Chapter 20
Carcinogenicity and Modification of Carcinogenic Response by Plant Phenols
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N . Ito, M . Hirose, and T. Shirai First Department of Pathology, Nagoya City University Medical School, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467, Japan
Since the carcinogenicity ofBHAin rodent forestomach was first shown, several plant phenols have been demonstrated to exert carcinogenic potential in rats, e.g., caffeic acid (forestomach and kidney), sesamol (forestomach), catechol (glandular stomach) and hydroquinone (kidney). In the second stage of 2-stage carcinogenesis, these compounds demonstrate different modifying activities depending on the organ. For example, catechol enhances forestomach, glandular stomach, tongue and esophageal carcinogenesis while inhibiting colon, lung and hepato-carcinogenesis. Hydroquinone and caffeic acid enhance kidney and forestomach carcinogenesis, respectively. Several other phenolic antioxidants have been examined for their modifying effects using multi-organ carcinogenesis models. The results indicate that catechins clearly inhibit rat small intestinal carcinogenesis with the effects being most obvious when applied during the initiation stage. On the other hand, combined treatment with sodium nitrite and phenolic compounds such as catechol and 3-methoxycatechol strongly induces forestomach proliferative lesions which are not observed with individual treatments. The results suggest forestomach carcinogenicity of these compounds in the presence of sodium nitrite.
The synthetic antioxidant butylated hydroxyanisole (BHA) has been shown to induce forestomach carcinomas in rats and hamsters (1-3). It strongly enhances forestomach carcinogenesis in rats pretreated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-methylnitrosourea (MNU) or N,N-dibutylnitrosamine (DBN), and enhances urinary bladder carcinogenesis induced by N-butyl-N-(4hydroxybutyl)nitrosamine or DBN. On the other hand, it inhibits liver, lung, and mammary carcinogenesis induced by carcinogens (4-6). BHA rapidly induces cell proliferation in the forestomach epithelium of both rats (3) and hamsters (7); this increase in DNA synthetic activity presumably plays an important role in the observed carcinogenic effect.
0097-6156/92/0507-0269$06.00/0 © 1992 American Chemical Society
Huang et al.; Phenolic Compounds in Food and Their Effects on Health II ACS Symposium Series; American Chemical Society: Washington, DC, 1992.
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PHENOLIC COMPOUNDS IN FOOD AND THEIR EFFECTS ON HEALTH II
Recently, some plant phenols, i.e. caffeic acid, sesamol and catechol, have been shown to also induce strong cell proliferation in either the forestomach or glandular stomach epithelium of rats (8) and hamsters (9). These antioxidants similarly appear to exert carcinogenic potential in their target organs, enhance stomach carcinogenesis, or modify carcinogenesis in other organs. Recently we developed multi-organ carcinogenesis models to examine modifying activity of chemicals (10-13). In these models complex (enhancing or inhibitory) modifying effects of chemicals in over ten organs could be detected in a single experiment. Thus these models could be applied to distinguish chemopreventors which do not exert enhancing effects in any organ. Catechins, which are major components of green tea tannins, have been suggested as potential chemopreventors since they inhibit teleocidine-induced promotion of mouse skin carcinogenesis initiated by D M B A (14), N-ethyl-W-nitro-Nnitrosoguanidine-induced mouse duodenal carcinogenesis (15) and spontaneouslyinduced mammary carcinomas in C3H/HeN mice (76). It is well known that the reaction of secondary amines and sodium nitrite in acidic conditions results in formation of carcinogenic nitroso compounds. Several phenolic compounds and antioxidants such as ascorbic acid and α-tocopherol have been demonstrated to block formation of carcinogenic nitroso compounds by reducing nitrous acid to nitric oxide (77). On the other hand, interaction of phenolic compounds such as phenol (18), butylated hydroxyanisole (79), catechol and 3methoxycatechol (20), and NaNC>2 under acidic conditions is itself associated with generation of genotoxic substances. These genotoxic compounds may produce tumors in the rat stomach, where they directly contact the epithelium. In the present paper carcinogenicities of caffeic acid, sesamol, catechol and hydroquinone (an isomer of catechol), modification of carcinogenesis by these phenolic compounds, modification of carcinogenesis by green tea catechins using rat multi-organ carcinogenesis model, and combined effects of phenolic compounds and NaN02 on rat gastric epithelium are reviewed. Carcinogenicities of Phenolic Compounds in Rats Groups of 30, 6-week-old F344 rats of both sexes (Charles River Japan Inc., Kanagawa) were treated with 2% caffeic acid (Tokyo Kasei Kogyo Co., Tokyo, purity > 98%), 2% sesamol (Fluka Chemie A G , Switzerland, purity >98%) or 0.8% catechol (Wako Pure Chemical Industies, Osaka, purity > 99%) in Oriental M F powdered basal diet (Oriental Yeast Co., Tokyo) or basal diet alone for 104 weeks. Food and water were given ad libitum. Chemicals were incorporated into powdered diet using a mixer and the diets stored at 4° C until use. Animals which died during the experiment were necropsied and all surviving animals were killed under anesthesia and subjected to complete autopsy at the end of week 104. Livers and kidneys were weighed before fixation in 10% buffered formalin solution. Formalin was injected into the stomachs, which were then opened via an incision along the greater curvative. Three sections each were cut from the anterior and posterior walls of the forestomach and six sections from each glandular stomach. Tissues were processed in the usual way for histopathologic^ examination. Animals which survived more than 77 weeks (caffeic acid, rat), 82 weeks (sesamol, rat), 26 weeks (catechol, male rats), 52 weeks (catechol, female rats), when the first tumor appeared, were included in the effective numbers. Student's t test and Fisher's exact probability test were used for statistical evaluation of the data.
Huang et al.; Phenolic Compounds in Food and Their Effects on Health II ACS Symposium Series; American Chemical Society: Washington, DC, 1992.
20. ITO E T A L .
Carcinogenicity & Modification of Carcinogenic Response
271
At the end of the experiment, body weights of animal treated with phenolic compounds were generally lower than in the controls, particularly for animals treated with catechol (17.1%-41.1% reduction compared to controls). The relative liver and kidney weights, however, were all higher in animals receiving the phenolic compounds. Histopathologically, changes in the forestomach were classified into papilloma and squamous cell carcinoma categories as previously reported. The results are summarized in Table I. Caffeic acid induced significant increases in the incidences of papillomas and squamous cell carcinomas in male and female rats. Sesamol also induced, 34 and 47%, incidences of papillomas in male and female rats, respectively. Significant increase in the development of squamous cell carcinoma was, however, observed only in male rats (p*** (34) (47)
» *** 17 ( 5 6 · & * 15 ( 5 0 ) * * * . 9 (31.0) 3 (10) 0 0 0 0 0 0
P t * t (100) 12 (42.9) 0 0 0 0
(3)
No. o f r a t s with (%) Forestomach G l . stomach Sex No. of Papilloma Squamous c e l l Adenoma Adenorats carcinoma carcinoma 22 6 0 0 0 0 30 2 1 0
4 (13.3) 0 0 0 0 (100)*** 14 (46.6)*** (7) 0 0 (3) 0
*** (73)* (20)*
Kidney Tubular Adenoma hyperplasia
Table I. Incidences of Lesions i n the Stomach and Kidney Treated with Chemicals f o r 104 Weeks i n F344 Rats
20. ITO E T A L .
Carcinogenicity & Modification of Carcinogenic Response
was not different from the control value (20%). The incidence of glandular stomach adenocarcinoma was significandy enhanced only in rats treated with M N N G followed by catechol (94.7% vs 0% in control) (7,27,22). In organs other than those targeted for carcinogenicity, different modifying effects (promotion and inhibition) were observed depending on the individual organ as shown in Table II. For example, catechol promoted esophageal carcinogenesis but inhibited colon, lung, and mammary gland carcinogenesis; hydroquinone enhanced kidney carcinogenesis and weakly promoted esophageal carcinogenesis (22,23). Effects of Green Tea Catechins in a Rat Multi-organ Carcinogenesis Model Groups of 15 male F344 rats, 6 weeks old at the commencement of the study (Charles River Japan, Inc., Atsugi, Kanagawa), were treated with combined single ip administration of 100 mg/kg bw N-diethylnitrosamine (DEN), 4 ip administrations of 20 mg/kg bw N-methylnitrosourea (MNU), 4 sc doses of 40 mg/kg bw NJJdimethylhydrazine (DMH), 0.05% B B N in drinking water for 2 weeks or 0.1% D H P N for 2 weeks during the initial 4 week period for wide spectrum initiation. Experimental groups were formed as follows: Group 1,1% catechins in Oriental M F powdered basal diet administered from one day before and during carcinogen exposure. Group 2, 1% catechins in diet administered after carcinogen exposure. Group 3, carcinogens alone. Group 4, 1% catechins in diet alone and group 5, basal diet alone during the experiment A l l animals were killed under ether anesthesia at the end of week 36, and complete autopsies were performed. Tissues were processed routinely for histopathological examination. Final average body weights of rats treated with catechins were slightly higher than those in the initiation alone group. Incidences and average number of tumors in the small intestine are summarized in Table ΙΠ. Incidences of adenomas in groups 1 and 2 were significantly lower (7 and 8%, respectively, p
