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Potent antimutagenic activity of white tea in

comparison with green tea in the Salmonella
assay. Mutat Res
ARTICLE in MUTATION RESEARCH/FUNDAMENTAL AND MOLECULAR MECHANISMS OF MUTAGENESIS SEPTEMBER
2001
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Mutation Research 495 (2001) 6174

Potent antimutagenic activity of white tea in comparison with

green tea in the Salmonella assay
Gilberto Santana-Rios a , Gayle A. Orner a , Adams Amantana a ,
Cynthia Provost b , Shiau-Yin Wu c , Roderick H. Dashwood a,
a

Linus Pauling Institute, Department of Environmental and Molecular Toxicology,

Oregon State University, 571 Weniger Hall, Corvallis, OR 97331-6512, USA
b Department of Pharmacy, Xavier University, New Orleans, LA 70125, USA
Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR 97331, USA
Received 17 May 2000; received in revised form 30 April 2001; accepted 3 May 2001

Abstract
There is growing interest in the potential health benefits of tea, including the antimutagenic properties. Four varieties of
white tea, which represent the least processed form of tea, were shown to have marked antimutagenic activity in the Salmonella
assay, particularly in the presence of S9. The most active of these teas, Exotica China white tea, was significantly more effective
than Premium green tea (Dragonwell special grade) against 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and four other
heterocyclic amine mutagens, namely 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethyl3H-imidazo[4,5-f]quinoxaline (4,8-DiMeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), and 3-amino-1methyl-5H-pyrido[4,3-b]indole (Trp-P-2). Mechanism studies were performed using rat liver S9 in assays for methoxyresorufin
O-demethylase (MROD), a marker for the enzyme cytochrome P4501A2 that activates heterocyclic amines, as well as
Salmonella assays with the direct-acting mutagen 2-hydroxyamino-3-methylimidazo[4,5-f]quinoline (N-hydroxy-IQ). White
tea at low concentrations in the assay inhibited MROD activity, and attenuated the mutagenic activity of N-hydroxy-IQ in
the absence of S9. Nine of the major constituents found in green tea also were detected in white tea, including high levels
of epigallocatechin-3-gallate (EGCG) and several other polyphenols. When these major constituents were mixed to produce
artificial teas, according to their relative levels in white and green teas, the complete tea exhibited higher antimutagenic
potency compared with the corresponding artificial tea. The results suggest that the greater inhibitory potency of white
versus green tea in the Salmonella assay might be related to the relative levels of the nine major constituents, perhaps
acting synergistically with other (minor) constituents, to inhibit mutagen activation as well as scavenging the reactive
intermediate(s). 2001 Elsevier Science B.V. All rights reserved.
Keywords: Salmonella assay; Tea polyphenols; Heterocyclic amines; Catechins; Caffeine; EGCG; IQ; PhIP

1. Introduction

Corresponding author. Tel.: +1-541-737-5086;

fax: +1-541-737-5077.
E-mail address: rod.dashwood@orst.edu (R.H. Dashwood).

Tea is the second most widely consumed beverage

in the world. Reasons for its great popularity range
from cultural traditions to purported health benefits.
Results from epidemiological studies as well as laboratory experiments suggest that consumption of tea

1383-5718/01/$ see front matter 2001 Elsevier Science B.V. All rights reserved.
PII: S 1 3 8 3 - 5 7 1 8 ( 0 1 ) 0 0 2 0 0 - 5

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G. Santana-Rios et al. / Mutation Research 495 (2001) 6174

confers protection against the development of chronic

diseases, such as cardiovascular disease and cancer
[14].
The health benefits of tea have been mainly
attributed to the relatively high levels of flavonoids,
including catechins and other polyphenols. Precisely
how tea polyphenols might provide health benefits remains unclear, but the following mechanisms
have been proposed: scavenging of reactive oxygen
species (ROS) [5], modification of signal transduction pathways, cell cycle checkpoints, and apoptosis
[69], and the induction of various enzyme activities
involved with drug metabolism and carcinogen activation/detoxification [1012]. Through these various
mechanisms, tea has demonstrated excellent chemoprotective effects in animal models of skin, lung,
esophageal, and gastrointestinal cancers [1318]. Included among the latter studies are investigations

in vitro and in vivo of heterocyclic amine-induced

mutagenesis and carcinogenesis [1618].
Heterocyclic amines are procarcinogens created
during the cooking of foods that contain sugars, amino
acids and creatinine, namely meats and fish [1921].
Certain heterocyclic amines induce tumors of the
small intestine and colon in experimental animals, and
for this reason they have been used as model compounds for the study of events that occur during colon
carcinogenesis [2226]. In our previous work, tea was
demonstrated to protect against the heterocyclic amine
2-amino-3-methylimidazo[4,5-f]quinoline (IQ), using
the colonic aberrant crypt focus as an intermediate biomarker [18]. The degree of protection by tea
appeared to be related to the extent of processing,
since green tea was generally more effective than
black tea in vitro and in vivo [1618]. This suggested the possibility that higher antimutagenic or

Fig. 1. Tea manufacturing processes, showing oxidation steps that increase as tea is converted from white green oolong black.
The minimal processing of white tea, plus the higher overall proportion of buds to leaf, gives a pale beverage with a slightly sweet, subtle
flavor compared with other teas.

G. Santana-Rios et al. / Mutation Research 495 (2001) 6174

anticarcinogenic activity might be expected from teas

that have undergone the least amount of processing.
Green tea, consumed mainly in Japan, China and
Korea, is produced when freshly harvested leaves of
Camellia sinensis are subjected to withering, and then
they are panfried/steamed prior to rolling/shaping and
drying (Fig. 1). Black tea, which represents >90% of
the total consumption worldwide, follows some of the
processing steps used for green tea, but with the critical difference that the leaves are bruised, crushed, or
broken, thus allowing polyphenol oxidases in the leaf
to generate theaflavins, thearubigins and other complex polyphenols from the endogenous catechins. Oolong tea, popular in China and Japan, goes through
an intermediate process involving withering, bruising,
brief oxidation, and firing/drying (Fig. 1). White tea,
which has received little if any attention for its health
benefits, represents the least processed of teas in that
it goes through steaming and drying without a prior
withering stage. Because the catechins are converted to
theaflavins, thearubigins, and more complex polyphenols as green tea is processed into oolong and black
teas, and the catechins generally are assumed to be
more active based on their antioxidant and other protective properties [512], we hypothesized that white
tea might prove to be more inhibitory than green tea
in the Salmonella assay.
Therefore, we examined the antimutagenic activity
of several white tea varieties in the Salmonella assay against IQ and other heterocyclic amines, including studies of the possible inhibitory mechanisms, and
compared the results with those for green tea. Subsequently, several of the major constituents in white tea
and green tea were reconstituted to form artificial
teas that were tested for antimutagenic activity. Finally, because white tea contains both leaves and buds,
these were tested separately and in combination for
their inhibitory activity in the Salmonella assay.

2. Materials and methods

2.1. Chemicals
The heterocyclic amines IQ, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,
8-trimethyl-3H-imidazo[4,5-f]quinoxaline (4,8-DiMeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyr-

63

idine (PhIP), and 3-amino-1-methyl-5H-pyrido

[4,3-b]indole (Trp-P-2) were from Toronto Research
Chemicals (Ont., Canada), whereas 2-hydroxyamino3-methylimidazo[4,5-f]quinoline (N-hydroxy-IQ) was
from SRI International (Menlo Park, CA). Tea standards (+)-catechin (CAT), ()-epicatechin (EC),
()-epigallocatechin (EGC), ()-epicatechin gallate
(ECG), epigallocatechin-3-gallate (EGCG), theobromine (TB), theophylline (TP), and monochloroacetic acid were purchased from Sigma Chemical
Co. (St. Louis, MO). Caffeine (CAF) and gallic acid
(GA) were from Acros (Fair Lawn, NJ). For chemical structures of tea constituents, see [17]. Methanol
(HPLC grade) was from Fisher Scientific (Fair Lawn,
NJ). Materials used for the mutagenicity assays were
from sources previously described [16,17].
2.2. Tea preparation
Three white teas (Exotica China white, Flowery
Pekoe, and Mutant white) and the green tea Premium
green (Dragonwell special grade) were generous
gifts of The Stash Tea Co. (Portland, OR). A fourth
white tea, Silver Needle, was obtained from Tea and
Company (San Francisco, CA). Teas were brewed
fresh immediately before each experiment using double distilled-deionized water. The concentration during brewing was 2 g tea leaves per 100 ml (2%, w/v)
and the brewing time was 5 min, unless indicated
otherwise. Teas were sterilized by passage through a
20 m filter before testing for inhibitory activity in
the Salmonella mutagenicity assay.
2.3. Analysis of teas
High performance liquid chromatography (HPLC)
analysis of teas was performed on a Shimadzu VP series instrument equipped with a 25 cm4.6 mm, 5 m
particle size, reverse-phase column (SupelcosilTM
LC-18). The mobile phase was composed of methanol
(buffer A) and water (buffer B). Chloroacetic acid
was added to both solvents at a final concentration of
0.3% (w/v) and the pH was adjusted to 4.5 using 1N
NaOH. The gradient program used for separation of
the tea components was as follows: 10% buffer A at
0 min, increased linearly to 40% buffer A at 50 min
and returned to 10% buffer A at 60 min. The flow rate
was 1 ml/min and detection was at 273 nm. The major

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G. Santana-Rios et al. / Mutation Research 495 (2001) 6174

fractions were identified by co-elution with authentic

standards, and subsequently confirmed by LC-MS using a Perkin-Elmer Sciex API III + triple-quadruple
mass spectrometer with an atmospheric pressure
chemical ionization (APCI) source.
2.4. Mutagenicity assays
The antimutagenic activities of white tea and green
tea were studied against IQ and other heterocyclic
amines using Salmonella typhimurium strain TA98 in
the presence of aroclor-induced rat liver S9, using the
method of Hernaez et al. [16,17]. Some experiments
used the direct-acting mutagen N-hydroxy-IQ in the
absence of an exogenous activation system. All tests
were performed under subdued lighting. Unless stated
otherwise, assays were conducted at 37 C for 30 min
by preincubation of the inhibitor (0.2 ml, adjusted
to 1 ml with water) with 10% S9 activation system
(0.2 ml, or the equivalent amount of buffer alone),
mutagen (0.01 ml, in DMSO), and bacteria (0.2 ml),
followed by the addition of 2 ml soft agar and pouring
onto minimal glucose plates. Each tea was brewed at
a concentration of 2% (w/v), but the results are given
for final concentrations in the preincubation assay,
expressed as % equivalent after taking into account
the dilution factor. All experiments were conducted
at least twice, and more often three times, using triplicate plates for each dose tested. Appropriate solvent
and vehicle controls were used in each assay; these
included experiments in which the tea volume was
varied from 0.05 to 1 ml and the volume was made up
to 1 ml in each test tube with water, for both positive
and negative (no mutagen, vehicle) controls. His+ revertant colonies were counted after 48 h of incubation
at 37 C in the dark. Under the conditions reported
here none of the tests gave evidence for toxicity, as
determined in three ways. First, background lawns
and spontaneous revertants counts were carefully
monitored in each experiment. Doses of inhibitor that
reduced the spontaneous count or produced thinning
of the background lawn when inspected under the
microscope were regarded as toxic, and were avoided
in subsequent experiments. Second, smaller suspect
colonies, if detected, were picked from the plate and
confirmed to be His+ revertants by streaking on agar
plates in the presence and absence of added histidine.
Third, to ensure against an effect on cell survival, the

highest dose of antimutagen was tested as reported

before [27], by serial dilution of the preincubation
mixtures and pouring onto plates containing histidine
and biotin. In addition to experiments with complete
tea, purified tea components were mixed to produce
artificial teas and tested with the appropriate solvent
and vehicle controls. None of the assays using tea or
purified tea constituents in the absence of mutagen
gave evidence for toxicity under the conditions reported here. Mutagen doses were selected from the
linear region of the dose-response curve, as determined in preliminary experiments (not presented).
2.5. MROD assays (inhibition of CYP1A2)
To test for inhibitory effects of tea on the isozyme
primarily responsible for the activation of heterocyclic
amines, namely cytochrome P4501A2 (CYP1A2), we
conducted methoxyresorufin O-demethylase (MROD)
assays, as described before [18]. Reactions contained
rat liver S9 plus co-factors, and they were started by
the addition of NADPH. To parallel the mutagenicity
assays, tea was added in increasing concentrations, and
the final incubation was brought up to 1 ml with water.
Resorufin product was determined by its fluorescence
(540 nm excitation, 580 nm emission filters); to test for
possible quenching, in some experiments the reaction
was allowed to proceed in the absence of tea, and
the appropriate concentration of tea was added after
stopping the reaction.
2.6. Statistics
Group comparisons were made using one-way analysis of variance (SAS version 8 or StatView 5.01).

3. Results
3.1. Antimutagenic activities of white and green
teas against heterocyclic amines
Initial assays compared the inhibitory activities of
four different white tea varieties brewed for various
times up to 5 min (Fig. 2). All four teas exhibited
good antimutagenic activity against IQ in the presence
of S9, with Mutant white and Exotica China white
varieties being more potent than Silver Needle and

G. Santana-Rios et al. / Mutation Research 495 (2001) 6174

65

Fig. 2. Antimutagenic activities of four white teas in Salmonella typhimurium strain TA98. Each tea was brewed for 5 min at 2% (w/v)
and 0.2 ml was added to the preincubation assay containing IQ + S9. See Section 2 for further details. Data points and bars indicate
mean S.D. from triplicate plates and they are actual plates counts. In this experiment, spontaneous counts were in the range of 3745
revertants per plate in the absence of mutagen (with DMSO alone) following each tea treatment, with normal growth of the background
lawn. Dashed line indicates spontaneous counts (mean) for DMSO in the absence of tea. The results are from a single assay, but are
representative of data obtained in separate tests with each tea individually.

Flowery Pekoe teas. In most cases, a brew time of

only 1 min appeared to be sufficient to release most
of the antimutagens, although Exotica China white tea
(referred to hereafter as Exotica) showed evidence
for increasing inhibition with brew time. Thus, Exotica tea was selected for further study, including direct
comparison with green tea.
Exotica white tea and Premium green tea, each
brewed for up to 10 min at 2% (w/v), were tested
against IQ in the presence of S9. Premium green tea is
a high-end variety with a large rolled leaf that opens
completely during brewing, allowing for the rapid
release of antimutagens, typically within 23 min of

brewing (Fig. 3a). Exotica white tea showed consistently greater inhibitory activity than green tea under
the same assay conditions (Fig. 3a, open symbols).
The antimutagenic activity of both teas increased with
concentration (Fig. 3b), but Exotica white tea was
consistently more potent than Premium green tea at
all concentrations tested. At the maximum concentration in the assay, neither tea gave evidence of overt
toxicity based on normal growth of the background
lawn and spontaneous counts in the normal range.
The lack of toxicity for each tea was confirmed both
in the presence and absence of mutagen, using DMSO
as the vehicle control.

66

G. Santana-Rios et al. / Mutation Research 495 (2001) 6174

Fig. 3. Antimutagenic activities of Exotica white tea and Premium green tea. Results for (a) brew time and (b) concentration (%, w/v
equivalent) are given as mean S.D. of actual plate counts from triplicate plates with Salmonella strain TA98, IQ and S9, and they are
representative of the data obtained from three separate experiments. In these assays, spontaneous counts were (a) 43 6 and (b) 38 4
in the presence of S9. Concurrent tests using the same amounts of tea in the absence of mutagen (DMSO alone) showed no evidence of
toxicity, based on normal growth of the background lawn and spontaneous counts in the normal range. The dashed line represents the
mean value for spontaneous counts with DMSO (no mutagen) and the maximum dose of tea.

In subsequent experiments, each tea was tested

against four other heterocyclic amines, namely,
MeIQx, Trp-P-2, 4,8-DiMeIQx, and PhIP (Fig. 4).
In all cases, white tea was more effective than green
tea under the conditions of the assay. Occasionally, a
slight enhancement was detected at the 0.1% green
tea concentration (e.g. Fig. 4b and c); we do not know
the significance, if any, of these increased responses.
However, this is certainly not a rare phenomenon,
since previous studies have shown slight enhancement
of mutagenesis, for example, with low but not high
doses of chlorophyllin against benzo[a]pyrene, copper
chlorin against Glu-P-2, retinoic acid against aflatoxin
B1, hemin against the tobacco-specific nitrosamine
4-(N-methyl-N-nitrosoamino)-1-(3-pyridinyl)-1-butanone (NNK), and chlorophyll, Ge-protoporphyrin,
and the food coloring agent Monascus red against
3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole
(N-hydroxy-Trp-P-2) [2733].

Because some inhibitors, such as chlorophyllin

[17], rely on complex formation for their antimutagenic activity, and this mechanism depends on a large
molar excess of inhibitor to mutagen, we included
higher mutagen doses in some experiments. For example, in assays using 1 mg PhIP per plate (Fig. 4d),
white tea and green tea both exhibited potent antimutagenic activity, without toxicity, implying that
mechanism(s) other than complex formation might be
important.
One alternative mechanism, namely direct
inhibition of the ultimate carcinogen, was tested using
N-hydroxy-IQ in the absence of an exogenous activation system. In contrast to the results from experiments in which S9 was included in the assay (Figs. 3
and 4), green tea and white tea exhibited more or less
equivalent inhibitory activity against N-hydroxy-IQ,
even at high concentrations (Fig. 5a). This suggested
that inhibition of the enzymes in S9 might be a factor

G. Santana-Rios et al. / Mutation Research 495 (2001) 6174

67

Fig. 4. Antimutagenic activities of Exotica white tea and Premium green tea against several heterocyclic amines. Assays were performed
with Salmonella strain TA98 in the presence of S9 under the conditions used for IQ (Fig. 3b). Results are given as mean S.D. of
actual plate counts from triplicate plates, and they are representative of the data obtained from two or more separate experiments for each
mutagen/tea combination. Concurrent tests using the same amounts of tea in the absence of mutagen (DMSO alone) showed no evidence
of toxicity, based on normal growth of the background lawn and spontaneous counts similar to those in the absence of tea. The dashed
line in each figure represents the mean value for spontaneous counts, using DMSO (no mutagen) plus the maximum dose of tea. Each
mutagen dose was selected from the approximate linear portion of the dose-response curve, based on preliminary assays conducted in the
absence of tea (not shown).

in explaining the more potent antimutagenic activity

of white tea versus green tea.
White tea at a concentration of 0.0125% inhibited
MROD activity by 50%, and 0.05% white tea inhibited enzyme activity completely (Fig. 5b). At these
concentrations of tea, there was minimal interference
via direct quenching of fluorescence (closed symbols,
Fig. 5b). When the data were corrected for quenching
and converted to percent inhibition, EROD activity was attenuated at concentrations similar to those
required for inhibition of IQ mutagenicity in the

Salmonella assay, but at concentrations below those

needed to inhibit the mutagenicity of N-hydroxy-IQ
(Fig. 5c).
One of the major polyphenols in tea, namely EGCG,
was tested in the MROD assay, together with a known
inhibitor of CYP1A2 (3,3 -diindolylmethane, I33 ).
Concentrations of EGCG required to give a similar
profile of MROD inhibition as seen for white tea were
in the range 10100 M, the highest concentration
being more effective than an equivalent level of I33
(Fig. 5d).

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G. Santana-Rios et al. / Mutation Research 495 (2001) 6174

Fig. 5. Mechanism studies with white tea and green tea. (a) Inhibition of N-hydroxy-IQ in the Salmonella assay in the absence of S9.
Assays were conducted with Salmonella strain TA98, using triplicate plates per dose; data are given as mean S.D. and are representative
of two separate experiments. Dotted line, spontaneous counts for vehicle (DMSO) plus the highest concentration of tea; no toxicity was
observed under the conditions of the assay, using the criteria stated in Section 2. (b) Inhibition of MROD activity, a marker for CYP1A2.
Assays were conducted with rat liver S9 and co-factors in the presence or absence of tea, including incubations with addition of tea after
stopping the reaction (test for quenching). Data are given as mean S.D. from triplicate test tubes, and are representative of results from
three such assays. (c) In order to directly compare results for white tea in three separate test systems, namely IQ-induced mutagenicity
(+S9), N-hydroxy-IQ-induced mutagenicity (S9), and CYP1A2 (MROD) enzyme assays, background counts or quenching results were
subtracted and the data were converted to percent inhibition using the following formulae: 1 [induced TA98 revertants + tea]/[induced
TA98 revertants tea] 100, or 1 [MROD activity + tea]/[MROD activity tea] 100. (d) Inhibition of MROD activity by EGCG, a
major polyphenol in tea, in comparison with a positive control inhibitor, I33 . Data are given as mean S.D., n = 3.

3.2. Tea composition

EGCG is a major polyphenol in green tea, but the
levels of this compound and other catechins were
not known in white tea. Therefore, quantitative and

qualitative analyses of white tea and green tea were

undertaken. Fig. 6 shows a typical HPLC chromatogram for Exotica white tea, with several peaks
detected at 273 nm. The identities of nine of the
major constituents were confirmed by UV spectral

G. Santana-Rios et al. / Mutation Research 495 (2001) 6174

69

Fig. 6. Analysis of the major constituents in Exotica white tea. (a) White tea was brewed for 5 min at 2% (w/v) and 20 ml were subjected
to separation by reverse-phase HPLC, using conditions described in Section 2. Identification was based on (i) UVVIS spectra for each of
the fractions collected; (ii) co-elution with authentic standards; (iii) mass spectral analysis (data not shown). (b) HPLC analysis of Exotica
white tea after different brew times. Bars indicate mean S.D. from three separate determinations. For the abbreviations used, see Section 2.

analyses, co-elution with authentic standards, and

CI mass spectrometry (not shown). When green tea
was separated using the same HPLC conditions (not
shown), there were higher levels of CAT and EC,
but lower concentrations GA, TB, ECG, and caffeine
compared with white tea (Table 1). Interestingly,
EGCG was present at equally high levels in both
teas. One peak with Rt 6.1 min (Fig. 6a) was detected at higher levels in white tea than green tea,
plus several minor peaks, but the identity of these

constituents could not be confirmed in the present

study.
The nine major tea components identified in Table 1
were detected after a brew time of only 0.5 min, and
each continued to increase in concentration with brew
times up to 5 min (Fig. 6b). These results suggested
that the overall ratios of the nine major constituents
were constant, and that the changes in tea composition
with brew time were quantitative rather than
qualitative.

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G. Santana-Rios et al. / Mutation Research 495 (2001) 6174

Table 1
Concentrations of major constituents in white tea (WT) and green tea (GT)a

GA
TB
TP
EGC
CAT
CAF
EGCG
EC
ECG

Rt

WT (g/ml)

WT (%)

GT (g/ml)

GT (%)

4.5
12.9
15.1
19.7
20.8
26.1
28.6
31.8
40.1

0.041
0.022
0.003
0.465
0.109
0.822
1.229
0.031
0.619

0.203
0.113
0.015
2.324
0.543
4.109
6.146
0.153
3.095

0.005
0.009
0.005
0.044
0.405
0.403
1.222
0.190
0.403

0.027
0.045
0.027
0.219
2.024
2.014
6.110
0.949
2.014

a Teas were brewed at 2% (w/v) for 5 min and separated using the HPLC conditions shown in Fig. 6a. Percent values in the table
(mg/100 mg dry wt.) were calculated by peak area integration, with reference to the standard curve for the corresponding compound (not
shown). Rt: retention time (minutes); for other abbreviations see Section 2.

3.3. Antimutagenic activities of artificial teas,

leaves and buds
Due to the different composition of white and green
teas (Table 1), the nine major constituents were mixed
in concentrations identical to those in each tea, and
these artificial teas were tested for inhibitory activity
in the MROD and Salmonella assays versus complete
tea. In the MROD assay (Fig. 7a), the artificial white
and green teas proved to be more effective than the
corresponding complete tea, whereas the reverse was
true in the Salmonella assay using IQ + S9 (Fig. 7b).
In both assays, however, white tea or artificial white
tea inhibited more effectively than the corresponding
green tea or artificial green tea.
Finally, because Exotica tea is a cut version of
a loose leaf variety (Mutant white), we obtained the
uncut tea and carefully separated the leaves and buds.
The antimutagenic activity of cut leaf tea was greater
than that of cut buds alone, or the mixture of cut leaves
and buds (Fig. 7c). The uncut tea was significantly
less effective than the cut variety against IQ (compare the final two bars in Fig. 7c). Testing of cut and
uncut Premium green tea showed only slightly higher
antimutagenic activity of the former (data not shown).

4. Discussion
In this report, we describe for the first time the
antimutagenic activities of four white tea varieties in
the Salmonella assay, and include results from studies of the inhibitory mechanisms and characterization

of the major constituents. The most effective of the

white teas, namely Exotica China white, showed better antimutagenic activity than Premium green tea
against IQ and against four other heterocyclic amines
in the presence of S9. Interestingly, both teas were
equally effective against the direct-acting mutagen
N-hydroxy-IQ (S9), but the maximum inhibition was
60% versus >95% in assays containing the promutagen in the presence of S9. In MROD assays containing
S9, inhibition by white tea occurred at concentrations
similar to those used in Salmonella assays with IQ
(+S9), suggesting that constituent(s) in white tea
might prevent the metabolic activation of heterocyclic
amines via interference with CYP1A2. A concentration of 0.05% white tea inhibited MROD activity by
approximately the same degree as 50100 M EGCG.
EGCG is a major polyphenol in green tea, but little
was known about the content of EGCG or other constituents in white tea. One hypothesis for the different
antimutagenic activities of white tea and green tea
was that the more limited processing of the former
tea (Fig. 1) might allow for higher levels of EGCG
and related catechins to be recovered after brewing.
Previous work indicated that green tea was more effective than black tea in inhibiting IQ-induced colonic
aberrant crypt foci in vivo [18], possibly due to the
conversion of catechins to theaflavins and higher
molecular weight polyphenols during tea processing.
A comparison of Exotica white and Premium green
teas by HPLC revealed similar overall profiles for
the major UV absorbing peaks, suggesting that the
greater antimutagenic activity of the former tea was
not due to a unique constituent, present solely in the

G. Santana-Rios et al. / Mutation Research 495 (2001) 6174

71

Fig. 7. Inhibitory activity of artificial tea (a mixture of nine major constituents) in comparison with complete tea in the MROD assay and
the Salmonella assay, and antimutagenic activity of white tea leaves and buds. Based on the data in Table 1, each of the nine major tea
constituents was mixed in the proportions and concentrations equivalent to those in green or white teas, and then tested (a) in the MROD
assay and (b) in the Salmonella assay according to the protocols used previously. Results are given as mean S.D., n = 3. Controls for
background counts and quenching were included as described before (Figs. 3b and 5b, respectively). (c) Leaves and buds from white tea
were separated by hand and tested separately for antimutagenic activity; the tea was cut to a consistency used in commercial teabags, or
in some cases left uncut. Results are given as mean S.D., n = 3; statistical analysis using ANOVA. Bars with different superscripts were
statistically different, P < 0.05.

white tea. However, certain quantitative differences

were detected, including higher levels of gallic acid,
theobromine, EGC, caffeine, and ECG in white tea
(Table 1).
When the nine major constituents were mixed
to produce artificial teas, according to their

concentrations in white and green teas (Table 1),

inhibitory activities in the MROD assay were generally higher than for the corresponding complete tea
(Fig. 7a). However, complete tea was more effective
than the corresponding artificial tea in the Salmonella
assay. One interpretation of these results is that

72

G. Santana-Rios et al. / Mutation Research 495 (2001) 6174

inhibition of IQ-induced mutagenicity involves all of

the constituents in tea, including minor constituents
not present in the artificial tea. The major constituents
might contribute primarily to inhibition of carcinogen activation via by effects on CYP1A2, but minor
constituents (not identified) could act synergistically to inhibit the activated carcinogen(s) (Fig. 5a).
The results are in general accordance with previous work showing inhibition of the mutagenicity of
N-hydroxy-IQ by EGCG and EGC concentrations
in the range 1050 M [17]. These concentrations
of tea polyphenols appear to be achievable in vivo,
and relevant to human exposures. Thus, in rats given
decaffeinated green tea (25 mg/kg body weight, i.v.),
EGCG reached a peak concentration of 55 M in
the intestine, and maintained steady state levels of
1520 M for 4 h [34]. In humans consuming green
tea, the circulating plasma levels of total catechins
were in the order of 110 M, but higher concentrations might be achieved in the tissues of the GI tract
after oral ingestion of tea [35].
In contrast to other teas, white tea has a relatively
high content of buds, which are covered with fine
silvery hair and impart a white/gray appearance to the
dried tea. It was possible that the more potent antimutagenic activity of white tea might be due to higher
levels of antimutagens in the buds. However, when
the leaves and buds were carefully separated and
tested for antimutagenic activity, leaves alone showed
better inhibition than buds alone, or a mixture of buds
and leaves (Fig. 7c). When white tea was cut to a
consistency found in commercially available tea bags,
this increased the antimutagenic activity, and HPLC
experiments revealed improved overall extraction of
the major UV absorbing constituents (not shown). Although the content of chlorophyll-related compounds
was not examined, these were most likely present
only in the beverage obtained from green tea leaves,
judging by the hues of the brewed beverage (Fig. 1).
Chlorophylls and chlorophyllins are potent antimutagens against heterocyclic amines in the Salmonella
assay [17,2932,36], and chlorophyll-related compounds isolated from the non-polyphenolic fraction
of green tea exhibit strong antioxidant activities in
vitro [37]. However, it is evident from the results
of the present investigation that chlorophyll-related
compounds did not contribute to a higher degree of
antimutagenic activity of green versus white teas.

Additional studies with white tea are needed, including testing for possible synergistic effects of the individual constituents, as reportedly recently for EGCG
in the presence of ()-epicatechin using a human
lung cancer cell line [38]. It also will be important
to compare the inhibitory activities of white tea and
green tea in in vivo and cell culture assays, focusing
on mechanisms such as induction of detoxification
enzymes, scavenging of reactive species (free radicals
and electrophilic intermediates of carcinogens), alterations in signal transduction pathways, and changes
in cell cycle regulation and apoptosis [512].
In summary, results from the present study demonstrate for the first time that white teas exhibited
potent antimutagenic activity against a number of
heterocyclic amines in the Salmonella assay. Exotica
China white tea was more effective than Premium
green tea in these assays, and mechanism studies
suggested inhibition of carcinogen activation via interference with the enzymes in S9, plus inhibition of
the direct-acting mutagen(s). EGCG was identified
as the major polyphenol in both white and green
teas, but caffeine, gallic acid, theobromine, EGC, and
ECG were present at higher concentrations in white
tea. Thus, the greater inhibitory activity of white
tea versus green tea appears to be related to higher
concentrations of several of the major constituents,
perhaps acting synergistically with minor constituents
to prevent mutagenicity via multiple mechanisms.

Acknowledgements
The authors would like to thank Dr. Bruce Ames
for kindly providing Salmonella strain TA98, and
Max Deinzer, Brian Arbogast, and Alan Taylor of
the Oregon State University Environmental Health
Sciences Center for LC-MS analyses. We also thank
Judith Hernaez for the gift of Silver Needle tea, and
Joy Edlund and The Stash Tea Co. for providing additional teas used in the studies reported here. This
work was supported in part by NIH Grants CA65525
and CA80176. GS-R and GAO were supported by a
Toxicology Training Grant from the National Institute of Environmental Health Sciences (contract T32
ES0707060). Color images in Fig. 1 are reproduced
with permission from Special Teas, Inc., Norwack, CT.

G. Santana-Rios et al. / Mutation Research 495 (2001) 6174

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