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Nutrition and Cancer
ISSN: 0163-5581 (Print) 1532-7914 (Online) Journal homepage: http://www.tandfonline.com/loi/hnuc20
Tea as a Potential Chemopreventive Agent in PhIP
Carcinogenesis: Effects of Green Tea and Black Tea
on PhIP-DNA Adduct Formation in Female F-344
Rats
Herman A. J. Schut & Ruisheng Yao
To cite this article: Herman A. J. Schut & Ruisheng Yao (2000) Tea as a Potential
Chemopreventive Agent in PhIP Carcinogenesis: Effects of Green Tea and Black Tea on PhIP-
DNA Adduct Formation in Female F-344 Rats, Nutrition and Cancer, 36:1, 52-58, DOI: 10.1207/
S15327914NC3601_8
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Date: 18 November 2015, At: 13:50

NUTRITION AND CANCER, 36(1), 52–58
Copyright © 2000, Lawrence Erlbaum Associates, Inc.
Tea as a Potential Chemopreventive Agent in PhIP
Carcinogenesis: Effects of Green Tea and Black Tea on
PhIP-DNA Adduct Formation in Female F-344 Rats
Herman A. J. Schut and Ruisheng Yao
Abstract: The heterocyclic amine 2-amino-1-methyl-6-
phenylimidazo[4,5-b]pyridine (PhIP) is formed during the
cooking of proteinaceous animal foods (meat, chicken, and
fish). PhIP is a carcinogen in the Fischer 344 (F-344) rat; it
induces mammary tumors in female rats and lymphomas and
colon and prostate tumors in male rats. In F-344 rats, PhIP
forms DNA adducts in various organs, including the target
organs. Inhibition of PhIP-DNA adduct formation is likely to
lead to inhibition of PhIP tumorigenicity. We have examined
the chemopreventive properties of green tea and black tea in
PhIP carcinogenesis by evaluating their effects on PhIP-DNA
adduct formation in the female F-344 rat. Young adult ani-
mals were maintained on powdered AIN-76A diet while receiv-
ing regular drinking water or 2% (wt/vol) infusions of green
tea or black tea for a total of six weeks. During Weeks 3, 4, and
5, all animals received PhIP by gavage (1 mg/kg/day). Three
rats per group were euthanized on Days 1 and 8 after termina-
tion of PhIP exposure. DNA was isolated from a number of or-
gans and analyzed for PhIP-DNA adducts by 32P-postlabeling
assays. Compared with animals on regular drinking water,
PhIP-DNA adduct formation was inhibited in small intestine,
colon, liver, and mammary epithelial cells (MECs) of animals
receiving green tea or black tea as the sole source of drinking
fluid. Green tea inhibited adduct formation in colon, liver, and
MECs (33.3–80.0%) on both days, but only on Day 8 (54.4%)
in small intestine. Black tea inhibited adduct formation on both
days in liver (71.4–80.0%), on Day 1 in colon (40.0%), and on
Day 8 in small intestine (81.8%); it had no effect on MEC ad-
ducts. Neither green tea nor black tea had an effect on adduct
levels in pancreas, lungs, white blood cells, heart, kidneys,
spleen, cecum, or stomach. Similarly, these teas did not affect
the rate of adduct removal (percent change from Day 1 to
Day 8) in any organ. It is concluded that green tea and black
tea are potential chemopreventive agents in PhIP-induced
tumorigenesis in the F-344 rat.
nents in cancer prevention. Although the results of epidemi-
ological studies are mixed and often inconclusive, the results
of studies in rodents have demonstrated significant chemo-
preventive effects of tea and tea components in a number of
experimental models (reviewed in References 1 and 2).
These effects have been ascribed to tea’s high content of
polyphenolic compounds, principally catechins and other
flavanols (3). Tea catechins not only suppress the in vitro
mutagenicity of chemical carcinogens (4) but are also potent
inhibitors of the initiation and promotion stages of chemical
carcinogenesis (1,2). The anticarcinogenic and chemo-
preventive properties of tea polyphenols are thought to be
due to their effects on carcinogen activation/deactivation en-
zymes (5–8), their growth-suppressing properties (9,10),
and their antioxidant properties (11–13).
Cooking of proteinaceous foods (meat, fish, and chicken)
results in the formation of a number of highly mutagenic
compounds (14), collectively called heterocyclic amines,
most of which belong to the subclass of aminoimidazoaza-
arenes (AIAs). All the AIAs tested are carcinogenic in animals
(reviewed in Reference 15). 2-Amino-1-methyl-6-phenyl-
imidazo[4,5-b]pyridine (PhIP), the most abundant AIA in
cooked meat (16), is carcinogenic in rats and mice (15). PhIP
induces principally mammary tumors as well as some colon
tumors in female Fischer 344 (F-344) rats and lymphomas
and prostate and colon tumors in male F-344 rats (15).
Most carcinogens, including PhIP, need to be enzymati-
cally activated to a DNA-interacting species before their car-
cinogenicity is expressed (17). PhIP is activated in two
steps: 1) by cytochrome P-450 (CYP) 1A1 or 1A2 to yield
N-hydroxy-PhIP and 2) by further esterification of N-hy-
droxy-PhIP (acetylation or sulfation) to generate a reactive
species, probably the nitrenium ion, which reacts with DNA
to form specific adducts (15). PhIP-DNA adducts have been
isolated from a number of animal organs and cells after treat-
ment with PhIP (15); the major adduct has been identified as
N-(deoxyguanosin-8-yl)-PhIP (18–20).
Introduction
Green tea and black tea, as well as a number of individual
tea polyphenols, have been shown to inhibit the bacterial
mutagenicity of PhIP (21–24). Although in the male F-344
The worldwide consumption of tea has prompted
searches for possible beneficial effects of tea and its compo-
The authors are affiliated with the Department of Pathology, Medical College of Ohio, Toledo, OH 43614-5806.

rat green tea and black tea have been shown to inhibit DNA
adduct formation of PhIP in the colon (25), as well as that of
the related AIA, 2-amino-3-methylimidazo[4,5-f]quinoline
(IQ) in the liver (26), such inhibition has not been tested in
the female F-344 rat. PhIP is a mammary carcinogen in fe-
male F-344 rats and, since human exposure to PhIP through
the diet is well established (27–30), PhIP is a suspect agent
in the etiology of human breast cancer. In the present stud-
ies, we have, therefore, examined the potential chemo-
preventive properties of tea in PhIP carcinogenesis by
evaluating PhIP-DNA adduct formation in female F-344 rats
given prolonged, low oral doses of PhIP while receiving in-
fusions of green tea or black tea as the sole source of drink-
ing fluid.
weighed once a week, and doses of PhIP were adjusted
weekly on the basis of weight gains.
On the first day of Week 6 (Day 1), as well as on the first
day of Week 7 (Day 8), three animals from each group were
euthanized, and blood [for isolation of white blood cells
(WBCs)], mammary glands [for isolation of mammary epi-
thelial cells (MECs)], liver, lungs, stomach, small intestine,
cecum, colon, kidneys, heart, spleen, and pancreas were col-
lected as described previously (33). Briefly, for the isolation
of MECs, pooled mammary glands were added to 10 ml of
phosphate-buffered saline, minced into small pieces (<2
mm3), and then incubated with collagenase type I (0.25%,
wt/vol) and collagenase type IV (0.35%, wt/vol) at 37°C for
two hours. During this period the mixture was pipetted up
and down every 15–20 minutes. At the end of the incubation
period, the mixture was filtered through a nylon cell strainer
(70 mm, Falcon) and then centrifuged for five minutes at 500
g. The pellet (MECs) was dissolved in 2 ml of nuclei lysis
buffer [10 mM tris(hydroxymethyl)aminomethane·HCl, 400
mM NaCl, 2 mM Na4-EDTA, pH 8.2] and stored at –70°C
(33). WBCs, isolated as described previously (33), were
stored similarly. All organs were added to a small volume of
phosphate-buffered saline and then immediately frozen at
–70°C until isolation of DNA.
Materials and Methods
Materials
PhIP (>98% pure) was purchased from Toronto Research
Chemicals (North York, ON, Canada). All materials for the
32P-postlabeling assay were from the same sources used in a
previous study (31). Green tea and black tea (World Blends)
were generously donated by Dr. Douglas A. Balentine
(Thomas J. Lipton, Englewood Cliffs, NJ). Infusions of 2%
(20 g/l) were prepared in the laboratory by steeping the tea in
boiling water for10minutesandfilteringthecooledproduct.
Isolation of DNA and 32P-Postlabeling Analysis
DNA was isolated from organs by a direct salt precipita-
tion method, as described previously (34). PhIP-DNA ad-
ducts were isolated and quantitated using the intensification
version of the 32P-postlabeling assay, as described elsewhere
(34), and the solvents for ion-exchange chromatography, as
described by Josyula and co-workers (33). Adduct levels,
expressed as relative adduct labeling (RAL) values, repre-
sent the sum of the individual PhIP-DNA adducts.
Animal Treatment
Eighteen young, adult female F-344 rats (6–7 wk old,
88–105 g) were purchased from Harlan Sprague Dawley (In-
dianapolis, IN). The animals were kept in polycarbonate
cages (3/cage) in a temperature- and humidity-controlled
room. Powdered AIN-76A diet was prepared in the labora-
tory as described previously (32), except the antioxidants
were omitted. The experimental design is outlined in Figure
1. Briefly, the animals were maintained on the AIN-76A diet
for a total of six weeks and given water, 2% green tea, or 2%
black tea (6 animals/group) as drinking fluid during this pe-
riod. Animals received fresh food twice a week (Tuesday
and Friday) and fresh tea three times a week (Monday,
Wednesday, and Friday). During Weeks 3, 4, and 5, PhIP
was administered daily by gavage (1 mg/kg/day) to all ani-
mals, with dimethyl sulfoxide—0.05 M sodium phosphate,
pH 4.5 (1:1), as the vehicle (0.5 ml/animal). Animals were
Statistical Analysis
The effect of the type of drinking fluid on adduct forma-
tion and rate of adduct removal was analyzed by one-way
analysis of variance, followed, in case of a significant effect,
by Fisher’s least significant difference post hoc test.
Results
Body weight gains were independent of the type of drink-
ing fluid during the entire six-week period of the study (p >
0.05 for each of Weeks 1–6, data not shown). During Weeks
1, 2, 3, 4, 5, and 6, body weights of the animals increased
(%gain/wk) as follows: 14.6–19.7%, 18.2–23.2%, 7.6–
10.1%, 3.9–6.8%, 3.7–6.3%, and 3.2–6.1%, respectively.
The PhIP-DNA adduct pattern was qualitatively similar
in all organs/cells and was identical to that observed in our
previous PhIP-DNA adduct studies in the F-344 rat (33–36).
As described previously (33–36), the major 32P-postlabeled
PhIP-DNA adduct was N-(deoxyguanosin-8-yl)-PhIP (data
not shown).
Figure 1. Experimental design. PhIP, 2-amino-1-methyl-6-phenylim-
idazo[4,5-b]pyridine.
Vol. 36, No. 1
53

Figure 2. Total PhIP-DNA adducts in pancreas, lungs, and white blood cells (WBCs) of female Fischer 344 (F-344) rats maintained on AIN-76A diet and
given water (control), 2% (wt/vol) green tea, or 2% (wt/vol) black tea to drink for 6 wk. All animals were treated with PhIP (1 mg/kg/day) by gavage for 21 days
(Weeks 3, 4, and 5). Three animals per time point per group were euthanized on Days 1 and 8 after cessation of PhIP treatment. Adduct levels were independent
(p > 0.05) of type of drinking fluid. RAL, relative adduct labeling.
Adduct levels in animals receiving regular drinking water
were highest in the pancreas (RAL = 1.59 × 10–7; Figure 2)
and lowest in the stomach (RAL = 0.02 × 10–7; Figure 5). In
the colon, adduct formation was inhibited by green tea (40.0%
on Day 1 and 75.0% on Day 8) and by black tea (40.0% on
Day 1; Figure 3). In the small intestine, green tea and black
tea inhibited adduct formation, but only on Day 8 (54.4–
81.8%; Figure 3). In the liver, green tea and black tea in-
hibited adduct formation at both time points (60.0–71.4%
inhibition with green tea and 71.4–80.0% inhibition with
black tea; Figure 5). Only green tea inhibited adduct forma-
tion in MECs (33.3% inhibition on Day 1 and 80.0% inhibi-
tion on Day 8; Figure 5). Neither green tea nor black tea had
any inhibitory effects on PhIP-DNA adduct formation in the
pancreas, lungs, WBCs (Figure 2), heart (Figure 3), kidneys,
spleen, cecum (Figure 4), or stomach (Figure 5).
It is possible that tea may affect the rate of adduct re-
moval (repair), and, therefore, adducts were measured im-
mediately after cessation of exposure to PhIP (Day 1), as
well as on Day 8, when animals had been on a PhIP-free diet
for one week to allow for repair (Figure 1). For all organs and
cells, the average change in adduct levels on Day 8 compared
with that on Day 1 was –38.7% (range –9.1 to –87.5%) in ani-
mals on regular drinking water, –31.5% (range –16.1 to
Figure 3. Total PhIP-DNA adducts in heart, small intestine, and colon. Experimental details are as outlined in Figure 2 legend. Adduct levels were dependent
(p < 0.05) on type of drinking fluid in small intestine (Day 8) and colon (Days 1 and 8) but not in heart (p > 0.05). *, Significantly different from corresponding
control (water) value.
54
Nutrition and Cancer 2000

Figure 4. Total PhIP-DNA adducts in kidneys, spleen, and cecum. Experimental details are as outlined in Figure 2 legend. In all 3 organs, adduct levels were
independent (p > 0.05) of type of drinking fluid.
–88.9%, with 0%, +7.9%, and +7.7% in the liver, pancreas,
and kidneys, respectively) in animals on 2% green tea, and
–33.2% (range –22.2% to –77.8%, with 0%, +28.6%, and
+42.9% in the pancreas, MECs, and WBCs, respectively) in
animals on 2% black tea. The overall differences between
the three groups were not significant (p > 0.05).
In these studies, the pancreas, a nontarget organ, has the
highest PhIP-DNA adduct levels; the stomach and the liver
have the lowest levels. In the female rats, MECs have rela-
tively low adduct levels (36) (Figure 5). Likewise, the colon
has relatively low adduct levels in the male rats (37). There-
fore, because the mammary gland and the colon are the main
target organs in the female and male F-344 rat, respectively
(15), these results mean that PhIP-DNA adduct levels in or-
gans/cells are not necessarily correlated with organ sensitiv-
ity to tumorigenesis. The results of an immunohistochemical
interorgan comparison of PhIP-DNA adducts in F-344 rats
(38) also confirm this conclusion. It should be emphasized
Discussion
Interorgan distribution of PhIP-DNA adducts (Figures
2–5) resembled that observed previously in male (37) and
female (36) F-344 rats treated with PhIP in the same manner.
Figure 5. Total PhIP-DNA adducts in liver, mammary epithelial cells (MECs), and stomach. Experimental details are as outlined in Figure 2 legend. For MEC
adduct levels, mammary glands from 3 animals were harvested and, to obtain sufficient DNA from isolated MECs, glands from 3rd animal were divided into 2
equal portions and added to that of other 2 animals (thus n = 2 for all MEC adduct levels). Where no variability is shown (MEC, control and 2% green tea), SDs
were too small to be visualized. Adduct levels were dependent (p < 0.05) on type of drinking fluid in liver (Days 1 and 8) and in MECs (Days 1 and 8), but not
in stomach (p > 0.05). *, Significantly different from corresponding control (water) value.
Vol. 36, No. 1
55

that interorgan distribution of PhIP-DNA adducts strongly
depends on the manner of administration of PhIP, i.e., high,
bolus dose or prolonged, low levels in the diet (39).
As we found previously with other potential chemo-
preventive agents such as conjugated linoleic acid (33),
menhaden oil (40), and n–3 fatty acids (41), green tea and
black tea inhibit DNA adduct formation only in certain or-
gans and, in some cases, only at certain time points (Figures
2–5). In this regard, these agents are different from
indole-3-carbinol, which inhibits AIA-DNA adduct forma-
tion in all organs/cells examined (36,42,43). It is likely that
inhibition of DNA adduct formation is not only a function of
the kinetics of absorption and elimination of the chemo-
preventive agent but is also related to the enzymatic mecha-
nisms by which the AIA is activated/deactivated in the
various organs. For instance, for PhIP it is known that these
mechanisms differ, not qualitatively, but quantitatively, in
liver, colon, and MECs of the F-344 rat (44,45) and that a
compound like indole-3-carbinol may induce phase I and
phase II enzymes (36,42).
The inhibition of PhIP-DNA adduct formation in MECs
by green tea (Figure 5) would suggest that green tea inhibits
initiation of PhIP-induced mammary carcinogenesis. In the
few mammary tumorigenesis studies involving chemo-
prevention by green tea catechins, the principal action of tea
was in inhibiting the size, not the incidence or multiplicity,
of rat mammary tumors induced by IQ, PhIP, or 7,12-di-
methylbenz[a]anthracene (46–48), suggesting an effect of
tea on promotion rather than initiation. This conclusion was
recently affirmed in a study using black tea as the chemo-
preventive agent in 7,12-dimethylbenz[a]anthracene-in-
duced mammary tumors in Sprague-Dawley rats maintained
on a high-fat (24.0%) diet (49). Thus, in inhibiting mam-
mary tumor formation, tea may act at the initiation and pro-
motion stages of the process.
In the colon, green tea and black tea inhibited PhIP-DNA
adduct formation (Figure 3), confirming a recent finding in
male F-344 rats with use of a similar protocol, except PhIP
was given as a single dose by gavage (25). Also, green tea
and black tea were found to inhibit hepatic IQ-DNA adduct
formation as well as colonic aberrant crypt foci in male
F-344 rats (26). Thus tea probably offers protection against
the initiation phase of colon carcinogenesis induced by
PhIP. This interpretation agrees with chemoprevention stud-
ies by green tea polyphenols in colon tumor induction by
non-AIA carcinogens in rats and mice (50,51). Green tea
polyphenols, however, may also inhibit the postinitiation
phase of colon carcinogenesis in rats (52). In a complex,
multiorgan rat carcinogenesis model, 1% (wt/wt) dietary
green tea catechins failed to inhibit colon carcinogenesis
(53).
(1,2). The observed inhibition of hepatic PhIP-DNA adducts
by green tea and black tea (Figure 5) implies a potential pro-
tective effect in PhIP-induced liver tumors. PhIP, however,
is not a liver carcinogen (15), but its hepatic metabolism, es-
pecially that by phase I enzymes, probably plays a major
role in determining its carcinogenicity in extrahepatic target
organs such as the colon and the mammary gland (44,45,54).
In a complex rat liver tumor initiation model, 1% (wt/wt) di-
etary green tea catechins were found to inhibit hepatic
glutathione S-transferase placental form (GST-P)-positive
foci induced by diethylnitrosamine, followed by a partial
hepatectomy and exposure to 0.03% (wt/wt) dietary
2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-
P-1), a heterocyclic amine structurally related to PhIP (55).
When the protocol was modified, however, by replacing the
dietary Glu-P-1 exposure with 0.02% (wt/wt) dietary
2-amino-3,8-dimethylimidazo[4,5-f ]quinoxaline, a dietary
AIA and liver carcinogen, the inhibitory effect of the 1% di-
etary green tea catechins could not be reproduced (56). It is
therefore not known whether hepatic AIA-DNA adduct for-
mation is related to induction of GST-P-positive foci, the lat-
ter being an excellent preneoplastic marker of the initiation
phase of hepatocarcinogenicity (57,58).
Our finding of an inhibitory effect of tea on PhIP-DNA
adducts in the small intestine (Figure 3) is in agreement with
the inhibitory effect of green tea polyphenols on small intes-
tine tumor formation in a multiorgan carcinogenesis model
in the F-344 rat (53).
In inhibiting PhIP-induced carcinogenesis, it appears that
tea may act by multiple mechanisms. On the basis of in vitro
studies (21–24,59), tea polyphenols may inhibit the hepatic
microsomal N-hydroxylation of PhIP, even though CYP
1A1 and 1A2, which catalyze this reaction, may be induced
in vivo by green tea and black tea (8,26,60). In view of our
present results on the inhibition of PhIP-DNA adducts by tea
(Figures 3 and 5), the effects of tea on other carcinogen-me-
tabolizing enzymes, such as induction of phase II detoxi-
fying enzymes (5,7), may predominate in vivo. The antioxi-
dant properties of tea polyphenols (11) are expected to exert
a principal inhibitory effect on the promotional phase of
carcinogenesis (1,2). Several antioxidants also exhibit
chemopreventive properties during the initiation phase of IQ
liver tumorigenesis, as measured by inhibition of IQ-in-
duced GST-P-positive foci in rat liver (61). Similar evidence
for the antioxidant role of green tea polyphenols has been in-
consistent (55,56), as discussed above. In an in vitro system,
however, it has been shown that the production of oxygen
free radicals from IQ in the presence of NADPH-cy-
tochrome P-450 reductase is quenched by green tea, black
tea, and (–)-epigallocatechin gallate (62), the latter being the
major catechin in green tea (3). Whether such a mechanism
is operative in vivo and whether PhIP is also a substrate for
this reaction remain to be investigated.
Most studies on the chemopreventive properties of tea
and tea polyphenols have been concentrated on experimen-
tal skin and lung carcinogenesis in the mouse (1,2).
Chemopreventive effects in the liver are not well established
In summary, we have shown that a 2% (wt/wt) infusion
of green tea or black tea, when given as the sole source of
56
Nutrition and Cancer 2000

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bitions imply that tea is a potential chemopreventive agent
in PhIP-induced mammary and colon tumors in the F-344
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Acknowledgments and Notes
These studies were supported by a grant from the American Institute
for Cancer Research. Address reprint requests to Herman A. J. Schut,
Ph.D., Dept. of Pathology, Health Education Bldg., Rm. 202, Medical
College of Ohio, 3055 Arlington Ave., Toledo, OH 43614-5806.
Submitted 17 August 1999; accepted in final form 1 October 1999.
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