Limited role for the bilirubin-biliverdin redox amplification cycle in the cellular antioxidant protection by biliverdin reductase

Abstract

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, H(2)O(2) 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 H(2)O(2)-mediated cell death, just as BVR overexpression failed to enhance protection of these cells against H(2)O(2)-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 H(2)O(2) 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.

FIGURE 1.

Peroxyl radical- and H₂O₂-mediated oxidation of protein-bound bilirubin and bilirubin conjugate. Bilirubin prepared freshly in 50 mm NaOH was added to human serum albumin (500 μm, dissolved in PBS and containing 2 mm linoleic acid) (A and D) or equine GST (50 μm, dissolved in PBS) (B and E) such that the final concentration of the pigment was ∼20 μm. C and F, bilirubin ditaurate (∼100 μm, prepared freshly in PBS) was used in the absence of protein. A–C, oxidation was initiated by incubating the reaction mixture under air at 37 °C in the presence of the aqueous peroxyl radical generator AAPH (50 mm). D–F, oxidation was initiated by incubating the reaction mixture under air at 37 °C in the presence of 5 mm d-glucose, 1.5 units/ml glucose oxidase, and 50 μm DTPA. At the time points indicated, aliquots of the reaction mixture were removed and analyzed by HPLC for bilirubin/bilirubin ditaurate (circles) or biliverdin/biliverdin ditaurate (diamonds). The concentration of bile pigments was expressed as a percentage of the initial bilirubin/bilirubin ditaurate concentration. Data represent mean ± S.E. of three independent experiments.

FIGURE 2.

Solvent effects on peroxyl radical-mediated conversion of bilirubin to biliverdin. Bilirubin dissolved in chloroform (A) or DMSO (B) at 100 or 20 μm final concentration, respectively, was oxidized by incubation under air at 37 °C in the presence of AAPH (50 mm) or the lipid-soluble peroxyl radical generator AMVN (50 mm). At the indicated time points, aliquots of the reaction mixture were removed and analyzed by HPLC for bilirubin (circles) and biliverdin (diamonds). The concentration of bile pigments was expressed as a percentage of the initial bilirubin concentration. Data represent mean ± S.E. of three independent experiments.

FIGURE 3.

Overexpression of human BVR in HeLa cells does not affect the rate of bilirubin oxidation. A, HeLa cells were transfected for 18 h with empty vector (EV; pcDNA3) or vector encoding human BVR, and lysates were analyzed by Western blotting for α-tubulin (inset, 1) and BVR protein (inset, 2). BVR activity was determined in lysates by monitoring the formation of bilirubin from added biliverdin (50 μm) at 450 nm. B and C, bilirubin (20 μm final concentration) prepared fresh in 50 mm NaOH was added to cell lysates (200–300 μg of protein) prepared from HeLa cells transfected with empty vector (filled symbols) or BVR (empty symbols) in the presence of 1 mm NADPH, 1 mm glucose-6-phosphate, and 1 unit of glucose-6-phosphate dehydrogenase. Oxidation was then initiated by incubating the reaction mixture under air at 37 °C in the presence of 50 mm AAPH (B) or 5 mm d-glucose (C), 1.5 units/ml glucose oxidase, and 50 μm DTPA. At the indicated time points, aliquots of the reaction mixture were removed and analyzed by HPLC for bilirubin (circles) or biliverdin (diamonds). The concentration of bile pigments was expressed as a percentage of the initial bilirubin concentration. Data represent mean ± S.E. of three independent experiments. *, p < 0.05 compared with empty vector-transfected cells (Wilcoxon Mann-Whitney rank test).

FIGURE 4.

Overexpression of human BVR in HeLa cells does not affect cell death. Shown is the viability of HeLa cells in the presence of 2 mm H₂O₂ (A), 2 mm H₂O₂ plus 50 μm biliverdin (B), and 2 mm H₂O₂ plus 20 μm bilirubin (C). Cells transfected with empty vector (●) or BVR (○) for 18 h were pretreated with the respective pigment for 1 h in DMEM plus 1% FBS and then washed with PBS. H₂O₂ was then added as a bolus to the cells in DMEM plus 10% FBS. At the indicated time points, cell viability was determined by trypan blue exclusion and expressed as the percentage of untreated cells at the zero time point. Data represent mean ± S.E. of three independent experiments, each done in triplicate.

FIGURE 5.

Overexpression of human BVR in HeLa cells does not decrease H₂O₂-induced ROS levels. HeLa cells transfected with empty vector (●) or BVR (○) for 18 h were loaded with 100 μm dichlorofluorescein diacetate for 30 min in DMEM plus 1% FBS and then washed with Krebs buffer. H₂O₂ in Krebs was then added as a bolus to the cells, and fluorescence was measured every 5 min for 30 min (excitation, 485 nm; emission, 538 nm). ROS levels are expressed as change in dichlorofluorescein diacetate fluorescence over 30 min compared with the respective untreated empty vector and BVR-transfected cells. Data represent mean ± S.E. of three independent experiments.

FIGURE 6.

Knockdown of BVR does not alter the susceptibility of HeLa cells to H₂O₂-induced death. A, HeLa cells were transfected for 48 h with scrambled or BVR-targeted RNAi, and lysates were analyzed by Western blotting for α-tubulin (inset, 1) and BVR protein (inset, 2) as described under “Experimental Procedures.” In addition, BVR activity was determined in the cell lysates by spectroscopy, monitoring the formation of bilirubin from added biliverdin (5 μm) at 450 nm. B, cells transfected with scrambled (●) or BVR-targeted (○) RNAi for 48 h were washed with PBS. Different H₂O₂ concentrations were then added as a bolus to the cells in DMEM plus 10% FBS. After 8 h, cell viability was determined by the MTT assay and expressed as the percentage of untreated cells. C, cells transfected with BVR-targeted RNAi for 48 h were pretreated with biliverdin (BV; 50 μm) or bilirubin (BR; 20 μm) for 1 h in DMEM plus 1% FBS and then washed with PBS. H₂O₂ (2 mm) was then added to the cells in DMEM plus 10% FBS, and cell viability was determined by the MTT assay after 4 h and expressed as a percentage of untreated cells. Data represent mean ± S.E. of at least three independent experiments.

FIGURE 7.

Overexpression of human HO-1 but not human BVR rescues the sensitivity of hmx1 mutant yeast to oxidant challenge. Null hmx1 mutant strains were transformed with pESC-LEU-HO-1 and pESC-LEU-HO-1-BVRA. Expression of HO-1 and BVR was induced with the addition of galactose to the growth medium. Cells extracts were prepared as described under “Experimental Procedures” for cDNA synthesis and reverse transcription-PCR (A) and Western blotting (B). In A, the presence of both the HO-1 (row 1) and BVR (row 2) transcript was verified by agarose gel electrophoresis and by sequencing (note that the presence of a band (in row 2) in the hmx1 transformed with pESC-LEU-HO-1 was investigated by sequencing and was confirmed to be a nonspecific sequence (data not shown)). Data are representative of three independent experiments. B, Western blot showing expression of human HO-1 and BVR protein at 32 and 39 kDa, respectively, in yeast cells transformed with pESC-LEU-HO-1 and pESC-LEU-HO-1-BVRA and cultured in the presence of galactose. Null hmx1 mutant yeast cells were transformed with galactose-inducible multicopy plasmids containing human HO-1 (C) or human HO-1 and human BVR (D). Cells were grown to exponential phase in medium containing raffinose (●) or galactose (○) to respectively prevent or induce expression of the gene(s) contained in the multicopy plasmid and treated for 1 h with H₂O₂ at the concentration indicated. Survival was determined as described under “Experimental Procedures,” and results are expressed relative to non-treated control cultures. Data represent the mean ± S.E. of three independent experiments performed in triplicate.