- Email: [email protected]
Inhibitory effects of lemon grass (Cymbopogon citratus, Stapf) extract on the early phase of hepatocarcinogenesis after initiation with diethylnitrosamine in male Fischer 344 rats
Cancer Letters 183 (2002) 9–15 www.elsevier.com/locate/canlet
Inhibitory effects of lemon grass (Cymbopogon citratus, Stapf) extract on the early phase of hepatocarcinogenesis after initiation with diethylnitrosamine in male Fischer 344 rats Rawiwan Puatanachokchai a,b, Hideki Kishida a,c,d, Ayumi Denda a, Nao Murata a, Yoichi Konishi a,d, Usanee Vinitketkumnuen b, Dai Nakae e,* a
Department of Oncological Pathology, Cancer Center, Nara Medical University, Kashihara, Nara 634-8521, Japan b Department of Biochemistry, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand c Third Department of Internal Medicine, Nara Medical University, Kashihara, Nara 634-8521, Japan d Free Radical Biology and Aging Research Program, Oklahoma Medical Foundation, Oklahoma City, OK 73104, USA e Department of Pathology, Sasaki Institute, Sasaki Foundation, Chiyoda, Tokyo 101-0062, Japan Received 27 November 2001; received in revised form 18 February 2002; accepted 18 February 2002
Abstract Effects of lemon grass extract (LGE) on hepatocarcinogenesis were examined in male Fischer 344 rats, administered diethylnitrosamine (DEN) at three weekly intraperitoneal doses of 100 mg/kg body weight and partially hepatectomized at the end of week 5. LGE was given at dietary concentrations of 0, 0.2, 0.6 or 1.8% from the end of week 4 for 10 weeks. All rats were sacrificed at the end of week 14. LGE reduced the number of putatively preneoplastic, glutathione S-transferase placental form-positive lesions and the level of oxidative hepatocyte nuclear DNA injury, as assessed in terms of 8-hydroxydeoxyguanosine production. In contrast, LGE did not affect the size of the preneoplastic lesions, hepatocyte proliferative activity, activities of phase II enzymes or hepatocyte extra-nuclear oxidative injury. These results suggest inhibitory effects of LGE on the early phase hepatocarcinogenesis in rats after initiation with DEN. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Lemon grass extract; Cancer chemoprevention; Diethylnitrosamine; Hepatocarcinogenesis; Rat
1. Introduction Chemoprevention has attracted attention as one of the most promising strategies to control cancers [1–3]. Great efforts are thus being made in the search for natural or synthesized agents capable of inhibiting carcinogenic processes [1–4]. Candidate agents need to be investigated in various animal models to explore * Corresponding author. Tel.: 181-3-3294-3286; fax: 181-35259-9301. E-mail address: [email protected] (D. Nakae).
the extent of inhibition, underlying mechanisms and possible adverse reactions, in coordination with epidemiological studies to evaluate responses in man [1–4]. Evidence has now accumulated of antimutagenic and/or anti-carcinogenic potential of a variety of food factors, including vitamin C, vitamin E, retinoids, carotinoids and flavonoids [3,4]. As these are natural compounds present in the human environment, they may serve as particularly good candidates as cancer chemopreventive agents [3–5]. It should be noted, however, that food factors can exert adverse
0304-3835/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(02)00111-8
10
R. Puatanachokchai et al. / Cancer Letters 183 (2002) 9–15
effects in particular situations [3,6]. Careful evaluation is therefore required. Lemon grass (Cymbopogon citratus, Stapf) is a widely used herb in tropical countries, especially in Southeast Asia, and also known as a source of ethnomedicines. Lemon grass extract (LGE), produced with ethanol, exhibits anti-mutagenic activity in various models [7–9] and retards the growth of fibrosarcoma cells transplanted in mice in association with prevention of lung metastasis [10]. It is thus conceivable that lemon grass contains some components that may be cancer chemopreventive. In fact, LGE has been shown to inhibit rat colon carcinogenesis initiated with azoxymethane [11]. The available data are, however, still limited, and more studies are required to evaluate the cancer chemopreventive properties of lemon grass or its components. The present study was conducted in order to generate information about effects of LGE on carcinogenesis. The liver was selected as a target organ, because hepatocellular carcinomas develop at high incidences in Asian countries, including Thailand and Japan [12,13].
2. Materials and methods 2.1. Animals and chemicals A total of 30 male Fisher 344 rats, 5 weeks old, were purchased from Japan SLC, Inc. (Hamamatsu, Shizuoka, Japan) and housed at five to a plastic cage with white flake bedding (Kansai Animal Corp., City of Kyoto, Kyoto, Japan) in an air-conditioned room, with a constant temperature of 25 ^ 3 8C, a relative humidity of 55 ^ 8% and a 12-h dark–light cycle. The rats were acclimatized on a basal diet (CE-2 diet: Clea Japan, Meguro, Tokyo, Japan) for 5 days before the experimentation. They were allowed free access to food and tap water throughout the acclimation and experimental periods. Body weight, water intake and food consumption were monitored weekly. Diethylnitrosamine (DEN) was obtained from Wako Pure Chemical Industries, Ltd. (City of Kyoto, Kyoto, Japan) and diluted with 0.9% sodium chloride solution at a concentration of 100 mg/ml. LGE was prepared from fresh lemon grass purchased twice, in summer and winter, from a local market in
Chiang Mai, Thailand. The stems were washed, sliced into small pieces and dried in a hot-air-oven drier at 40 8C overnight. The dried plants were finely powdered in a blender, suspended in 80% ethanol and filtered under suction pressure. The residue on the filter was re-extracted with 80% ethanol. The resultant filtrate was dried in a rotating evaporator under vacuum at 50 8C and then completely dried with a lyophilizer. The above procedure usually gave LGE at a weight of approximately 30 g from 1 kg of fresh plants. LGE was administered to rats by admixture into the basal diet after being dissolved in double-distilled water.
2.2. Experimental protocol Rats were divided into six groups each consisting of five animals. The experimental procedure was adapted from the medium-term rat liver bioassay protocol established by Ito et al. [14]. The modification chiefly involved the initiation stage in order to obtain more and larger putatively preneoplastic, glutathione S-transferase placental form (GST-P)positive lesions than with the original protocol. Briefly, rats were administered three weekly intraperitoneal doses of 100 mg/kg body weight (groups 1–4) DEN or the vehicle (groups 5 and 6), and partially hepatectomized by the method of Higgins and Anderson [15] at the end of week 5. LGE was administered at concentrations of 0 (groups 1 and 5), 0.2 (group 2), 0.6 (group 3) or 1.8% (groups 2 and 6) from the end of week 4 for 10 weeks. All surviving rats were sacrificed by exsanguination from the abdominal aorta under light ether anesthesia at the end of week 14. Upon sacrifice, the livers were excised and weighed. Three slices of 5-mm-thickness were taken from cranial and caudal parts of the right lateral lobe and caudate lobe from each liver, fixed in 10% neutrally buffered formalin and embedded in paraffin. Three serial sections of 4-mm-thickness were prepared from each liver specimen. One of them was employed for histological examination after being stained by a routine hematoxylin and eosin procedure, and the other two were utilized for the immunohistochemistry specified below. The remaining liver portions were immediately frozen under liquid nitrogen and stored at 280 8C until applied for biochemical tests.
R. Puatanachokchai et al. / Cancer Letters 183 (2002) 9–15
11
2.3. Immunohistochemistry
3. Results
GST-P-positive lesions were visualized immunohistochemically, and those comprising more than ten cells in cross-section were quantified using an IPAP image analyzing system (Sumika Technoservice Corp., City of Osaka, Osaka, Japan) as described elsewhere [16]. The data were corrected for three-dimensions according to Campbell et al. [17]. Hepatocyte proliferative activity was determined using the double-staining technique by combination of GST-P immunohistochemistry as above with the enhanced polymer one-step method for proliferating cell nuclear antigen (PCNA) [18]. Numbers of PCNA-positive hepatocytes among approximately 1000 cells were counted in both GST-P-positive lesions and in surrounding liver parenchyma under a light microscope to obtain percentages as PCNA labeling indices (PCNA L.I.).
During the experimental period, one, one, two and one rats died in groups 1, 2, 3 and 5, respectively, for unknown reasons. Final body weights did not differ among the groups, while relative liver weights in groups 4 and 6 were significantly lower than those in groups 1 and 5, respectively (Table 1). Water intake and food consumption were similar in all groups, and there were no particular macroscopic or microscopic alterations of note (data not shown). GST-P-positive lesions were significantly induced in groups 1–4, but were not observed in the vehicletreated groups 5 or 6 (Table 1). LGE significantly decreased the number of the lesions equally with all of the doses used in groups 2–4 (Table 1). In contrast, there was no influence on the size of the lesions (Table 1). Hepatocyte proliferation also did not differ in GSTP-positive lesions in groups 1–4 or in non-preneoplastic, surrounding liver tissues in groups 1–6 (Table 1). The 8-OHdG level in group 1 was significantly higher than that in group 5 (Table 2). LGE significantly reduced the 8-OHdG levels equally at all of the doses used (Table 2). However, there were no differences among groups in terms of the TBARS levels, cytosolic GST activity or cytosolic DTdiaphorase activity (Table 2).
2.4. Biochemical assessments Biochemical tests were conducted on frozen liver samples. Oxidative injury to hepatocyte nuclear DNA and extra-nuclear components was assessed using levels of 8-hydroxydeoxyguanosine (8-OHdG), standardized by deoxyguanosine (dG) contents [19], and 2-thiobarbituric acid-reacting substances (TBARS), expressed as malondialdehyde equivalent (MDA eq) values and standardized by protein contents [20], respectively. Hepatic status of phase II enzymes was assessed using cytosolic activity of glutathione Stransferase (GST), with 1-chloro-2,4-dinitrobenzene serving as the substrate [21], and DT-diaphorase, with 2,6-dichlorophenolindophenol [22], as parameters.
2.5. Statistical analysis Inter-group differences in quantitative data were considered significant, when P values smaller than 0.05 were obtained by the Student–Newman–Keuls multiple comparison test, employed after one-way analysis of variance to determine the variation among the group means followed by the Bartlett’s test to determine the homogeneity of variance.
4. Discussion The present results indicate that LGE can exert preventive effects on development of DEN-initiated hepatocellular lesions in rats, because LGE significantly decreased the numbers of putatively preneoplastic, GST-P-positive lesions. LGE did not, however, alter the sizes of such lesions, suggesting that LGE may not affect the growth of preneoplastic hepatocyte populations. In fact, neither preneoplastic nor non-neoplastic hepatocytes demonstrated any alteration in the level of proliferation with administration of LGE. Since the extract was administered during the post-initiation phase, the decrease in the numbers of GST-P-positive lesions was not due to inhibition of their initial induction. The question therefore arises of how LGE could have reduced the numbers of GST-P-positive lesions under the present experimental conditions.
12
Group
1 2 3 4 5 6
Treatment(s)
DEN/LGE (0%) DEN/LGE (0.2%) DEN/LGE (0.6%) DEN/LGE (1.8%) Vehicle/LGE (0%) Vehicle/LGE (1.8%) a b c
Effective number of rats
4 4 3 5 4 5
Values are the means ^ SD. Significantly different from the group 5 value. Significantly different from the group 1 value.
Final body weight (g) a
297 ^ 46 310 ^ 19 293 ^ 31 303 ^ 22 308 ^ 39 306 ^ 13
Relative liver weight (g/ 100 g body weight) a
3.23 ^ 0.12 2.90 ^ 0.10 2.95 ^ 0.21 2.80 ^ 0.21 2.88 ^ 0.25 2.79 ^ 0.40
GST-P-positive lesion a
PCNA L.I. (%) a
Number/cm 3
Size (mm 3)
In the lesions
In surroundings
0.014 ^ 0.004 0.007 ^ 0.004 0.009 ^ 0.004 0.015 ^ 0.006 – –
3.92 ^ 1.03 4.25 ^ 0.35 3.63 ^ 0.40 4.30 ^ 0.93 – –
3.35 ^ 0.95 3.43 ^ 0.17 3.43 ^ 0.21 2.88 ^ 0.45 2.38 ^ 0.26 2.38 ^ 0.19
24 ^ 10 12 ^ 4 c 11 ^ 3 c 9 ^ 3c 0 0
b
R. Puatanachokchai et al. / Cancer Letters 183 (2002) 9–15
Table 1 Final body weights, relative liver weights, numbers and sizes of GST-P-positive lesions and hepatocyte proliferative activity
R. Puatanachokchai et al. / Cancer Letters 183 (2002) 9–15
13
Table 2 8-OHdG and TBARS levels and GST and DT-diaphorase activity Group Treatment(s)
Effective number of rats
8-OHdG (/10 6 dG) a
TBARS (pmol MDA eq/mg protein) a
GST (units/mg protein) a
DT-diaphorase (mmol/ min per mg protein) a
1 2 3 4 5 6
4 4 3 5 4 5
2.00 ^ 0.38 b 1.32 ^ 0.61 c 1.42 ^ 0.26 c 1.08 ^ 0.27 c 0.67 ^ 0.26 0.52 ^ 0.06
8.8 ^ 1.2 10.0 ^ 2.2 9.9 ^ 4.1 7.1 ^ 1.5 9.9 ^ 2.2 12.1 ^ 2.2
9.9 ^ 1.4 8.5 ^ 0.5 10.7 ^ 3.4 9.1 ^ 3.0 9.3 ^ 2.1 9.2 ^ 2.0
16.7 ^ 2.7 17.7 ^ 3.2 17.9 ^ 1.4 19.5 ^ 5.3 19.3 ^ 5.2 15.8 ^ 2.2
DEN/LGE (0%) DEN/LGE (0.2%) DEN/LGE (0.6%) DEN/LGE (1.8%) Vehicle/LGE (0%) Vehicle/LGE (1.8%) a b c
Values are the means ^ SD. Significantly different from the group 5 value. Significantly different from the group 1 value.
A hint to the answer to this query may be provided by the data for 8-OHdG levels in hepatocyte nuclear DNA in the present study. Whereas 8-OHdG formation is apparently involved in the initiation phase of hepatocarcinogenesis in rats given DEN [23], LGE administered from 2 weeks after the last DEN administration could not have affected DEN-induced formation of the oxidative DNA injury in the present study. Furthermore, LGE did not alter TBARS generation, in contrast to the results of a previous investigation in which inhibition of the in vitro TBARS generation in human erythrocytes induced by tert-butyl hydroperoxide was found [11]. This discrepancy is presumably due to differences in the experimental conditions, but LGE did not appear to serve as an antioxidant in the present in vivo situation. One possible explanation for the reduction in 8-OHdG levels is that LGE might enhance the repair mechanisms [24,25] and/or the elimination of cells bearing 8-OHdG. As this might be the case for preneoplastic hepatocyte populations induced by DEN initiation [23], the decrease in both numbers of GST-P-positive lesions and 8-OHdG levels could thus be ascribed to the same mechanism, namely, enhanced elimination of preneoplastic hepatocyte populations. The most plausible candidate is apoptosis, well known to be involved in elimination of preneoplastic cell populations [26,27]. Such cell populations may acquire resistance to apoptosis because of dysregulation of the underlying signal machinery [28]. It is thus conceivable that LGE might affect these phenomena and in turn exert antihepatocarcinogenic effects, a possibility presently under investigation in our laboratories.
LGE has been shown to prevent rat colon carcinogenesis initiated with azoxymethane by inhibiting fecal b-glucuronidase to disturb the release of methylazoxymethane from glucuronide conjugates, the uptake of this active metabolite from the intestinal mucosa, and finally, the formation of azoxymethaneDNA adducts in the colon [11]. The potential chemopreventive effects have thus been attributed at least partly to LGE modification of the metabolism of xenobiotics. For instance, GST activity was found to be elevated in mouse intestinal mucosa by LGE [29]. The present study revealed, however, that neither GST nor DT-diaphorase activity was altered in the rat liver by its administration. Furthermore, it was recently demonstrated that the activities of these phase II enzymes in the liver and intestine of rats are elevated by dietarily administered LGE for 5 days but that the activities return to normal levels despite continued exposure and then remain unchanged thereafter [30]. It remains unclear which component(s) of lemon grass might have cancer chemopreventive potential. Among a variety of lemon grass ingredients, citral is the most abundant, and it has been shown to inhibit 12-O-tetradecanoylphorbol-13-acetate-derived promotion of mouse skin carcinogenesis initiated with 7,12-dimethylbenz[a]antharacene [31]. However, another constituent, geraniol, inhibits the growth of hepatoma and melanoma cells transplanted to mice and rats [32] and of colon cancer cells in vitro [33], while b-myrcene reduces the sister-chromatid exchange induced by aflatoxin B1 and cyclophosphamide in V79 and HTC cells in vitro [34]. By examin-
14
R. Puatanachokchai et al. / Cancer Letters 183 (2002) 9–15
ing effects of these and other lemon grass components on in vivo carcinogenesis in detail, it is to be hoped that the mechanisms underlying the anti-carcinogenic effects of LGE can be well elucidated. Such investigations are necessary to define any future strategy for the use of lemon grass as a cancer chemopreventive.
[8]
[9]
Acknowledgements [10]
The authors would like to express their sincere gratitude to Ms Sachiko Nakai, Akiko Nambu, Mariko Okuda, Janya Tipsri and Megumi Yamaguchi (alphabetical order of surnames) for technical support. The first author would especially like to thank Nara Medical University for providing financial support under the Young Scientist Exchange Program. This work was supported in part by a Research Grant from the Princess Takamatsu Cancer Research Fund, Japan, and Scientific Research Expenses for Health and Welfare Programs, 2nd-term Comprehensive 10-year Strategy for Cancer Control, Cancer Prevention, from the Ministry of Health, Labor and Welfare of Japan. References [1] Chemoprevention Working Group, Prevention of cancer in the next millennium: report of the Chemoprevention Working Group to the American Association for Cancer Research, Cancer Res. 59 (1999) 4743–4758. [2] S.D. Hursting, T.J. Slaga, S.M. Fischer, J.D. DiGioivanni, J.M. Phang, Mechanism-based cancer prevention approaches: targets, examples and the use of transgenic mice, J. Natl. Cancer Inst. 91 (1999) 215–225. [3] A. Murakami, H. Ohigashi, K. Koshimuzu, Chemoprevention: insights into biological mechanisms and promising food factors, Food Rev. Int. 15 (1999) 335–395. [4] A. Murakami, K. Koshimizu, H. Ohigashi, Chemoprevention with food phytochemicals: screening, recent studies, and action mechanisms, J. Med. Food 1 (1998) 29–38. [5] G.L. Kelloff, J.A. Crowell, V.E. Steele, R.A. Lubet, W.A. Malone, C.W. Boone, L. Kopelovich, E.T. Hawk, R. Lieberman, J.A. Lawrence, I. Ali, J.L. Viner, C.C. Sigman, Progress in cancer chemoprevention: development of diet-derived chemopreventive agents, J. Nutr. 130 (2S Suppl.) (2000) 467S–471S. [6] M. Paolini, S.Z. Abdel-Rahman, G. Cantelli-Forti, M.S. Legator, Chemoprevention or antichemoprevention? A salutary warning from the b-carotene experience, J. Natl. Cancer Inst. 93 (2001) 1110–1111. [7] U. Vinitketkumnuen, R. Puatanachokchai, P. Kongtawelert, N. Lertprasertsuke, T. Matsushima, Antimutagenicity of
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
lemon grass (Cymbopogon citratus, Stapf) to various known mutagens in Salmonella mutation assay, Mutat. Res. 341(1994) 71–75. U. Meevatee, S. Boontim, O. Keereeta, U. Vinitketkumnuen, N. O-ariyakul, Antimutagenic activity of lemon grass, in: S. Boot-in (Ed.), Man and Environment, Chiang Mai University Press, Chiang Mai, Thailand, 1993, p. 346. K. Pimsaeng, Anti-micronucleus formation of lemon grass extract, Master’s thesis, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand, 1993. R. Puatanachokchai, Antimutagenicity, cytotoxicity and antitumor activity from lemon grass (Cymbopogon citratus, Stapf) extract, Master’s thesis, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand, 1994. R. Suaeyun, T. Kinouchi, H. Arimochi, U. Vinitketkumnuen, Y. Ohnishi, Inhibitory effects of lemon grass (Cymbopogon citratus, Stapf) on formation of azoxymethane-induced DNA adducts and aberrant crypt foci in the rat colon, Carcinogenesis 18 (1997) 949–955. V. Vatanasapt, N. Martin, K. Sriplung, K. Chindavijak, S. Sontipong, S. Sriamporn, D.M. Parkin, J. Ferlay (Eds.), Cancer in Thailand 1988–1991, IARC Technical Report No. 16, International Agency for Research on Cancer, Lyon, France, 1993. D.M. Parkin, C.S. Muir, S.L. Whelan, Y.T. Gao, J. Ferlay, J. Powell (Eds.), Cancer Incidence in Five Continents, IARC Scientific Publications No. 120, Vol. 6, International Agency for Research on Cancer, Lyon, France, 1992. N. Ito, M. Tatematsu, R. Hasegawa, H. Tsuda, Medium-term bioassay system for detection of carcinogens and modifiers of hepatocarcinogenic substances utilizing the GST-P-positive liver cell focus as an endpoint marker, Toxicol. Pathol. 17 (1989) 630–641. G.M. Higgins, R.M. Anderson, Experimental pathology of the liver. 1. Restoration of the liver of the white rat following partial surgical removal, Arch. Pathol. Lab. 12 (1931) 1186– 1202. H. Kishida, D. Nakae, Y. Kobayashi, O. Kusuoka, W. Kitayama, A. Denda, H. Fukui, Y. Konishi, Enhancement of hepatocarcinogenesis initiated with diethylnitrosamine or Nnitrosobis(2-hydroxypropyl)amine by a choline-deficient, lamino acid-defined diet administered prior to the carcinogen exposure in rats, Exp. Toxicol. Pathol. 52 (2000) 405–412. H.A. Campbell, H.C. Pitot, V.R. Potter, B.A. Laishes, Application of quantitative stereology to the evaluation of enzymealter foci in the liver, Cancer Res. 42 (1982) 465–472. Y. Tsutsumi, A. Serizawa, K. Kawai, Enhanced polymer onestep staining (EPOS) for proliferating cell nuclear antigen (PCNA) and Ki-67 antigen: application to intra-operative frozen diagnosis, Pathol. Int. 45 (1995) 108–115. D. Nakae, Y. Mizumoto, E. Kobayashi, O. Noguchi, Y. Konishi, Improved genomic/nuclear DNA extraction for 8hydroxydeoxyguanosine analysis of small amounts of rat liver tissue, Cancer Lett. 97 (1995) 233–239. D. Nakae, K. Yamamoto, H. Yoshiji, T. Kinugasa, H. Maruyama, J.L. Farber, Y. Konishi, Liposome-encapsulated
R. Puatanachokchai et al. / Cancer Letters 183 (2002) 9–15
[21]
[22]
[23]
[24]
[25]
[26] [27]
superoxide dismutase prevents liver necrosis induced by acetaminophen, Am. J. Pathol. 136 (1990) 787–795. S. Tsuchida, Glutathione S-transferase, in: N. Taniguchi (Ed.), Experimental Protocols for Research on Reactive Oxygen Species, Shujunsha, Co., Ltd., Tokyo, 1994, pp. 110–112. A.M. Benson, M.J. Hunkeler, P. Talalay, Increase of NADPH:quinone reductase activity by dietary antioxidants: possible role in protection against carcinogenesis and toxicity, Proc. Natl. Acad. Sci. USA 77 (1980) 5216–5220. D. Nakae, Y. Kobayashi, H. Akai, N. Andoh, H. Satoh, K. Ohashi, M. Tsutsumi, Y. Konishi, Involvement of 8-hydroxyguanine formation in the initiation of rat liver carcinogenesis by low dose levels of N-nitrosodiethylamine, Cancer Res. 57 (1997) 1281–1287. J. Laval, J. Jurado, M. Saparbaev, O. Sidorkina, Antimutagenic role of base-excision repair enzymes upon free radical-induced DNA damage, Mutat. Res. 18 (1998) 93–102. S. Nishimura, Mammalian Ogg1/Mmh gene plays a major role in repair of the 8-hydroxyguanine lesions in DNA, Prog. Nucleic Acid Res. Mol. Biol. 68 (2001) 107–123. S.W. Lowe, A.W. Lin, Apoptosis in cancer, Carcinogenesis 21 (2000) 485–495. A.H. Wyllie, C.O. Bellamy, V.J. Bubb, A.R. Clarke, S. Corbet, L. Curtis, D.J. Harrison, M.L. Hooper, N. Toft, S.
[28] [29]
[30]
[31]
[32]
[33]
[34]
15
Webb, C.C. Bird, Apoptosis and carcinogenesis, Br. J. Cancer 80(Suppl. 1) (1999) 34–37. J.C. Reed, Dysregulation of apoptosis in cancer, J. Clin. Oncol. 17 (1999) 2941–2953. L.K.T. Lam, B. Zhang, Effects of essential oils on glutathione S-transferase activity in mice, J. Agric. Food Chem. 39 (1991) 660–662. S. Thhumvijit, Effect of lemon grass extract on rat hepatic and intestinal xenobiotic-metabolizing enzymes, Master’s thesis, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand, 1999. M.J. Connor, Modulation of tumor promotion in mouse skin by the food additive citral (3,7-dimethyl-2,6-octadienal), Cancer Lett. 56 (1991) 25–28. S.G. Yu, L.A. Hildebrandt, C.E. Elson, Geraniol, an inhibitor of mevalonate biosynthesis, suppresses the growth of hepatomas and melanomas transplanted to rats and mice, J. Nutr. 125 (1995) 2763–2767. F. Raul, Geraniol, a component of plant essential oils, inhibits growth and polyamine biosynthesis in human colon cancer cells, J. Pharmacol. Exp. Ther. 298 (2001) 197–200. C. Ro¨ scheisen, H. Zamith, F.J.R. Paumgartten, G. Speit, Influence of b-myrcene on sister-chromatid exchanges induced by mutagens in V79 and HTC cells, Mutat. Res. 264 (1991) 43–49.
Inhibitory effects of lemon grass (Cymbopogon citratus, Stapf) extract on the early phase of hepatocarcinogenesis after initiation with diethylnitrosamine in male Fischer 344 rats Rawiwan Puatanachokchai a,b, Hideki Kishida a,c,d, Ayumi Denda a, Nao Murata a, Yoichi Konishi a,d, Usanee Vinitketkumnuen b, Dai Nakae e,* a
Department of Oncological Pathology, Cancer Center, Nara Medical University, Kashihara, Nara 634-8521, Japan b Department of Biochemistry, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand c Third Department of Internal Medicine, Nara Medical University, Kashihara, Nara 634-8521, Japan d Free Radical Biology and Aging Research Program, Oklahoma Medical Foundation, Oklahoma City, OK 73104, USA e Department of Pathology, Sasaki Institute, Sasaki Foundation, Chiyoda, Tokyo 101-0062, Japan Received 27 November 2001; received in revised form 18 February 2002; accepted 18 February 2002
Abstract Effects of lemon grass extract (LGE) on hepatocarcinogenesis were examined in male Fischer 344 rats, administered diethylnitrosamine (DEN) at three weekly intraperitoneal doses of 100 mg/kg body weight and partially hepatectomized at the end of week 5. LGE was given at dietary concentrations of 0, 0.2, 0.6 or 1.8% from the end of week 4 for 10 weeks. All rats were sacrificed at the end of week 14. LGE reduced the number of putatively preneoplastic, glutathione S-transferase placental form-positive lesions and the level of oxidative hepatocyte nuclear DNA injury, as assessed in terms of 8-hydroxydeoxyguanosine production. In contrast, LGE did not affect the size of the preneoplastic lesions, hepatocyte proliferative activity, activities of phase II enzymes or hepatocyte extra-nuclear oxidative injury. These results suggest inhibitory effects of LGE on the early phase hepatocarcinogenesis in rats after initiation with DEN. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Lemon grass extract; Cancer chemoprevention; Diethylnitrosamine; Hepatocarcinogenesis; Rat
1. Introduction Chemoprevention has attracted attention as one of the most promising strategies to control cancers [1–3]. Great efforts are thus being made in the search for natural or synthesized agents capable of inhibiting carcinogenic processes [1–4]. Candidate agents need to be investigated in various animal models to explore * Corresponding author. Tel.: 181-3-3294-3286; fax: 181-35259-9301. E-mail address: [email protected] (D. Nakae).
the extent of inhibition, underlying mechanisms and possible adverse reactions, in coordination with epidemiological studies to evaluate responses in man [1–4]. Evidence has now accumulated of antimutagenic and/or anti-carcinogenic potential of a variety of food factors, including vitamin C, vitamin E, retinoids, carotinoids and flavonoids [3,4]. As these are natural compounds present in the human environment, they may serve as particularly good candidates as cancer chemopreventive agents [3–5]. It should be noted, however, that food factors can exert adverse
0304-3835/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(02)00111-8
10
R. Puatanachokchai et al. / Cancer Letters 183 (2002) 9–15
effects in particular situations [3,6]. Careful evaluation is therefore required. Lemon grass (Cymbopogon citratus, Stapf) is a widely used herb in tropical countries, especially in Southeast Asia, and also known as a source of ethnomedicines. Lemon grass extract (LGE), produced with ethanol, exhibits anti-mutagenic activity in various models [7–9] and retards the growth of fibrosarcoma cells transplanted in mice in association with prevention of lung metastasis [10]. It is thus conceivable that lemon grass contains some components that may be cancer chemopreventive. In fact, LGE has been shown to inhibit rat colon carcinogenesis initiated with azoxymethane [11]. The available data are, however, still limited, and more studies are required to evaluate the cancer chemopreventive properties of lemon grass or its components. The present study was conducted in order to generate information about effects of LGE on carcinogenesis. The liver was selected as a target organ, because hepatocellular carcinomas develop at high incidences in Asian countries, including Thailand and Japan [12,13].
2. Materials and methods 2.1. Animals and chemicals A total of 30 male Fisher 344 rats, 5 weeks old, were purchased from Japan SLC, Inc. (Hamamatsu, Shizuoka, Japan) and housed at five to a plastic cage with white flake bedding (Kansai Animal Corp., City of Kyoto, Kyoto, Japan) in an air-conditioned room, with a constant temperature of 25 ^ 3 8C, a relative humidity of 55 ^ 8% and a 12-h dark–light cycle. The rats were acclimatized on a basal diet (CE-2 diet: Clea Japan, Meguro, Tokyo, Japan) for 5 days before the experimentation. They were allowed free access to food and tap water throughout the acclimation and experimental periods. Body weight, water intake and food consumption were monitored weekly. Diethylnitrosamine (DEN) was obtained from Wako Pure Chemical Industries, Ltd. (City of Kyoto, Kyoto, Japan) and diluted with 0.9% sodium chloride solution at a concentration of 100 mg/ml. LGE was prepared from fresh lemon grass purchased twice, in summer and winter, from a local market in
Chiang Mai, Thailand. The stems were washed, sliced into small pieces and dried in a hot-air-oven drier at 40 8C overnight. The dried plants were finely powdered in a blender, suspended in 80% ethanol and filtered under suction pressure. The residue on the filter was re-extracted with 80% ethanol. The resultant filtrate was dried in a rotating evaporator under vacuum at 50 8C and then completely dried with a lyophilizer. The above procedure usually gave LGE at a weight of approximately 30 g from 1 kg of fresh plants. LGE was administered to rats by admixture into the basal diet after being dissolved in double-distilled water.
2.2. Experimental protocol Rats were divided into six groups each consisting of five animals. The experimental procedure was adapted from the medium-term rat liver bioassay protocol established by Ito et al. [14]. The modification chiefly involved the initiation stage in order to obtain more and larger putatively preneoplastic, glutathione S-transferase placental form (GST-P)positive lesions than with the original protocol. Briefly, rats were administered three weekly intraperitoneal doses of 100 mg/kg body weight (groups 1–4) DEN or the vehicle (groups 5 and 6), and partially hepatectomized by the method of Higgins and Anderson [15] at the end of week 5. LGE was administered at concentrations of 0 (groups 1 and 5), 0.2 (group 2), 0.6 (group 3) or 1.8% (groups 2 and 6) from the end of week 4 for 10 weeks. All surviving rats were sacrificed by exsanguination from the abdominal aorta under light ether anesthesia at the end of week 14. Upon sacrifice, the livers were excised and weighed. Three slices of 5-mm-thickness were taken from cranial and caudal parts of the right lateral lobe and caudate lobe from each liver, fixed in 10% neutrally buffered formalin and embedded in paraffin. Three serial sections of 4-mm-thickness were prepared from each liver specimen. One of them was employed for histological examination after being stained by a routine hematoxylin and eosin procedure, and the other two were utilized for the immunohistochemistry specified below. The remaining liver portions were immediately frozen under liquid nitrogen and stored at 280 8C until applied for biochemical tests.
R. Puatanachokchai et al. / Cancer Letters 183 (2002) 9–15
11
2.3. Immunohistochemistry
3. Results
GST-P-positive lesions were visualized immunohistochemically, and those comprising more than ten cells in cross-section were quantified using an IPAP image analyzing system (Sumika Technoservice Corp., City of Osaka, Osaka, Japan) as described elsewhere [16]. The data were corrected for three-dimensions according to Campbell et al. [17]. Hepatocyte proliferative activity was determined using the double-staining technique by combination of GST-P immunohistochemistry as above with the enhanced polymer one-step method for proliferating cell nuclear antigen (PCNA) [18]. Numbers of PCNA-positive hepatocytes among approximately 1000 cells were counted in both GST-P-positive lesions and in surrounding liver parenchyma under a light microscope to obtain percentages as PCNA labeling indices (PCNA L.I.).
During the experimental period, one, one, two and one rats died in groups 1, 2, 3 and 5, respectively, for unknown reasons. Final body weights did not differ among the groups, while relative liver weights in groups 4 and 6 were significantly lower than those in groups 1 and 5, respectively (Table 1). Water intake and food consumption were similar in all groups, and there were no particular macroscopic or microscopic alterations of note (data not shown). GST-P-positive lesions were significantly induced in groups 1–4, but were not observed in the vehicletreated groups 5 or 6 (Table 1). LGE significantly decreased the number of the lesions equally with all of the doses used in groups 2–4 (Table 1). In contrast, there was no influence on the size of the lesions (Table 1). Hepatocyte proliferation also did not differ in GSTP-positive lesions in groups 1–4 or in non-preneoplastic, surrounding liver tissues in groups 1–6 (Table 1). The 8-OHdG level in group 1 was significantly higher than that in group 5 (Table 2). LGE significantly reduced the 8-OHdG levels equally at all of the doses used (Table 2). However, there were no differences among groups in terms of the TBARS levels, cytosolic GST activity or cytosolic DTdiaphorase activity (Table 2).
2.4. Biochemical assessments Biochemical tests were conducted on frozen liver samples. Oxidative injury to hepatocyte nuclear DNA and extra-nuclear components was assessed using levels of 8-hydroxydeoxyguanosine (8-OHdG), standardized by deoxyguanosine (dG) contents [19], and 2-thiobarbituric acid-reacting substances (TBARS), expressed as malondialdehyde equivalent (MDA eq) values and standardized by protein contents [20], respectively. Hepatic status of phase II enzymes was assessed using cytosolic activity of glutathione Stransferase (GST), with 1-chloro-2,4-dinitrobenzene serving as the substrate [21], and DT-diaphorase, with 2,6-dichlorophenolindophenol [22], as parameters.
2.5. Statistical analysis Inter-group differences in quantitative data were considered significant, when P values smaller than 0.05 were obtained by the Student–Newman–Keuls multiple comparison test, employed after one-way analysis of variance to determine the variation among the group means followed by the Bartlett’s test to determine the homogeneity of variance.
4. Discussion The present results indicate that LGE can exert preventive effects on development of DEN-initiated hepatocellular lesions in rats, because LGE significantly decreased the numbers of putatively preneoplastic, GST-P-positive lesions. LGE did not, however, alter the sizes of such lesions, suggesting that LGE may not affect the growth of preneoplastic hepatocyte populations. In fact, neither preneoplastic nor non-neoplastic hepatocytes demonstrated any alteration in the level of proliferation with administration of LGE. Since the extract was administered during the post-initiation phase, the decrease in the numbers of GST-P-positive lesions was not due to inhibition of their initial induction. The question therefore arises of how LGE could have reduced the numbers of GST-P-positive lesions under the present experimental conditions.
12
Group
1 2 3 4 5 6
Treatment(s)
DEN/LGE (0%) DEN/LGE (0.2%) DEN/LGE (0.6%) DEN/LGE (1.8%) Vehicle/LGE (0%) Vehicle/LGE (1.8%) a b c
Effective number of rats
4 4 3 5 4 5
Values are the means ^ SD. Significantly different from the group 5 value. Significantly different from the group 1 value.
Final body weight (g) a
297 ^ 46 310 ^ 19 293 ^ 31 303 ^ 22 308 ^ 39 306 ^ 13
Relative liver weight (g/ 100 g body weight) a
3.23 ^ 0.12 2.90 ^ 0.10 2.95 ^ 0.21 2.80 ^ 0.21 2.88 ^ 0.25 2.79 ^ 0.40
GST-P-positive lesion a
PCNA L.I. (%) a
Number/cm 3
Size (mm 3)
In the lesions
In surroundings
0.014 ^ 0.004 0.007 ^ 0.004 0.009 ^ 0.004 0.015 ^ 0.006 – –
3.92 ^ 1.03 4.25 ^ 0.35 3.63 ^ 0.40 4.30 ^ 0.93 – –
3.35 ^ 0.95 3.43 ^ 0.17 3.43 ^ 0.21 2.88 ^ 0.45 2.38 ^ 0.26 2.38 ^ 0.19
24 ^ 10 12 ^ 4 c 11 ^ 3 c 9 ^ 3c 0 0
b
R. Puatanachokchai et al. / Cancer Letters 183 (2002) 9–15
Table 1 Final body weights, relative liver weights, numbers and sizes of GST-P-positive lesions and hepatocyte proliferative activity
R. Puatanachokchai et al. / Cancer Letters 183 (2002) 9–15
13
Table 2 8-OHdG and TBARS levels and GST and DT-diaphorase activity Group Treatment(s)
Effective number of rats
8-OHdG (/10 6 dG) a
TBARS (pmol MDA eq/mg protein) a
GST (units/mg protein) a
DT-diaphorase (mmol/ min per mg protein) a
1 2 3 4 5 6
4 4 3 5 4 5
2.00 ^ 0.38 b 1.32 ^ 0.61 c 1.42 ^ 0.26 c 1.08 ^ 0.27 c 0.67 ^ 0.26 0.52 ^ 0.06
8.8 ^ 1.2 10.0 ^ 2.2 9.9 ^ 4.1 7.1 ^ 1.5 9.9 ^ 2.2 12.1 ^ 2.2
9.9 ^ 1.4 8.5 ^ 0.5 10.7 ^ 3.4 9.1 ^ 3.0 9.3 ^ 2.1 9.2 ^ 2.0
16.7 ^ 2.7 17.7 ^ 3.2 17.9 ^ 1.4 19.5 ^ 5.3 19.3 ^ 5.2 15.8 ^ 2.2
DEN/LGE (0%) DEN/LGE (0.2%) DEN/LGE (0.6%) DEN/LGE (1.8%) Vehicle/LGE (0%) Vehicle/LGE (1.8%) a b c
Values are the means ^ SD. Significantly different from the group 5 value. Significantly different from the group 1 value.
A hint to the answer to this query may be provided by the data for 8-OHdG levels in hepatocyte nuclear DNA in the present study. Whereas 8-OHdG formation is apparently involved in the initiation phase of hepatocarcinogenesis in rats given DEN [23], LGE administered from 2 weeks after the last DEN administration could not have affected DEN-induced formation of the oxidative DNA injury in the present study. Furthermore, LGE did not alter TBARS generation, in contrast to the results of a previous investigation in which inhibition of the in vitro TBARS generation in human erythrocytes induced by tert-butyl hydroperoxide was found [11]. This discrepancy is presumably due to differences in the experimental conditions, but LGE did not appear to serve as an antioxidant in the present in vivo situation. One possible explanation for the reduction in 8-OHdG levels is that LGE might enhance the repair mechanisms [24,25] and/or the elimination of cells bearing 8-OHdG. As this might be the case for preneoplastic hepatocyte populations induced by DEN initiation [23], the decrease in both numbers of GST-P-positive lesions and 8-OHdG levels could thus be ascribed to the same mechanism, namely, enhanced elimination of preneoplastic hepatocyte populations. The most plausible candidate is apoptosis, well known to be involved in elimination of preneoplastic cell populations [26,27]. Such cell populations may acquire resistance to apoptosis because of dysregulation of the underlying signal machinery [28]. It is thus conceivable that LGE might affect these phenomena and in turn exert antihepatocarcinogenic effects, a possibility presently under investigation in our laboratories.
LGE has been shown to prevent rat colon carcinogenesis initiated with azoxymethane by inhibiting fecal b-glucuronidase to disturb the release of methylazoxymethane from glucuronide conjugates, the uptake of this active metabolite from the intestinal mucosa, and finally, the formation of azoxymethaneDNA adducts in the colon [11]. The potential chemopreventive effects have thus been attributed at least partly to LGE modification of the metabolism of xenobiotics. For instance, GST activity was found to be elevated in mouse intestinal mucosa by LGE [29]. The present study revealed, however, that neither GST nor DT-diaphorase activity was altered in the rat liver by its administration. Furthermore, it was recently demonstrated that the activities of these phase II enzymes in the liver and intestine of rats are elevated by dietarily administered LGE for 5 days but that the activities return to normal levels despite continued exposure and then remain unchanged thereafter [30]. It remains unclear which component(s) of lemon grass might have cancer chemopreventive potential. Among a variety of lemon grass ingredients, citral is the most abundant, and it has been shown to inhibit 12-O-tetradecanoylphorbol-13-acetate-derived promotion of mouse skin carcinogenesis initiated with 7,12-dimethylbenz[a]antharacene [31]. However, another constituent, geraniol, inhibits the growth of hepatoma and melanoma cells transplanted to mice and rats [32] and of colon cancer cells in vitro [33], while b-myrcene reduces the sister-chromatid exchange induced by aflatoxin B1 and cyclophosphamide in V79 and HTC cells in vitro [34]. By examin-
14
R. Puatanachokchai et al. / Cancer Letters 183 (2002) 9–15
ing effects of these and other lemon grass components on in vivo carcinogenesis in detail, it is to be hoped that the mechanisms underlying the anti-carcinogenic effects of LGE can be well elucidated. Such investigations are necessary to define any future strategy for the use of lemon grass as a cancer chemopreventive.
[8]
[9]
Acknowledgements [10]
The authors would like to express their sincere gratitude to Ms Sachiko Nakai, Akiko Nambu, Mariko Okuda, Janya Tipsri and Megumi Yamaguchi (alphabetical order of surnames) for technical support. The first author would especially like to thank Nara Medical University for providing financial support under the Young Scientist Exchange Program. This work was supported in part by a Research Grant from the Princess Takamatsu Cancer Research Fund, Japan, and Scientific Research Expenses for Health and Welfare Programs, 2nd-term Comprehensive 10-year Strategy for Cancer Control, Cancer Prevention, from the Ministry of Health, Labor and Welfare of Japan. References [1] Chemoprevention Working Group, Prevention of cancer in the next millennium: report of the Chemoprevention Working Group to the American Association for Cancer Research, Cancer Res. 59 (1999) 4743–4758. [2] S.D. Hursting, T.J. Slaga, S.M. Fischer, J.D. DiGioivanni, J.M. Phang, Mechanism-based cancer prevention approaches: targets, examples and the use of transgenic mice, J. Natl. Cancer Inst. 91 (1999) 215–225. [3] A. Murakami, H. Ohigashi, K. Koshimuzu, Chemoprevention: insights into biological mechanisms and promising food factors, Food Rev. Int. 15 (1999) 335–395. [4] A. Murakami, K. Koshimizu, H. Ohigashi, Chemoprevention with food phytochemicals: screening, recent studies, and action mechanisms, J. Med. Food 1 (1998) 29–38. [5] G.L. Kelloff, J.A. Crowell, V.E. Steele, R.A. Lubet, W.A. Malone, C.W. Boone, L. Kopelovich, E.T. Hawk, R. Lieberman, J.A. Lawrence, I. Ali, J.L. Viner, C.C. Sigman, Progress in cancer chemoprevention: development of diet-derived chemopreventive agents, J. Nutr. 130 (2S Suppl.) (2000) 467S–471S. [6] M. Paolini, S.Z. Abdel-Rahman, G. Cantelli-Forti, M.S. Legator, Chemoprevention or antichemoprevention? A salutary warning from the b-carotene experience, J. Natl. Cancer Inst. 93 (2001) 1110–1111. [7] U. Vinitketkumnuen, R. Puatanachokchai, P. Kongtawelert, N. Lertprasertsuke, T. Matsushima, Antimutagenicity of
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
lemon grass (Cymbopogon citratus, Stapf) to various known mutagens in Salmonella mutation assay, Mutat. Res. 341(1994) 71–75. U. Meevatee, S. Boontim, O. Keereeta, U. Vinitketkumnuen, N. O-ariyakul, Antimutagenic activity of lemon grass, in: S. Boot-in (Ed.), Man and Environment, Chiang Mai University Press, Chiang Mai, Thailand, 1993, p. 346. K. Pimsaeng, Anti-micronucleus formation of lemon grass extract, Master’s thesis, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand, 1993. R. Puatanachokchai, Antimutagenicity, cytotoxicity and antitumor activity from lemon grass (Cymbopogon citratus, Stapf) extract, Master’s thesis, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand, 1994. R. Suaeyun, T. Kinouchi, H. Arimochi, U. Vinitketkumnuen, Y. Ohnishi, Inhibitory effects of lemon grass (Cymbopogon citratus, Stapf) on formation of azoxymethane-induced DNA adducts and aberrant crypt foci in the rat colon, Carcinogenesis 18 (1997) 949–955. V. Vatanasapt, N. Martin, K. Sriplung, K. Chindavijak, S. Sontipong, S. Sriamporn, D.M. Parkin, J. Ferlay (Eds.), Cancer in Thailand 1988–1991, IARC Technical Report No. 16, International Agency for Research on Cancer, Lyon, France, 1993. D.M. Parkin, C.S. Muir, S.L. Whelan, Y.T. Gao, J. Ferlay, J. Powell (Eds.), Cancer Incidence in Five Continents, IARC Scientific Publications No. 120, Vol. 6, International Agency for Research on Cancer, Lyon, France, 1992. N. Ito, M. Tatematsu, R. Hasegawa, H. Tsuda, Medium-term bioassay system for detection of carcinogens and modifiers of hepatocarcinogenic substances utilizing the GST-P-positive liver cell focus as an endpoint marker, Toxicol. Pathol. 17 (1989) 630–641. G.M. Higgins, R.M. Anderson, Experimental pathology of the liver. 1. Restoration of the liver of the white rat following partial surgical removal, Arch. Pathol. Lab. 12 (1931) 1186– 1202. H. Kishida, D. Nakae, Y. Kobayashi, O. Kusuoka, W. Kitayama, A. Denda, H. Fukui, Y. Konishi, Enhancement of hepatocarcinogenesis initiated with diethylnitrosamine or Nnitrosobis(2-hydroxypropyl)amine by a choline-deficient, lamino acid-defined diet administered prior to the carcinogen exposure in rats, Exp. Toxicol. Pathol. 52 (2000) 405–412. H.A. Campbell, H.C. Pitot, V.R. Potter, B.A. Laishes, Application of quantitative stereology to the evaluation of enzymealter foci in the liver, Cancer Res. 42 (1982) 465–472. Y. Tsutsumi, A. Serizawa, K. Kawai, Enhanced polymer onestep staining (EPOS) for proliferating cell nuclear antigen (PCNA) and Ki-67 antigen: application to intra-operative frozen diagnosis, Pathol. Int. 45 (1995) 108–115. D. Nakae, Y. Mizumoto, E. Kobayashi, O. Noguchi, Y. Konishi, Improved genomic/nuclear DNA extraction for 8hydroxydeoxyguanosine analysis of small amounts of rat liver tissue, Cancer Lett. 97 (1995) 233–239. D. Nakae, K. Yamamoto, H. Yoshiji, T. Kinugasa, H. Maruyama, J.L. Farber, Y. Konishi, Liposome-encapsulated
R. Puatanachokchai et al. / Cancer Letters 183 (2002) 9–15
[21]
[22]
[23]
[24]
[25]
[26] [27]
superoxide dismutase prevents liver necrosis induced by acetaminophen, Am. J. Pathol. 136 (1990) 787–795. S. Tsuchida, Glutathione S-transferase, in: N. Taniguchi (Ed.), Experimental Protocols for Research on Reactive Oxygen Species, Shujunsha, Co., Ltd., Tokyo, 1994, pp. 110–112. A.M. Benson, M.J. Hunkeler, P. Talalay, Increase of NADPH:quinone reductase activity by dietary antioxidants: possible role in protection against carcinogenesis and toxicity, Proc. Natl. Acad. Sci. USA 77 (1980) 5216–5220. D. Nakae, Y. Kobayashi, H. Akai, N. Andoh, H. Satoh, K. Ohashi, M. Tsutsumi, Y. Konishi, Involvement of 8-hydroxyguanine formation in the initiation of rat liver carcinogenesis by low dose levels of N-nitrosodiethylamine, Cancer Res. 57 (1997) 1281–1287. J. Laval, J. Jurado, M. Saparbaev, O. Sidorkina, Antimutagenic role of base-excision repair enzymes upon free radical-induced DNA damage, Mutat. Res. 18 (1998) 93–102. S. Nishimura, Mammalian Ogg1/Mmh gene plays a major role in repair of the 8-hydroxyguanine lesions in DNA, Prog. Nucleic Acid Res. Mol. Biol. 68 (2001) 107–123. S.W. Lowe, A.W. Lin, Apoptosis in cancer, Carcinogenesis 21 (2000) 485–495. A.H. Wyllie, C.O. Bellamy, V.J. Bubb, A.R. Clarke, S. Corbet, L. Curtis, D.J. Harrison, M.L. Hooper, N. Toft, S.
[28] [29]
[30]
[31]
[32]
[33]
[34]
15
Webb, C.C. Bird, Apoptosis and carcinogenesis, Br. J. Cancer 80(Suppl. 1) (1999) 34–37. J.C. Reed, Dysregulation of apoptosis in cancer, J. Clin. Oncol. 17 (1999) 2941–2953. L.K.T. Lam, B. Zhang, Effects of essential oils on glutathione S-transferase activity in mice, J. Agric. Food Chem. 39 (1991) 660–662. S. Thhumvijit, Effect of lemon grass extract on rat hepatic and intestinal xenobiotic-metabolizing enzymes, Master’s thesis, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand, 1999. M.J. Connor, Modulation of tumor promotion in mouse skin by the food additive citral (3,7-dimethyl-2,6-octadienal), Cancer Lett. 56 (1991) 25–28. S.G. Yu, L.A. Hildebrandt, C.E. Elson, Geraniol, an inhibitor of mevalonate biosynthesis, suppresses the growth of hepatomas and melanomas transplanted to rats and mice, J. Nutr. 125 (1995) 2763–2767. F. Raul, Geraniol, a component of plant essential oils, inhibits growth and polyamine biosynthesis in human colon cancer cells, J. Pharmacol. Exp. Ther. 298 (2001) 197–200. C. Ro¨ scheisen, H. Zamith, F.J.R. Paumgartten, G. Speit, Influence of b-myrcene on sister-chromatid exchanges induced by mutagens in V79 and HTC cells, Mutat. Res. 264 (1991) 43–49.