Heme oxygenase protects hippocampal neurons from ethanol-induced neurotoxicity

Neuroscience Letters 405 (2006) 168–171

Heme oxygenase protects hippocampal neurons from ethanol-induced neurotoxicity Bo Mi Ku, Yeon Joo, Jihye Mun, Gu Seub Roh, Sang Soo Kang, Gyeong Jae Cho, Wan Sung Choi, Hyun Joon Kim ∗ Department of Anatomy and Neurobiology, Institute of Health Sciences, College of Medicine, Gyeongsang National University, 92 Chilam-dong, Jinju, Gyeongnam, 660-751, Korea Received 6 April 2006; received in revised form 23 June 2006; accepted 27 June 2006

Abstract Ethanol has deleterious effects on neuronal cells both in vivo and in vitro, but the mechanisms are unknown. Here, treatment with increasing doses of ethanol (from 20 up to 600 mM) decreased the viability of a mouse hippocampal neuroblastoma cell line, HT22. The glutathione concentration decreased and intracellular reactive oxygen species (ROS) increased in a dose-and time-dependent manner, suggesting that the neurotoxicity was due to oxidative stress. Expression of heme oxygenase (HO)-1, a redox regulator and heat shock protein, increased with time after ethanol treatment, but HO-2 was expressed constitutively. The addition of 5 ␮M zinc protoporphyrin IX (ZnPP IX), a competitive HO inhibitor, with the ethanol further reduced cell viability and increased intracellular ROS, but these effects were reversed by co-treatment with 50 nM bilirubin, a well-known antioxidant and a product of HO catalysis. These results suggest that HO has a protective role in hippocampal neurons as an intrinsic factor against ethanol-induced oxidative stress and the protection depends on the degree of oxidative stress. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Ethanol; Heme oxygenase; Neurotoxicity; Oxidative stress; HT22

Ethanol has deleterious effects on neuronal cells both in vivo and in vitro [9,10,18]. Ethanol is converted into acetaldehyde via intracellular oxidation, eventually generating reactive oxygen species (ROS) such as superoxide anion (O2 − ), hydrogen peroxide (H2 O2 ), and hydrogen radical (HO• ). These ROS create oxidative conditions, resulting in alterations of DNA, lipids, and proteins that affect cell survival [14]. In addition, ethanol suppresses antioxidant enzymes such as glutathione peroxidase/glutathione reductase (GSH-Px/GSSG-R) [17]. Thus, the deleterious effects of ethanol on neuronal cells have been associated with oxidative stress [12], and many studies have shown that oxidative stress in neuronal cells, including that due to ethanol, causes both apoptosis and necrosis [9,12,18]. However, the pathways involved in ethanol-induced neuronal cell death are not known in detail. Heme oxygenase (HO) has an important role in controlling the redox state of the cell by functioning as a rate-limiting

∗

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0304-3940/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2006.06.052

enzyme in the heme degradation process. Heme degradation produces biliverdin, which is converted into bilirubin, free iron, and carbon monoxide. Biliverdin and bilirubin are known to be potent antioxidants [5]. Three isoforms of HO have been identified. HO-1 is an inducible isoform that is responsive to various stimuli, including oxidative stress. HO-2 is a constitutive isoform that is highly concentrated in the brain and testes and is not induced by oxidative stress. The remaining isoform, HO3, has been less well characterized [6]. Overexpression of HO protects neuronal and non-neuronal cells from oxidative stress [2,15]. However, it is not clear yet how HO-1 induction protects neuronal cells from the toxicity induced by ethanol. We performed the present study to elucidate the mechanism underlying ethanol-induced neurotoxicity and its relationship with HO. We used the mouse hippocampal cell line HT22 because the hippocampus is the area most closely associated with ethanol toxicity in the brain [10], and the HT22 line is frequently used as a good model system for studying neuronal cell death related to oxidative stress [14]. All media, sera, and media supplements were obtained from HyClone (Logan, UT). HT22 cells were cultured in high-glucose

B.M. Ku et al. / Neuroscience Letters 405 (2006) 168–171

DMEM containing 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 ␮g/ml streptomycin at 37 ◦ C and 5% CO2 . For ethanol treatment, cells were cultured in medium containing different concentrations of ethanol (20 ∼ 600 mM). Cultures were exposed to ethanol for 24 h to measure cell viability. Zinc protoporphyrin IX (ZnPP IX; ICN Medical, Geneva, Switzerland; solubilized in dimethylsulfoxide), and bilirubin (Sigma, St. Louis, Missouri; in PBS) were added to the culture media at the same time as the ethanol. To avoid ethanol volatility, experiments were carried out as described by Mitchell et al. [7]. As a preliminary experiment, the ethanol concentration in the medium was measured using an Alcohol Reagent Set (Pointe Scientific, INC, Belgium), and the results revealed adequate stability (data not shown). Cell viability was assessed at 37 ◦ C by measuring the ability of cells to metabolize 3-(4, 5-dimethyldiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT; Sigma). For RT-PCR, total RNA (1 ␮g) was used for cDNA synthesis. The sequences of the primers used for PCR were as follows: HO-1 forward, 5 -GCACTATGTAAAGCGTCTCC3 ; HO-1 reverse, 5 -TCTGGTCTTTGTGTTCCTCT-3 ; HO-2 forward, 5 -CCACTGCACTTTACTTCACA-3 ; HO-2 reverse, 5 -CACAATCCTCTCTTTGGTCT-3 ; GAPDH forward, 5 TGCCGCCTGGAGAAACCTGC-3 ; and GAPDH reverse, 5 TGAGAGCAATGCCAGCCCCA-3 . The amplified DNA fragments were 350, 449, and 172 bp for HO-1, HO-2, and GAPDH, respectively. For western blotting, cells were lysed in lysis buffer containing 50 mM Tris-Cl (pH 8.0), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.02% sodium azide, and proteinase inhibitor cocktail (PMSF, 100 ␮g/ml; Aprotinin, 1 ␮g/ml; Leupeptin, 0.5 ␮g/ml). Equivalent amounts of total protein (30 ␮g) were separated by SDS-PAGE and then transferred to a nitrocellulose membrane. The membrane was incubated with polyclonal rabbit anti–HO-1 (1:2000; Stressgen, Victoria, BC, Canada), rabbit anti–HO-2 (1:5000; Stressgen), and ␣-tubulin (1:10,000; Sigma) antibodies. Immunoreactive bands were detected using chemiluminescence (ECL, Amersham Biosciences, Piscataway, NJ) and x-ray film. Total intracellular glutathione (reduced form GSH + oxidized form GSSG) was measured with a glutathione assay kit (Sigma) according to the manufacturer’s instructions. The amount of GSH in the sample was estimated by measuring the absorbance at 412 nm for 5 min using a microplate reader in kinetic mode (Zenyth 340st, Anthos, Salzburg, Austria). The standard curve was obtained from absorbance of diluted commercial GSH. ROS production was detected using the fluorescent dye 2 , 7 -dichlorodihydrofluorescein diacetate (DCFH-DA, Sigma), whose fluorescence intensity is directly proportional to cellular oxidative stress. After treatment, the cells were harvested, washed with PBS, and suspended in PBS. DCFH-DA was added to the cell suspension at a final concentration of 30 ␮M. The suspension was incubated at 37 ◦ C for 10 min and then kept on ice until measurement. ROS generation was measured by fluorescence intensity (FL-1, 530 nm) of 10,000 cells using FACS (Becton Dickinson, Tokyo, Japan). Data are expressed as mean ± S.E. Statistical significance was determined using the Student’s t-test, taking P < 0.05 to be significant.

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To examine the effect of ethanol on neuronal cells, we first used an MTT assay to measure cell survival after incubation with several concentrations of ethanol (20–600 mM) for 24 h. Cell viability decreased linearly with increasing doses of ethanol,

Fig. 1. Effect of ethanol on HT22 cells. (A) HT22 cells were treated with various concentrations of ethanol (20 ∼ 600 mM) for 24 h. Cell viability was determined with an MTT assay. Mean ± S.E. of percent cell survival are presented (n = 8). (B) Time-dependent glutathione (GSH) depletion. Cells were treated with 100 and 400 mM ethanol for 2 or 24 h. The cellular levels of total GSH were measured and are presented as a percentage of the control value. GSH data are the mean + S.E. values of three trials. (C) Time-dependent reactive oxygen species (ROS) accumulation. Cells were exposed to 100 or 400 mM ethanol for 2 or 24 h and then treated with DCFH-DA. ROS generation was measured with a FACS as fluorescence intensity (FL-1). ROS data are mean + S.E. values of three trials. * P < 0.05, ** P < 0.01 vs. control (CTL).

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up to 400 mM (Fig. 1A); ethanol concentrations higher than 400 mM did not have any further effect on cell viability. Two concentrations (100 and 400 mM) were chosen for the later experiments because cells had distinct levels of viability at these concentrations (80% at 100 mM and 40% at 400 mM). Both intracellular GSH depletion and ROS accumulation are hallmarks of oxidative stress [4]. In cells treated with 100 and 400 mM ethanol, GSH levels decreased (Fig. 1B) and intracellular ROS increased (Fig. 1C) in a dose-and timedependent manner. These results indicate that ethanol exerts its neurotoxicity on the HT22 cells via the oxidative pathway. In addition, the degree of oxidative stress depends on ethanol concentration and this damage was more severe at high concentrations. Cells usually express heat shock proteins (HSP) upon exposure to stressful conditions to protect themselves from damage [1,16]. HO-1, which is also called HSP 32, is barely detectable

Fig. 2. Heme oxygenase (HO) expression after ethanol exposure in HT22 cells. (A) RT-PCR analysis. Cells were exposed to 100 or 400 mM ethanol, and total RNA was extracted at the times indicated. The expression level of HO-1 mRNA (upper panel) was normalized to that of GAPDH (lower panel) and is represented as arbitrary units (A.U.). (B) Western blot analysis of HO-1 protein (upper panel) and ␣-tubulin (lower panel). (C) RT-PCR analysis of HO-2 mRNA (upper panel) and GAPDH (lower panel). (D) Western blot analysis of HO-2 protein (upper panel) and ␣-tubulin (lower panel).

in the brain under basal conditions but is up-regulated by various stressful conditions, including intracellular GSH depletion and ROS accumulation [16]. Thus up-regulation of HO-1 is thought to play a key role in regulating intracellular ROS and neuronal resistance to oxidative stress [2,8]. HO-2 is the most abundant

Fig. 3. Effect of zinc-protoporphyrin IX (ZnPP IX) and bilirubin on ethanolinduced cell death. (A) Cells were co-treated with ethanol and various concentrations of ZnPP IX (5 ∼ 20 ␮M) for 24 h (n = 4). ** P < 0.01 versus ethanol alone. (B) Cells were exposed to ethanol with ZnPP IX (5 ␮M) for 24 h in the presence or absence of 50 nM bilirubin. Percent cell survival is presented as mean + SE (n = 4). (C) ROS production in cells exposed to ethanol and 5 ␮M ZnPP IX in the presence or absence of 50 nM bilirubin and treated with DCFH-DA. ROS data are mean + S.E. values of three trials. * P < 0.05, ** P < 0.01 between EtOH and EtOH + ZnPP IX; # P < 0.05, ## P < 0.01 between EtOH + ZnPP IX and EtOH + ZnPP IX + Bilirubin.

B.M. Ku et al. / Neuroscience Letters 405 (2006) 168–171

HO isoform in the brain and is associated with protection of neuronal cells [13]. To investigate the relationship between ethanolinduced neurotoxicity and HO, we examined HO expression after ethanol exposure using RT-PCR and western blot analysis. HO-1 mRNA expression increased rapidly within 30 min of treatment with either 100 or 400 mM ethanol (Fig. 2A), and HO1 protein increased within 2 h (Fig. 2B). However, HO-2 mRNA and protein were expressed constitutively, and expression levels were not changed by either concentration of ethanol (Fig. 2C, D). No significant differences were found between the effects of 100 mM and 400 mM ethanol on the expression of HO-1 or HO-2. These results suggest that HO might protect HT22 cells against ethanol-induced oxidative stress. To verify whether HO protects HT22 cells from ethanolinduced neurotoxicity, we used an HO inhibitor (ZnPP IX) and an HO catalytic product (bilirubin). ZnPP IX is a competitive inhibitor of HO that blocks HO activity in neuronal cells [3]. After co-treatment with ethanol and 5 ∼ 20 ␮M ZnPP IX, the viability of HT22 cells was lower than after treatment with ethanol alone (Fig. 3A). All subsequent experiments involving ZnPP IX were performed using a concentration of 5 ␮M ZnPP IX. No toxicity was found with 5 ␮M ZnPP IX alone. Addition of 5 ␮M ZnPP IX to ethanol increased HT22 cell death compared to ethanol alone, confirming the protective role of HO in ethanolinduced neurotoxicity (Fig. 3B). In the presence of 5 ␮M ZnPP IX, both 100 and 400 mM ethanol also increased ROS accumulation compared to ethanol alone (Fig. 3C). Because these data seemed to suggest that HO activity is needed for protection against ethanol in HT22 cells, we added bilirubin to the cultures to mimic HO activity. Bilirubin is an end product of heme catabolism and probably the best-known endogenous antioxidant in mammals. Nanomolar concentrations of bilirubin have been shown to be protective in rat hippocampal and cortical neurons [19], HT22 cells [15], and cerebral glial cells [11]. In this study, 50 nM bilirubin significantly reduced the ZnPP IX–mediated increase in cell death and ROS accumulation in HT22 cells (Fig. 3B, C). It implicates that endogenous antioxidant system in neuronal cells, including HO, has an ability to prevent neurons from ethanol induced oxidative stress. In summary, this study shows that ethanol induces oxidative stress in HT22 cells, and induces HO-1. The increased HO activity may reduce the toxic effect of ethanol through bilirubin production. Therefore, the addition of exogenous anti-oxidants may help to protect hippocampal neurons from ethanol-induced oxidative stress. Acknowledgement This work supported by the Korea Research Foundation Grant (KRF-2004-003-E00005).

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