Protective Effects Against Liver, Colon, and Tongue Carcinogenesis

Chapter 26

Protective Effects Against Liver, Colon, and Tongue Carcinogenesis by Plant Phenols T. Tanaka, N. Yoshimi, S. Sugie, and H. Mori

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First Department of Pathology, Gifu University School of Medicine, 40 Tsukasa-machi, Gifu 500, Japan

Three different experiments were performed in order to examine the potential inhibitory effects of the plant phenolic compounds ellagic acid (EA) and chlorogenic acid (CA) on chemical carcinogenesis. Experiment I: The effect of dietary EA (400 ppm for 18 weeks) on the carcinogenicity to rats of concurrently administered N-2-fluorenylacetamide (FAA, 200 ppm in diet for 16 weeks) was determined. Experiment II: The effect of EA in diet (400 ppm for 10 weeks) on the rat tongue carcinogenesis by 4-nitroquinoline 1-oxide (4-NQO, 10 ppm in drinking water for 8 weeks) during the initiation stage was investigated. Experiment III: The protective effect of dietary CA (250 ppm for 24 weeks) on methylazoxymethanol (MAM) acetate (a single i.v. injection, 20 mg./kg body weight)-induced colon and liver carcinogenesis during the post-initiation stage was examined in Syrian golden hamsters. In the rats receiving EA together with FAA or 4-NQO, the incidence of neoplasm in the liver (30%) or tongue (20%) was significantly decreased compared with those of rats given the carcinogen alone (100% in liver; 71% in tongue). Similarly, colon tumor (13%) and liver cell foci (6.0 ± 2.7/cm ) incidence in hamsters given MAM acetate and CA was significantly smaller than those of hamsters given MAM acetate alone (50% and 10.8±2.7/cm ). These results suggest that the plant phenolics EA and CA are potential chemopreventive agents in carcinogenesis. The number and area of AgNORs in epithelial cell nuclei of liver, tongue, and colon of animals were also quantified in order to evaluate the proliferative activity of cells. The number and area of AgNORs of nuclei of the target organs from the animals treated with the carcinogens and test chemicals were significantly 2

2

0097-6156/92/0507-0326$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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Inhibition of Liver, Colon, & Tongue Carcinogenesis 327


lower than those of animals given the carcinogen alone, indicating that EA and CA suppressed the proliferative activity of targeted epithelial cells and AgNOR enumeration could be a useful method for estimating the chemopreventive potential of certain chemicals in carcinogenesis.

Chemical carcinogenesis i s now generally recognized as a multistage process (initiation, promotion, and progression) (1). Environmental factors (mutagens, carcinogens, and promoters) are important f o r occurrence of human cancers (2). There i s growing evidence f o r the involvement o f genetic a l t e r a t i o n s o f various types of cancers i n mammals (3,4). However, the process leading from the i n i t i a t i o n stage to the overt cancer stage i s less understood. Although recent advances i n cancer diagnosis and therapy have improved the short-term prognosis and q u a l i t y of l i f e f o r many cancer patients, there has been l i t t l e or no e f f e c t s on the m o r t a l i t y rate f o r most cancers. Thus, i t i s considered that primary and secondary prevention of cancer development i n humans i s very important (5). Recently, much a t t e n t i o n has been given to chemopreventive agents that may act as protection against mutagens or carcinogens i n the environment. Several compounds have been found to i n h i b i t development o f neoplasms induced by chemical carcinogens when administered p r i o r t o and/or simultaneously with the carcinogen (β). These include n a t u r a l l y occurring constituents of food, p a r t i c u l a r l y edible plants, vegetables and f r u i t s , and a l s o synthetic compounds (6-9). We present here our recent studies (10,11) on the i n h i b i t o r y e f f e c t s of the plant phenolics e l l a g i c a c i d (EA) and chlorogenic a c i d (CA) on l i v e r , colon, and tongue carcinogenesis. Materials and Methods Chemicals. Test chemicals, e l l a g i c a c i d (EA, >97% pure) and chlorogenic a c i d (CA) were purchased from Tokyo Chem. Ind. Co., Ltd., Tokyo, Japan and Sigma Chem. Co., St. Louis, MO, U.S.A., respectively. N-2-Fluorenylacetamide (FAA, Nacalai Tesque, Inc., Kyoto, Japan), 4-nitroquinoline 1-oxide (4-NQO), and methylazoxymethanol (MAM) acetate (Ash Stevens, Milwaukee, WI, U.S.A.) were used as a chemical carcinogen. EA and CA were mixed with powdered basal d i e t , CE-2 (Japan CLEA Inc., Tokyo, Japan) at concentrations 400 ppm and 250 ppm, respectively. These d i e t were made every two weeks. Animals. Male inbrd ACI/N r a t s (6 weeks of age), which were maintained i n our laboratory, were used f o r examining the i n h i b i t o r y e f f e c t of EA on l i v e r carcinogenesis induced by FAA (Experiment I) and on tongue carcinogenesis induced by 4-NQO (Experiment I I ) . Syrian golden hamsters of both sexes (Japan CLEA Inc., Tokyo, Japan), 2 months o l d were used f o r examining the suppressing e f f e c t s of CA on large i n t e s t i n a l and l i v e r carcinogenesis induced by MAM acetate (Experiment I I I ) .



328

PHENOLIC COMPOUNDS IN FOOD AND THEIR EFFECTS ON HEALTH II

Experiment I : I n h i b i t o r y E f f e c t o f EA on FAA-Induced Hepatocarcinogenesis. A t o t a l of 70 male ACI/N r a t s were divided into 4 groups as shown i n Figure 1. Group 1 (19 r a t s ) was f e d the d i e t containing 200 ppm FAA f o r 16 weeks and maintained on the basal d i e t , CE-2 (Japan CLEA Inc., Tokyo, Japan), f o r 20 weeks. Group 2 (19 r a t s ) was f e d the d i e t containing 200 ppm FAA and 400 ppm EA f o r 16 weeks. Rats i n t h i s group were fed 400 ppm EA containing d i e t f o r one week before and a f t e r the carcinogen exposure, and then maintained on the basal d i e t f o r 19 weeks. Group 3 (16 r a t s ) was fed the d i e t containing 400 ppm EA begining one week before the s t a r t of the study and continued f o r 18 weeks. Group 4 (16 r a t s ) was fed only the basal d i e t during the experiment. At weeks 17, 20, and 24, three r a t s from each group were s a c r i f i c e d to determine the i n h i b i t o r y e f f e c t of EA on the occurrence of T-glutemyltranspeptidase (GGT)-positive l i v e r c e l l f o c i . The remaining animals were k i l l e d a t the end of the study (week 36) i n order to obtain the f i n a l incidence of l i v e r neoplasms. At autopsy, two s l i c e s were taken from each sublobe of l i v e r and f i x e d i n 10% buffered formalin f o r hematoxylin and eosin (H & E) s t a i n i n g and i n 95% c o l d ethanol (4C) f o r GGT reaction, respectively. L i v e r c e l l focus, adenoma, and carcinoma were diagnosed according to the c r i t e r i a described by Stewart et al. (12). The incidence of a l t e r e d l i v e r c e l l f o c i (no./cm ) was q u a n t i f i e d on the H & Ε-stained and GGT-reacted sections using a microscope. 2

Experiment I I : Inhibitory E f f e c t of EA on 4-NQQ-Induced Tongue Carcinogenesis. A t o t a l o f 48 male ACI/N rats were d i v i d e d into 4 experimental groups as shown i n Figure 2. Groups 1 (17 r a t s ) and 2 (15 r a t s ) were given 4-NQO i n the d r i n k i n g water (10 ppm) f o r 8 weeks. Group 2 was given the d i e t c o n t a i n i n g 400 ppm EA, s t a r t i n g one week before the commencement of the study u n t i l one week a f t e r the carcinpgen exposure. Rats were then switched t o the basal d i e t , CE-2, and maintained on t h i s d i e t f o r 27 weeks. Group 3 (8 r a t s ) was f e d EA d i e t f o r 10 weeks as f o r group 2. Group 4 (8 r a t s ) was f e d the basal d i e t alone during the experiment and served as an untreated c o n t r o l . A l l animals were s a c r i f i c e d 36 weeks a f t e r the s t a r t of the experiment i n order to evaluate the incidence of preneoplastic and n e o p l a s t i c l e s i o n s of the tongue. A f t e r complete necropsy of animals, a l l organs were f i x e d i n 10% buffered formalin and a l l t i s s u e s and gross lesions were processed f o r histology by the conventional methods. E p i t h e l i a l lesions (hyperplasia, d y s p l a s i a and neoplasia) o f the tongue were diagnosed on the Η & Ε stained sections according to the c r i t e r i a described by Rubio (13). Experiment I I I : I n h i b i t o r y E f f e c t o f CA on MAM Acetate-Induced Large Bowel and L i v e r Carcinogenesis. A t o t a l of 94 Syrian golden hamsters of both sexes (47 each) were used and d i v i d e d into 4 groups as shown i n Figure 3.


26.

TANAKA ET AL.

Inhibition of Liver, Colon, & Tongue Carcinogenesis

36 vjks

24

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Group 4


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Figure

1. Experimental FAA l~1

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Basal

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(400

36 wks

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ppm)

diet

Group 1 Group 2 Group 3 Group 4

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Figure

2. Experimental WÊÊ 4 - N Q O •

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Figure

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protocol (10

(Experiment

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3. Experimental Ο •

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MAM a c e t a t e •

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329


330

PHENOLIC COMPOUNDS IN FOOD AND THEIR EFFECTS ON HEALTH II

Groups 1 (10 males and 10 females) and 2 (12 males and 12 females) were given a s i n g l e i.v. i n j e c t i o n of MAM acetate (20 mg/kg body weight). A f t e r the i n j e c t i o n , animals i n group 1 were kept on the basal d i e t , CE-2, and those i n group 2 were fed the d i e t containing 250 ppm CA f o r 24 weeks. Group 3 (10 males and 10 females) was fed the d i e t containing 250 ppm CA f o r 24 weeks. Group 4 (15 males and 15 females) was fed the basal d i e t alone throughout the experiment. On termination of the study, a l l animals were s a c r i f i c e d and autopsied. At autospy, the g a s t r o i n t e s t i n a l t r a c t was removed and a longitudinal i n c i s i o n was made from the stomach to the rectum. A f t e r gross examination of a l l organs, a l l tissues, including the d i g e s t i v e t r a c t and l i v e r , were f i x e d i n 10% buffered formalin, stained with Η & Ε, and examined h i s t o l o g i c a l l y according to the c r i t e r i a described by Ward f o r colon neoplasms (14) and those described by Stewart et al. f o r l i v e r lesions (12). Enumeration of S i l v e r - S t a i n e d Nucleolar Organizer Regions (AgNORs). For assessment of p r o l i f e r a t i v e a c t i v i t y of e p i t h e l i a l c e l l s , number and area of AgNORs were q u a t i f i e d i n three animals from each group i n Experiments I, I I , and I I I . One-step s i l v e r c o l l o i d method f o r AgNOR s t a i n i n g was c a r r i e d out on the l i v e r (Experiment I ) , tongue (Experiment I I ) , and colon (Experiment I I I ) specimens from the animals s a c r i f i c e d at the end of the study. Computer-assisted image analysis quantitation of the number and area of AgNORs i n 300 interphase c e l l s from each nonlesional areas of these organs was performed using the image analysis system SPICCA I I (Japan Abionics, Co., Tokyo, Japan) with a Olympus BH-2 microscope and a c o l o r CCD camera (Hamamatsu Photonics, Co., Hamamatsu City, Japan). Results Experiment I. The r e s u l t s are summarized i n Table I. Administration of EA to r a t s i n group 3 d i d not a f f e c t the body weights compared with the controls i n group 4. Treatment with FAA increased the l i v e r weight i n group 1 due to the development of tumors. The l i v e r weight of rats i n group 2 was s i g n i f i c a n t l y greater than that i n group 3, but was s l i g h t l y smaller than that i n group 1 without s t a t i s t i c a l s i g n i f i c a n c e . Altered l i v e r c e l l f o c i and hepatocellular neoplasms developed i n r a t s of groups 1 and 2. The number of f o c i i n group 2 was almost h a l f of that i n group 1 during and a t the end of the experiment. In r a t s of group 1, the incidence and m u l t i p l i c i t y were 100% and 4.0 neoplasms/rat, respectively. Feeding of EA together with FAA (group 2) reduced the incidence (30%) and m u l t i p l i c i t y (0.6/rat) of l i v e r c e l l neoplasms. In other organs, no preneoplastic and neoplastic lesions were found i n any group. Experiment I I . The r e s u l t s are given i n Table I I . The weights, l i v e r weights, and r e l a t i v e l i v e r weights of r a t s

average body i n a l l groups


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Inhibition of Liver, Colon, & Tongue Carcinogenesis

331

Table I. Results o f Experiment I Group 1 (FAA) e

Body wt (g) L i v e r wt (g) R e l a t i v e l i v e r wt

246 i 24 11.7 + 1.3 4.75 + 0.31

L i v e r c e l l tumors Incidence: Total Adenoma Carcinoma Multiplicity Downloaded by IOWA STATE UNIV on April 12, 2017 | http://pubs.acs.org Publication Date: October 1, 1992 | doi: 10.1021/bk-1992-0507.ch026

Group 2 (FAA + EA)

10/10 7/10 8/10 4.00

(100%) (70%) (80%) i 2.41

269 ί 18 10.9 + 0.4 4.06 + 0.34

3/10 2/10 2/10 0.60

(30%)' (20%)° (20%) i 1.02' d

2

GGT-positive l i v e r c e l l f o c i (/cm ) Week 16 Week 20 Week 24 Week 36

45.7 + 54.0 + 55.7 + 59.8 +

5.4 0.8 3.3 6.3

21.5 + 5.4' 20.0 + 4.1 23.7 + 4.2 24.3 + 0.9 e

e

e

'Mean + SD. * " S i g n i f i c a n t l y d i f f r e n t from group 1 by Fisher's exact p r o b a b i l i t y test ( P=0.02, P=0.004, ^=0.01). " S i g n i f i c a n t l y d i f f e r e n t from group 1 by Student's ί-test ( P

Protective Effects Against Liver, Colon, and Tongue Carcinogenesis

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