Mutagenesis vol.13 no.6 pp.631-636, 1998
Contribution of theafulvins to the antimutagenicity of black tea:
their mechanism of action
Fenton Catterall, Emma Copeland, Michael N.Clifford
and Costas Ioannides1
School of Biological Sciences, University of Surrey, Guildford, Surrey
GU2 5XH, UK
Theafulvins were isolated from black tea aqueous infusions
and their antimutagenic activity was evaluated against a
number of food carcinogens. Theafulvins gave rise to a
concentration-dependent inhibition of the mutagenicity
of 2-amino-3-methyumidazo-[4,5-/|quinoline, 2-amino-lmethyl-6-phenylimidazo[4,5-fc]pyriduie,
benzo[a]pyrene,
7,12-dimethylbenz[a]anthracene, nitrosopyrrolidine and
nitrosopiperidine, but, in contrast, the mutagenicity of
aflatoxin B, was enhanced. The mutagenicity exhibited by
N'-methyl-./V'-nitro-iV-iiitrosoguanidine and 9-aminoacridine was not influenced and weakly inhibited by theafulvins,
respectively. The p-hydroxylation of aniline and the
O-dealkylations of methoxy-, ethoxy- and, to a lesser extent,
pentoxyresorufln were inhibited by theafulvins in a concentration-dependent manner. When microsomal metabolism
was terminated after metabolic activation of the promutagens, incorporation of the theafulvins into the activation
system did not modulate the mutagenic response. It is
concluded that theafulvins play an important role in the
antimutagenic activity of black tea by inhibiting cytochrome
P450-dependent bioactivation of the carcinogens.
Introduction
Numerous experimental studies in animal models have shown
that green tea acts as an effective anticarcinogen against
chemical- and radiation-induced cancers (Ahmad et al., 1998).
Black tea, preferred in Europe and in North America, has not
received as much attention, but in studies where it was
employed as a possible anticarcinogen against 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone (NNK)-, N-nitrosodiethylamine-, N-nitrosomethylbenzylamine- and UVB-induced (in
7,12-dimethylbenz[a]anthracene-initiated) tumourigenesis in
mice, it performed with similar efficiency as green tea (Wang
et al., 1992, 1993, 1994). In more recent studies, black tea
theaflavins have been shown to effectively antagonize the
carcinogenicity of NNK in mice (Yang et al., 1997). Thus, it
is becoming increasingly evident that both black and green tea
are potential human anticarcinogens. The precise mechanisms
through which tea exerts its anticarcinogenic effects have not
been fully clarified, but it is evident that it can act at all stages
of chemical carcinogenesis, including initiation. Similarly,
the constituent(s) of tea responsible for the anticarcinogenic
activity has not been identified, but, at least in the case of
green tea, flavanoids are believed to play an important role.
In studies concerned with the antimutagenic potential of
tea, it has been reported that green tea and black tea display
the same degree of antimutagenic activity against dietary
carcinogens, despite the much higher content of flavanols
present in green tea (Bu-Abbas et al., 1996). Subsequent
studies failed to reveal any relationship between antimutagenic
activity and content of individual flavanols in fractions of green
tea (Bu-Abbas et al., 1997). Consequently, the contribution of
flavanoids to the ability of green tea to impair the initiation
stage of carcinogenesis has been questioned (Ioannides, 1998).
In black tea, flavanoids are oxidised by polyphenol oxidases
to yield the more complex reddish-brown thearubigins and
theaflavins and, consequently, the levels of flavanoids in black
tea are low compared with green tea (Wang et al., 1994). One
fraction of the black tea thearubigins is the theafulvins (Bailey
et al., 1992; Powell, 1995), whose biological activity has not
been evaluated. The theafulvins are the major polyphenolic
fraction in black tea and occur at concentrations in the range
0.3—3.0 mg/ml, depending largely on the method of brewing
employed. Preliminary work using a polyphenolic fraction
from black tea, comprising largely thearubigins, revealed
antimutagenic activity when used at concentrations of 1-3 mg/
plate against a number of indirect-acting mutagens, but, in
contrast, did not modulate the mutagenic response elicited by
direct-acting mutagens (Weisburger et al., 1996).
In the present study we have isolated the theafulvin fraction
of thearubigins from black tea and evaluated its antimutagenic
potential against the major classes of dietary carcinogens.
Moreover, the underlying mechanisms have been investigated.
Materials and methods
N-Nitrosopyrrolidine, Af-nitrosopiperidine, JV-methyl-A''-nitro-A'-nitrosoguanidine (MNNG), 9-aminoacridine, benzo[a]pyrene, isoniazid, all cofactors and
cytochrome c (Sigma Chemical Co., Poole, Dorset), 2-amino-3-methylimidazo[4,5-/]quinoline (IQ) and 2-amino-l-methyl-6-phenylimidazo[4,5-b]pyridine
(PhIP) (Toronto Research Chemicals, Toronto, Canada), 7,12-dimethylbenz[a]anthracene (111, Research Institute, Chicago, IL) and resorufin and
all alkoxyresorufins (Molecular Probes, Eugene, OR) were all purchased.
Theafulvins were isolated from black tea (Lattakari Assam) as previously
described (Bailey et al., 1992). Infusions of tea were first decaffeinated using
chloroform and then partitioned against ethyl acetate. Theaflavins were
extracted into the organic solvent while the thearubigins remained in the
aqueous phase. Theafulvins were separated from the other thearubigins by
passing the crude thearubigin fraction through a Solka floe cellulose column
from which the theafulvins are eluted with 50% acetone in water.
Male Wlstar albino rats (120-150 g), obtained from Harlan Olac (Bicester,
Oxon, UK), were used in all studies. Induction of the CYP1 family was
achieved by administration of a single i.p. dose of Aroclor 1254 (500 mg/
kg), the animals being killed on diefifthday following administration. Induction
of the CYP2E1 isoform was achieved by daily intragastric administration for
3 days of isoniazid (100 mg/kg), the animals being killed 24 h after the last
administration. Hepatic post-mitochondrial (S9) and microsomal fractions
were prepared as previously described (Ioannides and Parke, 1975). The
following assays were performed using isolated microsomes: the O-dealkylations of methoxyresorufin (Burke and Mayer, 1983), ethoxyresorufin
(Burke and Mayer, 1974) and pentoxyresorufin (Lubet et al., 1985),
/>-hydroxylation of aniline (Guarino et al., 1969) and protein determination
(Lowry et al, 1951).
Mutagenic activity was monitored using the Ames mutagenicity assay (Maron
snd Ames, 1983). When isolated microsomes were used, the activation system
was supplemented with glucose 6-phosphate dehydrogenase (1 U/plate). In
'To whom correspondence should be addressed. Tel: -"-44 1483 2597C9; Fax: +44 1483 576978; Email: cioannidesSsurrey.ac.uk
© UK Environmental Mutagen Society/Oxford University Press 1998
631
F.Catterall et al.
order to assess whether the aqueous tea extracts scavenge the electrophilic
intermediates of the food carcinogens, the Ames procedure was modified. Initially, the bacteria, carcinogen and activation system were preincubated for 20
min in a shaking water bath at 37°C. Miciosomal metabolism was terminated
by the addition of 100 nl menadione (900 nM) and a second 20 mm preincubation
was carried out in the presence of the aqueous tea extract. Top agar was added
and the mixture was poured onto minimal agar plates that were incubated for 48
h at 37°C to allow revertants to develop into colonies AJ1 mutagenicity studies
were repeated and results were reproducible.
Results
Black tea theafulvins, at concentrations >100 jig/plate, caused
a concentration-dependent inhibition of the mutagenic response
PhIP
5000 -i
100
200
300
400
500
100
Theafulvin (u,g / plate)
Benzo[a]pyrene
100
200
300
200
300
400
500
Theafulvin (u.g / plate)
Dimethylbenz(a)anthracene
100
400
200
300
400
Theafulvin ^ g / pUte)
Theafulvin (u.g / plite)
Nitrosopyrrolidine
Nitrosopiperidine
250 -i
375
100
200
300
400
Theafulvin (jig / plate)
500
100
200
300
400
Theafulvin (ng / plate)
Fig. 1. Inhibition of the mutageniaty of indirect-acting mutagens by black tea theafulvins. The study was conducted using Salmonella typhimurium strains
TA98 (50 ng IQ and 2 |ig PhIP), TA100 (25 \x.g benzola]pyrene and 25 ng dimethylbenz[a]anthracene) and TA1530 (4 mg nitrosopyrrolidine and 4 mg
nitrosopiperidine) Hepatic S9 preparations from Aroclor 1254-induced rats (10% v/v) served as the activation system for IQ. PhIP. benzo[a]pyrene and
dimethylbcnzJa]anthiaccne and from isoniazid-treated rats (10% v/v) for nitrosopiperidine and nitrosopyrrolidine Results are presented as mean x SD for
triplicates The spontaneous reversion rates were 38 ± 5. 113 ± 8 and 14 ± 2 for TA98, TAIOO and TA1530. respectively, arid have already been subtracted
632
Antimutagenic activity of theafuMns
elicited by the heterocyclic amines IQ and PhIP, with mutagenicity being completely suppressed at a concentration of 500
Hg/plate (Figure 1). When the polycyclic aromatic hydrocarbons
benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene served as
the model carcinogens, significant antimutagenic response was
seen only at the highest concentration studied of 500 ng/plate
(Figure 1). The mutagenicity displayed by the two nitrosamines,
namely nitrosopiperidine and nitrosopyrrolidine, was inhibited
by theafulvins in a concentration-dependent fashion when the
concentrations exceeded 100 ng/plate (Figure 1). Surprisingly,
tea theafulvins at concentrations above 50 ug/plate potentiated
the mutagenic response induced by the mycotoxin aflatoxin B (
(Figure 2). The stimulating effect of theafulvins reached a plateau at concentrations of 150 |ig/plate and then started to decrease,
but, at the concentrations studied, never attained control levels.
At the same concentrations theafulvins had no effect on the
mutagenicity of the direct-acting mutagen MNNG and only a
weak inhibitory effect in the case of 9-aminoacridine, another
direct-acting mutagen (Figure 3).
Theafulvins caused a concentration-dependent decrease in the
hepatic microsomal O-dealkylation of methoxy-, ethoxy- and
pentoxyresorufin in Aroclor 1254-induced microsomes and in
Methoxyrewrnfin O-demethyla**
002
00)
OH
00)
0J6
TFU (mf per mnrh*tVT*)
Ethoxyresorofin O-deethylase
li
I•
0 02
001
•
0.03
0.04
0.03
0.06
TFU (mf per *Tmihilw*p)
Pentotyretornfin O-depcntyiase
Aflatoxin B,
J500
0 125
200
300
400
500
TbeafoMa (fig / pint)
Fig. 2. Effect of black tea theafulvins on the mutagenicity of aflatoxin B|.
The study was carried out using Salmonella typhimurium strain TA98,
aflatoxin B, (3 |ig/plate) and an activation system (10% v/v) derived from
Aroclor 1254-induced rats. Results are presented as mean ± SD for
triplicates. The spontaneous reversion rate was 27 ± 1 and has already been
subtracted.
9-Aminoacridine
1500 i
0 25
0J7J
0J
TFU (mf per Incubation)
Fig. 4. Impairment of mixed function oxidase activity by black tea
theafulvins. Studies were conducted using pooled microsomes from Aroclor
1254-induced rats, except in the case of aniline, where isoniazid-induced
microsomes were employed. Remits reflect the mean of two determinations.
MNNG
5000
1200 -
100
200
300
400
Theafurvin (jig / plate)
500
100
200
300
400
500
Tbeafulvin (ng / plate)
Fig. 3. Effect of black tea theafulvins on the mutagenicity of direct-acting mutagens. The study was carried out using Salmonella typhimurium strains TA100
(1 |ig MNNG) and TA97 (50 ng 9-aminoacridine). Results are presented as mean ± SD for triplicates. The spontaneous reversion rates were 209 ± 3 and
148 ± 19 for TA100 and TA97, respectively, and have already been subtracted.
633
F.Catterall et al.
Table I. Interaction iof black tea theafulvins with the microsome-denved
genotoxic species of IQ
First preincubation
Experiment 1
IQ
IQ + TFu (10 HO
IQ + TFu (20 HD
IQ + TFu (50 HO
IQ + TFu (100 HO
IQ + TFu (150 HO
IQ + TFu (200 HO
IQ + TFu (500 ul)
Experiment 2
IQ
IQ
IQ
IQ
IQ
IQ
IQ
IQ
Second preincubalion
Histidine revertants/plate
Menadione
Menadione
Menadione
Menadione
Menadione
Menadione
Menadione
987
1012
980
922
404
268
186
19
±
±
±
±
±
±
±
±
75
65
13
36
19
10
8
2
1560
1506
1480
1574
1658
1458
(200 ul) 1688
(500 ul) 1571
±
±
±
±
±
±
±
±
161
81
78
51
62
70
100
117
Menadione
Menadione
Menadione
Menadione
Menadione
Menadione
Menadione
-h
-1-h
-1-h
-h
TFu
TFu
TFu
TFu
TFu
TFu
Menadione HV TFu
(10 Hi)
(20 HO
(50 HO
(100 HD
(150 HO
The study was earned out using IQ (50 ng/plate), the TA98 bacterial strain
and hepatic microsomes (10% v/v) derived from Aroclor 1254-induced rats
Theafulvins (TFu) were added either during the first preincubation
(experiment 1) or during the second preincubation, after microsomal
metabolism was terminated by addition of 100 H1 menadione (900 uM) to
the activation system (experiment 2) The spontaneous reversion rates were
26 ± 3 and 23 ± 5 for experiments I and 2. respectively Results are
presented as mean ± SD of triplicates
the p-hydroxylation of aniline in isoniazid-treated microsomes
(Figure 4).
In studies aimed at establishing whether theafulvins exert their
antimutagenic activity by scavenging the reactive intermediates,
the bacteria, activation system and IQ were incubated at 37°C
for 20 min to allow their metabolic generation and microsomal
metabolism was terminated by addition of menadione. Theafulvins or buffer was added and a second incubation was earned
out. Under such conditions the theafulvins failed to modulate
the mutagenic response (Table I), fn order to distinguish between
the ability of theafulvins to inhibit microsomal bioactivation of
IQ and to scavenge the reactive intermediates, incubations were
carried out concurrently where the theafulvins were present
in the activation system. Under such conditions, as expected,
theafulvins suppressed the mutagenic response (Table I).
Discussion
Previous studies have indicated that commercial preparations
of theaflavins and thearubigins could suppress the mutagenicity
of promutagens in the Ames test (Weisburger et al.. 1996;
Apostolides et al.. 1997). In the present study theafulvins were
isolated from black tea aqueous infusions. Thearubigins were
initially separated from the theaflavins by ethyl acetate extraction and the crude thearubigin fraction was passed through a
cellulose column allowing the separation of theafulvins, free
from known polyphenols of known structure such as flavanols,
flavanol glycosides, fiavanol gallates (characteristic of green
tea and present in varying amounts in black tea) and theaflavins/
theaflavin gallates (characteristic of black tea). We have shown
(Powell. 1995; Powell et al., 1995) that this isolated crude
theafulvin is free from caffeine and low in total nitrogen. Ft
elutes as a convex hump from reverse phase HPLC packings
but can be partially resolved by size exclusion HPLC (Clifford
and Powell. 1996; Clifford et al.. 1996). These peaks have
634
apparent masses in the range 900-2300 Da when the column
is calibrated with pure neutral condensed and hydrolysable
tannins. The recently characterized theacitrins are minor components of the isolated theafulvins (Davis et al., 1997).
Theafulvins caused a concentration-dependent decrease in
the mutagenicity of indirect-acting food carcinogens such as
heterocyclic amines, polycyclic aromatic hydrocarbons and
nitrosamines. The heterocyclic amines IQ and PhIP and the
nitrosamines nitrosopiperidine and nitrosopyrrolidine were the
most sensitive to the theafulvins, virtually no mutagenic
response being seen at a concentration of 0.5 mg/plate. It is
thus feasible that theafulvins are, at least partly, responsible
for the reported marked antimutagenic effect of black tea
against the same promutagens (Bu-Abbas et al., 1996).
The antimutagenic activity of black tea involves two distinct
mechanisms, namely inhibition of the cytochrome P450dependent bioactivation of the promutagens and, to a lesser
extent, scavenging of the reactive intermediates (Bu-Abbas
et al., 1996). Theafulvins were shown in the present study to
inhibit, in a concentration-dependent manner, the O-dealkylation of methoxy-, ethoxy- and pentoxyresorufin, these
being respectively chemical probes for the CYP1A2, Al and
2B proteins of the cytochrome P450 system (Lubet et al.,
1985; Namkung et al., 1988). Similarly, theafulvins inhibited
the p-hydroxylation of aniline, an activity associated largely
with CYP2E1 (Ryan et al., 1985). Bioactivation of the heterocyclic amines is selectively catalysed by the CYPI family,
particularly CYP1A2 (Kleman and Overvik, 1995) and that of
polycyclic aromatic hydrocarbons by CYPI A1 (Ioannides and
Parke, 1990; Gonzalez and Gelboin, 1994), whereas the
bioactivation of nitrosamines involves primarily CYP2E1 and
the CYP2B subfamily (Yang et al., 1985; Flammang et al.,
1993; Shu and Holleberg, 1997). In the mutagenicity studies,
the source of the activation system employed in the case of
nitrosamines was from rats treated with isoniazid, a potent
inducer of CYP2EI (Ryan et al., 1985), whereas in the case
of the polycyclic aromatic hydrocarbons and heterocyclic
amines the activation system was derived from rats treated
with Aroclor 1254, a potent inducer of CYPI A and CYP2B
activities (Parkinson et al.. 1983). Clearly, these studies demonstrate that the theafulvins, by virtue of their ability to decrease
cytochrome P450 activity, suppress the mutagenicity of promutagens.
A second mechanism through which the theafulvins may
exert their antimutagenic effect is by scavenging the reactive
intermediates of carcinogens, thus protecting the DNA. In
contrast to the effects of black tea (Bu-Abbas et al., 1996),
theafulvins failed to scavenge the reactive intermediates of IQ.
indicating that another component(s) of tea is responsible for
its scavenging characteristics. In concert with this conclusion
is the observation that theafulvins failed to suppress the
mutagenic activity of the direct-acting mutagen MNNG and
had only a relatively weak effect against 9-aminoacridine,
another direct-acting mutagen. In contrast, black tea itself
effectively antagonized the mutagenic response displayed by
9-aminoacridine (Bu-Abbas et al., 1996).
We have reported that black tea at a concentration of about
100 (il/plate of a 2.5% aqueous infusion (w/v), representing
an average strong brew, completely abolished the mutagenic
activity in the Ames test of carcinogens such as benzo[a]pyrene.
2-amino-6-methyldipyrido[l,2-fl:3',2'-d]imidazole and nitrosopyrrolidine (Bu-Abbas et al., 1996). We have shown that
black tea brews vary markedly in composition depending on
Antimutagenic activity of theafulvins
the method of brewing and, to a lesser extent, with the origin
of the leaf used. Using isolated theafulvins to prepare a
calibration curve we have found by HPLC some 0.3 mg/ml
theafulvins in brews prepared by dipping a tea bag for some
60-90 s in 200 ml freshly boiled water (currently a common
practice in the UK) but as much as 3 mg/ml in brews allowed
to steep for up to 10 min (M.N.Clifford, G.Harden and
E.M.Copeland, unpublished results). Thus, the minimum level
of theafulvins in the antimutagenic studies was ~0.03 mg/plate
and the maximum level was ~0.3 mg/plate. Clearly, at such
concentrations theafulvins are major contributors to the antimutagenic activity of black tea.
An unexpected finding was the behaviour of the theafulvins
in potentiating the mutagenicity of aflatoxin B i in Salmonella
typhimurium TA98. This cannot be attributed to any additive
mutagenic response, since the theafulvins did not elicit a
mutagenic response either in the presence or absence of an
activation system (results not shown). Such a peculiar response
of aflatoxin B] has also been reported in the presence of
other phenolic antioxidants, such as butylated hydroxyanisole,
butylated hydroxytoluene and, to a lesser extent, propyl gallate
(Shelef and Chin, 1980). Aflatoxin Bj mutagenicity in the
Ames test was enhanced in the presence of these phenolics
in a concentration-dependent fashion, the potentiating effect
decreasing at the highest concentrations of the phenolics, the
same picture as observed in the present studies employing
theafulvins. Two mechanisms are likely to mediate the effect
of theafulvins, and these are currently being investigated.
Theafulvins may influence the metabolism of aflatoxin Bj so
as to facilitate an increase in bioactivation, for example through
inhibition of pathways of metabolism that lead to formation
of inactive metabolites, directing more metabolism towards
bioactivation. Alternatively, theafulvins may interact with bacterial DNA, making it more susceptible to the genotoxic effects
of the ultimate mutagens of aflatoxin B,.
In summary, we have demonstrated in the present study
that theafulvins isolated from black tea can suppress the
mutagenicity of food carcinogens such as heterocyclic amines,
polycyclic aromatic hydrocarbons and nitrosamines. The mechanism of their antimutagenic activity involves inhibition of the
cytochrome P450-mediated bioactivation of these carcinogens.
It is concluded that theafulvins make an important contribution
to the antimutagenic activity of black tea.
Acknowledgement
The authors gratefully acknowledge financial support for this study from the
European Union under Project PL0653.
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Received on March 31, 1998; accepted on June 3, 199S
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