www.elsevier.com/locate/ynbdi Neurobiology of Disease 29 (2008) 30 – 40 Unconjugated bilirubin differentially affects the redox status of neuronal and astroglial cells Maria A. Brito,a,⁎ Alexandra I. Rosa,a Ana S. Falcão,a Adelaide Fernandes,a Rui F.M. Silva,a D. Allan Butterfield,b and Dora Britesa a Centro de Patogénese Molecular - Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculdade de Farmácia da Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal b Department of Chemistry, Center of Membrane Sciences and Sanders-Brown Center on Aging, University of Kentucky, Lexington, KY 40506, USA Received 24 April 2007; revised 11 July 2007; accepted 23 July 2007 Available online 1 August 2007 We investigated whether nerve cell damage by unconjugated bilirubin (UCB) is mediated by oxidative stress and ascertained the neuronal and astroglial susceptibility to injury. Several oxidative stress biomarkers and cell death were determined following incubation of neurons and astrocytes isolated from rat cortical cerebrum with UCB (0.01–1.0 μM). We show that UCB induces a dose-dependent increase in neuronal death in parallel with the oxidation of cell components and a decrease in the intracellular glutathione content. Comparison of the results obtained in both cell types demonstrates that neurons are more vulnerable than astrocytes to oxidative injury by UCB, for which accounts the lower glutathione stores in neuronal cells. Moreover, neuronal oxidative injury is prevented by supplementation with Nacetylcysteine, a glutathione precursor, whereas astroglial sensitivity to UCB is enhanced by inhibition of glutathione synthesis, using buthionine sulfoximine. Collectively, we demonstrate that oxidative stress is involved in UCB neurotoxicity and depict a new therapeutic approach for UCB-induced oxidative damage. © 2007 Elsevier Inc. All rights reserved. Keywords: Astrocytes; Cell-type vulnerability; Cell death; Glutathione; Lipid peroxidation; Neurons; Oxidative stress; Protein oxidation; Reactive oxygen species; Unconjugated bilirubin Introduction Unconjugated bilirubin (UCB) is the principal end product of heme catabolism. Increased levels of UCB are responsible for the clinical manifestation of jaundice, a common condition in the neonatal period usually referred as physiologic jaundice of the newborn. UCB has been regarded as a natural antioxidant since the pioneer studies of Stocker et al. (1987). Because of the antioxidant properties of low UCB concentrations, it is nowadays believed that ⁎ Corresponding author. Fax: +351 217946491. E-mail address: abrito@ff.ul.pt (M.A. Brito). Available online on ScienceDirect (www.sciencedirect.com). 0969-9961/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.nbd.2007.07.023 physiologic jaundice may have inherent benefits (Sedlak and Snyder, 2004). However, in some newborn infants, plasma UCB levels can increase dramatically owing to impaired postnatal maturation of hepatic transport or conjugation of UCB, and/or enhanced entero-hepatic circulation of UCB, or augmented hemolysis (Brito et al., 2006b). If untreated, and depending on the level of severity, hyperbilirubinemia can lead to minor brain deficits, acute bilirubin encephalopathy, kernicterus, or even death (American Academy of Pediatrics, 2004; Harris et al., 2001; Kaplan and Hammerman, 2005; Soorani-Lunsing et al., 2001). Different brain cells, as neurons and astrocytes, have their own functions and specialized machinery, and tend to have unique responses towards a stimulus (Brito et al., in press). Comparative data on the cell susceptibility to UCB showed that neurons are more sensitive than astrocytes to cytoskeleton disruption and to necrotic or apoptotic cell death, while astrocytes are more disturbed than neurons regarding both MTT [(3-(4,5-dymethylthiazol, 2-yl)2,5-diphenyltetrazolium bromide] metabolism and glutamate uptake (Silva et al., 2002). In addition, astrocytes are more competent in releasing glutamate and display a more marked inflammatory response than neurons, as indicated by the higher secretion of pro-inflammatory cytokines and the highest activation of the nuclear factor-κB (Falcão et al., 2006). Although the primary concern with respect to hyperbilirubinemia is the potential for neurotoxic effects, general cellular injury also occurs. Our previous studies conducted in erythrocytes, isolated brain mitochondria and synaptosomal membrane systems indicated that UCB-induced cytotoxicity is mediated, at least in part, by perturbation of cell membranes (Brito et al., 2000, 2001, 2002, 2004; Rodrigues et al., 2002a). In fact, morphological changes, alterations in membrane composition and assembly, with the accompanying calcium intrusion and cytochrome c release, are among the UCB effects. An increase in protein mobility, and an elevation of lipid polarity and fluidity, as well as a disorder of the redox status also result from the physical interaction of UCB with membranes. In addition, reactive oxygen species (ROS) production, protein oxidation and lipid peroxidation, together with a disruption of M.A. Brito et al. / Neurobiology of Disease 29 (2008) 30–40 glutathione homeostasis occur by UCB exposure. When taken together, these observations point to oxidative injury as a relevant component of UCB toxicity in several experimental systems. However, the contribution of oxidative stress in the mechanisms of neurotoxicity by UCB, namely the spectrum of the oxidative effects in different nerve cell types, remains to be characterized. Oxidative stress occurs when the physiological balance between radical-generating and radical-scavenging is disrupted in favour of the former (Sies, 1997). In this condition, oxidized nucleic acids, proteins and lipids accumulate as a result of the ROS attack to cell components. Protein carbonyls and 4-hydroxy-2nonenal (HNE) are widely used as reliable markers of protein oxidation and lipid peroxidation, respectively (Butterfield et al., 2002; Butterfield and Stadtman, 1997; Stadtman, 1992). The antioxidant capacity of tissues can also be assessed by quantification of oxidative stress markers, as glutathione, which is one of the major cell antioxidants and reflects the chain-breaking thiol antioxidant capacity (Dringen, 2000). Glutathione is a tripeptide, γ-L-glutamyl-L-cysteinylglycine, synthesized by the action of two enzymes, γ-glutamylcysteine (γGluCys) synthetase and glutathione synthetase, at expenses of ATP. Although this two-steps synthesis takes place in both neurons and astrocytes, neurons cannot use cysteine as a cysteine donor, relying on astrocytes as a cysteine donor in vivo. Moreover, intracellular glutathione stores are greater in astroglial cells than in neuronal ones, as well as in neurons cocultures with astrocytes, as compared to isolate neuron cultures. This fact, together with the greater antioxidant enzymatic machinery of astrocytes, renders astroglial cells more resistant than neuronal ones to free radicals and oxidant insults and, thus, to stressful situations (Brito et al., in press). In this study, we evaluate several oxidative stress biomarkers, as well as cell death, following incubation of primary culture of rat neurons and astrocytes obtained from cerebrum cortex with free UCB (0.01–1.0 μM). We show that the UCB-induced loss of cell viability is associated with a disruption of the redox status, which involves oxidative damage of cell components, as well as impairment of the cellular antioxidant defense system provided by glutathione. Comparison of the results obtained in neurons and astrocytes demonstrate that neurons are more vulnerable than astrocytes to oxidative injury by UCB, to which contributes the lower intracellular levels of glutathione in neuronal cells. Finally, by demonstrating that oxidative injury to neurons is prevented by supplementation with a glutathione precursor, whereas astroglial sensitivity to the pro-oxidant effects of UCB is enhanced by inhibition of the thiol synthesis, the present results point to the restoration of glutathione homeostasis as a new potential therapeutical approach for UCB-induced oxidative damage. Materials and methods Chemicals Dulbecco’s modified Eagle’s medium (DMEM) and fetal calf serum (FCS) were purchased from Biochrom AG (Berlin, Germany). Neurobasal medium, B-27 Supplement (50×), Hanks’ balanced salt solution (HBSS-1), Hanks’ balanced salt solution without Ca2+ and Mg2+ (HBSS-2), gentamicin (50 mg/ml) and trypsin (0.025%) were acquired from Invitrogen (Carlsbad, CA, USA). Cell culture clusters were from Orange Scientific (Brainel’Alleud, Belgium). Slot blotting materials including apparatus, nitrocellulose membranes (0.45-μm pore size), and transfer filter 31 papers, as well as the protein assay kit were from Bio-Rad (Hercules, CA, USA). The OxyBlot kit used for protein carbonyl determination was from Chemicon (Temecula, CA, USA) and the HNE antibody used for estimation of lipid peroxidation was from Alpha Diagnostic International (San Antonio, USA). Antibiotic antimycotic solution (20×), bilirubin, goat anti-rabbit alkaline phosphatase-conjugate secondary antibody, Sigma Fast Tablets (BCIP/NBT substrate), reduced and oxidized glutathione (GSH and GSSG, respectively), dihydrorhodamine 123 (DHR), N-acetylcysteine (NAC) and buthionine sulfoximine (BSO) were acquired from Sigma Chemical Co. (St. Louis, MO, USA). Stock solutions of DHR 123 (577 μM in DMSO), NAC (100 mM in PBS) and BSO (10 mM in PBS) were prepared and stored at −20 °C. Lactate dehydrogenase (LDH)-cytotoxicity detection kit was obtained from Roche Molecular Biochemicals (Mannheim, Germany). All other chemicals were of analytical grade and were purchased from Merck (Darmstadt, Germany). Primary neuronal and astroglial cell cultures Animal care followed the recommendations of European Convention for the Protection of Vertebrate Animals Used for Experimental and other Scientific Purposes (Council Directive 86/ 609/EEC) and National Law 1005/92 (rules for protection of experimental animals). All animal procedures were approved by the Institutional Animal Care and Use Committee. Every effort was made to minimize the number of animals used and their suffering. Both neuronal and astroglial cell cultures were prepared from the cerebrum cortical region of Wistar rats. Primary cultures of neurons were prepared from fetuses of 17- to 18-day pregnant rats as previously described (Brewer et al., 1993) with minor modifications (Silva et al., 2002). In short, pregnant rats were anesthetized and decapitated. The fetuses were collected in HBSS-1 and rapidly decapitated. After removal of meninges and white matter, the brain cortex was collected in HBSS-2 and mechanically fragmented. The cortex fragments were transferred to a 0.025% trypsin in HBSS-2 solution and incubated for 15 min at 37 °C. Following trypsinization, cells were washed twice in HBSS-2 containing 10% FCS, and resuspended in Neurobasal medium supplemented with 0.5 mM L-glutamine, 25 μM L-glutamic acid, 2% B-27 Supplement, and 0.12 mg/ml gentamicin. Aliquots of 1 × 105 cells/cm2 were plated on tissue culture plates precoated with poly-D-lysine and maintained at 37 °C in a humidified atmosphere of 5% CO2. Every 3 days, 0.5 ml of old medium was removed by aspiration and replaced by the same volume of fresh medium without L-glutamic acid. Cells were morphologically characterized by phase contrast microscopy and used after 8 days in culture. Primary cultures of astrocytes were prepared from 2-day-old rats as previously described (Blondeau et al., 1993) with minor modifications (Silva et al., 1999). In brief, rats were decapitated and the brains collected in DMEM containing 11 mM sodium bicarbonate, 38.9 mM glucose and 1% antibiotic antimycotic solution. Following removal of meninges, blood vessels and white matter, the cortical fraction was homogenized by mechanical fragmentation and the cells collected by centrifugation at 700×g for 10 min. Finally, cells were resuspended in culture medium supplemented with 10% FCS, plated (2.0 × 105 cells/cm2) on tissue culture plates and cultured for 10 days, at 37 °C in a humidified atmosphere of 5% CO2. The use of 17–18-day fetal brains raises the efficiency of neuronal cultures due to the fewer cell connections that exists in 32 M.A. Brito et al. / Neurobiology of Disease 29 (2008) 30–40 embryonic tissue as compared to older tissue, which would be damaged if older brains were used (Brewer et al., 1993). Furthermore, contamination by glial cells, already observed in cultures from brain cortex of 1-day post-natal rats, is greatly avoided by the use of fetal brains. In contrast, the preparation of astrocyte cultures from brain cortex of 2-day-old rats, together with the use of uncoated plates, minimizes the contamination by cortical neurons (Silva et al., 2006). Comparable differentiation and confluence of glial and neuronal cell cultures were achieved at 10 and 8 days in vitro, respectively, as was observed by morphological analysis and immunolabeling of specific neuronal and astrocytic markers (Brewer, 1997; McCarthy and de Vellis, 1980). Moreover, the use of this time in culture will match up to a full-term neonate, at least for glial cells (Silva et al., 2006). To exclude the interference of contaminant astrocytes and microglia in our primary cultures of neurons and astrocytes, respectively, that could interfere with the specific cell-type response to UCB, we evaluated the purity of our nerve-cell cultures. The immunochemical staining using primary antibodies raised against glial fibrillary acidic protein of astrocytes, against neurofilaments of neurons and against the CR3 complement receptor of microglia, OX-42 (Silva et al., 2006) assured that highly pure cultures of neurons as well as of astrocytes were obtained. In fact, in neuronal primary cultures the contaminant glial cells was ∼ 2%, whereas in astrocyte cultures the contaminant microglial cells were ∼2.5%. Cell treatment Cells were exposed to UCB at concentrations of 0.01, 0.1 and 1.0 μM, previously purified according to McDonagh (1979). Appropriate aliquots of a 10 mM stock solution, prepared in 0.1 N NaOH immediately prior to use, were added to neurons and astrocytes, and the restoration of the pH value to 7.4 was achieved by addition of 0.1 N HCl. UCB-treated cells, as well as untreated cells (controls), were incubated for 4 h at 37 °C. In another set of experiments, neurons were treated with NAC (25 μM and 100 μM) for 1 h and astrocytes were exposed to BSO (25 μM and 100 μM) for 18 h, prior to the 4 h incubation with UCB (0.1 μM), at 37 °C. NAC is an acetylated analog of cysteine that easily crosses the cell membrane and is rapidly deacetylated inside the cell and utilized for GSH synthesis (Zafarullah et al., 2003), whereas BSO is an irreversible inhibitor of GSH synthesis through blockage of the rate-limiting enzyme, γ-glutamylcysteine synthetase (Dringen, 2000). The concentrations of NAC and BSO, as well as the preincubation periods, were based on previous reports (James et al., 2005; Vexler et al., 2003). All the experiments were performed under light protection (tin foil wrapping of the vials and dim light) to avoid UCB photodegradation. At the end of the incubation period, the cellfree medium was collected for determination of LDH activity, while attached cells were used for evaluation of ROS production and determination of protein oxidation, lipid peroxidation and intracellular glutathione levels. Cell death Release of LDH by cells with a disrupted membrane is considered an indicator of cell death by necrosis/oncosis. In this study, we measured the activity of LDH released by nonviable cells as a tool to characterize UCB cytotoxicity in the present experimental conditions and to establish its correlation with the eventual oxidative effects of UCB. LDH was determined in the incubation medium using the Cytotoxicity Detection kit, LDH, as previously described (Silva et al., 2002). The reaction was performed in a 96-well microplate and the absorbance measured at 490 nm, using a reference filter of 620 nm. The results were expressed as percent of the maximum amount of releasable LDH, obtained by lysing non-incubated cells with 2% Triton® X-100 in Neurobasal or DMEM medium (for neurons and astrocytes, respectively) for 30 min. Assessment of ROS formation The nonfluorescent DHR easily crosses cell membranes due to its lipophilicity and is converted by ROS to rhodamine 123, a fluorescent compound that accumulates in mitochondria and is considered as a sensitive indicator of ROS production in cell systems (Gomes et al., 2005; Royall and Ischiropoulos, 1993). To evaluate the production of ROS in neuronal and astroglial cultures, cells were seeded on glass coverslips placed in the 12-well culture plates. Cells were loaded, under light protection, with 3 μM DHR in DMSO (0.5% final concentration) for 30 min at 37 °C, prior to incubation with UCB or no addition (control). At the end of the incubation period, cells were fixed with freshly prepared 4% paraformaldehyde in phosphate-buffered saline (PBS), washed with PBS, and mounted using DPX. Cellular fluorescence was observed using a fluorescence microscope (Axioskop®, ZEISS, Germany) and the intensity of the fluorescence emission was quantified in at least five microscopic fields (×400) per sample with an image analyzer software (ImageJ 1.29×, National Institutes of Heath, USA). Since UCB was referred as an autofluorescent molecule (Özkan et al., 1995), a set of experiments was performed in parallel, with no addition of DHR 123. The fact that no variations in the fluorescence intensity were noticed in these control experiments guarantees that the rise in the fluorescence intensity observed in the UCB-treated samples was due to ROS formation and not to UCB interference. Measurement of protein carbonyls Formation of protein carbonyls is an indication of oxidative stress and a key marker of protein oxidation (Butterfield and Stadtman, 1997; Stadtman, 1992). The method of detection used in the present study was the slot blot analysis of the 2,4dinitrophenylhydrazone (DNP-hydrazone) adduct of the carbonyls formed by reaction with 2,4-dinitrophenylhydrazine (DNPH). The formation of protein carbonyls was measured using the OxyBlot kit, according to the manufacturer’s instructions, with some modifications. Cells were detached from the 12-well culture plate by scraping with 50 μl PBS and the suspension was sonicated. A 5 μl aliquot was mixed with an equal volume of 12% SDS, and derivatized with 10 μl of DNPH solution. After 20 min, the reaction was stopped by neutralization with 7.5 μl of a 2 M Tris in 30% glycerol solution. A 250 ng protein aliquot of the sample solution was subjected to slot blot analysis and standard immunochemical techniques were performed, as previously described (Brito et al., 2004). A rabbit-anti DNP-hydrazone protein adduct polyclonal primary antibody (1:133) and a goat anti-rabbit IgG alkaline phosphatase secondary antibody (1:8000) were used. The protein stain was developed using Sigma Fast Tablets, a colorimetric substrate for alkaline phosphatase, and stain intensity was quantified with ImageJ software, after scanning into Adobe Photoshop (Adobe Systems Software, Uxbridge, UK). M.A. Brito et al. / Neurobiology of Disease 29 (2008) 30–40 Evaluation of lipid peroxidation One valuable tool to assess lipid peroxidation is the estimation of HNE, one of the major reactive products and a specific marker of lipid peroxidation (Butterfield et al., 2002). Thus, levels of protein-bound HNE were determined by slot blot analysis, using 5 μl aliquots of the cell suspensions in PBS, obtained as described above for protein oxidation. HNE–protein adducts were detected on the nitrocellulose membrane using a rabbit anti-HNE primary antibody (1:5000) and a goat anti-rabbit alkaline phosphataseconjugate secondary antibody (1:8000), as previously described (Brito et al., 2004; Lauderback et al., 2002). Sigma Fast Tablets were used as the colorimetric substrate for alkaline phosphatase and the blots were quantified with ImageJ, as referred for protein oxidation. Glutathione determinations The tripeptide glutathione is the most abundant thiol present in mammalian cells, playing an important part in the cellular detoxification of ROS (Dringen, 2000). Total glutathione (GSt) was determined by an enzymatic recycling procedure: the sulphydryl group of the molecule reacts with 5,5′-dithiobis-2nitrobenzoic acid (DTNB; Ellman’s reagent) producing a yellow colored 5-thio-2-nitrobenzoic acid (TNB), and the disulfide is reduced by NADPH in the presence of glutathione reductase (Griffith, 1980; Tietze, 1969). Following addition of 600 μl icecold 5% perchloric acid (6-well culture plates), cells were detached by scraping and transferred to eppendorf tubes. The suspension was sonicated and then neutralized with a 0.76 M KHCO3 solution. GSH levels were calculated from those of GSt and GSSG [GSt = GSH + 2GSSG]. GSSG was determined by derivatization of GSH by reaction with 2-vinylpyridine, for 1 h, at 4 °C, under shaking, prior to neutralization. After removal of the formed potassium perchlorate by centrifugation, supernatants were used for quantification of total and oxidized glutathione. Supernatant aliquots (300 μl) were assayed in 100 mM sodium phosphate buffer, containing 0.62 mM EDTA, 1.7 mM NADPH and 20.2 mM DTNB (total reaction volume of 765 μl). The rate of TNB formation was monitored following addition of 1.2 U of glutathione reductase, in a thermostated cuvette (30 °C), at 415 nm, for 1 min, in a Unicam UV2 spectrophotometer (Unicam Limited, UV2, Cambridge, UK). Glutathione concentrations were calculated using appropriate standards, treated as samples. 33 plasmatic enzyme, indicates the loss of cell viability and, thus, necrosis after exposure to UCB (Fig. 1). This effect was observed in both types of cells, even though it was considerably more marked in neurons than in astrocytes. In fact, exposure of neurons to a concentration of UCB of 1 μM resulted in a 4.5% increase of LDH release (P b 0.01 vs. control) while in astrocytes the obtained value for the same UCB concentration was only 2.4% (P b 0.05 vs. control). No significant difference was, however, found between control values for the different cell types (P N 0.1). Comparison of the extent of cell death between the two cell types after exposure to the highest UCB concentration revealed that neuronal cells are 1.9fold more susceptible to UCB-induced loss of cell viability than astrocytes (P b 0.05). The present findings are in line with our previous results, obtained in a different experimental model where cells were exposed to UCB in the presence of human serum albumin, that have demonstrated a greater loss of cell viability in neurons than in astrocytes after exposure to UCB (Silva et al., 2002). UCB triggers ROS production, which is more marked in neurons than in astrocytes We proceeded to determine if UCB induces ROS formation in neurons and astrocytes. Changes in mitochondrial ROS levels in both types of cells were assessed, after treatment with UCB for 4 h, by evaluation of fluorescence intensity of rhodamine 123 (Fig. 2A). The elevation of fluorescence emission observed indicates that UCB triggers ROS formation in a concentrationdependent manner, an effect that was markedly higher in neurons than in astrocytes. As shown in Fig. 2B, exposure of neurons to 0.1 μM UCB resulted in a 77% increase of fluorescence intensity (P b 0.05 vs. control), which further augmented to 109% by 1 μM UCB (P b 0.01 vs. control). Interestingly, an increase (though not statistically significant) in the fluorescence emission of rhodamine 123 was already observed after neuronal exposure to 0.01 μM UCB, a concentration referred to protect neurons from injury induced by H2O2 (Doré et al., 1999). In accord with the less noticeable production of ROS in astrocytes, Statistics All data are expressed as mean ± S.E.M. from at least 3 separate experiments. Differences between groups were compared using the two-tailed Student’s t test, performed on the basis of equal or unequal variance, as appropriate. Statistical significance was considered when P values were lower than 0.05. Results UCB induces cell death, which is greater in neurons than in astrocytes In initial studies, we determined whether UCB induces necrotic-like cell death in neurons and astrocytes in the tested experimental conditions. The elevation of released LDH, a cyto- Fig. 1. Cell death increases with unconjugated bilirubin (UCB) levels and is greater in neurons than in astrocytes. Both cell types were incubated with growing concentrations of UCB, or no addition (0 μM; control), for 4 h at 37 °C. Cell death was evaluated by quantification of the lactate dehydrogenase (LDH) released by nonviable cells and results were expressed as percentage of total LDH release. Each bar represents the mean ± S.E.M. of at least three independent experiments. ⁎P b 0.05, ⁎⁎P b 0.01 vs. control; † P b 0.05 vs. neurons. 34 M.A. Brito et al. / Neurobiology of Disease 29 (2008) 30–40 Fig. 2. Production of reactive oxygen species (ROS) increases with unconjugated bilirubin (UCB) levels and is more marked in neurons than in astrocytes. Both cell types were incubated with growing concentrations of UCB, or no addition (0 μM; control), for 4 h at 37 °C. Cells were pre-loaded with dihydrorhodamine 123 and ROS were assessed by detection of rhodamine 123 by fluorescence microscopy. (A) Representative photographs of one experiment are shown. Original magnification: 516×. (B) The fluorescence intensity was quantified by scanning densitometry and results were expressed as arbitrary units (A.U.). Each bar represents the mean ± S.E.M. of at least three independent experiments. ⁎P b 0.05, ⁎⁎P b 0.01 vs. control; †P b 0.05, ††P b 0.01 vs. neurons. a significant increase of fluorescence intensity was only achieved by incubation of glial cells with 1 μM UCB (27%, P b 0.05 vs. control). The comparison of UCB-induced ROS production between the two cell types revealed a higher propensity of neurons to the formation of oxidant species, which was 6.9-fold higher in neurons than in astrocytes (P b 0.01) after incubation with the highest UCB concentration. Interestingly, the more marked formation of oxidant species in UCB-treated neuronal cells is in line with a much higher production of ROS in neurons than in astrocytes by exposure to the redox-cycling compound paraquat (Schmuck et al., 2002). The present results indicate that exposure of nerve cells to UCB leads to ROS production, which may be involved in the induced loss of cell viability. This assumption is supported by the correlation coefficients obtained between the LDH release and the ROS produced in neurons (r = 0.953, P b 0.05) and astrocytes (r = 0.986, P b 0.05). UCB promotes protein oxidation in neurons, but not in astrocytes To determine whether exposure to UCB alters the levels of membrane protein oxidation in neurons and astrocytes, cells were incubated for 4 h and the levels of carbonyls were assessed as a marker of protein oxidation. As shown in Fig. 3, in the absence of UCB cultured neurons and astrocytes displayed similar levels of protein carbonyls. Exposure of neurons to UCB led to protein oxidation, as revealed by the 18% (P b 0.05 vs. control) and 31% (P b 0.01 vs. control) increase in protein carbonyls induced by treatment with 0.1 and 1 μM UCB, respectively. Curiously, treatment of neurons with UCB, at a concentration as low as 0.01 μM, already induced a slight elevation in protein carbonyls (12%, not statistically different from control). Contrasting with the oxidative injury to neuronal proteins, treatment with UCB in the same experimental conditions did not lead to significant changes in protein oxidation in astroglial cells. The oxidative disruption of M.A. Brito et al. / Neurobiology of Disease 29 (2008) 30–40 35 Fig. 3. Protein oxidation increases with unconjugated bilirubin (UCB) levels in neurons, but not in astrocytes. Both cell types were incubated with growing concentrations of UCB, or no addition (0 μM; control), for 4 h at 37 °C. Protein oxidation was evaluated by measuring protein carbonyls by slot blot analysis using rabbit anti-2,4-dinitrophenylhydrazine primary and goat anti-IgG secondary antibodies. The line staining was quantified by scanning densitometry and the results were expressed as arbitrary units (A.U.). Each bar represents the mean ± S.E.M. of at least three independent experiments. ⁎P b 0.05, ⁎⁎P b 0.01 vs. control; †P b 0.05 vs. neurons. Fig. 4. Lipid peroxidation increases with unconjugated bilirubin (UCB) levels in neurons, but not in astrocytes. Both cell types were incubated with growing concentrations of UCB, or no addition (0 μM; control), for 4 h at 37 °C. Lipid peroxidation was evaluated by measuring 4-hydroxy-2-nonenal (HNE) by slot blot analysis using rabbit anti-HNE primary and goat anti-IgG secondary antibodies. The line staining was quantified by scanning densitometry and the results were expressed as arbitrary units (A.U.). Each bar represents the mean ± S.E.M. of at least three independent experiments. ⁎P b 0.05 vs. control, †P b 0.05 vs. neurons. proteins observed in neurons appears to be implicated in the neuronal cell viability impairment induced by UCB, as indicated by the correlation coefficient obtained between the levels of protein carbonyls and those of released LDH (r = 0.982, P b 0.05). values decreased from 13.7 ± 0.9 in controls to 10.9 ± 1.0 nmol/mg protein (P b 0.05) in neurons treated with 1 μM UCB and from 26.1 ± 1.0 to 16.7 ± 2.9 (P b 0.05) in equivalent experiments performed in astrocytes. In agreement with the literature (Almeida et al., 2002; Bolaños et al., 1995), control values of total glutathione were lower in neurons than in astrocytes (∼ 50%, P b 0.01), a difference that can account, at least in part, for the higher vulnerability of neurons towards UCB-induced oxidative injury and even cell death. Interestingly, whereas 0.01 and 0.1 μM UCB induced a ∼ 10 and ∼ 20% decline in GSt levels in either neurons or astrocytes, the highest UCB concentration led to a more marked drop in GSt in astrocytes (∼ 37%) than in neurons (∼ 21%). Accordingly, in this latest condition, the difference in the thiol antioxidant content between neurons and astrocytes was attenuated, with neuronal values approaching those found in astrocytes (∼ 70%, P b 0.05). It is meaningful that the pronounced decline in astroglial GSt levels was accompanied by a significant increase in UCB induces lipid peroxidation in neurons, but not in astrocytes To further investigate the role of UCB on the redox status of nervous cells, HNE, a lipid peroxidation marker (Butterfield et al., 2002), was evaluated in neurons and astrocytes after exposure of these cells to UCB. As indicated in Fig. 4, protein-bound HNE levels in untreated neurons and astrocytes were similar. Interaction of UCB with neuronal membranes caused a dose-dependent increase in HNE–protein adducts, which was not observed in astrocytes. In fact, treatment of neurons with 0.1 μM UCB for 4 h led to a 23% increase in HNE levels, which further augmented to 43% (P b 0.05 vs. control) by exposure to 1 μM UCB, whereas no statistically significant changes were induced by the lowest UCB concentration assayed. The UCB-induced formation of HNE– protein adducts in neuronal cells corroborate previous indications pointing to the role of lipid peroxidation in the neurotoxicity resulting from the pigment (Brito et al., 2004; Park et al., 2001, 2002), which are further supported by the correlation coefficient obtained in the present study between HNE and LDH levels (r = 0.989, P b 0.01). Different intracellular levels of glutathione contributes to the variable susceptibility of neurons and astrocytes towards UCB-induced oxidative damage Having established that neurons and astrocytes present a different vulnerability to oxidative injury by UCB exposure, it appeared relevant to analyze the antioxidant defense mechanism accounted by the small molecular weight molecule, glutathione, in both cell types. To this end, intracellular content of the thiol compound was determined at the end of the 4 h incubation with the tested concentrations of UCB. As shown in Fig. 5, cell treatment with UCB caused a dose-dependent depletion in the intracellular stores of GSt in neurons and even more in astrocytes. In fact, GSt Fig. 5. Disruption of glutathione stores increases with unconjugated bilirubin (UCB) levels and is more marked in astrocytes than in neurons. Both cell types were incubated with growing concentrations of UCB, or no addition (0 μM; control), for 4 h at 37 °C. Intracellular content of total glutathione was evaluated by an enzymatic recycling assay and the results were expressed as nmol/mg protein. Each bar represents the mean ± S.E.M. of at least three independent experiments. ⁎P b 0.05 vs. control; †P b 0.05, †† P b 0.01 vs. neurons. 36 M.A. Brito et al. / Neurobiology of Disease 29 (2008) 30–40 ROS production following UCB exposure, reflecting the breakdown of the scavenging capacity of glutathione. The levels of GSH corresponded to approximately 95% of GSt and followed an equivalent profile by exposure to UCB. These results show that hyperbilirubinemia leads to the consumption of the main intracellular antioxidant thiol molecule, therefore pointing to the disruption of glutathione homeostasis as a relevant event in cell damage by UCB. Glutathione is a key molecule in the prevention of oxidative injury induced by UCB to nerve cells Having confirmed that astrocytes possess more relevant intracellular pools of glutathione than neurons, we hypothesized that this difference accounts for the unequal susceptibilities of the two cell types to UCB. To test our hypothesis, we evaluated whether augmenting the intracellular levels of GSH counteracts neuronal vulnerability to oxidative damage by UCB. On the other hand, we induced the depletion of GSH in astrocytes and tested their resistance to UCB. For this purpose, neurons were preincubated with NAC, a cysteine donor (Dringen, 2000), and astrocytes were treated with BSO, a GSH synthesis inhibitor (Dringen, 2000), and then both cell types were exposed to 0.1 μM UCB. Control experiments, performed with no addition of UCB, confirmed the increment in GSt levels in neurons by treatment with NAC (Fig. 6A), as well as the thiol depletion in astrocytes by BSO (Fig. 6B). In fact, GSt content increased from roughly 14 nmol/mg Fig. 7. Protein oxidation by unconjugated bilirubin (UCB) in neurons is prevented by N-acetylcysteine (NAC), whereas it is enhanced in astrocytes by buthionine sulfoximine (BSO). Both cell types were incubated with 0.1 μM UCB, or no addition (0 μM; control), for 4 h at 37 °C. (A) Neurons were pre-treated with 0, 25 or 100 μM NAC, a precursor of glutathione, for 1 h at 37 °C. (B) Astrocytes were pre-treated with 0, 25 or 100 μM BSO, an inhibitor of glutathione synthesis, for 18 h at 37 °C. Protein oxidation was evaluated by measuring protein carbonyls by slot blot analysis using rabbit anti-2,4-dinitrophenylhydrazine primary and goat anti-IgG secondary antibodies. The line staining was quantified by scanning densitometry and the results were expressed as percent change from control. §P b 0.05 vs. UCB alone. Fig. 6. N-acetylcysteine (NAC), a precursor of glutathione, increases glutathione levels in neurons, whereas buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis, decreases the thiol content of astrocytes. (A) Neurons were treated with 0, 25 or 100 μM NAC, for 1 h at 37 °C. (B) Astrocytes were treated with 0, 25 or 100 μM BSO, for 18 h at 37 °C. Intracellular levels of total glutathione were evaluated by an enzymatic recycling assay and the results were expressed as nmol/mg protein. §P b 0.05, §§ P b 0.01 vs. no addition (0 μM NAC or BSO). protein to nearly 15 (N.S.) and 18 (P b 0.05) after treatment of neurons with 25 or 100 μM NAC, respectively, while those levels decreased from around 26 to 4 and 3 nmol/mg protein (P b 0.01) by exposure of astrocytes to 25 or 100 μM BSO, respectively. At the end of the 4 h incubation period with UCB, damage of membrane components was assessed using quantification of protein carbonyls formation as a tool. As shown in Fig. 7A, pre-treatment of neurons with NAC reduced UCB-induced protein oxidation. This protection was more pronounced at a NAC concentration of 100 μM, which was able to reduce the 18.8 ± 0.9% change induced by UCB to a marginal 1.4 ± 0.1% increase over control values (P b 0.05 vs. UCB alone). It is interesting to note that treatment with NAC at 25 μM, which did not significantly elevate the intracellular GSt stores, was only able to counteract the UCB-induced formation of protein carbonyls by nearly 3%. It is then conceivable that NAC protects neurons towards UCB-induced oxidative damage. Pretreatment of astrocytes with BSO led to an augment in protein oxidation (Fig. 7B), which depends on the concentration of BSO. M.A. Brito et al. / Neurobiology of Disease 29 (2008) 30–40 In fact, the levels of carbonyls increased from 5.7 ± 0.2% over control values in UCB-treated cells not exposed to BSO to 11.3 ± 0.2% in cells exposed to 25 μM BSO and to 15.2 ± 0.3% by exposure to 100 μM BSO (P b 0.05 vs. UCB alone). It is worthwhile to point out that the extent of protein oxidation nearly doubled by treatment with BSO at 25 μM, a concentration that significantly depletes the stores of the thiol compound. These findings show that BSO aggravates oxidative stress induced by UCB in astrocytes, probably by promoting glutathione depletion. Moreover it seems that when the thiol is depleted, astrocytes are only slightly less sensitive to protein oxidation than neurons, a fact that confirms the key role of glutathione in the prevention of UCB oxidative injury to nerve cells. Discussion UCB induces toxicity to the nervous system by multiple pathways, involving different events. These include morphological changes, structural and cytoskeleton disruption (Brito et al., 2006a, b; Silva et al., 2002), as well as energetic breakdown (Grojean et al., 2001; Park et al., 2001), ionic imbalance (Brito et al., 2004) and extracellular accumulation of glutamate (Falcão et al., 2006; Fernandes et al., 2004). Release of inflammatory cytokines (Falcão et al., 2006; Gordo et al., 2006) and impairment of neurite development are also among the UCB-induced effects (Falcão et al., 2007). Additionally, the involvement of oxidative stress in the pathways of UCB cytotoxicity has been suggested in several experimental models and using different assay methodologies (Brito et al., 2004; Chroni et al., 2006; Oakes and Bend, 2005; Rodrigues et al., 2002a,b). The UCB-induced dysfunction may culminate in nerve cell death (Falcão et al., 2006; Gordo et al., 2006; Hankø et al., 2005; Silva et al., 2002). In the present work, oxidative action of UCB was assessed in both neurons and astrocytes and related with the extent of cell death. For that purpose, intracellular levels of ROS, as well as of HNE and carbonyls, indicators of lipid peroxidation and protein oxidation, respectively, were determined. To correlate UCB-induced oxidative stress with nerve cell demise, cytotoxicity was assessed by measuring the release of LDH by cells with a disrupted membrane, which is attributed to necrosis/oncosis. Finally, glutathione stores were evaluated to assess the response capacity of each cell type regarding the major cellular antioxidant defense system. The results obtained in neurons showed that UCB induces a significant increase in all the oxidative markers, as well as a decrease in the intracellular thiol content. Interestingly, data indicated that oxidation of cell components occurs in parallel with UCB-induced neuronal cell death. Therefore, it seems likely that oxidative stress is involved in UCB neurotoxicity, a notion that is corroborated by previous findings obtained in isolated synaptosomes and brain mitochondria, as well as in whole nerve cells (Brito et al., 2004; Genc et al., 2003; Rodrigues et al., 2002a,b). This assumption is further supported by the recently provided evidence of the involvement of oxidative stress in jaundiceinduced encephalopathy (Chroni et al., 2006), as occurs in several other neuropathological conditions, namely Alzheimer’s and Parkinson’s diseases (Brito et al., in press). Oxidative stress in brain is highly associated with both these and several other agedependent neurodegenerative disorders (Butterfield, 2006; Sultana et al., 2006). It is meaningful that oxidative stress manifestations are usually associated with disruption of the cytoskeleton network, excitotoxicity, compromise of ionic balance, inflammatory reac- 37 tions, impairment of neurite outgrowth and loss of cell viability (Chinopoulos and Adam-Vizi, 2006; Mhatre et al., 2004; Neely et al., 1999, 2005; Valencia and Morán, 2004), events that are also observed following exposure to UCB (Brito et al., 2004; Falcão et al., 2006, 2007; Grojean et al., 2000; Silva et al., 2002). Thus, it is conceivable that oxidative stress is a common denominator between different molecular events implicated in neuronal damage by UCB. Nevertheless, it is still important to clarify whether oxidative injury is a primary event resulting from the physical interaction of the pigment with neurons in culture or is secondary to the cell injury induced by UCB. In contrast with the UCB effects in neurons, production of ROS in astroglial cultures was only mildly increased, while carbonyl and HNE levels revealed no increase at all. It is, conceivable that the absence of HNE–protein adducts in astrocytes results from the capacity of these cells to eliminate the HNE as a GSH conjugate and the action of the multidrug resistant protein transporter (Schmuck et al., 2002; Sultana and Butterfield, 2004). Thus, it seems that UCB induces ROS production in both types of cells but oxidative damage only occurs in neurons. This assumption is corroborated by the correlations between ROS production and protein oxidation (r = 0.993, P b 0.01) or lipid peroxidation (r = 0.982, P b 0.05) found in neurons, but not in astrocytes. Interestingly, the higher vulnerability of neurons towards UCBinduced oxidation of cell components was further amplified by endotoxin, a bacterial lipopolysaccharide (LPS) used to mimic sepsis, a frequent aggravating condition of neonatal hyperbilirubinemia. In fact, co-incubation with UCB (0.1 μM) and LPS (10 ng/ml) led to a nearly 10% aggravation of the UCB-induced protein oxidation in neurons, whereas less than 5% variation was observed in astrocytes (unpublished observations). The present findings are in line with previous reports demonstrating that neurons are more sensitive to paraquat toxicity than astrocytes (Schmuck et al., 2002) and that exposure to NO and to peroxynitrite selectively damages neurons, whereas astrocytes remain unaffected (Almeida et al., 2001; Bolaños et al., 1995). The different susceptibility of neurons and astrocytes to oxidative insults may be explained by the fact that astrocytes possess detoxification mechanisms that do not exist or are less important in neurons (Aschner et al., 2002; Chen and Swanson, 2003; Dringen, 2000). In effect, neurons rely on the availability of cysteine for their glutathione synthesis, since they can not use cysteine, as astrocytes do. Moreover, astroglial cells possess higher levels of enzymes involved in the defense against ROS, as well as of various antioxidants, namely glutathione. Accordingly, the thiol stores in non-treated cells were higher in astrocytes than in neurons and suffered a more drastic decrease in the former than in the latter cells following exposure to UCB. Therefore, our data indicate that UCB disrupts the thiol antioxidant defense system and that astrocytes use more efficiently GSH in the defense against UCB. Intracellular GSH is consumed in non-enzymatic reactions with radicals or in the reduction of peroxides catalyzed by GPx. In these reactions, GSH becomes oxidized to GSSG, which is recycled to GSH through a reaction catalyzed by GR, at expenses of NADPH (Joshi et al., 2007). The decrease in intracellular levels of GSH can also result from its release to the extracellular medium or consumption in reactions catalyzed by GST. Since brain cells cannot synthesize cysteine, the rate limiting amino acid for glutathione synthesis, the maintenance of the intracellular concentrations depends on the availability of the precursors. Whether the GSH decrease observed following exposure of nerve 38 M.A. Brito et al. / Neurobiology of Disease 29 (2008) 30–40 cells to UCB is secondary to a depletion of NADPH, cysteine, or both, remains to be elucidated. To confirm the relevance of oxidative stress in the pathways of neuronal damage by UCB, as well as the role of the thiol antioxidant defenses in the prevention of the injurious effects, we evaluated the ability of NAC to protect neurons from oxidative disruption induced by UCB. NAC is a cysteine donor, which in turn is an important precursor of cellular GSH (Dringen, 2000). Apart from the increase in the intracellular thiol stores, NAC also scavenges ROS (Estany et al., 2007; Pocernich et al., 2001), further accounting to its antioxidant properties. Our results showed that neuronal supplementation with NAC led to a reduction of UCBinduced oxidation of cell components to values comparable to those of controls, a fact attributed to the enrichment in the glutathione levels. This is consistent with the hypothesis that UCB pathogenic mechanisms involve oxidative stress and suggests that NAC may become an important molecule in the treatment of UCB encephalopathy. Accordingly, NAC already is potentially useful in the prevention of neurotoxicity associated with oxidative stress, namely in experimental models of Parkinson’s disease (BahatStroomza et al., 2005), Alzheimer’s disease (Pocernich et al., 2001; Fu et al., 2006), methyl mercury intoxication (Kaur et al., 2006) and hypoxia (Jayalakshmi et al., 2005). Our assumptions are supported by a recent study showing the NAC capability to counteract jaundice-induced brain oxidative stress in rats (Karageorgos et al., 2006) and assume a particular relevance since this thiol molecule appears to be able to cross the blood–brain barrier after intraperitoneal injection (Karageorgos et al., 2006). The neuroprotective potential of NAC is additionally supported by other studies showing that this molecule prevents from neuronal injury induced by hypoxia (Jayalakshmi et al., 2005) and hydroxyl free radicals (Pocernich et al., 2000), as well as from the lipid peroxidation products acrolein and HNE (Arakawa et al., 2007; Neely et al., 1999; Pocernich et al., 2001). Thus, the present findings, when taken together with the reports available in the literature, open new perspectives regarding the potential usefulness of glutathione supplementation for the prevention of UCB-induced neuronal damage. Contrasting with the beneficial effects resulting from the thiol supplementation, it is well established that agents that deplete cells of glutathione increase ROS formation and promote oxidative damage (Dringen, 2000; Hall et al., 1997; Shanker et al., 2005). Thus, the final stage of this study consisted in evaluating the UCB effects in astrocytes previously treated with BSO, an irreversible inhibitor of GSH synthesis (Dringen, 2000). As expected, the BSO-induced drop in intracellular GSH promoted oxidative injury of astroglial proteins by UCB exposure. In fact, GSH-depleted astrocytes demonstrated protein oxidation values similar to the ones encountered in neurons, consistent with the fact that astrocyte resistance to oxidative stress relevantly relies in high GSH pools. In conclusion, our results show that oxidative stress is involved in UCB-induced neurotoxicity. 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