Ingredients contribute to variation in production of reactive oxygen species by areca quid

Article abstract of DOI:10.1080/15287390500360224

Areca quid (AQ) chewing has been implicated an independent risk factor for the development of oral cancer. Taiwanese areca quid (AQ) refers to a combination of areca nut (AN), lime, and inflorescence of Piper betle Linn. (IPB) or Piper betle leaf (PBL). Studies of AQ in other countries reported that AN extract combined with lime generates reactive oxygen species (ROS), such as hydroxyl radical (HO^? ), known to be a contributing factor in oral mucosa damage. To determine whether HO^? is formed in the oral cavity during AQ chewing, the formation of meta -tyrosine ( m -Tyr) and ortho -tyrosine ( o -Tyr) from l -phenylalanine (Phe) was confirmed. It was demonstrated that combined aqueous extracts of AN, lime, metal ions (such as Cu 2+ and Fe 2+ ), and IPB or PBL produced HO^?. Thus, the yield of HO^? significantly increases when higher amounts of IPB or lime are added and also when Cu 2+ and Fe 2+ are increased. Further, the omission of any one of these ingredients significantly reduces the formation of HO ^?. Our results found that chewing AQ with IPB generated significantly higher HO^? than chewing AQ with PBL, and may result in greater oxidative damage to the surrounding oral mucosa. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/15287390500360224

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Ingredients Contribute to Variation in
Production of Reactive Oxygen Species
by Areca Quid
a d
a b
a
c
Ping-Ho Chen
, Chi-Cheng Tsai
, Ying-Chu Lin , Ying-Chin Ko
d
e
e f
g
d
,
Yi-Hsin Yang , Tien-Yu Shieh
, Pei-Shan Ho , Chien-Ming Li ,
h
a f
Albert Min-Shan Ko & Chung-Ho Chen
a
Graduate Institute of Dental Sciences, College of Dental Medicine,
Kaohsiung Medical University , Kaohsiung, Taiwan
b
Division of Periodontics, Department of Dentistry , Kaohsiung
Medical University, Chung-Ho Memorial Hospital, Kaohsiung Medical
University , Kaohsiung, Taiwan
c
Graduate Institute of Medicine, College of Medicine, Kaohsiung
Medical University , Kaohsiung, Taiwan
d
Division of Environmental Health and Occupational Medicine,
National Health Research Institutes , Kaohsiung, Taiwan
e
Graduate Institute of Oral Health Sciences, College of Dental
Medicine, Kaohsiung Medical University , Kaohsiung, Taiwan
f
Division of Oral and Maxillofacial Surgery, Department of
Dentistry , Kaohsiung Medical University Chung-Ho Memorial
Hospital, Kaohsiung Medical University , Kaohsiung, Taiwan
g
Faculty of Dental Hygiene, College of Dental Medicine, Kaohsiung
Medical University , Kaohsiung, Taiwan
h
School of Medicine, University of Western Australia , Perth,
Australia
Published online: 24 Feb 2007.
To cite this article: Ping-Ho Chen , Chi-Cheng Tsai , Ying-Chu Lin , Ying-Chin Ko , Yi-Hsin Yang ,
Tien-Yu Shieh , Pei-Shan Ho , Chien-Ming Li , Albert Min-Shan Ko & Chung-Ho Chen (2006)
Ingredients Contribute to Variation in Production of Reactive Oxygen Species by Areca Quid,
Journal of Toxicology and Environmental Health, Part A: Current Issues, 69:11, 1055-1069, DOI:
10.1080/15287390500360224
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Journal of Toxicology and Environmental Health, Part A, 69:1055–1069, 2006
Copyright© Taylor & Francis Group, LLC
ISSN: 1528–7394 print / 1087–2620 online
DOI: 10.1080/15287390500360224
INGREDIENTS CONTRIBUTE TO VARIATION IN PRODUCTION
OF REACTIVE OXYGEN SPECIES BY ARECA QUID
1
,4
1,2
1
3,4
Ping-Ho Chen , Chi-Cheng Tsai , Ying-Chu Lin , Ying-Chin Ko ,
5
5,6
7
4
Yi-Hsin Yang , Tien-Yu Shieh , Pei-Shan Ho , Chien-Ming Li ,
Albert Min-Shan Ko , Chung-Ho Chen
8
1,6
1
Graduate Institute of Dental Sciences, College of Dental Medicine,
Kaohsiung Medical University, Kaohsiung, Taiwan
2
Division of Periodontics, Department of Dentistry, Kaohsiung Medical
University, Chung-Ho Memorial Hospital, Kaohsiung Medical University,
Kaohsiung, Taiwan
3
Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical
University, Kaohsiung, Taiwan
4
Division of Environmental Health and Occupational Medicine, National
Health Research Institutes, Kaohsiung, Taiwan
5
Graduate Institute of Oral Health Sciences, College of Dental Medicine,
Kaohsiung Medical University, Kaohsiung, Taiwan
6
Division of Oral and Maxillofacial Surgery, Department of Dentistry,
Kaohsiung Medical University Chung-Ho Memorial Hospital, Kaohsiung
Medical University, Kaohsiung, Taiwan
7
Faculty of Dental Hygiene, College of Dental Medicine, Kaohsiung
Medical University, Kaohsiung, Taiwan
8
School of Medicine, University of Western Australia, Perth, Australia
Areca quid (AQ) chewing has been implicated an independent risk factor for the development
of oral cancer. Taiwanese areca quid (AQ) refers to a combination of areca nut (AN), lime, and
inflorescence of Piper betle Linn. (IPB) or Piper betle leaf (PBL). Studies of AQ in other coun-
tries reported that AN extract combined with lime generates reactive oxygen species (ROS),
·
such as hydroxyl radical (HO ), known to be a contributing factor in oral mucosa damage. To
·
determine whether HO is formed in the oral cavity during AQ chewing, the formation of meta-
tyrosine (m-Tyr) and ortho-tyrosine (o-Tyr) from L-phenylalanine (Phe) was confirmed. It was
2+
2+
demonstrated that combined aqueous extracts of AN, lime, metal ions (such as Cu and Fe ),
·
·
and IPB or PBL produced HO . Thus, the yield of HO significantly increases when higher
amounts of IPB or lime are added and also when Cu2+ and Fe are increased. Further, the
2+
·
omission of any one of these ingredients significantly reduces the formation of HO . Our results
·
found that chewing AQ with IPB generated significantly higher HO than chewing AQ with PBL,
and may result in greater oxidative damage to the surrounding oral mucosa.
Received 18 January 2005; accepted 22 April 2005.
This study was supported by a grant from the Division of Environmental Health and Occupational
Medicine, National Health Research Institutes, Kaohsiung 807, Taiwan (NHRI-CN-IN-9006P).
Address correspondence to Chung-Ho Chen, Graduate Institute of Dental Sciences, No. 100 Shin-
Chuan 1st Road, Kaohsiung Medical University, Kaohsiung 807, Taiwan. E-mail: chench@cc.kmu.edu.tw
1055

1056
P.-H. CHEN ET AL.
The chewing of areca quid (AQ) and tobacco has been closely linked to
oral cancer in parts of Southeast Asia and India (IARC, 1985). Oral cancer in
Taiwan is the fifth leading cause of cancer death for males (Department of
Health [R.O.C.], 2000). Although areca quid (AQ) is not chewed normally with
tobacco in Taiwan, a strong link has been found in areca quid (AQ) use with
oral cancer (Ko et al., 1995; Yang et al., 2001; Lee et al., 2003). Epidemiolog-
ical studies demonstrated that areca quid (AQ) chewing was an independent
risk factor for oral cancer and the development of oral precancerous diseases
(
Ko et al., 1995; Lee et al., 2003; IARC, 2003).
In Taiwan, where the practice of betel-quid chewing is widespread, approxi-
mately 2 million people chew areca quid (AQ) habitually (Ko et al., 1992). The
two major types of areca quid (AQ) are Lao-hwa quid, a mixture of tender areca
nut (AN), an inflorescence of Piper betle Linn. (IPB), and red lime paste, and “betel
quid,” a mixture of tender areca nut (AN), Piper betle leaf (PBL), and white lime
paste (Ko et al., 1992; Yang et al., 2001). Under an alkaline condition of pH ≥ 9.5,
areca quid (AQ) extract and lime generate reactive oxygen species (ROS) such as
•
hydroxyl radical (HO) (Nair et al., 1987, 1990, 1992, 1995). When catalyzed by
•
2+
2+
metal ions such as Cu and Fe , HO yields are increased, and 8-hydroxy-2′-
deoxyguanosine (8-OH-dG) is formed in vitro, leading to possible oxidative DNA
damage (Nair et al., 1987, 1995; Liu et al., 1996; Chen et al., 2002). Indeed,
genetic damage in oral mucosa is known to be associated with oral cancer (Nair
et al., 1987, 1990, 1992, 1995; Liu et al., 1996; Chen et al., 2002).
Although the relative risks of oral cancer for the two types of areca quid (AQ)
have been studied (Ko et al., 1995; Lee et al., 2003), their relative oxidative effects
•
have not been compared. Two markers of HO, meta-tyrosine (m-Tyr) and ortho-
tyrosine (o-Tyr) from L-phenylalanine (Phe), were measured in the oral cavity (Nair
et al., 1995; Chen et al., 2002). The formation of m-Tyr and o-Tyr from Phe in
vitro has been demonstrated via a metal-catalyzed oxidation (Huggins et al., 1993)
•
or via the HO-generating systems such as ultraviolet (UV) irradiation (Ishimitsu
et al., 1990) and ionizing radiation (Karam & Simic, 1990). Therefore, m-Tyr and
•
o-Tyr formation are useful markers of HO and have been shown to induce oxida-
tive damage in the human oral cavity (Nair et al., 1995; Chen et al., 2002). In this
study, m-Tyr and o-Tyr were measured in the two types of areca quid (AQ), and
•
HO was measured and compared in the saliva of their users.
METHODS
Subjects Collection
The present study was reviewed and approved by the Human Study Commit-
tee of the Institutional Review Board (IRB) of Kaohsiung Medical University (KMU).
Materials and Chemicals
Lao-hwa quid and betel quid samples were simultaneously obtained from
two different vendors during the August areca quid (AQ) season in Kaohsiung

REACTIVE OXYGEN SPECIES BY ARECA QUID
1057
City, Taiwan. Phe (Ajinomoto, Japan) was obtained from the China Chemical
Company (Taiwan). The m- and o-Tyr and para-tyrosine (p-Tyr) were pur-
chased from Sigma (Sigma Chemical Company, St. Louis, MO). All other
chemicals were of analytical grade, and solutions were prepared with double-
distilled water.
Analytical Quantification
Multiple level calibrations with m-, o-, and p-Tyr standards were per-
formed by linear regression. The concentration of m- and o-Tyr ranged from
0
.1 to 100 μM, and peak areas were increased linearly. The correlation coeffi-
cients (R) of m- and o-Tyr were .998 and .999, respectively. The concentra-
tions of m- and o-Tyr (means ± standard deviation [SD]) in AQ samples were
estimated from peak areas by comparison with m- and o-Tyr standards.
m- and o-Tyr Formation From Phe in Lao-hwa Quid
and Betel Quid Samples
Samples of different compositions of Lao-hwa quid and betel quid were
prepared for analysis of m- and o-Tyr. Five quids of Lao-hwa quid and betel
2+
quid were prepared with Phe and transition metal ions, such as Cu and
2+
Fe , in 100 ml of 37°C distilled water and powdered in a blender. The solu-
tion was incubated in a shaker bath at 37°C for 2 h and used to determine pH.
A reaction was commenced by adding lime to each 100-ml sample, and
stopped by adding 2 ml glacial acetic acid. After centrifugation (8000 × g) and
filtration through a 0.45-μm membrane filter, the solution was frozen and lyo-
philized immediately. The lyophilized samples were stored at −20°C prior
analysis by high-performance liquid chromatography (HPLC).
m- and o-Tyr Formation From Phe in Human Saliva
During Areca Quid Chewing
Fifteen healthy volunteers (8 males and 7 females, aged 25–30 yr) with no
previous history of habitual areca quid (AQ) use were recruited, and randomly
divided into three groups. The first group chewed 2 quids of Lao-hwa with 20
mg Phe (n = 5) added. The second group chewed 2 quids of betel quid with
20 mg Phe (n = 5) added. The third group chewed gum with 20 mg Phe (n = 5).
Saliva samples were collected from each subject after 15 min of chewing to
determine pH. The saliva samples were deproteinated by adding 25% trich-
loroacetic acid, and supernatant was frozen and lyophilized immediately.
The lyophilized samples were prepared at −20°C prior to analysis by high-
performance liquid chromatography (HPLC).
Analysis of m-, o-, and p-Tyr Formation
The formation of m-, o-, and p-Tyr was measured according to methods
described in a previous study, with minor modifications (Nair et al., 1995). Sam-
ples were separated from Lichrospher RP-18(e) (5 μm, 4.0 mm × 250 mm;
Merck, Germany) with aqueous solutions containing 0.5% acetic acid and

1058
P.-H. CHEN ET AL.
0
.1% NaCl at a 1 ml/min flow rate. The effluent was monitored with excitation at
2
75 nm and emission at 305 nm using a fluorescence detector (Jasco FP-1520,
Japan). Analyses carried out in four samples of each group and their mean
concentrations (means ± SD, n = 4) are shown in the Results section.
The occurrence of m- and o-Tyr in human saliva was confirmed by liquid
chromatography–mass spectroscopy (LC-MS). Analytes (m- and o-Tyr) were
separated on a C-18 analytical column (3 μm, 4.0 mm × 55 mm; Merck,
Darmstadt, Germany). The mobile phase of high-performance liquid chroma-
tography (HPLC) was delivered using two micro pumps (PE series 200, Perkin-
Elmer, Elmer, Norfolk, CT) with 5% acetonitrile in 0.1% formic acid at a flow
rate of 0.6 ml/min. An API 3000 mass spectrometer (PE-SCIEX, Concord,
Ontario, Canada) was used in the present study. A TurboIon Spray source was
operated in positive ionization mode with typical settings as follows: nebulizer
gas 10, curtain gas 10, CAD gas 4, drying gas 8 L/min, electrospray voltage
4
500 V, ring voltage 300 V, orifice voltage 30 V, collision energy 30 eV, and a
drying temperature of 400°C. Qualitation of each analyte was achieved using
the product’s ion scan mode with a precursor ion of m/z 182 selected.
Statistical Analysis
The general linear model (GLM) procedure with statistical multiple com-
parison by Tukey’s method was used for the primary analysis. Group means
are presented with SD. The differences in means ± SD between groups and
subsets were assessed by one-way analysis of variance (ANOVA) or Mann–
Whitney nonparametric test. The p value for significance was set at p < .05.
The SPSS v11.0.1 software package (SPSS, Inc.) was employed for statistical
analysis of all data.
RESULTS
Quantifying Areca Quid Samples
According to an IARC report, habitual areca quid (AQ) users chewed an
average of 5–20 quids a day (IARC, 1985). Each sample in this in vitro study
contained 5 quids to simulate the chewing habit. Each Lao-hwa sample (5
quids) dissolved in 100 ml water contained a mean concentration of 0.15 g/ml
of areca nut (AN), 0.02 g/ml of IPB, and 0.02 g/ml of red lime. On the other
hand, the betel quid sample (5 quids) dissolved in 100 ml water contained areca
nut (AN) (0.15 g/ml), PBL (0.05 g/ml), and white lime (0.005 g/ml) (Table 1).
Formation of m- and o-Tyr From Phe in Lao-hwa Quid Groups 1–6
In the Lao-hwa quid groups (groups 1–6), significant differences in m- and
o-Tyr from Phe were observed in all variations of ingredients (Figure 1). In Lao-hwa
quid group 1 [AN (0.15 g/ml) + IPB (0.02 g/ml) + red lime (0.02 g/ml)], the
reaction generated 68.01 ± 7.99 μM m-Tyr and 65.51 ± 6.59 μM o-Tyr at pH
2+
8
.85. With the addition of Cu (2 mM) under the same condition, the formation

REACTIVE OXYGEN SPECIES BY ARECA QUID
1059
TABLE 1. Mean Concentration of Ingredients Contained in Lao-hwa Quid and
Betel Quid From Two Different Vendors in Kaohsiung Region of Taiwan
Ingredient
Mean concentration (g/ml) of 5 quids
Lao-hwa quid sample
a
AN
0.15
0.02
0.02
b
IPB
Red lime
Betel quid sample
AN
0.15
0.05
0.005
c
PBL
White lime
Note. Different types of Lao-hwa quid and betel quid were mixed with 100 ml of
distilled water and powdered in a blender for 5 min.
a
Areca nut (AN).
Inflorescence of Piper betle Linn. (IPB).
Piper betle leaf (PBL).
b
c
FIGURE 1. Formation of m- and o-Tyr from Phe with various Lao-hwa quid ingredients (1–6): 1, AN + IPB +
2
+
2+
red lime; 2, AN + IPB + red lime + Cu (2 mM); 3, AN + IPB + red lime + Cu (5 mM); 4, AN + IPB +
2+
red lime + Fe (5 mM); 5, AN + 5 × IPB (0.10 g/ml) + red lime; 6, AN + IPB + 5 × red lime (0.10 g/ml);
means ± S.D. (n = 4) are presented. An asterisk denotes significance at p < .05 as compared with group 1.

1060
P.-H. CHEN ET AL.
of m- and o-Tyr was significantly higher in group 2 (pH 8.82) than in group 1.
2
+
The higher concentration of Cu (5 mM) was found to increase m- and o-Tyr
2
+
formation significantly at pH 8.84 (group 3 vs. group 1). The presence of Fe
(
5 mM) also enhanced m- and o-Tyr formation at pH 8.95, as seen in group 4.
Compared with group 1 (IPB = 0.02 g/ml), a significant increase in the forma-
tion of m-Tyr and o-Tyr was observed for group 5 (IPB = 0.08 g/ml) at pH
8
.42. In addition, group 6 (pH = 10.93), exposed to four-fold concentrations
of red lime (0.08 g/ml), exhibited no marked change.
Formation of m- and o-Tyr From Phe in Lao-hwa Quid Groups A–D
The omission of any one of the ingredients [areca nut (AN), IPB, or red
lime] from the Lao-hwa quid mixture significantly reduced the formation of
m- and o-Tyr (Figure 2). The absence of red lime in the reaction significantly
reduced the formation of m- and o-Tyr, as shown in group B (pH 4.93). In
the absence of IPB, group C (pH 9.46) demonstrated a significant decrease
in the formation of m- and o-Tyr. Formation of m- and o-Tyr at pH 11.89
was significantly decreased when the mixture did not contain areca nut (AN)
(
group D).
FIGURE 2. Formation of m- and o-Tyr from Phe with various Lao-hwa quid ingredients (A–D): A, AN + IPB +
red lime; B, AN + IPB; C, AN + red lime; D, IPB + red lime; means ± SD (n = 4) are presented. An aster-
isk denotes significance at p < .05 as compared with group A.

REACTIVE OXYGEN SPECIES BY ARECA QUID
1061
Formation of m- and o-Tyr From Phe in Betel Quid Groups 1–6
The significant differences in m- and o-Tyr formation arising from various com-
positions of betel quid are shown in Figure 3. Betel quid group 1 [AN (0.15 g/ml) +
PBL (0.05 g/ml) + white lime (0.005 g/ml)] generated 9.91 ± 5.83 μM m-Tyr and
2
+
7
.36 ± 3.52 μM o-Tyr at pH 9.45. When Cu (2 mM) was added under the same
conditions (group 2, pH 9.46), the formation of m-Tyr was significantly higher, but
2+
that of o-Tyr was not (group 2 vs. group 1). Higher concentrations of Cu (5 mM)
significantly increased the formation of m- and o-Tyr at pH 9.50 (group 3 vs. group
2+
1
). The addition of Fe (5 mM) also significantly increased m- and o-Tyr formation
at pH 9.54 (group 4 vs. group 1). When 0.05 g/ml of PBL was added in group 5
(
pH 9.14), the concentration of m- and o-Tyr fell below detection limits (0.01 μM).
The levels of m- and o-Tyr were significantly elevated with a twofold increase in the
concentration of white lime (0.01 g/ml) at pH 11.38 (group 6 vs. group 1).
Formation of m- and o-Tyr From Phe in Betel Quid Groups A–D
Exclusion of either white lime or areca nut (AN) from the reaction mixture
significantly reduced the formation of m- and o-Tyr (Figure 4). In the absence
FIGURE 3. Formation of m- and o-Tyr from Phe with various betel quid ingredients (1–6): 1, AN + PBL +
2
+
2+
white lime; 2, AN + PBL + white lime + Cu (2 mM); 3, AN + PBL + white lime + Cu (5 mM); 4, AN +
2+
PBL + white lime + Fe (5 mM); 5, AN + 2 × PBL (0.10 g/ml) + white lime; 6, AN + PBL + 3 × white lime
0.015 g/ml); means ± SD (n = 4) are presented. Nondetection (nd) indicates the m- and o-Tyr concentrations
below detection limit (0.01 μM). An asterisk denotes significance at p < .05 as compared with group 1.
(

1062
P.-H. CHEN ET AL.
FIGURE 4. Formation of m- and o-Tyr from Phe with various betel quid ingredients (A-D): A, AN + PBL +
white lime; B, AN + PBL; C, AN + white lime; D, PBL + white lime; means ± SD (n = 4) are presented.
Nondetection (nd) indicates the m- and o-Tyr concentrations below detection limit (0.01 μM). An asterisk
denotes significance at p < .05 as compared with group A.
of white lime, the formation of m- and o-Tyr was below detection limits, as
shown in group B. However, group C, which was not exposed to PBL, exhib-
ited significantly higher levels of m- and o-Tyr formation than group A, which
was exposed to PBL. Even with higher alkaline levels (group D, pH 12.18),
exclusion of areca nut (AN) reduced m- and o-Tyr formation to below detection
limits.
Effects of IPB and PBL on the Formation of m- and o-Tyr From Phe
The Lao-hwa quid group [AN (0.15 g/ml) + IPB (0.02 g/ml) + red lime
(
0.02 g/ml)] generated 68.59 ± 11.33 μM m-Tyr and 60.67 ± 9.63 μM o-Tyr.
The formation of m- and o-Tyr was significantly inhibited by adding twofold
amounts of PBL (0.1 g/ml) (Figure 5A). On the other hand, the betel quid
group containing areca nut (AN) (0.15 g/ml), PBL (0.05 g/ml), and white lime
(
0.005 g/ml) generated 11.84 ± 2.66 μM m-Tyr and 14.04 ± 4.59 μM o-Tyr.
The addition of fivefold amounts (0.1 g/ml) of IPB significantly increased
formation of m- and o-Tyr (Figure 5B).

REACTIVE OXYGEN SPECIES BY ARECA QUID
1063
FIGURE 5. The effects of PBL and IPB on the formation of m- and o-Tyr from Phe in two types of AQ. (A)
Effects of Piper betle leaf (PBL) on the Lao-hwa quid. (B) Effects of inflorescence of Piper betle Linn. (IPB) on
the betel quid; means ± SD (n = 4) are presented. An asterisk denotes significance at p < .05.

1064
P.-H. CHEN ET AL.
Formation of m- and o-Tyr From Phe in Human Saliva
During Areca Quid Chewing
The formed m-, o-, and p-Tyr in the saliva samples collected after areca quid
AQ) chewing were separated using HPLC and then detected through a fluores-
(
cence detector (Figure 6). Human saliva, especially from Lao-hwa quid chewers
with 20 mg Phe, contained high concentrations of m- and o-Tyr (Figure 6B). On
the other hand, a small amount of m- and o-Tyr was detected in human saliva
after chewing betel quid with 20 mg Phe (Fig. 6C). No m- and o-Tyr was
detected above the detection limit of 10 nM in the human saliva of subjects who
chewed gum with 20 mg Phe (Figure 6D). The concentrations of m- and o-Tyr
detected in subjects who chewed Lao-hwa quid are given in Table 2. Further,
these levels were significantly higher than in the subjects who chewed betel
quid. The presence of m- and o-Tyr in the human saliva of subjects was con-
firmed by LC-MS, as described in the Methods section (data not shown).
DISCUSSION
Studies have shown that areca quid (AQ) combined with lime will result in
•
the formation of ROS such as HO (Nair et al., 1987; Stich & Anders, 1989).
•
HO is formed by the auto-oxidation of polyphenols of areca quid (AQ) in the
presence of transition metals via Haber–Weiss or Fenton reactions. In the pres-
•
ence of HO-generating systems, m- and o-Tyr formation from Phe was pro-
•
posed as a useful marker to measure HO-induced tissue damage (Nair et al.,
1
995). In accordance with this system, our study demonstrated that aqueous
•
extracts of Taiwanese areca quid (AQ) are capable of producing HO at alka-
line pH conditions.
Areca quid (AQ) ingredients and chewing methods vary widely by country
and season (Wei & Chung, 1997; Gupta & Warnakulasuriya, 2002). In India,
for example, areca quid (AQ) generally contains areca nut (AN), lime, and
tobacco, with or without PBL, whereas in Taiwan and Papua New Guinea, IPB
is used instead of tobacco (Thomas & MacLennan, 1992; Yang et al., 2001). In
Papua New Guinea, a paste of areca quid (AQ) with lime and IPB is used. This
form elevates the salivary pH level, creating an alkaline condition conducive to
•
stimulating ROS generation, such as HO, in the corner of the mouth. The for-
mation of ROS suggests a possible mechanism for oral cancer (Thomas &
MacLennan, 1992). In this in vivo study, the human saliva from Lao-hwa quid
chewed with IPB displayed significantly higher amounts of m- and o-Tyr than
for betel quid chewed with PBL. The presence of m- and o-Tyr in human
•
saliva, confirmed by LC-MS, may implicate the presence of HO. In vitro stud-
•
ies also demonstrated that Lao-hwa quid containing IPB generated higher HO
than betel quid containing PBL. These results are compatible with the study by
Chen et al. (2002), which indicated that chewing areca quid (AQ) containing
IPB generated a higher concentration of m-Tyr than chewing areca quid (AQ)
containing PBL.

REACTIVE OXYGEN SPECIES BY ARECA QUID
1065
FIGURE 6. (A) HPLC fluorescence chromatograms of p-, m-, and o-Tyr standards. (B) Human saliva created
by chewing Lao-hwa quid with 20 mg Phe. (C) Human saliva created by chewing betel quid with 20 mg
Phe. (D) human saliva created by chewing gum with 20 mg Phe. Peaks: (1), p-Tyr; (2), m-Tyr; (3), o-Tyr.

1066
P.-H. CHEN ET AL.
TABLE 2. Formation of m- and o-Tyr (Means ± SD) in Human Saliva From Phe After Chewing Lao-hwa
Quid, Betel Quid, and Chewing Gum
Formation of Tyr (nM)
n
pHa
m-Tyr
o-Tyr
c
c
Chewing 2 quids of Lao-hwa containing AN
5
7.57 ± 0.22
809 ± 38
643 ± 88
(
(
0.06 g/ml) + IPB (0.008 g/ml) +red lime
0.008 g/ml)
Chewing 2 quids of betel quid containing AN
5
5
8.32 ± 0.26
6.87 ± 0.34
187 ± 60
178 ± 38
(
(
0.06 g/ml) + PBL (0.02 g/ml) + white lime
0.002 g/ml)
Chewing gum
ndb
nd
a
The pH level of human saliva was assessed after chewing AQ for 15 min.
nd: m- and o-Tyr concentrations below detection limits (10 nM).
Significant at p < .05 as compared with the subjects chewing betel quid, by Mann–Whitney nonpara-
b
c
metric test.
•
The major differences in HO formation may be produced by different
ingredients in Lao-hwa quid and betel quid. The ingredients of the Lao-hwa
quid chewed in Taiwan include areca nut (AN), IPB, and red lime. Thomas
and MacLennan (1992) found that a combination of areca nut (AN), IPB, and
•
powdered lime is required for generation of HO during the areca quid (AQ)
chewing process. Our in vitro studies showed that the omission of any one of
areca nut (AN), IPB, or red lime from the Lao-hwa quid significantly reduced
the formation of m- and o-Tyr, in accordance with previous studies (Nair et al.,
1
987, 1990, 1995). When fourfold amounts (0.08 g/ml) of IPB were added to
Lao-hwa quid, m- and o-Tyr formation was enhanced significantly. On the
other hand, higher amounts of IPB (0.1 g/ml) in betel quid mixtures [areca nut
(
AN) + PBL + white lime] also significantly enhanced m- and o-Tyr formation.
•
Thus, IPB as an ingredient seems to contribute to HO formation.
The preparation of Lao-hwa quid in Taiwan is somewhat dissimilar from
other countries in that fresh IPB is added; IPB contains a high concentration of
safrole (15.4 mg/g), 9.7 mg/g hydroxychavicol (HC), and 2.5 mg/g eugenol
(
(
Hwang et al., 1992). These ingredients may contribute to formation of safrole
420 μM) in human saliva (Wang & Hwang, 1993). Safrole is metabolized to
dihydroxychavicol (DHAB) and induces oxidative DNA damage (Lee-Chen
et al., 1996; M. J. Chang et al., 2002). On the other hand, safrole is a docu-
mented animal liver carcinogen and generates DNA adducts (8-OH-dG) that
have been shown to produce oxidative DNA damage in the rat liver (Liu et al.,
1
999). The safrole–DNA adducts derived from IPB may contribute to oral car-
cinogenesis in areca quid (AQ) chewers (Chen et al., 1999). HC is the major
urinary metabolite of safrole in humans and rats, and the minor metabolite is
eugenol (Benedetti et al., 1977; Klungsoyr & Scheline, 1983). Both HC and
eugenol may oxidize to form DNA adducts (8-OH-dG) and induce oxidative

REACTIVE OXYGEN SPECIES BY ARECA QUID
1067
damage in culture cells (Lee-Chen et al., 1996; Bodell et al., 1998; Chen
et al., 2000; Jeng et al., 2004). These properties may at least partially explain
•
the increased HO exposure and oxidative damage from IPB use. Accordingly,
epidemiological studies also suggested that chewing areca quid (AQ) with IPB
increases the potential for oral cancer (Ko et al., 1995; Lee et al., 2003).
The PBL of betel quid, sometimes a substitute for IPB, is composed of a
small amount of HC and eugenol, but without safrole (Hwang et al., 1992;
Chen et al., 2002). The high concentration of m- and o-Tyr in Lao-hwa quid
groups may result from higher concentrations of safrole, HC, and eugenol.
•
These agents known to yield HO formation are found in IPB in greater quanti-
ties than in PBL. Recent studies also indicated that HC of PBL extract is an effi-
•
•
−
cient scavenger of ROS (H O , O , and HO) at low concentrations, whereas
2
2
2
at high concentrations (HC > 0.1 mM), HC may induce oxidative damage
(
M. C. Chang et al., 2002). Our results also demonstrate that a significant
decline in m- and o-Tyr formation occurs with the addition of PBL (0.1 g/ml) to
the Lao-hwa quid mixture [areca nut (AN) + IPB + red lime]. The betel quid
group without PBL significantly generates higher levels of m- and o-Tyr forma-
tion than betel quid with PBL.
2
+
2+
Transition metal ions such as Cu and Fe are known to enhance the
•
production of HO by areca quid (AQ) (Nair et al., 1987, 1990). Consistent
•
with this, our studies showed that the formation of HO is enhanced by both
2
+
2+
2+
Cu and Fe , where Cu is more effective. Our results also concur with a
2+
previous study that revealed that the addition of Cu to an areca quid (AQ)
2+
mixture had generated higher amounts of m- and o-Tyr than addition of Fe
Nair et al., 1995). In contrast, Nair et al. (1987) found ROS formation was
(
2
+
3+
2+
2+
enhanced in the following order: Fe > Fe > Mg > Cu . According to
2+
2+
previous studies, Cu and Fe levels are higher in the oral epithelial and con-
nective tissues of AQ chewers (Paul et al., 1996; Trivedy et al., 2000). Since
2
+
2+
transition metal ions such as Cu and Fe have been shown to play an impor-
tant role in accelerating oxidative damage (Nair et al., 1987; Liu et al., 1996),
•
2+
2+
the effect of Cu and Fe on the formation of HO during areca quid (AQ)
chewing merits further elucidation.
•
In conclusion, these in vitro studies indicate that the generation of HO
was inhibited by the addition of PBL. In contrast, the markedly increased oxi-
dative effects of human saliva after chewing areca quid (AQ) with IPB are likely
risk factors for oral mucosa damage. Therefore, the difference in risks between
areca quid (AQ) with and without IPB implies IPB might induce additional
oxidative damage on oral mucosa tissue. The oxidative damage produced by
•
HO during areca quid (AQ) chewing is probably implicated in the development
of oral cancer.
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