Dietary Protocatechuic Acid During Initiation and Postinitiation Phases1

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[CANCER RESEARCH54, 2359â€”2365,May1, 19941 Chemoprevention of 4-.Nitroquinoline 1-Oxide-induced Oral Carcinogenesis by Dietary Protocatechuic Acid during Initiation and Postinitiation Phases1 Takuji Tanaka,2 Toshihiko Kawamori, Masami Ohnishi, Kiyohisa Okamoto, Hideki Mon, and Aidra Hara First Department of Pathology, Gift University School of Medicine, 40 Tsukasa-machi, Gifu City 500 fT. T, T. K, M. 0., K 0., H. M./, and Department of Biochemistry, Gifu Pharmaceutical University, Gifu City 502 [A. H.J, Japan

ABSTRACT 10) and contaminants such as nitrosamines (1 1), polycyclic hydrocar bons (12), or urethans (13) are also suggested to be causative factors. The modifying effects of three doses of dietary protocatechuic acid Although Japan has one of the lowest incidences of oral and pharyn (PCA) given the initiation and postinitiation phases of oral carcinogenesis Initiated with 4-nitroquinoline 1-oxide (4-NQO) were investigated in male geal cancer in the world, the number of patients with these malignan F344rats At 6 weeksof age,rats weredividedinto experimentaland cies has been increasing, accounting for 4,900 new cases and 1,825 control groups and fed the diet containing PCA at various doses ofO glkg deaths in 1980 and 11,000 new cases and 2,607 deaths in 1990 (14). diet (basal diet alone), 0.5 g/kg diet (500 ppm), 1 g/kg diet (1000 ppm), and It has been reported that patients with oral cancer have an increased 2 g/kg diet (2000 ppm). At 7 weeks of age, all animals except PCA alone incidence of second primary tumors of the oral cavity (15, 16). In fact, and control groups were given 4-NQO (20 ppm) in the drinking water for patients with early lesions have a high cure rate of their primary tumor 8 weeksto induceoral cancer.Sevendaysafter the 4-NQOexposure, but go on to succumb to the second malignancy. Approximately groups of animals fed the PCA diets were Switched to the basal diet and 10â€”40%of such patients will develop second primary tumors, a rate continued on this diet until the end ofthe study. Starting 1 week after the end of 4-NQO exposure, the groups given 4-NQO and a basal diet were that is related to continued exposure to carcinogens or promoters (17). switched to the diets containing PCA and maintained on these diets for 22 This is considered to be the result of a diffuse mucosal abnormality, weeks. The other groups consisted of rats given 2000 ppm PCA alone or often referred as â€œfieldcancerizationâ€•resulting from carcinogen ex untreated rats. All animals were necropsied at the termination of the posure (18). Oral cancer is a multifocal disease and experimental experiment (week 32). The Incidences of tongue neoplasms and preneo studies indicated that such lesions develop through a multistage proc plastic lesions, polyamine levels in the tongue tissue, and cell proliferation ess (19, 20). Because of easy accessibility to examine and follow-up activity estimated by bromodeoxyurldine-labeling mdcx and by morpho of the lesions in the oral cavity, the oral cavity is one of the excellent metric analysis of silver-stained nucleolar organizer regions' protein were target organs for experimental chemoprevention studies. compared among the groups. Feeding ofPCA at all doses during initiation or postiaitiatioa phase significantly decreased the development of tongue The term â€œcancerchemopreventionâ€• refers to the prevention of neoplasms (squamous cell papilloma and carcinoma) and preneoplasia cancer by intervention using nontoxic synthetic chemicals or chemi (hyperplasia and dysplasia) (P < 0.05). There were no such lesions in rats cals from natural substances before malignancy (21). A number of treated with 2000 ppm PCA alone or those in an untreated control group. micronutrients (22), macronutrients (23), and nonnutrients (24) have Dietary administration of PCA also caused significant decreases in the been reported as inhibiting or chemopreventive agents in chemical labeling index of bromodeoxyuridlne and the number and area of silver carcinogenesis in rodents. In our search for chemopreventive agents stained nucleolar organizer regions per cell nucleus, known as cell prolif for cancer development using several experimental animal models, eration indices, ofthe tongue squamous epithelium (P < 0.05). In addition, some natural products with antioxidative property from edible plants, PCA exposure during either Initiation or postinitiation phase decreased herbs, or fruits have appeared to exert tumor-inhibiting effects in polyamine levels in the oral mucosa (P < 0.05). These results cleady digestive organs (25, 26). Experimental studies on cancer chemopre Indicated that PCA inhibited rat oral carcinogenesis in both initiation and postinitiation phases, when administered in these respective phases to vention in the oral cavity have mainly been conducted using hamster gether with or following treatment with 4-NQO, and such Inhibition might buccal pouch carcinogenesis model with a carcinogen, 7,12-dimeth be related to suppression of cell proliferation by PCA. ylbenz[a]anthracene, and chemopreventives have been limited to some vitamins (27â€”29).Our previous works revealed inhibitory and chemopreventive effects of several natural and synthetic compounds INTRODUCTION on 4-NQO3-induced oral carcinogenesis (30â€”33).These include in Oral cancer is a common neoplasm in some regions in the world. dole-3-carbinol, sinigrin, plant phenolics (caffeic, ellagic, chloro The highest rates occur in developing countries, particularly in the genic, and ferulic acids), synthetic antioxidants (butylated hydroxy Southern Asia (India, Sri Lanka, South Vietnam, Papua New Guinea, toluene and butylated hydroxyanisole), nonsteroidal antiinflammatory the Philippines, Hong Kong, or Taiwan), China, and parts of Brazil drugs (indomethacin and piroxicam), and disulfiram. An ODC inhib (1â€”4).In the Southern Asian countries, up to 25% of all cancers are itor, DL-a-difluoromethylormthine, also exerted remarkable suppress present in the oral cavity, and tobacco and betel nut chewing are ing effects on 4-NQO-induced rat carcinogenesis (34). Some of these responsible for this malignancy (5). In Europe, France has the highest agents have been shown to inhibit cell proliferation (32, 33), which incidence of oral and pharyngeal cancer. This variation in incidence is plays an important role in the multistage carcinogenesis (35â€”37),in related to exposure to known etiological agents (6). Epidemiological the target organs including oral cavity (38), and such property is data provide strong support for exogenous factors such as tobacco and suggested to be one of the mechanisms of their tumor-inhibitory alcohol use as being major causative agents (7, 8). Viral infection (9, potential (39). More recently, we have proposed a new chemopreven tive agent, PCA, in liver and colon carcinogenesis (40, 41). Epidemi ological observations suggest a statistically significant inverse asso Received 10/23/93; accepted 3/3/94. Thecostsof publicationofthisarticleweredefrayedinpartby thepaymentofpage ciation between oral cancer and the consumption of fruits and/or charges. This article must therefore be hereby marked advertisement in accordance with vegetables (42, 43). PCA is one of the constituents in edible plants, 18 U.S.C. Section 1734 solely to indicate this fact.

I This work was supported in part by a grant from the Ministry of Health and Welfare and a Grants-in-Aid for Scientific Research (No. 05671568) from the Ministry of Edu 3Theabbreviationsusedare:4-NQO,4-nitroquinolineI-oxide;ODC,ornithinede cation, Science and Culture of Japan, and a 1993 grant from the Sagawa Foundation for carboxylase; PCA, protocatechuic acid (3,4-dihydmxybenzoic acid); BrdUrd, 5-bromo Promotion of Cancer Research in Japan. deoxyuridine; AgNORS,silver-stained nucleolar organizer regions protein; MTD, maxi 2 To whom requests for reprints should be addressed. mum tolerated dose; 4-HAQO, 4-hydroxyaminoquinoline 1-oxide. 2359

fruits, and vegetables. Recently, PCA from the rind of Citrus reticu 0 1 9 10 32wks lata BLANCO has been reported to have a strong antioxidative II II I property (44). Therefore, a modifying (possibly inhibiting) effect of Group PCA on oral carcinogenesis might be suspected. iI@@////@/@ I In the present study, a possible inhibitory effect of dietary exposure of PCA during initiation and postinitiation stage on 4-NQO-induced oral carcinogenesis was investigated in male F344 rats to obtain @ 2â€”4 I further evidence that PCA possesses cancer chemopreventive prop erty. Dose-related efficacy of PCA, the effects of PCA on polyamine levels in the oral cavity, and the alteration of proliferative potential of @ 5..7 I the oral mucosa by measuring BrdUrd labeling index and AgNORS number were also assessed to clarify underlying mechanism(s), if @@@@@@ PCA possessed an inhibitory effect on oral tumorigenesis. 8 L :::. :.. â€¢:.: :.: ..::: :1::j

MATERIALS AND METHODS 9 I I Animals, Diets, and Carcinogen. Male F344 rats, 4 weeks old, were purchased from Japan SLC, Inc. (Hamamatsu City, Japan). After quarantine for 2 weeks, a total of 193 rats (6 weeks of age) for determining MiD and the : 4-NOO;20 ppm indrinkingwater modifying effect of PCA were transferred to the holding room under controlled : PCA;500. 1000or 200 ppm indiet condition at 23 Â± 2Â°C (SD) temperature, 50 Â± 10% humidity, and a 12-h light/dark cycle and randomized into experimental and control groups. They I I : Basalduetandtapwater were housed three or four to a wire cage. Powdered CE-2 (CLEA Japan, Inc., Tokyo, Japan) was used as basal diet during the experiment. It contained Fig. 1. Experimental protocol. 50.4% crude carbohydrate, 24.8% crude protein, 4.6% crude fat, 7.2% ash, 4.2% crude cellulose, and 8.8% water but did not plant phenolics present from plant foods. 4-NQO (CAS: 56-57-5, 98% pure) was obtained from Wako Pure 2000 ppm PCA alone during the experiment. Group 9 was given the basal diet Chemical Ind. Co., Ltd., Osaka, Japan. PCA (CAS: 99-50-3, 97% pure) was and tap water throughout the experiment and served as an untreated control. purchased from Aldrich Chemical Co., Milwaukee, WI. Experimental diets All rats were carefully observed daily and consumption of the drinking mixed with PCA at various concentrations and 4-NQO solution (20 ppm) were water containing 4-NQO or the diets mixed with PCA was recorded to estimate prepared on a weekly basis and stored in a cold room (4Â°C)until used. The intake of chemicals. The experiment was terminated at 32 weeks and all stability test of PCA in the diet at room temperature was not done since this animals were sacrificed by decapitation between 9 a.m. and noon, avoiding chemical is quite stable as was confirmed in the previous studies (the recovery artificial changes from polyamine assay, to evaluate the incidences of preneo of PCA was more than 98% under the bioassay feeding conditions) (40, 41). plastic and neoplastic lesions in the oral cavity. At necropsy, all organs, Determination of M'FD of PCA Before the start of the chemopreventive especially the oral cavity, were examined grossly and all organs except for experiment, the MTD of PCA was determined. At 5 weeks of age, groups of tongue were fixed in 10% buffered formalin. Tongues were cut approximately male F344 rats (10 rats/group) were fed the basal diet (CE-2) containing into two halves; one portion was used for polyamine assays and the other was various levels of PCA (0, 0.3, 0.6, 1.2, 2.5, 5.0, and 10 g/kg diet). Body used for histopathology and cell proliferation counts. For histopathological weights were recorded weekly for 6 weeks. All animals were inspected daily confirmation, tissues and gross lesions were fixed in 10% buffered formalin, for any symptoms of toxicity. At the end of 6 weeks, all animals were embedded in paraffin blocks, and processed by the conventional histological sacrificed, and several organs (stomach, intestine, liver, kidney, and lung) were methods using hematoxylin and eosin stain. Epitheial lesions (hyperplasia, examined grossly and histopathologically for any abnormalities. Also, clinical dysplasia, and neoplasm) in the oral cavity were diagnosed according to the chemistry was performed for aspartate transaminase, alanine transaminase, criteria described by BÃ¡nÃ³czyandCsiba (45) and WHO (46). alkaline phosphatase, lactic dehydrogenase, and glucose-6-phosphatase. The Polyamine Levels of Tongue Tissue. The polyamines in the oral cavity MTD is defined as the highest dose that causes no more than a 10% weight tissues were measured by means of a new enzymatic method developed by decrement as compared to the control diet group and does not produce Koide et aL (47). At sacrifice, one-half of the tongues of all rats were collected mortality or any external signs of toxicity that would be predicted to shorten and the amounts of diamine, spermine, and spermidine were determined by an the natural life span of the animal. The MiD value of PCA was over 10 g/kg enzymatic differential assay. The results obtained by this method correlated PCA diet (10,000 ppm),since no clinical signs andhistopathologicalchanges well with those obtained by high performance liquid chromatography. for toxicity, weight gain retardation, and abnormalities of chemical profiles Determination of Proliferative Activity in the Tongue Epithelium by were noted in rats fed at various levels (data not shown). AgNORsEnumeration and BrdUrd-labelingIndeL To assessthe prolifer Experimental Procedure for Chemopreventive Efficacy of PCA. The ative activity of squamous epithelium of the tongue, the number and area of experiment was designed to examine the modifying effect of three doses of AgNORS per nucleus and BrdUrd-labeling index of randomly selected five PCA during the initiationand postinitiationphases of 4-NQO-inducedoral animals from each experimental group were quantified according to the meth carcinogenesis in male F344 rats (Fig. 1). Based on the results of MTD study ods described previously (19). For measurement of BrdUrd-incorporated nu and those in previous studies (40, 41), the highest dose of PCA used in the dci, the animals were given an i.p. injection of 50 mg/kg body weight BrdUrd present study was 2 g PCA/kg diet (<20% MTh). (Sigma Chemical Co., Ltd., St. Louis, MO) 1 h prior to killing. The tongue was A total of 173 rats were divided into nine groups as shown in the tables. At removed and cut into two. One half was used for polyamine assay and the other 7 weeks of age, rats in groups 1 through 7 were given 4-NQO (20 ppm) in the was fixed in 10% buffered formalin for histopathology, AgNORs counting, drinking water for 8 weeks. Groups 2, 3, and 4 were, respectively, given the and BrdUrd-labeling index. Three serial sections (3 mm thick) were made after diets containing 0.5 g PCAIkg diet (500 ppm), 1 g PCA/kg diet (1000 ppm), embedding in paraffin. On the one section, a one-step silver colloid method for and 2 g PCA/kg diet (2000 ppm), starting at 6 weeks of age until 1 week after AgNORs staining (19) was carried out and computer-assisted image analysis the stop of the carcinogen exposure. They were then switched to the basal diet quantification using an image analysis system SPICCA II(Japan Abionics Co., and maintained on this diet for 22 weeks. Groups 5, 6, and 7 were, respectively, Tokyo, Japan) with a Olympus BH-2 microscope (Olympus Optical Ind. Co., fed the diets mixed with PCA at concentrations of 0.5 g/kg diet, 1 g/kg diet, Ltd., Tokyo, Japan) and a color-charged coupled device camera (Hamamatsu and 2 g/kg diet, starting 1 week after the cessation of 4-NQO treatment and Photonics Co., Hamamatsu City, Japan) was performed on 100 nuclei of continued on these diets for 22 weeks. Group 8 was fed the diet containing interphase cells from nonlesional areas (25). The other section was used for 2360

Table1 Body,liver,andgroupNo. relativeliverweights in each WIRelativeGroupTreatmentexamined(g)(g)liver of ratsBody wtLiver wt1 Â± 23â€• Â± 2.5â€• Â± 0.57' O.58'@34-NQO24-NQO 4-NQO + 500 ppm PCA19 20360 356 Â± 3915.7 13.9 Â± 2.9â€•4.37 3.87 Â± 0.6144-NQO + 1000ppmPCA19369 Â± 4615.2 Â± 3.24.08 Â± 0.455 + 2000 ppm PCA21359 Â± 2815.8 Â± 2.24.40 Â± -@ 500ppmPCA Â± 26 Â± 1.7 Â± 0.24 6 4-NQO â€”â€˜1000ppm PCA 19 327 Â± 3ff 13.1 Â± il 4.01 Â± 0.43â€• 0.50â€•8200074-NQO 4-NQO â€”â€˜2000ppm PCA21 20355 335 Â± 38e15.1 13.4 Â± 2.Y4.24 3.99 Â± 0.359No ppm PCA16344 Â± 2713.3 Â± 1.83.87 Â± treatment18348 Â±2113.6 Â± 1.53.91 Â± 0.32 a Mean Â± SD. b@cSignificantly different from group 9 by Student's t test. bp < o.oo4

Cp < dâ€”gSignificantly different from group 1 by Student's t test. dP< 0.05. e p < 0.02. fP< 0.001. g p < o.oo4. immunohistochemical detection of BrdUrd incorporation using an immuno tumors between group 1 and groups 2â€”7(P< 0.05-P < 0.001). Besides histochemical analysis kit (Amersham, United Kingdom). The labeling indices these neoplasms, a number of preneoplastic lesions (hyperplasia and of BrdUrd (by percentage) were calculated by counting for labeled nuclei of dysplasia) were present in the tongue of rats in groups 1â€”7.The 100 cells from each rat at X400. The remaining section was used for his incidences of such lesions are indicated in Table 3. The incidences of topathological diagnosis. rats with hyperplasia and dysplasia in group I were 89 and 58%, Statistical Analysis. Statistical analysis on the incidence of lesions was respectively. In rats fed PCA together with or after 4-NQO exposure performed using Fisher's exact probability test, and the data of measurements of body weight, liver weight, polyamine assay, AgNORs enumeration, and (groups 2â€”7),the incidences of these lesions were smaller compared BrdUrd-labeling index were compared by Student's t test for paired samples or with those of group 1 (4-NQO alone). Significant differences in the a two-sample t-test with Welch's correction. The results were considered incidence of preneoplasia were obtained between group 1 and groups statistically significant if P was 0.05 or less. 2 through 7 (hyperplasia, P < 0.05â€”P< 0.001), and between group 1 and group 3, 4, 6, or 7 (dysplasia, P < 0.05â€”P < 0.001) Also, RESULTS significant differences in the combined incidence of hyperplasia and dysplasia were obtained between group 1 and groups 2 through 7 General Observations. Animals in groups 1â€”8tolerated well the (P < 0.05-P < 0.001). Dysplasia present in the present study could be p.o. administration of 4-NQO and/or PCA. There were no significant classified into three degrees (mild, moderate, and severe dysplasia) differences on total intake of 4-NQO/rat among seven groups (groups and the frequencies of various grades of dysplasia are shown in 1â€”7;data not shown). Total intakes of PCA/rat in groups 2â€”4or Table 4. The incidences of moderate and severe dysplasia in groups groups 5â€”7were increased in proportion as the dose increased (data 2â€”7were lower than those of group 1 and in rats of group 4 there were not shown). The mean body and liver weights at the end of the study no moderate and severe dysplasia (group 1 versus group 4: P < 0.05). are indicated in Table 1. The mean body weights of rats in all groups Also no moderate dysplasia were found in rats of groups 6 and 7 given 4-NQO and/or PCA except groups 6 and 7 were comparable (group 1 versus group 6 or 7: P < 0.05). On the other hand, the with that of a control group (group 9). The mean body weights of rats incidences of mild dysplasia in groups 5 and 6 were higher than that in groups 6 and 7 were significantly lower than that of group 1 of group 1 with no statistical differences. (P < 0.001 and P < 0.02). The mean liver weight of animals in group Polyamine Levels The results of polyamine assay of tongue epi 1 wassignificantlyhigherthanthat of group9 (P < 0.004).The thelium are summarized in Table 5. Total polyamine and spermine average liver weights of groups 2, 6, and 7 were significantly smaller levels in rats of group 1 were significantly greater than those of group than that of group 1 (P < 0.05, P < 0.001, and P < 0.004) and the 9 (P < 0.01). Total polyamine, spermidine, and spermine levels in values were similar to that of group 9. The mean relative liver weight (g/100 g body weight) in group 1 was significantly larger than that groupGroupTreatmentNo.Table 2 incidence of tongue neoplasms in rats of each of group 9 (P < 0.01). The mean relative liver weights in groups 2, 6 tongueneoplasmsTotalof rats with and 7 were significantly lower than that of group 1 (P < 0.02 and of rats P < 0.05). PapillomaCarcinoma1 examinedNo. Incidences of Tumors and Preneoplastic Lesions. In this study, (58y' 3(16) (58) 2 4-NQO + 500 ppm PCA 20 3 (15)â€• 3 (15) tumors occurred only in the oral cavity, especially the tongue. They 0'@4 1 (Sf 34-NOO4-NQO + 1000ppm PCA19 1911 2 (1 1)â€• 2 (1 1)11 were located at the dorsal site of the tongue and mostly were well +2000ppmPCA 2(10) differentiated squamous cell carcinoma, microscopically. The mci 5 4-NQO â€”+500ppmPCA 21 4 (19/ 3 (14) 1 (Sf dences of tongue tumors (squamous cell papilloma and carcinoma) in 64-NQO2(11)â€•74-NQO4-NOO â€”@l000ppmPCA21 192(1O)c S(26)@ 4(21)0'@ 2(10)0'@82000ppmPCA160â€”@2O0OppmPCA202(10)' all groups are shown in Table 2. In group 1 (4-NQO alone), the 009No incidences of tongue squamous cell carcinoma and squamous cell treatment180 00 papilloma were 58 and 16%, respectively. Combined incidence of a Numbers in parentheses, percentage. these tumors in this group was 58%. On the other hand, only a few rats bâ€”gSignificantly different from group 1 by Fisher's exact probability lest. bp<001 given PCA during 4-NQO administration (groups 2â€”4)or those fed C p < o.oo@. PCA diets after 4-NQO exposure (groups 5â€”7)possessedtongue â€œP<0.005. ep <0.001. neoplasms. No neoplasms developed in rats of groups 8 and 9. â€˜P<0.02. Statistical analysis revealed significant differences on the incidence of g p < 0.05. 2361

Table3 Incidenceofpreneoplasticlesionsofthetongueofrats in eachgroup AgNORs in group 8 (2000 ppm PCA alone) were almost similar to No. of rats with preneoplastic lesions those of group 9 (untreated control). No. of rats GroupTreatmentexaminedTotalHPâ€•DYS1 DISCUSSION (95)b (89) (58) 2 4-NQO + SOOppmPCA 20 10 (SO)c 12 (@)d 6 (30) The results in the present study indicated that dietary PCA admin 4(21)â€•434-NQO4-NQO + l000ppmPCA19 1918 8 (42f17 11 (58)d11 + 2000ppmPCA (33)e (33)e (Sf istration during the initiation or postinitiation phase effectively sup 5 4-NQO â€”â€˜SOOppmPCA 21 12 (57@f 14 (67) 7 (33) pressed oral carcinogenesis initiated with 4-NQO as revealed by 6 4-NQO â€”â€˜l000ppmPCA 19 10 (53)C 9 (47)f 5 (26)d (1Sf82000ppmPCA160009No74-NQO4-NQO â€”@2000ppmPCA21 207 8 (40f7 7 (35f1 3 reduced the incidences of neoplasms and preneoplasia in the tongue. Such inhibition was more remarkable when rats were given PCA treatment18000 together with 4-NQO. In particular, no malignant tongue neoplasms a @jp squamous cell hyperplasia; DYS, squamous cell dysplasia. developed in rats given 4-NQO together with 1000 or 2000 ppm PCA b Numbers in parentheses, percentage. @1Significantly different from group 1 by Fisher's exact probability test. diet. Also, no malignant tumors occurred in the tongue of rats fed the Cp<0 005 diet mixed with 2000 ppm PCA after 4-NQO exposure. The lowest dp < ep <0.001. dose of PCA used in the present study is almost 4 times larger than fP < 0.01. that consumed by humans, if humans daily comsume 10 g lettuce and/or strawberry that contains 10â€”40mg PCA/100 g. The results in the current study are in agreement with those in our groups 2â€”7were significantly larger than those of group 1, except for earlier works showing a suppressing effect of PCA on chemically spermidine level, between groups 6 and 1 (P < 0.001, P < 0.05, or induced liver and colon carcinogenesis in rats (40, 41). In liver P < 0.01). carcinogenesis, feeding of PCA at 500 and 1000 ppm during initiation AgNORS Enumeration and BrdUrd-labeling Index. The results and postinitiation phases significantly reduced the occurrence of pre of morphometric analysis of AgNORs and BrdUrd-labeling indices in neoplasia (liver cell foci) and hepatocellular neoplasms induced by the squamous epithelium are listed in Table 6. The mean number of diethylnitrosamine in rats (40). In addition, livers of rats fed PCA had AgNORs and BrdUrd-labeling index in the tongue epithelium cx lowered ODC activity compared with those treated with diethylnitro posed to 4-NQO alone (group 1) were significantly larger than those samine alone (40). In azoxymethane-initiated rat colon carcinogene of untreated control (group 9) (P < 0.05 and P < 0.001). Dietary sis, PCA feeding at three dose levels (250, 500, and 1000 ppm) administration of PCA (groups 2â€”7)significantly decreased those significantly inhibited the development of intestinal tumors when fed values (P < 0.05â€”P< 0.001). The mean total areas of AgNORs/ at a dose of 1000 ppm during initiation and fed at levels of 500 and nucleus in groups 2â€”7were smaller than that in group 1, but the 1000 ppm after azoxymethane exposure (41). The inhibitory effect of differences were not significant. The average number and total area of PCA in the present study was more remarkable than that found in liver

Table 4 Incidence of various grades of dysplasia of the tongue

No. of rats with dysplasia GroupModerateSevere1 Treatment examined Total Mild 5(26)4(21)2 4-NQO 19 11(S8)a 2(11) 3(15)2(10)3 4-NQO+ SOOppmPCA 20 6(30) 1(5) 4-NQO+1000ppmPCA 19 4(211â€• 1(5) 2(11) 40â€•S 4-NQO + 2000 ppm PCA 21 1 (5) 1 (5) 0â€•1(5) 2(10)1(5)6 4-NQO--' SOOppmPCA 21 7(33) 4(19) (5)7 4-NQO â€”â€˜1000ppm PCA 19 S (26f 4 (21) 0â€•1 0â€•1(5)a 4-NOO--+l000ppmPCA 20 3(lS)c 2(10) Numbers in parentheses, percentage. bâ€”d Significantly different from group 1 by Fisher's exact probability test. b p

<0.001.dp<001Table

5 Polyamine levels of tongue tissues at the end of theexperimentPolyamine levels (nmol/g protein) Treatment GroupSpermidineSpermine1 (no. of ratsexamined) Total Diamine 4-NQO(19) 3.18 Â±0.4Oâ€•@ 0.16 Â±0.12 1.59 Â±0.28 Â±0.21â€• 2 4-NOO + 500 ppm PCA (20) 2.64 Â±03S'@ 0.11 Â±0.11 1.33 Â±0.19â€• 1.19 Â±0.22â€• 3 4-NQO + 1000 ppm PCA (19) 2.54 Â±0.33c 0.12 Â±0.10 1.28 Â±0@d 1.13 Â±O.21c 4 4-NQO + 2000 ppm PCA (21) 2.59 Â±0,32c 0.12 Â±0.11 1.35 Â±0.22â€• 1.12 Â±0.22'@ S 4-NOO â€”â€˜500ppm PCA (21) 2.70 Â±030c 0.15 Â±0.10 1.37 Â±O.30e 1.19 Â± 6 4-NQO â€”â€˜1000ppm PCA (19) 2.78 Â±0.48e 0.16 Â±0.18 1.41 Â±0.35 1.22 Â±0.22â€• 7O.25'@8 4-NQO â€”*2000 ppm PCA (20) 2.68 Â±0.41'@ 0.14 Â±0.12 1.35 Â±[email protected] 1.20 Â± 0.239 2000 ppm PCA (16) 2.65 Â±0.40 0.10 Â±0.10 1.39 Â±0.391.16 Â± No treatment (18) 2.81 Â±0.28 0.15 Â±0.12 1.43 Â±0.281.23 Â±0.19 â€œMeanÂ±SD. b Significantly different from group 9 by two-sample t test with Welch's correction (P < 0.01). câ€”eSignificantly different from group 1 by two-sample t test with Welch's correction. Cp <0.001. dp<001

e p < o.os. 2362

cellsGroupTreatmentNo. Table 6 Number and total area ofAgNORs and BrdUrd-labeling index of tongue squamous of AgNORs/ area of AgNORs/ (%)1 nucleusTotal nucleus (@.m2)BrdUrd-labeling indices

2 4-NQO + 500 ppm PCA 2.22 Â±0.84â€• 2.88 Â±0.91 9.2 Â±2.8â€• 3 4-NQO + 1000 ppm PCA 2.21 Â±0.61â€• 2.75 Â±1.21 8.9 Â±26d 4 4-NQO + 2000 ppm PCA 2.03 Â±O.92e 2.63 Â±0.65 7.6 Â±2.3k S 4-NQO â€”â€˜500ppm PCA 2.28 Â±oW 2.92 Â±1.20 9.1 Â±36d 6 4-NQO â€”p1000ppm PCA 2.16 Â±O.50e 2.80 Â±0.98 7.3 Â±2.9k 7 4-NQO---'2000ppmPCA 2.11 Â±O.32e 2.71 Â±0.77 6.4Â±3.3k 8 2000 ppm PCA 2.36 Â±0.11 2.60 Â±0.32 6.3 Â±1.4 94-NQO No treatment2.%Â±l.Ol@ 2.38 Â±0.133.14Â±1.26 2.61 Â±0.3011.7Â±3.7' 6.5 Â±1.2 a Mean Â±SD. b.c Significantly different from group 9 by two-sample t test with Welch's correction. bp <

Cp <0.001. â€œ@â€˜Significantlydifferentfrom group 1 by two-sample t test with Welch's correction. dp < ep < fP < 0.001.

and colon carcinogenesis (40, 41), since the highest dose of PCA lung, and stomach to 4-HAQO which is considered to be a proximate (2000 ppm) completely inhibited tongue cancer occurrence when fed carcinogen (59); adduct formation of 4-HAQO with DNA (N-2- during either the initiation or the postinitiation phase. Such tumor guanine, C-8-guanine, and N-6-adenine adducts) (60); and accesses of reducing effect by PCA exposure during either initiation or postini 4-NQO and/or 4-HAQO to the target tissues are necessary. Therefore, tiation stage was observed parallel with decreased ODC activity of the mechanism(s) by which PCA administration during initiation colonic mucosa and AgNORs counts in crypt cell nuclei (41). Thus, stage suppressed tongue neoplasms might be its blocking capability of the results in the current study provide the additional evidences that a one or more of such processes. natural product, PCA, could effectively inhibit tumor development In the present study, the proliferation activity of the tongue without any toxicity and PCA might be a possible cancer chemopre mucosal epithelium was estimated and PCA feeding lowered the ventive agent in the oral cavity in addition to other organs (colon and cell proliferation activity. These results were comparable to our liver). earlier experiments testing the chemopreventive efficacy of several Various types of inhibitors of mutagenesis or carcinogenesis and natural phenolic antioxidants (25, 33) and a synthetic compound their mechanisms have been postulated in available reviews (24, 39, DL-a-difluoromethylornithine (34). In rodent and human oral 48â€”52).Initiation might be inhibited by preventing the formation of carcinogenesis, increased polyamine levels and/or ODC activity that carcinogens from precursors, blocking the metabolic activation of are essential for cellular proliferation (61) were reported (34, 62). carcinogens, increasing the detoxification of carcinogens by increas Cell proliferation is suggested to play an important role in multistage ing the level of the enzymes involved (e.g., glutathione S-transferase), carcinogenesis including oral tumorigenesis (35â€”38).Presently, di interception of carcinogens before their reaction with DNA, stimula etary PCA administration in either the initiation or the postinitiation tion of error-free DNA repair, or suppression of cell proliferation. stage reduced cell proliferation in the tongue epithelium and poly Chemopreventive agents that inhibit such processes have been cate amine levels in the tongue tissue with or without preneoplastic and gorized as â€œblockingagentsâ€•byWattenberg (21, 49). Other types of neoplastic lesions. Indole-3-carbinol and sinigrin, which possess some chemopreventives are â€œsuppressingagentsâ€•that prevent the develop antioxidative property and antitumor activity in oral carcinogenesis, ment of neoplasms after carcinogen exposure (21, 49). The results in also exert their antitumor effects through suppression of cell prolif the current and previous studies may suggest that PCA possesses both eration in the oral squamous epithelium (32). Such an effect of PCA blocking and suppressing properties in carcinogenesis (21, 49), al might also partly contribute to the inhibition of tongue tumorigenesis, though biochemical studies on modifying effects of dietary PCA on especially when PCA diet was fed during the postinitiation phase. An the phase I and phase II enzymes that affect the metabolism, detoxi isomer of PCA, 2,3-dihydroxybenzoic acid diacetate (dipyrocetyl), is fication, and elimination of carcinogens should be done. Such che a known analgesic or antipyretic agent, presumably by inhibiting mopreventives acting as blocking and suppressing agents from envi ronment have been reported. These include an w-3-fatty acid arachidonic metabolism to prostaglandins, leukotrienes, thrombox docosahexaenoic acid (53), benzyl isothiocyanate (54), and 1,4-phe anes, etc., Thus, PCA may act as a tumor antipromoter by inhibition nylenebis(methylene)selenocyanate (55) in colon carcinogenesis, of arachidonic acid metabolism in oral epithelial cells, like other D-limonene in mammary carcinogenesis (21), and garlic or garlic nonsteroidal antiinflammatory agents (31). Ongoing study using two compounds (56). Our series of chemopreventive study in oral carci stage mouse skin carcinogenesis model will clarify such an antipro nogenesis revealed similar properties of indole-3-carbinol and sinigrin moter effect of PCA. (32). Indole-3-carbinol could also suppress the occurrence of sponta Since an important element in the evaluation of the possible role of neous endometrial cancer in female Donryu rats by altering estradiol chemopreventive compounds is the assessment of preclinical toxicity 2-hydroxylation (57). Although the mechanisms by which PCA exert (48), the safety and toxicity of the possible chemopreventives deserve its inhibitory effect on 4-NQO-induced rat oral carcinogenesis remain more attention. The results in this study and those in previous exper to be elucidated, several mechanisms could be considered. The results iments in our laboratory (40, 41) indicated that a dose as low as 500 that PCA feeding during the initiation phase inhibited tongue tumor ppm (<5% MTD) of PCA in diet was found to be effective. Moreover, development may support the hypothesis that some plant phenolics no toxicity was observed in rats fed the diet mixed with PCA at a dose with an antioxidative effect act as blocking agents (52, 58). As for of 10,000 ppm. PCA is a widely distributed phenolic acid. Several 4-NQO-induced carcinogenesis, metabolic activation by DT-diapho fruits and plants like citrus fruit, Illicium anisatum, and Illicium verum rase [NAD(P)H dehydrogenasel in several organs including liver, contain a small amount of PCA. However, very few epidemiological 2363

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1994 American Association for Cancer Research. INHiBITIONOF ORAL CANCERBY PROTOCATECHUICACID studies on the relationship between the intake of foods rich in PCA Symposium Series 507, pp. 326â€”337.Washington,DC: American Chemical Society, 1992. and cancer incidence are available. 26. Yoshinii, N., Wang, A., Morishita, Y., Tanaka, T., Sugie, S., Kawai, K., Yamahara, The significant reduction in the incidence of tongue preneoplastic J., and Moti, H. Modifying effects of fungal and herb metabolites on azoxymethane lesions by PCA feeding may also indicate that PCA could be applied induced intestinal carcinogenesis in rats. Jpn. J. Cancer Res., 83: 1273â€”1278,1992. 27. Trickier, D., and Shklar, G. Prevention by vitamin E of experimental oral carcino for the prevention of second primary neoplasms in the oral cavity, genesis. J. Natl. Cancer Inst., 78: 165â€”169,1987. since such lesions are believed to precede tumor development in the 28. Shklar, G., Schwartz, J., Grau, D., TrickIer, D., and Wallace, K. Inhibition of hamster oral cavity and to account for the development of local recurrence and buccal pouch carcinogenesis by 13-cis-retinoic acid. Oral Surg., 50: 45â€”52,1980. second primary malignancies (18). Additional study on cancer che 29. Suda, D., Schwartz, J., and Shklar, G. Inhibition of experimental oral carcinogenesis by topical 13carotene. Carcinogenesis (Lond.), 7: 71 1â€”715,1986. moprevention of PCA is now under way in our laboratory using a rat 30. Tanaka, T., Iwata, H., Kanai, N., Nishikawa, A., and Mori, H. Inhibitory effect of bladder carcinogenesis model that is also considered to be a â€œfield butylated hydroxytoluene, butylated hydroxyanisole and disulfiram on 4-nitroquino line 1-oxide-induced tongue carcinogenesis in male ACt rats. J. Nutr. Growth Cancer, cancerizationâ€• model. 4: 239â€”248,1987. In summary, dietary administration of PCA during the initiation and 31. Tanaka, T., Nishikawa, A., Moit Y., Morishita, Y., and Mori, H. Inhibitory effects of postinitiation phases significantly inhibited 4-NQO-induced oral car non-steroidal anti-inflammatory drugs, piroxicam and indomethacin on 4-nitroquino line 1-oxide-induced tongue carcinogenesis in male ACI/N rats. Cancer Left., 48: cinogenesis in rats, although the exact mechanisms by which PCA 177â€”182,1989. exerts its chemopreventive property are not known. The available 32. Tanaka, T., Kojima, T., Morishita, Y., and Mon, H. Inhibitory effects of the natural evidences in this study warranted further research on the possible role products indole-3-carbinol and sinigrmnduring initiation and promotion phases of 4-nitroquinoline 1-oxide-induced rat tongue carcinogenesis. Jpn. J. Cancer Res., 83: of PCA in the prevention of cancer development. 835â€”842,1992. 33. Tanaka, T., Kojima, T., Kawamori, T., Wang, A., Suzui, M., Okamoto, K., and Mori, H. Inhibition of 4-nitroquinoline 1-oxide-induced rat tongue carcinogenesis by the naturally occurring plant phenolics caffeic, ellagic, clilorogenic and ferulic acids. REFERENCES Carcinogenesis (Lond.), 14: 1321â€”1325,1993. 34. Tanaka, T., Kojima, T., Hara, A., Sawada, H., and Mon. H. Chemoprevention of oral 1. Dunham, L. J. A geographic study of a relationship between oral cancer and plants. carcinogenesis by DL-a-difluOrOmethylOrnithine,anornithine decarboxylase inhibi Cancer Res., 28: 2369â€”2371, 1968. tor: dose-dependent reduction in 4-nitroquinoline 1-oxide-induced tongue neoplasms 2. Baden, E. Tobacco and oropharyngeal and bronchial cancer. Latest data. Rev. Med. in rats. Cancer Res., 53: 772â€”776,1993. Toulouse, 14: 549â€”560,1968. 35. Cayama, E., Tsuda, H., Sarma, D. S. R., and Farber, E. Initiation of chemical 3. Magrath, I., and Litvak, J. Cancer in developing countries: opportunity and challenge. carcinogenesis requires cell proliferation. Nature (Lond.), 275: 60â€”62,1978. J. Natl. Cancer Inst., 85: 862â€”874,1993. 36. Ames, B. N., and Gold, L S. Chemical carcinogenesis: too many rodent carcinogens. 4. Parkin, D. M., Pisani, P., and Ferlay, J. Estimates of the worldwide incidence of Proc. Natl. Acad. Sci. USA, 87: 7772â€”7776,1990. eighteen major cancers in 1985. Int. J. Cancer, 54: 594-606, 1993. 37. Cohen, S. M., and Ellwein, L. B. Cell proliferation and carcinogenesis. Science S. Muir, C. S., and Kirk, R. Betel, tobacco and cancer of the mouth. Br. J. Cancer, 14: (Washington, DC), 249: 1007â€”1011,1990. 597â€”608,1960. 38. Preston-Martin, S., Pike, M. C., Ross, R. K., Jones, P. A., and Henderson, B. E. 6. Blot, W. J., McLaughlin, J. K., Winn, D. M., Austin, D. F., Greenberg, R. S., Increased cell division as a cause of human cancer. Cancer Res., 50: 7415â€”7421, Preston-Martin, S., Bernstein, L., Schoenberg, J. B., Stemhagen, A., and Fraumeni, J. 1990. E., Jr. Smoking and drinking in relation to oral and pharyngeal cancer. Cancer Res., 39. Tanaka, T. Cancer chemoprevention. Cancer J., 5: 11â€”16,1992. 48: 3282â€”3287,1988. 40. Tanaka, T., Kojima, T., Kawamori, T., Yoshimi, N., and Mori, H. Chemoprevention 7. Sankaranarayanan, R. Oral cancer in India: an epidemiologic and clinical review. Oral of diethylnitrosamine-induced hepatocarcinogenesis by a simple phenolic acid pro Surg. Oral Med. Oral Pathol., 69: 325â€”330,1990. tocatechuic acid in rats. Cancer Res., 53: 2775â€”2779,1993. 8. Smith, C. J. Oral cancer and precancer: background, epidemiology and aetiology. Br. 41. Tanaka, T., Kojima, T., Suzui, M., and Mon. H. Chemoprevention of colon carci Dent. J., 167: 377â€”383,1989. nogenesis by the natural product of a simple phenolic compound, protocatechuic acid: 9. Eskinazi, D. P. Oncogenic potential of sexually transmitted viruses with special suppressing effects on tumor development and biomarkers expression of colon reference to oral cancer. Oral Surg. Oral Med. Oral Pathol., 64: 35â€”40,1987. tumorigenesis. Cancer Res., 53: 3908â€”3913,1993. 10. Cox, M. F., Scully, C., and Maitland, N. Viruses in the etiology of oral carcinoma? 42. Block, G., Patterson, B., and Subar, A. Fruit, vegetables, and cancer prevention: a Examination of the evidence. Br. J. Oral Maxillofac. Surg., 29: 381â€”387,1991. review of the epidemiological evidence. Nutr. Cancer, 18: 1â€”29,1992. 11. Hecht, S. S., and Hoffmann, D. The relevance of tobacco-specific nitrosamines to 43. Hebert, J. R., Landon, J., and Miller, D. R. Consumption of meat and fruit in relation human cancer. Cancer Sun'., 8: 273-294, 1989. to oral and esophageal cancer: a cross-national study. Nutr. Cancer, 19: 169â€”179, 12. Felton, J. S., and Knize, M. G. Heterocyclic amine mutagens/carcinogens in foods. In: 1993. C. S. Cooper and P. L. Grover (cdx.), Handbook of Experimental Pharmacology, pp. 44. Ueno, A. The studies on the constituents and the utilization in Citrus species (in 471â€”502.Berlin: Springer-Verlag, 1990. Japanese). Food & Food Ingredients J., 157: 46â€”53,1993. 13. Ng, S. K. C., Kabat, G. C., and Wynder, E. L. Oral cavity cancer in non-users of 45. BÃ¡ndczy,J., and Csiba, A. Occurrence of epithelial dysplasia in oral leukoplakia. Oral tobacco. J. Natl. Cancer Inst., 85: 743â€”745,1993. Surg., 42: 766â€”774,1976. 14. Health and Welfare Statistics Association. J. Health Welfare Statistics (in Japanese), 46. WHO. WHO Collaborating Center for oral precancerous lesions: definition of leu 40: 29â€”35,1993. koptakia and related lesions: an aid to studies on oral precancer. Oral Surg., 46: 15. Kotwall, C., Razack, M. S., Sako, K., and Rao, U. Multiple primary cancers in 518â€”539,1978. squamous cell cancer of the head and neck. J. Surg. Oncol., 40: 97â€”99,1989. 47. Koide, T., Sakai, S., Kawada, Y., Hara, A., and Sawada, H. Detection of polyamines 16. Lippman, S. M., and Hong, W. K. Second primary tumors in head and neck squamous by a new enzymatic differential assay. (7) Fundamental study on a new enzymatic cellcarcinoma:theovershadowingtreatforpatientswithearly-stagedisease.mt.J. differential assay of tissue polyamines (in Japanese). Acts Urol. Jpn., 36: 1103â€”1108, Radial. Oncol. Biol. Phys., 17: 691â€”694, 1989. 1990. 17. Garewal, H. S. Potential role of @3-carotenein prevention of oral cancer. Am. J. Clin. 48. Malone, W. F. Chemoprevention research. In: E. K. Weisburger (ed), Mechanisms of Nutr., 53: 2945-2975, 1991. Carcinogenesis, Vol. 2, pp. 31â€”42.Dordrecht, the Netherlands: Kiuwere Academic 18. Slaughter, D. P., Southwick, H. W., and Smejkal, W. â€œFieldcancerizationâ€•in oral Press, 1989. stratified squamous epithelium: clinical implications of multicentric origin. Cancer 49. Wattenberg, L. W. Chemoprevention of cancer. Cancer Res., 45: 1â€”8,1985. (Phila.), 5: 963â€”968,1953. So. Fiala, E. S., Reddy, B. S., and Weisburger, J. H. Naturally occurring anticarcinogenic 19. Tanaka, T., Kojima, T., Okumura, A., Yoshimi, N., and Mon, H. Alterations of the substances in food stuffs. Annu. Rev. Nutr., 5: 295â€”321,1985. nucleolar organizer regions during 4-nitroquinoline 1-oxide-induced tongue carcino 51. Wattenberg, L. W. Inhibition of carcinogenesis by minor dietary constituents. Cancer genesis in rats. Carcinogenesis (Lond.), 12: 329â€”333,1991. Res., 52: 2O8Ss-2O91s, 1992. 20. Weinberg, R. Oncogenes, antioncogenes and the molecular basis of multistep carci 52. Tanaka, T. Chemoprevention of huma cancer. Biology and therapy. Crit. Rev. nogenesis. Cancer Res., 49: 3713â€”3721, 1989. Oncol./Hematol., in press, 1994. 21 . Wattenberg, L. w. Chemoprevention of cancer by naturally occurring and synthetic 53. Narisawa, T., Takahashi, M., Niwa, M., Kusaka, H., Yamazaki, Y., Nishizawa, Y., compounds. In: L. W. Wattenberg, M. Lipkin, C. W. Boone, and G. J. Kelloff(eds.), Ozawa, K., Kotsugai, M., Hirano, J., and Isoda, Y. Effect of w-3 polyunsaturated fatty Cancer Chemoprevention, pp. 19â€”39.Boca Raton, FL: CRC Press, 1992. acids in fish oil on carcinogenesis of the large bowel in rats (in Japanese). J. Chin.Exp. 22. Moon, T. E., and Micozzi, M. S., cdx. Nutrition and Cancer Prevention: Investigating Med.,145:911â€”912,1988. the Role of Micronutrients. New York: Marcel Dekker, Inc., 1988. 54. Nayini, J. R., Sugie, S., El-Bayoumy, K., Rao, C. V., Rigotty, J., Sohn, 0-S., and 23. Micozzi, M. S., and Moon, T. E., eds. Macronutrients: Investigating Their Role in Reddy, B. S. Effect of dietary benzylselenocyanate on azoxymethane-induced colon Cancer. New York: Marcel Dekker, Inc., 1992. carcinogenesis in male F344 rats. Nutr. Cancer, 15: 129â€”139,1991. 24. Wattenberg, L W. Inhibition of carcinogenesis by minor anutrient constituents of the 55. Reddy, B. S., Rivenson, A., Kulkarni, N., Upadhyaya, P., and El-Bayoumy, K. diet. Proc. Nutr. Soc., 49: 173â€”183,1990. Chemoprevention of colon carcinogenesis by the synthetic organoselenium com 25. Tanaka, T., Yoshimi, N., Sugie, S., and Mon, H. Protective effects against liver, pound 1,4-phenylenebis(methylene)selenocyanate. Cancer Res., 52: 5635â€”5640, colon, and tongue carcinogenesis by plant phenols. In: M-T. Huang, C-T. Ho, and C. 1992. Y. Lee (eds.), Phenolic Compounds in Food and Their Effects on Health II, ACS 56. Dorant, E., van den Brandt, P. A., Goldbohm, R. A., Hermits, R. J. J., and Sturmans, 2364

F. Garlic and its significance for the prevention of cancer in humans: a critical view. 60. Bailleul, B., Daubersies, P., GaliÃ¨gue-Zouitina, S., and Loucheux-Lefebvre, M-H. Br. J. Cancer, 67: 424â€”429, 1993. Molecular basis of 4-nitroquinoline 1-oxide carcinogenesis. Jpn. J. Cancer Res., 80: 57. Kojima, T., Tanaka, T., and Mori, H. Chemoprevention of spontaneous endometrial 691â€”697,1989. cancer in female Donryu rats by dietary indole-3-carbinol. Cancer Res., 54: 1446â€” 61. Russell, D. H., and Dune, B. G. M. Polyamines as biochemical markers of normal and 1449, 1994. malignant growth. In: D. H. Russell and B. G. M. Dune (eds.), Progress in Cancer 58. Newmark, H. L. A hypothesis for dietary components as blocking agents ofchemical Research and Therapy, Vol. 8, pp. 1â€”178.New York, NY: Raven Press, 1978. carcinogenesis: plant phenolics and pyrrole pigments. Nutr. Cancer, 6: 58â€”70,1984. 62. Dimery, I. W., Nishioka, K., Bruce Grossie, V., Jr., Ota, D. M., Schantz, S. P., Byers, 59. Sugimura, T., Okabe, K., and Endo, K. The metabolism of 4-nitroquinoline 1-oxide. R., Robbins, K. T., and Hong, w. K. Polyamine metabolism in carcinoma of the oral 3. An enzyme catalyzing the conversion of 4-nitroquinoline 1-oxide to 4-hydroxy- cavity compared with adjacent normal oral mucosa. Am. J. Surg., 154: 429â€”433, aminoquinoline 1-oxide in rat liver and hepatoma. Cancer Res., 26: 1717-1721, 1966. 1987.

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Takuji Tanaka, Toshihiko Kawamori, Masami Ohnishi, et al.

Cancer Res 1994;54:2359-2365.

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