CANCER RESEARCH57. 246-252. January 5. 19971

INTRODUCTION

The modifying effects of the two flavonoids diosmin and hesperidingiven during the initiation and postinitiation phases oforal carcinogenesisinitiated with 4-nitroquinoline 1-oxide (4-NQO) were investigated in male

F344 rats. The compounds were tested alone and in combination. At 6weeks of age, animals were divided into experimental and control groupsand fed diets containing 1000 ppm diosmin and 1000 ppm hesperidin anda diet containing both compounds (900 ppm diosmin and 100 ppm hesperidin). At 7 weeks of age, all animals except those treated with each test

chemical alone and control groups were given 4-NQO (20 ppm) in thedrinking water for 8 weeks to induce oral cancer. Starting 7 dayS beforethe 4-NQO exposure, groups of animals were fed the diets containing testchemicals for 10 weeks and then switched to the basal diet. Starting 1 week

after the cessation of 4-NQO exposure, the groups given 4.NQO and abasal diet were switched to the diets containing diosmin, hesperidin, ordiosmin combined with hesperidin and maintained on these diets for 22weeks. The other groups consisted of rats given diosmin (1000 ppm),hesperidin (1000 ppm), and the combination regimen of these two compounds (900 ppm diosmin with 100 ppm hesperidin) alone, and untreated

rats. All animals were necropsied at the termination of the study (week32). The incidences of tongue lesions (neoplasms and preneoplasms),polyamine levels in the tongue tissue, and cell proliferation activity estimated by a 5-bromodeoxyuridine-Iabeling index and by morphometricanalysis of silver-stained nucleolar organizer regions protein were compared among the groups Feeding of both compounds singly or in combination during the initiation phase caused a significant reduction in thefrequency of tongue carcinoma [diosmin, 68% reduction (P < 0.01);hesperidin, 75% reduction (P < 0.005); and the combination regimen,

69% (P < 0.05)1.When fed the test compounds singly or the combinationregimen after 4-NQO exposure, the frequency of tongue cancer was alsodecreased [diosmin, 77% reduction (P < 0.005); hesperidln, 62% reduc

tion (P < 0.05); and the combination regimen, 77% (P < 0.005)]. Theincidences of oral preneoplasia (hyperplasia and dysplasia) in thesegroups were also decreased when compared with carcinogen controls(P < 0.05 - P < 0.001). There were no pathological alterations in ratstreated with test compounds or the combined regimen alone or those in anuntreated control group. Dietary administration of these compounds sig

nificantly decreased the expression of cell proliferation blomarkers (5-bromodeoxyuridine.labellng index and silver-stained nucleolar organizerregions protein number) of the nonlesional tongue squamous epithelium(P < 0.05). Also, polyamine concentrations in the oral mucosa werelowered in rats given the carcinogen and test compounds, alone and incombination, compared with those of rats given 4-NQO alone (P < 0.05).These findings suggest that supplementation with the flavonoids diosminand hesperidin, individually and in combination, is effective in inhibitingthe development oforal neoplasms induced by 4-NQO, and such inhibitionmight be related to suppression of increased cell proliferation caused by4-NQO in the oral mucosa.

Received 5120/96; accepted I 1/8/96.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by Grants-in-Aid from the Ministry of Education,

Science, Sports and Culture of Japan and for the 2nd Term Comprehensive 10-yearStrategy for Cancer Control from the Ministry of Health and Welfare of Japan.

2 To whom requests for reprints should be addressed.

Oral cancer is a common neoplasm in certain areas, such as Asia,the Pacific Islands, parts of Europe, and parts of Brazil (1). AlthoughJapan has one of the lowest incidences of oral, lip, and pharyngealcancers in the world (2), the patients with these malignancies haverecently been increasing from 3.6/100,000 population in 1985 to6.0/100,000 population in 1987 (3). Moreover, recent studies mdicated that the use of smokeless tobacco increased the incidence of oralcancer, especially tongue cancer, among young adults (4). Neoplasmsin the head and neck, including the oral cavity, possess severalbiological characteristics that are multistage and multifocal carcinogenesis (5).

In a large number of epidemiological data on the relationshipsbetween diet and cancer, a protective effect of the consumption ofvegetables and fruits on various forms of cancer including oral canceris found (6—8). A number of micronutrients, macronutnents, andnonnutrients in diets have been reported as inhibiting or chemopreventive agents in chemical carcinogenesis (9). One of the nonnutritivechemopreventive agents is flavonoids ( I0, 11). Previously, averageintake of all flavonoids was estimated to be 1 g/day (12). However,recent reports suggest that estimates of the amounts consumed dailyvary widely: the mean flavonoid intake varied from 2.6 mg/day inFinland to 68.2 mg/day in Japan (13, 14). A major flavonoid, quercetin, is reported to inhibit chemical carcinogenesis in various organs(15—17).We recently found the cancer-protective effects of flavonoidsquercetin, chalcone, and hesperidin on oral carcinogenesis in rats (18,19). The flavonoids display a variety ofbiological actions (see Ref. 20for review). They are antioxidants and scavengers of free radicals withvarious degrees of activity. Free radicals are possibly involved in celldamage and tumor promotion (21). Other biological functions, such asanti-inflammatory, antiallergic, antiviral, and antiproliferative activities have long been recognized. In addition, flavonoids could affectdrug-metabolizing enzyme activities, xanthine oxidase, and cellularprotein phosphorylation (see Ref. 20 for review). A protective effectof a xanthine oxidase inhibitor, 1‘-acetoxychavicolacetate, was foundin our laboratory (22).

All Citrus fruits contain variable quantities of flavonoids. The7-j3-rutinoside of the flavonone hesperitin, hesperidin (3'-5,7-trihydroxy-4'-methoxyflavanone 7-rhamnoglucoside; Fig. 1), the most important flavonoid of orange peel, is known to increase capillary

resistance in various conditions. Hesperidin is an abundant and inexpensive by-product of citrus cultivation. It is isolated in large amountsfrom the discarded rinds of the ordinary orange Citrus aurantium L.The amount of hesperidin is estimated as 0.81 ±0.14 mg/g Amanatsu(C. aurantium) and 4.86 ± 1.3 1 mg/g Kijitsu (immature orange).Hesperidin possesses significant anti-inflammatory and analgesic effects (23, 24). The anti-inflammatory activity of hesperidin may berelated partially to inhibition of prostaglandin biosynthesis (24). The7-j3-rutinoside of the flavone diosmetin, diosmin (3'-5,7-trihydroxy4'-methoxyflavone-7-rutinoside, Fig. 2) is used for the treatment ofhuman diseases such as chronic venous insufficiency (25). Diosmininhibits the production of oxygen radicals in vivo and in vitro (26) and

246

Chemoprevention of 4-Nitroquinoline 1-Oxide-induced Oral Carcinogenesis in Ratsby Flavonoids Diosmin and Hesperidin, Each Alone and in Combination'

Takuji Tanaka,2 Hiroki Makita, Masami Ohnishi, Hideki Mori, Kumiko Satoh, Akira Hara, Takashi Sumida,Keisou Fukutani, Tsukasa Tanaka, and Hiroshi Ogawa

First Department of Pathology. Gifu Universits School of Medicine. 40 Tsukasa-machi. Gifu City 500 (Ta. T., H. Ma., M. 0.. H. Mo.J: Department of Biochemistry. GiftsPharmaceutical University, Gifu City 502 (K. S., A. HI: Ehime Citrus Research Institute. Inc., 478 Anjoji, Matsuyama City 791 (T. S.. K. Fl. and Ehinse-ken Fruit GrowerCooperative Association, Matsuyaya City 79/ (l's. T., H. 0.1, Japan

ABSTRACT

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p 1-

I

INHIBITION OF ORAL CANCER BY DIOSMIN AND HESPERIDIN

(2) Hesperidin

Fig. 1. Chemical structures of test compounds diosmin (top) and hesperidin (bottom).

01II910 II32wks I

Group 1

Groups 2-4

Groups 5-7@

Groups 8-1 0 I

Group 11 I3 The thbreviations used are: 4-NQO. 4-nitroquinoline 1-oxide; BrclUrd, 5-bromode

oxyuiidine; AgNORS. silver-stained nucleolar organizer regions protein; ODC. ornithinedecarboxylase;4-HAQO,4-hydroxyaminoquinoline1-oxide.

Fig. 2. Experimental protocol. I, 4-NQO, 20 ppm in drinking water. @,diosmin andhesperidin.alone(1000ppm)or in combination(900ppmdiosminand 100ppmhesperidin) in diet. 0. basal diet and tap water.

247

MATERIALS AND METHODS

Animals, Diets, and Carcinogen. Male F344 rats, 4 weeks old, werepurchased from Japan SLC, Inc. (Hamamatsu City, Japan). After 2 weeks ofquarantine, they were transferred to the holding room under controlled conditions at a temperature of 23 ±2°C(SD), 50 ±10% humidity, and a 12 hlight/dark cycle and randomized into experimental and control groups. Theywere housed three or four to a wire cage. Powdered CE-2 (CLEA Japan, Inc.,Tokyo, Japan) was used as a basal diet during the experiment. CE-2 (50.4%crude carbohydrate, 24.8% crude protein, 4.6% crude fat, 7.2% ash, 4.2%crude cellulose, and 8.8% water) did not contain diosmin and hesperidin.

. ocH@ 4-NQO (CAS, 56-57-5; 98% pure) was obtained from Wako Pure Chemicals,

Inc. (Osaka, Japan). Diosmin (CAS, 520-27-4; >95% pure) and hesperidin(CAS, 520-26-3; >80% pure)werepurchasedrespectivelyfromSigmaChemical Co. (St. Louis, MO) and Fluka Chemie AG, (Buchs, Switzerland). Experimentaldietsmixedwith testcompoundsatadoseof 1000ppmwerepreparedweekly. The combination regimen contained 900 ppm diosmin and 100 ppm

hesperidin. The 4-NQO solution (20 ppm) was prepared on a weekly basis. The

diets and 4-NQO solution were stored in a cold room (4°C)until used. Theywere freely available.

Experimental Procedures@ The experiment was designed to examine themodifying effects ofdiosmin and hesperidin, singly and in combination, duringthe initiation and postinitiation phases of4-NQO-induced oral tumorigenesis inmale F344 rats (Fig. 2).

A totalof 161 ratswere dividedinto 11 groupsas shown in the tables(seeTables 1—5).At 7 weeks of age, rats in groups 1—7were given 4-NQO (20ppm) in the drinking water for 8 weeks. Groups 2, 3, and 4 were given the dietscontaining 1000 ppm diosmin, 1000 ppm hesperidin, and both compounds(900ppmdiosminand 100ppmhesperidin),respectively,startingat 6 weeksof age until 1 week after the stop of the carcinogen exposure. They were thenswitched to the basal diet and maintained on this diet for 22 weeks. Groups 5,

6, and 7 were fed the diets mixed with diosmin, hespendin, and both chemicals, respectively, starting 1 week after the cessation of 4-NQO treatment andcontinued on these diets for 22 weeks. Groups 8—10 were fed the diets

containing test compounds alone during the experiment. Group I I was giventhe basal diet and tap water throughout the experiment and served as anuntreated control.

All rats were carefully inspected daily and consumption of the drinkingwater containing 4-NQO or the diets mixed with test compounds was recorded

Rhamnoglucosyl—@

cL g (1) Diosmin

RhamnoglucosyL

reduces the inflammatory reaction by affecting the synthesis of prostaglandins (prostaglandins E@and F2a) and thromboxane (thromboxane B2; Ref. 27). Diosmetin is present 608 mg/kg Daphne pseudomezerewn (28), which is used for the treatment of chronic skin diseaseandrheumatoidarthritis.Diosminandhesperidinareinhibitorsof thenonenzymatic lipid peroxidation induced by either ascorbic acid orferrous sulfate (29). These compounds either exist in the form ofisolated aglycons or are bound to a sugar to form glycosides. Amicronized complex of 90% diosmin + 10% hesperidin (Daflon 500mg) was launched commercially in France in 1971 for the treatmentof venous insufficiency of the lower limb and of acute symptoms ofhemorrhoids. Its pharmacological characteristics are now well established (30).

Oral lesions produced by 4-NQO3 are comparable to human lesions, because many ulcerated and endophytic tongue tumors anddysplasia develop when 4-NQO in the drinking water is given to ratsor 4-NQO is applied topically to the oral mucosa (31, 32). Because ofeasy accessibility to examination and follow-up of the lesions in theoral cavity, the oral cavity is one of the excellent target organs forexperimental chemoprevention studies (32). Using a 4-NQO-inducedrat oral carcinogenesis model, inhibitory and chemopreventive effectsof several natural products were found in a series in which wesearched for nonnutritive cancer chemopreventive agents in humandiets (32—38).In our previous study, the chemopreventive effect ofhesperidin at a dose level of 500 ppm was not so remarkable whencompared with those of protocatechuic acid, curcumin, and @-carotene (18). Hesperidin, hesperetin, diosmin, and diosmetin have antimutagenic (39—41)and/or antipromotional (42) properties. However,there were no studieson the modifying effect of diosminon in vivoexperimental carcinogenesis. Recent studies showed inhibitory effectof diosmetin (and diosmin) on the amine re-uptake system (43). Thesefindings led us to investigate the modifying effects of diosmin andhesperidin on experimental carcinogenesis.

In the present study, possible inhibitory effects of dietary exposureof diosmin and hesperidin, each alone or in combination (diosmin:hesperidin ratio, 9:1), during the initiation and postinitiation stages of4-NQO-induced oral carcinogenesis were investigated in male F344rats. Also, the effects of these compounds on the expression ofproliferation biomarkers, such as tissue polyamine levels, BrdUrdlabeling index, and AgNORs number were assessed to clarify underlying mechanism(s) of modification.

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Table I Body. liver. and relative liver weights ofrats fed diosmin (DI0) and/or Isesperidin (HPD) during or after 4-NQOexposureGroup

No.TreatmentNo. of ratsBody weight (g)Liver weight (g)Relative liver weight(%)I4-NQO

aloneI 7347 ±I7―1 1.6 ±I . “3.34 ±0.27@2

34

56784-NQO

+ 1000 ppm DIO4-NQO + 1000 ppm HPD4-NQO + 900 ppm DIO and 100 ppm HPD4-NQO —@1000 ppm DlO4-NQO —@1000 ppm HPD4-NQO —s900 ppm DIO and 100 ppm HPD1000 ppm DIO19

19152020208348

±20330 ±15―334 ±5―335 ±19―330 ±20―348 ±25349 ±2012.1

±1.511.7 ±1.211.6 ±2.012.4 ± 1.911.9 ±1.813.4 ±l.9'@12.1± @.?3.48

±0.353.55 ±0.353.49 ±0.503.70 ±0.59'S3.61 ±0.47'3.85 ±0.50's3.48±0.46―9

101000ppm HPD

900 ppm DIO and 100 ppm HPD7 8362±10

338 ±17―13.9±2.6

13.1 ±1.83.83±0.69

3.88±0.49IINo treatment8362 ±2414.5 ±2.43.99 ±0.47

INHIBITIONOF ORAL CANCER BY DIOSMINAND HESPERIDIN

to estimate intake of chemicals. The experiment was terminated at 32 weeks,and all animals were sacrificed by decapitation to evaluate the frequencies ofpreneoplastic and neoplastic lesions in the oral cavity. At necropsy, all organs,especially the oral cavity, were examined grossly, and all organs except for the

tonguewerefixed in 10%bufferedformalin.Tongueswerecut approximatelyinto two halves; one portion was used for a polyamine assay and the other forhistopathology and cell proliferation counts. The tongues used for these measurements mostly did not contain tumorous lesions. For histopathological contIrmation, tissues and gross lesions were fixed in 10% buffered formalin,

embedded in paraffin blocks, and processed by the conventional histologicalmethods using H&E stain. Epithelial lesions (hyperplasia, dysplasia, andneoplasia) in the oral cavity were diagnosed according to the criteria describedby Bánóczyand Csiba (44) and Kramer et a!. (45).

Polyamine Levels of Tongue Tissue. The polyamines in the oral cavitytissues were measured by the method of Koide et a!. (46). The results obtained

were confirmed to be correlated well with those by high-performance liquidchromatography. At sacrifice, one-half of the tongues of all rats were collected,and the amounts of diamine, spermine, and spermidine were determined by anenzymatic differential assay.

Determination of Proliferative Activity in the Tongue Epithelium byAgNORs Enumeration and BrdUrd-labeling Index. To assess the proliferative activity of squamous epithelium of the tongue, the number of AgNORsper nucleus and BrdUrd-labeling index of all animals were quantified accord

ing to the methods described previously (47). For measurement of BrdUrd

incorporated nuclei, the animals were given an i.p. injection of 50 mg/kg bodyweight BrdUrd (Sigma Chemical Co.) I h prior to killing. The tongue was

removed and cut into two. One-half was used for the polyamine assay, and theotherwasfixed in 10%bufferedformalin for histopathology,AgNORscounting, and BrdUrd-labeling index. Three serial sections (3 p.m in thickness) were

made in each tongue half after it was embedded in paraffin. On one section, aone-step silver colloid method for AgNORs staining (47) was carried out, andcomputer-assisted image analysis quantification using the image analysis systern SPICCA II (Japan Abionics Co., Tokyo, Japan) with a Olympus BH-2microscope (Olympus Optical Ind. Co., Ltd., Tokyo, Japan) and a colorcharged-coupled device camera (Hamamatsu Photonics Co., Hamarnatsu City,

Japan) was performed on 100 nuclei of interphase cells from nonlesional areas(38). Another section was used for immunohistochernical detection of BrdUrd

incorporation using an immunohistochemical analysis kit (Amersham Corp.,Little Chalfont, United Kingdom). The labeling indices of BrdUrd (percentages) were calculated by counting for labeled nuclei of 100cells from each ratunder X400 magnification. The remaining section was used for histopathological diagnosis.

Statistical Analysis. Statistical analysis on the incidence of lesions was

performed using Fisher's exact probability test or @,and the data includingbody weight, liver weight, polyamine assay, AgNORs enumeration, and

BrdUrd-labeling index were compared by Student's t-test. The results wereconsidered statistically significant if the P value was 0.05 or less.

a Mean ±SD.b Significantly different from group I I by Student's t test (P < 0.05).(. Significantly different from group I I by Student's t test (P < 0.001).

d Significantly different from group I by Student's t test (P < 0.01).e Significantly different from group 1 by Student's t test (P < 0.05).‘@Significantlydifferent from group I by Student's t test (P < 0.005).S Significantly different from group I by Student's : test (P < 0.001).

248

RESULTS

General Observations. Rats in groups 1—10tolerated well the oraladministration of 4-NQO, diosmin, and/or hesperidin. There were nosignificant differences on a mean intake of 4-NQO/day/rat amongseven groups (groups 1—7:0.214-0.230 mg). The mean body andliver weights at the end of the study are indicated in Table 1. The

mean body weights of rats in groups 3 (4-NQO + hesperidin), 5(4-NQO —4diosmin), and 6 (4-NQO —@hesperidin) were significantlylower than that of group 1 (P < 0.01). The mean body weight of ratsin group 10 (combined regimen of diosmin and hesperidin alone) wassignificantly smaller than that of group I 1 (untreated control;P < 0.05). The mean liver weights of animals in groups 1 (4-NQOalone) and 8 (diosmin alone) were significantly smaller than that ofgroup I l(P < 0.05). The average liver weights of animals in group 7(4-NQO —@diosmin and hesperidin) was significantly greater thanthat of group l(P < 0.005). The average percentage weights (g liverweightll00 g body weight) of groups I and 8 were significantlysmaller than that of group 11 (P < 0.001 and P < 0.05), and thevalues of groups 5—7were significantly larger than that of group 1

(P < 0.05 and P < 0.001).Incidences of Tumors and Preneoplastic Lesions. In the present

study, endophytic or exophytic tumors occurred only in the dorsal siteof the tongue of rats in groups 1—7,the former being microscopically

well-differentiated squamous cell carcinoma and the latter, squamouscell papilloma. The incidences of tongue neoplasms (squamous cell

papilloma and carcinoma) in each group are shown in Table 2. Ingroup I (4-NQO alone), the incidences of tongue squamous cellcarcinoma and squamous cell papilloma were 65% ( 1 1 of I 7 rats) and

47% (8 of 17 rats), respectively. On the other hand, only a few ratsgiven test compounds during 4-NQO administration or those fed dietsmixed with test chemicals after 4-NQO exposure possessed tongueneoplasms [papilloma: 5% (groups 2, 3, and 7), 13% (group 4), and10% (groups 5 and 6); carcinoma: 21% (group 2), 16% (group 3),20% (group 4), 15% (groups 5 and 7), and 25% (group 6)1. Theseincidences were significantly smaller than those of group 1 (P < 0.01,P < 0.05, and P < 0.005). No such neoplasms developed in theanimals of groups 8—II.

In addition to these neoplasms, a number of hyperplasia and dysplasia that are considered to be preneoplastic lesions for oral cancerwere present in the tongue of rats in groups 1—7,but not in rats ofgroups 8—I1. The incidences of such lesions are also listed in Table2. Hyperplasia could be classified into two categories (simple andpapillary) and three types of dysplasia (mild, moderate, and severe)according to the degree of atypism present (Table 3). As for squamous

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Table 2 incidence of tongue neoplasms and pleneoplastic lesions of rats feddiosmin (DIO) and/orhesperidin (HPD) during or after 4-NQOexposureGroup

No. Treatment No. of ratsHyperplasia (%)Dysplasia (%)Papilloma (%)Squamouscell

carcinoma(%)I

4-NQO alone 172 4-NQO + 1000ppm DIO 193 4-NQO + 1000 ppm HPD 194 4-NQO + 900 ppm DIO and 100 ppm HPD 155 4-NQO —s1000 ppm DIO 206 4-NQO —@1000 ppm HPD 207 4-NQO —*900 ppm DIO and 100 ppm HPD 208 l000ppmDIO 89 1000ppm HPD 7

I0 900 ppm DIO and I00 ppm HPD 81 1 No treatment 817

(100)9 (47)a

12(63)b10(67)―16(80)12(60)'14 (70)d0(0)0 (0)0 (0)0 (0)17

(100)10 (53)―10(53t'9 (f,@)b

13(65)@'10(50)―13 (65)b

0(0)0 (0)0 (0)0 (0)8

(47,@1 (5)I (5)―2 (13)d2 (l0)'@2 (10)dI (5)C0(0)0 (0)0 (0)0 (0)I

I (65)4 (21 )“3 (l6f3 (20)d3 (l5)@5 (25)d3 (15)'0(0)0(0)0(0)0(0)a

Significanfly different from group I by Fisher's exact probability test (P < 0.001).

b Significantly different from group I by Fisher's exact probability test (P < 0.01).

C Significantly different from group I by Fisher's exact probability test (P < 0.005).

d Significantly different from group I by Fisher's exact probability test (P < 0.05).

Table 3 Incidence ofpreneoplastic lesions of rats fed diosmin (DIO) and/or hesperidin (HPD) duringor after4-NQOexposureGroup

No. Treatment No. of ratsHyperplasia

(%)

Simple PapillaryDysplasia

(%)MildModerateSevereI

4-NQOalone 172 4-NQO + 1000 ppm DIO 193 4-NQO + 1000 ppm HPD 194 4-NQO + 900 ppm DIG and 100 ppm HPD 155 4-NQO —s1000 ppm DIO 206 4-NQO ‘-s1000 ppm HPD 207 4-NQO —s900 ppm DIO and 100 ppm HPD 208 l000ppmDIO 89 l000ppmHPD 7

10 900ppmDIOand lOOppmHPD 8I I No treatment 817(100)

10(59)9 (47)― 2 (II)“

11 (58)― 5 (26)10(67)' 6 (40)16(80) 4 (20)'11(55)― 4 (20)'13 (65)d 6 (30)0 00 00 00 00(0)

3 ( 16)5 (26)'3 (20)0 (0)2 ( 10)3 ( I5)00008(47)

4 (2 1)4 (21)3 (20)6 (30)6 (30)5 (25)000012(71)

2 ( I I )“2(11?3 (20)―8 (40)2 (10)―6 (30)'000

0a

Significantly different from group I by Fisher's exact probability test (P < 0.005).

b Significantly different from group 1 by Fisher's exact probability test (P < 0.001).C Significantly different from group 1 by Fisher's exact probability test (P < 0.05).

d Significantly different from group 1 by Fisher's exact probability test (P < 0.01).

INHIBITION OF ORAL CANCER BY DIOSMIN AND HESPERIDIN

cell hyperplasia, the incidence in group 1 was 100% (100% withsimple hyperplasia and 59% with papillary hyperplasia). In rats ingroups 2—7,such incidences were smaller than in group 1, withstatistical differences between group I and group 2, 3, 4, 6, and 7(P < 0.05—P< 0.001). The incidence of dysplasia of group I was also100% (0% with mild dysplasia, 47% with moderate dysplasia, and71 % with severe dysplasia). The frequencies of dysplasia in groups2—7(50—65%)were significantly lower than that of group 1 (P < 0.01or P < 0.001). The incidences of severe dysplasia in rats of groups 2,3, 4, 6, and 7 were significantly smaller than in group 1 (P < 0.05—P < 0.001).

Polyamine Levels. The results of the polyamine assay of tonguemucosa are indicated in Table 4. Total polyamine (diamine + spermidine + spermine) and spermine levels in rats of group 1 weresignificantly greater than those of group I I (P < 0.05). The meanamounts of total polyamine in groups 2—7were significantly smallerthan that of group 1 (P < 0.05 or P < 0.01). Similarly, averagespermidine levels in groups 3, 5, 6, and 7 and mean spermine contentsin groups 3—6were significantly lower than those of group 1(P < 0.05—P< 0.001). Those values in groups 8 and 9 were almostcomparable to those in group 11.

Enumeration of AgNORS Number and BrdUrd-labeled Cells.The results of morphometric analysis of AgNORs and BrdUrd-labeling indices in the nonlesional squamous epithelium are summarized inTable 5. The mean number of AgNORs/nucleus and BrdUrd-labelingindex in the nonlesional tongue epithelium exposed to 4-NQO alone(group I) were significantly larger than those of the untreated control(group 11; P < 0.01). Dietary administration of two test chemicalssingly or in combination during initiation or postinitiation (groups2—7)significantly decreased those values (P < 0.05—P< 0.001). The

average numbers of AgNORs/nucleus and BrdUrd-labeling indices ingroup 8—lOswere almost similar to those of group 11.

DISCUSSION

The results in the present study demonstrated that dietary diosminand hesperidin, each alone or in combination, administered duringeither the initiation or postinitiation phase, effectively suppressed4-NQO-induced oral carcinogenesis as revealed by reduction in theincidences of neoplasms and preneoplasms in the tongue. The combination of diosmin with a low level of hesperidin caused a similarinhibitory effect to that of each single agent. Feeding of the dietsmixed with these compounds also suppressed the polyamine levelsand cell proliferation biomarkers' expression. This is the first reportdemonstrating the cancer-inhibitory effects of diosmin and diosmincombined with hesperidin.

Suppressing effect of hesperidin in the present study confirmed ourprevious findings (18), in which rats were p.o. administered at a levelof 500 ppm hesperidin in diet during the initiation or postinitiationphase of 4-NQO-initiated oral carcinogenesis, and a significant reduction in the tongue cancer incidence was found when fed during theinitiation stage. The results of previous and present studies indicatethat dietary administration of 1000 ppm hesperidin could effectivelyinhibit two different stages of oral tumorigenesis induced by 4-NQO.This might suggest the presence of the threshold effect of hesperidinon carcinogenesis. Treatment with the combination of 900 ppm diosmm and 100 ppm hesperidin was also efficacious, but the combinationof diosmin and hesperidin did not show any synergetic effect, suggesting that the combination of two flavonoids did not produceadditional benefit. However, this is quite of interest, because this

249

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Table 4 Polyaminelevels of oral macosa of rats in eachgroupGroup

No. TreatmentNo.of rats

examinedPolyamine

levels (mean± SD, nmol/mgtissue)TotalDiamineSpermidineSpermine1

4-NQO alone2 4-NQO + DIO (1000ppm)17 193.51

±0.31―3.35 ±0.10―0.05

±0.050.07 ±0.1 11.73

±0.241.67 ±0.291.74

±0.17°1.61 ±0.203

4-NQO + HPD (1000 ppm)4 4-NQO + DIO (900 ppm) and HPD (100 ppm)5 4-NQO-'+DIO(lOOOppm)6 4-NQO —@HPD (1000ppm)7 4-NQO —.DIO (900 ppm) and HPD (100 ppm)19

152020202.92

±0.75c3.05 ±081b3.11 ±0.38'3.10±068b3.18 ±0,57―0.05

±0.100.04 ±0.1 10.06±0.070.09 ±0.330.10 ±oW1.38

±0.41c1.49 ±0.451.53±0.22―1.50±038b1.47 ±0.29'1.48

±0,38―1.52 ±0.39―153÷018d

1.51 ±0.43―1.60 ±0.278

DIO (1000 ppm)83.00 ±0.250.07 ±0.071.43 ±0.241.50 ±0.129HPD(1000ppm)73.09 ±0.170.03 ±0.091.46 ±0.221 .61 ±0.2510DIO (900 ppm) and HPD (100 ppm)83.07 ±0.410.02 ±0.041.55 ±0.361.51 ±0.0811

Notreatment83.07±0.500.04±0.061.49±0.431.54±0.13a

Significantly different from group I 1 by Student's t test (P < 0.05).b Significantly different from group 1 by Student's t test (P <0.05).C

Significantly different from group 1 by Student's t test (P < 0.01).

d Significantly different from group 1 by Student's t test (P < 0.001).

Table 5 Number ofAgNORs and BrdU-labelingindex of nonlesional area of tongue squamousepitheliumGroup

No. TreatmentNo. of AgNORslnucleus°

3.33±l.20@―@(l7)2.51±114d(19)2.25 ±l.l9―(l9)2.06 ±o.85e (15)2.22 ±l.07―(20)2.17 ±0.91(20)2.13±l.00―(20)2.10 ±0.22 (8)2.08 ±0.20(7)2.05±0.31(8)2.07 ±0.22 (8)BrdU-labeing

index (%Y'

10.95 ±2,41c(5)7.26±2,l4'@(5)7.16 ±I.92―(S)7.45±1.59―(5)7.56 ±2,l3―(5)7.17 ±2.68―(S)7.11±218d(5)5.97 ±1.06(4)5.70 ±1.10(4)5.98±1.03(4)5.49 ±1.14(4)

INHlB@ON OF ORAL CANCER BY DIO5MIN AND HESPERIDIN

combination regimen is similar to a micronized flavonoid complex(Daflon), which is used for the treatment of chronic venous insufficiency and related disorders in Europe (30). This drug is known tohave several pharmacological properties, including inhibition of prostanoids biosynthesis, anti-inflammation, and protection against freeradicals (48). Because the safety of Daflon (49) and the efficacy of thecombined regimen against carcinogenesis (1 1, 50) are known, and thedrug is already in use for other disorders (48, 49), it is of interest tostudy the relationship between the use of Daflon and the risk of cancerdevelopment in an epidemiological context,

Although a protective effect of the consumption of vegetables andfruits on various types of cancer has been found (6, 7), epidemiological studies on the effects of flavonoids on human health have notbeen carried out because of a lack of accurate data on flavonoidcontent of common foods and intake in humans. A recent crosscultural correlation study suggested that average flavonoid intake maycontribute partly to differences in coronary heart disease mortalityacrosspopulationbutdidnotassociateit withlung,gastrointestinal,orall-cause cancer mortality incidence in a male cohort followed for 5years (14). Unfortunately, it is difficult to evaluate the results of thestudy because of the modest size and short follow-up period of thestudy (51). In this study, we have observed the evidence of tumorinhibiting effects of two dietary flavonoids for doses of 1000 ppm inthe diet. It is considered that humans ingest 1 g of flavonoids daily(12); although this is overestimated, it represents approximately

50,000 ppm in the diet. Thus, the effective doses used are likely occurin normal diets.

Several mechanisms by which diosmin and hesperidin exert theiranticarcinogenesis property in oral tumorigenesis could be considered.Cell proliferation is suggested to play an important role in multistage

carcinogenesis, including oral tumorigenesis (32, 33, 52—54).In thisstudy, diosmin and hesperidin inhibited polyamine levels and cellproliferation induced by 4-NQO. These results were comparable to

our earlier experiments demonstrating the chemopreventive efficacyof several natural antioxidants (18, 34, 36—38,55) and the syntheticcompounds butylated hydroxytoluene, butylated hydroxyanisole, andD,L-a-difluoromethylomithine (56, 57). In rodent and human oralcarcinogenesis, increased polyamine levels and/or ODC activity essential for cellular proliferation were reported (58). ODC, a ratelimiting enzyme in polyamine biosynthesis, has been correlated withthe rate of DNA synthesis and cell proliferation in several tissues (58),and an important role for ODC in tumor promotion has been indicatedin various carcinogenesis models (58). It is also known that agents thatinhibit ODC activity or polyamine levels are effective tumor inhibitom (32, 33, 52, 57—59).Thus, the suppressing effects of diosmin andhesperidin in the present study might be due to lowered cell proliferation by feeding of these chemicals. Other possible mechanisms arealso considered. As for 4-NQO-induced carcinogenesis, metabolicactivation of4-NQO by DT-diaphorase [NAD(P)H:dehydrogenase] toconvert a proximate carcinogen 4-HAQO in several organs, includingliver, lung, and stomach (60), adduct formation of 4-HAQO withDNA (N2-guanine, C8-guanine, and N6-adenine adducts; Ref. 61),and accessof 4-NQO and/or 4-HAQO to the target tissuesare necessary. Unlike other chemical carcinogens, 4-NQO does not requiremetabolic activation by cytochrome P450 enzymes but by DT-diaphorase (phase H enzyme) to exert its carcinogenic potency. It was

reported that certain flavonoids used for anticoagulants could inhibitDT-diaphorase activity (62, 63), suggesting the possibility that diosmm and hesperidin may modulate (inhibit) the activity of DT-diaphoruse. The water-soluble carcinogen 4-NQO also produces intracellular

I 4-NQO alone2 4-NQO + 1000 ppm DIO3 4-NQO+ 1000ppmHPD4 4-NQO + 900 ppm DIO and 100 ppm HPDS 4-NQO-@l000ppmDIO6 4-NQO--@l000ppmHPD7 4-NQO -@900 ppm DIO and 100 ppm HPD8 1000 ppm DIO9 1000ppm HPD10 900 ppm DIO and 100ppm HPD

__________i_i_ Notreatmenta Numbera in parentheses are numbers of rats examined.

M@ ±SD.C Significantly different from group I I by Student's t test (P < 01).

d Significantly different from group I by Student's t test (P < 05).e Significantly different from group I by Student's t test (P < 001).

‘SignificantlydifferentfromgroupI by Student'st test (P < 01).250

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16. Mukhtar, H., Das, M., Khan, W. A., Wang, Z. Y., Bik, D. P., and Bickers, D. R.Exceptional activity of tannic acid among naturally occurring plant phenols inprotecting against 7,l2-dimethylbenz(a)anthracene-, benzo(a)pyrene-, 3-methylcholanthrene-, and N-methyl-N-nitrosourea-induced skin tumongenesis in mice. CancerRes.,48:2361—2365,1988.

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7,l2-dimethylbenz(a)anthracene- and N-nitrosourea-induced rat mammary cancer bydietary flavonol quercetin. Cancer Res., 48: 5754—5788, 1988.

18. Tanaka, T., Makita, H., Ohnishi, M., Hirose, Y., Wang, A., Mon. H., Satoh, K., Hara,A., and Ogawa, H. Chemoprevention of 4-nitroquinoline 1-oxide-inducedoral carcinogenesis by dietary curcumin and hesperidin: comparison with the protective effectof f3-carotene.Cancer Res., 54: 4653—4659,1994.

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24. Da Silva Emim, J. A., Oliviera, A. B., and Lapa, A. J. Pharmacological evaluation ofthe anti-inflammatory activity of a citrus bioflavonoid, hesperidin, and the isoflavonoids, duartin and claussequinone, in rats and mice. J. Pharm. Pharmacol., 46:118—122,1994.

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vivo and in vitro study. Arzneim.-Forsch., 39: 882—885,1989.27. Damon, P., Flandre, 0., Michel, F., Perdrix, L., Labrid, C., and Crastes de Paulet, A.

Effect of chronic treatment with a purified flavonoid fraction on inflammatorygranuloma in the rat. Study of prostaglandin E2 and F2a and thromboxane B2 releaseandhistologicalchanges.Arzneim.-Forsch.,37: 1149—1153,1987.

28. Konishi, T., Wads, S., and Kiyosawa, S. Constituents of leaves of Daphne pseudome@ereuna(in Japanese). Yakugaku Zasshi, 113: 670—675, 1993.

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30. Labrid, C. Pharmacologic properties of Daflon 500 mg. Angiology, 45: 524—530,I994.

31. Gerson, S. J. Oral cancer. Crit. Rev. Oral Biol. Med., 1: 153—166.1990.32. Tanaka, T. Chemoprevention of oral carcinogenesis. Oral Oncol. Eur. J. Cancer, 31B:

3—15,1995.33. Tanaka, T. Cancer chemoprevention by natural products (Review). Oncol. Rep.. I:

1139—1155,1994.34. Tanaka, 1., Kojima, T., Morishita, Y.. and Mon, H. Inhibitory effects of the natural

products indole-3-carbinol and sinigrin during initiation and promotion phases of4-nitroquinoline I-oxide-induced rat tongue carcinogenesis. Jpn. J. Cancer Res., 83:835—842, 1992.

35. Tanaka, T., Kojima, T., Kawamon, T., and Mon. H. Chemoprevention of digestiveorgans carcinogenesis by natural product protocatechuic acid. Cancer (Phila.), 75:1433—1439,1995.

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251

oxidative stress (64). Therefore, the mechanism(s) by which diosminor hesperidin feeding during the initiation stage suppressed tongueneoplasms might be its blocking capability of one or more of such

processes. Also, diosmetin or diosmin might be conjugated by aspecific isoform of UDP-glucuronyltransferase using liver microsomal preparations (65).

The products and intermediates (hydroxyeicosatetraenoic acids andprostaglandin E2) of the lipoxygenase and cyclo-oxygenase pathwayshave been implicated in tumor promotion (66, 67). Moreover, apositive correlation exists between the levels of 8-(S)-hydroxyeicosatetraenoic acid and degree of inflammation, hyperproliferation, clastogenicity, and tumor development in two-stage mouse skin carcinogenesis (68). Diosmin and hesperidin could affect the arachidonic acidpathway (lipoxygenase and cyclo-oxygenase; Refs. 27, 69, and 70).Previously, we reported that nonsteroidal anti-inflammatory agents,

indomethacin and piroxicam, which are potent inhibitors of arachidonic acid cascade, are effective for inhibiting oral carcinogenesisinduced by 4-NQO (71). Thus, the results in the current study giveadditional evidence that some arachidonic cascade and/or ODC inhibitors cause the inhibition of 4-NQO-induced neoplasms. Reactiveoxygen species produced also play a role in several stages of carcinogenesis (72).

Recent research suggests new biological functions of certain flavonoids. These include inhibition of protein kinase C (73) and protooncogene expression (74), antiproliferative activity of human breastcancer cell line (75), enhancement of gap-junctional intercellularcommunication (76), induction of cellular differentiation and G2-Marrest in rat neuronal cells (77), and reversion of transformed phenotypes of v-Ha-ras NIH 3T3 cells (78). Therefore, these novel functionsof the flavonoids may also contribute their antitumor effects.

Morse and Stoner (79) suggested that a chemopreventive agentshould have five qualities: (a) few or no untoward side effects; (b)high efficacy; (c) capability of oral administration; (d) a knownmechanism of action, and (e) low cost. The compounds discussed heremay fulfill these criteria, except for (4 Although additional studieson dose-dependent efficacy and the mechanistic basis of inhibitionshould be done, the results described here indicate that diosmin andhesperidin might be candidates for a chemopreventive agent againstoral carcinogenesis.

ACKNOWLEDGMENTS

We thank Kyoko Takahashi, Tomomi Hirose, Chikako Usui, Suzuyo Satoh,and Keiko Nakanishi for their excellent technical assistance and KazumasaSatoh for animal care.

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