Chapter 19
Dietary Quercetin and Rutin Inhibitors of Experimental Colonic Neoplasia Eleanor Ε. Deschner
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Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, N Y 10021
The phenolic flavonoids quercetin (QU) and rutin ( R U ) found in the outer leaves of vegetables and fruits were examined as modifiers of an experimental model of colon cancer. CF1 mice were fed an AIN-76A diet to which was added various levels of QU or RU. Acute studies revealed that, in control mice, QU and RU had no effect on colonic epithelial cell proliferation but 2% QU and 4% RU significantly reduced azoxymethanol ( A O M ) induced hyperproliferation of these cells. The same doses also significantly inhibited the development of focal areas of dysplasia (FAJDs). Compared with controls, 2% QU for 50 weeks significantly depressed colon tumor incidence while both 2% QU and 4% RU suppressed tumor multiplicity. Thus, under conditions of low dietary fat intake, these flavonoids have considerable activity to suppress the hyperproliferation of colonic epithelial cells, thereby reducing FADs and ultimately colon tumor incidence.
Flavonoids such as QU andRUoccur widely in edible fruits and vegetables. To date, over two thousand flavonoids have been chemically characterized and most occur in nature in a glycoside form. Dietary intake of QU is thought to range between 50 and 500 mg per day (1). Some i n vitro assays employing QU have erroneously reported the mutagenicity of this compound due to the production of hydrogen peroxide by its interaction with trace metals (i.e. copper and iron) in the media. The latter act on the catechol group of the flavonoids exposed to atmospheric oxygen in a classical reaction producing the toxic hydrogen peroxide (2). This reaction does not occur in biological in vivo systems.
0097-6156/92A)507-0265$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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Dietary Quercitin. Ingestion of a known quantity of Q U revealed that less than 1% was absorbed in the gut. More than 50% of the dose given was degraded by microbes in the colon while the remainder was lost in the feces (3). Urine samples over 24 hours in volunteers given 4g of oral Q U contained no Q U or Q U conjugates. The presence of unabsorbed dietary Q U in the colon or alternatively the release of Q U from the glycoside R U when it is cleaved by microflora in the colon (4) suggest that, in either case, Q U in close proximity with the colonic mucosa could act as an agent which might protect, prevent or inhibit carcinogenesis there. Furthermore, studies of experimentally induced mammary cancer in rats fed dietary Q U revealed a diminution in tumor incidence (5) although the amount of Q U delivered to the mammary gland by this route in this instance is questionable. Model Testing Efficacy. To evaluate the possible protective effect of dietary Q U (0.1, 0.5 and 2% of diet) and R U (1.0 and 4.0% of diet), a model for experimental colon cancer in CF1 mice was employed using the carcinogen A O M (6). 2% Q U is approximately equimolecular to 4% R U . Three tests carried out helped determine the efficacy of Q U and R U against A O M induced carcinogenesis. Two were short term studies and one a long term investigation. The two acute studies evaluated the incidence of F A D s and the modification of carcinogen induced elevated levels of colonic epithelial cell proliferation. The chronic study with Q U and R U assessed the tumor incidence and tumor multiplicity levels when compared to mice not fed the flavonoids. Acute Studies. A O M is an active metabolite of the well studied carcinogen 1,2-dimethylhydrazine ( D M H ) (7). Six injections of D M H , once weekly at a dose of 20 mg/kg body weight, induce F A D s in the colon. They appear as early as 27 days after delivery of the first few injections. These F A D s arise in the upper two-thirds of the colonic crypts of the otherwise normal appearing mucosa. F A D s are composed of epithelial cells exhibiting a loss of mucin, loss of polarity of nuclei as well as nuclei of various size and shape (8,9). The number of F A D s present in 500 microns of serially sectioned distal colon was determined. In mice after 6 weekly injections of A O M at a dose of 10 mg/kg body weight and concomitant dietary intake of 2% Q U and 4% R U , a significantly reduced incidence of F A D s was observed compared with the number present in mice fed the control diet (10). Prior to and simultaneously with the development of A O M induced F A D s , alterations of colonic epithelial cell proliferation are initiated by the first and repeated injections of the carcinogen (9). To analyze these induced abnormalities in cell proliferation, tritiated thymidine is injected one hour before sacrifice of the mice to allow incorporation into newly synthesized D N A . Microscopic observation of slides treated by autoradiography allow the number and position of tritiated thymidine labeled cells to be noted as
Huang et al.; Phenolic Compounds in Food and Their Effects on Health II ACS Symposium Series; American Chemical Society: Washington, DC, 1992.
19. DESCHNER
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well as the total number of cells per crypt column or one half of a crypt or gland. Dietary Q U and R U had no effect on colonic epithelial cell proliferation in mice not injected with the carcinogen. However, A O M treated mice fed the 2% Q U and 4% R U had significantly fewer cells/crypt column (i.e. reduction of hyperplasia) than carcinogen treated mice receiving the control diet. Moreover, the population of D N A synthesizing cells was significantly reduced (i.e. reduction of hyperproliferation) in the colonic mucosa of mice fed 2% Q U and 4% R U compared with those treated by A O M alone (20). Chronic Dietary Study. The long term tumor incidence investigation involved feeding Q U and R U diets for a total of fifty weeks (6). Diets were provided two weeks before carcinogen treatment was initiated and protocol called for three weekly subcutaneous injections of A O M at a level of 10 mg/kg body weight. The highest level of dietary Q U (2%) significantly reduced colonic tumor incidence, a four fold inhibition, when compared with those mice receiving the control diet. When the 3 Q U dietary groups were combined and 2 R U groups combined and then compared with the A O M treated control group, it was determined that both Q U and R U significantly reduced the number or multiplicity of tumors/tumor bearing mouse (10). General Conclusions. Previous studies examining the ability of Q U to inhibit the formation of nuclear aberrations 24 hours following D M H injection demonstrated that Q U was inactive at this stage of carcinogenesis (11). Rather than the initiation phase, the acute studies with Q U and R U described here, showing reduced F A D incidence and a depression in A O M induced hyperproliferation, suggest instead that it is the promotional stage which is more intensely affected by dietary consumption of these phenolics. These experiments have not elucidated a mechanism by which Q U inhibits or modifies the development of neoplasia in the colon. Nevertheless it has become apparent that this flavonoid has significant antitumoral activity there and that further experimental studies with it should be undertaken. Literature Cited 1. Jones, E.; Hughes, R.E. Expt. Gerontol. 1982, 117, 213-217. 2. Ueno, I.; Haraikama, K.; Kohno, M.; Hinomoto, T.; Ohya-Nishiguchi, H.; Tomatsuri T.; Yoshihira K. In: Plant Flavonoids in Biology and Medicine; Cody, V., Middleton E., Harborne J.B., Eds; Alan R. Liss: New York, NY, 1986, pp 425-428. 3. Gugler, R.; Leschick. M., Dengler. Europe. J. Clin. Pharmacol. 1975, 9, 229-234. 4. Goldin, B.R.; Lichtenstein, A.H.; Gorbach, S.L. In: Modern Nutrition in Health and Disease; Shils, M.E., Young, V.R. 7th edition. Lea and Febiger: Philadelphia, PA, 1988; pp 503.
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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