Chapter 14
Cancer Chemoprevention by Antioxidants Masao Hirose, Katsumi Imaida, Seiko Tamano, and Nobuyuki Ito
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First Department of Pathology, Nagoya City University Medical School, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467, Japan
Modulation effects of antioxidants on chemical carcinogenesis were investigated using three experimental approaches. First, naturally occurring antioxidants such as N-tritriacontane-16,18-dione (TTAD), tannic acid, phytic acid, green tea catechins (GTC), diallyl sulfide (DAS) and diallyl disulfide (DDS) were examined in two rat multi-organ carcinogenesis models in which both enhancing and inhibitory effects of chemicals could be investigated at the whole body level. In one model, T T A D and phytic acid inhibited hepatocarcinogenesis, tannic acid inhibited large intestinal carcinogenesis and phytic acid enhanced bladder carcinogenesis when they were administered after carcinogen treatment. In the second multi-organ model, G T C clearly inhibited small intestinal carcinogenesis when it was given either during or after carcinogen treatment. DDS potently inhibited large intestine and kidney carcinogenesis, but DAS en hanced hepatocarcinogenesis when applied subsequent to carcinogen treatment. Another approach used the analysis of chemical carcinogen enhancement of liver preneoplastic GST-P positive foci in partially hepatectomized rats. GTC, phenethyl isothiocyanate, α-tocopherol and β-carotene were found to strongly inhibit the Glu-P-1-enhancement of GST-P positive foci, but not dimethylnitrosamine enhancement. In an third approach, female SD rats fed 1% GTC in the diet for 35 weeks, starting one week after a single intragastric administration of D M B A , did not have significantly different incidences or average numbers of mammary carcinomas, but the survival rate was clearly higher in the G T C group (94%) as compared with controls (33%).
It is generally accepted that environmental factors are appreciable causes of human cancer. This is partly reflected in the increasing number of man-made or naturally occurring chemicals which have been shown to have carcinogenic potential. Since cancer mortality is rising with prolongation of the life span, it is important to detect
0097-6156/94/0547-0122$06.00/0 © 1994 American Chemical Society
In Food Phytochemicals for Cancer Prevention II; Ho, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.
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14. HIROSE ET AL.
Cancer Chemoprevention by Antioxidants
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human carcinogens and to eliminate them, wherever possible, from our environ ment. This approach, however, is clearly limited by the number of experimental facilities, and the involved political and social expense. Another important possible way to decrease human cancer mortality is through cancer chemoprevention. Although much attention has recently been paid to this area, ideal chemopreventors have not yet been developed. This is largely because chemicals inhibit or enhance carcinogenesis depending on the organ site, timing of administration (before carcinogen exposure, with carcinogen or after carcinogen exposure), and species used (1-4). Therefore, the development or detection of chemopreventive com pounds in the case of natural agents, requires analysis of potential at the whole body level. The present investigations of possible chemopreventive antioxidants were conducted using different three experimental systems with this requirement in mind. Materials and Methods Effects of Antioxidants in Rat Multi-Organ Carcinogenesis Models. In the D E D model, groups of 6-week-old F344 male rats (Charles River Japan, Inc.) were given, at intervals of 3-4 days, two i.p. injections of 1000 mg/kg body weight (bw) 2,2 -dihydroxy-di-n-propylnitrosamine (DHPN), two i.g. administrations of 1500 mg/kg bw Λ^-ethyl-N-hydroxyethylnitrosamine (EHEN) and three s.c. injections of 75 mg/kg bw 3,2 -dimethyl-4-aminobiphenyl (DMAB) over the initial 3 week period. The animals were housed 5 or 6 to a plastic cage with wood chips for bedding in an air-conditioned room at 24 ± 2°C with a 12 hr light-dark cycle. In the first experiment, 4 groups of 15 to 17 animals each were fed Oriental M F powdered basal diet containing 0.2% N-tritriacontane-16,18-dione (TTAD, Eisai Co. Ltd., Tsukuba, Japan), 1% tannic acid (Wako Pure Chemical Industries Ltd., Osaka, Japan), 2% phytic acid (Tokyo Kasei Kogyo Co. Ltd., Tokyo, Japan) or basal diet alone starting one day before the E H E N treatment until one week after the last D M A B treatment. The animals were then placed on basal diet until sacrifice at week 36. In the second experiment, starting one week after the last D M A B treatment, 4 groups of 15 rats were treated with basal diet containing 0.2% TTAD, 1% tannic acid, 2% phytic acid or basal diet alone for 32 weeks. A further 4 groups of 10 animals each were treated with antioxidants or basal diet alone without carcinogen treatment. Animals were killed at week 36. In the D M B D D model, animals were given combined treatment with a single i.p. administration of 100 mg/kg bw diethylnitrosamine (DEN), 4 i.p. administrations of 20 mg/kg bw methylnitrosourea (MNU), 4 s.c. doses of 40 mg/kg bw 1,2-dimethylhydrazine (DMH), 0.05% iV-butyl-N-(hydroxybutyl)nitrosamine (BBN) in the drinking water for 2 weeks and 0.1% D H P N in drinking water for 2 weeks during the initial 4 week period for initiation. In the first experiment, 3 groups of animals each were continuously administered 1% green tea catechins (GTC, Mitsui Norin Co. Ltd., Japan) in powdered basal diet starting one day before until the completion of carcinogen exposure, or starting 3 days after the carcinogen exposure until week 36. Further groups of animals were given the D M B D D treatment alone, or 1% GTC alone throughout the experiment without carcinogen exposure. In the second experiment, 3 groups of 20 animals each were administered i.g. 200 mg/kg bw diallyl sulfide (DAS), 50 mg/kg bw diallyl disulfide (DDS) or corn oil alone 3 times a week after D M B D D treatment. D A S and DDS were obtained from Tokyo Kasei Kogyo Co. Ltd., Tokyo. Further groups of animals /
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In Food Phytochemicals for Cancer Prevention II; Ho, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.
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FOOD PHYTOCHEMICALS II: TEAS, SPICES, AND HERBS
were treated with D A S , DDS or basal diet alone without D M B D D treatment. A l l animals were killed for autopsy at week 28. In each of these multi-organ carcinogenesis models, all major organs were removed, fixed in neutral buffered formalin solution, or cold acetone for liver, and routinely processed for H & E staining or immunohistochemical staining with anti-glutathione S-transferase placental form (GST-P) antibody. Effects of Antioxidants in a Rat Medium-term Bioassay for the Detection of Hepatocarcinogens. Six-week-old F344 male rats were given a single i.p. injection of 100 mg/kg bw D E N and starting 2 weeks later received diet containing 300 ppm 2-amino-6-methyldipyrido[l,2-a:3 ,2 -ui]imidazole (Glu-P-1) or 20 ppm dimethylnitrosamine (DMN) in the drinking water, alone, or in combination with 1% GTC, 0.1% 2-phenylethyl isothiocyanate (PEITC), 1.5% α-tocopherol or 0.1% β-carotene for 6 weeks. Glu-P-1 was kindly supplied by Drs. Takashi Sugimura and Minako Nagao of the National Cancer Center Research Institute, Tokyo, Japan. PEITC was obtained from Tokyo Kasei Kogyo Co. Ltd., Tokyo, α-tocopherol from Eisai Co. Ltd., Tokyo, and β-carotene from Riken Vitamins Co. Ltd., Tokyo. Animals underwent partial hepatectomy at week 3 and the experiment was terminated at week 8. Livers were removed, fixed in cold acetone, and paraffin-embedded sections were stained immunohistochemically with anti-GST-P antibody.
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Effects of an Antioxidant in a DMBA-induced Rat Mammary Carcinogenesis Model. S even-week-old female SD rats (Charles River Japan Inc., Kanagawa, Japan) were given a single i.g. administration of 7,12-dimethylbenz[a]anthracene (DMBA) at a dose of 50 mg/kg bw. Starting one week after D M B A administration, groups of 15 or 16 animals each were fed powdered basal diet containing 1% GTC, or basal diet alone for 35 weeks. Further groups of 10 animals each were fed G T C or basal diet alone without the carcinogen treatment. Presence of palpable mammary tumors was recorded once every 2 weeks. Animals were killed at week 36, when mammary tumors, Zymbal's glands, liver, kidneys and spleen were removed and routinely processed for examination of sections stained with H & E . Student's t test, cumulative χ test and Fisher's exact test were used for statistical analysis of the data. 2
Results Effects of Antioxidants in Rat Multi-organ Carcinogenesis Models. DED Model. The final body weights of animals treated with carcinogens simultaneously with antioxidants were not significantly different from those receiving D E D treatment alone. Those treated with D E D followed by T T A D , however, were significantly lower. The incidences of histopathologically assessed preneoplastic and neoplastic lesions in the thyroid, esophagus, large intestine, liver, pancreas, lung, kidney and urinary bladder of rats treated with antioxidants during D E D treatment were not significandy different from those with DED alone. Incidences of preneoplastic and neoplastic lesions in major organs of rats treated with antioxidants after D E D treatment are shown in Table I. In the group given phytic acid, the incidence (50%, p
