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Original Contribution
Graduate Institute of Natural Products, School of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan
Department of Psychiatry, Hospital and College of Medicine, National Cheng Kung University, Tainan, Taiwan
c
Department of Pharmacology, School of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan
d
Cancer Center, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan
e
Center of Excellence for Environmental Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan
b
a r t i c l e
i n f o
Article history:
Received 9 April 2010
Revised 1 July 2010
Accepted 21 July 2010
Available online 4 August 2010
Keywords:
Arecoline
Neurotoxicity
NADPH oxidase
Glutathione
Oxidative stress
Apoptosis
Free radicals
a b s t r a c t
Arecoline, an areca nut alkaloid, has been noted for its potential cognition-enhancing effects in patients with
Alzheimer dementia. However, it has been conrmed that areca nut use is associated with oral and pharyngeal
cancers. In addition, arecoline is genotoxic and cytotoxic both in vitro and in vivo through oxidative stressdependent mechanisms. The aim of this study was to investigate whether arecoline would interfere with the
antioxidant defense system and induce cytotoxicity in rat primary cortical neurons. Results indicate that arecoline
(50200 M) induces neuronal cell death, and catalase, NADPH oxidase inhibitors (diphenyleneiodonium
chloride and apocynin), and a caspase inhibitor (z-VAD-fmk) can prevent arecoline-induced cell death.
Furthermore, arecoline increased reactive oxygen species production and upregulated protein expression and
mRNA levels of NADPH oxidase 2, which could be attenuated by catalase and NADPH oxidase inhibitors. Arecoline
also attenuated neuronal antioxidant defense by decreasing glutathione (GSH) level and superoxide dismutase
activity. In addition, arecoline enhanced the expression of proapoptotic proteins (cytochrome c, Bax, caspase-9,
and caspase-3) and attenuated the expression of the antiapoptotic protein Bcl-2. Moreover, NADPH oxidase
inhibitors could attenuate the arecoline-induced GSH depletion and reverse arecoline-induced changes in
proapoptotic and antiapoptotic proteins. In conclusion, the results indicate that arecoline could induce neuronal
apoptotic death by attenuating antioxidant defense and enhancing oxidative stress.
2010 Elsevier Inc. All rights reserved.
Introduction
Areca nut is one of the mostly widely used substances in Asia, and
arecoline, a cholinergic agonist, is the major areca nut alkaloid present
in the saliva of areca nut chewers [14]. Moreover, arecoline could
cross the bloodbrain barrier with a brain/plasma concentration ratio
close to unity [5,6]. Because of its effect on the cholinergic system, its
potential benecial effects in patients with Alzheimer dementia (AD)
have been noted [79]. In addition, the potentially therapeutic roles of
arecoline on both positive and negative symptoms of schizophrenia
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Fig. 1. Effects of arecoline on viability of rat primary cortical neurons. Cell viability was assessed by measuring the levels of (A) MTT reduction and (B) LDH activity and (C) the
numbers of MAP-2-positive neurons in primary cortical neuron cultures incubated with 1 to 200 M arecoline for 24 h. By immunouorescence assay, (D) primary cortical neurons
(control) treated with (E) 100 M arecoline, (F) 200 M arecoline, or (G) DPI (1 M) plus 200 M arecoline were further conrmed by staining with anti-MAP-2 antibody (green) and
the nucleus was visualized with DAPI (blue). Numbers of MAP-2-positive neurons were counted under a uorescence microscope. (H) Cultures treated without anti-MAP-2 antibody
were used for negative control for immunohistochemistry. Data are expressed as percentages of control (no arecoline). Bars represent the means SEM from six independent
experiments. *p b 0.05, **p b 0.01 versus control group. #p b 0.05 versus arecoline (200 M) only. Scale bar, 50 m.
Y.-T. Shih et al. / Free Radical Biology & Medicine 49 (2010) 14711479
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Glutathione quantication
A GSH measurement kit was purchased from Assay Designs.
Neurons were grown on six-well plates for 6 days. After arecoline
treatment, the cells were collected and washed with PBS. After
centrifugation, the pellets were suspended and deproteinated in 5%
metaphosphoric acid by brief sonication. After centrifugation at
13,000 g for 5 min, the supernatants were collected for total
glutathione measurement. GSH was determined by adding the
reaction mix and GSH reductase supplied in the kit, followed by
incubation and measurement by ELISA reader at 405 nm for 20 min at
1-min intervals. The total amount of GSH was determined by means of
a calibration curve and normalized to the protein concentration,
which was quantied using a Bio-Rad protein assay kit.
Superoxide dismutase activity assay
A superoxide dismutase (SOD) activity kit was purchased from
Assay Designs. This activity was assayed by xanthine oxidase and
conversion of WST-1 to WST-1 formazan. Neurons were grown on 12well plates for 6 days. After arecoline treatment, the cells were
harvested and cytosolic protein was extracted. SOD activity was
determined by adding the master mix and xanthine supplied in the
kit, followed by incubation and measurement by ELISA reader at
450 nm for 10 min at 1-min intervals. Protein concentration was
quantied using a Bio-Rad protein assay kit. Then, SOD activity in the
cell extracts was calculated versus a SOD standard curve and
normalize to the protein concentration.
Statistical analysis
Y.-T. Shih et al. / Free Radical Biology & Medicine 49 (2010) 14711479
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Fig. 2. Effects of arecoline on (A) iROS production, (B) NOX2 protein expression, and (C)
mRNA levels of NOX1 and NOX2 in primary cortical neurons. Neurons were treated
with arecoline for 24 h for ROS and protein expression measurement and treated with
arecoline for 8 h for mRNA detection. iROS production was measured by H2DCF-DA
staining. Protein expression and mRNA levels were detected by Western blot analysis
and RT-PCR, respectively, as described under Materials and methods. Densitometry
analyses are presented as the relative ratio of protein/-actin or mRNA/GAPDH, and
data are presented as percentages of the control group (no arecoline). Bars represent
the means SEM from six independent experiments. *p b 0.05, **p b 0.01, ***p b 0.001
versus control group.
Fig. 3. Effects of DPI, apocynin, and catalase on arecoline-induced (A) iROS formation
and (B) NOX2 expression. Neurons were treated with DPI (1 M), apocynin (100 M),
or catalase (200 U/ml) 30 min before arecoline (200 M) treatment. The iROS level was
measured by H2DCF-DA staining. Protein expression was detected by Western blotting.
Data are presented as percentages of the control group (no arecoline). Bars represent
the means SEM from six independent experiments. **p b 0.01 versus control group.
#
p b 0.05 versus arecoline only.
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Fig. 4. Effects of arecoline on (A) SOD activity and (B) GSH level in primary cortical
neurons. Cells were treated with arecoline (1200 M) for 24 h. (C) Effects of DPI,
apocynin, catalase, and z-VAD-fmk on arecoline-induced cell death. Cells were treated
with DPI (1 M), apocynin (100 M), catalase (200 U/ml), or z-VAD-fmk (20 M)
30 min before arecoline (200 M) treatment. Cell viability was determined by MTT
assay. Data are presented as percentages of the control group (no arecoline). Bars
represent the means SEM from six independent experiments. *p b 0.05, **p b 0.01
versus control group. #p b 0.05 versus arecoline only.
Fig. 5. (A) Flow-cytometric analysis of primary cortical neurons stained with annexin V
and PI performed 24 h after cells were exposed to arecoline (1200 M). The degree of
apoptosis in each group is shown as the apoptosis index evaluated by counting the
percentage of apoptotic cells (annexin V-positive cells). (B) Effects of arecoline on the
release of cytochrome c in primary cortical neurons. Cells were treated with arecoline
for 24 h. Protein expression was detected by Western blotting. Densitometry analyses
are presented as the relative ratio of protein/-actin protein. Data are presented as
percentages of control group (no arecoline) from six independent experiments.
*p b 0.05, **p b 0.01 versus control group.
Fig. 6. Effects of arecoline on the ratio of Bax to Bcl-2 in primary cortical neurons. Cells
were treated with arecoline for 24 h. Protein expression was detected by Western
blotting. Densitometry analyses are presented as the relative ratio of protein/-actin
protein, and data are presented as percentages of the control group (no arecoline). Bars
represent the means SEM from six independent experiments. *p b 0.05, **p b 0.01,
***p b 0.001 versus control group.
Y.-T. Shih et al. / Free Radical Biology & Medicine 49 (2010) 14711479
Fig. 7. Effects of arecoline on the expression of (A) caspase-9 and (B) caspase-3 in
primary cortical neurons. Neurons were treated with arecoline for 24 h. Protein
expression was detected by Western blotting. Densitometry analyses are presented as
the relative ratio of protein/-actin protein, and data are presented as percentages of
the control group (no arecoline). Bars represent the means SEM from six independent
experiments. *p b 0.05, **p b 0.01, ***p b 0.001 versus control group.
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Fig. 8. Effects of (A) DPI and (B) catalase on the expression of Bax, Bcl-2, cytochrome c,
caspase-9, and caspase-3 in arecoline-treated primary cortical neurons. DPI (1 M) or
catalase (200 U/ml) was added 30 min before arecoline (200 M) treatment. Protein
expression was detected by Western blot in six independent experiments.
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treatment will increase cell viability and inhibit mitochondriadependent apoptotic signaling in arecoline-treated neuronal cells.
In conclusion, exposure to arecoline (50 to 200 M) caused
oxidative stress-dependent neurotoxicity in primary cortical neurons
(Fig. 9). It must be noted that arecoline disrupts the redox status by
reducing antioxidant defense and enhancing ROS production. This
study also suggests that antioxidants could attenuate or prevent
neurotoxicity in people exposed to arecoline (e.g., areca nut chewers).
Acknowledgments
We appreciate the reviewers constructive suggestions. This study
was supported by Grant NSC 96-2320-B-037-039-MY3 from the
National Science Council, Taiwan, and supported in part by Grant
DOH99-TD-C-111-002 from the Excellence for Cancer Research
Center, Department of Health, Executive Yuan, Taiwan.
References
[1] IARC Tobacco habits other than smoking; betel-quid and areca-nut chewing; and
some related nitrosamines. IARC Working Group: Lyon, 2330 October 1984. IARC
Monogr. Eval. Carcinog. Risk Chem. Hum. 37:1268; 1985.
[2] Boucher, B. J.; Mannan, N. Metabolic effects of the consumption of Areca catechu.
Addict. Biol. 7:103110; 2002.
[3] Guh, J. Y.; Chuang, L. Y.; Chen, H. C. Betel-quid use is associated with the risk of the
metabolic syndrome in adults. Am. J. Clin. Nutr. 83:13131320; 2006.
[4] Ko, Y. C.; Chiang, T. A.; Chang, S. J.; Hsieh, S. F. Prevalence of betel quid chewing
habit in Taiwan and related sociodemographic factors. J. Oral Pathol. Med. 21:
261264; 1992.
[5] Herz, A.; Fraling, F.; Niedner, I.; Farber, G. Pharmacologically induced alterations of
cortical and subcortical evoked potentials compared with physiological changes
during the awakesleep cycle in cats. Electroencephalogr. Clin. Neurophysiol. Suppl.
26:164; 1967.
[6] Soncrant, T. T.; Holloway, H. W.; Greig, N. H.; Rapoport, S. I. Regional brain
metabolic responsivity to the muscarinic cholinergic agonist arecoline is similar in
young and aged Fischer-344 rats. Brain Res. 487:255266; 1989.
[7] Asthana, S.; Greig, N. H.; Holloway, H. W.; Raffaele, K. C.; Berardi, A.; Schapiro, M. B.;
Rapoport, S. I.; Soncrant, T. T. Clinical pharmacokinetics of arecoline in subjects
with Alzheimer's disease. Clin. Pharmacol. Ther. 60:276282; 1996.
[8] Freedman, S. B.; Harley, E. A.; Marwood, R. S.; Patel, S. In vivo characterisation of
novel efcacious muscarinic receptor agonists. Eur. J. Pharmacol. 187:193199;
1990.
[9] Raffaele, K. C.; Asthana, S.; Berardi, A.; Haxby, J. V.; Morris, P. P.; Schapiro, M. B.;
Soncrant, T. T. Differential response to the cholinergic agonist arecoline among
different cognitive modalities in Alzheimer's disease. Neuropsychopharmacology
15:163170; 1996.
[10] Sullivan, R. J.; Andres, S.; Otto, C.; Miles, W.; Kydd, R. The effects of an indigenous
muscarinic drug, betel nut (Areca catechu), on the symptoms of schizophrenia: a
longitudinal study in Palau. Micronesia. Am. J. Psychiatry 164:670673; 2007.
[11] Bales, A.; Peterson, M. J.; Ojha, S.; Upadhaya, K.; Adhikari, B.; Barrett, B.
Associations between betel nut (Areca catechu) and symptoms of schizophrenia
among patients in Nepal: a longitudinal study. Psychiatry Res. 169:203211; 2009.
[12] Panigrahi, G. B.; Rao, A. R. Chromosome-breaking ability of arecoline, a major
betel-nut alkaloid, in mouse bone-marrow cells in vivo. Mutat. Res 103:197204;
1982.
[13] Chou, W. W.; Guh, J. Y.; Tsai, J. F.; Hwang, C. C.; Chiou, S. J.; Chuang, L. Y. Arecolineinduced phosphorylated p53 and p21(WAF1) protein expression is dependent on
ATM/ATR and phosphatidylinositol-3-kinase in clone-9 cells. J. Cell. Biochem. 107:
408417; 2009.
[14] Stich, H. F.; Stich, W.; Lam, P. P. Potentiation of genotoxicity by concurrent
application of compounds found in betel quid: arecoline, eugenol, quercetin,
chlorogenic acid and Mn2+. Mutat. Res. 90:355363; 1981.
[15] Jeng, J. H.; Chang, M. C.; Hahn, L. J. Role of areca nut in betel quid-associated
chemical carcinogenesis: current awareness and future perspectives. Oral Oncol.
37:477492; 2001.
[16] Dasgupta, R.; Saha, I.; Pal, S.; Bhattacharyya, A.; Sa, G.; Nag, T. C.; Das, T.; Maiti, B. R.
Immunosuppression, hepatotoxicity and depression of antioxidant status by
arecoline in albino mice. Toxicology 227:94104; 2006.
[17] Tilakaratne, W. M.; Klinikowski, M. F.; Saku, T.; Peters, T. J.; Warnakulasuriya, S.
Oral submucous brosis: review on aetiology and pathogenesis. Oral Oncol. 42:
561568; 2006.
[18] Walvekar, R. R.; Chaukar, D. A.; Deshpande, M. S.; Pai, P. S.; Chaturvedi, P.; Kakade,
A.; Kane, S. V.; D'Cruz, A. K. Verrucous carcinoma of the oral cavity: a clinical and
pathological study of 101 cases. Oral Oncol. 45:4751; 2009.
[19] Thomas, S. J.; Bain, C. J.; Battistutta, D.; Ness, A. R.; Paissat, D.; Maclennan, R. Betel
quid not containing tobacco and oral cancer: a report on a casecontrol study in
Papua New Guinea and a meta-analysis of current evidence. Int. J. Cancer 120:
13181323; 2007.
[20] Ko, Y. C.; Huang, Y. L.; Lee, C. H.; Chen, M. J.; Lin, L. M.; Tsai, C. C. Betel quid
chewing, cigarette smoking and alcohol consumption related to oral cancer in
Taiwan. J. Oral Pathol. Med. 24:450453; 1995.
Y.-T. Shih et al. / Free Radical Biology & Medicine 49 (2010) 14711479
[21] Hung, C. R.; Cheng, J. T.; Shih, C. S. Gastric mucosal damage induced by arecoline
seizure in rats. Life Sci. 66:23372349; 2000.
[22] Thangjam, G. S.; Kondaiah, P. Regulation of oxidative-stress responsive genes by
arecoline in human keratinocytes. J. Periodontal Res. 44:673682; 2009.
[23] Lai, K. C.; Lee, T. C. Genetic damage in cultured human keratinocytes stressed by
long-term exposure to areca nut extracts. Mutat. Res. 599:6675; 2006.
[24] Chang, M. C.; Ho, Y. S.; Lee, P. H.; Chan, C. P.; Lee, J. J.; Hahn, L. J.; Wang, Y. J.; Jeng, J. H.
Areca nut extract and arecoline induced the cell cycle arrest but not apoptosis of
cultured oral KB epithelial cells: association of glutathione, reactive oxygen species
and mitochondrial membrane potential. Carcinogenesis 22:15271535; 2001.
[25] Sundqvist, K.; Liu, Y.; Nair, J.; Bartsch, H.; Arvidson, K.; Grafstrom, R. C. Cytotoxic
and genotoxic effects of areca nut-related compounds in cultured human buccal
epithelial cells. Cancer Res. 49:52945298; 1989.
[26] Winstock, A. Areca nut-abuse liability, dependence and public health. Addict. Biol.
7:133138; 2002.
[27] Shirname, L. P.; Menon, M. M.; Nair, J.; Bhide, S. V. Correlation of mutagenicity and
tumorigenicity of betel quid and its ingredients. Nutr. Cancer 5:8791; 1983.
[28] Cox, S.; Vickers, E. R.; Ghu, S.; Zoellner, H. Salivary arecoline levels during areca
nut chewing in human volunteers. J. Oral Pathol. Med. 39:465469; 2010.
[29] Chang, M. C.; Wu, H. L.; Lee, J. J.; Lee, P. H.; Chang, H. H.; Hahn, L. J.; Lin, B. R.; Chen,
Y. J.; Jeng, J. H. The induction of prostaglandin E2 production, interleukin-6
production, cell cycle arrest, and cytotoxicity in primary oral keratinocytes and KB
cancer cells by areca nut ingredients is differentially regulated by MEK/ERK
activation. J. Biol. Chem. 279:5067650683; 2004.
[30] Jeng, J. H.; Wang, Y. J.; Chiang, B. L.; Lee, P. H.; Chan, C. P.; Ho, Y. S.; Wang, T. M.; Lee, J. J.;
Hahn, L. J.; Chang, M. C. Roles of keratinocyte inammation in oral cancer: regulating
the prostaglandin E2, interleukin-6 and TNF-alpha production of oral epithelial cells
by areca nut extract and arecoline. Carcinogenesis 24:13011315; 2003.
[31] Yap, L. P.; Garcia, J. V.; Han, D.; Cadenas, E. The energyredox axis in aging and
age-related neurodegeneration. Adv. Drug Delivery Rev. 61:12831298; 2009.
[32] Jenner, P.; Olanow, C. W. Oxidative stress and the pathogenesis of Parkinson's
disease. Neurology 47:S161S170; 1996.
[33] Calabrese, V.; Sultana, R.; Scapagnini, G.; Guagliano, E.; Sapienza, M.; Bella, R.;
Kanski, J.; Pennisi, G.; Mancuso, C.; Stella, A. M.; Buttereld, D. A. Nitrosative
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
[45]
[46]
1479
stress, cellular stress response, and thiol homeostasis in patients with Alzheimer's
disease. Antioxid. Redox Signaling 8:19751986; 2006.
Sorce, S.; Krause, K. H. NOX enzymes in the central nervous system: from signaling
to disease. Antioxid. Redox Signaling 11:24812504; 2009.
Shelat, P. B.; Chalimoniuk, M.; Wang, J. H.; Strosznajder, J. B.; Lee, J. C.; Sun, A. Y.;
Simonyi, A.; Sun, G. Y. Amyloid beta peptide and NMDA induce ROS from NADPH
oxidase and AA release from cytosolic phospholipase A2 in cortical neurons.
J. Neurochem. 106:4555; 2008.
Loh, K. P.; Huang, S. H.; De Silva, R.; Tan, B. K.; Zhu, Y. Z. Oxidative stress: apoptosis
in neuronal injury. Curr. Alzheimer Res. 3:327337; 2006.
Brown, D. I.; Griendling, K. K. Nox proteins in signal transduction. Free Radic. Biol.
Med. 47:12391253; 2009.
Circu, M. L.; Aw, T. Y. Reactive oxygen species, cellular redox systems, and
apoptosis. Free Radic. Biol. Med. 48:749762; 2010.
Liu, B.; Du, L.; Hong, J. S. Naloxone protects rat dopaminergic neurons against
inammatory damage through inhibition of microglia activation and superoxide
generation. J. Pharmacol. Exp. Ther. 293:607617; 2000.
Chu, N. S. Neurological aspects of areca and betel chewing. Addict. Biol. 7:111114;
2002.
Cawte, J. Psychoactive substances of the South Seas: betel, kava and pituri. Aust. N. Z. J.
Psychiatry 19:8387; 1985.
Liu, B.; Andrieu-Abadie, N.; Levade, T.; Zhang, P.; Obeid, L. M.; Hannun, Y. A.
Glutathione regulation of neutral sphingomyelinase in tumor necrosis factoralpha-induced cell death. J. Biol. Chem. 273:1131311320; 1998.
Reiter, R. J. Oxidative processes and antioxidative defense mechanisms in the
aging brain. FASEB J. 9:526533; 1995.
Choi, J.; Rees, H. D.; Weintraub, S. T.; Levey, A. I.; Chin, L. S.; Li, L. Oxidative
modications and aggregation of Cu,Zn-superoxide dismutase associated with
Alzheimer and Parkinson diseases. J. Biol. Chem. 280:1164811655; 2005.
Trachootham, D.; Lu, W.; Ogasawara, M. A.; Nilsa, R. D.; Huang, P. Redox regulation
of cell survival. Antioxid. Redox Signaling 10:13431374; 2008.
Niizuma, K.; Endo, H.; Chan, P. H. Oxidative stress and mitochondrial dysfunction
as determinants of ischemic neuronal death and survival. J. Neurochem. 109
(Suppl. 1):133138; 2009.