Objectives: We have previously reported that an antioxidant, auraptene (AUR), isolated from citrus fruit effectively inhibits chemically induced carcinogenesis in digestive tracts, such as the oral cavity, esophagus and large bowel. In this study, we investigated the modifying effects of dietary supplementation with AUR on N,N-diethylnitrosamine (DEN)-initiated hepatocarcinogenesis in male F344 rats in two different experiments to determine whether the compound exerts a cancer-chemopreventive action in other organs. Methods: In the first experiment, animals were fed diets containing AUR at dose levels of 100 and 500 ppm for 7 weeks 1 week before, during, and 1 week after the start of liver carcinogenesis induced by DEN (40 ppm in drinking water for 5 weeks) to predict the modulatory effect on hepatocarcinogenesis. After 7 weeks, the numbers of hepatocellular enzyme-altered foci (EAF; cm2) which stained positive for the placental form of glutathione S-transferase (GST-P) and transforming growth factor (TGF)-α were determined on immunohistochemically stained sections. In the second experiment conducted to confirm the findings, animals subjected to DEN treatment were fed AUR-containing diets (100 and 500 ppm) during either the initiation stage (‘initiation’ feeding for 7 weeks) or post-initiation phase (‘post-initiation’ feeding for 25 weeks) of DEN-induced hepatocarcinogenesis. Results: In the first experiment, feeding with AUR at both doses during DEN exposure decreased the mean numbers of GST-P-positive and TGF-α-positive EAF/cm2, and the reduction in the number of TGF-α-positive EAF by feeding 500 ppm AUR was statistically significant (p < 0.005). In the second experiment, the ‘initiation’ feeding with 500 ppm AUR significantly inhibited the incidence (33 vs. 83%, p = 0.000511) and multiplicity (0.67 ± 1.09 vs. 1.96 ± 1.85, p < 0.005) of liver cell carcinoma. Also, the ‘post-initiation’ feeding with AUR at both doses significantly reduced the development of hepatocellular carcinoma (100 ppm: incidence, 15%, p = 0.000006; multiplicity: 0.25 ± 0.64, p < 0.001; 500 ppm: incidence, 11%, p = 0.000002; multiplicity, 0.26 ± 0.81, p < 0.001). In addition, AUR feeding reduced cell proliferation and the apoptotic index in liver cell neoplasms. Conclusions: The results suggest that the citrus antioxidant AUR is a potential chemopreventive agent against DEN-induced hepatocarcinogenesis in rats.

1.
Kiyosawa K, Tanaka E: Characteristics of hepatocellular carcinoma in Japan. Oncology 2002;62(suppl 1):5–7.
2.
Okuno M, Kojima S, Moriwaki H: Chemoprevention of hepatocellular carcinoma: Concept, progress and perspectives. J Gastroenterol Hepatol 2001;16:1329–1335.
3.
Muto Y, Moriwaki H, Ninomiya M, Adachi S, Saito A, Takasaki KT, Tanaka T, Tsurumi K, Okuno M, Tomita E, Nakamura T, Kojima T: Prevention of second primary tumors by an acyclic retinoid, polyprenoic acid, in patients with hepatocellular carcinoma. N Engl J Med 1996;334:1561–1567.
4.
Okita K, Sakaida I, Hino K: Current strategies for chemoprevention of hepatocellular carcinoma. Oncology 2002;62(suppl 1):24–28.
5.
Sakaida I, Matsumura Y, Akiyama S, Hayashi K, Ishige A, Okita K: Herbal medicine Sho-saiko-to (TJ-9) prevents liver fibrosis and enzyme-altered lesions in rat liver cirrhosis induced by a choline-deficient L-amino acid-defined diet. J Hepatol 1998;29:298–306.
6.
Eaton EA, Walle UK, Lewis AJ, Hudson T, Wilson AA, Walle T: Flavonoids, potent inhibitors of the human P-form phenolsulfotransferase. Potential role in drug metabolism and chemoprevention. Drug Metab Dispos 1996;24:232–237.
7.
Ghazali RA, Waring RH: The effects of flavonoids on human phenolsulphotransferases: Potential in drug metabolism and chemoprevention. Life Sci 1999;65:1625–1632.
8.
Siess MH, Le Bon AM, Canivenc-Lavier MC, Suschetet M: Mechanisms involved in the chemoprevention of flavonoids. Biofactors 2000;12:193–199.
9.
Ogawa K, Kawasaki A, Yoshida T, Nesumi H, Nakano M, Ikoma Y, Yano M: Evaluation of auraptene content in citrus fruits and their products. J Agric Food Chem 2000;48:1763–1769.
10.
Murakami A, Kuki W, Takahashi Y, Yonei H, Nakamura Y, Ohto Y, Ohigashi H, Koshimizu K: Auraptene, a citrus coumarin, inhibits 12-O-tetradecanoylphorbol-13-acetate-induced tumor promotion in ICR mouse skin, possibly through suppression of superoxide generation in leukocytes. Jpn J Cancer Res 1997;88:443–452.
11.
Tanaka T, Kawabata K, Kakumoto M, Matsunaga K, Mori H, Murakami A, Kuki W, Takahashi Y, Yonei H, Satoh K, Hara A, Maeda M, Ota T, Odashima S, Koshimizu K, Ohigashi H: Chemoprevention of 4-nitroquinoline 1-oxide-induced oral carcinogenesis by citrus auraptene in rats. Carcinogenesis 1998;19:425–431.
12.
Kawabata K, Tanaka T, Yamamoto T, Hara A, Murakami A, Koshimizu K, Ohigashi H, Stoner GD, Mori H: Suppression of N-nitrosomethylbenzylamine-induced rat esophageal tumorigenesis by dietary feeding of auraptene. J Exp Clin Cancer Res 2000;19:45–52.
13.
Tanaka T, Kawabata K, Kakumoto M, Makita H, Hara A, Mori H, Satoh K, Hara A, Murakami A, Kuki W, Takahashi Y, Yonei H, Koshimizu K, Ohigashi H: Citrus auraptene inhibits chemically induced colonic aberrant crypt foci in male F344 rats. Carcinogenesis 1997;18:2155–2161.
14.
Tanaka T, Kawabata K, Kakumoto M, Hara A, Murakami A, Kuki W, Takahashi Y, Yonei H, Maeda M, Ota T, Odashima S, Yamane T, Koshimizu K, Ohigashi H: Citrus auraptene exerts dose-dependent chemopreventive activity in rat large bowel tumorigenesis: The inhibition correlates with suppression of cell proliferation and lipid peroxidation and with induction of phase II drug-metabolizing enzymes. Cancer Res 1998;58:2550–2556.
15.
Tanaka T, Kohno H, Murakami M, Kagami S, El-Bayoumy K: Suppressing effects of dietary supplementation of the organoselenium 1,4-phenylenebis(methylene)selenocyanate and the Citrus antioxidant auraptene on lung metastasis of melanoma cells in mice. Cancer Res 2000;60:3713–3716.
16.
Umeda T, Hino O: Molecular aspects of human hepatocarcinogenesis mediated by inflammation: From hypercarcinogenic state to normo- or hypocarcinogenic state. Oncology 2002;62(suppl 1):38–42.
17.
Chuang SE, Cheng AL, Lin JK, Kuo ML: Inhibition by curcumin of diethylnitrosamine-induced hepatic hyperplasia, inflammation, cellular gene products and cell-cycle-related proteins in rats. Food Chem Toxicol 2000;38:991–995.
18.
Ito N, Tsuda H, Tatematsu M, Inoue T, Tagawa Y, Aoki T, Uwagawa S, Kagawa M, Ogiso T, Masui T, Imaida K, Fukushima S, Asamoto M: Enhancing effect of various hepatocarcinogens on induction of preneoplastic glutathione S-transferase placental form positive foci in rats – An approach for a new medium-term bioassay system. Carcinogenesis 1988;9:387–394.
19.
Dragan Y, Teeguarden J, Campbell H, Hsia S, Pitot H: The quantitation of altered hepatic foci during multistage hepatocarcinogenesis in the rat: Transforming growth factor α expression as a marker for the stage of progression. Cancer Lett 1995;93:73–83.
20.
Kaufmann WK, Zhang Y, Kaufman DG: Association between expression of transforming growth factor-α and progression of hepatocellular foci to neoplasms. Carcinogenesis 1992;13:1481–1483.
21.
Steinmetz KL, Klaunig JE: Transforming growth factor-α in carcinogen-induced F344 rat hepatic foci. Toxicol Appl Pharmacol 1996;140:131–145.
22.
Chuang SE, Kuo ML, Hsu CH, Chen CR, Lin JK, Lai GM, Hsieh CY, Cheng AL: Curcumin-containing diet inhibits diethylnitrosamine-induced murine hepatocarcinogenesis. Carcinogenesis 2000;21:331–335.
23.
Risio M: Methodological aspects of using immunohistochemical cell proliferation biomarkers in colorectal carcinoma chemoprevention. J Cell Biochem 1994;19(suppl):61–67.
24.
Galati G, Teng S, Moridani MY, Chan TS, O’Brien PJ: Cancer chemoprevention and apoptosis mechanisms induced by dietary polyphenolics. Drug Metabol Drug Interact 2000;17:11–49.
25.
Oridate N, Lotan D, Mitchell MF, Hong WK, Lotan R: Inhibition of proliferation and induction of apoptosis in cervical carcinoma cells by retinoids: Implications for chemoprevention. J Cell Biochem 1995;23(suppl): 80–86.
26.
Hosoda A, Miyake Y, Nomura E, Mizuno K, Taniguchi H: Facile preparation of ethyl 3- (4-geranyloxy-3-methoxyphenyl)-2-propenoate (EGMP) and related compounds: For the practical use of EGMP as a cancer chemopreventive agent. ITE Lett Batt New Tech Med 2001;2:659–662.
27.
Nakatani N, Yamada Y, Fuwa H: 7-Geranyloxycoumarin from juice of hassaku (Citrus hassaku) and antimicrobial effects of related coumarins. Agric Biol Chem 1987;51:419–423.
28.
Harada T, Maronpot RR, Morris RW, Boorman GA: Observations on altered hepatocellular foci in National Toxicology Program two-year carcinogenicity studies in rats. Toxicol Pathol 1989;17:690–706.
29.
Harada T, Maronpot RR, Morris RW, Stitzel KA, Boorman GA: Morphological and stereological characterization of hepatic foci of cellular alteration in control Fischer 344 rats. Toxicol Pathol 1989;17:579–593.
30.
Mohr U: 10. Digestive system; in Mohr U (ed): International Classification of Rodent Tumours. I: The Rat. Lyon, IARC Press, 1997, IARC Scientific Publications No. 122, pp 65–83.
31.
Kawaii S, Tomono Y, Ogawa K, Sugiura M, Yano M, Yoshizawa Y: The antiproliferative effect of coumarins on several cancer cell lines. Anticancer Res 2001;21:917–923.
32.
Kawaii S, Tomono Y, Ogawa K, Sugiura M, Yano M, Yoshizawa Y, Ito C, Furukawa H: Antiproliferative effect of isopentenylated coumarins on several cancer cell lines. Anticancer Res 2001;21:1905–1911.
33.
Nair RV, Fisher EP, Safe SH, Cortez C, Harvey RG, DiGiovanni J: Novel coumarins as potential anticarcinogenic agents. Carcinogenesis 1991;12:65–69.
34.
Lu HQ, Schmitz U, Niggemann B, Zanker KS: Computer-assisted molecular design for the determination of structure-activity relationships for chemopreventive agents. Ann NY Acad Sci 1997;833:147–153.
35.
Mori H, Sugie S, Yoshimi N, Hara A, Tanaka T: Control of cell proliferation in cancer prevention. Mutat Res 1999;428:291–298.
36.
Mori H, Niwa K, Zheng Q, Yamada Y, Sakata K, Yoshimi N: Cell proliferation in cancer prevention: Effects of preventive agents on estrogen-related endometrial carcinogenesis model and on an in vitro model in human colorectal cells. Mutat Res 2001;480–481:201–207.
37.
Kelly VP, Ellis EM, Manson MM, Chanas SA, Moffat GJ, McLeod R, Judah DJ, Neal GE, Hayes JD: Chemoprevention of aflatoxin B1 hepatocarcinogenesis by coumarin, a natural benzopyrone that is a potent inducer of aflatoxin B1-aldehyde reductase, the glutathione S-transferase A5 and P1 subunits, and NAD(P)H:quinone oxidoreductase in rat liver. Cancer Res 2000;60:957–969.
38.
Kojima T, Tanaka T, Kawamori T, Hara A, Mori H: Chemopreventive effects of dietary D,L-α-difluoromethylornithine, an ornithine decarboxylase inhibitor, on initiation and postinitiation stages of diethylnitrosamine-induced rat hepatocarcinogenesis. Cancer Res 1993;53:3903–3907.
39.
Rahman KM, Sugie S, Okamoto K, Watanabe T, Tanaka T, Mori H: Modulating effects of diets high in omega-3 and omega-6 fatty acids in initiation and postinitiation stages of diethylnitrosamine-induced hepatocarcinogenesis in rats. Jpn J Cancer Res 1999;90:31–39.
40.
Jung SH, Lee YS, Lee S, Lim SS, Kim YS, Ohuchi K, Shin KH: Anti-angiogenic and anti-tumor activities of isoflavonoids from the rhizomes of Belamcanda chinensis. Planta Med 2003;69:617–622.
41.
Kojima-Yuasa A, Hua JJ, Kennedy DO, Matsui-Yuasa I: Green tea extract inhibits angiogenesis of human umbilical vein endothelial cells through reduction of expression of VEGF receptors. Life Sci 2003;73:1299–1313.
42.
Maeda H, Akaike T: Nitric oxide and oxygen radicals in infection, inflammation, and cancer. Biochemistry (Mosc) 1998;63:854–865.
43.
Murakami A, Wada K, Ueda N, Sasaki K, Haga M, Kuki W, Takahashi Y, Yonei H, Koshimizu K, Ohigashi H: In vitro absorption and metabolism of a citrus chemopreventive agent, auraptene, and its modifying effects on xenobiotic enzyme activities in mouse livers. Nutr Cancer 2000;36:191–199.
44.
Gupta S, Sundarrajan M, Rao K: Tumor promotion by metanil yellow and malachite green during rat hepatocarcinogenesis is associated with dysregulated expression of cell cycle regulatory proteins. Teratog Carcinog Mutagen 2003;23(suppl 1):301–312.
45.
Murakami A, Nakamura Y, Tanaka T, Kawabata K, Takahashi D, Koshimizu K, Ohigashi H: Suppression by citrus auraptene of phorbol ester- and endotoxin-induced inflammatory responses: Role of attenuation of leukocyte activation. Carcinogenesis 2000;21:1843–1850.
46.
Boitier E, Merad-Boudia M, Guguen-Guillouzo C, Defer N, Ceballos-Picot I, Leroux JP, Marsac C: Impairment of the mitochondrial respiratory chain activity in diethylnitrosamine-induced rat hepatomas: Possible involvement of oxygen free radicals. Cancer Res 1995;55:3028–3035.
47.
Thirunavukkarasu C, Sakthisekaran D: Effect of selenium on N-nitrosodiethylamine-induced multistage hepatocarcinogenesis with reference to lipid peroxidation and enzymic antioxidants. Cell Biochem Funct 2001;19:27–35.
48.
Shiota G, Maeta Y, Mukoyama T, Yanagidani A, Udagawa A, Oyama K, Yashima K, Kishimoto Y, Nakai Y, Miura T, Ito H, Murawaki Y, Kawasaki H: Effects of Sho-Saiko-to on hepatocarcinogenesis and 8-hydroxy-2′-deoxyguanosine formation. Hepatology 2002;35:1125–1133.
49.
Sarkar A, Bishayee A, Chatterjee M: β-Carotene prevents lipid peroxidation and red blood cell membrane protein damage in experimental hepatocarcinogenesis. Cancer Biochem Biophys 1995;15:111–125.
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