[CANCER RESEARCH 52, 3875-3879, July 15, 1992]

Inhibition of Tobacco-specific Nitrosamine-induced Lung Tumorigenesisin A/J Mice by Green Tea and Its Major Polyphenol as Antioxidants1

Yong Xu,2 Chi-Tang Ho, Shantu G. Amin, Chi Han, and Fung-Lung Chung3American Health Foundation, Valhalla, New York 10595 fY. A'., F-L. C./, Department of Food Science, Rutgers University, New Brunswick, New Jersey 08903

[C-T. H.J, and Institute of Nutrition and Food Hygiene, Chinese Academy of Preventive Medicine, Beijing, China ¡C.HJ

ABSTRACT

In this study we examined the effects of green tea and its majorcomponents, (-)-epigallocatechin gallate (EGCG) and caffeine, onthe tobacco-specific nitrosamine 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone (NNK)-induced lung tumorigenesis in A/J mice. We alsostudied the effects of green tea and EGCG on C^-methylguanine and8-hydroxydeoxyguanosine (8-OH-dGuo) formation in lung tissuescaused by NNK treatment. Mice were given 2% tea, 560 ppm EGCG, or1120 ppm caffeine in drinking water for 13 weeks. During this time,NNK (11.65 mg/kg body weight) was administered by gavage threetimes weekly for 10 weeks from weeks 3 to 12. The bioassay was terminated 6 weeks after the last NNK treatment. Mice treated with NNKdeveloped 22.5 lung adenomas per mouse, whereas NNK-treated micethat drank green tea or EGCG as drinking water developed only 12.2(/' < 0.01) and 16.1 (P< 0.05) tumors per mouse, respectively. Mice that

drank green tea or caffeine solution showed lower body weight gains,although little difference in water and diet consumption was noted inthese groups. While green tea and EGCG exerted little effect on theformation of CP-methylguanine, a critical DNA lesion in NNK lungtumorigenesis, both treatments suppressed the increase of 8-OH-dGuolevels in mouse lung DNA. The inhibition of 8-OH-dGuo formation inlung DNA by green tea and EGCG is consistent with their ability toinhibit lung tumorigenesis by NNK. Because 8-OH-dGuo is a DNAlesion caused by oxidative damage, these results suggest that the mechanism of inhibition by green tea and EGCG in NNK-induced lungtumorigenesis is due at least partly to their antioxidant properties.

INTRODUCTION

Among the carcinogens found in cigarette smoke, NNK4 ap

pears highly specific for lung cancer induction in various laboratory animals (1, 2). NNK is also one of the most potentcarcinogenic nitrosamines found in tobacco (2). These activitiessuggest that NNK may play a significant role in the development of lung cancer in smokers. Our studies showed that dietary compounds such as isothiocyanates and Õndolesin cruciferous vegetables inhibited lung tumor induction in rats andmice treated with NNK (3, 4). Furthermore, NNK-treated micefed the cereal-based diet (NIH-07) developed significantly fewerlung tumors than those fed the semipurified diet (AIN-76A) (5).These results indicate that NNK-induced lung carcinogenesis isinfluenced by diet. Epidemiological studies have shown thatlung cancer mortality among males in Japan is considerablylower than it is in the United States even though the prevalenceof cigarette smoking during the last 40 years among Japanese

Received 1/24/92; accepted 5/7/92.The costs of publication of this article were defrayed in part by the payment of

page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This study is supported by the National Cancer Institute Grant CA-465.15 andthe Chinese Academy of Preventive Medicine. This is Paper 17 in the series"Dietary Inhibitors of Chemical Carcinogenesis."

2 Present address: Institute of Nutrition and Food Hygiene. Chinese Academy ofPreventive Medicine, Nan Wei Road 29, Beijing 100050, China.

1To whom requests for reprints should be addressed.4 The abbreviations used are: NNK, 4-(methylnitrosamino)-l-(3-pyridyl)-l-

butanone; O6-mGua. O6-methylguanine; 8-OH-dGuo, 8-hydroxydeoxyguanosine: EGCG, (—)-epigallocatechin gallate: HPLC, high-performance liquid chromatography.

males is much higher than in the United States (6). Diet couldbe an important factor contributing to the difference in lungcancer risk between these two countries, although other factorssuch as race and smoking patterns may also be involved.Considering the dietary differences between these two countries, the prevalence of green tea consumption in Japan is anintriguing one because numerous studies have shown that greentea and its polyphenol extract inhibited mutagenesis and carcinogenesis (7-11). In order to assess the possible role of greentea in the protection against lung cancer, in this study we examined the effects of green tea and its major components,EGCG and caffeine (structures in Fig. 1), on NNK-inducedlung tumorigenesis in A/J mice. In addition, we studied theeffects of green tea and EGCG on liver and lung O6-mGua and8-OH-dGuo formation, both of which are DNA lesions causedby NNK treatment (12, 13).

MATERIALS AND METHODS

Chemicals. NNK was synthesized by a previously published method(14). Green tea was procured from the Tea Research Institute of TheChinese Academy of Agricultural Sciences in Hangzhou, China, andstored in a sealed plastic bag at 4°Cbefore use. EGCG was obtained by

fractionation of a crude catechin mixture, which was prepared fromgreen tea by a method described by Matsuzaki and Hará(15), usingSephadex LH-20 (Pharmacia, Inc.) column chromatography accordingto a method described by Takino el al. (16). The purified EGCG wasstored at -20°Cand its purity was determined to be greater than 99%

by HPLC analysis. Caffeine was purchased from Sigma Chemical Co.(St. Louis, MO). The 2% tea was prepared daily by adding 50 ml ofboiling water to l g of green tea leaves followed by filtration after lettingthe mixture stand at room temperature for 30 min. The concentrationsof EGCG and caffeine in the tea infusion were determined by thereverse-phase HPLC described below. EGCG and caffeine solutionswere prepared daily with tap water. The concentrations of EGCG andcaffeine, 560 and 1120 ppm, respectively, were identical to those foundin the tea. O6-mGua was purchased from Chemsyn Science Laborato

ries (Lenexa, KS). Guanine, deoxyguanosine, nuclease PI, RNase, alkaline phosphatase (type III), and proteinase K were purchased fromSigma. 8-OH-dGuo, prepared by the method of Kasai and Nishimura(17) and purified by HPLC as described by Floyd et al. (18), was kindlyprovided by Dr. Emerich S. Fiala.

HPLC Chromatography. Concentrations of EGCG and caffeine intea were determined on a Varian 5000 HPLC system and a Varian 2050UV detector (Sunnyvale, CA) using a Whatman Partisil-5 ODS-2 column (Whatman, Inc., Clifton, NJ) eluted ¡socraticallywith H2O/dimethylformamide/acetic acid/acetonitrile (81/15/3/1) at a flow rateof 1.0 ml/min. O6-mGua was analyzed using a HPLC system coupled toa Perkin Elmer LS40 fluorescence detector (Norwalk, CT) and a LC290UV spectrophotometric detector as reported previously (12). TheHPLC system consists of a Perkin Elmer binary LC pump 250 and twoWhatman Partisil-10 SCX (25 x 0.45 cm) columns eluted with ammonium phosphate buffer in 10% methanol (O.I M, pH 2.0) at a flow rateof 2.0 ml/min. Analysis of 8-OH-dGuo was performed on a HPLCsystem connected to a model 11IB UV detector and a model LCI7A/LC-4B amperometric detector from Bioanalytical Systems (WestLafayette, IN), set at 1.0 nA range, +600 mV (13). The HPLC systemconsists of a Waters model 510 HPLC pump equipped with a Waters

3875

Research. on November 1, 2020. © 1992 American Association for Cancercancerres.aacrjournals.org Downloaded from

EFFECTS OF GREEN TEA AND EGCG ON LUNG TUMORIGENESIS AS ANTIOXIDANTS

CH3x P

UOH

"NC^Q-OH

° >W

(-) Epigallocatechin-3-gallate(EGCG)

'rVkVQ

CH,

Caffeine

Fig. 1. Structures of (-)-epigallocatechin gallate and caffeine.

model U6K injector (Milford, MA). The column used was a 0.46-x 25-cm Altex Ultrasphere ODS column (Beckman Instruments, Inc.,Berkeley, CA) protected with a C|8 guard column (Alltech Associates,Inc., Deerfield, IL). The eluant was a 10% aqueous methanol containing 12.5 HIMcitric acid, 25 HIMsodium acetate, and 10 HIMacetic acid(pH 5.1) and run at a flow rate of 1 ml/min. Guanine was quantitatedwith the HPLC-UV detection.

Animal Bioassay. Six-week-old female A/J mice were purchasedfrom The Jackson Laboratory (Bar Harbor, ME) and kept under quarantine for 2 weeks prior to the experiment. Animals were fed AIN-76A

diet (5% corn oil) and kept in plastic cages, with 5 mice/cage. They weremaintained under a 12-h light/dark cycle, at 20 ±2°C(SD) and a

relative humidity of 50 ±10%. Mice were divided into 7 groups asshown in Table 1. Tea, EGCG, and caffeine solutions were test substances consumed as drinking water for 13 weeks. After consumption oftest substances in drinking water for 2 weeks, mice were given NNK bygavage (56 Mmol/kg body weight or 11.65 mg/kg body weight in cornoil) 3 times weekly for 10 weeks. The daily water consumption wasmeasured and weekly body weights were recorded. Food consumptionwas determined only for weeks 6 and 7 after NNK treatment began.One week after the last NNK treatment, all groups were given tap wateruntil sacrifice. Mice were sacrificed 6 weeks after the last NNK administration. Lung adenomas were counted. Representative tissues wereprocessed for histopathological examination of tumors. Statistical significance was determined by the 2-sample Student t test.

DNA Adduct Formation. Groups of 10 to 12 six-week-old femaleA/J mice were given 2% tea solution, EGCG solution, or water asdrinking water for 5 weeks. The concentration of EGCG solution wasidentical to that used in the bioassay (560 ppm EGCG in water). Twoweeks after these treatments, NNK was administered in corn oil bygavage at a dose of 112 ^mol/kg body weight or 23 mg/kg body weight3 times weekly for 3 weeks. This dose, twice that of the bioassay dose,facilitated the detection of elevated 8-OH-dGuo levels in tissue DNA.Mice were sacrificed 2 h after last NNK administration and lung andliver DNA were isolated for the analysis of O6-mGua and 8-OH-dGuo.

In a separate but similar experiment, mice that drank water, greentea, or EGCG solution of the same concentration as in the above experiment were treated with NNK in corn oil by gavage (56 ¿imol/kgbody weight or 11.65 mg/kg body weight) 3 times weekly for 3 weeks.Five mice were sacrificed from each group 4 and 24 h after the lastNNK administration and the lung DNA was isolated for O6-mGua

analysis.DNA was isolated by a modified procedure of Marmur's (19).

Briefly, individual lung and liver DNA in 6 or 8 ml cold 15 mm NaCl-50HIMtrisodium citrate buffer, pH 7.0, was homogenized. After centrifu-gation at 9000 x g for 20 min the pellet was dispersed in equal volumeof 10 mM Tris-I mMEDTA-1% sodium dodecyl sulfate buffer, pH 7.0,and incubated with 10 units of proteinase K at 37°Cfor 10 min. The

mixture was extracted twice with chloroform: isoamyl alcohol (24:1,v/v). DNA was precipitated from the aqueous phase by adding 0.1volume of 2 M NaCl and 2 volumes of cold ethyl alcohol. DNA wasredispersed in 10 mM Tris-HCI buffer, pH 7.0, and RNA and proteinwere removed by digesting with RNase (10 units) at 37°Cfor 10 min

followed by proteinase K (10 units) for 30 min and extracted withchloroform-isoamyl alcohol. The precipitated DNA was kept at -20°C

overnight and was solubili/ed in 0.5 ml (lung) and 0.6 ml (liver) 10 IHMTris-HCI buffer, pH 7.O. A portion of this DNA (0.2 ml) was immediately used for 8-OH-dGuo analysis. The other portion of DNA wasstored at -20°C until O6-mGua analysis. Statistical significance

was determined by the 2-sample Student t test.For O6-mGua analysis, DNA was hydrolyzed with 0.1 NHC1 at 80°C

for 60 min. The hydrolysate was filtered with Gelman Acrodisc toobtain samples for HPLC analysis as previously described ( 12).

For 8-OH-dGuo analysis, 10 n\ 0.5 Msodium acetate buffer, pH 5.1,was added to the DNA solution. DNA was then denatured by heating at90-95°C for 2 min. An aliquot containing 100 to 200 MgDNA wasincubated with nuclease PI at 37°Cfor 30 min. After addition of 40 n\

0.4 M Tris-HCI, pH 7.5, DNA was further digested with 8 units ofalkaline phosphatase at 37°Cfor 1 h. The resulting hydrolysate wascentrifuged and the supernatant was stored at 0°Cprior to analysis by

HPLC. The DNA samples were analyzed within 4 to 5 h after enzymatic hydrolysis.

RESULTS

Inhibition of NNK Lung Tumorigenesis. In the present studyNNK was given by gavage in 30 doses over a period of 10 weeks.Each dose was 11.65 mg/kg body weight which equals a totalNNK dose of 1.7 /¿mol/kgbody weight (350 mg/kg bodyweight). This dose regimen induced a 100% tumor incidencewith an average of 22.5 lung adenomas/mouse at week 16 afterNNK treatment began. Table 1 shows the inhibitory effects ofgreen tea, EGCG, and caffeine on the formation of lung adenomas in NNK-treated mice. While the tumor incidences werenot affected by green tea and EGCG, mice that drank tea orEGCG solution developed an average of only 12.2 or 16.1tumors/mouse. These tumor multiplicities correspond to a 45and 30% reduction of lung tumor formation in these groups ascompared with the NNK-treated group that drank water. Interestingly, the caffeine group also showed a marginal but significant inhibition. Groups that drank tea, EGCG, or caffeinewithout NNK treatment showed the tumor incidence and multiplicity comparable to the background levels commonly seen inthe control groups of our previous studies (3).

Fig. 2 shows the body weight curves of different groups during bioassay. Independent of NNK treatment, groups thatdrank tea or caffeine solution showed consistently lower weightgains as compared with groups that drank water or EGCGsolution. Since comparable body weight gains were observed inthe tea and caffeine groups, it appears that caffeine is responsible for the decrease in weight gain in the tea group. In contrastto the tea and caffeine groups, mice that drank EGCG solutionhad normal growth. The weekly water consumption, 0.6-0.7ml/g body weight, was similar among most of the groups, withonly the exception of the tea group which consumed an averageof 0.82 ml/g body weight. No difference in diet consumptionbetween groups was found.

Table 1 Effectof tea. EGCG,and caffeineon NNK-inducedlung adenomasin A/J mice

TreatmentgroupNNKTea

+NNKEGCG+NNKCaffeine

+NNKTeaEGCGCaffeine%

of micewithtumors10010010010072020No.

ofanimals302525IS15IS15Tumors/mouse±SD22.5

±4.712.2±4.3»16.1±5.3»19.2±4.8»0.1±0.20.3

±0.60.3±0.6

" Statisticallydifferentfrom NNK group. P < 0.001.*Statisticallydifferentfrom NNK group, P < 0.05.

3876

Research. on November 1, 2020. © 1992 American Association for Cancercancerres.aacrjournals.org Downloaded from

EFFECTS OF GREEN TEA AND EGCG ON LUNG TUMORIGENESIS AS ANTIOXIDANTS

r 15-ANNK+EGCG0 NNK-fTEA«NNK+Caffeine

3 30

25-

18

Fig. 2. Growth curves of female A/J mice during tumor bioassay.

Table 2 Effects of green lea and EGCG on O6-mGuo formation in lungand liver DNA of A/J mice 2 h after treatment with NNK"

O6-mGuo (/¿mol/molguanine)

TreatmentNNK

NNK + TeaNNK + EGCGNo.

ofmice10

1212Lung55.3

±11.0*57.6 ±16.757.5 ±12.3Liver107.7

±29.8110.1 ±38.294.2 ±36.7

°Mice that drank water, green tea, or EGCG solution were NNK in corn oil(23 mg/kg body weight) by gavage 3 times weekly for 3 weeks. Mice were sacrificed2 h after the last NNK treatment.

* Mean ±SD.

Effects of O6-mGua and 8-OH-dGuo Formation. MultipleNNK doses resulted in an accumulation of O6-mGua in both

lung and liver DNA. Table 2 shows that, 2 h after the last NNKtreatment at a dose of 23 mg/kg body weight, levels of O6-

mGua in mouse lung and liver were 55.3 and 107 ^mol/molguanine, respectively. Despite the fact that O6-mGua is a critical lesion in NNK tumorigenesis in A/J mice (20-22), tea orEGCG solution given as drinking water before and during NNKadministration exhibited little effect on the formation of thisDNA lesion in either lung or liver. In a separate study, O6-

mGua in lung DNA was measured at 4 and 24 h after NNKtreatment. Table 3 shows that levels of O6-mGua were not

significantly affected at both time points by green tea or EGCGtreatment. In fact, a small increase of O6-mGua in the tea group

was seen as compared with the NNK control group 4 h afterNNK treatment. Little difference in O6-mGua levels between 4and 24 h in both tea and EGCG groups suggests that the O6-

mGua methyltransferase activity was not altered by these treatments. Table 4 shows that multiple doses of NNK caused asignificant increase in the 8-OH-dGuo levels in lung DNA from1.7 ±1.2 to 3.2 ±1.7 adducts/105 deoxyguanosine, an approximately 2-fold elevation from the basal levels. Only a slightincrease from 3.9 ±0.6 to 4.7 ±1.1 adducts/105 deoxygua

nosine was seen in liver DNA; however, this increase was notstatistically significant. Green tea and EGCG suppressed theelevated 8-OH-dGuo levels in lungs of NNK-treated mice. Thedecrease in 8-OH-dGuo levels in lungs of NNK-treated micethat drank green tea or EGCG is consistent with their inhibitory activity against lung tumor formation.

DISCUSSION

This study showed that green tea has a protective effectagainst NNK-induced lung tumorigenesis in A/J mice and thepolyphenol EGCG in green tea appears to be a major activecomponent for this activity. Green tea is composed of at least10 to 20% poly phenols (23). These compounds are powerfulantioxidants, capable of scavenging H2O2 and Superoxide an-ion, thus preventing H2O2 and oxygen free radical-induced cy-totoxicity and mutagenicity (24,25). Green tea polyphenol fractions have been shown to be protective in mice against skintumor induction by polycyclic aromatic hydrocarbons (7). Theyalso inhibited aflatoxin B, hepatocarcinogenesis (26). The administration of several types of Chinese tea as drinking waterresulted in a significant reduction in esophageal tumors inducedby yV-nitrosobenzylmethylamine (8). Total green tea extractgiven p.o. also protected mice from UV-induced skin tumorigenesis (27). Because either tea or total tea polyphenol fractionwas used in these studies, it is not possible to identify which arethe active compounds in green tea responsible for the inhibitoryeffect. EGCG, the main polyphenol in green tea, is an antimu-tagen (9). Topical application of EGCG-inhibited teleocidinepromoted 7,12-dimethylbenz[a]anthracene-induced skin tumors in mice (28). More recently, the antipromoting activity ofEGCG was demonstrated in mouse duodenum carcinogenesisinduced by /V-ethyU/V-nitro-N-nitrosoguanidine (10). Our results showed that EGCG, given in a concentration identical tothat found in tea infusion, inhibited NNK-induced lung tumorigenesis in mice. Furthermore, EGCG, unlike caffeine, exertedno adverse effect on growth. Therefore, it may be considered asa potential chemopreventive agent. It is estimated that an average smoker is exposed to roughly 2 mg of NNK annually (29).In our bioassay, mice consumed an average of 100 mg of EGCGand at the same time were exposed to 7 mg of NNK(EGCGrNNK, 14:3). Thus, one may hypothesize that if theinhibitory activities of EGCG are important in humans, a cup

Table 3 Effects of green tea and EGCG on (P-mGuo levels in lung DNAof A/J mice 4 and 24 h after treatment with NNK"

O'-mGiia (fimol/mol guanine)

Treatment 4h 24 h

NNKNNK + teaNNK + EGCG

16.8 ±2.7»23.3 ±2.819.6 ±1.0

17.8 ±6.219.2 ±5.015.0 ±5.5

" Mice that drank water, green tea, or EGCG solution were given NNK in comoil by gavage (11.65 mg/kg body weight) 3 times weekly for 3 weeks. Mice weresacrificed 4 and 24 h after the last NNK treatment.

* Mean ±SD from 5 mice.

Table 4 Effects of green tea and EGCG on the 8-OH-dGuo levels in lungand liver DNA of A/J mice 2 h after treatment with NNKa

8-OH-dGn 10-Vdeoxyguanosine

TreatmentControlNNK

NNK + teaNNK + EGCGEGCGTeaNo.

ofmice11

1011121011Lung1.7

±1.2»3.2 ±\.1C1.9 ±1.0''2.1 ±1.1"1.8 + 0.9'1.8 ±0.7'Liver3.9

±0.64.7 ±1.14.3 ±1.44.3 ±1.24.1 ±1.24.0 ±1.4

" Mice that drank water, green tea, or EGCG solution as drinking water weregiven NNK in corn oil (23 mg/kg body weight) by gavage 3 times weekly for 3 timesweekly for 3 weeks. Mice were sacrificed 2 h after the last NNK treatment.

»Mean ±SDe Statistically different from control group, P < 0.01.•¿�'Statisticallydifferent from NNK group, P < 0.05.' Statistically different from NNK group, P < 0.01.

3877

Research. on November 1, 2020. © 1992 American Association for Cancercancerres.aacrjournals.org Downloaded from

EFFECTS OF GREEN TEA AND EGCG ON LUNG TUMOR1GENESIS AS ANTIOXIDANTS

of green tea per day consumed by humans would appear tocontain a sufficient amount of EGCG to alleviate the potentialcarcinogenic action of NNK.

In the tea preparation used in the present study, caffeineconstitutes about 5.6% of tea leaves by dry weight. It is a majorcomponent in tea and has been shown to inhibit chemical carcinogenesis in other studies (30-32). The exact mechanisms bywhich caffeine inhibits the induction of tumors are not clear.Recently, Lagopoulos et al. (33) found that the reduced bodyweight gains in caffeine-treated groups correlated with decreased hepatocarcinogenesis by diethylnitrosamine. Vander-Ploeg et al. (34) suggested that the inhibition of 7,12-dimeth-ylbenz[a]anthracene-induced mammary gland tumorigenesis inrats by caffeine was due to the ability of caffeine to suppress itsmetabolism. In the present study, although diet consumptionappeared to be unaffected by caffeine treatment, the bodyweight gains in the caffeine group were considerably lower thanthose in the EGCG and NNK control groups. Therefore, theslight but significant reduction in lung tumor multiplicity bycaffeine treatment could be related to its negative effect on bodyweight. Regardless of the mechanism, the potential protectiveeffect of caffeine should not be ignored because of its widespread consumption by humans.

Several lines of evidence indicated that O6-mGua is a criticallesion in NNK lung tumorigenesis (20-22). Previously weshowed that a decrease in lung O6-mGua formation correlatedwith a decrease of lung tumor formation in NNK-treated A/Jmice pretreated with arylalkyl isothiocyanates or indole-3-carbinol (4,35). In the present study, however, neither green teanor EGCG treatment inhibited NNK-induced iX'-methylgua-

nine formation or stimulated its repair, although both treatments inhibited the lung tumor induction by NNK. These results suggested that additional mechanisms other than DNAmethylation or repair are likely to be involved in lung tumorigenesis by NNK. NNK treatment also resulted in pyridyloxobu-tylation of DNA. However, the exact role of this adduci inNNK lung tumorigenesis is not yel known (36). Our resultsshowed lhal multiple NNK doses caused a significanl increasein 8-OH-dGuo levels in the lung but not in the liver and theelevation of this lesion in lung DNA was suppressed by greentea treatment. Since 8-OH-dGuo is a DNA lesion caused byoxidative stress commonly associated with tumor promotion(37), these results suggest that the inhibition of lung tumorinduction by green lea could be due in pari lo antipromolingactivity of its polyphenols such as EGCG. The anlioxidanlsellagic acid and butylaied hydroxyanisole given in the diel inhibited lung adenoma formalion in NNK-irealed A/J mice (38).Togelher wilh these results, the presenl study provides evidencesupporting the roles of green lea and ils major polyphenolEGCG as antioxidants in the inhibition of NNK lung lumori-

genesis.

ACKNOWLEDGMENTS

The authors thank Dr. E. S. Fiala for providing the 8-OH-dGuo 26.

standard.27.

REFERENCES1. Hoffmann. D., Rivenson, A., Chung, F-L., and Hecht, S. S. Nicotine-derived

íV-nitrosamines(TSNA) and their relevance in tobacco carcinogenesis. Crit.Rev. Toxicol., 21: 305-311. 1991.

2. International Agency for Research on Cancer. Tobacco Smoking, Vol 38.Lyon. France: IARC, 1986.

3. Chung, F-L., Morse, M. A., and Eklind, K. I. New potential chemopreventiveagents for lung carcinogenesis of tobacco-specific nitrosamine. Cancer Res.,

3878

52:2719s-2722s, 1992.4. Morse, M. A., LaGreca, S. D., Amin, S. G., and Chung, F-L. Effects of

indole-3-carbinol on lung tumorigenesis and DNA methylation inducedby 4-(methylnitrosamino)-l-(3-pyridyl)-I-butanone (NNK) and on the metabolism and disposition of NNK in A/J mice. Cancer Res., SO:2613-2617,1990.

5. Hecht. S. S., Morse, M. A., Amin, S. G., Stoner, G. D., Jordan, K. G.. Choi,C-I., and Chung. F-L. Rapid single dose model for lung tumor induction inA/J mice by 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone and the effectof diet. Carcinogenesis (Lond.), 10: 1901-1904, 1989.

6. Wynder, E. L., Fujita, Y., Harris, R. E., Hiraguma, T., and Hiyarnor. T.Comparative epidemiology of cancer between the United States and Japan: asecond look. //;: R. Sasaki and K. Aoki (eds.). Epidemiology and Preventionof Cancer, pp. 103-127. Nagoya, Japan: University of Nagoya Press, 1990.

7. Wang. Z. Y., Khan. W. A., Bickers. D. R., and Mukhtar, H. Protectionagainst polycyclic aromatic hydrocarbon-induced skin tumor initiation inmice by green tea polyphenols. Carcinogenesis (Lond.), 10: 411-415, 1989.

8. Han, C., and Xu, Y. The effect of Chinese tea on occurrence of esophagealtumor induced by iV-nitrosomethylbenzylamine in rats. Biomed. Environ.Sci., J: 35-42. 1990.

9. Kada, T., Kaneko, K., Matsuzaki, S., Matsuzaki, T., and Hará,Y. Detectionand chemical identification of natural bio-antimutagens, a case of the greentea factor. Mutât.Res., ISO: 127-132, 1985.

10. Fujita, Y.. Yamane. T., Tanaka. M., Kuwata. K.. Okuzumi, J., Takahashi, T.,and Fujiki, H. Inhibitory effect of (-)-epigallocatechin gallate on carcinogenesis with iV-ethyl-iV-nitro-A'-nitrosoguanidine in mouse duodenum. Jpn. J.Cancer Res.. 80: 503-505, 1989.

It. Wang, Z. Y.. Huang, M. T.. Hong, J-Y.. Conney, A. H., and Yang, C. S.Inhibition of A'-nitrosodiethylamine (NDEA)-induced lung and forestomach

tumorigenesis in A/J mice by green tea. Proc. Am. Assoc. Cancer Res., 32:125, 1991.

12. Chung, F-L., Wang, M., and Hecht, S. S. Effects of dietary Õndolesandisothiocyanates on V-nitrosodimelh> lamine and 4-(methylnitrosamino)-1 -(3-pyridyl)-l-butanone n-hydroxylation and DNA methylation in rat liver. Carcinogenesis (Lond.). 6: 539-543. 1985.

13. Chung, F-L.. and Xu, Y. Increased 8-hydroxydeoxyguanosine in DNA ofmice and rats treated with the tobacco-specific nitrosamines 4-(methylnitro-samine)-l-(3-pyridyl)-l-butanone. Carcinogenesis (Lond.), in press, 1992.

14. Hecht, S. S., Chen, C. B., Dong, M., Ornaf, R. M.. Hoffmann, D., and Tso,T. C. Studies on non-volatile nitrosamines in tobacco. Beitr. Tabakforsch., 9:1-6, 1977.

15. Matsuzaki, T., and Hará, Y. Antioxidative activity of tea leaf catechins.Nippon Nogeigaku Kaishi, 59: 129-134, 1985.

16. Takino, Y., Tanizawa, H., Ikeda. H., and Fujiki, S. Japanese Patent, 59:26385, 1984.

17. Kasai, H., and Nishimura, S. Hydroxylation of deoxyguanosine at the C-8position by ascorbic acid and other reducing agents. Nucleic Acids Res., 12:2137-2145, 1984.

18. Floyd, R. A., Watson, J. J., Wong, P. K.. Altmiller, D. H., and Rickard, R.C. Hydroxyl free radical adduci of deoxyguanosine: sensitive detection andmechanism of formation. Free Radical Res. Commun., /: 163-172, 1986.

19. Marmur. J. A procedure for the isolation of deoxyribonucleic acid frommicroorganisms. J. Mol. Biol.. 3: 208-216, 1961.

20. Belinsky. S. A., Foley, J. F., White, C. M., Anderson. M. W., and Maronpot,R. R. Dose-response relationship between O6-methylguanine formation inClara cells and induction of pulmonary neoplasia in the rat by 4--(methylni-trosamino)-l-(3-pyridyl)-l-butanone. Cancer Res., 50: 3772-3780. 1990.

21. Peterson, L. A., and Hecht, S. S. O'-Methylguanine is a critical determinantof 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone tumorigenesis in A/Jmouse lung. Cancer Res., SI: 5557-5564, 1991.

22. Devereux, T. R., Anderson. M. W., and Belinsky. S. A. Role of ras protooncogene activation in the formation of spontaneous and nitrosamine-inducedlung tumors in the resistant C3H mouse. Carcinogenesis (Lond.). 12: 299-303, 1991.

23. Graham, H. N. Tea, the plant and its manufacture, chemistry and consumption of the beverage. In: G. A. Spiller (ed.). The Methylxanthine Beveragesand Foods. Chemistry, Consumption and Health Effects, pp. 29-74. NewYork: Alan R. Liss. 1984.

24. Rush, R. J., Cheng, S. J., and Klaunig. J. E. Prevention of cytotoxicity andinhibition of intercellular communication by antioxidant catechins isolatedfrom Chinese green tea. Carcinogenesis (Lond.). 10: 1003-1008, 1989.

25. Osawa, T., Namiki, M., and Kawakishi, S. Role of dietary antioxidants inprotection against oxidative damage. Basic Life Sci., 52: 139-153, 1990.Chen, Z. Y., Yan, P. Q., Qin, G. Z., and Qin, L. L. Effect of six edible plantson the development of AFB,-induced 5-glutamyl transpeptidase positive hep-atocyte foci in rats. Chin. J. Oncol.. 9: 109-111. 1987.Wang, Z. Y., Huang, M-T.. Ferraro, T., Wong, C. Q., Newmark, H., Yang,C. S., and Conney, A. H. Inhibitory effect of orally administered green tea onultraviolet B light (UV-B)-induced carcinogenesis in skin of SKIM mice.Proc. Am. Assoc. Cancer Res.. 32: 129, 1991.

28. Yoshizawa, S.. Horiuchi. T., Fujiki, H., Yoshida. T.. Okuda, T.. and Sug-imura, T. Antitumor promoting activity of (—)-epigallocatechin gallate, themain constituent of "tannin" in green tea. Phytother. Res., /: 44-47, 1987.

29. Hoffmann, D., and Hecht, S. S. Nicotine-derived A'-nitrosamines and tobacco related cancer: current status and future directions. Cancer Res., 45:935-944, 1985.

Research. on November 1, 2020. © 1992 American Association for Cancercancerres.aacrjournals.org Downloaded from

EFFECTS OF GREEN TEA AND EGCG ON LUNG TUMORIGENESIS AS ANTIOXIDANTS

30. Rothwell, K. Dose-related inhibition of chemical carcinogenesis in mouseskin by caffeine. Nature (Lond.), 252: 69-70, 1974.

31. Nomura, T., Diminution of tumorigenesis initiated by 4-nitroquinoline-l-oxide by post-treatment with caffeine in mice. Nature (Lond.), 260: 547-549,1976.

32. Kakunaga, T. Caffeine inhibits cell transformation by 4-nitroquinoline-l-oxide. Nature (Lond.), 258: 248-250, 1975.

33. Lagopoulos, L., Sunahara. G. I., Wurzner, H., Fliesen, T., and Stalder.R. The correlation of body growth with diethylnitrosamine-induced hepato-carcinogenesis in relation to serum insulin and somatomedin-C. Carcinogenesis (Lond.), 12: 211-215. 1991.

34. VanderPloeg, L. V. C, Wolfrom, D. M., and Welch. C. W. Influence ofcaffeine on development of benign and carcinomatous mammary gland tumors in female rats treated with carcinogens 7,12-dimethylbenz(a)anthracene

and /V-methyl-A'-nitrosourea. Cancer Res., 51: 3399-3404. 1991.35. Morse, M. A., Amin, S. G., Hecht, S. S., and Chung, F-L. Effects of aromatic

isothiocyanates on tumorigenicity, O6-methylguanine formation, and metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-l-(3-py-ridyl)-l-butanone in A/J mouse lung. Cancer Res., 49: 2894-2897, 1989.

36. Hecht, S. S.. Spratt, T. E., and Trushin, N. Evidence for 4-(3-pyridyl)-4-oxobutylation of DNA in F344 rats treated with the tobacco specific nitro-samines 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone and /V-nitroso-nornicotine. Carcinogenesis (Lond.), 9: 161-165, 1988.

37. Cerutti, P. A., Prooxidant states and tumor promotion. Science (WashingtonDC), 277:375-381. 1985.

38. Pépin.P.. Rossignol, G., and Castonguay, A. Inhibition of NNK-inducedlung tumorigenesis in A/J mice by ellagic acid and butylated hydroxyanisole.Cancer ¡.,3: 266-271, 1990.

3879

Research. on November 1, 2020. © 1992 American Association for Cancercancerres.aacrjournals.org Downloaded from

1992;52:3875-3879. Cancer Res   Yong Xu, Chi-Tang Ho, Shantu G. Amin, et al.   Polyphenol as AntioxidantsTumorigenesis in A/J Mice by Green Tea and Its Major Inhibition of Tobacco-specific Nitrosamine-induced Lung

  Updated version

  http://cancerres.aacrjournals.org/content/52/14/3875

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/52/14/3875To request permission to re-use all or part of this article, use this link

Research. on November 1, 2020. © 1992 American Association for Cancercancerres.aacrjournals.org Downloaded from