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Inhibition by licorice flavonoid oil of glutathione S-transferase–positive foci in the medium-term rat hepatocarcinogenesis bioassay
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Nutrition Research 30 (2010) 74 – 81 www.nrjournal.com
Inhibition by licorice flavonoid oil of glutathione S-transferase–positive foci in the medium-term rat hepatocarcinogenesis bioassay Kaku Nakagawa a , Kazunori Hosoe a , Takayoshi Hidaka b , Kyoko Nabae c , Mayumi Kawabe c , Mitsuaki Kitano d,⁎ b
a QOL Division, Kaneka Corporation, Kita-ku, Osaka 530-8288, Japan Department of Health Sciences and Social Pharmacy, Faculty of Pharmaceutical Sciences, Kobe Gakuin University, Chuo-ku, Kobe 650-8586, Japan c DIMS Institute of Medical Science, Inc., Azai-cho, Ichinomiya, Aichi 491-0113, Japan d Pharmacology and Toxicology Laboratory, Frontier Biochemical and Medical Research Laboratories, Kaneka Corporation, Takasago, Hyogo 676-8688, Japan Received 19 October 2009; revised 5 December 2009; accepted 17 December 2009
Abstract Licorice flavonoid oil (LFO) is a new functional food ingredient consisting of hydrophobic licorice polyphenols in medium-chain triglycerides. Recently, it was reported that licorice and its derivatives have anticarcinogenic activity in some types of tumors. However, the anticarcinogenic activity has not been identified in the liver, which is a major target organ for carcinogenesis in human. Therefore, we hypothesized that LFO has antihepatocarcinogenic activity, and we tested this hypothesis using the rat medium-term liver bioassay for carcinogens. Six-week-old male F344 rats (15 animals/group) received N-diethylnitrosamine (200 mg/kg by intraperitoneal injection) to initiate carcinogenesis. From the second week after initiation, animals received a 6-week regimen of either LFO concentrate solution (0, 150, 300, or 600 mg/kg) intragastrically or phenobarbital sodium salt in the diet (500 ppm) as a positive control. During the third week after initiation, animals were subjected to a two-thirds partial hepatectomy. During the eighth week of the treatment period, liver samples were taken from animals and examined immunohistochemically for expression of glutathione S-transferase placental form. No increase in the number of glutathione S-transferase placental form–positive liver foci was observed in all LFO groups compared with the negative control (solvent) group, and the number of foci in the 600 mg/kg LFO group was significantly lower than that in the negative control group. These results indicate that LFO concentrate solution has a significant inhibitory effect on liver carcinogenesis at 600 mg/kg. © 2010 Elsevier Inc. All rights reserved. Keywords: Abbreviations:
Licorice flavonoid oil; Glycyrrhiza glabra; Medium-term liver bioassay; Rat; Carcinogenesis DEN, nitrosodiethylamine; GST-P, glutathione S-transferase placental form; LFO, licorice flavonoid oil; MCTs, medium-chain triglycerides; S.PB, phenobarbital sodium salt.
1. Introduction Licorice flavonoid oil (LFO), derived from licorice, Glycyrrhiza glabra L., is a new functional food ingredient consisting of licorice hydrophobic polyphenols in medium⁎ Corresponding author. Tel.: +81 79 445 2427; fax: +81 79 445 2699. E-mail address: [email protected] (M. Kitano). 0271-5317/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.nutres.2009.12.005
chain triglycerides (MCTs). Licorice flavonoid oil was developed by Kaneka Corporation (Osaka, Japan) under the brand name Kaneka Glavonoid. Recently, we found that LFO is effective in reducing visceral fat accumulation and in suppressing elevated blood glucose levels in obese diabetic KK-Ay mice [1]. The mechanism of the antiobesity effect of LFO was investigated using a high-fat diet–induced obese C57BL/6J mouse model, and the data suggested that this
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effect was mediated by modification of the expression levels of lipid metabolism-related genes in the liver [2]. Furthermore, a double-blind, placebo-controlled clinical trial showed that LFO was effective in suppressing body weight gain by reducing body fat mass in overweight volunteers [3,4]. These studies indicate that LFO may help to prevent lifestyle-related diseases, such as obesity associated with metabolic syndrome. Although licorice has a long history of human consumption, information on the safety of the hydrophobic fraction of G glabra is limited. Clinical studies showed that LFO is safe, because no clinically significant adverse events occurred when it was given daily to healthy or overweight subjects for up to 12 weeks [3,5]. Furthermore, in a 90-day subchronic toxicity study in rats that was part of a series of preclinical safety assessments of LFO, we demonstrated that LFO had low toxicity [6]. In addition, the genotoxicity of LFO was investigated using a bacterial reverse mutation assay, a chromosomal aberration assay, and a micronucleus in vivo assay [7]. The results of the bacterial reverse mutation and micronucleus in vivo assays were all negative; however, at higher concentrations, LFO induced clastogenic activity during a short period with an S9 mix in a chromosomal aberration test. The results obtained using all 3 methods of genotoxicity evaluation indicated that LFO was substantially nongenotoxic in vivo. On the other hand, there are indications that licorice extract and its components have antitumor activity on several types of tumors [8,9]. Furthermore, the anticarcinogenic activity has been identified in 1,2-dimethylhydrazine– induced colon and lung tumors in mice [10]. In this present study, we hypothesized that LFO, hydrophobic polyphenols from licorice glabra, has an anticarcinogenic effect in the liver, which is a major target organ for carcinogenesis in human. If this hypothesis is correct, LFO may be useful in human nutrition, not only for prevention of metabolic syndrome but also for prevention of liver cancer development. This hypothesis was tested using the rat medium-term liver bioassay for carcinogens (Ito test). This bioassay is considered to be reliable in predicting the promoting or inhibitory activity of chemicals with respect to hepatocarcinogenesis, based on background data for more than 300 chemicals [11-14], and was recommended as an alternative to long-term carcinogenicity testing at the International Conference on Harmonization [15].
2. Methods and materials 2.1. Study design (Good Laboratory Practice and Test Guidance) This study was performed at the DIMS Institute of Medical Science, Inc (Ichinomiya, Japan), in accordance with the Good Laboratory Practice standards of the Japanese Ministry of Health and Welfare Ordinance No. 21 (March 26, 1997) and in compliance with the Guidelines for
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Carcinogenicity Studies of Drugs 3.2 (In Vivo Additional Tests for Detection of Carcinogenicity) of the Japanese Ministry of Health and Welfare (Notification Iyakushin No. 1607; November 1, 1999). The use of animals and the experimental protocol were performed in accordance with the Guidelines for Animal Experimentation, Japanese Association for Laboratory Animal Science [16]. The inhouse guidelines for the Care and Use of Laboratory Animals were used, and the protocol was approved by the DIMS Institute of Medical Science, Inc. 2.2. LFO test substance An ethanol extract of licorice (G glabra) root was obtained to prepare the LFO test substance. After filtration and concentration, the ethanolic layer was mixed with MCTs with a fatty-acid composition of C8:C10 = 99:1. The concentration of glabridin, the major component of the solution, was adjusted to 3% (wt/wt). This solution was named “LFO concentrate solution” and given the brand name Kaneka Glavonoid. The LFO concentrate solution comprised 30% licorice ethanol extract and 70% MCTs, with the latter functioning as a carrier/diluent. The licorice ethanol extract consisted almost entirely of hydrophobic polyphenols, including licorice prenyl flavonoids such as glabridin, which are active components of LFO. In the present study, an LFO concentrate solution containing 3.0% glabridin (Lot No. 51217002; Kaneka Corporation) was used as the LFO test substance. 2.3. Preparation of test substance solution The LFO test substance (LFO concentrate solution) was diluted with MCTs (Lot No. C030401; Riken Vitamin Co, Ltd, Tokyo, Japan) to adjust it to the intended concentration. Working solutions were stored in a refrigerator (recorded temperature, 4-7°C), and their stability during the treatment period (8 weeks) was analyzed at the termination of treatment and was confirmed to be appropriate. 2.4. Other chemicals N-nitrosodiethylamine (DEN), an initiator of carcinogenesis, and phenobarbital sodium salt (S.PB), used as a positive control for a promoter of carcinogenesis, were purchased from Tokyo Kasei Kogyo (Tokyo, Japan). 2.5. Animals One hundred 5-week-old male F344 rats were purchased from Charles River Japan, Inc. (Ibaraki, Japan) and used in the study after a 6-day quarantine/acclimation period. The rats were housed in groups of 3 in transparent plastic cages with chipped hardwood bedding and received an irradiated powder diet (MF diet; Oriental Yeast Co, Ltd, Tokyo, Japan) and water ad libitum. The animal room was maintained on a 12-hour light/12-hour dark cycle; temperature and relative humidity were maintained within the ranges of 19 to 24°C and 51% to 60%, respectively.
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2.6. Groups and numbers of animals
2.8. Gross pathology
Of the 100 rats, 93 were allocated to 7 groups (15 rats/ group for groups 1-5, 9 rats/group for groups 6 and 7) using a randomized block design. The mean body weights of the 7 groups at the time of group allocation were similar. Grouping was performed on the day before administration of the initiator.
All surviving animals were fasted overnight before the day of their termination. Blood samples were drawn from the abdominal aorta under ether anesthesia; the organs and tissues in the thoracic and abdominal cavities were examined macroscopically, lesions were recorded individually, and the livers were quickly removed. At necropsy, the liver weight of all surviving animals was measured, and the relative liver weight (ratio of absolute liver weight to final body weight) was calculated.
2.7. Experimental design The experiment was performed according to the protocol of the medium-term liver bioassay (Ito test) [14,17-19]. As shown in Fig. 1, animals in groups 1 to 5 were treated with DEN (200 mg/kg body weight/5 ml saline, IP, single injection) at the commencement of the experiment. Rats in groups 6 and 7 were given the vehicle alone instead of the carcinogen. Starting 2 weeks later, the rats were treated with LFO working solution by oral gavage (2 mL/kg body weight) once a day, or fed a diet containing S.PB at 500 ppm as a positive control until week 8. Animals in control groups 1 and 6 were also treated with MCTs, the vehicle for LFO, by gavage for 6 weeks. Three weeks after the beginning of the experiment (1 week after the beginning of the administration of LFO test substance), a two-thirds partial hepatectomy was performed on all animals under anesthesia to maximize any interactions between cellular proliferation and the effects of the test chemicals. All animals were checked for their general condition, and findings on behavior, toxicity, and mortality were individually recorded. Body weight was measured for all animals at the initiation of the experiment and at weekly intervals thereafter. At the termination of treatment, body weight was determined after overnight fasting (final body weight). Food and water consumption per cage was measured weekly after initiation of the experiment. All surviving rats in each group were euthanized by whole blood drawing under ether anesthesia at week 8 for pathologic and immunohistochemical examination.
2.9. Quantitative immunohistochemical analysis of hepatocyte foci For all surviving animals, roughly 5-mm-thick sections from the 3 liver lobes (right lateral lobe, cranial part; right lateral lobe, caudal part; and caudate lobe, caudal part) were fixed in 10% buffered formalin solution, embedded in paraffin wax, serially sectioned, and stained immunohistochemically for glutathione S-transferase placental form (GST-P; avidin-biotin complex [ABC] method). The numbers and areas of GST-P–positive foci larger than 0.2 mm in diameter per square centimeter (cm2) of liver section were measured using a color video image processor (IPAPWIN; Sumika Technos, Co, Osaka, Japan) [20]. 2.10. Electron microscopic analysis of liver Three rats each in groups 6 and 7 were selected for electron microscopic examination. Small portions of the liver (right lateral lobe, cranial lobe) were fixed in glutaraldehyde solution. The samples were analyzed at Bozo Research Center (Shizuoka, Japan) under Good Laboratory Practice conditions. 2.11. Statistical analyses The statistical significance of the differences between the DEN-initiated control (group 1) and treated groups for each parameter (excluding general condition and food and water consumption) was analyzed. Comparisons of group 1 and the
Fig. 1. Protocol for the study, based on the medium-term liver bioassay. The bioassay was performed using 6-week-old F344 male rats. Glutathione LFO– positive foci were counted at week 8. Values in parentheses are numbers of animals. Animals in groups 1 and 6 were given MCT; those in groups 2, 3, 4, and 7 were given the LFO concentrate solution at 150, 300, 600, and 600 mg kg−1 d−1, respectively; and those in group 5 were given S.PB at 500 ppm in a basal diet for 6 weeks. ▾, DEN 200 mg/kg body weight IP; ▿, saline IP; O, two-thirds partial hepatectomy.
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LFO-treated groups (groups 2, 3, and 4) for numerical data on body weight, organ weight, and quantitative values for GST-P–positive hepatocytic foci were performed using Bartlett test. If the data were found to be homogeneous, they were analyzed with Dunnett multiple comparison test (1-sided); if not, the data were analyzed with Steel test (1-sided). The significance of the differences between the DEN-initiated group 1 (control) and group 5 (S.PB) and the noninitiated group 6 (control, not treated with LFO or S.PB) and group 7 (LFO 600 mg/kg) for numerical data were assessed using the F test. If the data were found to be homogeneous, they were analyzed with the Student t test (1sided), and otherwise, they were tested with the Welch test (1-sided). The significance of the differences in the incidence data for gross pathology was analyzed using Fisher exact probability test (1-sided). The number of animals per group was determined according to the past successful results of this model [17,21]. These analyses were conducted with Stat Light software 2000 (Yukms Co Ltd, Tokyo, Japan). 3. Results 3.1. Survival rate and general condition No mortality or changes in general conditions related to administration of the test material were seen in any group. One rat in the group receiving 600 mg/kg LFO (group 4) was found supine on day 24 and dead on day 25. The cause of death was considered to be related to surgical failure because the liver was found to be discolored on macroscopic examination. 3.2. Food and water consumption The range of values for food intake during study period were as follows: 13.3 to 15.2 g animal−1 d−1 in the control group initiated with DEN (group 1), 13.1 to 14.6 g animal−1 d−1 in the 150 mg/kg LFO group (group 2), 14.0 to 16.6 g animal−1 d−1 in the 300 mg/kg LFO group (group 3), 13.9 to 18.2 g animal−1 d−1 in the 600 mg/kg LFO group (group 4), 13.4 to 18.4 g animal−1 d−1 in the S.PB group
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(group 5), 13.7 to 16.3 g animal d in the control group not initiated with DEN (group 6), and 14.1 to 16.7 g animal−1 d−1 in the 600 mg/kg LFO group not initiated with DEN (group 7). In the DEN-initiated groups, there was a tendency toward greater food consumption in the 300 mg/ kg LFO group (group 3) at week 3 and weeks 6 to 8 and in the 600 mg/kg LFO group (group 4) at weeks 5 and 7, compared with the control group (group 1). A tendency toward greater food consumption was also observed in the S.PB group (group 5) from weeks 3 to 7 compared with the control group (group 1). No change in food consumption was observed in the 2 groups not initiated with DEN (groups 6 and 7). The range of values for water consumption during study period were as follows: 18.6 to 20.8 g animal−1 d−1 in the control group initiated with DEN (group 1), 18.2 to 20.7 g animal−1 d−1 in the 150 mg/kg LFO group (group 2), 20.1 to 22.0 g animal−1 d−1 in the 300 mg/kg LFO group (group 3), 20.5 to 22.6 g animal−1 d−1 in the 600 mg/kg LFO group (group 4), 19.3 to 23.5 g animal−1 d−1 in the S.PB group (group 5), 19.6 to 21.9 g animal−1 d−1 in the control group not initiated with DEN (group 6), and 20.1 to 23.2 g animal−1 d−1 in the 600 mg/kg LFO group not initiated with DEN (group 7). In the DEN-initiated groups, there was a tendency toward greater water consumption in the 300 mg/ kg LFO group (group 3) from weeks 6 to 8 and in the 600 mg/kg LFO (group 4) from weeks 4 to 8 compared with the control group (group 1). A tendency toward greater water consumption was also observed in the S.PB group (group 5) from weeks 3 to 5 and at week 7 compared with the control group (group 1). In the non–DEN-initiated groups, water consumption from weeks 4 to 7 tended to be greater in the 600 mg/kg LFO group (group 7) than in the control group (group 6). 3.3. Gross pathology At necropsy, shrunken caudate lobes of the liver were observed in 1 animal in each of the groups given 300 mg/kg LFO (group 3) and S.PB (group 5). The cause of the finding
Table 1 Final body and liver weight of rats Group no.
1 2 3 4 5 6 7
Treatment DEN
Test Chemical
+ + + + + − −
LFO LFO LFO LFO S.PB LFO LFO
Level (mg/kg)
Final BW (g)
0 150 300 600 500 a 0 600
265.2 ± 12.8 267.6 ± 9.6 271.7 ± 12.5 266.8 ± 15.0 276.6 ± 8.2 ⁎⁎ 289.4 ± 12.3 285.7 ± 13.0
Liver weight Absolute (g)
Relative (%)
6.7311 ± 0.3642 7.0276 ± 0.4059 7.4460 ± 0.5865 ⁎⁎ 7.5464 ± 0.4357 ⁎⁎ 9.2781 ± 0.4913 ⁎⁎ 7.6091 ± 0.5606 8.1869 ± 0.5380 ⁎
2.5378 ± 0.0454 2.6255 ± 0.0946 ⁎⁎ 2.7456 ± 0.2559 ⁎⁎ 2.8291 ± 0.0714 ⁎⁎ 3.3527 ± 0.1032 ⁎⁎ 2.6264 ± 0.0989 2.8647 ± 0.1065 ⁎⁎⁎
Values are means ± SD. BW indicate body weight. a In parts per million. ⁎ Significantly different from control group (group 6) at P b .05 and 0.01, respectively (Student t test). ⁎⁎ Significantly different from control group (group 1) at P b .01 (Dunnett multiple comparison or Steel test). ⁎⁎⁎ Significantly different from control group (group 6) at P b .05 and 0.01, respectively (Student t test).
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Table 2 Quantitative measurements for GST-P–positive foci in rats Group no.
1 2 3 4 5 6 7
Treatment DEN
Test chemical
+ + + + + − −
LFO LFO LFO LFO S.PB LFO LFO
Level (mg/kg)
0 150 300 600 500 a 0 600
No. examined
15 15 15 14 15 9 9
GST-P–positive foci Number (no./cm2)
Area (mm2/cm2)
3.763 ± 1.503 2.779 ± 1.228 3.083 ± 1.287 2.484 ± 1.054 ⁎ 8.328 ± 2.467 ⁎⁎ 0.000 ± 0.000 0.000 ± 0.000
0.277 ± 0.159 0.164 ± 0.078 ⁎ 0.175 ± 0.076 0.134 ± 0.055 ⁎ 0.541 ± 0.160 ⁎⁎ 0.000 ± 0.000 0.000 ± 0.000
Values are means ± SD. a In parts per million. ⁎ Significantly different from control group (group 1) at P b .05 and 0.01, respectively (Dunnett multiple comparison or Steel test). ⁎⁎ Significantly different from control group (group 1) at P b .05 and 0.01, respectively (Dunnett multiple comparison or Steel test).
was considered to be related to partial hepatectomy. No gross findings were noted in the other groups. 3.4. Final body and liver weights Final body and liver weight data are shown in Table 1. None of the LFO-treated groups, with or without DEN initiation, differed significantly from the control groups in final body weight. Final body weight was significantly greater in the S.PB group (group 5) than in the control group (group 1). Absolute and relative liver weights in the 300 and 600 mg/kg LFO groups with DEN initiation (groups 3 and 4) were significantly greater than those in the control group (group 1). Relative liver weight in the 150 mg/kg LFO group with DEN initiation (group 2) was also significantly greater than that in the control group (group 1). Furthermore, absolute and relative liver weights in the 600 mg/kg LFO group without DEN initiation (group 7) were significantly greater than those in the control group (group 6). Absolute and relative liver weights in the S.PB-treated group (group 5) were significantly higher than those in the control group (group 1).
3.5. Immunohistochemical analysis of the liver Quantitative data on GST-P–positive foci are shown in Table 2. Glutathione S-transferase placental form–positive foci (Fig. 2) developed in all DEN initiation groups (groups 1-5) but not in the non-DEN initiation groups (groups 6 and 7). In the DEN initiation groups, the density (number/cm2) of GST-P–positive foci in the 600 mg/kg LFO group (group 4) was significantly lower than that in the control group (group 1) and tended to be lower in the 150 and 300 mg/kg groups (groups 2 and 3) than that in the control group (group 1). The area of GST-P–positive foci per cm2 was significantly lower in the 150 and 600 mg/kg LFO groups (groups 2 and 4) than in the control group (group 1) and tended to be lower in the 300 mg/kg group (group 3) than in the control group (group 1). On microscopic examination, no hepatocellular hypertrophy was observed in any group except group 5. The number and area of GST-P–positive foci in the S.PBtreated group (group 5) were significantly greater than that in the control group (group 1). 3.6. Electron microscopic examination of liver No changes were observed when comparing electron microscopic images of the livers of 3 rats in the control group (group 6) to images from the 600 mg/kg LFO group (group 7) without DEN treatment. Furthermore, there were no qualitative or quantitative differences in peroxisomes between the control group (group 6) and the 600 mg/kg LFO group (group 7), and no proliferation of peroxisomes was apparent. 4. Discussion
Fig. 2. Glutathione LFO–positive foci in the liver. Scale bar = 200 μm.
The purposes of this study was to investigate the anticarcinogenic potential of LFO using the rat mediumterm liver bioassay for carcinogens, which is based on the 2-stage mechanism of tumor production in combination with two-thirds partial hepatectomy [17,22]. This bioassay
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enables the rapid detection of carcinogenic agents by measuring GST-P–positive foci as end-point lesions in Caesarean Derived Fischer (F344) CrlBR male rats [19,23], because GST-P positivity has been shown to be a highly sensitive marker of preneoplastic hepatic lesions and for the detection of the carcinogenicity or cancer-promoting potential of chemicals [24-26]. Furthermore, the quantitative traits of GST-P–positive foci determined using this bioassay system have been reported to correlate with hepatocellular carcinoma development in a dose-dependent manner in 2year long-term studies [21,27,28]. Because the expression of GST-P in the liver can be suppressed by peroxisome proliferators [29], we confirmed before the present study that the administration of an excessive amount (800 mg/kg) of the LFO test substance (LFO concentrate solution) did not elicit peroxisome proliferation activity in the rat liver (data not shown). In addition, we failed to detect peroxisome proliferation in the 600 mg/kg LFO group without DEN initiation on electron microscopic examination in the present study. These results indicate that LFO is not a peroxisome proliferator and does not inhibit the induction of GST-P in the rat liver. Therefore, a false-negative result for the LFO test substance in this medium-term liver bioassay can be ruled out. During the study period, no mortality or changes in general condition related to administration of the test material except the death of 1 animal due to the surgical failure were seen in any group. No significant differences were observed in body weight or gross pathologic findings between any of the groups given LFO concentrate solution and the control groups with or without DEN. However, we found significant increases in absolute and relative liver weights in the 300 and 600 mg/kg LFO groups with DEN initiation and a significant increase in relative liver weight in the 150 mg/kg LFO group with DEN initiation. In addition, significant increases in absolute and relative liver weights were observed in the 600 mg/kg LFO group without DEN initiation. Although these changes were considered to be related to the administration of the test material, they were slight, and no microscopic changes were observed in the liver. Furthermore, no changes in peroxisomes, mitochondria, or other organelles of hepatocytes, were detected by electron microscopy in the 600 mg/kg LFO group without DEN initiation. Therefore, we consider that the increases in absolute and relative liver weights in the 600 mg/kg LFO groups with or without DEN initiation were not toxicologically significant. The similar changes in liver weights were not seen in the 90-day subchronic toxicity study in rats [6]. The number and area in the 600 mg/kg LFO group and the area in the 150 mg/kg LFO group with DEN initiation were significantly lower than those in the control group. The number and area in the 300 mg/kg LFO group with DEN initiation tended to be lower than those in the control group. In contrast, the S.PB-positive control group showed significantly greater values for quantitative traits of GST-
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P–positive foci and for body weight, food consumption, water consumption, and liver weight. Consequently, we conclude that the present experiment was adequate to evaluate the carcinogenetic promotion potential of test substances. This means that the LFO concentrate solution may be expected to have a suppressive effect on carcinogenesis at the dose of 150 mg/kg or higher. Several studies have demonstrated anticarcinogenic effects of dietary phytochemicals such as LFO in this bioassay; examples include Chlorella pyrenoidosa phytochemicals [30], nobiletin [31], curcumin [32], Porphyra tenera (asakusa-nori) [33] phytochemicals, and garlic [34]. Interestingly, some oil and water-soluble organosulfur compounds in garlic and onion showed suppressive activities for carcinogenesis, but some allicin-derived oil-soluble organosulfur compounds including diallylsulfide, diallyltrisuldide, and allylmethyltrisulfide showed promoting activities, which indicates that the carcinogenic potential of phytochemicals may differ considerably depending on the fractions or compounds examined, even though they are derived from the same plant [35-37]. In this regard, the carcinogenic potential of purified or fortified dietary ingredients derived from plants should be evaluated using the finished product, such as LFO concentrate solution, for which there is an established manufacturing process. In earlier studies, a body of evidence has been accumulated to suggest that licorice and its derivatives have antitumor potential. For example, Tamir et al [8] showed that glabridin, a major hydrophobic component of licorice (G glabra), which is a raw material used in the preparation of LFO concentrate solution, has a growth-inhibitory action on breast cancer cells in vitro. In addition, using a mouse xenograft model of colon carcinoma, Lee et al [9,38] demonstrated the antitumor activity of an ethanolic extract of licorice (Glycyrrhiza inflata) and of isoliquiritigenin, a flavonoid with a chalcone structure found in several Glycyrrhiza species. Furthermore, Chin et al [10] have identified anticarcinogenic activity of licorice in colon and lung tumors. They sought new cancer chemopreventive agents in chloroform extracts using G glabra, a raw material used in the preparation of LFO concentrate solution, and isolated 9 phenolic compounds with potent antioxidant activity; these included glabridin and isoliquiritigenin. Subsequently, they demonstrated that 300 mg/kg of isoliquiritigenin significantly prevented the incidence of 1,2-dimethylhydrazine–induced colon and lung tumors in mice. In this present study, we could accept our hypothesis by demonstrating for the first time the anticarcinogenic activity of G. glabra polyphenols in a liver, which is a major target organ for carcinogenesis in human using the reliable medium-term liver bioassay based on the 2stage mechanism of tumor production. Further studies are required, for example, to determine which phenolic compounds in the LFO concentrate solution contribute to the anticarcinogenic action. It is interesting to estimate what dose of LFO concentrate solution would be necessary to exert an anticarcinogenic action in humans. Our previous reports demonstrated that
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LFO suppresses elevated blood glucose levels at the dose of 500 mg/kg in obese diabetic KK-Ay mice (corresponding to approximately 170 mg/kg of the LFO concentrate solution as defined in the “Methods and materials”) [1] and that LFO reduces visceral fat accumulation at the dose of 800 mg/kg in diet-induced obese mice (corresponding to approximately 270 mg/kg of LFO concentrate solution) [2]. As mentioned above, based on the results of the present study, the LFO concentrate solution was expected to have carcinogenesissuppressing activity at the dose of 150 mg/kg or higher, the same dose range as that demonstrated in mice for antidiabetic and antivisceral obese effects; however, the difference in species has to be considered. Furthermore, in 2 randomized, double-blind, placebo-controlled clinical trials in humans [3,4], LFO significantly suppressed body weight gain at doses of 300 to 900 mg/d (corresponding to approximately 100-300 mg/d of LFO concentrate solution), as a result of a decrease in body fat mass. Therefore, repeated intake of LFO concentrate solution in humans at doses of 100 to 300 mg/d may be effective not only in preventing metabolic syndrome by maintaining a healthy body fat mass but also in preventing cancer by suppressing tumor promotion. Although it may be the limitation of the study, furthermore expanded studies such as an epidemiologic study with consumers of LFO are necessary in the future to confirm that repeated intake of LFO concentrate solution prevents liver cancer development. Acknowledgment The study was supported by the internal funds of Kaneka Corporation (Osaka, Japan). There are no conflicts of interest for any of the researchers conducting these animal studies. References [1] Nakagawa K, Kishida H, Arai N, Nishiyama T, Mae T. Licorice flavonoids suppress abdominal fat accumulation and increase in blood glucose level in obese diabetic KK-Ay mice. Biol Pharm Bull 2004;27: 1775-8. [2] Aoki F, Honda S, Kishida H, Kitano M, Arai N, Tanaka H, et al. Suppression by licorice flavonoids of abdominal fat accumulation and body weight gain in high-fat diet-induced obese C57BL/6J mice. Biosci Biotechnol Biochem 2007;71:206-14. [3] Tominaga Y, Mae T, Kitano M, Sakamoto Y, Ikematsu H, Nakagawa K. Licorice flavonoid oil effects body weight loss by reduction of body fat mass in overweight subjects. J Health Sci 2006;52:672-83. [4] Tominaga Y, Nakagawa K, Mae T, Kitano M, Yokota S, Arai T, et al. Licorice flavonoid oil reduces total body fat and visceral fat in overweight subjects. Obes Res Clin Pract 2009;3:169-78. [5] Aoki F, Nakagawa K, Kitano M, Ikematsu H, Nakamura K, Yokota S, et al. Clinical safety of licorice flavonoid oil (LFO) and pharmacokinetics of glabridin in healthy humans. J Am Coll Nutr 2007;26: 209-18. [6] Nakagawa K, Kitano M, Kishida H, Hidaka T, Nabae K, Kawabe M, et al. 90-Day repeated-dose toxicity study of licorice flavonoid oil (LFO) in rats. Food Chem Toxicol 2008;46:2349-57. [7] Nakagawa K, Hidaka T, Kitano M, Asakura M, Kamigaito T, Noguchi T, et al. Genotoxicity studies on licorice flavonoid oil (LFO). Food Chem Toxicol 2008;46:2525-32.
[8] Tamir S, Eizenberg M, Somjen D, Stern N, Shelach R, Kaye A, et al. Estrogenic and antiproliferative properties of glabridin from licorice in human breast cancer cells. Cancer Res 2000;60:5704-9. [9] Lee CK, Park KK, Lim SS, Park JH, Chung WY. Effects of the licorice extract against tumor growth and cisplatin-induced toxicity in a mouse xenograft model of colon cancer. Biol Pharm Bull 2007;30:2191-5. [10] Chin YW, Jung HA, Liu Y, Su BN, Castoro JA, Keller WJ, et al. Antioxidant constituents of the roots and stolons of licorice (Glycyrrhiza glabra). J Agric Food Chem 2007;55:691-7. [11] Shirai TA. Medium-term rat liver bioassay as a rapid in vivo test for carcinogenic potential: a historical review of model development and summary of results from 291 tests. Toxicologic Pathology 1997;25: 453-60. [12] Shirai T, Hirose M, Ito N. Medium-term bioassays in rats for rapid detection of the carcinogenic potential of chemicals. In: Mcgregor DB, Rice JM, Venitt S, editors. The use of short- and medium-term tests for carcinogens and data on genetic effects in carcinogenic hazard evaluation. Lyon: IARC; 1999. p. 251-2. [13] Pitot HC, Dragan YP. Chemical carcinogenesis. In: Klaassen CD, editor. Casarett and Doull's toxicology (the basic science of poisons). 6th ed. NY: McGraw-Hill Medical Publishing Division; 2001. p. 241-319. [Chapter 8]. [14] Ito N, Tamano S, Shirai T. A medium-term rat liver bioassay for rapid in vivo detection of carcinogenic potential of chemicals. Cancer Sci 2003;94:3-8. [15] International Conference on Harmonization of Technical Requirements for the Registration of Pharmaceuticals for Human Use. Testing for carcinogenicity of pharmaceuticals. ICH Harmonised Tripartite Guideline; 1997. [16] Japanese Association for Laboratory Animal Science. Guideline for animal experimentation. Exp Anim 1987;36:285-8. [17] Ito N, Hasegawa R, Imaida K, Hirose M, Shirai T. Medium-term rat liver bioassay for rapid detection of hepatocarcinogenic substances. J Toxicol Pathol 1997;10:1-11. [18] Ito N, Tsuda H, Tatematsu M, Inoue T, Tagawa Y, Aoki T, et al. 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;9:387-94. [19] 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. [20] Watanabe T, Katsura Y, Yoshitake A. IPAP: Image processor for analytical pathology. J Toxicol Pathol 1994;7:353-61. [21] Ogiso T, Tatematsu M, Tamano S, Hasegawa R, Ito N. Correlation between medium-term liver bioassay system data and results of longterm testing in rats. Carcinogenesis 1990;11:561-6. [22] Berenblum I. The mechanism of carcinogenesis: a study of the significance of cocarcinogenic action and related phenomena. Cancer Res 1941;1:807-14. [23] Tatematsu M, Mera Y, Ito N, Satoh K, Sato K. Relative merits of immunohistochemical demonstrations of placental A, B and C forms of glutathione S-transferase and histochemical demonstration of γglutamyl transferase as markers of altered foci during liver carcinogenesis in rats. Carcinogenesis 1985;6:1621-6. [24] Hasegawa R, Shirai T, Hakoi K, Takaba K, Iwasaki S, Hoshiya T, et al. Synergistic enhancement of glutathione S-transferase placental formpositive hepatic foci development in diethylnitrosamine-treated rats by combined administration of five heterocyclic amines at low doses. Jpn J Cancer Res 1991;82:1378-84. [25] Kitano M, Ichihara T, Matsuda T, Wanibuchi H, Tamano S, Hagiwara A, et al. Presence of a threshold for promoting effects of Phenobarbital on diethylnitrosamine-induced hepatic foci in the rat. Carcinogeneis 1998;19:1475-80. [26] Masuda C, Wanibuchi H, Otori K, Wei M, Yamamoto S, Hiroi T, et al. Presence of a no-observed effect level for enhancing effects
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[32] Shukla Y, Arora A. Suppression of altered hepatic foci development by curcumin in Wistar rats. Nutr Cancer 2003;45:53-9. [33] Ichihara T, Wanibuchi H, Taniyama T, Okai Y, Yano Y, Otani S, et al. Inhibition of liver glutathione S-transferase placental form-positive foci development in the rat hepatocarcinogenesis by Porphyra tenera (Aswakusa-nori). Cancer Lett 1999;141:211-8. [34] Samaranayake MD, Wickramasinghe SM, Angunawela P, Jayasekera S, Iwai S, Fukushima S. Inhibition of chemically induced liver carcinogenesis in Wister rats by garlic (Allium sativum). Phytother Res 2000;14:564-7. [35] Takada N, Matsuda T, Otoshi T, Yano Y, Otani S, Hasegawa T, et al. 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. [36] Fukushima S, Takada N, Hori T, Wanibuchi H. Cancer prevention by organosulfur compounds from garlic and onion. J Cell Biochem Suppl 1997;27:100-5. [37] Uda N, Kashimoto N, Sumioka I, Kyo E, Sumi S, Fukushima S. Aged garlic extract inhibits development of putative preneoplastic lesions in rat hepatocarcinogenesis. J Nutr 2006;136(3 Suppl):855S-60S. [38] Lee CK, Son SH, Park KK, Park JH, Lim SS, Chung WY. Isoliquiritigenin inhibits tumor growth and protects the kidney and liver against chemotherapy-induced toxicity in a mouse xenograft model of colon carcinoma. J Pharmacol Sci 2008;106:444-51.
Nutrition Research 30 (2010) 74 – 81 www.nrjournal.com
Inhibition by licorice flavonoid oil of glutathione S-transferase–positive foci in the medium-term rat hepatocarcinogenesis bioassay Kaku Nakagawa a , Kazunori Hosoe a , Takayoshi Hidaka b , Kyoko Nabae c , Mayumi Kawabe c , Mitsuaki Kitano d,⁎ b
a QOL Division, Kaneka Corporation, Kita-ku, Osaka 530-8288, Japan Department of Health Sciences and Social Pharmacy, Faculty of Pharmaceutical Sciences, Kobe Gakuin University, Chuo-ku, Kobe 650-8586, Japan c DIMS Institute of Medical Science, Inc., Azai-cho, Ichinomiya, Aichi 491-0113, Japan d Pharmacology and Toxicology Laboratory, Frontier Biochemical and Medical Research Laboratories, Kaneka Corporation, Takasago, Hyogo 676-8688, Japan Received 19 October 2009; revised 5 December 2009; accepted 17 December 2009
Abstract Licorice flavonoid oil (LFO) is a new functional food ingredient consisting of hydrophobic licorice polyphenols in medium-chain triglycerides. Recently, it was reported that licorice and its derivatives have anticarcinogenic activity in some types of tumors. However, the anticarcinogenic activity has not been identified in the liver, which is a major target organ for carcinogenesis in human. Therefore, we hypothesized that LFO has antihepatocarcinogenic activity, and we tested this hypothesis using the rat medium-term liver bioassay for carcinogens. Six-week-old male F344 rats (15 animals/group) received N-diethylnitrosamine (200 mg/kg by intraperitoneal injection) to initiate carcinogenesis. From the second week after initiation, animals received a 6-week regimen of either LFO concentrate solution (0, 150, 300, or 600 mg/kg) intragastrically or phenobarbital sodium salt in the diet (500 ppm) as a positive control. During the third week after initiation, animals were subjected to a two-thirds partial hepatectomy. During the eighth week of the treatment period, liver samples were taken from animals and examined immunohistochemically for expression of glutathione S-transferase placental form. No increase in the number of glutathione S-transferase placental form–positive liver foci was observed in all LFO groups compared with the negative control (solvent) group, and the number of foci in the 600 mg/kg LFO group was significantly lower than that in the negative control group. These results indicate that LFO concentrate solution has a significant inhibitory effect on liver carcinogenesis at 600 mg/kg. © 2010 Elsevier Inc. All rights reserved. Keywords: Abbreviations:
Licorice flavonoid oil; Glycyrrhiza glabra; Medium-term liver bioassay; Rat; Carcinogenesis DEN, nitrosodiethylamine; GST-P, glutathione S-transferase placental form; LFO, licorice flavonoid oil; MCTs, medium-chain triglycerides; S.PB, phenobarbital sodium salt.
1. Introduction Licorice flavonoid oil (LFO), derived from licorice, Glycyrrhiza glabra L., is a new functional food ingredient consisting of licorice hydrophobic polyphenols in medium⁎ Corresponding author. Tel.: +81 79 445 2427; fax: +81 79 445 2699. E-mail address: [email protected] (M. Kitano). 0271-5317/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.nutres.2009.12.005
chain triglycerides (MCTs). Licorice flavonoid oil was developed by Kaneka Corporation (Osaka, Japan) under the brand name Kaneka Glavonoid. Recently, we found that LFO is effective in reducing visceral fat accumulation and in suppressing elevated blood glucose levels in obese diabetic KK-Ay mice [1]. The mechanism of the antiobesity effect of LFO was investigated using a high-fat diet–induced obese C57BL/6J mouse model, and the data suggested that this
K. Nakagawa et al. / Nutrition Research 30 (2010) 74–81
effect was mediated by modification of the expression levels of lipid metabolism-related genes in the liver [2]. Furthermore, a double-blind, placebo-controlled clinical trial showed that LFO was effective in suppressing body weight gain by reducing body fat mass in overweight volunteers [3,4]. These studies indicate that LFO may help to prevent lifestyle-related diseases, such as obesity associated with metabolic syndrome. Although licorice has a long history of human consumption, information on the safety of the hydrophobic fraction of G glabra is limited. Clinical studies showed that LFO is safe, because no clinically significant adverse events occurred when it was given daily to healthy or overweight subjects for up to 12 weeks [3,5]. Furthermore, in a 90-day subchronic toxicity study in rats that was part of a series of preclinical safety assessments of LFO, we demonstrated that LFO had low toxicity [6]. In addition, the genotoxicity of LFO was investigated using a bacterial reverse mutation assay, a chromosomal aberration assay, and a micronucleus in vivo assay [7]. The results of the bacterial reverse mutation and micronucleus in vivo assays were all negative; however, at higher concentrations, LFO induced clastogenic activity during a short period with an S9 mix in a chromosomal aberration test. The results obtained using all 3 methods of genotoxicity evaluation indicated that LFO was substantially nongenotoxic in vivo. On the other hand, there are indications that licorice extract and its components have antitumor activity on several types of tumors [8,9]. Furthermore, the anticarcinogenic activity has been identified in 1,2-dimethylhydrazine– induced colon and lung tumors in mice [10]. In this present study, we hypothesized that LFO, hydrophobic polyphenols from licorice glabra, has an anticarcinogenic effect in the liver, which is a major target organ for carcinogenesis in human. If this hypothesis is correct, LFO may be useful in human nutrition, not only for prevention of metabolic syndrome but also for prevention of liver cancer development. This hypothesis was tested using the rat medium-term liver bioassay for carcinogens (Ito test). This bioassay is considered to be reliable in predicting the promoting or inhibitory activity of chemicals with respect to hepatocarcinogenesis, based on background data for more than 300 chemicals [11-14], and was recommended as an alternative to long-term carcinogenicity testing at the International Conference on Harmonization [15].
2. Methods and materials 2.1. Study design (Good Laboratory Practice and Test Guidance) This study was performed at the DIMS Institute of Medical Science, Inc (Ichinomiya, Japan), in accordance with the Good Laboratory Practice standards of the Japanese Ministry of Health and Welfare Ordinance No. 21 (March 26, 1997) and in compliance with the Guidelines for
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Carcinogenicity Studies of Drugs 3.2 (In Vivo Additional Tests for Detection of Carcinogenicity) of the Japanese Ministry of Health and Welfare (Notification Iyakushin No. 1607; November 1, 1999). The use of animals and the experimental protocol were performed in accordance with the Guidelines for Animal Experimentation, Japanese Association for Laboratory Animal Science [16]. The inhouse guidelines for the Care and Use of Laboratory Animals were used, and the protocol was approved by the DIMS Institute of Medical Science, Inc. 2.2. LFO test substance An ethanol extract of licorice (G glabra) root was obtained to prepare the LFO test substance. After filtration and concentration, the ethanolic layer was mixed with MCTs with a fatty-acid composition of C8:C10 = 99:1. The concentration of glabridin, the major component of the solution, was adjusted to 3% (wt/wt). This solution was named “LFO concentrate solution” and given the brand name Kaneka Glavonoid. The LFO concentrate solution comprised 30% licorice ethanol extract and 70% MCTs, with the latter functioning as a carrier/diluent. The licorice ethanol extract consisted almost entirely of hydrophobic polyphenols, including licorice prenyl flavonoids such as glabridin, which are active components of LFO. In the present study, an LFO concentrate solution containing 3.0% glabridin (Lot No. 51217002; Kaneka Corporation) was used as the LFO test substance. 2.3. Preparation of test substance solution The LFO test substance (LFO concentrate solution) was diluted with MCTs (Lot No. C030401; Riken Vitamin Co, Ltd, Tokyo, Japan) to adjust it to the intended concentration. Working solutions were stored in a refrigerator (recorded temperature, 4-7°C), and their stability during the treatment period (8 weeks) was analyzed at the termination of treatment and was confirmed to be appropriate. 2.4. Other chemicals N-nitrosodiethylamine (DEN), an initiator of carcinogenesis, and phenobarbital sodium salt (S.PB), used as a positive control for a promoter of carcinogenesis, were purchased from Tokyo Kasei Kogyo (Tokyo, Japan). 2.5. Animals One hundred 5-week-old male F344 rats were purchased from Charles River Japan, Inc. (Ibaraki, Japan) and used in the study after a 6-day quarantine/acclimation period. The rats were housed in groups of 3 in transparent plastic cages with chipped hardwood bedding and received an irradiated powder diet (MF diet; Oriental Yeast Co, Ltd, Tokyo, Japan) and water ad libitum. The animal room was maintained on a 12-hour light/12-hour dark cycle; temperature and relative humidity were maintained within the ranges of 19 to 24°C and 51% to 60%, respectively.
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2.6. Groups and numbers of animals
2.8. Gross pathology
Of the 100 rats, 93 were allocated to 7 groups (15 rats/ group for groups 1-5, 9 rats/group for groups 6 and 7) using a randomized block design. The mean body weights of the 7 groups at the time of group allocation were similar. Grouping was performed on the day before administration of the initiator.
All surviving animals were fasted overnight before the day of their termination. Blood samples were drawn from the abdominal aorta under ether anesthesia; the organs and tissues in the thoracic and abdominal cavities were examined macroscopically, lesions were recorded individually, and the livers were quickly removed. At necropsy, the liver weight of all surviving animals was measured, and the relative liver weight (ratio of absolute liver weight to final body weight) was calculated.
2.7. Experimental design The experiment was performed according to the protocol of the medium-term liver bioassay (Ito test) [14,17-19]. As shown in Fig. 1, animals in groups 1 to 5 were treated with DEN (200 mg/kg body weight/5 ml saline, IP, single injection) at the commencement of the experiment. Rats in groups 6 and 7 were given the vehicle alone instead of the carcinogen. Starting 2 weeks later, the rats were treated with LFO working solution by oral gavage (2 mL/kg body weight) once a day, or fed a diet containing S.PB at 500 ppm as a positive control until week 8. Animals in control groups 1 and 6 were also treated with MCTs, the vehicle for LFO, by gavage for 6 weeks. Three weeks after the beginning of the experiment (1 week after the beginning of the administration of LFO test substance), a two-thirds partial hepatectomy was performed on all animals under anesthesia to maximize any interactions between cellular proliferation and the effects of the test chemicals. All animals were checked for their general condition, and findings on behavior, toxicity, and mortality were individually recorded. Body weight was measured for all animals at the initiation of the experiment and at weekly intervals thereafter. At the termination of treatment, body weight was determined after overnight fasting (final body weight). Food and water consumption per cage was measured weekly after initiation of the experiment. All surviving rats in each group were euthanized by whole blood drawing under ether anesthesia at week 8 for pathologic and immunohistochemical examination.
2.9. Quantitative immunohistochemical analysis of hepatocyte foci For all surviving animals, roughly 5-mm-thick sections from the 3 liver lobes (right lateral lobe, cranial part; right lateral lobe, caudal part; and caudate lobe, caudal part) were fixed in 10% buffered formalin solution, embedded in paraffin wax, serially sectioned, and stained immunohistochemically for glutathione S-transferase placental form (GST-P; avidin-biotin complex [ABC] method). The numbers and areas of GST-P–positive foci larger than 0.2 mm in diameter per square centimeter (cm2) of liver section were measured using a color video image processor (IPAPWIN; Sumika Technos, Co, Osaka, Japan) [20]. 2.10. Electron microscopic analysis of liver Three rats each in groups 6 and 7 were selected for electron microscopic examination. Small portions of the liver (right lateral lobe, cranial lobe) were fixed in glutaraldehyde solution. The samples were analyzed at Bozo Research Center (Shizuoka, Japan) under Good Laboratory Practice conditions. 2.11. Statistical analyses The statistical significance of the differences between the DEN-initiated control (group 1) and treated groups for each parameter (excluding general condition and food and water consumption) was analyzed. Comparisons of group 1 and the
Fig. 1. Protocol for the study, based on the medium-term liver bioassay. The bioassay was performed using 6-week-old F344 male rats. Glutathione LFO– positive foci were counted at week 8. Values in parentheses are numbers of animals. Animals in groups 1 and 6 were given MCT; those in groups 2, 3, 4, and 7 were given the LFO concentrate solution at 150, 300, 600, and 600 mg kg−1 d−1, respectively; and those in group 5 were given S.PB at 500 ppm in a basal diet for 6 weeks. ▾, DEN 200 mg/kg body weight IP; ▿, saline IP; O, two-thirds partial hepatectomy.
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LFO-treated groups (groups 2, 3, and 4) for numerical data on body weight, organ weight, and quantitative values for GST-P–positive hepatocytic foci were performed using Bartlett test. If the data were found to be homogeneous, they were analyzed with Dunnett multiple comparison test (1-sided); if not, the data were analyzed with Steel test (1-sided). The significance of the differences between the DEN-initiated group 1 (control) and group 5 (S.PB) and the noninitiated group 6 (control, not treated with LFO or S.PB) and group 7 (LFO 600 mg/kg) for numerical data were assessed using the F test. If the data were found to be homogeneous, they were analyzed with the Student t test (1sided), and otherwise, they were tested with the Welch test (1-sided). The significance of the differences in the incidence data for gross pathology was analyzed using Fisher exact probability test (1-sided). The number of animals per group was determined according to the past successful results of this model [17,21]. These analyses were conducted with Stat Light software 2000 (Yukms Co Ltd, Tokyo, Japan). 3. Results 3.1. Survival rate and general condition No mortality or changes in general conditions related to administration of the test material were seen in any group. One rat in the group receiving 600 mg/kg LFO (group 4) was found supine on day 24 and dead on day 25. The cause of death was considered to be related to surgical failure because the liver was found to be discolored on macroscopic examination. 3.2. Food and water consumption The range of values for food intake during study period were as follows: 13.3 to 15.2 g animal−1 d−1 in the control group initiated with DEN (group 1), 13.1 to 14.6 g animal−1 d−1 in the 150 mg/kg LFO group (group 2), 14.0 to 16.6 g animal−1 d−1 in the 300 mg/kg LFO group (group 3), 13.9 to 18.2 g animal−1 d−1 in the 600 mg/kg LFO group (group 4), 13.4 to 18.4 g animal−1 d−1 in the S.PB group
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(group 5), 13.7 to 16.3 g animal d in the control group not initiated with DEN (group 6), and 14.1 to 16.7 g animal−1 d−1 in the 600 mg/kg LFO group not initiated with DEN (group 7). In the DEN-initiated groups, there was a tendency toward greater food consumption in the 300 mg/ kg LFO group (group 3) at week 3 and weeks 6 to 8 and in the 600 mg/kg LFO group (group 4) at weeks 5 and 7, compared with the control group (group 1). A tendency toward greater food consumption was also observed in the S.PB group (group 5) from weeks 3 to 7 compared with the control group (group 1). No change in food consumption was observed in the 2 groups not initiated with DEN (groups 6 and 7). The range of values for water consumption during study period were as follows: 18.6 to 20.8 g animal−1 d−1 in the control group initiated with DEN (group 1), 18.2 to 20.7 g animal−1 d−1 in the 150 mg/kg LFO group (group 2), 20.1 to 22.0 g animal−1 d−1 in the 300 mg/kg LFO group (group 3), 20.5 to 22.6 g animal−1 d−1 in the 600 mg/kg LFO group (group 4), 19.3 to 23.5 g animal−1 d−1 in the S.PB group (group 5), 19.6 to 21.9 g animal−1 d−1 in the control group not initiated with DEN (group 6), and 20.1 to 23.2 g animal−1 d−1 in the 600 mg/kg LFO group not initiated with DEN (group 7). In the DEN-initiated groups, there was a tendency toward greater water consumption in the 300 mg/ kg LFO group (group 3) from weeks 6 to 8 and in the 600 mg/kg LFO (group 4) from weeks 4 to 8 compared with the control group (group 1). A tendency toward greater water consumption was also observed in the S.PB group (group 5) from weeks 3 to 5 and at week 7 compared with the control group (group 1). In the non–DEN-initiated groups, water consumption from weeks 4 to 7 tended to be greater in the 600 mg/kg LFO group (group 7) than in the control group (group 6). 3.3. Gross pathology At necropsy, shrunken caudate lobes of the liver were observed in 1 animal in each of the groups given 300 mg/kg LFO (group 3) and S.PB (group 5). The cause of the finding
Table 1 Final body and liver weight of rats Group no.
1 2 3 4 5 6 7
Treatment DEN
Test Chemical
+ + + + + − −
LFO LFO LFO LFO S.PB LFO LFO
Level (mg/kg)
Final BW (g)
0 150 300 600 500 a 0 600
265.2 ± 12.8 267.6 ± 9.6 271.7 ± 12.5 266.8 ± 15.0 276.6 ± 8.2 ⁎⁎ 289.4 ± 12.3 285.7 ± 13.0
Liver weight Absolute (g)
Relative (%)
6.7311 ± 0.3642 7.0276 ± 0.4059 7.4460 ± 0.5865 ⁎⁎ 7.5464 ± 0.4357 ⁎⁎ 9.2781 ± 0.4913 ⁎⁎ 7.6091 ± 0.5606 8.1869 ± 0.5380 ⁎
2.5378 ± 0.0454 2.6255 ± 0.0946 ⁎⁎ 2.7456 ± 0.2559 ⁎⁎ 2.8291 ± 0.0714 ⁎⁎ 3.3527 ± 0.1032 ⁎⁎ 2.6264 ± 0.0989 2.8647 ± 0.1065 ⁎⁎⁎
Values are means ± SD. BW indicate body weight. a In parts per million. ⁎ Significantly different from control group (group 6) at P b .05 and 0.01, respectively (Student t test). ⁎⁎ Significantly different from control group (group 1) at P b .01 (Dunnett multiple comparison or Steel test). ⁎⁎⁎ Significantly different from control group (group 6) at P b .05 and 0.01, respectively (Student t test).
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Table 2 Quantitative measurements for GST-P–positive foci in rats Group no.
1 2 3 4 5 6 7
Treatment DEN
Test chemical
+ + + + + − −
LFO LFO LFO LFO S.PB LFO LFO
Level (mg/kg)
0 150 300 600 500 a 0 600
No. examined
15 15 15 14 15 9 9
GST-P–positive foci Number (no./cm2)
Area (mm2/cm2)
3.763 ± 1.503 2.779 ± 1.228 3.083 ± 1.287 2.484 ± 1.054 ⁎ 8.328 ± 2.467 ⁎⁎ 0.000 ± 0.000 0.000 ± 0.000
0.277 ± 0.159 0.164 ± 0.078 ⁎ 0.175 ± 0.076 0.134 ± 0.055 ⁎ 0.541 ± 0.160 ⁎⁎ 0.000 ± 0.000 0.000 ± 0.000
Values are means ± SD. a In parts per million. ⁎ Significantly different from control group (group 1) at P b .05 and 0.01, respectively (Dunnett multiple comparison or Steel test). ⁎⁎ Significantly different from control group (group 1) at P b .05 and 0.01, respectively (Dunnett multiple comparison or Steel test).
was considered to be related to partial hepatectomy. No gross findings were noted in the other groups. 3.4. Final body and liver weights Final body and liver weight data are shown in Table 1. None of the LFO-treated groups, with or without DEN initiation, differed significantly from the control groups in final body weight. Final body weight was significantly greater in the S.PB group (group 5) than in the control group (group 1). Absolute and relative liver weights in the 300 and 600 mg/kg LFO groups with DEN initiation (groups 3 and 4) were significantly greater than those in the control group (group 1). Relative liver weight in the 150 mg/kg LFO group with DEN initiation (group 2) was also significantly greater than that in the control group (group 1). Furthermore, absolute and relative liver weights in the 600 mg/kg LFO group without DEN initiation (group 7) were significantly greater than those in the control group (group 6). Absolute and relative liver weights in the S.PB-treated group (group 5) were significantly higher than those in the control group (group 1).
3.5. Immunohistochemical analysis of the liver Quantitative data on GST-P–positive foci are shown in Table 2. Glutathione S-transferase placental form–positive foci (Fig. 2) developed in all DEN initiation groups (groups 1-5) but not in the non-DEN initiation groups (groups 6 and 7). In the DEN initiation groups, the density (number/cm2) of GST-P–positive foci in the 600 mg/kg LFO group (group 4) was significantly lower than that in the control group (group 1) and tended to be lower in the 150 and 300 mg/kg groups (groups 2 and 3) than that in the control group (group 1). The area of GST-P–positive foci per cm2 was significantly lower in the 150 and 600 mg/kg LFO groups (groups 2 and 4) than in the control group (group 1) and tended to be lower in the 300 mg/kg group (group 3) than in the control group (group 1). On microscopic examination, no hepatocellular hypertrophy was observed in any group except group 5. The number and area of GST-P–positive foci in the S.PBtreated group (group 5) were significantly greater than that in the control group (group 1). 3.6. Electron microscopic examination of liver No changes were observed when comparing electron microscopic images of the livers of 3 rats in the control group (group 6) to images from the 600 mg/kg LFO group (group 7) without DEN treatment. Furthermore, there were no qualitative or quantitative differences in peroxisomes between the control group (group 6) and the 600 mg/kg LFO group (group 7), and no proliferation of peroxisomes was apparent. 4. Discussion
Fig. 2. Glutathione LFO–positive foci in the liver. Scale bar = 200 μm.
The purposes of this study was to investigate the anticarcinogenic potential of LFO using the rat mediumterm liver bioassay for carcinogens, which is based on the 2-stage mechanism of tumor production in combination with two-thirds partial hepatectomy [17,22]. This bioassay
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enables the rapid detection of carcinogenic agents by measuring GST-P–positive foci as end-point lesions in Caesarean Derived Fischer (F344) CrlBR male rats [19,23], because GST-P positivity has been shown to be a highly sensitive marker of preneoplastic hepatic lesions and for the detection of the carcinogenicity or cancer-promoting potential of chemicals [24-26]. Furthermore, the quantitative traits of GST-P–positive foci determined using this bioassay system have been reported to correlate with hepatocellular carcinoma development in a dose-dependent manner in 2year long-term studies [21,27,28]. Because the expression of GST-P in the liver can be suppressed by peroxisome proliferators [29], we confirmed before the present study that the administration of an excessive amount (800 mg/kg) of the LFO test substance (LFO concentrate solution) did not elicit peroxisome proliferation activity in the rat liver (data not shown). In addition, we failed to detect peroxisome proliferation in the 600 mg/kg LFO group without DEN initiation on electron microscopic examination in the present study. These results indicate that LFO is not a peroxisome proliferator and does not inhibit the induction of GST-P in the rat liver. Therefore, a false-negative result for the LFO test substance in this medium-term liver bioassay can be ruled out. During the study period, no mortality or changes in general condition related to administration of the test material except the death of 1 animal due to the surgical failure were seen in any group. No significant differences were observed in body weight or gross pathologic findings between any of the groups given LFO concentrate solution and the control groups with or without DEN. However, we found significant increases in absolute and relative liver weights in the 300 and 600 mg/kg LFO groups with DEN initiation and a significant increase in relative liver weight in the 150 mg/kg LFO group with DEN initiation. In addition, significant increases in absolute and relative liver weights were observed in the 600 mg/kg LFO group without DEN initiation. Although these changes were considered to be related to the administration of the test material, they were slight, and no microscopic changes were observed in the liver. Furthermore, no changes in peroxisomes, mitochondria, or other organelles of hepatocytes, were detected by electron microscopy in the 600 mg/kg LFO group without DEN initiation. Therefore, we consider that the increases in absolute and relative liver weights in the 600 mg/kg LFO groups with or without DEN initiation were not toxicologically significant. The similar changes in liver weights were not seen in the 90-day subchronic toxicity study in rats [6]. The number and area in the 600 mg/kg LFO group and the area in the 150 mg/kg LFO group with DEN initiation were significantly lower than those in the control group. The number and area in the 300 mg/kg LFO group with DEN initiation tended to be lower than those in the control group. In contrast, the S.PB-positive control group showed significantly greater values for quantitative traits of GST-
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P–positive foci and for body weight, food consumption, water consumption, and liver weight. Consequently, we conclude that the present experiment was adequate to evaluate the carcinogenetic promotion potential of test substances. This means that the LFO concentrate solution may be expected to have a suppressive effect on carcinogenesis at the dose of 150 mg/kg or higher. Several studies have demonstrated anticarcinogenic effects of dietary phytochemicals such as LFO in this bioassay; examples include Chlorella pyrenoidosa phytochemicals [30], nobiletin [31], curcumin [32], Porphyra tenera (asakusa-nori) [33] phytochemicals, and garlic [34]. Interestingly, some oil and water-soluble organosulfur compounds in garlic and onion showed suppressive activities for carcinogenesis, but some allicin-derived oil-soluble organosulfur compounds including diallylsulfide, diallyltrisuldide, and allylmethyltrisulfide showed promoting activities, which indicates that the carcinogenic potential of phytochemicals may differ considerably depending on the fractions or compounds examined, even though they are derived from the same plant [35-37]. In this regard, the carcinogenic potential of purified or fortified dietary ingredients derived from plants should be evaluated using the finished product, such as LFO concentrate solution, for which there is an established manufacturing process. In earlier studies, a body of evidence has been accumulated to suggest that licorice and its derivatives have antitumor potential. For example, Tamir et al [8] showed that glabridin, a major hydrophobic component of licorice (G glabra), which is a raw material used in the preparation of LFO concentrate solution, has a growth-inhibitory action on breast cancer cells in vitro. In addition, using a mouse xenograft model of colon carcinoma, Lee et al [9,38] demonstrated the antitumor activity of an ethanolic extract of licorice (Glycyrrhiza inflata) and of isoliquiritigenin, a flavonoid with a chalcone structure found in several Glycyrrhiza species. Furthermore, Chin et al [10] have identified anticarcinogenic activity of licorice in colon and lung tumors. They sought new cancer chemopreventive agents in chloroform extracts using G glabra, a raw material used in the preparation of LFO concentrate solution, and isolated 9 phenolic compounds with potent antioxidant activity; these included glabridin and isoliquiritigenin. Subsequently, they demonstrated that 300 mg/kg of isoliquiritigenin significantly prevented the incidence of 1,2-dimethylhydrazine–induced colon and lung tumors in mice. In this present study, we could accept our hypothesis by demonstrating for the first time the anticarcinogenic activity of G. glabra polyphenols in a liver, which is a major target organ for carcinogenesis in human using the reliable medium-term liver bioassay based on the 2stage mechanism of tumor production. Further studies are required, for example, to determine which phenolic compounds in the LFO concentrate solution contribute to the anticarcinogenic action. It is interesting to estimate what dose of LFO concentrate solution would be necessary to exert an anticarcinogenic action in humans. Our previous reports demonstrated that
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LFO suppresses elevated blood glucose levels at the dose of 500 mg/kg in obese diabetic KK-Ay mice (corresponding to approximately 170 mg/kg of the LFO concentrate solution as defined in the “Methods and materials”) [1] and that LFO reduces visceral fat accumulation at the dose of 800 mg/kg in diet-induced obese mice (corresponding to approximately 270 mg/kg of LFO concentrate solution) [2]. As mentioned above, based on the results of the present study, the LFO concentrate solution was expected to have carcinogenesissuppressing activity at the dose of 150 mg/kg or higher, the same dose range as that demonstrated in mice for antidiabetic and antivisceral obese effects; however, the difference in species has to be considered. Furthermore, in 2 randomized, double-blind, placebo-controlled clinical trials in humans [3,4], LFO significantly suppressed body weight gain at doses of 300 to 900 mg/d (corresponding to approximately 100-300 mg/d of LFO concentrate solution), as a result of a decrease in body fat mass. Therefore, repeated intake of LFO concentrate solution in humans at doses of 100 to 300 mg/d may be effective not only in preventing metabolic syndrome by maintaining a healthy body fat mass but also in preventing cancer by suppressing tumor promotion. Although it may be the limitation of the study, furthermore expanded studies such as an epidemiologic study with consumers of LFO are necessary in the future to confirm that repeated intake of LFO concentrate solution prevents liver cancer development. Acknowledgment The study was supported by the internal funds of Kaneka Corporation (Osaka, Japan). There are no conflicts of interest for any of the researchers conducting these animal studies. References [1] Nakagawa K, Kishida H, Arai N, Nishiyama T, Mae T. Licorice flavonoids suppress abdominal fat accumulation and increase in blood glucose level in obese diabetic KK-Ay mice. Biol Pharm Bull 2004;27: 1775-8. [2] Aoki F, Honda S, Kishida H, Kitano M, Arai N, Tanaka H, et al. Suppression by licorice flavonoids of abdominal fat accumulation and body weight gain in high-fat diet-induced obese C57BL/6J mice. Biosci Biotechnol Biochem 2007;71:206-14. [3] Tominaga Y, Mae T, Kitano M, Sakamoto Y, Ikematsu H, Nakagawa K. Licorice flavonoid oil effects body weight loss by reduction of body fat mass in overweight subjects. J Health Sci 2006;52:672-83. [4] Tominaga Y, Nakagawa K, Mae T, Kitano M, Yokota S, Arai T, et al. Licorice flavonoid oil reduces total body fat and visceral fat in overweight subjects. Obes Res Clin Pract 2009;3:169-78. [5] Aoki F, Nakagawa K, Kitano M, Ikematsu H, Nakamura K, Yokota S, et al. Clinical safety of licorice flavonoid oil (LFO) and pharmacokinetics of glabridin in healthy humans. J Am Coll Nutr 2007;26: 209-18. [6] Nakagawa K, Kitano M, Kishida H, Hidaka T, Nabae K, Kawabe M, et al. 90-Day repeated-dose toxicity study of licorice flavonoid oil (LFO) in rats. Food Chem Toxicol 2008;46:2349-57. [7] Nakagawa K, Hidaka T, Kitano M, Asakura M, Kamigaito T, Noguchi T, et al. Genotoxicity studies on licorice flavonoid oil (LFO). Food Chem Toxicol 2008;46:2525-32.
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