Bilirubin Links Heme Metabolism to Neuroprotection by Scavenging Superoxide

Abstract

Bilirubin is one of the most frequently measured metabolites in medicine, yet its physiologic roles remain unclear. Bilirubin can act as an antioxidant in vitro, but whether its redox activity is physiologically relevant is unclear because many other antioxidants are far more abundant in vivo. Here, we report that depleting endogenous bilirubin renders mice hypersensitive to oxidative stress. We find that mice lacking bilirubin are particularly vulnerable to superoxide (O₂^⋅-) over other tested reactive oxidants and electrophiles. Whereas major antioxidants such as glutathione and cysteine exhibit little to no reactivity toward O₂^⋅-, bilirubin readily scavenges O₂^⋅-. We find that bilirubin's redox activity is particularly important in the brain, where it prevents excitotoxicity and neuronal death by scavenging O₂^⋅- during NMDA neurotransmission. Bilirubin's unique redox activity toward O₂^⋅- may underlie a prominent physiologic role despite being significantly less abundant than other endogenous and exogenous antioxidants.

Keywords: NMDA receptor; bilirubin; biliverdin; heme; metabolism; neuroprotection; oxidative stress; superoxide.

Figure 1.. BVR^−/− mice lack bilirubin and accumulate biliverdin.

a, Schematic of heme catabolism. b, Strategy for deleting exon 3 of mus musculus Blvra gene. c-d, PCR from DNA for exon 3 (c) and RNA for full-length Blvra (d) from WT and BVR^−/− mice. e, Photographs WT and BVR^−/− mouse gallbladders. f-h, HPLC chromatograms of (f) 10 M biliverdin, bilirubin, and hemin standards (λ=405 nm), (g) bile pooled from 6 WT and BVR^−/− mice (λ=405 nm), and (h) plasma from WT and BVR^−/− mice (λ=450 nm). i, Blvra RNA levels in different regions of the brain measured by quantitative PCR. Points represent individual mice. j, Bilirubin immunostaining in cerebellar sections from WT and BVR^−/− mice. Scale bar = 100 μm. k, Coomassie blue gel of purified UnaG. l, Normalized fluorescence intensities of 1 μM apoUnaG incubated with various heme metabolites. m-p, Live-cell images of neurons co-expressing UnaG (m) or IRFP (o) and mCherry. Scale bar = 50 μm. Mean UnaG fluorescence (n) and IRFP fluorescence (p) normalized to mCherry fluorescence as a transfection control. Points represent individual neurons. n = 9–12 neurons per genotype per co-transfection. (i, l, n, p) Mean ± SEM depicted. ** = P < 0.01 by two-way ANOVA followed by a post hoc Tukey test (i) or two-tailed Student’s t-test (n, p).

Figure 2.. BVR^−/− cells are hypersensitive to exogenous and endogenous O2•− over other ROS and electrophiles.

a, WT and BVR^−/− MEF viability after 8 h exposure to varying concentrations of oxidants/electrophiles. n = 2–4 in triplicate. b, Live-cell images of WT and BVR^−/− MEFs loaded with dihydroethidium (DHE) after exposure to 25 μM menadione for 1 h. Scale bar = 100 μm. c, Representative HPLC chromatograms of DHE alone, DHE + KO₂, authentic ethidium (E⁺), and DHE + KO₂ spiked with authentic E⁺. d-f, Representative normalized HPLC chromatograms (d) and quantification of E⁺ and 2-OH-E⁺ (e-f) of organic extracts from WT and BVR^−/− MEFs loaded with DHE at baseline or after 1 h treatment with 25 μM menadione with or without bilirubin pre-treatment. g-h, Live-cell images (g) and quantification (h) of MitoSOX Red fluorescence WT and BVR^−/− MEFs after 4 h exposure to 25 μM rotenone with or without bilirubin pre-treatment. Scale bar = 25 μm. (a, e, f, h) Mean ± SEM depicted. * = P < 0.05 and ** = P < 0.01 by two-way ANOVA followed by a post hoc Tukey test (e, f, h).

Figure 3.. Bilirubin selectively scavenges O2•− at physiologically-relevant rates.

a, EPR spectrum KO₂ without (blue) and with (green) equimolar bilirubin in 0.2 M 18-crown-6 in DMSO at 100 K. b, EPR spectra of DMPO-spin adducts of radicals from solutions of KO₂ with or without 1 equivalent of bilirubin, bilirubin ditaurate, or biliverdin or 100 equivalents of glutathione at 298 K. c, Change in pmoles of bilirubin per second with xanthine/xanthine oxidase (XO) with or without excess 100 nM MnSOD or 1 μM catalase. d, Normalized change in UnaG fluorescence from 1 μM bilirubin and 1 μM apoUnaG with either 1 mM H₂O₂ (red), 100 μM pyrogallol (green), or 100 μM pyrogallol and 100 nM MnSOD (orange). e, Representative HPLC chromatograms of bilirubin or bilirubin + KO₂ with the detector set to (left) 440 nm or (right) 320 nm. f, UV-visible difference spectrum of bilirubin subtracted from bilirubin + KO₂. g, Average fold change in chemiluminescence upon mixing 5 μM bilirubin with varying equivalents of KO₂. h, Average competitive kinetic inhibition curves for bilirubin, glutathione, cysteine, and ascorbic acid against p-nitro blue tetrazolium (NBT) and XO-derived O₂^•−. i, LUMO densities (|LUMO|) and change in Gibbs free energy (ΔG) for reducing indicated metabolites as calculated by the B3LYP functional and 6–31+G* diffuse basis set. Blue illustrates greater LUMO density, whereas red indicates lower density. (c, g, h) Mean ± SEM depicted. ** = P < 0.01 and ns = P > 0.05 by two-way ANOVA followed by a post hoc Tukey test (c).

Figure 4.. NMDA receptors signal via O2•−, and bilirubin prevents NMDA excitotoxicity by scavenging O2•−.

a-c, WT and BVR^−/− male mouse open field locomotor activity over time at (a) baseline or after treatment with either (b) NMDA or (c) MK-801. n = 5 WT and 6 BVR^−/− mice (baseline and NMDA) and 9 WT and 5 BVR^−/− mice (MK-801). d-f, Normalized DHE fluorescence (d) or live images (e) and traces (f) of UnaG fluorescence from primary cortical neurons over time. Neurons were treated (arrow) with 100 μM NMDA + 10 μM glycine, 50 μM (S)-AMPA, or 50 μM kainate. Scale bar = 25 μm. n = 6 neurons in (d) and n = 5 (NMDA), 3 (S-AMPA), and 3 (kainate) neurons in (f). g-i, Representative normalized HPLC chromatograms (g) and quantification of E⁺ and 2-OH-E⁺ (h-i) of organic extracts from WT and BVR^−/− ex vivo brain slices loaded with DHE at baseline or after treatment with 100 μM NMDA + 10 μM glycine for 30 min. Points represent normalized average of 2–3 slices from individual mice. j, Average thiobarbituric acid-reactive substances (TBARS) in WT and BVR^−/− neurons 24 h after treatment with vehicle or 100 μM NMDA + 10 μM glycine for 20 min with or without bilirubin pre-treatment. k, Schematic illustrating NMDA microinjections. l-m, Nissl stained coronal sections (l) and lesion volumes (m) from WT and BVR^−/− mice 48 h after injection of vehicle or 20 nmoles NMDA. Scale bar = 2 mm. Points represent individual mice. (a-d, f, h-j, m) Mean ± SEM depicted. * = P < 0.05, ** = P < 0.01, and ns = P > 0.05 by one-way (d, f) or two-way ANOVA (h-j) followed by a post hoc Tukey test or two-tailed Student’s t-test (a-c, m).