Mutagenesis vol.11 no.5 pp.
519-523, 1996
Role of oxidative DNA damage in hydroxychavicol-induced
genotoxicity
Shwu-Fei Lee-Chen1, Chiu-Lan Chen2, Liang-Yi Ho1,
Pi-Chen Hsu1, Jung-H Chang1, Chang-Ming Sun3,
Chin-Wen Chi2-4 and Tsung-Yun Liu2-4-5
'Developmental Center for Biotechnology, 2Institute of Pharmacology,
National Yang-Ming University, ^National Research Institute of Chinese
Medicine and 4Department of Medical Research, Veterans General Hospital-
Taipei, Taipei, Taiwan, Republic of China
^ o whom correspondence should be addressed at: Department of Medical
Research, Veterans Genera] Hospital-Taipei, Taipei, Taiwan, Republic of
China
Chewing betel quid has been linked to the development of
oral cancer. In Taiwan, fresh Piper be tie inflorescence is
uniquely added to betel quid, and hydroxychavicol is the
major phenolic components of P.betle inflorescence. In this
study, we tested the mutagenic potential of hydroxychavicol
in Salmonella typhimurium TA97, TA98, TA100 and TA102
with and without Aroclor-1254 induced S9 fraction. The
results showed that hydroxychavicol was positive in S.typhi-
murium TA102 without metabolic activation. This increase
in revertants was partially inhibited by catalase and super-
oxide dismutase. In Chinese hamster ovary (CHO-K1)
cells, hydroxychavicol induced chromosome aberrations in
a dose-dependent manner (10-50 uM) and the majority
were chromosome-type aberrations. Hydroxychavicol also
significantly increased the frequency of micronuclei in
CHO-K1 cells up to 3-fold at a concentration of 40 uM. In
addition, hydroxychavicol dose-dependently (0.1-20 uM)
induced copper-dependent strand breaks in plasmid DNA.
We further tested the oxidative DNA damage potential of
hydroxychavicol by measuring 8-hydroxydeoxyguanosine
(8-OH-dG) formation in CHO-K1 cells following an 18-h
incubation and found that hydroxychavicol (6.25-100 uJVf)
induced 8-OH-dG levels dose-dependently. The increase of
8-OH-dG formation was positively correlated (r = 0.79)
with the hydroxychavicol-induced cytotoxicity. In conclu-
sion, hydroxychavicol may exert its genotoxic potential
through oxidative DNA damage.
Introduction
Epidemiological studies have demonstrated a clear causal
association between chewing betel quid containing tobacco
and an increased risk of oral cancer [International Agency for
Research on Cancer (IARC), 1986]. In Taiwan, the number of
betel quid chewers was estimated at one-tenth of the 20 million
inhabitants (Ko et al, 1992). The betel quid chewed in Taiwan
contains Piper betle inflorescence which is not used elsewhere
except in Guam and Papua New Guinea (Thomas and
MacLennan, 1992). Fresh P.betle inflorescence is added to
betel quid for its aromatic flavor, which contains many phenolic
compounds such as hydroxychavicol (9.74 mg/g wet weight),
eugenol (2.51 mg/g wet weight), quercetin (1.11 mg/g wet
weight), isoeugenol (1.81 mg/g wet weight), eugenol methyl
ester (1.81 mg/g wet weight) and safrole (15.35 mg/g wet
UK Environmental Mutagen Society/Oxford University Press 1996
weight) (Hwang et al., 1993). Among these, safrole is a known
weak carcinogen (Miller et al., 1983). The phenolic compounds
such as quercetin (Bjeldanes and Chang, 1977; Ito, 1992)
and eugenol (Ellahuefle et al., 1994) have well documented
mutagenic and carcinogenic effects. The mutagenic potential
of the major phenolic component in P.betle inflorescence,
hydroxychavicol (Figure 1), has not been documented. To
better understand the risk of the locally chewed betel quid
containing fresh P.betle inflorescence, we have isolated hydro-
xychavicol and investigated its genotoxic effects in four strains
of Salmonella typhimurium and in CHO-K1 cells. We further
examined whether the effect of hydroxychavicol was metal
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dependent by assessing plasmid DNA strand breaks in the
presence of Cu(II). In addition, we tested the hydroxychavicol
induced oxidative DNA damage, as evidenced by the formation
of 8-hydroxydeoxyguanosine (8-OH-dG) in CHO-K1 cells.
Materials and methods
Chemicals
8-OH-dG was custom synthesized by Chemsyn Science Laboratory (USA).
All other chemicals were of the highest grade available from commercial
sources. Ultra pure water was used for reagent preparation.
Preparation of hydroxychavicol
Fresh P.betle inflorescence was obtained from the local market in Taipei. The
preparation of hydroxychavicol was according to the published method (Hwang
et al, 1993). Briefly, 100 g fresh P.betle inflorescence was minced and
extracted with 1000 ml of 80% acetone in water. This filtrate was successively
partitioned three times with equal volume of n-hexane and chloroform. The
combined chloroform extracts were concentrated in vacuum and passed
through a silica gel 60 (E. Merck, Germany) column, eluted with chloroform
and monitored by UV. The desired fractions which contain hydroxychavicol
were combined, concentrated and confirmed by nuclear magnetic resonance
(NMR) and mass spectrometry.
Bacterial mutagenicity assay
Salmonella typhimurium TA97, TA98, TA100 and TA102 were obtained from
the Culture Collection and Research Center, Food Industry Research and
Development Institute, Hsin-Chu, Taiwan, Republic of China. The muta-
genicity test was based on the method described by Maron and Ames (1983).
Basically, the reaction mixture contained IX 10s S.typhimurium TA97, TA98,
TA100 or TA102, and different concentrations of hydroxychavicol in 0.1 ml
dimethyl sulphoxide (DMSO). For the metabolic activation, 2 mg Aroclor
1254 (Chem Service, USA)-induced rat liver protein which was prepared
according to Ames et al. (1975) from male Sprague-Dawley rats (200 g) was
added to the incubation mixture. All experiments were performed three times
and each assay was done in triplicate.
CHrCK=CH2 = CH- CH,
OCHj
OH
OH
Hydroxychavicol Eugenol
Fig. 1. The molecular structures of hydroxychavicol and the related
phenolic compounds, eugenol and isoeugenol, in P.betle inflorescence.
519
S.-F.Lee-Chen el al

Assay for DNA strand breaks

DNA strand breaks were measured by the conversion of supercoiled pRSVcat Table I. Mutagenicity of hydroxychavicol ini different strains of
(kindly provided by Dr C.C K Chao, Chang Gung Medical College. Taiwan, S.typhimunuiT:
i in the absence of S9 mix
Republic of China) to open circular and linear forms as described by Li and
Trush (1994) with modifications. In brief, 0.3 )ig of DNA was incubated with Dose Number of revertants per plate
the indicated concentrations of H 2 OT or hydroxychavicol in the presence of (Hg/plate)
100 |iM Cu(II) in phosphate-buffered saline (PBS), at 37C for 30 mm. The TA97 TA98 TA100 TAI02
samples were immediately electrophoresed in a 1% agarose gel at 25 V for
SC. 266 27" 37 2 160 6 275 21
15 h The stained gels were photographed under UV light using Polaroid Type
55 film. DNA quantification was earned out by Computing Densitometer 0 1 370 11 29 5 173 2 428 91
(Molecular Dynamics. USA). 1 383 1 25 2 173 41 437 77
10 180 52 18 0 194 7 835 87
Micronucleus assay 25 241 10 42 11 158 7 617 116
The cytokinesis-blocked micronucleus assay of French (1993) was followed 50 132 13 24 4 131 6 348 46
with minor modifications After treatment. 1 |ig/ml cytochalasin B (Sigma. St 100 toxic toxic toxic toxic
Louis, MO. USA) was added to cell culture and incubated for another 21 h. PC. 308 11 416 37 236 42 508 3<)
At the end of incubation, cells were treated with 0.59fc KG for 5 min. The
cells were then fixed in a 20:1 mixture of methanol-acetic acid for 3 min J
Data are represented as mean SD from three independent experiments
The dishes were air dried and stained with 5% Giemsa for 10 mm. The
and each experiment was run in triplicate.
criteria for scoring the micronuclei within binucleated cells were according
S.C = solvent control
to Lynch and Parry (1993). There are > 8 0 * of cells in untreated control
PC. = positive control; 2,4.7-tnnitro-9-fluorenone is the positive control for
group containing two or more main nuclei. In each treatment group, 1000
TA97 and TA98, at 0 02 and 0.04 |ig/plate respectively: sodium azide at 0 4
binucleated cells were analysed for the presence of micronuclei on coded slides
|ig/plate is the positive control for TA100, mitomycin C at 0 5 |ig/plate is
Chromosome aberration assav the positive control for TA102
Exponentially growing CHO-KI cells (ATCC, USA) were used for assay of

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chromosome aberrations (Galloway et al., 1985). Briefly, cells were incubated
with different concentrations of hydroxychavicol (20-50 |iM) at 37C for Table II. Mutagenicity of hydroxychavicol in different strains of
18 h with the last 2 h in the presence of colcemid (0 2 |ig/ml) (Sigma) The S typhimurtum in the presence of S9 mix
cells were collected by shake-off, resuspended in 0.5% KG for 8 mm, and
fixed in methanol-acetic acid (3:1). The cells were then dropped on slides Dose Number of revertants per plate
and stained with Giemsa. A total of 100 metaphases were analysed for the
presence of chromosome aberrations ((ig/plate) TA97 TA98 TA100 TA102
DNA isolation, digestion and 8-OH-dG determination S.C. 141 20" 42 8 97 13 390 74
For 8-OH-dG determination, CHO-KI cells were treated with different 0.1 106 11 58 9 158 52 411 36
concentrations of hydroxychavicol (6.25100 |lM) for 18 h and harvested 1 128 36 66 3 116 8 357 25
Cytotoxicity was determined by the Trypan Blue dye exclusion method. DNA 10 128 6 58 11 125 9 375 64
from cell pellets was purified by the standard proteinase K digestion and 50 124 2 58 1 Toxic 386 39
followed by the phenol-chloroform extraction. The isolated DNA, 100 |ig. 100 148 20 37 10 Toxic 480 81
was digested to nucleoside level with subsequent treatment of 40 U DNase I 200 101 10 47 14 Toxic 381 54
(Boehnnger Mannheim, Germany), 2.5 U nuclease PI (Sigma) and 25 U PC 357 84 301 56 340 29 1029 67
alkaline phosphatase (Sigma) (Frenkel, 1993: Liu et al., 1995). Nucleosides
(20 |il) were then separated on a Cosmosil CI8 reversed-phase column a
Data are represented as mean SD from three independent expenments
(250X4.6 mm, 5 |im particles. Nacalai, Japan) using 10% methanol in 50 mM and each experiment was run in triplicate.
potassium phosphate, pH 5.5, as the mobile phase at I ml/min for 30 min S.C. = solvent control
(Park et al.. 1989; Frenkel et al., 1991). The samples were detected at 260 PC = positive control: aminofluorene at 4 |ig/plate is the positive control
nm by a diode array detector module 168 (Beckman, USA) and a LC-4B for TA97. TA98 and TAI00: 1,8-dihydroxyanthranquinone at 25 ng/plate is
amperometric detector (Bioanalytical Systems, USA) with a glassy-carbon the positive control for TAI02.
electrode (+0.6 V. 20 nA) Mixtures of authentic deoxyguanosine (dG)
and 8-OH-dG were also chromatographed to generate standard curves for
quantitative analysis The amount of 8-OH-dG was expressed as the number
of 8-OH-dG for every I0 5 dG in DNA. However, no increase in mutation frequency was detected
following incubation with Aroclor 1254-induced rat liver S9
Results mix. This protein mixture contains the induced microsomal
Hydroxychavicol (4-allylpyrocatechol) was isolated and identi- enzymes and phase n conjugating enzymes which may block
fied from fresh P.betle inflorescence. The mutagenic response the genotoxic effect of hydroxychavicol.
of hydroxychavicol was measured in S.typhimurium TA97, In agarose gel electrophoresis, the closed circular DNA
TA98, TA100 and TA102 with and without metabolic activa- migrated faster than its linear form, while the open circular
tion. The number of revertants in these strains of S.typhimurium DNA migrated appreciably slower than either the closed
using five to six doses ranging from 0.1 to 200 |ig/plate were circular or the linear form. Interaction between H2O2 and
presented in Tables I and II. An increase in mutation frequency Cu(II) induced the formation of both open circular and linear
above background was observed in TA102 without S9 activa- forms from the closed circular pRSVcat DNA at 1-10 (0.M of
tion. At 10 (ig/plate, hydroxychavicol induced a 3-fold increase H2O2. Hydroxychavicol (10 (lM) alone did not produce any
in TA102 revertant numbers above background levels. Concen- nick on plasmid DNA; however, in the presence of Cu(II),
trations of hydroxychavicol >100 ^ig/plate in the absence of hydroxychavicol greatly increased its ability in inducing the
S9 protein were cytotoxic, as noticed by a thinning of the formation of open circular and linear forms DNA (Figure 3).
bacterial lawn. Coincubating hydroxychavicol with catalase At 5 U.M hydroxychavicol, the closed circular DNA completely
(5-50 U/plate) in TA102 partially reduced the number of disappeared resulting in open circular and linear forms of
revertants. At 10 U/plate, catalase inhibited the number of DNA. At 20 (J.M hydroxychavicol, most DNA were degraded
revertants up to 67% induced by 10 (o.g/plate hydroxychavicol. and left faint bands. At higher dose, 40 |iM, all the DNA was
On the other hand, the addition of similar amount of superoxide degraded and no band was found on gel (data not shown). On
dismutase (SOD) also inhibited the number of revertants but the other hand, H2O2 up to 20 ^M caused complete disappear-
to a lesser extent as compared with catalase (Figure 2). ance of the closed circular form DNA. Figure 4 shows the
520
 Role of oxidative DNA damage in hydroxychavicoHnduced genotoxicity

comparative effects of hydroxychavicol and H2O2 on the 5.17 0.54 per 105 dG. When incubating with CHO-K1 for
plasmid DNA in the presence of Cu(IT). 18 h, hydroxychavicol (6.25-100 [M) steadily induced 8-OH-
Hydroxychavicol at concentrations ranging from 6.25 to dG levels from 6.% to 10.84 per lO'dG (Figure 5). The formation
100 \iM increased the cytotoxicity of CHO-K1 cells in a dose-
dependent manner (Figure 5). In the analysis of micronucleus
_
formation, a significant and dose-dependent induction of 100 h

9 Hydroxychavicol
micronuclei was observed at concentrations of 20-40 |iM of

lined
80 Hydrogen peroxide
hydroxychavicol for 18 h treatment in CHO-K1 cells (Table
III). The chromosome aberration data under similar treatment 1 60
2
condition is shown in Table FV, a noticeable increase in the
frequencies of chromosome aberrations was found in these
o

40 1
cells. Of particular interest was the observation that the type
of chromosome aberration was dominated by the chromosome-
d
5
20 \
1

type damage. n
U
The background level of 8-OH-dG in CHO-K1 cells was 1 1 t 1 1 1

6 8 10
Concentration, uM

Fig. 4. Quantitative analyis of DNA strand breaks of pRSVcat plasmid

DNA incubated with the indicated concentrations of H2O2 (solid square) and

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hydroxychavicol (solid circle) in the presence of 100 11M Cu(Il). The DNA
strand breaks were determined by measuring the remaining closed circular
o (C.C.) forms of DNA following incubation. Data were averaged from two
U independent assays.

Inhibitor (Units/Plates)

Fig. 2. The effects of various concentrations of superoxide dismutase

(SOD; open circles) and catalase (solid circles) on the mutagenicity of
hydroxychavicol, 10 |ig/plate, in S.typhimurium TA102. Fifty (il of
hydroxychavicol (10 |ig) in dimethyl sulphoxide (DMSO) was mixed with
50 \l\ of varying concentrations (0, 5, 10, 25 and 50 U) of SOD or catalase,
and the mutagenicity of 100 u.1 mixture was determined by the pre-
incubation method. SOD and catalase had no mutagenicity at the tested dose
level. Each point represents the mean of triplicate results obtained from a 0 to 40 eo eo 100
representative experiment. Positive control (mitomycin C) at Hydroxychavicol cone. (uM)
0.5 (lg/plate gave 636 revertants.

Fig. S. The effects of different concentrations of hydroxychavicol on the

- 4 5 6 7 8
8-OH-dG formation and cytotoxicity in CHO-K1 cells following an 18 h
exposure. Cytotoxicity was determined by the Trypan Blue dye exclusion
Open Circular method. Solid circle denotes the 8-OH-dG level and open circle is the
surviving fraction of CHO-K1 cells following treatment with
I. incur hydroxychavicol. *P < 0.05 by Student's f-test when compared with
control.
Closed Circular

Table i n . Effect of hydroxychavicol on the micronucleus induction in

CHO-K1 cells
Lane I: Control
Lane 2: 10 uM hydroxychavicol
Treatment Concentration Number of micronuclei/
Lane 3: I(K) uM Cu(ll)
1000 binucleated cells
Lane 4: 1 uM hydroxychavicol + 100 uM Cu(II)
Lane 5: 2 uM hydroxychavicol + 100 uM Cu(II)
Lane 6: 5 uM hydroxychavicol + 100 jiM Cu(II) Solvent control (DMSO) 32.0 1.41
Mitomycin C (1 h) 1.0 67.3 7.0b
Lane 7: 10 uM hydroxychavicol + 100 \iM Cu(II)
Hydroxychavicol (18 h) 20.0 54.0 2.9b
Lane 8: 20 UM hydroxychavicol + 100 uM Cu(II)
30.0 72.3 7.1 b
40.0 %.O 6.8b

Fig. 3. Photograph of a DXA gel following incubation of pRSVcat plasmid DMSO = dimethyl sulphoxide.
DNA with different concentrations of hydroxychavicol in the presence of Data are represented as mean SD from three independent experiments
100 uM Cu(II) in phosphate-buffered saline (PBS), at 37C for 30 min. and each experiment was run in triplicate.
Data were from a representative experiment of four separate assays. "P < 0.005 by Student's /-test

521
S.-F.Lee-Chen el aL

comparable H2O2/Cu(II) system up to 5-fold (Figure 4). In the

Table IV. Effect of hydroxychavicol on tJie induction of chromosome
aberrations in CHO-KI cells H2O2/Cu(n) system, reactive oxygen species generated from
a Fenton-type reaction have been suggested to be responsible
Treatment Aberrant Numbei of aberrations/100 for the site-specific DNA cleavage (Sagripanti and Kraemer,
cells (%) cells 1989; Prutz et al, 1990; Li and Trash, 1993). Similarily, the
G B D R g b e reactive oxygen species may result from the interaction between
hydroxychavicol and DNA-associated copper, and con-
Solvent control (DMSO) 0 0 0 0 0 0 0 0 sequently activate hydroxychavicol by copper-redox mechan-
Hydroxychavicol (uM) ism. Such formed reactive oxygen species may elicit the in
20 5 1 2 1 0 0 2 0
25 6 5 1 2 0 0 3 0
situ DNA strand breaks in pRSVcat (Li and Trush, 1994).
30 11 2 6 1 0 3 5 0 This result also supports the prediction of Li and Trush (1994)
35 16 4 7 3 0 2 5 1 that by using the structure-activity analysis, the demethylation
40 27 3 21 2 1 0 8 0 product of eugenol, which is hydroxychavicol, exhibits signi-
50 too few metaphases ficant DNA cleaving activity in the presence of Cu(II).
MMC 1 uM 23 0 0 0 2 3 8 15
A significant induction of micronuclei and dominant chromo-
G = chromosome gap, B = chromosome break; D = dicentnc; R = nng some-type aberration following hydroxychavicol treatment in
g = chromatid gap, b = chromatid break; e = exchange. CHO-K1 cells indicate the possible association of a strong
Note: Hydroxychavicol was dissolved in dimethyl sulphoxide (DMSO)
Treatment duration for hydroxychavicol and mitomycin C (MMC) were 18
oxidative damaging agent. To further test the oxidative DNA
and 3 h respectively Both chromosome gap (G) and chromatid gap (g) are damage potential of hydroxychavicol in CHO-K1 cells, we
not included in the percentage of aberrant cells. extended our observation to the 8-OH-dG formation in these
cells following the same 18 h treatment protocol. Formation

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of 8-OH-dG was positively correlated with the cytotoxicity of
hydroxychavicol-treated CHO-K1 cells (r = 0.79).
Discussion
Previous study has documented that hydroxychavicol and
eugenol are the two main phenolic constituents in P.betle
leaves, and both of them are not mutagenic when tested in
S.typhimurium TA98, TA100, TA1535 and TA1538 with and
without metabolic activation (Amonkar et al., 1986). High
concentration of hydroxychavicol (9.74 mg/g wet weight) is
also found in fresh P.betle inflorescence (Hwang et al, 1993)
which is added to betel quid uniquely in Taiwan, Guam and
Papua New Guinea (Thomas and MacLennan, 1992).
In this study, hydroxychavicol was tested positive in S.typhi-
murium TA102 without metabolic activation (Table I). TA102
is specifically constructed to contain conservative A-T base
pairs at the site of reversion and oxidative mutagens preferen-
tially act at these sites (Levin et al., 1982). Mutagens with
oxidative nature are inactive in the standard tester strains as
used in this and previous (Amokar et al, 1986) investigations
in assaying hydroxychavicol, because the standard strains
contains G-C base pairs at the site of reversion. Strain TA102
is uvr + that is different from the other three tester strains used
(uvrB). Our results might indicate that the kind of lesions
induced by hydroxychavicol is not recognized and repaired by
the uvrABC-dependent nucleotide excision pathway. Coincub-
ating superoxide dismutase and catalase inhibited hydroxy-
chavicol-induced mutation in S.typhimurium TA102 suggests
that reactive oxygen species, such as O2~ and H2O2 are involved
in the observed mutagenicity of hydroxychavicol. This result
further demonstrates the oxidative potential of hydroxychavicol
in S.typhimurium TA102.
In order to elucidate the oxidative nature of hydroxychavicol,
we incubated hydroxychavicol with supercoiled plasmid DNA
in the presence of Cu(ET). The result of the DNA strand breaks
clearly showed that the interaction of hydroxychavicol with
Cu(II) induced DNA strand breaks in pRSVcat in a concentra-
tion-dependent manner (Figure 3), while the Cu(II), H2O2 or
hydroxychavicol alone did not induce DNA cleavage. In this
assay system, the DNA strand breaks induced by hydroxychav-
icol/Cu(II) were dependent on the presence of both hydroxy-
chavicol and Cu(H), and were much more extensive than a
of 8-OH-dG is the adduction of the reactive hydroxyl radical
on deoxyguanosine (Floyd et al, 1986). 8-OH-dG is a muta-
tion-prone product of deoxyguanosine oxidation (Kehrer, 1993)
and a molecular hallmark of many carcinogenic insults includ-
ing ionizing radiation, UV, asbestos, Cr(III) etc. (Faux et al,
1994; Floyd et al, 1988; Tsou et al, 1996). This study
demonstrates that hydroxychavicol (6.25-100 U.M) induced 8-
OH-dG formation dose-dependently in CHO-K1 cells when
no metal ions were added to the culture medium (Figure 5).
The hydroxychavicol-induced 8-OH-dG levels were positively
correlated (r = 0.79) with the cytotoxicity in CHO-K1 cells.
In addition, hydroxychavicol dose-dependently (20-40 U.M)
induced micronucleus formation and chromosome-type DNA
damages, whereas high dose (s=50 uM) of hydroxychavicol
arrested cell progresssion at G ( /G| (Tables HI and IV). These
results suggest that hydroxychavicol-induced oxidative DNA
damage, as evidenced by the 8-OH-dG formation, may be the
major determinant of chromosome aberration, micronucleus
fomation and cytotoxicity in CHO-K1 cells.
Collectively, the results presented here strongly suggest that
the reactive oxygen species such as H2O2 are generated from
hydroxychavicol and participated in the Fenton-type reaction
which in turn is responsible for the increased revertants in
TA102 and the induction of plasmid DNA strand breaks.
Whereas the formation of oxidative DNA base modification,
8-OH-dG, by hydroxychavicol may be responsible for the
chromosome aberration, micronucleus formation and cytotox-
icity in CH0-K1 cells.
Acknowledgements
We thank Dr K.YJan (Institute of Zoology, Academia Sinica, Taiwan. Republic
of China) for his critical reading of this manuscript. This study was supported
in part by grants 84-O4I2-B075-O21-M14 and 85-233I-B-O75-O9O-MI4 from
the National Science Council of the Republic of China.
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Received on March 15, 1996; accepted on May 8, 1996

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