Bilirubin is cytotoxic to rat oligodendrocytes in vitro

Brain Research 985 (2003) 135–141 www.elsevier.com / locate / brainres

Research report

Bilirubin is cytotoxic to rat oligodendrocytes in vitro Sermin Genc a , *, Kursad Genc b , Abdullah Kumral c , Huseyin Baskin d , Hasan Ozkan c a

Department of Medical Biology and Genetics, School of Medicine, Dokuz Eylul University, Inciralti, 35340 Izmir, Turkey b Department of Physiology, School of Medicine, Dokuz Eylul University, Inciralti, 35340 Izmir, Turkey c Department of Pediatrics, School of Medicine, Dokuz Eylul University, Inciralti, 35340 Izmir, Turkey d Department of Microbiology, School of Medicine, Dokuz Eylul University, Inciralti, 35340 Izmir, Turkey Accepted 19 May 2003

Abstract High levels of unconjugated bilirubin can be neurotoxic and gliotoxic. However, the effect of bilirubin on oligodendrocyte viability has never been investigated. In the present study, we searched the possible toxic effect of bilirubin on differentiated rat oligodendrocytes. Bilirubin was added to oligodendrocyte cultures at different concentrations varied between 10 and 100 mM, and cultures were incubated for different times (24, 48 and 72 h). Cell viability was evaluated by trypan blue exclusion. The results showed that bilirubin decreased oligodendroglial cell viability in a concentration and time-dependent manner. Bilirubin induced apoptotic cell death as revealed by TUNEL staining and poly(ADP-ribose) polymerase cleavage. We found that bilirubin induced inducible nitric oxide synthase (NOS) mRNA expression in rat oligodendrocytes. Bilirubin also increased oligodendroglial nitrite production in a concentration-dependent manner and NOS inhibitor partly blocked bilirubin-induced cytotoxicity. These results suggest that bilirubin induces cytotoxicity, at least partly, via the induction of nitric oxide production in oligodendrocytes.  2003 Elsevier B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Neurotoxicity Keywords: Bilirubin; Oligodendrocyte; Apoptosis; Rat; Nitric oxide; Nitric oxide synthase

1. Introduction Unconjugated bilirubin is a pigment resulted from the degradation of heme and deposition of this pigment in the central nervous system (CNS) is the major factor causing bilirubin encephalopathy during severe neonatal hyperbilirubinemia [7]. Neurological manifestations of bilirubin toxicity vary from transient or sometimes definitive auditory and visual impairment to death [7]. However, the exact mechanisms of bilirubin encephalopathy still remain unclear. Neural and glial cells are the main target for bilirubin toxicity. Bilirubin toxicity to neurons have been reported in previous studies [2,10,11,20]. Recent studies have *Corresponding author. Tel.: 190-232-412-4604; fax: 190-232-2590541. E-mail address: [email protected] (S. Genc). 0006-8993 / 03 / $ – see front matter  2003 Elsevier B.V. All rights reserved. doi:10.1016 / S0006-8993(03)03037-3

described compromised astrocyte function following bilirubin exposure [5,19,25,26]. Astrocytes are glial cells that provide metabolic support to neurons and contribute to the formation of blood–brain barrier. A recent study has also reported the in vitro toxic effect of bilirubin on endothelial cells another cellular element of blood–brain barrier [1]. Another cell type of macroglia, oligodendrocytes, are the myelin-forming cells in the CNS and therefore play a pivotal role in the proper execution of neural functions [3,6]. Relatively high concentrations of bilirubin have been found in the myelin fraction of rat brain after i.v. administration of radiolabelled bilirubin, suggesting that myelin and myelin forming cells might be another target of bilirubin in the CNS [12]. Furthermore, myelin alterations have been reported in hyperbilirubinemic Gunn rats [14,17,28] and rat cerebellum cultures exposed to toxic concentrations of bilirubin [23]. Cerebral white matter changes have also been observed in magnetic resonance

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imaging studies on infants with kernicterus [22]. However, the cytotoxic effect of bilirubin has been investigated mostly in neuronal and astroglial cells and to our knowledge the in vitro effect of bilirubin on oligodendrocytes has not been reported. For this reason, we aimed to search whether bilirubin has cytotoxic effect on rat oligodendrocyte cultures. Recent evidence suggests that oligodendrocytes, apart from being a target, may be a source of cytokines and nitric oxide (NO) in inflammatory conditions [13,16]. Among cytotoxic effector molecules evoked by the proinflammatory stimuli, increasing evidence supports a role for NO in oligodendrocyte damage [4,15]. Conditions that alter the blood–brain barrier, such as infection and sepsis may affect the entry of bilirubin into the brain and some additional factors are known to promote bilirubin toxicity in neuronal cells such as cytokines [7,30]. The results of a recent study on bilirubin-induced alterations in brain cell membrane fraction suggest that NO produced by neuronal NO synthase is involved in mediating bilirubin-induced cerebral dysfunction [18]. Similarly, NO produced by inducible NO synthase (iNOS) may play a role in bilirubin-induced cytotoxicity. However, the effect of bilirubin on iNOS expression has never been reported. Thus, we searched the effect of bilirubin on iNOS mRNA expression and NO production. The possible role of NO on oligodendroglial cell viability in bilirubin-treated and control cultures was also evaluated in the present study.

astrocytes were dislodged by shaking at 150 rpm for 24 h. The medium containing floating cells from the different flasks was combined and filtered through a nylon mesh (pore size of 28 mm; Millipore) to eliminate clumps of astrocytes. Then, contaminating microglia were removed by differential adhesion on plastic dishes. Non-adherent cells were collected and cell suspension was spun at 200 g for 5 min. The cell pellets were resuspended in plating medium containing DMEM / F12, 0.5% FBS, and insulintransferrin-selenite (Gibco) and plated in 100-cm 2 PDLcoated petri dishes (Greiner) for Western blotting, 25-cm 2 PDL-coated flasks (Greiner) for reverse transcriptase-polymerase chain reaction (RT-PCR), 24-well PDL-coated plates (Greiner) for cell viability assay and nitrite measurement, and PDL-coated glass coverslips in 35-mm petri dishes for immunocytochemistry and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labelling (TUNEL) staining at a density of 5310 4 cells / cm 2 . Seven days later, cultures were used for experiments. Stock bilirubin solution (10 M) was prepared in 0.1 M NaOH and stored in the dark at 4 8C until use. Stock bilirubin solution was further diluted with serum-free and insulin-transferrin-selenite-supplemented medium under sterile conditions and added to cultures at various concentrations. The cell cultures were kept in dark conditions to prevent light degradation of the bilirubin. Control cells were not exposed to bilirubin. Cultures were incubated for another 24–72 h.

2.2. Immunocytochemistry 2. Materials and methods

2.1. Cell cultures This study was approved by the Local Ethical Committee for Experimental Research at Dokuz Eylul University. Oligodendrocyte cultures were prepared from brains of newborn (1–2 day old) Wistar rats by a modification of the shakeoff method of De Vellis and Espinosa de los Monteros [8]. After decapitation, brains were retrieved and meninges were completely trimmed. The brains were minced and dissociated mechanically and then sieved through a nylon mesh (pore size of 140 mm; Millipore). Cells were spun at 200 g for 5 min in a benchtop centrifuge, and the cell pellets were resuspended in Dulbecco’s modified Eagle’s medium (DMEM) / F12 containing 10% heat-inactivated fetal bovine serum (FBS), 100 U / ml penicillin and 0.1 mg / ml streptomycin. The resuspended cells were seeded in 75-cm 2 flasks previously coated with 10 mg / ml of poly-D-lysine (PDL) (Sigma), using one flask for three brains dissociated. Cells were incubated in a humidified CO 2 incubator at 37 8C (5% CO 2 ), with changes of medium every 2 days. After 8–10 days of culture, macrophages and loosely attached cells were removed from the astrocyte monolayer by shaking cultures at 150 rpm for 1 h. Then, oligodendrocyte progenitors present on the top of a confluent monolayer of

For immunocytochemistry, the cells plated onto PDLcoated glass coverslips were rinsed in phosphated-buffered saline (PBS) and fixed in 4% paraformaldehyde in PBS for 10 min at room temperature (RT). After rinsing, cells were incubated for 30 min at RT with anti-galactocerebrosid C (GalC) antibody (Roche). Coverslips were rinsed twice in PBS containing 0.1% bovine serum albumin (BSA) and then incubated for 30 min at RT with secondary antibodies (Texas Red-conjugated goat anti-mouse IgG; Santa Cruz) diluted 1:100 in PBS containing 0.1% BSA. After rinsing, coverslips were mounted with fluorescence mounting medium and nuclei were labelled with 49,6-diamidino-2phenylindole dihydrochloride (DAPI)-antifade (Oncor). Slides were examined under a Nikon fluorescence microscope. Negative control coverslips were stained by omitting primary antibody. The ratio of GalC and DAPI double positive stained cells to total cells was calculated by counting cells in each of five randomly-selected low power fields under fluorescence microscope. In these cultures more than 95% of cells were GalC positive stained oligodendrocytes.

2.3. Cell viability assay Cell viability was evaluated using trypan blue exclusion assay. Cells were seeded at a density of 5310 4 cells / cm 2

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in 24-well plates. After 7 days, the cells were incubated with bilirubin (10, 50, and 100 mM) for periods of varying duration (24, 48, 72 h). After incubation, non-adherent, floating cells were collected. Adherent cells were harvested by trypsinization and pooled with spontaneously detached cells. After centrifugation, cell pellet was resuspended with PBS. Trypan blue (0.05 g / dl) was added to cell suspension for 5 min at a 1:5 dilution. Dead cells were stained blue. The number of cells excluding trypan blue was counted in a hemocytometer. Cell viability was presented as a percentage of viable cells in each condition. Cytotoxic effect of bilirubin in the presence or absence of the iNOS inhibitor, Nitro-L-arginine methyl ester ( L-NAME; Sigma) also was evaluated.

2.4. TUNEL staining For TUNEL staining, an In Situ Cell Death Detection Kit with fluorescein (Roche Molecular Biochemicals) was used. Cells seeded on PDL-coated coverslips were fixed by 4% paraformaldehyde and permeabilized incubating in 0.1% Triton X-100 solution for 2 min. Labelling was carried out in accordance with the manufacturer’s instructions, in association with nuclear DAPI staining to identify apoptotic cells. After labelling with DAPI-antifade, slides were examined under a Nikon fluorescence microscope. Negative control coverslips for TUNEL immunostaining were stained by omitting enzyme solution in reaction mixture.

2.5. Western blotting Oligodendroglial cells were harvested and lysed in cold lysis buffer containing non-ionic detergents and protease inhibitors. After centrifugation at 12,000 g for 20 min at 4 8C, the cell lysates were collected and stored at 270 8C until use. The amount of protein was determined by the Bio-Rad Detergent Compatible protein assay kit following the manufacturer’s instructions. Equal amounts of protein were separated by a 12.5% sodium dodecyl sulphate– polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Membranes were blocked by 5% nonfat dry milk in Tris-buffered saline (TBS)–Tween 20, and then hybridized with anti-poly(ADP-ribose) polymerase (PARP) antibody (Santa Cruz; 1:1000 dilution) for 1 h at room temperature. After three washes with TBS–Tween 20 and incubation with horseradish peroxidase-conjugated anti-rabbit secondary antibody (Santa Cruz) for 1 h, the blot was washed three times in TBS–Tween 20. Immunoreactive proteins were visualized using a chemiluminescent peroxidase substrate.

2.6. RT-PCR analysis For RT-PCR analysis of iNOS mRNA, cells grown in 25-cm 2 flasks were treated with 100 mM bilirubin for 24 h. Rat oligodendrocytes treated with 100 U / ml rat interferon

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gamma (IFNg; GIBCO) plus 1 mg / ml lipopolysaccharide (LPS; Sigma) for 24 h were used as positive control for iNOS mRNA expression. Then, cells were rinsed with PBS and total RNA was isolated using Nucleospin RNA isolation kit. RNA concentration was quantified spectrophotometrically. Isolated RNA was treated with DNase to digest any contaminant genomic DNA. RT-PCR amplification was carried out with 5 mg RNA using the rat primers for iNOS (sense 59-CCA CAA TAG TAC AAT ACT ACT TGG-39; antisense 59-ACG AGG TGT TCA GCG TGC TCC ACG-39) The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an internal standard (sense 59-ACCACAGTCCATGCCATCAC-39; antisense 59-TCCACCACCCTGTTGCTGTA-39). PCRs were carried out in a thermal cycler (Perkin-Elmer Cetus). The steps of amplification were 95 8C for 3 min, 95 8C for 30 s, 55 8C 30 s, 72 8C 1 min, 72 8C 5 min for 35 cycles. PCR products were resolved on 2% agarose gels.

2.7. Nitrite measurement The activity of iNOS was also evaluated by determination of nitrite levels in the supernatants. Nitrite is a stable product of NO and generated by the rapid oxidation of NO. To assay nitrite amount we used a modification of a previously published method [9,29]. Aliquots of 100 ml culture supernatants were mixed with equal volumes of Griess reagent (0.1% naphthyethylenediamine dihydrochloride, 1% sulphanilamide and 2.5% phosphoric acid) mixture in a 96-well microtitre plate (Maxisorb Immunoplate, NUNC). After 10 min of incubation at room temperature, the absorbance at a wavelength of 540 nm was measured in a microplate reader (Model 230S; Organon Technica). A range of twofold dilutions of sodium nitrite (0–128 mM) in PBS were run in each assay to generate a standard curve.

2.8. Statistical analysis Results were presented as mean6S.E.M. of three different separate experiments performed with separate cell cultures. Each condition was triplicated in each experiment. Analysis of variance (ANOVA) was used to compare the data from multiple groups. Comparison between two experimental groups was based on two-tailed t-test. P, 0.05 was considered to be significant.

3. Results In the present study, the effect of bilirubin on viability has been evaluated in cells at only a single stage of oligodendrocyte lineage, a differentiated GalC-positive stage. The purity of rat oligodendrocyte cultures was above 95% in our cultures as revealed by anti-GalC and DAPI double immunostaining (Fig. 1). High GalC positivity and morphological appearance can exclude the presence of

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Fig. 1. The purity of rat oligodendrocyte cultures was high in the study as revealed by anti-GalC and DAPI double immunostaining. DAPI staining reveals all nuclei as blue (10003).

oligodendroglial precursor cells, and other contaminant glia (i.e., microglia and astrocytes) in cultures.

3.1. Cell viability Trypan blue exclusion assay showed that bilirubin induced oligodendroglial cell death in a time and concentration-dependent manner. When oligodendrocyte cultures were treated with bilirubin at increasing concentrations, a marked concentration-dependent decrease in cell viability was found (Fig. 2). The maximum effect was observed at a concentration of 100 mM bilirubin. The cytotoxic effect of bilirubin was also time-dependent (Fig. 2). Low bilirubin concentration (10 mM) induced cell injury only after 72 h incubation. All bilirubin doses tested in this study induce significantly higher cell injury than control cells and vehicle-treated cells did not show any significant cell death as compared with control cells (data not shown).

3.2. TUNEL staining Fig. 3 shows the nuclei of oligodendrocytes treated with bilirubin or saline, immunostained with TUNEL and counterstained with DAPI. Virtually no staining was seen in control cultures (Fig. 3A). Nuclei of cells treated with bilirubin stain abundantly TUNEL positive (Fig. 3B). These findings are compatible with oligodendrocyte death by an apoptotic mechanism.

3.3. Western blotting A measure of caspase-3 activation is the cleavage of specific substrates for caspase-3. PARP is a 118 kDa polypeptide that is cleaved by activated caspase-3 to yield a cleaved product. Exposure of rat oligodendrocytes to 100 mM bilirubin for 24 h led to a decrease in PARP (116 kDa)

Fig. 2. Concentration- and time-dependence of bilirubin-induced oligodendrocyte death. Trypan blue assay was performed after 24, 48 and 72 h exposure of rat oligodendrocyte cultures to various concentrations of bilirubin (10, 50, and 100 mM). Cytotoxicity appears to increase in a dose- and time-dependent manner. Cytotoxicity values from three different time points for a constant concentration (50 or 100 mM) are significantly different from each other (P,0.05). A 100 mM concentration of bilirubin induces significantly higher cytotoxicity than 50 and 10 mM (P,0.05 at all different time points). Similarly, 50 mM bilirubin induces significantly higher cytotoxicity than 10 mM (P,0.05 at all different time points). The results are means6S.E.M. of triplicate conditions obtained from three independent experiments. Error bars represent S.E.M.

and a clear increase in the 85 kDa product (Fig. 4), indicating that caspase-3 is activated during bilirubininduced cell death of rat oligodendrocytes.

3.4. Inducible nitric oxide synthase mRNA Differentiated rat oligodendrocytes were exposed to 100 mM bilirubin for 24 h and iNOS mRNA expression was evaluated by RT-PCR technique. No iNOS mRNA expression was found in control condition (Fig. 5). Bilirubin apparently induced iNOS mRNA (Fig. 5). Rat oligodendrocytes treated with 100 U / ml rat IFNg plus 1 mg / ml LPS for 24 h were used as positive control for iNOS mRNA expression.

3.5. Nitric oxide production The activity of iNOS was also evaluated by determination of nitrite levels (a stable product of nitric oxide) in the supernatants. Nitrite (a stable oxidation product of NO) released into the culture medium was determined by using a colorimetric method. As shown in Fig. 6, 24 h exposure of cells to increasing concentrations of bilirubin resulted in induced nitrite production in a concentration-dependent manner. Low levels of nitrite were found in control cultures. Co-treatment of cultures with 100 mM bilirubin

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Fig. 3. Pictures show the nuclei of oligodendrocytes immunostained with TUNEL staining, and counterstained with DAPI. Virtually no positive TUNEL staining was seen in control cultures (A). Oligodendroglial cells treated with 100 mM bilirubin for 24 h stained abundantly with TUNEL staining (B).

condition and control cells, respectively. Similar results were obtained for 24 h incubation. Cell viability values were 57.363.5, 77.164.2 and 97.764.3 for bilirubintreated condition, bilirubin plus L-NAME condition and control cells, respectively. Fig. 4. Bilirubin-induced PARP-cleavage in rat oligodendrocytes as determined by Western blotting. The 116 kDa band is the uncleaved full-length PARP, whereas the 85 kDa band represents the apoptotic cleavage after exposure of cells to100 mM bilirubin for 24 h. Untreated cultures were used as control.

and 1 mM L-NAME produced significant reduction in induced-nitrite levels (near control values) (Fig. 6). To determine the possible role of NO in bilirubin induced toxicity, cell viability assay was also performed after addition of a combination of bilirubin and L-NAME to cultures. The presence of the L-NAME in cultures significantly increased cell viability suggesting a partly prevention of cells against high dose (100 mM) of bilirubin-induced toxicity after 48 h incubation. Mean viability values were 23.862.5, 56.863.8 and 96.863.9 for bilirubin-treated condition, bilirubin plus L-NAME

Fig. 5. Qualitative evaluation of iNOS mRNA expression upon bilirubin exposure. Cells were exposed to 100 mM bilirubin for 24 h, and total RNA was extracted, and subjected to RT-PCR. Concomitantly, analysis of GAPDH mRNA was carried out. The agarose gels were photographed and scanned. Bilirubin exposure significantly induces iNOS mRNA. Rat oligodendrocytes treated with 100 U / ml rat IFNg plus 1 mg / ml LPS for 24 h were used as positive control for iNOS mRNA expression.

4. Discussion Unconjugated bilirubin is a pigment resulting from the degradation of heme. The cytotoxic effect of bilirubin has been investigated mostly in neuronal and astroglial cells to date. A recent study has also reported the in vitro toxic effect of bilirubin on endothelial cells [1]. The present

Fig. 6. The effect of bilirubin on endogenous nitrite production in differentiated rat oligodendrocytes. Production of nitrite was determined in 100 ml aliquots of culture supernatant and the values represent the mean6S.E.M. of three independent experiments performed in triplicate. After 24 h incubation, bilirubin stimulates the release of NO measured as nitrite in a concentration-dependent manner (as compared with mean control value, * P,0.05). iNOS inhibitor, L-NAME (1 mM) significantly reverses bilirubin-induced nitrite production (** P,0.05).

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study is the first in vitro demonstration of oligodendroglial cytotoxicity induced by bilirubin. Here, we found that bilirubin induced death of differentiated rat oligodendrocytes in a concentration and time-dependent manner. The range of bilirubin concentrations used in our experiments was similar to that used in previous studies on astrocytes and endothelial cells [1,5,19,26]. Oligodendrocytes are the responsible cells for myelin synthesis in the CNS and therefore play a pivotal role in the proper execution of neural functions [3,6]. The direct cytotoxic effects of bilirubin on oligodendrocytes raises the possibility that bilirubin may affect myelin integrity and thus contribute to white matter dysfunction. This in particular seems an attractive basis for the myelin alterations observed in hereditary hyperbilirubinemic Gunn rats [14,17,28]. Alterations of gliotypic proteins in cerebellum that were consistent with the neuropathological effects have been shown to be associated with development of hyperbilirubinemia in the Gunn rat [17]. Region specific significant decrease of glial cells has also been reported in Gunn rats [28]. Additional compromising factors such as cytotoxicity due to activation of microglia or protection due to astroglia those may contaminate cultures were excluded by the high purity of cultures in this study as confirmed by anti-GalC immunostaining. We have examined the vulnerability of cells at only a single stage of oligodendrocyte lineage, a differentiated GalC-positive stage. High GalC positivity and morphological appearance can exclude the presence of oligodendroglial precursor cells in our cultures. The sensitivity of oligodendrocytes from different developmental stages (progenitors or immature oligodendrocytes) to bilirubin toxicity was not focused in this study and further studies are needed to clarify this issue. This is important because of the differential sensitivity of oligodendroglial cells from different developmental stages to toxic factors [3]. Bilirubin toxicity in cultured astrocytes and neurons may be attributed to apoptotic processes [21,24], but whether it can induce similar changes in oligodendrocytes is not known. Here, we next examined whether bilirubin-induced oligodendrocyte death implicates an apoptotic mechanism. Our results showing an increase in DNA fragmentation in bilirubin-treated cultures have suggested that the nature of cell death induced by bilirubin is apoptotic as revealed by TUNEL staining. The present study suggests that the involvement of caspase-3 activation in bilirubin-induced apoptotic oligodendrocyte death as revealed by PARP cleavage (Fig. 4). Bilirubin has been shown to disturb several essential aspects of cell function, including inhibition of DNA and protein synthesis, impairment of neurotransmitter synthesis, release, and uptake, and inhibition of protein phosphorylation [7,10,11,26]. The present results suggest that an another mechanism, NO-induced cell injury can mediate bilirubin toxicity. Recent evidence suggests that

oligodendrocytes, apart from being a target, may be a source of cytokines and NO in inflammatory conditions [13,16]. NO, a short-lived, highly reactive free radical, has been implicated in several inflammatory and degenerative disease of the CNS [27]. Among cytotoxic effector molecules evoked by the proinflammatory stimuli, increasing evidence supports a role for NO in oligodendrocyte damage [4,15]. For this reason, we focused here to clarify whether bilirubin induces oligodendroglial NO production, and if so, whether NO mediates the cytotoxic effect of bilirubin in this cell culture system. The results of the present study confirm the stimulating effect of bilirubin on endogenous NO production in oligodendrocytes. The incomplete reversibility of the bilirubin toxicity after the addition of NOS inhibitor, L-NAME, suggests that mechanisms other than NO are also involved in bilirubin-induced cytotoxicity. These mechanisms and the effect of bilirubin on iNOS expression, NO production and proinflammatory cytokine production in main cellular sources of NO derived from iNOS in the brain (i.e., microglia and astrocytes) needed to be clarified. Since the sensitivity of oligodendrocytes from different strains to various toxic agents, this study should be replicated with human oligodendrocytes. Regardless of underlying mechanisms, present results raise the possibility that the clinical syndrome of bilirubin toxicity may, at least in part, reflect direct induction of oligodendrocyte death. This situation might have some impacts in clinical area. Combining of neuroprotective strategies with oligodendroglioprotective strategies may provide to reach more efficient results in the treatment of bilirubin toxicity.

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