Taurine protects against bilirubin-induced neurotoxicity in vitro

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Research Report

Taurine protects against bilirubin-induced neurotoxicity in vitro Benzhong Zhang a , Xi Yang b , Xiaoling Gao c,⁎ a

Institute of Toxicology, School of Public Health, Lanzhou University, Lanzhou 730000, Gansu, China Department of Medical Microbiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada R3E 0W3 c Center of Clinical Laboratory, The Gansu People's Hospital, Lanzhou 730000, Gansu, China b

A R T I C LE I N FO

AB S T R A C T

Article history:

Kernicterus is a bilirubin-induced encephalopathy in newborn. Its spectrum ranges from

Accepted 13 January 2010

subtle extrapyramidal to acute encephalopathy and chronic posticteric sequelae. Current

Available online 21 January 2010

treatment of this serious problem is far from optimal. Taurine has been documented to have protective effects on neuronal cells against ischemia in vivo and in vitro. This study used

Keywords:

primary neuronal culture to investigate the toxicological effects of unconjugated bilirubin

Taurine

(UCB) and the protection of taurine against UCB-mediated neuron damage. Dose-dependent

Bilirubin

reduction of cell viability was found. Changes in neurite outgrowth preceded the reduction

Hyperbilirubinemia

of cell viability. The bilirubin-mediated neurotoxicity is mainly due to increased rate of cell

Primary cultured neuron

apoptosis and higher levels of intracellular free calcium ion level. Taurine dramatically

Apoptosis

improved cell viability in cultured neurons exposed to 12.5 µM UCB. Taurine pretreatment

Intracellular free calcium

reduced UCB-mediated apoptotic cell death in primary cultured neurons in a concentration-

concentration

dependent manner, which was associated with reversal of the increased intracellular free calcium ion levels caused by UCB. This study suggests the potential of taurine as a broadspectrum agent for preventing and/or treating neuronal damage in neonatal jaundice. © 2010 Elsevier B.V. All rights reserved.

1.

Introduction

Almost all newborn infants demonstrate high serum unconjugated bilirubin (UCB) level, a form of hyperbilirubinemia clinically called neonatal jaundice. In the vast majority of cases, neonatal jaundice is a benign, transient phenomenon. However, under certain conditions, UCB may induce adverse effects in the CNS because of the neurotoxic effect of hyperbilirubin. Kernicterus has long been recognized as the pathologic sequela of severe hyperbilirubinemia. Effective screening for Rh incompatibility and the accessibility of phototherapy to treat hyperbilirubinemia has reduced its

prevalence. However, the annual incidence of kernicterus varies among different countries and areas. In China, the kerniterus is still the number one reason for hospitalization of infants. The neuronal injury, associated with kernicterus, also called as bilirubin-induced neurologic dysfunction (BIND), is mainly demonstrated as 1) a movement disorder consisting of athetosis, dystonia, choreoathetosis, spasticity and hypotonia, 2) auditory dysfunction consisting of deafness or hearing loss and 3) oculomotor impairments, especially of upward gaze (Johnson et al., 2002). Severe kernicterus may result in death or serious brain damage. The risk factors for UCBinduced brain injury and kernicterus include lower level and

⁎ Corresponding author. Molecular Immunology, Central of Laboratory, Gansu People's Hospital, China. Fax: +86 931 8281673. E-mail address: [email protected] (X. Gao). Abbreviations: UCB, unconjugated bilirubin; i[Ca2+], intracellular free calcium ion concentration; BSS, basal salt solution 0006-8993/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2010.01.036

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ability of albumin to bind with UCB, hemolysis, immature and genetic vulnerability (Neonatal jaundice and kernicterus, 2001). Failure to promptly recognize jaundice by pediatricians, especially the improper estimation of its severity contributes to the development of kernicterus (Bhutani et al., 2004). Novel strategies to protect the CNS against UCB-induced injury are urgently required (Tiribelli and Ostrow, 2005). Taurine, 2-amino-ethanesulfonic acid, is one of the most abundant amino acids in mammals (Jacobsen and Smith, 1968). The physiological role of taurine has received considerable attention since it is found that cats fed a taurinedeficient diet developed central retinal degeneration (Hayes et al., 1975). Taurine may function as a neuromodulator or neurotransmitter in CNS which could maintain the structural integrity of the membrane, regulate calcium binding and transport (Lombardini, 1992), and protect neuron against L-Glu-induced neurotoxicity (El Idrissi and Trenkner, 1999). Taurine can improve the recovery of neuronal functions in brain slices after hypoxic conditions (Schurr et al., 1987). Taurine also has neuroinhibitory properties and neuroprotection in cerebral ischemia (reviewed in Shuaib, 2003). Taurine and its rate-limiting synthetic enzyme, cysteine sulfinate decarboxylase (CSD), located in neurons in the cerebellum, hippocampus and retina (Magnusson et al., 1989; Saransaari and Oja, 2000; Taber et al., 1986). Taurine biosynthesis and transport play an important role in maintaining high intracellular levels of taurine in the brain. Extracellular taurine inhibits Glu-induced Ca2+ accumulation to prevent Ca2+ influx with no effect on efflux (Chen et al., 2001). Despite its importance in neuronal function, the effects on the reversibility of UCB-mediated neurotoxicity and involved mechanisms remain unclear. In order to understand the neurotoxicity of bilirubin and the potential role of neuroprotection of taurine, we used a primary cultured neuron to test UCB-mediated neurotoxicity and the effect of pretreatment with taurine on UCB-induced neurotoxicity. Our data has shown that taurine efficiently protect the neuron against the UCB-induced neurotoxicity through downregulation of intracellular calcium concentration and attenuation of neuron apoptosis. The data suggested the potential of taurine as a novel pharmaceutical for the prevention and/or treatment of neuronal pathology in kernicterus.

2.

Results

Fig. 1 – Effects of UCB on neuronal cell variability. Different doses of UCB were added to primary cultured neuron cells for 12 h (A) and 24 h (B). The neuronal cell viabilities were evaluated by MTT assay as described in the Experimental procedures. The percentage of viable cells was calculated as follows: (A of experimental group/A of control group) × 100%. Control cells (sham) underwent identical experimental procedures as cells treated with UCB for each experimental condition. That is, control cells were exposed to control solution (NaOH and BSA) instead of UCB-containing solutions. Results presented are representative of 3 independent experiments. Data represent mean ± SD, *p < 0.05; **p < 0.01, ***p < 0.001.

shown from 12.5 to 100 µmol/L UCB. Extended culture to 24 h showed similar effect (Fig. 1B). These data indicated that UCB significantly decreased viability of primary neuronal cells in a dose-dependent manner.

2.1. UCB-induced neuronal injury: analysis of cell viability by MTT measurement

2.1.1. UCB-induced neuronal injury: analysis of neurite outgrowth

The initial study determined neuronal injuries after the UCB exposure for 12 h or 24 h by measuring neuronal viability. The number of viable cells was measured by the MTT assay and the effect of UCB on cell viability was expressed as percentage to the UCB untreated controls. To check dose–responses, cells were treated with varying concentrations of UCB. As shown in Fig. 1A, UCB at 5 µmol/L led to a 2–8% reduction of cell viability at 12 h and 24 h, no statistical significance compared to control. However, a statistically significant decrease in cellular viability was found starting from 12.5 µmol/L UCB after a 12 h treatment. A dose-dependent reduction of cell viability was

Neurite outgrowth and neuronal sprouting are significantly histological hallmarks of kernicterus disease (Falcao et al., 2007). In parallel with measurements of cell viability, we also examined the effects of UCB on general morphology of neuronal cells and neurite length. Interestingly, although exposure of cultured neurons to 5 µM UCB did not significantly reduce cell viability in 12 h or 24 h culture (Fig. 1), it caused neuronal damage as judged from neurite outgrowth, showing a reduction in neurite length by quantitative observation (Fig. 2). Higher UCB concentration led to a more severe reduction of neurite outgrowth. Hence, it suggests that the morphological effect preceded the reduction of cell viability.

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Fig. 3 – Apoptotic analysis of primary cultured neurons treated with UCB. Primary cultured neurons were exposed to UCB for 12 h and incubated with propidium iodide and Annexin V for 2 h at room temperature and cell apoptosis was analyzed by FACs. Quantitative analysis of the fraction of apoptotic cells treated with UCB at 5.0, 12.5, 25 µM. Data are given as the mean± SD of four independent experiments each performed in duplicate. *p < 0.01 vs. untreated control cells.

2.1.3. The intracellular free calcium ion concentration i[Ca2+] after the UCB exposure Fig. 2 – The reduced length of neurite outgrowth of neurons cultured with various doses of UCB. The cells were seeded in 35 mm culture dishes at the density of 1 × 105 cells/ dishes. After 10 days of culture, the cells were exposed to UCB concentrated from 5.0 to 100 µmol/L. The length of neurite outgrowth was measured under a microscope equipped with a ruler. Data represent mean ± SD, n = 150, *p < 0.05; **p < 0.01, ***p < 0.001.

2.1.2.

UCB mediated neuron apoptosis

It is reported that UCB may cause apoptosis in cultured neurons (Hanko et al., 2005). To verify whether the reduced cell viability is due to apoptosis, the primary neuron cells were collected and analyzed by flow cytometry. The cells were co-stained with propidium iodide (PI) and Annexin V (50 µg/mL). Apoptotic cells were distinguished from nonapoptotic, intact cells by the intensity of their propidium iodide (PI) and Annexin V staining. PI labels the DNA in cells where the cell membrane had been compromised, while the Annexin V-FITC cell membrane-labelling assay detects the translocation of phosphatidylserine from the inner face of the cell membrane to the outer surface, as an early marker of apoptosis. Double positive cells were defined as apoptotic cells. As shown in Fig. 3, without UCB exposure, almost no neurons (1.4%) were undergoing apoptosis, while 12.5 µM UCB exposure increased the rate of cell apoptosis to 21%. UCB at 25 µM in the culture increased the rate of apoptosis to 27%. Our data confirmed that 5–25 µM UCB exposure for 12 h can cause dose-dependent elevation of apoptosis in cultured neurons (Fig. 3). Morphological changes in apoptotic characteristics, such as cellular shrinkage, rounding, poor adherence, and round floating shapes were also observed in some of the 5 µM UCB exposed neurons under light microscopy (data not shown).

These studies focused on the effects of UCB on i[Ca2+] concentrations in the 10-day cultured viable neuronal cells. Cells were treated with 5, 12.5, 25 µM UCB for 4 h and i[Ca2+] was monitored. The images in the Fig. 4 represent changes in i [Ca2+] in four repeating experiments at different concentration of UCB with similar results. In all experiment, UCB caused a rapid elevation of i[Ca2+] levels in a concentration-dependent manner. Elevated i[Ca2+] was observed within 4 h after UCB exposure and remained until 8 h (Fig. 4E). Our data suggested that UCB had little effect on the release of intracellular calcium from ER. In the presence of 3 mM EGTA in the medium to block all the extracellular calcium (Fig.4F), the elevation of calcium induced by the UCB is totally inhibited. It is concluded that Ca2+ influx was the major contributor to the elevation of intracellular Ca2+ level mediated by UCB. Similarly, addition of TMB-8 (300 µM), an intracellular Ca2+ release blocker in the culture system also support the promoting effect of UCB on Ca2+ influx (data not shown).

2.1.4. Taurine alleviated UCB-induced loss of cell viability and inhibition of neurite outgrowth in primary cultured neurons Stimulation with 12.5 µM UCB for 12 h resulted in about 30% loss of neuron viability (Fig. 1A) and had a remarkable effect on neurite outgrowth (Fig. 2). Therefore, exposure to 12.5 µM UCB for 12 h was used in the current experiment to examine the effect of taurine on UCB-induced neural cell toxicity. As shown in Fig. 5, in the concentration of as low as 0.4 mM taurine, significantly reversed UCB-induced reduction of neuron viability (Fig. 5A) and neurite outgrowth (Fig. 5B). Taurine pretreatment alone exhibited no effect on neurite outgrowth changes in primary cultured neurons (data not shown), suggesting a protective function of taurine against UCB-induced neuronal injury.

2.1.5. Taurine attenuated UCB-induced apoptosis and intracellular free calcium ion concentration of primary cultured neurons To further examine whether the reversal effect of taurine on UCBinduced loss of cell viability is related to changes in apoptosis, we

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Fig. 4 – UCB exposure dramatically increase i[Ca2+] and extracellular Ca2+ influx is the major contributor to the UCB-stimulated elevation of intracellular Ca2+ in cultured neuronal cells. Fluorescent intensity comparison of cultured neurons subjected to the UCB as described in Experimental procedures. The images of intracellular [Ca2+] of neurons after 4 hour UCB exposure were shown in A–D. A. Normal neuron without UCB; B. neurons subjected to 5 µM UCB; C. neurons subjected to 12.5 µM UCB; D. neurons subjected to 25 µM UCB; E. analysis of fluorescent intensity in different UCB doses at 4 h and 8 h exposures. F. Neuronal cells were insulted with UCB in the absence or in the presence of 3 mM EGTA for 4 h. Data are mean±SD for each point. Statistical significance: **p<0.01, ***p<0.001.

determined the effect of taurine on increased cell apoptosis in primary cultured neurons exposed to UCB. 12.5 µM UCB exposure for 12 h was chosen to demonstrate the effects of taurine on the UCB-mediated apoptosis. The data shown in Fig. 6 confirmed the concentration-dependent protective effects of taurine evidenced by the changes in percentage of cell apoptosis. Pretreatment with 0.4 mM and 1.6 mM taurine reduced the percentage of cell apoptosis about 34% and 66%, respectively in UCB exposed neurons (Fig. 6). This observation suggests that taurine can prevent UCB-induced neuron cell death by preventing apoptosis. For measurement of intracellular free calcium ion concentration, we used 5.0 µM and 25 µM UCB exposed neuronal cells to investigate the modulation of taurine on the intracellular calcium homeostasis because 5.0 µM UCB significantly increased i[Ca2+] (Fig. 4). As shown in Fig. 7, the addition of 0.4 mM and 1.6 mM taurine in the medium before 5.0 µM UCB exposure reduced i[Ca2+] by about 20% and 45% respectively,

both significantly lower than control cells. However, taurine did not attenuate i[Ca2+] further upon prolonged stimulation (8 h). The reducing effect of taurine on i[Ca2+] was also observed in the condition of high concentration, that is 25 µM of UCB caused higher calcium accumulation intracellularly which is reversed by the pretreatment with 0.4 mM taurine, further effects were observed in 1.6 mM taurine pretreatment groups (Fig. 7B).

2.2. The mechanisms of taurine mediated restore the calcium homeostasis in the UCB insulted neurons To further clarify the role of taurine in the calcium homeostasis, we added the EGTA, a free Ca2+ chelator, to the culture medium to deplete the calcium in culture. As shown in Fig. 8, the EGTA almost abate the elevation of intracellular calcium ion level. Taurine can partly inhibit the elevation of intracellular calcium concentration mediated by 12.5 µM UCB. The data demonstrate that

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Fig. 5 – Treatment effects of taurine on UCB toxicity. Effects of treatment with the specific doses of taurine for 4 h on cell viability and neurite outgrowth before exposure to 12.5 µM UCB. Panel A showed the effects of taurine on cell viability as measured with MTT reduction assays after 12 h 12.5 µM UCB exposure. The cells without any treatment used as controls and cell viability were designated as 100% (n = 3–4). Panel B showed neurite outgrowth in total viable cells. *Denotes significant differences (*p< 0.05, **p < 0.01) from neurons exposed to 12.5 µM UCB alone under otherwise identical experimental conditions. Values are given as mean± SD (n = 150).

Fig. 7 – Modulation of taurine on the increased i[Ca2+] level induced by the UCB. The primary cultured neurons were subjected to 5 µM UCB for 4 h or 8 h with or without pretreatment of taurine at the 0.4 or 1.6 mM concentration as indicated in Experimental procedures. The neurons were labeled with Furo-3/AM 1 h. Selected cells in each chamber were scanned by the TCS SP2 (EλX = 488 nm EλM = 530/30 nm) and fluorescence emission was monitored.

3. taurine mainly influences the influx of calcium from external microenvironment with limited effect on the release of calcium from ER.

Fig. 6 – Protection of taurine on cell apoptosis in primary cultured neurons. Cell apoptosis was assessed after 12 h UCB (12.5 µM) stimulation as indicated in Experimental procedures. Different doses of taurine were added to culture 4 h before the UCB exposure. All data were expressed as mean± SD of three experiments. *p < 0.05, **p < 0.01.

163

Discussion

Neonatal jaundice is the result of an increase in serum-free bilirubin and is present in two thirds of all newborns during

Fig. 8 – The modulation of taurine on the UCB-mediated elevation of intracellular calcium ion in the presence or absence of EGTA. The primary cultured neurons were exposure to 12.5 µM UCB for 4 h in the medium with or without EGTA. The taurine was added to the culture before the treatment of UCB and EGTA. The data was representative of two independent experiments with similar results. **p < 0.01, ***p < 0.001.

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the first week of life (Gartner, 1994). In one study, UCB concentrations of 5 µmol/L were found in the cerebrospinal fluid of infants (Meisel et al., 1981). Also, clinical evidence suggests that the duration of UCB-mediated stress is an important factor in the pathogenesis of bilirubin-induced brain damage (de Vries et al., 1985). So far, the underlying mechanisms of the neurotoxicity of bilirubin and pharmacological effects of taurine in the hyperbilirubinemia remain unclear. In this study, primary neuron culture was used as a model for investigating the neurotoxicity of bilirubin (Provan and Yokel, 1992). Our work presented evidence to support the notion that bilirubin-mediated neurotoxicity is related to an increase in neuronal cell apoptosis and intracellular free calcium ion. Moreover, this study showed a protective effect of taurine against the UCB-mediated neurotoxicity. Taurine can efficiently increase cell viability and neurite outgrowth, inhibit the cell apoptosis and restore the intracellular calcium homeostasis in the presence of low to high concentration of UCB. To the best of our knowledge, this is the first study that showed a protective role of taurine in the prevention of neuropathology of kernicterus. MTT assay is a well-established method to evaluate cell viability and proliferation. MTT is a tetrazolium salt that is actively transported into cells by endocytosis (Bhutani et al., 2004; Liu et al., 1997) and can be reduced predominantly by an NADH dependent mechanism (Berridge et al., 2005). Impairment of any of these steps may affect the MTT reduction assays (Liu et al., 1997). Astrocytes exposed to moderate UCB (17 µM) impaired endocytosis and MTT reduction without affecting the cytoskeleton, suggesting an effect of UCB primarily at the plasma membrane (Silva et al., 2001). In the present study, neuronal viability is dramatically reduced in the condition of high dose of UCB exposure (12.5, 25, 50, 100 µM) for 12 h and 24 h. The neuron incubated with 100 µM UCB for 24 h caused a nearly complete loss of cell viability as measurement of MTT assays, leading to the conclusion that bilirubin toxicity is a progressive and dose-dependent process. The level of cell viability appeared higher than that reported in the literature which used the human NT2-N neuron cell line (Hanko et al., 2006). The reason for the difference is likely due to the use of different cells and culture medium. Instead of using long term cultured cell line and serum-free medium, our study used primary cultured neuron and serum supplemental culture medium. We believe that serum supplement can better mimic physiological condition and avoid the neuron damage caused by a serum-free condition itself. In the current study, we found that bilirubin at low concentration (5 µM) significantly reduced neurite outgrowth, which occurred before the decline of cell viability (Figs. 1 and 2). This finding indicates that bilirubin-induced reduction of neurite outgrowth is an early event in bilirubin-induced neurotoxicity. NF-160 may be related to the bilirubin-induced reduction of neurite outgrowth since studies showed that NF-160 expression is closely related to the neurite sprout (Qian et al., 2009). More work is necessary to understand this mechanism. Increases in of apoptosis rate and intracellular calcium ion level were both found in neurons that were insulted with UCB. However, the disruption of calcium homeostasis appeared prior to the initiation of cell apoptosis. Intracellular calcium concen-

tration is important in the maintenance of cell stability. i[Ca2+] overload leads to the activation of many pivotal cellular processes that can alter the cellular functions and even lead to cell injury and death. The results in Fig. 4 show the concentrationdependent increase in i[Ca2+] in bilirubin-exposed neuronal cells. Calcium is an important physiological player as a second messenger and triggers neurotransmitter release. It is likely that the break of intracellular calcium homeostasis is one of the important initiators of cell apoptosis. The reverse of the inhibition of neuronal growth is a major goal in the treatment of neurological diseases. Our finding on the ability of taurine to quickly restore the neurite outgrowth and prevent the loss of neuron viability is very exciting. The neuroprotective function of taurine is related to reestablishment of calcium homeostasis in UCB insulted cell and downregulation of increased apoptosis level caused by high bilirubin exposure. The finding in the present study is consistent with previous reports in rodents and rodent neuronal cell line studies showing that taurine treatment promotes neuronal proliferation and neurite outgrowth (Lima and Cubillos, 2006). UCB-mediated neurotoxicity is related to an accumulation of i[Ca2+]. The intracellular free calcium ion overload can be a consequence of either calcium influx from extracellular microenvironment or the release of additional calcium from intracellular stores. Taurine has been documented to have protective effects on neuronal cells by reducing cell apoptosis (Chen et al., 2009; Shuaib, 2003) and regulation of calcium homeostasis in many tissues, including the heart (Schurr et al., 1987); brain (Lehmann et al., 1985; Shuaib, 2003), retina (Lombardini, 1985) and cultured neuronal cells (El Idrissi and Trenkner, 1999). To determine whether taurine affects the i [Ca2+] in bilirubin-exposed neurons, Fura-3/AM fluorescence dying method was used. As shown in Fig. 7, a significant increase in i[Ca2+] was found in taurine-untreated neurons after bilirubin exposure. In contrast, a relative lower i[Ca2+] was detected in taurine-pretreated neuronal cells. This finding is consistent with the observation that bilirubin stimulates i [Ca2+] accumulation in astrocyte in the brain (Ostrow et al., 2003) and taurine efficiently keeps intracellular calcium homeostasis (Chen et al., 2009; El Idrissi and Trenkner, 1999). Our data support a role for taurine in modulating the intracellular calcium homeostasis in response to hyperbilirubin exposure. The quantitative study of i[Ca2+] in cultured neurons under UCB stress indicates that an extracellular calcium chelator, EGTA, can substantially prohibit the UCBinduced alteration of i[Ca2+]. The i[Ca2+] alteration shown in Fig. 8 indicated that UCB-induced opening of the calcium channel caused calcium influx, which is a main reason for the elevation of intracellular calcium concentration. Taurine may function as a modulator to membrane calcium channel to restore intracellular calcium homeostasis. In summary, we propose that taurine protects neurons against bilirubin-induced neurotoxicity, in part, by downregulation of elevated i[Ca2+] and reduction of cell apoptosis. The disturbed Ca2+ homeostasis under UCB stress in cultured neurons induced the opening of various types of calcium channels, thus ensuing the influx of i[Ca2+]. Our result also suggested that taurine may act as a regulator on the membrane calcium channels so that it can efficiently prevent the calcium influx. Further studies are needed to determine which calcium

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ion channels are involved in the disruption of calcium homeostasis in the UCB stress and how taurine restore the intracellular calcium stability. This study sheds light on key mechanisms responsible for taurine neuroprotection. Further investigation may lead to better understanding of taurine as a great therapeutic potential and possible treatment for neurodegenerative diseases.

protein (Lee et al., 1994). The use of fetal brains at 3–4 days before delivery is suitable for neuronal culture due to avoidance of damage to cell connections that exists in the older brains and contamination of glial cells observed in cultures from brain cortex of 1-day postnatal mice.

4.

Experimental procedures

4.1.

Mice

Healthy Kun ming mice (male: 25 ± 0.3 g, female: 28 ± 0.3 g) were bred at the Experimental Animal Center in Lanzhou University and hosted under specific-pathogen-free conditions. The pregnant mice were sacrificed at 16–17 days of gestation for preparation of neuronal cultures. The animal experimental protocol was approved by the Lanzhou University Committee on Animal Research.

After 10 days of culture, the neurons were washed in basic salt solution (BSS) [130 mM NaCl, 2.5 mM KCl, 2.0 mM MgCl2, 10 mM HEPES-KOH, and 10 mM D-glucose (pH 7.3)] twice. Neurons were suspended in BSS and preincubated with different concentrations of taurine (0.4 mM, 1.6 mM, and 6.4 mM) for 4 h or 8 h. Then, the UCB was added to the buffer at different concentration for 12 h or 24 h in the presence of taurine or absence of taurine. The intercellular calcium ion concentration and apoptosis level were checked. Control groups were treated under the same conditions without UCB and/or taurine.

4.2.

4.5.

UCB preparation

Bilirubin purchased from Sigma Chemical Co.-Aldrich (Milan, Italy) was purified (McDonagh and Assisi, 1972) and the crystalline precipitate was collected and used for the further preparation of unconjugated bilirubin (UCB). UCB preparation is performed according to the literature (Hanko et al., 2006). Briefly, UCB was freshly dissolved in 0.1 M NaOH to different concentrations and supplied with bovine serum albumin (BSA) solution to obtain a UCB/BSA molar ratio of 1.5. The ready UCB was immediately added to cultured cells. All handling of bilirubin was performed in dim light and pH is around 8.0. The slightly supra-physiological pH is to minimize the UCB precipitation. Cells exposed to control solution only (same concentration of NaOH) was served as controls in all experiments testing UCB toxicity.

4.3.

Primary culture of mice neurons

Primary neuronal cultures were prepared from fetal mice brains as previously described (Lee et al., 1994; Yarom et al., 1985). Briefly, the brains dissected from fetal mice were mechanically dissociated in DMEM, supplemented with 2 mM glutamine, and 20% heat inactivated fetal bovine serum. Cell suspensions were centrifuged at 200 g for 3 min, and the pellet was resuspended in DMEM. Single cell suspension was collected after the cell transfluxed through 70 µm cell strainer. The viability of the isolated neural cells was over 90% as determined by 0.2% trypan blue exclusion. 1 × 105 neurons were plated in either 35-mm tissue culture dishes, or 96-well plate for cell culture. The plates were precoated with 5 mg/mL poly-L-lysine if desired. Neurons were cultured for 3 days before the supplement of 5 mg/mL cytarabine. The medium had been refreshed every 2 days. It had been shown that neurons are morphologically and physiologically mature after 12–14 days in vitro (Lee et al., 1994). Cultures prepared under these conditions usually contain about 80–85% neurons as estimated by immunohistochemical staining using antibodies against neurofilament

4.4. Exposure of neurons with UCB in the presence and absence of taurine

Cell viability assay

The viability of neurons was examined using 3-(4, 5Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay on a microplate reader (DG-3022). Primary neuron cells were seeded in a 96-well plate (5× 104 cells/well) after 10 days of culture in BSS. After 24 h seeding, cells were treated with UCB (5, 12.5, 25, 50, 100 µmol/L) for 12 h or 24 h in 3 parallel wells each, then the MTT assay was performed as described by Xiao et al. (2007). Briefly, 20 mL of MTT solution (5 mg/mL) was added to each well and incubated for further 4 h. The medium was removed and 200 mL of DMSO was added to each well to dissolve the formazan crystals produced by MTT reduction. Absorbance (A) at 570 nm was measured using a microplate reader. The percentage of viable cells was calculated as follows: (A of experimental group/A of control group) × 100%. The effects of taurine on the cell viability were analyzed. The seeded primary neuron in 96-well plates (5 × 104/well) was incubated with different concentrations of taurine (0.4, 1.6, 6.4 mM) for 4 h before 12.5 µmol/L of UCB was added to the cultured cells for further 12 h, then the MTT assay was performed as described above.

4.6.

Apoptosis assay by flow cytometry (FACS analysis)

4 × 105 cells/well were seeded in 96-well plates after 10 days of culture, and after a pre-equilibration period of 24 h, they were exposed to different doses of UCB for 12 h or 24 h. The measurement of taurine protection was performed by the supplement of taurine to culture for 4 or 8 h before exposure to UCB. To analyze cell apoptosis, cells were resuspended in 1 mL PBS supplemented with propidium iodide and Annexin V (50 µg/mL), and incubated for 2 h at room temperature. Analyses were performed using a FACScan Benchtop Cytometer (Beckman Dickinson, New York, USA). A total of 10,000 cells were counted per sample. The data were interpreted using the CellQuest software. Apoptotic cells were distinguished from non-apoptotic, intact cells by the determination

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by propidium iodide (PI) and Annexin V co-staining. Propidium iodide (PI) was used to label the DNA in cells where the cell membrane had been compromised. In addition, we used the Annexin V-cell membrane-labelling assay to detect the translocation of phosphatidylserine from the inner face of the cell membrane to the outer surface, as an early marker of apoptosis.

4.7. The intracellular calcium examined by the confocal laser scanning microscopy The measurement of i[Ca2+] levels was conduced according to literature (Paredes et al., 2008). The cultured neurons were washed in the basal salt solution (BSS) twice. Then the neuronal cells were non-invasively labeled at 37 °C with Furo-3/AM at a concentration of 3 pM in phenol red-free BSS for 1 h. This membrane permanent, non-fluorescent acetoxymethyl ester is converted to fluorescent form by intracellular esterases and exhibits 40-fold increased fluorescence intensity upon Ca2+ binding. Control experiments indicated that the probe concentration and dye loading were optimal and that no residual unconjugated dye was present when cells were analyzed. Selected cells in each chamber were scanned by the TCS SP2 (EλX = 488 nm EλM = 530/30 nm) and fluorescence emission was monitored. Excitation and detection parameters were kept constant in all experiments. In the designed experiment, the medium was supplemented with 3 mM EGTA to block the influx of extracellular calcium ion.

4.8.

Statistical analysis

All data were expressed as mean± SD. Statistical analysis was performed with analysis of variance (ANOVA). Newman–Keuls multiple comparison test was used for further assessment of the statistical significance of the difference between two sets of data. p-values <0.05 were regarded as statistically significant.

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