The effects of dietary administration of green tea catechins. (GTC) were examined using a multi-organ carcinogenesis model. Groups of 15 F344 male rats were ...
Carcinogenesis vol.14 no.8 pp.1549-1553, 1993
Effects of green tea catechins in a rat multi-organ carcinogenesis model
Masao Hirose, Toru Hoshiya, Keisuke Akagi, Satoru Takahashi, Yukihiko Hara1 and Nobuyuki Ito First Department of Pathology, Nagoya City University Medical School, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467 and 'Food Research Laboratories, Mitsui Norin Co. Ltd, Fujieda Shizuoka 426, Japan
The effects of dietary administration of green tea catechins (GTC) were examined using a multi-organ carcinogenesis model. Groups of 15 F344 male rats were initially treated with a single i.p. administration of 100 mg/kg body wt A'-diethylnitrosamine, 4 i.p. administrations of 20 mg/kg body wt iV-methylnirrosourea, 4 s.c. doses of 40 mg/kg body wt 1,2-dimethylhydrazine, together with 0.05% W-butyl-W(4-hydroxybutyl)nitrosamine for 2 weeks and then 0.1% 2,2'-dihydroxy-di-n-propymitrosamme for 2 weeks, both in the drinking water, for a total initiation period of 4 weeks. GTC in the diet, at doses of 1.0 or 0.1%, was administered from 1 day before and during carcinogen exposure, after carcinogen exposure or both during and after carcinogen exposure. Further groups of animals were treated with carcinogen, 1% GTC or basal diet alone as controls. All animals were killed at the end of week 36, and all major organs examined histopathologically. The numbers of small intestinal tumors (adenomas and carcinomas) per rat were significantly reduced in the groups treated with 1% GTC during (0.13 ± 0.35) and after carcinogen exposure (0.31 ± 0.48) and in those receiving 1% and 0.1% GTC both during and after carcinogen exposure (0.14 ± 0.36, 0.46 ± 0.97 respectively) as compared with the carcinogen alone group (1.07 ± 1.21). On the other hand, numbers of glutathione S-transferase placenta] form positive liver foci per cm2 were slightly but significantly increased in the groups treated with 1 and 0.1% GTC during carcinogen exposure, 1% GTC after carcinogen exposure and 1% GTC both during and after carcinogen exposure. The results indicated that while GTC inhibits small intestinal carcinogenesis it slightly enhances hepatocarcinogenesis in a dose dependent manner when applied both during and after carcinogen exposure.
Introduction Green tea catechins (GTC*) are major components of the polyphenols contained in green tea infusion. Japanese green tea contains up to 15% of a mixture of catechins, i.e. (-)-epicatechin (EC), (-)-epigallocatechin (EGQ, (-)-epicatechin gallate (EGQ
and (-y-epigallocatechin gallate (EGCG). These chemicals possess strong antioxidant activity (1) and epidemiological data have indicated that the standardized mortality ratios for cancer of all sites and stomach cancer in the mid-west area of Shizuoka Prefecture, where green tea is a staple product, are much lower than the national averages for both sexes (2). Inhibitory effects of green tea or GTC in experimental carcinogenesis have also been reported by some investigators. For example, i.g. or i.p. applications of GTC inhibited the growth of transplantable sarcoma 180 in mice or 20-methylcholanthrene-induced transplantable tumors in rats (3). Continuous oral administration of 0.005% EGCG after AZ-ethyl-A^-nitro-A^-nitrosoguanidine exposure also significandy inhibited the development of duodenal tumors in mice (4). Furthermore, treatment with an aluminiumEGCG complex at a dose of 0.2% in diet inhibited spontaneous mouse mammary carcinogenesis in female C3H/HeN mice (5). In the two stage mouse skin carcinogenesis model, EGCG and green tea extract potently inhibited the promotion activity of teleocidin (6) and 12-Otetradecanoylphorbol-13-acetate (TPA) (7) respectively. In addition, green tea polyphenols inhibited the initiating activity of both poly cyclic aromatic hydrocarbons and ultraviolet in mouse skin carcinogenesis (8 — 10). Green tea infusion clearly inhibited Akiiethylnitrosamine (DEN) and 4^memylnitrosamino)-l-(3-pyridyl)-l-butanone (NNK) induced mouse lung and forestomach tumorigenesis, and mouse lung carcinogenesis either during initiation or promotion stage (11). In line with these effects, EGCG, (+)-catechin or green tea polyphenol exert anti-mutagenic activity against benzo[a]pyrene aflatoxin Bi, 2-aminofluorene, 4-aminobiphenyl or benzo[a]pyrene diol-epoxide in Salmonella typhimurium TA 98 and TA 100 (12 — 14). Similarly, the green tea polyphenol (+)-catechin inhibited DNA damage induced by NNK in rat hepatocytes in culture or in vivo (15). These anti-tumorigenic activities in different experimental systems and extending over various organs promoted the present investigation using our recently-developed modified multi-organ carcinogenesis model (16—20) and effects of GTC during carcinogen exposure, after carcinogen exposure, and both during and after carcinogen exposure were examined, hi the present multi-organ model, 1,2-dimethylhydrazine (DMH) and Af-butyl-7V-(hydroxybutyl)nitrosamine (BBN) were further added to the previous model (16,18,19) as carcinogens in order to assess complex modifying effects of chemicals in over 10 organs in a single experiment. Enhancing effects of diallyl disulfide on hepatocarcinogenesis and inhibitory effects of the diallyl disulfide on colon and renal carcinogenesis have been shown using the same multi-organ carcinogenesis model (21). Materials and methods
•Abbreviations: GTC, green tea catechins; EC, (-)-epicatechin; EGC, ( - ) epigallocatechin; ECG, (-)-epicatechin gallate; EGCG, (-)-epigallocatechin gallnte; TPA, 12-Otetradecanoylphorbol-13-acetate; DEN, N-diethylnitrosaniine; NNK, 4^methylrutrosamino)-l-utyl-AAgallocatechin 1.4%, EGC 17.6%, EC 5.8%, EGCG 53.9% and ECG 12.5%] (22). Treatment Tbe experimental design is presented in Figure 1. Animals in groups 1—7 were given combined treatment with a single i.p. administration of 100 mg/kg body wt DEN, 4 i.p. administrations of 20 mg/kg body wt MNU, 4 s.c. doses of
r Group ,r V V V V T 1
1 2
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JED Fig. 1. Experimental protocol for the rat multi-organ carcinogenesis model. Animals: male F344 rats, 6 week old. I: DEN, 100 mg/kg body wt i.p., V: MNU, 20 mg/kg body wt i.p., T : DMH, 40 mg/kg body wt s.c, • : BBN, 0.05% in drinking water, B: DHPN, 0.1% in drinking water, H : GTC, 1.0 or 0.1% in diet, D : basal diet.
40 mg/kg body wt DMH, 0.05% BBN in the drinking water for 2 weeks and 0.1% DHPN in drinking water for 2 weeks during the initial 4 week period for initiation. Animals in groups 1 and 2 were administered 1 and 0.1% GTC respectively, in Oriental MF powdered basal diet one day before and during carcinogen exposure. Those in groups 3 and 4 were administered 1 and 0.1 % GTCrespectively,starting 3 days after the period of carcinogen exposure to prevent die interaction of carcinogens and GTC. Those in groups 5 and 6 were administered 1 and 0.1% GTC respectively, throughout tbe experiment. Animals in group 7 were treated with carcinogens alone. The further two groups of 10 animals each were treated with 1 % GTC and basal diet alone respectively, throughout the experiment without carcinogen exposure. Animals were weighed once a week for 4 weeks after the cessation of carcinogen treatment, and once every 4 weeks until the end of the experiment Animals which died during the experiment were autopsied and those which became moribund were killed for autopsy. Animals surviving 18 weeks, when the first tumor appeared were included in the effective numbers. All survivors were killed under ether anesthesia at the end of week 36 and were subjected to complete autopsy. Liver and kidneys were weighed. Neutral buffered formalin solution was injected into the lungs, esophagus, stomach, intestines and urinary bladder. One section from each lung lobule, three sections from liver, four sections from esophagus, four sections from urinary bladder and one section each from the other organs were made. Swiss roll preparations were made from the large and small intestines. In addition, two sections each were cut from liver and fixed in ice cold acetone for immunohistocbemical staining of glutathione S-transferase placental form (GST-P) positive foci. Tissues were processed routinely for histopathological examination using H&E and anti-GST-P immunohistochemical stainings (23). Numbers and areas of GST-P positive foci and numbers of lung lesions per cm2 were measured with the aid of an image analyzer (Olympus VIP-21C). Student's (-test and Fisher's exact probability test were used for stntiViral analysis of the data.
Results Final average body weights of animals treated with catechins simultaneously with or after carcinogen exposure tended to be higher than those receiving carcinogen alone, except in group 6. Relative liver and kidney weights were not significantly different between groups 1-6 and group 7, except in the group 1 kidney case (Table I). Incidences of tumors and preneoplastic
Table I. Final body and organ weights Group
Dose (%)
No. of rats
Body wt* (g)
Organ weights (g/100 g body wt)*
1.0 0.1 1.0 0.1 1.0 0.1
12 10 13 8 13 11 12 10 10
336 318 342 321 311 290 304 406 410
2.71 2.80 2.74 2.60 2.77 2.49 2.67 2.77 2.46
1.0
± ± ± ± ± ± ± ± ±
Liver
17 20 39 33 35 41 15 15 25
Kidney ± ± ± ± ± ± ± ± ±
0.15 0.20 0.42 0.16 0.20 0.25 0.26 0.15 0.07
0.63 0.79 0.65 0.63 0.70 0.75 0.80 0.50 0.49
± ± ± ± ± ± ± ± ±
0.05* 0.22 0.21 0.12 0.23 0.20 0.30 0.02 0.02
•Mean ± SD. *P < 0.05 versus group 7. Table £1. Incidences and numbers of tumors in the small intestine Group
1 2 3 4 5 6 7
Dose No. of (%) rats
Adenoma Incidence (%)
No./rat*
1.0 0.1 1.0 0.1 1.0 0.1 —
1 (7)* 7(50) 1 (8)* 4(31) 1 (7)* 2(15) 6(43)
0.07 0.57 O.Ofi 0.38 0.07 0.15 0.57
15 14 13 13 14 13 14
•Mean ± SD. P < *0.05, **0.02, ***0.01 versus group 7.
1550
Carcinoma
± ± ± ± ± ± ±
0.26* 0.65 0.28* 0.65 0.27* 0.38* 0.76
Total
Incidence (%)
No./rat*
1 (7) 2(14) 3(23) 4(31) 1 (7) 3(23) 5(36)
0.07 0.14 0.23 0.31 0.07 0.31 0.50
± ± ± ± ± ± ±
0.26** 0.36 0.44 0.48 0.27* 0.63 0.76
Incidence (%)
No./rat*
2 (13)" 7(50) 4(31) 5(38) 2 (14)** 3(23) 8(57)
0.13 0.71 0.31 0.69 0.14 0.46 1.07
± =t ± ± ± ± ±
0.35*** 0.83 0.48* 1.03 0.36** 0.97 1.21
Green tea mtcrtilns and cardnogenesis
Table HI. Incidences and numbers of colon tumors Group
1 2 3 4 5 6 7
Carcinoma
Dose No. of (%) rats
Adenoma Incidence (%)
No./rar1
Incidence (%)
No./rar*
1.0 0.1 1.0 0.1 1.0 0.1 —
9(60) 7(50) 7(54) 6 (46) 5(36) 6(46) 7(50)
1.07 0.79 1.08 0.85 0.64 0.54 0.57
3(20) 5(36) 2(15) 2(15) 4(29) 6 (46) 3(21)
0.20 0.43 0.15 0.15 0.29 0.46 0.43
15 14 13 13 14 13 14
± 1.22 ± 0.97 ± 1.50 ± 1.21 ± 1.01 0.66 ± 0.65
Total
± 0.41
± 0.65 ± 0.38 ± 0.38 ± 0.47 ± 0.52 ± 0.85
Incidence (%)
No./rat*
9(60) 8(57) 9(69) 6(46) 8(57) 8(62) 7(50)
1.27 1.21 1.23 1.00 0.93 1.00 1.00
± ± ± ± ± ± ±
.49 .31 .42 .40 .00 .00 .36
•Mean ± SD.
Table IV. Incidences and numbers of lung tumors Group
1
2 3 4 5 6 7
Dose No. of (%) rats
Adenoma Incidence (%)
No./rat"
1.0 0.1 1.0 0.1 1.0 0.1 -
9(60) 9(64) 9(69) 5(38) 7(50) 5(38) 6(43)
1.00 0.93 1.08 0.54 0.80 0.46 0.64
15 14 13 13 14 13 14
Carcinoma
± ± ± ± ± ± ±
1.00 1.00 1.12 0.78 1.15 0.66 0.93
Total
Incidence (%)
No./rat1
Incidence (%)
No./rat1
2 (13) 2 (14) 1 (8) 2 (15) 2 (14) 3 (23) 0
0.13 0.14 0.08 0.15 0.14 0.23 0
9 9 10 6 9 7 6
1.21 1.14 1.23 0.69 1.00 0.69 0.64
± 0.35 ± 0.36 ± 0.28 ± 0.38 ± 0.36 ± 0.44
(60) (64) (77) (46) (64) (54) (43)
± ± ± ±
1.19 1.17 1.01 0.85 ± 1.11 ± 0.75 ± 0.93
•Mean ± SD.
lesions and average numbers of tumors per rat or per unit area are presented in Tables II-VI. Significant decreases in the incidences of small intestinal adenomas were evident in groups 1, 3 and 5 (see Table II). In these groups and group 6, me average number of adenomas was also significantly decreased. Significant reduction in the number of carcinomas was found in groups 1 and 5. Incidences of total tumors, including both adenomas and carcinomas, were significantly lower in groups 1 and 5 and numbers of tumors per rat were significantly reduced in groups 1, 3 and 5 as compared with group 7. On the other hand, incidences and numbers of colon adenomas and adenocarcinomas did not differ between the groups (see Table m). Incidences and/or numbers of lung adenomas, carcinomas and total tumors tended to be increased in groups treated with GTC, particularly groups 1, 2, 3 and 5, but the values were not significantly different from those in group 7 (see Table IV). In the liver, although the incidences of hyperplastic nodules did not differ, the numbers of GST-P positive foci were significantly increased in groups 1, 2, 3 and 5. In addition the area occupied by foci was increased in group 1 (see Table V). In the other organs examined, no significant alteration in the incidences of neoplastic or preneoplastic lesions was found in the forestomach, esophagus, kidney, urinary bladder, thyroid or lymphoreticular system (see Table VI). No neoplastic or toxic changes were observed in animals not receiving the carcinogen treatment. Discussion The present study clearly showed that GTC in the diet can significantly inhibit rat small intestinal cardnogenesis, when appbed during the initiation stage, after carcinogen exposure and both during and after carcinogen exposure. However, the observed inhibition was more apparent when GTC was given in the initiation stage at high dose level.
Table V. Quantitative analyses of GST-P positive foci in tbe liver Group
1 2 3 4 5 6 7
Dose (%)
No. of rats
GST-P positive foci1
1.0 0.1 1.0 0.1 1.0 0.1 -
12 10 13 8 13 11 12
15.6 11.9 10.7 10.8 11.4 9.6 7.8
No./cm2 ± ± ± ± ± ± ±
Area (mnrVcm2
4.0«* 4.0" 2.9* 3.8
3.6** 4.2 2.3
1.5 1.4 0.8 1.0 1.2 0.8 0.9
± ± ± ± ± ± ±
0.5** 0.8 0.2 0.5 0.4 0.4 0.5
•Mean ± SD. P < *0.05, **0.01, •••0.001 versus group 7.
In the present multi-organ carcinogenesis model, the carcinogens used which target the small bowel were DMH and MNU. MNU induces alkylation of target tissue DNA without metabolic activation. DMH is oxidized in the liver to form the ultimate carcinogen methylazoxymethanol (MAM), then forming a conjugate (/3-glucuronide or sulfate) which is transported by the bile or die blood to the target organs. At these sites the conjugate is split to reform MAM, then enzymatically metabolized to methylating species (24). Several possibilities might be considered to explain how GTC inhibits small bowel carcinogenesis. Firstly GTC might hinder reactivation of conjugate in the small intestine. However, considering diat catechins inhibit small bowel carcinogenesis even after carcinogen exposure, it might be considered more likely that they lower cell proliferation of small bowel epidielium or of small bowel tumor cells. It is not likely that catechins stimulate detoxification of DMH or inhibit formation of DNA adducts, normally the major suggested mechanism of chemoprevention (25), since the observed effects were limited to small bowel carcinogenesis and no inhibition was evident in the colon or kidney which are odier targets of DMH. 1551
M.Hirose et al.
Table VI. Incidences of lesions in other organs Group 1
2
3
4
5
6
7
Dose
1
0.1
1
0.1
1
0.1
-
n
15
14
13
13
14
13
14
Forestomach PN hyperplasia Papilloma Carcinoma Esophagus PN hyperplasia Papilloma Liver Hyperplastic nodule Kidney Adenoma Nephroblastoma Urinary bladder PN hyperplasia Papilloma Thyroid gland Adenoma Adenocarcinoma Lymphoma
4 (27) 1 (7) 1 (7)
0 0 0
2 (15) 0 0
1 (7) 1 (7)
3 (21) 2 (15) 0 0
1 (8) 0
2 (14) 1 (8) 0 0
0 0
1 (7)
0
0
0
1 (7)
0
2 (15) 1 (7) 1 (8) 0 0 0
3 (23) 3 (21) 1 (8) 0 0 0
0
5 (33) 3 (21) 4 (31) 1 (8) 2 (14) 3 (23) 5 (36) 6 (40) 10 (71) 7 (54) 10 (77) 6 (43) 8 (54) 7 (50) 3 (20) 0
1 (7) 1 (7)
0 3 (23)
3 (23) 5 (36) 3 (23) 1 (7) 1 (8) 2 (14) 2 (15) 2 (14)
5 (33) 2 (13) 4 (27)
3 (21) 3 (23) 1 (7) 0 1 (7) 1 (8)
1 (8) 3 (21) 4 (31) 2 (14) 1 (8) 4 (29) 1 (8) 1 (7) 0 2 (15) 1 (7) 0
Percentages in parentheses.
Simultaneous treatment with quercetin and/or rutin, which are structurally similar to GTCs, also inhibited azoxymethane (AOM)-induced colon carcinogenesis in female CF1 mice (26). Similar results were reported for DMBA-induced mammary carcinogenesis in female CD rats when they were applied during or after carcinogen exposure (27,28). Quercetin may influence colon carcinogenesis by inhibiting arachinoid acid metabolism via the lipoxygenese or cycloxygenase pathways (29) or by altering carcinogen metabolism by modulating microsome cytochrome P-450-dependent enzymes (30). Moreover, recendy Yamane et al. reported that continuous oral administration of 0.1% catechins in drinking water potently inhibited AOMinitiated rat colon carcinogenesis (31). Thus GTC was in fact expected to inhibit colon carcinogenesis in the present study. The fact that no influence was observed with either high or low dose levels with application at any stage might indicate a basic difference in the agents tested. With regard to the GTC used, polyphenolic compounds comprised 92% in the present experiment versus 74.5% in Yamane's study. From the chemopreventive point of view, it is clearly important to elucidate the differences in these results. It is possible that differences in the experimental system may be responsible for the discrepancy. In other organs, catechins significandy enhanced the number and area of GST-P positive foci in the liver, particularly when applied during the initiation stage. However, the effects were only weak, and in addition, a hyperplastic nodule was found in one rat of the carcinogen alone group but none were evident in the catechin-treated groups. Therefore, any enhancing effects of catechins on hepatocarcinogenesis can be expected to be very slight, if present at all. In conclusion, in the present experimental system, GTC exerted chemoprevention effects on small intestinal but not colon carcinogenesis. Acknowledgements This work was supported in part by a grant from the Ministry of Health and Welfare of Japan, Grant-in Aid for Cancer Research from the Ministry of
1552
Education, Science and Culture, a grant from the Society for Promotion of Cancer Research of Aichi Prefecture, Japan and a grant from the Society for Promotion of Pathology of Nagoya, Japan.
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