Neuroprotective Action of Bilirubin against Oxidative Stress in Primary Hippocampal Cultures SYLVAIN DORÉa AND SOLOMON H. SNYDERb Johns Hopkins University, School of Medicine, Department of Neuroscience, 725 North Wolfe Street, Baltimore, Maryland 21205, USA INTRODUCTION Bilirubin (BR) elicits substantial antioxidant effects and is probably one of the most abundant endogenous antioxidants in mammalian tissues.1–3 Very little is known about its role in the nervous system. Heme oxygenase, the enzyme responsible for the synthesis of BR, is highly expressed in the brain, being enriched in the hippocampus.4 We wondered if BR applied to primary hippocampal neurons would be protective against hydrogen peroxide-induced toxicity. MATERIALS AND METHODS We prepared cultures of hippocampal neuronal cells isolated from 17-day-old embryos of timed pregnant Sprague-Dawley rats. Unless stated otherwise, all compounds used for cell culture were from Gibco BRL (Gaithersburg, MD). Neurons were cultured in serum-free conditions with the B-27 supplement, as previously described.5 Experimental treatments were conducted in the N-2 supplement N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES)-buffered high glucose neurobasal medium. Following the different treatments, neurons were maintained for an additional period of 24 hr, and their survival was assessed by phase-contrast microscopy with Trypan Blue exclusion assay and quantified using MTT [(3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)] colorimetric assay. Survival of control vehicle treated neuronal cells not exposed to H2O2 was set at 100%, and treated groups were represented as percentage of control values. All experiments were conducted under a dim light to avoid heme pigment photodegradation. BR (1 mM, Sigma, St-Louis MO) was freshly dissolved in NaOH. Bovine and human serum albumin (Sigma; fraction V) were dissolved in 0.1 M phosphate-buffered saline, pH 7.4 mixed in a ratio of 1:5 as described.1 Addition of BR to albumin in this way results in binding of the pigment to its primary physiological binding site.6 All experiments were repeated with at least three separate batches of cultures, and the aPhone, 410/955-3083; fax, 410/614-6249. e-mail, sdore@welchlink.welch.jhu.edu bPhone, 410/955-3024; fax, 410/955-3623. e-mail, s.snyder@jhmi.edu 167 168 ANNALS NEW YORK ACADEMY OF SCIENCES FIGURE 1. Neuronal toxicity induced by hydrogen peroxyde (H2O2). Rat primary hippocampal neurons were exposed to H2O2 (75 µM) for different periods of time, then replaced with fresh culture medium, and neuron survival was estimated 24 hr after the beginning of the experiment. data are represented as the mean ± SEM with *p < 0.05, **p < 0.01 being considered significant. RESULTS FIGURE 1 depicts the time course for the influence of H2O2 on survival of primary hippocampal neurons estimated after 24 hr of the initial treatment. A significant decrease is observed very rapidly, with only 15 min exposure. This decrease becomes more prominent and reaches a plateau of toxicity at approximately 1 hr. It is known that the effect of H2O2 is very rapid, and its half-life in petri dishes is approximately 15 min. Because of the very short half-life of H2O2 and the sensitivity of neurons to medium changes, we left the H2O2 for the entire experiment. To ascertain whether BR is neuroprotective, we examined its effects upon neurons treated with H2O2 (FIG . 2). As a first step, we tested different concentrations of BR and showed that 25–50 nM were the optimal concentrations for neuroprotection against H2O2-induced toxicity (FIG . 2A). Knowing that BR has low water solubility, estimated as <100 nM at pH 7.4,6 we coupled it to serum albumin in order to increase it solubility. We first looked at the effect of BR coupled to bovine serum albumin (BSA) and found significant neuroprotection at 10- and 25-nM concentrations (FIG . 2B). There is a primary and a secondary binding site on albumin for BR. Since human serum albumin (HSA) has higher affinity for those binding sites, we subsequently used BR coupled to HSA and found that as little as 10 nM BR almost completely reverses the neurotoxic actions of H2O2 (FIG . 2C), while lesser protection occurs at 1 and 3 nM. The neuroprotective effect diminishes at higher concentrations of BR, presumably because higher levels of BR are themselves neurotoxic. This is suggested by the diminished neuronal survival of control cultures treated with 100 and DORÉ & SNYDER: BILIRUBIN 169 FIGURE 2. Protective effect of bilirubin, BR-BSA, and BR-HSA on H2O2-induced toxicity on neurons. Induction of toxicity by H2O2 (75 µM) started after the addition BR and its complexes. Neuron survival was estimated 24 hr after the beginning of the experiment. Increasing concentrations of free BR (A) or BR complexed with bovine serum albumin (BRBSA) (B) or human serum albumin (BR-HSA) (C) were added to neurons. Addition of equivalent amounts of albumin alone were without effect. Control experiments were done without H2O2. 170 ANNALS NEW YORK ACADEMY OF SCIENCES 250 nM BR-SA. No significant effects are observed with the equivalent concentrations of BSA or HSA alone. DISCUSSION Hydrogen peroxide (H2O2) is normally detoxified in the cell by catalase and glutathione peroxidase, whose levels do not change after brief application of H2O2. The highly reactive hydroxyl radical (in the presence of transition metal cations) can initiate lipid peroxidation and damage proteins and DNA. BR, which is toxic at high concentrations, has antioxidant properties at low concentrations.1,7 One of the most interesting findings of the present study is the very potent protective effect of BR on primary hippocampal neurons with complete neuroprotection evident at 10 nM. BR actions have been mostly characterized in the high micromolar range where toxic effects also occur. How can nM concentrations of BR protect against higher H2O2 concentrations? The most likely explanation is a cycle of oxidation-reduction between BR and biliverdin (BV), the major oxidation product of BR.8,9 In mediating its antioxidant actions, BR would be transformed to BV. Biliverdin reductase, present in large functional excess in all tissues, would immediately regenerate BR. BR is one of the most abundant endogenous antioxidants in mammalian tissues, accounting for the majority of the antioxidant activity of human serum.10 In an extensive series of antioxidants, BR displayed the most potent superoxide and peroxide radical scavenger activity.11 In the circulation, BR is largely complexed with albumin. We have observed more extensive neuroprotection with BR complexed with HSA compared to BSA, perhaps because human albumin has higher affinity for the primary and secondary binding sites for BR than its bovine homolog. In most cells, BR is stored in a complex with various isoforms of glutathione-S-transferase (GST).12 Thermodynamic parameters for BR dissociation from GST are similar to those for HSA.13 Binding of BR to these proteins keeps BR in solution and inhibits its efflux from the cell thereby increasing the net accumulation. GSTs play a role in cellular uptake and the intracellular transport of BR.14 Certain GST isoforms are selectively present in neurons.15 A GST isoenzyme-specific distribution was also found in cytoplasm, microsome, nuclei and nucleoli suggesting the possibility of scavenging free radicals in different cell compartments. BR is best known as a potentially toxic agent that accumulates in the serum of neonates to cause jaundice. In high concentrations, BR can deposit in selected brain regions to elicit the neurotoxicity associated with kernicterus.16 The “physiologic jaundice” of normal neonates with BR levels fairly close to toxic levels has been puzzling. Conceivably, physiologic jaundice has a protective effect. It could represent a transitional antioxidative mechanism in the neonatal circulation. Serum antioxidant activities are selectively associated with BR in neonatal Gunn rats17 and jaundiced newborn infants.2 In preterm infants, higher bilirubin levels are associated with a lower incidence of oxygen radical-mediated injury.18 BR administration protects against retinopathy in premature babies.19 Moreover, beneficial effects of breast feeding are often accompanied with high BR levels.20 BR may be particularly important as a cytoprotector for tissues with relatively weak endogenous antioxidant defenses such as the myocardium and the nervous system.21 Interestingly, a de- DORÉ & SNYDER: BILIRUBIN 171 creased risk for coronary artery disease is associated with mildly elevated serum BR, with a protective effect comparable to that of high-density lipoprotein (HDL)cholesterol.3 Our findings imply that BR affords physiologic neuroprotection. This conclusion is supported by our recent observations that neuronal damage following middle cerebral artery occlusion is substantially worsened in heme oxygenase 2 knockout mice, the rate limiting enzyme for the BR synthesis in the brain.22 REFERENCES 1. N EUZIL , J. & R. S TOCKER. 1994. Free and albumin-bound bilirubin are efficient coantioxidants for alpha-tocopherol, inhibiting plasma and low density lipoprotein lipid peroxidation. J. Biol. Chem. 269: 16712–16719. 2. B ÉLANGER , S., J.C. L AVOIE & P. CHESSEX . 1997. Influence of bilirubin on the antioxidant capacity of plasma in newborn infants. Biol. Neonate 71: 233–238. 3. H OPKINS , P.N., L.L. W U , S.C. H UNT , B.C. J AMES , G.M. V INCENT et al. 1996. Higher serum bilirubin is associated with decreased risk for early familial coronary artery disease. Arterioscler. Thromb. Biol. 16: 250–255. 4. V ERMA , A., D.J. H IRSCH , C.E. G LATT , G.V. RONNETT & S.H. SNYDER. 1993. Carbon monoxide: a putative neural messenger. 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