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. 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. Cycling the Wagons for Biliverdin ReductaseJournal of Biological ChemistryVol. 284Issue 46PreviewThis letter was prompted by our reading of the article by Maghzal et al. (1). Bilirubin (BR) is a major physiologic antioxidant cytoprotectant. Tissue levels are only in nanomolar concentrations, because high levels are neurotoxic, yet BR protects against micromolar levels of oxidants because of a cycle wherein biliverdin (BV) generated by BR antioxidant action is efficiently reconverted by biliverdin reductase (BVR) to BR (2). Additional evidence for the cycle includes the extraordinary antioxidant potency of BR and augmented levels of reactive oxygen species (ROS), lipid oxidation, and susceptibility to cell death in BVR-depleted tissues (2, 3). Full-Text PDF Open AccessReply to Sedlak and Snyder: The Little Bighorn of the Biliverdin Reductase Amplification CycleJournal of Biological ChemistryVol. 284Issue 46PreviewThis is a response to a letter by Sedlak and Snyder (1). Full-Text PDF Open Access Biliverdin reductase (BVR) 3The abbreviations used are: AAPH2,2′-azobis(2-amidinopropane)hydrochlorideAMVN2,2′-azobis(2,4-dimethylvaleronitrite)BVRbiliverdin reductaseDMEMDulbecco's modified Eagle's mediumDTPAdiethylene triamine pentaacetic acidFBSfetal bovine serumGSTglutathione S-transferaseMTT3-(4,5-dimethylthiazl-2-yl)-2,5-diphenyl tetraxolium bromidePBSphosphate-buffered salineROSreactive oxygen speciesHPLChigh performance liquid chromatographyRNAiRNA interferenceDCFdichlorofluoresceinsiRNAsmall interfering RNA. 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 (1McDonagh A.F. The Porphyrins.in: Dolphin D. Academic Press, Inc., New York1979: 293-491Crossref Google Scholar). Hence, bilirubin is usually tightly bound to albumin in order to be transported within the blood circulation (2Brodersen R. CRC Crit. Rev. Clin. Lab. Invest. 1980; 11: 305-399Crossref Scopus (210) Google Scholar), 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 (3Fevery J. Van de Vijver M. Michiels R. Heirwegh K.P. Biochem. J. 1977; 164: 737-746Crossref PubMed Scopus (109) Google Scholar). 2,2′-azobis(2-amidinopropane)hydrochloride 2,2′-azobis(2,4-dimethylvaleronitrite) biliverdin reductase Dulbecco's modified Eagle's medium diethylene triamine pentaacetic acid fetal bovine serum glutathione S-transferase 3-(4,5-dimethylthiazl-2-yl)-2,5-diphenyl tetraxolium bromide phosphate-buffered saline reactive oxygen species high performance liquid chromatography RNA interference dichlorofluorescein small interfering RNA. Under physiological conditions, plasma bilirubin concentrations in humans range from ∼5 to 20 μm, practically all of which is unconjugated pigment bound to albumin (1McDonagh A.F. The Porphyrins.in: Dolphin D. Academic Press, Inc., New York1979: 293-491Crossref Google Scholar). 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 (4Bernhard K. Ritzel G. Steiner K.U. Helv. Chim. Acta. 1954; 37: 306-313Crossref Scopus (55) Google Scholar, 5Kaufmann H.P. Garloff H. Fette Seifen Anstrichm. 1961; 63: 334-344Crossref Scopus (15) Google Scholar). In a series of in vitro studies, Stocker et al. (6Stocker R. Ames B.N. Proc. Natl. Acad. Sci. U.S.A. 1987; 84: 8130-8134Crossref PubMed Scopus (129) Google Scholar, 7Stocker R. Glazer A.N. Ames B.N. Proc. Natl. Acad. Sci. U.S.A. 1987; 84: 5918-5922Crossref PubMed Scopus (664) Google Scholar, 8Stocker R. Yamamoto Y. McDonagh A.F. Glazer A.N. Ames B.N. Science. 1987; 235: 1043-1046Crossref PubMed Scopus (2921) Google Scholar) 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 (8Stocker R. Yamamoto Y. McDonagh A.F. Glazer A.N. Ames B.N. Science. 1987; 235: 1043-1046Crossref PubMed Scopus (2921) Google Scholar), and it is thought to protect plasma proteins and lipids from many but not all oxidants (9Stocker R. Antioxid. Redox Signal. PubMed Scopus Google Scholar). it is whether this antioxidant activity to in or protection of cells from oxidative produced in all cells, the range of cellular bilirubin concentrations is it is in the low nanomolar that of cellular antioxidants, as glutathione and against bilirubin being an important cellular in vitro with showed that the of bilirubin in the medium cells against higher concentrations of hydrogen M. R. D. Snyder Proc. Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar). et al. M. Snyder Proc. Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar) in HeLa cells and demonstrated that BVR reactive oxygen species and cell This to the of the redox cycle. as an antioxidant, bilirubin is oxidized to biliverdin that is then reduced to bilirubin by the and BVR. important of this cycle is that bilirubin oxidation in cells is and yields not of biliverdin. with this earlier showed that high yields of biliverdin are to oxidants peroxyl and albumin-bound In cells, bilirubin is in bound to proteins or in we reexamined the putative redox cycle. results that of these forms of bilirubin with or at modest of biliverdin. overexpression of BVR not protect mammalian or yeast cells from hydrogen damage, on the of the putative BVR redox cycle for cellular antioxidant and acid were from and 2,2′-azobis(2,4-dimethylvaleronitrite) were from Biliverdin bilirubin and bilirubin were from serum albumin was from Biological and were from reductase was from and with was from and were from Science. were from of were from were from and phosphate-buffered saline were from Dulbecco's modified Eagle's medium was from and fetal bovine serum was from and were from The of H2O2 was using and then to the concentrations Bilirubin in was added to glutathione S-transferase or human serum albumin in phosphate-buffered saline pH human serum in was added as an and the solution was at it as R. Glazer A.N. Ames B.N. Proc. Natl. Acad. Sci. U.S.A. 1987; 84: 5918-5922Crossref PubMed Scopus (664) Google Scholar). of was then The concentrations of were as or human serum albumin 20 bilirubin, and The oxidation was initiated by the in a water at oxidation by we used in the of to H2O2 at a from in and diethylene triamine pentaacetic acid was added to or a solution of bilirubin bound to human serum albumin acid and as was initiated by and the at of the were removed at the and the pigments were with of at for of the was to was added to bilirubin in and oxidation was and at was added to bilirubin in chloroform, and the oxidation at oxidation of bilirubin, or and in was added to bilirubin in was initiated by the at At the of the were removed and to of biliverdin and bilirubin was using an with in and water at as R. McDonagh A.F. Glazer A.N. Ames B.N. PubMed Scopus Google Scholar). Under these conditions, biliverdin and bilirubin at and Biliverdin was at or bilirubin was at pigments were by with from the of bilirubin and biliverdin in water was used as the at The of bilirubin was and that of its putative biliverdin Bilirubin and biliverdin were at and biliverdin biliverdin was used as due to the of biliverdin HeLa cells were in low with and were in a of at and the medium was At cells were with and for and in and for RNA was from cells by and to with a was then by with using the and The used was as cycle of for of for for and for cycle of for The was on a and using a from The was then into and transformed into cells The was with the and with to the BVR was with and with intestinal Both the BVR and were and from a and then with The was with to with The was to A human was in the yeast A was with and with to the was with and with intestinal Both the and were and from a and then with to A was in the yeast The was with the and with to the was with and with intestinal Both the and were and from a and then with to overexpression of BVR in HeLa cells was using cell to the or was with to the of The was a at the were added to the cells in with and were then for before being to BVR was using small interfering RNA. HeLa cells in were in with for were then with an of against human BVR and using to the were then for in with before being to a cells were with The was not a to in the human was by the or the 3-(4,5-dimethylthiazl-2-yl)-2,5-diphenyl tetraxolium bromide were at and into a and for cell by the of cells were for one the medium with cells and the cells to the medium and one with The cell was at for and cells were in an of of cell were then with of cells were and cell was as of cell cells the cells in were in and as At the the medium was and the cells were with medium and were then and the cells were at for in a of before medium was The in the cells were then by of by at for 20 of the were in of and its at was using a was as of cell of of were to in medium at and then for with H2O2 of cells were in and in on and the of was of BVR activity was by the of bilirubin using R. McDonagh A.F. Glazer A.N. Ames B.N. PubMed Scopus Google Scholar). cells with of were and into an and at for at of the to of protein by the acid was then added to an in a at the of cellular biliverdin added as of in A 20 and one pH The of the was The was in a for at The bilirubin generated was by reading at and using levels were using as H. PubMed Scopus Google Scholar) with HeLa cells in were for with or human as the of the cells were with and then for at with dichlorofluorescein μm, in in cells were with and with the of H2O2 in of the cells in was then for in a at was at and was at levels in cells were as the in with cells. cells were in with cell were by cells in with on a in the of for and then on for was on by of protein using as was at for using a and proteins were transferred at for using a The were in with 20 in saline for at were at with the at in and against human BVR against human against human were then for at with or in and 20 in were by to the yeast cell and BVR were the as but and were in bovine serum albumin and cells were to cells were in on a in the of for by on for and the RNA was then to the was using the for and for BVR activity were using the were using a of with was at important of the cycle by et al. M. Snyder Proc. Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar) is that oxidation of bilirubin is and yields not of biliverdin. bilirubin is and in the pigment with proteins or We the yield of biliverdin using bilirubin bound to albumin or and peroxyl radicals from as the and are and proteins of bilirubin, R. Google Scholar), peroxyl radicals were used as a model for radicals the bilirubin was oxidized by peroxyl radicals as earlier R. Glazer A.N. Ames B.N. Proc. Natl. Acad. Sci. U.S.A. 1987; 84: 5918-5922Crossref PubMed Scopus (664) Google Scholar). Under the peroxyl radicals were produced at and bilirubin oxidation was in 20 This with of biliverdin to of the bilirubin biliverdin as by the in the of that pigment radicals oxidized bilirubin, with observed within In to the with albumin-bound bilirubin, was only a modest of biliverdin, to of the bilirubin Similarly, peroxyl radicals oxidized bilirubin a model of the bilirubin, but the biliverdin was a oxidation only We the oxidation of bilirubin by since bilirubin to protect cells from by a of H2O2 M. R. D. Snyder Proc. Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar, M. Snyder Proc. Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar). In to peroxyl albumin-bound bilirubin was to a that was generated at using with biliverdin being We that the of H2O2 was to the at which we peroxyl radicals that oxidized bilirubin bound to at a with peroxyl radicals and with biliverdin to of the bilirubin of bilirubin by H2O2 was and failed to of the biliverdin results that the oxidation of bilirubin to biliverdin by peroxyl radicals or H2O2 is modest and on the of the protein to which bilirubin We whether the of bilirubin the of its oxidation to biliverdin. bilirubin is soluble in water (1McDonagh A.F. The Porphyrins.in: Dolphin D. Academic Press, Inc., New York1979: 293-491Crossref Google Scholar) due to intramolecular hydrogen that the pigment its R. Proc. R. Sci. PubMed Scopus Google Scholar). hydrogen are bilirubin with cell or is in but are the pigment is bound to albumin or in D. J. PDF PubMed Scopus Google Scholar). radicals oxidized bilirubin, irrespective of whether or was used as the was a in the of biliverdin with in and yield in results that of intramolecular hydrogen biliverdin during peroxyl oxidation of bilirubin and that membrane-bound bilirubin is to biliverdin these We whether overexpression of BVR cells from we added bilirubin to from HeLa cells and with BVR overexpression and then these to peroxyl radicals or BVR overexpression BVR protein and activity In from cells with bilirubin, to peroxyl radicals in oxidation of the pigment in This was associated with of biliverdin, to of the BVR as a the redox one its overexpression to the of bilirubin by to this the of peroxyl of bilirubin biliverdin was by BVR overexpression with in H2O2 was used as the bilirubin oxidation was biliverdin and both were by BVR overexpression results that in HeLa cells, the of the redox is by the low yield of biliverdin during oxidation of bilirubin by peroxyl radicals or by the with BVR cells against oxidative one its overexpression to protect against cell of HeLa cells with H2O2 in a of as by of BVR failed to protect cells against H2O2-mediated death whether this of protection was due to the of bile pigments for the redox cycle to we cells with biliverdin or bilirubin by with pigment to H2O2 of cells with the pigments failed to H2O2-mediated of cell and that of cells with biliverdin or bilirubin did not cell not work by et al. M. Snyder Proc. Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar) showed that of BVR to cellular levels of We whether BVR overexpression cellular as by dichlorofluorescein of HeLa cells with H2O2 for an in of cellular levels of BVR overexpression did not H2O2 that BVR not provide cells with protection against H2O2-mediated We BVR in HeLa cells and the response of these cells to H2O2 with that of cells, with of cells with to the human BVR the of BVR protein and BVR activity by yet this did not cell death by as by the Similarly, cells with biliverdin or bilirubin did not H2O2-mediated in cell is that BVR of the used above to BVR activity in HeLa cells, since in mammalian cells heme by heme oxygenase is tightly with BVR activity Biochem. PubMed Scopus Google Scholar). In yeast cells not BVR activity and only a heme oxygenase, for by D. PubMed Scopus Google Scholar, J. PDF PubMed Scopus Google Scholar). we transformed a hmx1 mutant yeast with human (heme A and or with human BVR A and and then in response to and to the in mammalian cells, overexpression of human of the hmx1 mutant yeast and BVR were was in to that seen with overexpression in the of overexpression of BVR not protection against that by heme the evidence that BVR important in to its function in heme of cell and activity Antioxid. Redox Signal. PubMed Scopus Google Scholar). 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In this we that Sedlak et al. of BVR to lipid in cells a in cell in cells, of BVR cell death lipid and in M. Snyder Proc. Natl. Acad. Sci. U.S.A. PubMed Scopus Google We these as evidence for a of cellular BVR levels and cell In our that bilirubin oxidation not generally biliverdin and that BVR is not an important cellular the antioxidant redox cycle by Snyder and M. Snyder Proc. Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar). our not the that BVR an this are We for with the of bile pigments and McDonagh for of the