Clin Oral Invest (2001) 5:5156

Springer-Verlag 2001

O R I G I N A L A RT I C L E

Yu-Chao Chang Chao-Chin Hu Chong-Kuei Lii

Kuo-Wei Tai Shyh-Hwang Yang Ming-Yung Chou

Cytotoxicity and arecoline mechanisms in human

gingival fibroblasts in vitro

Received: 16 February 2000 / Accepted: 25 July 2000

Abstract Betel nut chewing, like cigarette smoking, is a

popular oral habit which impinges on the daily lives of a
population of approximately 200 million. People who
chew betel nuts have a higher prevalence of periodontal
diseases than those who do not. Many of the undesirable
effects of betel nuts have been attributed to arecoline, a
major component of the particular alkaloid in betel nuts.
In this in vitro study, we have focused on the effects of
arecoline and the role it could play in periodontal breakdown via its direct effects on human gingival fibroblasts.
Human gingival fibroblasts were derived from three
healthy individuals undergoing crown-lengthening procedures. We found that arecoline is cytotoxic to human
gingival fibroblasts at a concentration higher than
50 g/ml by depleting intracellular thiols and inhibiting
mitochondrial activity (P<0.05). In addition, the cells
displayed a marked arrest at G2/M phase in a dose-dependent manner. Repeated and long-term exposure to
arecoline could impair the gingival fibroblast functions.
As they are cytotoxic, the use of betel nut products in
conjunction with periodontal therapy may interfere with
optimal healing and/or lead to further periodontal breakdown.

Yu-Chao Chang Ming-Yung Chou ()

Institute of Stomatology,
Chung Shan Medical and Dental College, 23 Section 1,
Taichung-Kang Rd, Taichung, Taiwan,
Tel.: +886-4-22015111 Ext. 6268, Fax: +886-4-22024349
Chao-Chin Hu
Department of Biochemistry,
Chung Shan Medical and Dental College,
Taichung, Taiwan
Chong-Kuei Lii
Institute of Nutritional Science,
Chung Shan Medical and Dental College, Taichung,
Taiwan
Kuo-Wei Tai Shyh-Hwang Yang
School of Dentistry, Chung Shan Medical and Dental College,
Taichung, Taiwan

Keywords Arecoline Fibroblasts Thiols depletion

Mitochondrial function Cell cycle

Introduction
The habit of chewing betel nuts is a common custom
among peoples from Taiwan, Southwest Asia and India
[17]. Important oral manifestations attributed to the habit
are submucous fibrosis and oral cancer [18]. In addition,
people who chew betel nuts may have a higher incidence
and severity of periodontal diseases. These have been attributed to inadequate levels of oral hygiene, increasing
accumulation of dental plaque and calculus, and more
severe gingivitis [12,28].
Most of the literature on the oral ramifications of betel nut use is based on clinical observations and measurements. However, the underlying mechanisms by which
betel nut chewing induces these diseases are complex
and not fully understood. The cytotoxic and genotoxic
effects of betel nut ingredients are considered to be the
main causative factors. Arecoline, the main betel nut alkaloid, has recently been found to exert cytotoxicity and
inhibit the growth of various cultured human cells including oral epithelial cells [8,24] and fibroblasts
[2,3,6,7,9,26]. Moreover, little is known about the actual
biological effects of betel nut chewing or its constituents
on the periodontium.
Betel nuts contain cytotoxic substances, such as arecoline, which might be important for the long-term effects
of betel nut chewing on the periodontium. Previously,
about 140 g/ml arecoline has been reported in saliva
while chewing betel nuts [16]. Betel nuts contain several
alkaloids of which arecoline is the most abundant and
many of the undesirable effects have been attributed to it
[5]. In Taiwan, two million people chew betel nuts habitually [10], which is suspected of elevating the incidence
and severity of periodontitis [29]. This implies that betel
nut chewing might be an additional risk factor for periodontal disease in Taiwan as normal gingival fibroblast
functioning is essential for the maintenance of periodon-

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tal connective tissue, as well as wound healing. The potential toxicological implications of arecoline on the gingival fibroblasts remain elucidated. The present study
was undertaken to investigate in vitro whether arecoline
causes pathobiological effects on cultured human gingival fibroblasts.

Materials and methods

Arecoline was purchased from Sigma Chemical Co. (St.
Louis, Mo., USA), dissolved in distilled water and added
to the culture medium. The final concentrations tested
in this study were 0400 g/ml. All culture materials
were obtained from Gibco (Grand Island, N.Y., USA).
Hoechst 33258 (H33258), alamar blue dye, propidium
iodide and perchloric acid were purchased from Sigma
Chemical Co..
Cell Culture
Healthy gingival connective tissues were obtained from
three healthy individuals (aged 2132 years, average 26
years) during crown-lengthening procedures with informed consent. The tissues were minced using sterile
techniques and washed twice in phosphate buffer saline
supplemented with antibiotics (100 U/ml penicillin,
100 g/ml streptomycin and 0.25 g/ml of fungizone).
Explants were placed into 60-mm Petri dishes and maintained in Dulbeccos modified Eagles medium (DMEM)
supplemented with 10% fetal calf serum (FCS) and antibiotics as described above. The cultures were maintained
at 37 C in a humidified atmosphere of 5% CO2 and 95%
air. Confluent cells were detached by treating them with
0.25% typsin and 0.05% EDTA for 5 minutes, and aliquots of separated cells were subcultured with the same
medium. Cell cultures between the third and eighth passages were used in this study.
Cytotoxicity assay
Cytotoxicity was assessed with H33258 fluorescent dye
which intercalates in the DNA of viable cells[19]. The
cells were plated at an initial density of 5104 cells/well
into 24-well culture plates and allowed to attach for 24 h.
Then cells were exposed to various concentrations of
arecoline for a further 24 h. Finally, the medium was removed and the plates frozen at -80 C and stored frozen
until ready for further processing. After thawing, plates
were added to each well with 100 l distilled water and
incubated for 1 h at room temperature before being refrozen at -80 C for 90 mins. and thawed at room temperature. Then 100 l TNE buffer (10 mM Tris, 1 mM
EDTA, 2 M NaCl, pH 7.4) containing 20 g/ml H33258
dye was added. After a 90 min. incubation period in
dark, the fluorescence intensity of the wells was measured using a fluorescent plate reader (CytoFluor 2300,

Millipore, USA) at an excitation wavelength of 365 nm

and an emission wavelength of 450 nm.
Measurement of cellular thiols
A rapid and sensitive high liquid performance chromatography (HPLC) method was used for determination of
nanomole levels of glutathione (GSH) and glutathione
disulfide (GSSG) as described by Reed et al.[20]. Fibroblasts were incubated at a concentration of 1106
cells/ml with different concentrations of arecoline for
24 h. Intracellular thiols were determined by adding 1 ml
5% perchloric acid containing 2.5 mM phenanthroline to
each Petri dish. The plates were then scraped and homogenates centrifuged at 10,000 g for 10 min. The acidsoluble thiols were in the supernatant fraction and the
pellets were used for the protein assay. The acid-soluble
thiols were measured using the HPLC method. Briefly,
the procedure is based upon the initial formation of
S-carboxymethyl derivatives of free thiols with iodoacetic acid followed by conversion of free amino groups to
2,4-dinitrophenyl derivatives by reaction with 1-fluoro2,4-dinitrobenzene. Chromatography of the reaction
mixture without sample isolation is carried out on a
3-aminopropylsilane derivatized silica column and elution with a sodium or ammonium acetate gradient in a
water-methanol-acetic acid solvent at pH 4.5. The
amounts of thiols were expressed as nmol/mg protein.
Measurement of cellular mitochondrial activity
The effects of arecoline on mitochondrial functioning
were measured by means of a colorimetric assay using
alamar blue dye [27]. Briefly, fibroblasts were seeded
5104 cells per well into 24-well culture plates and incubated to attach for 24 h. The culture medium was replaced with fresh DMEM and various concentrations of
arecoline. After trypsining, alamar blue dye was added to
a 250 l aliquot of cell suspensions for 2 h at 37 C. Colorimetric determination was carried out at 570 nm and
600 nm on a plate reader (CytoFluor 4500, Millipore,
USA). The percent inhibition of mitochondria activity in
response to a test agent was compared to that of untreated cells.
Cell cycle analysis
Cell cycle analysis was performed on the cells stained
with propidium iodine as described by Sorenson and
Eastman[23], but modified slightly [25]. Controlled cells
and arecoline-treated cells were detached by treating
them with 0.05% trypsin and then washed twice with
cold PBS and fixed in 80% ethanol in PBS at 20 C.
After 12 h, fixed cells were pelleted, gently re-suspended
in cold PBS and supplemented with 0.5% propidium iodide plus 50 g/ml RNase. Then, the samples were incubated at 37 C for 30 mins., stored in the dark at 4 C,
and analyzed using a flow cytometer on a Becton Dick-

53

inson FACScan (Becton Dickinson Immunocytochemistry Systems, San Jose, CA, USA).
Statistical analysis

and 56% of GSH, respectively. Fibroblasts in the control

group maintained their original GSH levels during the
incubation period.
During oxidative stress, GSH peroxidase oxidizing
reduced the GSH to its oxidizing form GSSG. As shown

Three replicates of each concentration were performed

for each test. All assays were repeated three times to ensure reproducibility. Statistical analysis was by one-way
analysis of variance (ANOVA). Tests showing differences in the treatments were analyzed by Duncans test
and a value of P<0.05 was considered statistically significant.

Results
Figure 1 shows the effects of arecoline on incorporation
of H33258 for DNA content by fluorescence intensity.
Arecoline inhibited the DNA content of gingival fibroblasts in a dose-dependent manner. However, arecoline
was cytotoxic at a concentration level higher than
50 g/ml wherea at a concentration of 200 g/ml arecoline inhibited the DNA contents by only 50% of those in
the untreated control. Moreover, numerous floating cells
were seen with a phase-contrast microscope.
The amounts of thiols in human gingival fibroblasts
were determined using the HPLC method (Fig. 2). In
this study, arecoline significantly depleted intracellular
GSH in a dose-dependent manner. At a concentration of
50 g/ml and 100 g/ml, arecoline depleted about 22.5%

Fig. 1 Cellular toxicity measured using H33258 fluorescent dye

on gingival fibroblasts after exposure to various concentrations of
arecoline for 24 h. Results are expressed as percentages of fluorescence intensity relative to the untreated control. Data are shown as
meanSD (bars). * denotes significant differences from control
values with P<0.05.

Fig. 2 Effects of arecoline on intracellular thiol levels in human

gingival fibroblasts detected by the HPLC method. The intracellular GSH and GSSG levels were expressed as nanomoles per milligram ofprotein. All values are shown as meanSD. * denotes significant differences from control values with P<0.05

Fig. 3 Effects of arecoline on the functional mitochondrial activities by alamar blue dye assay. Results are expressed as percentages of untreated controls. All values are shown as meanSD. * denotes significant differences from control values with P<0.05

54

Fig. 4 Cell cycle analysis was performed on the cells stained with
propidium iodine using flow cytometric DNA analysis of gingival
fibroblasts during a 48-h incubation period. Cells absent of arecoline display a normal profile, peaking at 99.83 units of fluorescence representing cells in G0/G1 and at 193.66 units of fluorescence representing cells in G2/M. Arecoline-treated cells displayed
a marked arrest during the G2/M phase of the cell cycle

in Fig. 2, thiol depletion was not coupled with oxidation

of GSH to GSSG.
An alamar blue dye assay was used to demonstrate
that arecoline inhibits mitochondrial activity on human
gingival fibroblasts (Fig. 3). It reduced the activity of the

mitochondrial cytochromoxidase system. At concentrations of 50200 g/ml, arecoline inhibited 16% through
56% of functional mitochondrial activities.
Figure 4 presents the effects of various concentrations
of arecoline on the cell cycle of human gingival fibroblasts during a 48-h incubation period. As shown by
flow cytometric analysis, in the absence of arecoline
cells display a normal profile, peaking at 99.83 units of
fluorescence representing cells in G0/G1 and at 193.66
units of fluorescence represented in G2/M. The proportion of arecoline-treated cells arrested at G2/M phase depends directly on the arecoline concentration.

55

Discussion
The gingival fibroblast is an important component of the
periodontal connective tissues involved both in wound
healing responses and in normal metabolic processes.
Studies have suggested that the cell growth, proliferation, and matrix synthesis of fibroblasts are necessary for
regeneration of a connective tissue attachment [1,11].
Previous studies reported that arecoline inhibited cell
growth, proliferation and collagen synthesis in human
gingival fibroblasts in a dose dependent manner [3,7].
However, the potential cytotoxicity mechanisms of arecoline on human gingival fibroblasts still remain to be
elucidated.
In the present study, we have focused on the effects of
arecoline and the role it could play in periodontal breakdown via its direct effects on human gingival fibroblasts.
We discovered that arecoline is cytotoxic to human gingival fibroblasts by depleting intracellular thiols and inhibiting mitochondrial activity. Furthermore, cell displayed a G2-M blockage of the cell cycle. The use of areca nut products in conjunction with periodontal therapy
may interfere with optimal healing and/or lead to further
periodontal breakdown via its cytotoxicity.
Thiols are involved in a variety of intercellular functions and also play an important role in cellular defense
against many reactive foreign compounds [13]. During
the present study, at a concentration of up to 50 g/ml,
arecoline depleted intracellular thiols. This finding indicated that thiol depletion might be one of the mechanisms underlying arecoline cytotoxicity. These results
confirm our previous study [2] and that of Jeng et al. [8],
i.e., cytotoxicity caused by arecoline can be prevented by
adding extracellular thiols.
GSH is a predominant physiological thiol that serves
many important biological functions. It helps maintain
membrane integrity, optimal transport of amino acids
and enzyme activity. In addition, GSH has been documented as having regulatory effects on cell proliferation
activity [22]. Since cell proliferation is critical for the
maintenance of periodontal tissues and for optimal
wound healing responses, intracellular GSH depletion by
arecoline might impair the reparative and regenerative
potential of periodontal tissues of persons who chew betel nuts.
During oxidative stress, GSH peroxidase oxidizing reduces GSH to its oxidizing form GSSG. In this study,
when the cellular content of GSH was measured, a similar
decrease was observed without any concomitant oxidative
formation of GSSG. These results are in general agreement with those reported by Sundqvist et al.[24] who
found that arecoline could delete free low-molecularweight thiols in human buccal epithelial cells and that thiol depletion was not coupled with oxidative stress in these
cells. The metabolism of arecoline is not a free-radicalgeneration system in human gingival fibroblasts.
The alamar blue dye technique has been shown to detect changes in cellular metabolic activity [15]. In addition, this assay is based on the reduction of tetrazolium

salts by a mitochondrial cytochromoxidase system [14].

Such activity does not take place in non-viable cells. In
the present study, arecoline was found to decrease mitochondrial function. Due to impairment of mitochondrial
function, gingival fibroblasts might lack of energy for
wound repair and regeneration during periodontal therapy.
In this study, a H33258 fluorescence assay was used
to show that arecoline inhibits the DNA content of gingival fibroblasts . A previous study demonstrated that arecoline inhibited total DNA synthesis and unscheduled
DNA synthesis in gingival keratinocytes [8]. Accordingly, in response to various types of DNA damage, the cell
cycle checkpoints and cell death signals are activated to
stop cell growth and eliminate multiplication of the generally altered cells. Two checkpoints in the cell cycle, G1
and G2, play a very important role in the regulation of
cells proceeding to S and M phases, respectively [17].
Damaged cells stop DNA replication at the G1 or G2
phase, presumably allowing the repair systems to function before the next round of cell cycle. Our findings on
cell arrest at the G2/M phase in human gingival fibroblasts exposed to arecoline, and its direct dependence on
the concentration, are novel and provide direct evidence
of phase-specific cell cycle regulation. In addition, a
similar result was found by Chatterjee and Deb [4], i.e.,
that arecoline induced a greater delay in the cell cycle of
murine bone marrow cells in vivo.
In the present study, arecoline could impair gingival
fibroblasts through various mechanisms. Normally the
gingival epithelium would act as an effective barrier
against arecoline, preventing its accumulation in connective tissue layers. It is very difficult, if not possible, to
measure how much arecoline affects underlying fibroblasts after permeating the epithelial barrier. Moreover,
the inflammatory change would enhance the permeability of the epithelium [21]. Since periodontitis is a chronic
inflammatory condition, arecoline might pass through
the epithelium more easily while the patient is chewing a
betel nut. In addition, recent study has shown that betel
quid ingredients, including arecoline, could induce cytotoxicity in human gingival keratinocytes [8]. While
chewing betel nuts, the gingival epithelium might not be
an effective barrier against various cytotoxic substances
leaching from areca nuts because normal gingival fibroblast functioning is critical to the maintenance of periodontal health and wound healing. These detrimental effects of arecoline might impair the reparative and regenerative potential of periodontal tissues of people who
chew betel nuts. The inhibitory effects of arecoline on
human gingival fibroblasts may be an important issue for
us to understand how betel nut chewing affects periodontal health. However, more detailed studies should be undertaken to clarify its effects in vitro as well as in vivo.

56

Conclusions
The present investigation presents evidence showing the
cytotoxicity and mechanism used by arecoline on human
gingival fibroblasts under experimental conditions. We
found that arecoline is cytotoxic to human gingival fibroblasts at a concentration higher than 50 g/ml by depleting intracellular thiols and inhibiting mitochondrial
activity. In addition, cells displayed a marked arrest at
the G2/M phase in a dose-dependent manner. Therefore,
our data indicate that the use of betel nut products in
conjunction with periodontal therapy may interfere with
optimal healing and/or lead to further periodontal breakdown due to its cytotoxicity.
Acknowledgments This work was supported by a research grant
from CSMC 86-OM-B-010 and CSMC 87-OM-B-019 from the
Chung Shan Medical and Dental College.

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