Journal of Nutrition OpenSOurce Diets- www.ResearchDiets.com

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Uda, N.
Right arrow Articles by Fukushima, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Uda, N.
Right arrow Articles by Fukushima, S.
© 2006 American Society for Nutrition J. Nutr. 136:855S-860S, March 2006

Supplement: Significance of Garlic and Its Constituents in Cancer and Cardiovascular Disease

Aged Garlic Extract Inhibits Development of Putative Preneoplastic Lesions in Rat Hepatocarcinogenesis1,2

Naoto Uda*,3, Naoki Kashimoto*, Isao Sumioka*, Eikai Kyo*, Shin-ichiro Sumi* and Shoji Fukushima{dagger}

* Healthcare Research Institute, Wakunaga Pharmaceutical Co., Ltd., Hiroshima 739-1195, Japan and {dagger} First Department of Pathology, Osaka City University Medical School, Osaka 545-8585, Japan

3 To whom correspondence should be addressed. E-mail: uda_n{at}wakunaga.co.jp.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
A unique garlic preparation, aged garlic extract (AGE), was examined for its modifying effect on diethylnitrosamine (DEN)-induced neoplasia of the liver in male F344 rats, using the medium-term bioassay system based on the 2-step model of hepatocarcinogenesis. Carcinogenic potential was scored by comparing the numbers and areas of induced glutathione S-transferase placental form (GST-P)-positive hepatocellular foci. GST-P-positive foci were significantly decreased in rats treated with AGE at doses of 2, 5, and 10 mL/kg, i.g., 5 times per week during the promotion phase. In addition, to clarify the mechanism underlying the inhibitory effect of AGE, the effect of AGE on hepatocellular proliferation was evaluated using partially hepatectomized rats as a liver-regeneration model. The bromodeoxyuridine-labeling indices in the livers of the AGE group were significantly lower than those in the control group at 24 h, the maximum proliferation period after partial hepatectomy. These findings indicate that AGE inhibited the development of putative preneoplastic lesions in rat hepatocarcinogenesis, involving a slowing in the proliferation rate of liver cells after partial hepatectomy.

KEY WORDS: • hepatocarcinogenesis • aged garlic extract • chemoprevention • medium-term bioassay system • glutathione S-transferase

Many epidemiologic studies have suggested that certain natural foods could have anticancer effects and may prevent tumor development. Garlic (Allium sativum), used worldwide as a food and folk medicine since ancient times, is one such natural food (1,2). The anticarcinogenic actions of garlic have been reported in numerous in-vitro and in-vivo studies; the latter have been primarily in experimental animals, but epidemiologic studies also have been reported (3–5).

Manufacturing processes significantly affect the chemical constituents of garlic preparations. Different forms contain different phytochemicals and may have different effects on human health. We have shown that a unique garlic preparation, called aged garlic extract (AGE),4 has a variety of pharmacological effects including tumor-cell growth inhibition and chemopreventive activity. These include an anti-stress effect (6), prevention of cardiovascular disease (7), senescence (8), and a protecting action against carbon tetrachloride–induced liver damage (9), liver injury (10), and immunomodulation (11–15). Moreover, AGE and its constituents inhibited the development of chemically induced tumors in the bladder (16), mammary gland (17), colon (18), esophagus (19), lung (20), skin (21), and stomach (22) in rodents.

Chronic feeding of raw garlic to rodents causes anemia, weight loss, and failure to grow (23). Shashikanth et al. (24) also reported that long-term feeding of raw-garlic extract resulted in decreased bacterial flora in the intestines as well as a reduction in serum globulins in rodents. AGE, extracted for >10 mo, was less irritating and did not produce the above-mentioned changes (23,25). Considering the low toxicity of AGE after chronic administration and its multiple biological effects, prospects for the prophylactic use of this drug in chemoprevention of cancer appear exciting.

Recently, liver cancer incidence has increased and is projected to increase further in the Japanese population (26). The medium-term bioassay system of Ito is beneficial for carcinogen risk assessment, especially in the liver, and is also useful for identifying promising chemoprevention agents (27,28). In the present study, the modifying effect of AGE, a candidate chemopreventive agent in various organs, against hepatocarcinogenesis was examined on diethylnitrosamine-induced neoplasia of the liver in male F344 rats using the Ito model.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
    Chemicals and diet. Diethylnitrosamine (DEN) and phenobarbital sodium salt were purchased from Tokyo Chemical Industry. A 0.05% phenobarbital-containing diet was prepared by Clea Japan, Inc. S-Methylcysteine was prepared by the Wakunaga Pharmaceutical Co. AGE was manufactured by Wakunaga Pharmaceutical Co., Ltd. (Osaka, Japan) in the following steps: Garlic cloves were sliced, soaked in a water–ethanol mixture, and naturally extracted and aged for >10 mo at room temperature. AGE used for the studies contained ~28.4% solid materials and 0.1% S-allylcysteine (calculated on a dried basis), a marker compound for standardization (8).

    Animals. Male Fischer 344 (F344) DuCrj rats, obtained at 5 wk of age (Charles River Japan, Inc.), were housed individually in an air-conditioned room at a temperature of 23 ± 3°C, a relative humidity of 55 ± 10%, and a 12-h light/12-h dark cycle and were given a normal diet (Clea Rodent Diet CE-2; Clea Japan Inc.) and tap water ad libitum. Rats were acclimated for 1 wk before use.

    Experiment 1. This experiment was performed to investigate the modifying effect of AGE on the second stage of hepatocarcinogenesis using the Ito test. The experimental design is shown in Table 1. A total of 55 rats were divided into 5 groups. The rats in groups 1–3 and 5 were given a single intraperitoneal (i.p.) injection of DEN (200 mg/kg body wt) dissolved in saline to initiate hepatocarcinogenesis. Rats in group 4 were administered a saline injection instead of the DEN solution. After 2 wk on a basal diet, the rats in groups 2–4 received AGE at 2 or 10 mL/kg body wt i.g. 5 times per week. Rats in group 5 were fed the pellet diet containing 0.05% phenobarbital for 6 wk. In group 1, animals were treated with distilled water instead of AGE. Animals were subjected to two-thirds partial hepatectomy (PH) (29) at wk 3 to promote the development of putative preneoplastic lesions. Animals in group 4 received saline instead of DEN solution, and they were administered PH and AGE. Food consumption and water intake were measured twice a week during the experimental period. Surviving rats in each group were killed under mild anesthesia for examination at wk 8. The liver samples, the remaining 3 lobes (right anterior lobe, right posterior lobe, caudate lobe), were immunohistochemically examined for glutathione S-transferase placental form (GST-P) expression. The in vivo experiments were approved by the Wakunaga Pharmaceutical Co. Institutional Animal Care and Use Committee.


View this table:
[in this window]
[in a new window]
  		TABLE 1 Experimental design

 
    Experiment 2. Experiment 2 was conducted to confirm the results of experiment 1. A total of 66 rats were divided into 5 groups. The rats in each group were treated using the same medium-term bioassay system as in experiment 1. They received AGE (5 or 10 mL/kg body wt, groups 2–4) or S-methylcysteine (100 mg/kg, group 5), by i.g. administration 5 times per week. Food consumption and water intake were measured twice a week during the experimental period. At wk 8, blood samples were collected from the abdominal artery under mild anesthesia, and biochemical findings and hematologic findings were observed. Removed livers were immunohistochemically examined for GST-P expression.

    Bromodeoxyuridine (BrdU) labeling indices. Experiment 3 examined sequential changes from AGE treatment in the appearance of BrdU-positive cells after PH. Twelve rats received AGE at 0, 24, and 48 h before PH. The control group (12 rats) received distilled water before PH. The liver tissue removed at PH was used as material for zero time. All rats were administered BrdU dissolved in saline (200 mg/kg, i.p.) 2 h before death. Six rats in each group were killed at 6 h and 24 h after the final i.g. administration. BrdU-positive cells were immunohistochemically stained and scored (over 4,000 hepatocytes per rat). To confirm the antiproliferating effect of AGE, experiment 4 measured the change in the appearance of BrdU-positive cells in response to AGE treatment at 24 h after the final i.g. administration. Two groups of 12 rats received either AGE or distilled water at 0, 24, and 48 h before PH, and the same procedure described above was performed.

    Tissue processing. At autopsy, livers were excised, and 4- to 5-mm-thick sections were cut with a razor blade. Three slices, 1 each from the right posterior, anterior, and caudate lobes, were fixed in buffered formalin for immunohistochemical examination of GST-P in experiments 1 and 2 and BrdU in experiments 3 and 4.

    Immunohistochemical staining of GST-P and BrdU. The avidin-biotin-peroxidase complex (ABC) method described by Hsu et al. (30) was used to demonstrate GST-P-positive foci, putative preneoplastic lesions (31–33), and proliferating cells. After deparaffinization (hydration), liver sections were treated sequentially with normal goat serum or horse serum, rabbit anti-GST-P antibody (1:1,000) (Medical & Biological Laboratories) or mouse anti-bromodeoxyuridine (1:30) (Dako Japan), biotin-labeled goat anti-rabbit or -mouse IgG, and ABC (Elite Vectastain, Vector Labs). The sites of peroxidase binding were demonstrated by the diaminobenzidine method. Sections were then counterstained with hematoxylin for microscopic examination.

The numbers and areas of GST-P-positive foci >0.2 mm in diameter and the total areas of the examined liver sections were measured using a Fujix Digital Camera System HC-2500 (Fuji Photo Film), Adobe Photoshop version 5.0J, and Image-Pro Plus version 3.0.1J.

The numbers of BrdU-positive cells were measured over 4,000 cells at random per rat, and the mean and BrdU labeling index (LI) were calculated.

    Statistical analysis. Values are expressed as means ± SD. Statistical analysis of the observed values was performed using SAS release 6.12 (SAS Institute Inc.). ANOVA and Dunnett's multiple comparison, or F-test and Student's t-test were used to determine significant differences among means.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
    General observations. Body-weight, food-consumption, and water-intake changes in experiment 1 are indicated in Figure 1. Immediately after DEN administration, body weight, food consumption, and water intake decreased in groups 1–3 and 5. From wk 2 to wk 8, body weight gain was observed in animals administered AGE (groups 2 and 3) or distilled water (group 4), but food consumption and water intake decreased. No significant differences in body weight, food consumption, or water intake among the control (group 1) and AGE groups (groups 2–4) were observed during the experimental period.


Figure 1
View larger version (24K):
[in this window]
[in a new window]
  		FIGURE 1  Changes in rat body weight (A), food consumption (B), and water intake (C). Each point represents the mean ± SEM. Asterisks indicate significantly different from control-group values at *P < 0.05 and **P < 0.01.

 
In experiment 2, no significant differences in body weight, food consumption, or water intake were observed among the experimental groups (data not shown). No biochemical or hematologic changes such as in numbers of leukocytes, erythrocytes, or platelets in blood, or activities of glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, alkaline phosphatase, lactate dehydrogenase, creatinine phosphokinase, and so on in response to AGE were observed (data not shown).

    Body and liver weights, food consumption, and water intake. Final body weights, relative liver weight (g/100 g body wt), food consumption, and water intake in each group are summarized in Table 2. In experiment 1, the final body weights of group 5 were higher than those in group 1. Liver weights of group 5 were also significantly higher than those in group 1. No significant differences in food consumption or water intake among the control (group 1) and AGE groups (groups 2–4) were observed during the experimental period.


View this table:
[in this window]
[in a new window]
  		TABLE 2 Mean final body weight, liver weight, food consumption and water-intake data1–3

 
In experiment 2, no significant differences in final body weights, relative liver weight, food consumption, or water intake among the groups were observed during the experimental period.

    Number and areas of GST-P-positive foci in the liver. In experiment 1, phenobarbital (group 5), as a promoter of hepatocarcinogenesis, enhanced the numbers (33.9%) and areas (105.3%) of GST-P-positive foci as reported previously (34). However, the numbers of GST-P-positive foci in the livers of rats treated with AGE doses of 2 mL/kg (group 2) or 10 mL/kg (group 3) decreased 29.5% and 55.1%, respectively. The areas of GST-P-positive foci also decreased 34.2% and 63.2%, respectively. These results indicate that AGE reduced the numbers and areas of GST-P-positive foci in a dose-dependent manner. In addition, AGE without DEN (group 4) produced no significant change, suggesting noninitiation (Fig. 2).


Figure 2
View larger version (40K):
[in this window]
[in a new window]
  		FIGURE 2  Quantitative values of GST-P liver cell foci in experiments 1 and 2. Asterisks indicate significantly different from the DEN-alone group at *P < 0.05 and **P < 0.01.

 
In experiment 2, SMC (group 5), as a positive control, reduced the numbers (41.5%) and area (41.5%) of GST-P-positive foci as reported previously (35). Moreover, AGE (groups 2 and 3) reduced the numbers and areas of GST-P-positive foci in a dose-dependent manner (Fig. 2).

    BrdU labeling indices. To examine the effect of AGE on hepatocellular regeneration, BrdU immunopositive cells after PH were counted per >4,000 liver cells. BrdU LI increased several hours after PH. BrdU LI of the control group was 0.16%, 0.17%, and 22.37% at 0, 6, 24 h after PH, respectively. BrdU LI of the AGE group was lower than that of the control group (Fig. 3A). The significant reduction in BrdU incorporation by AGE was reproducibly observed in hepatic cells 24 h after PH, when the regeneration rate reached a maximum (Fig. 3B).


Figure 3
View larger version (14K):
[in this window]
[in a new window]
  		FIGURE 3  BrdU labeling index in the liver of rats treated with AGE. Circle, control; square, AGE. Asterisk indicates significantly different from the control at *P < 0.05.

 
    Histological examination of the liver and other organs. Gross and microscopic examination of the liver revealed no histological changes in experiments 1 and 2 (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
We investigated the modifying effects of AGE on rat-liver carcinogenesis induced by DEN, using the medium-term hepatocarcinogenesis assay (36). AGE significantly suppressed the development of GST-P-positive foci, or preneoplastic lesions, thereby preventing cancer induction.

This assay protocol was established to evaluate modifying activities of various compounds on hepatocarcinogenesis, and it confirmed that the degree of induction of GST-P-positive foci was directly correlated with the incidence of hepatocellular carcinomas confirmed by a long-term in vivo experiment (37,38). In addition, the assay system enabled identification of the step—the initiation or promotion phase—at which an active chemical was functional during carcinogenesis.

AGE was administered in this study during the promotion stage of hepatocarcinogenesis, and the results suggest that AGE has antipromotion activities to inhibit the development of GST-P-positive foci.

Food components and natural products can modify carcinogenesis in different ways, such as modification of Phase 1 enzymes for carcinogen activation, detoxification of carcinogen through Phase 2 enzymes, scavenging DNA agents, suppressing proliferation of early, preneoplastic lesions, or inhibition of certain properties of cancer cells. Administration of AGE significantly suppressed the incorporation of BrdU in liver cells after PH. This finding suggested that inhibition of hepatocellular proliferation in DEN-induced rat liver carcinogenesis is a possible mechanisms by which AGE prevents hepatocarcinogenesis.

AGE contains a variety of organosulfur compounds (OCSs), such as S-allylcysteine, a marker compound for standardization of AGE, S-allylmercaptocysteine (9,10), diallylsulfide, allylmethylsulfide (39), fluctosylarginine (40), and allixin (41–43). Several OCSs were analyzed for their chemopreventive activities on the rat liver medium-term bioassay. Oil-soluble OCSs such as methylpropyldisulfide and propylenesulfide, and water-soluble OCSs, such as S-methylcysteine and cysteine, were shown to inhibit the development of GST-P-positive foci (44–46). However, allylsulfides, representative of allicin-derived oil-soluble OCSs, including diallylsulfide, diallyltrisulfide, and allylmethyltrisulfide, conversely, enhanced GST-P focus formation (44,45).

Garlic contains a limited number of characteristic S-containing precursor peptides including S-allyl cysteinesulfoxide (alliin), which are enzymatically converted to alk(en)yl thiosulfinates such as allylthiosulfinate (allicin). The resultant thiosulfinates are then rapidly transformed during processing to various volatile S-compounds, including allylmercaptocysteine, allylsulfides, and vinyldithiins, by nonenzymatic chemical cascade reactions. Moreover, the formation of these volatile S-compounds depends on the methods of processing (47). Indeed, dehydrated garlic powder retains S-containing precursor peptides such as {gamma}-glutamyl-S-allylcysteine and alliin, but these completely disappear in garlic oil produced by steam distillation and oil macerate. Garlic oil exclusively contains allylsulfides, and oil macerate contains vinyldithiins and ajoene in addition to allylsulfides. AGE used in this study was rich in water-soluble S-compounds such as SAC and SAMC but had little of the oil-soluble compounds (39). These findings suggest that garlic products differ in their biological activities and toxicity. Accordingly, confirmation of the safety, not to mention activity, of individual garlic preparations through intensive toxicity studies is important.

Epidemiologic studies indicate that the anticarcinogenic action of biologically active substances is mostly limited to an early stage of carcinogenesis. Therefore, it appears to be important to start prevention of cancer by nutrition as early as possible and to adhere to it over a long period (48). Low-toxicity compounds are desirable for continual intake to prevent onset of chronic disorders and cancer with a relatively long latent period. Sumiyoshi et al. reported no toxic symptoms from AGE even in the case of continual administration at a dose of 2,000 mg/kg in rats 5 times per week for 6 mo (25). Indeed, no apparent biochemical or hematologic changes were observed in experiments 1 and 2 (data not shown). The safety of AGE has been confirmed by several toxicity studies in animals (23,25,49) and through human use as a medicinal material and a health food for a considerable time.

In conclusion, this study demonstrates that AGE shows a significant inhibitory effect on the development of GST-P-positive foci in the rat liver medium-term bioassay system. The findings suggest that daily intake of AGE could exert a chemopreventive effect on hepatocarcinogenesis in addition to, as previously reported, carcinogenesis of the bladder, mammary gland, colon, esophagus, lung, skin, and stomach.


   FOOTNOTES
 
1 Published in a supplement to The Journal of Nutrition. Presented at the symposium "Significance of Garlic and Its Constituents in Cancer and Cardiovascular Disease" held April 9–11, 2005 at Georgetown University, Washington, DC. The symposium was sponsored by Strang Cancer Prevention Center, affiliated with Weill Medical College of Cornell University, and Harbor-UCLA Medical Center, and co-sponsored by American Botanical Council, American Institute for Cancer Research, American Society for Nutrition, Life Extension Foundation, General Nutrition Centers, National Nutritional Foods Association, Society of Atherosclerosis Imaging, Susan Samueli Center for Integrative Medicine at the University of California, Irvine. The symposium was supported by Alan James Group, LLC, Agencias Motta, S.A., Antistress AG, Armal, Birger Ledin AB, Ecolandia Internacional, Essential Sterolin Products (PTY) Ltd., Grand Quality LLC, IC Vietnam, Intervec Ltd., Jenn Health, Kernpharm BV, Laboratori Mizar SAS, Magna Trade, Manavita B.V.B.A., MaxiPharm A/S, Nature's Farm, Naturkost S. Rui a.s., Nichea Company Limited, Nutra-Life Health & Fitness Ltd., Oy Valioravinto Ab, Panax, PT. Nutriprima Jayasakti, Purity Life Health Products Limited, Quest Vitamins, Ltd., Sabinco S.A., The AIM Companies, Valosun Ltd., Wakunaga of America Co. Ltd., and Wakunaga Pharmaceutical Co., Ltd. Guest editors for the supplement publication were Richard Rivlin, Matthew Budoff, and Harunobu Amagase. Guest Editor Disclosure: R. Rivlin has been awarded research grants from Wakunaga of America, Ltd. and received an honorarium for serving as co-chair of the conference; M. Budoff has been awarded research grants from Wakunaga of America, Ltd. and received an honorarium for serving as co-chair of the conference; and Harunobu Amagase is employed by Wakunaga of America, Ltd. Back

2 Author disclosure: N. Uda, N. Kashimoto, I. Sumioka, and S. Sumi are employed by Wakunaga Pharaceutical Co. Back

4 Abbreviations used: AGE, aged garlic extract; BrdU, 5-bromo-2'-deoxyuridine; DEN, N-diethylnitrosamine; GST-P, glutathione S-transferase placental form; OCSs, organosulfur compounds; PH, two-thirds partial hepatectomy; SMC, S-methylcysteine. Back


   LITERATURE CITED
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 

1. Wargovich MJ, Uda N, Woods C, Velasco M, McKee K. Allium vegetables: their role in the prevention of cancer. Biochem Soc Trans. 1996;24:811–4.[Medline]

2. Wargovich MJ, Uda N. Allium vegetables and the potential for chemoprevention of cancer. Adv Exp Med Biol. 1996;401:171–7.[Medline]

3. Dausch JG, Nixon DW. Garlic: A review of its relationship to malignant disease. Prev Med. 1990;19:346–61.[Medline]

4. Stavric B. Role of chemopreventers in human diet. Clin Biochem. 1994;27:319–32.[Medline]

5. Dorant E, van den Brandt PA, Goldbohm RA, Hermus RJJ, Sturmans F. Garlic and its significance for the prevention of cancer in humans: A critical view. Br J Cancer. 1993;67:424–9.[Medline]

6. Takasugi N, Kotoo K, Fuwa T, Saito H. Effect of garlic on mice exposed to various stresses. Oyo Yakuri. 1984;28:991–1002.

7. Ide N, Lau BHS. Garlic compounds protect vascular endothelial cells from oxidized low density lipoprotein-induced injury. J Pharm Pharmacol. 1997;49:908–11.[Medline]

8. Moriguchi T, Saito H, Nishiyama N. Aged garlic extract prolongs longevity and improves spatial memory deficit in senescence-accelerated mouse. Biol Pharm Bull. 1996;19:305–7.[Medline]

9. Nakagawa S, Kasuga S, Matsuura H. Prevention of liver damage by aged garlic extract and its components in mice. Phytother Res. 1989;3:50–3.

10. Sumioka I, Matsuura T, Kasuga S, Itakura Y, Yamada K. Mechanisms of protection by S-allylmercaptocysteine against acetaminophen-induced liver injury on mice. Jpn J Pharmacol. 1998;78:199–207.[Medline]

11. Lau BH, Yamasaki T, Gridley DS. Garlic compounds modulate macrophage and T-lymphocyte functions. Mol Biother. 1991;3:103–7.[Medline]

12. Morioka N, Sze LL, Morton DL, Irie RF. A protein fraction from aged garlic extract enhances cytotoxicity and proliferation of human lymphocytes mediated by interleukin-2 and concanavalin-A. Cancer Immunol Immunother. 1993;37:316–22.[Medline]

13. Kyo E, Uda N, Kakimoto M, Yokoyama K, Ushijima M, Sumioka I, Kasuga S, Itakura Y. Anti-allergic effects of aged garlic extract. Phytomedicine. 1997;4:335–40.

14. Kyo E, Uda N, Suzuki A, Kakimoto M, Ushijima M, Kasuga S, Itakura Y. Immunomodulation and anti-tumor activities of aged garlic extract. Phytomedicine. 1998;5:259–67.

15. Kyo E, Uda N, Kasuga S, Itakura Y, Sumiyoshi H. Immunomodulatory agents from plants. In: In: Wagner H, editor. Garlic as an immunostimulant. Switzerland: Birkhäuser Verlag Basel; 1999. p. 273–288.

16. Lau BH, Woolly JL, Marsh CL, Barker GR, Koobs DH, Torrey RR. Superiority of intralesional immunotherapy with Corynebacterium parvum and Allium sativum in control of murine transitional cell carcinoma. J Urol. 1986;136:701–5.[Medline]

17. Amagase H, Milner JA. Impact of various sources of garlic and their constituents on 7,12-dimethylbenz[a]anthracene binding to mammary cell DNA. Carcinogenesis. 1993;14:1627–31.[Abstract/Free Full Text]

18. Sumiyoshi H, Wargovich MJ. Chemoprevention of 1,2-dimethylhydrazine-induced colon cancer in mice by naturally occurring organosulfur compounds. Cancer Res. 1990;50:5084–7.[Abstract/Free Full Text]

19. Wargovich MJ, Woods C, Eng VWS, Stephens LC, Gray K. Chemoprevention of n-nitrosomethyl-benzylamine–induced esophageal cancer in rats by the naturally occurring thioether, diallyl sulfide. Cancer Res. 1988;48:6872–5.[Abstract/Free Full Text]

20. Sparnins VL, Mott AW, Barany G, Wattenberg LW. Effects of allyl methyl trisulfide on glutathione S-transferase activity and BP-induced neoplasia in the mouse. Nutr Cancer. 1986;8:211–5.[Medline]

21. Nishino H, Iwashima A, Itakura Y, Matsuura H, Fuwa T. Antitumor-promoting activity of garlic extracts. Oncology. 1989;46:277–80.[Medline]

22. Wattenberg LW, Sparnins VL, Barany G. Inhibition of N-nitrosodiethylamine carcinogenesis in mice by naturally occurring organosulfur compounds and monoterpenes. Cancer Res. 1989;49:2689–92.[Abstract/Free Full Text]

23. Nakagawa S, Masamoto K, Sumiyoshi H, Kunihiro K, Fuwa T. Effect of raw garlic and extracted-aged garlic juice on growth of young rats and their organs after per oral administration. J Toxicol Sci. 1980;5:91–112.[Medline]

24. Shashikanth KN, Basappa SC, Murthy VS. Effect of feeding raw and boiled garlic (A. sativum L.) extracts on the growth, caecal microflora and serum proteins of albino rats. Nutr Rep Int. 1986;33:313–9.

25. Sumiyoshi H, Kanezawa A, Masamoto K, Harada H, Nakagami S, Yokota A, Nishikawa M, Nakagawa S. Chronic toxicity test of garlic extract in rats. J Toxicol Sci. 1984;9:61–75.[Medline]

26. The Research Group for Population-based Cancer Registration in JapanCancer incidence and incidence rates in Japan in 1997: estimates based on data from 12 population-based cancer registries. Jpn J Clin Oncol. 2002;32:318–22.[Free Full Text]

27. Ito N, Imaida K, Asamoto M, Shirai T. Early detection of carcinogenic substances and modifiers in rats. Mutat Res. 2000;462:209–17.[Medline]

28. Moore MA, Tsuda H, Tamano S, Hagiwara A, Imaida K, Shirai T, Ito N. Marriage of a medium-term liver model to surrogate markers–a practical approach for risk and benefit assessment. Toxicol Pathol. 1999;27:237–42.[Abstract/Free Full Text]

29. Higgins GM, Anderson RM. Experimental pathology of the liver. I. Restoration of the liver of the white rat following partial surgical removal. Arch Pathol. 1931;12:186–202.

30. Hsu SM, Raine L, Fanger H. Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem. 1981;29:577–80.[Abstract]

31. Ito N, Tsuda H, Tatematsu M, Inoue T, Tagawa Y, Aoki T, Uwagawa S, Kagawa M, Ogiso T, et al. Enhancing effects of various hepatocarcinogenesis on induction of preneoplastic glutathione S-transferase placental form positive foci in rats-an approach for a new medium-term bioassay system. Carcinogenesis. 1988;9:387–94.[Abstract/Free Full Text]

32. Sato K, Kitahara A, Satoh K, Ishikawa T, Tatematsu M, Ito N. The placental form of glutathione S-transferase as a new marker protein for preneoplasia in rat chemical hepatocarcinogenesis. Gann. 1984;75:199–202.[Medline]

33. Satoh K, Kitahara A, Soma Y, Inaba Y, Hatayama I, Sato K. Purification, induction and distribution of placental glutathione transferase: a new marker enzyme for preneoplastic cells in rat chemical carcinogenesis. Proc Natl Acad Sci USA. 1985;82:3964–8.[Abstract/Free Full Text]

34. Hagiwara A, Matsuda T, Tamano S, Kitano M, Imaoka S, Funae Y, Takesada Y, Shirai T, Fukushima S. Dose-related increases in quantitative values for altered hepatocytic foci and cytochrome P-450 levels in the livers of rats exposed to phenobarbital in a medium-term bioassay. Cancer Lett. 1996;110:155–62.[Medline]

35. Takada N, Yano Y, Wanibuchi H, Otani S, Fukushima S. S-Methylcysteine and cysteine are inhibitors of induction of glutathione S-transferase placental form-positive foci during initiation and promotion phases of rat hepatocarcinogenesis. Jpn J Cancer Res. 1997;88:435–42.

36. Ito N, Hasegawa R, Imaida K, Takahashi S, Shirai T. Medium-term rat liver bioassay for rapid detection of carcinogens and modifiers of hepatocarcinogenesis. Drug Metab Rev. 1994;26:431–42.[Medline]

37. Ogiso T, Tatematsu M, Tamano S, Tsuda H, Ito N. Comparative effects of carcinogens on the induction of placental glutathione S-transferase-positive liver nodules in a short-term assay and of hepatocellular carcinomas in a long-term assay. Toxicol Pathol. 1985;13:257–65.[Medline]

38. Ogiso T, Tatematsu M, Tamano S, Hasegawa R, Ito N. Correlation between medium-term liver bioassay system data and results of long-term testing in rats. Carcinogenesis. 1990;11:561–6.[Abstract/Free Full Text]

39. Weinberg DS, Manier ML, Richardson MD, Haibach FG. Identification and quantification of organosulfur compliance markers in a garlic extract. J Agric Food Chem. 1993;41:37–41.

40. Ide N, Lau BHS, Ryu K, Matsuura H, Itakura Y. Antioxidant effects of fructosyl arginine, a maillard reaction product in aged garlic extract. J Nutr Biochem. 1999;10:372–6.[Medline]

41. Kodera Y, Matuura H, Yoshida S, Sumida T, Itakura Y, Fuwa T, Nishino H. Allixin, a stress compound from garlic. Chem Pharm Bull (Tokyo). 1989;37:1656–8.

42. Nishino H, Nishino A, Takayasu J, Iwashima A, Itakura Y, Kodera Y, Matsuura H, Fuwa T. Antitumor-promoting activity of allixin, a stress compound produced by garlic. Cancer J. 1990;3:20–1.

43. Yamasaki T, Teel RW, Lau BH. Effect of allixin, a phytoalexin produced by garlic, on mutagenesis, DNA-binding and metabolism of aflatoxin B1. Cancer Lett. 1991;59:89–94.[Medline]

44. Takada N, Matsuda T, Otoshi T, Yano Y, Otani S, Hasegawa T, Nakae D, Konishi Y, Fukushima S. Enhancement by organosulfur compounds from garlic and onions of diethylnitrosamine-induced glutathione S-transferase positive foci in the rat liver. Cancer Res. 1994;54:2895–9.[Abstract/Free Full Text]

45. Takada N, Kitano M, Chen T, Yano Y, Otani S, Fukushima S. Enhancing effects of organosulfur compounds from garlic and onions on hepatocarcinogenesis in rats: Association with increased cell proliferation and elevated ornithine decarboxylase activity. Jpn J Cancer Res. 1994;85:1067–72.

46. Fukushima S, Takada N, Hori T, Wanibuchi H. Cancer prevention by organosulfur compounds from garlic and onion. J Cell Biochem. 1997;Suppl 27:100–5.

47. Iberl E, Winkler G, Knobloch K. Products of allicin transformation: Ajoenes and dithiins, characterization and their determination by HPLC. Planta Med. 1990;56:202–11.[Medline]

48. Frohlich RH, Kunze M, Kiefer I. Cancer preventive value of natural, non-nutritive food constituents. Acta Med Austriaca. 1997;24:108–13.[Medline]

49. Yoshida S, Hirao Y, Nakagawa S. Mutagenicity and cytotoxicity tests of garlic. J Toxicol Sci. 1984;9:77–86.[Medline]





This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Uda, N.
Right arrow Articles by Fukushima, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Uda, N.
Right arrow Articles by Fukushima, S.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]