and 37.0+/-1.6x10(-7) cm/s (mean+/-SD), respectively, while biliverdin diglucosamine (4), and biliverdin dioctylamine (5) were impermeable. Unconjugated bilirubin, biliverdin, bilirubin ditaurate and biliverdin diglucosamine did not decompose when incubated in Caco-2 cell homogenates, whereas biliverdin dioctylamine decomposed over time. Only unconjugated bilirubin showed toxicity towards red blood cells (> or = 1000 microM), an effect that was abolished by the addition of 40 g/L serum albumin. The data presented here suggest that bile pigments are absorbed across the Caco-2 cell monolayer and that conjugation of biliverdin to hydrophilic or lipophilic moieties decreases their absorption and can reduce their metabolic stability. In summary, exogenous bilirubin and biliverdin supplements could be absorbed across the intestinal epithelium in vivo and potentially increase circulating concentrations of these antioxidant compounds.     

Rv2074 is a novel F420H2‐dependent biliverdin reductase in Mycobacterium tuberculosis

Bilirubin is a potent antioxidant that is produced from the reduction of the heme degradation product biliverdin. In mammalian cells and Cyanobacteria, NADH/NADPH‐dependent biliverdin reductases (BVRs) of the Rossmann‐fold have been shown to catalyze this reaction. Here, we describe the characterization of Rv2074 from Mycobacterium tuberculosis, which belongs to a structurally and mechanistically distinct family of F₄₂₀H₂‐dependent BVRs (F‐BVRs) that are exclusively found in Actinobacteria. We have solved the crystal structure of Rv2074 bound to its cofactor, F₄₂₀, and used this alongside molecular dynamics simulations, site‐directed mutagenesis and NMR spectroscopy to elucidate its catalytic mechanism. The production of bilirubin by Rv2074 could exploit the anti‐oxidative properties of bilirubin and contribute to the range of immuno‐evasive mechanisms that have evolved in M. tuberculosis to allow persistent infection.     

The excretion pattern of biliverdin and bilirubin in bile of the small skate (Raja erinacea)

Summary Bile pigment composition (biliverdin, bilirubin and their conjugates) was analyzed in stored gallbladder bile and newly synthesized hepatic bile from the small skate (Raja erinacea). During a five day period of captivity, gallbladder volume remained relatively constant while bilirubin and biliverdin content increased two to three fold. Biliverdin which accounted for 50% of the pigments did not increase as a percentage of tetrapyrroles during this period. The relative proportion of bilirubin and its conjugates also remained constant, averaging 65% for bilirubin monoglucuronide, 30% for bilirubin diglucuronide and 5% for unconjugated bilirubin as measured by HPLC methods. Intravenous administration of biliverdin resulted in significant increases in the biliary excretion of both biliverdin and all bilirubin tetrapyrroles. Insignificant quantities of³H-biliverdin were detected in hepatic bile following the intravenous administration of³H-bilirubin. These studies indicate that the small skate excreted both biliverdin and bilirubin conjugates in bile and that the biliverdin was not produced by in vitro oxidation of bilirubin or its metabolites.     

Melatonin Ameliorates Cerebral Vasospasm After Experimental Subarachnoidal Haemorrhage Correcting Imbalance of Nitric Oxide Levels in Rats

In the present study, we investigated the in vivo effects of melatonin on SAH-induced cerebral vasospasm and oxidative stress, resulting from SAH in an experimental rat model. Twenty-eight rats (225–250 g) were divided into four groups equally: group 1; control, group 2; SAH, group 3; SAH plus placebo, and group 4; SAH plus melatonin. We used double haemorrhage method for SAH groups. Beginning 6 h after SAH, 20 mg/kg melatonin or equal volume of 0.9% saline was administered intraperitoneally twice daily for 5 days to groups 3 and 4, respectively. Melatonin or 0.9% saline injections were continued up to fifth day after SAH and rats were sacrificed at the end of this period. Brain sections at the level of the pons were examined by light microscopy. The lumen diameter and the vessel wall thickness of basilar artery were measured using a micrometer. The serum levels of cerebral vasodilator nitric oxide (NO), the brain levels of an intrinsic antioxidant superoxide dismutase (SOD) and a NO regulator arginase activities were measured. The brain levels of inducible nitric oxide (iNOS) and nitrotyrosine, a nitrosative stress parameter immunohistochemiacally determined. In conclusion, melatonin administration ameliorated cerebral vasospasm by increasing serum NO level and decreasing the brain the levels of arginase and oxidative stress. It is therefore possible that increased brain arginase activity after SAH may also have a significant role in the pathogenesis of vasospasm by limiting the availability of arginine for NO production.     

The heme synthesis and degradation pathways: role in oxidant sensitivity. Heme oxygenase has both pro- and antioxidant properties

The heme biosynthetic and catabolic pathways generate pro- and antioxidant compounds, and consequently, influence cellular sensitivity to oxidants. Heme precursors (delta-aminolevulinic acid, porphyrins) generate reactive oxygen species (ROS), from autoxidation and photochemical reactions, respectively. Heme, an essential iron chelate, serves in respiration, oxygen transport, detoxification, and signal transduction processes. The potential toxicity of heme and hemoproteins points to a critical role for heme degradation in cellular metabolism. The heme oxygenases (HOs) provide this function and participate in cellular defense. This hypothesis emerges from the observation that the activation of HO-1 is an ubiquitous cellular response to oxidative stress. The reaction products of HO activity, biliverdin, and its subsequent metabolite bilirubin, have antioxidant properties. Furthermore, iron released from HO activity stimulates ferritin synthesis, which ultimately provides an iron detoxification mechanism that may account for long-term cytoprotection observed after HO induction. However, such models have overlooked potential pro-oxidant consequences of HO activity. The HO reaction releases iron, which could be involved in deleterious reactions that compete with iron reutilization and sequestration pathways. Indeed, the induction of HO activity may have both pro- and antioxidant sequelae depending on cellular redox potential, and the metabolic fate of the heme iron.     

Heme oxygenase-1 expression in disease states

Heme oxygenase-1 (HO-1) is an enzyme which catalyzes the rate-limiting step in heme degradation resulting in the formation of iron, carbon monoxide and biliverdin, which is subsequently converted to bilirubin by biliverdin reductase. The biological effects exerted by the products of this enzymatic reaction have gained much attention. The anti-oxidant, anti-inflammatory and cytoprotective functions associated with HO-1 are attributable to one or more of its degradation products. Induction of HO-1 occurs as an adaptive and beneficial response to several injurious stimuli including heme and this inducible nature of HO-1 signifies its importance in several pathophysiological disease states. The beneficial role of HO-1 has been implicated in several clinically relevant disease states involving multiple organ systems as well as significant biological processes such as ischemia-reperfusion injury, inflammation/immune dysfunction and transplantation. HO-1 has thus emerged as a key target molecule with therapeutic implications.     

Limited Role for the Bilirubin-Biliverdin Redox Amplification Cycle in the Cellular Antioxidant Protection by Biliverdin Reductase

In mammalian cells, heme is degraded by heme oxygenase to biliverdin, which is then reduced to bilirubin by biliverdin reductase (BVR). Both bile pigments have reducing properties, and bilirubin is now generally considered to be a potent antioxidant, yet it remains unclear how it protects cells against oxidative damage. A presently popular explanation for the antioxidant function of bilirubin is a redox cycle in which bilirubin is oxidized to biliverdin and then recycled by BVR. Here, we reexamined this putative BVR-mediated redox cycle. We observed that lipid peroxidation-mediated oxidation of bilirubin in chloroform, a model of cell membrane-bound bilirubin, did not yield biliverdin, a prerequisite for the putative redox cycle. Similarly, H₂O₂ did not oxidize albumin-bound bilirubin to biliverdin, and in vitro oxidation of albumin or ligandin-bound bilirubin by peroxyl radicals gave modest yields of biliverdin. In addition, decreasing cellular BVR protein and activity in HeLa cells using RNA interference did not alter H₂O₂-mediated cell death, just as BVR overexpression failed to enhance protection of these cells against H₂O₂-mediated damage, irrespective of whether bilirubin or biliverdin were added to the cells as substrate for the putative redox cycle. Similarly, transformation of human BVR into hmx1 (heme oxygenase) mutant yeast did not provide protection against H₂O₂ toxicity above that seen in hmx1 mutant yeast expressing human heme oxygenase-1. Together, these results argue against the BVR-mediated redox cycle playing a general or important role as cellular antioxidant defense mechanism.Biliverdin reductase (BVR)³ forms part of the major pathway for the disposition of cellular heme in mammalian cells. This pathway is initiated by heme oxygenase, which converts heme to carbon monoxide, iron, and biliverdin, which in turn is reduced to bilirubin by BVR at the expense of NADPH. Because of its intramolecular hydrogen bonding, the bilirubin produced is sparingly soluble in water at physiological pH and ionic strength (1). Hence, bilirubin is usually tightly bound to albumin in order to be transported within the blood circulation (2), from which it is removed mainly through uptake by hepatocytes. Once bilirubin is transferred across the cell membrane of hepatocytes, it binds glutathione S-transferases before being transformed to water-soluble derivatives by conjugation of one or both of its propionyl groups before its excretion into bile and then the intestine (3).Under physiological conditions, plasma bilirubin concentrations in humans range from ∼5 to 20 μm, practically all of which is unconjugated pigment bound to albumin (1). Abnormally high plasma concentrations are associated with the risk of developing neurologic dysfunction due to preferential deposition of bilirubin in brain and its toxic effects on cell functions. In fact for many years, biliverdin and bilirubin were generally regarded as waste products of heme metabolism in higher animals, although earlier work suggested that these bile pigments might play a role as natural antioxidants, since small quantities of the pigment stabilize vitamin A and β-carotene during intestinal uptake, and animals with low plasma bilirubin showed early signs of vitamin E deficiency (4, 5).In a series of in vitro studies, Stocker et al. (6–8) demonstrated that unconjugated bilirubin, at micromolar concentrations, efficiently scavenged peroxyl radicals in homogenous solution or multilamellar liposomes. At physiologically relevant oxygen tension, bilirubin surpassed α-tocopherol as an antioxidant in liposomes (8), and it is thought to protect plasma proteins and lipids from many but not all oxidants (9). However, it is less clear whether this antioxidant activity extends to in vivo situations or protection of cells from oxidative stress. Although produced in essentially all cells, the normal range of cellular bilirubin concentrations is unknown. However, it is probably in the low nanomolar range, well below that of established cellular antioxidants, such as glutathione and ascorbate, arguing against bilirubin being an important cellular antioxidant. Nonetheless, in vitro studies with rat neuronal cultures showed that the presence of 10 nm bilirubin in the culture medium protected cells against 10,000-fold higher concentrations of hydrogen peroxide (10). Later, Barañano et al. (11) confirmed such observations in HeLa cells and demonstrated that BVR depletion increased reactive oxygen species (ROS) and cell death. This led to the following proposal of the BVR-amplified redox cycle. While acting as an antioxidant, bilirubin is oxidized to biliverdin that is then reduced back to bilirubin by the ubiquitous and abundant BVR.An important underlying assumption of this amplification cycle is that ROS-mediated bilirubin oxidation in cells is specific and yields substantial if not stoichiometric amounts of biliverdin. Inconsistent with this assumption, however, earlier studies showed that high yields of biliverdin formation are limited to certain oxidants (i.e. peroxyl radicals) and albumin-bound bilirubin. In cells, bilirubin is probably present in membranes, bound to proteins other than albumin, or present in conjugated form. Therefore, we reexamined the putative redox amplification cycle. Our results show that reaction of these forms of bilirubin with 1e- or 2e-oxidants at best generates modest amounts of biliverdin. Furthermore, overexpression of BVR does not protect mammalian or yeast cells from hydrogen peroxide-mediated damage, thereby casting doubt on the importance of the putative BVR redox cycle for cellular antioxidant protection.