A Possible Mechanism That Flavonoids Exert Anticarcinogenesis with

Chapter 10

Downloaded by INDIANA UNIV BLOOMINGTON on March 23, 2017 | http://pubs.acs.org Publication Date: September 19, 2008 | doi: 10.1021/bk-2008-0993.ch010

A Possible Mechanism That Flavonoids Exert Anticarcinogenesis with Activation of β-Glucuronidase in Cancerous Tissues Naomi Oi, Takashi Hashimoto, and Kazuki Kanazawa

Department of Biosystems Science, Graduate School of Science and Technology, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan

Flavonoids are immediately metabolized into their inactive forms as conjugates with glucuronic acid and/or sulfate during intestinal absorption. To exercise biological activity in our body, conjugated flavonoids should be de-conjugated into their aglycons. In the present study, the activity of βglucuronidase, which can de-conjugate flavonoid glucuronides into their aglycons, was compared between normal and hepatocarcinogenic Fisher 344 rats induced by N-diethylnitrosamine and phenobarbital. In the liver of the hepatocar cinogenic rats, the β-glucronidase activity significantly increased compared to that in the normal rats. On the other hand, the activity in the kidney, lung, heart, thymus and plasma only slightly changed. These results suggest that βglucuronidase is specifically activated in the inflammatory tissues such as cancerous tissues. This is a possible mechanism that flavonoids are able to exert biological activities in the carcinogenic tissue of our body.

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Shibamoto et al.; Functional Food and Health ACS Symposium Series; American Chemical Society: Washington, DC, 2008.


103 Flavonoids occur abundantly in plant foods such as vegetables, fruits and tea. Flavonoids, in vitro, render strong biological effects including antioxidative, anti-inflammatory and anti-carcinogenic properties (1-3). Many epidemiological studies also suggest that the daily intake of flavonoids is inversely associated with the risk of certain cancers and coronary heart diseases (4-6). However, ingested flavonoids are immediately metabolized and conjugated with glucuronic acid and/or sulfate during intestinal absorption (7, 8). It is wellknown that conjugated flavonoids have low biological activities compared with the aglycons due to their hydrophilicity and molecular size (9). Thus, the metabolism and bioavailability of flavonoids contradict the epidemiological studies (4-8). On the other hand, P-glucuronidase, an exo-glycosidase, hydrolyzes glucuronide-conjugated flavonoids into their aglycons (10). Recent reports (11, 12) have indicated the activation of P-glucuronidase in several cancerous tissues. These reports suggest that flavonoid glucuronides would be de-conjugated into the active form in cancerous tissues and prevent cancer development. However, it is not known whether these events are specific to cancerous tissues and not to normal tissues. In the present study, Fisher 344 rats were induced the early stage of carcinogenesis using a two-stage hepatocarcinogenesis model by N-diethylnitrosamine (DEN) and phenobarbital (PB). The P-glucuronidase activity in the liver and other tissues was compared between the normal and hepatocarcinogenic rats.

Materials and Methods Animal Treatments This study was approved by the Institutional Animal Care and Use Committee (Permission number: 17-03-02) and carried out according to the Guidelines of Animal Experimentation of Kobe University. Male Fisher 344 rats (5 weeks old; Clea Japan, Tokyo, Japan) were acclimatized for 1 week. They were housed in an animal facility maintained on a 12 h light/dark cycle at a constant temperature of 23 ± 1 °C. They were given free access to diet (Oriental MF diet, Oriental Yeast, Tokyo, Japan) and drinking water. As shown in Figure 1,10 rats were divided into 2 groups; i.e., the Control and DEN/PB groups. The DEN/PB group was intraperitoneally injected with DEN (100 mg/kg body weight) dissolved in saline once a week for 3 weeks. One week after the 3rd injection, the rats received 500 ppm of PB in their drinking water for 10 weeks, and then killed. The Control group was injected with saline instead of DEN, and given drinking water.


104 weeks

0

1 2

I

I

3

4

l

I

I

14

Control

TRKF

Group

Saline

DEN/PB Downloaded by INDIANA UNIV BLOOMINGTON on March 23, 2017 | http://pubs.acs.org Publication Date: September 19, 2008 | doi: 10.1021/bk-2008-0993.ch010

Water

Water

ITT

DEN in saline Figure 1. Animal experimental schedule. Fisher 344 rats were intraperitoneal injected with DEN (100 mg/kg body weight), dissolved in saline followed by receiving PB (500 ppm), which is contained in drinking water after 1 week interval. The Control group was injected saline and given drinking water. At week 14, all rats were killed.

Immunohistochemical Staining of Glutathione ^-Transferase Placental Form (GST-P) The liver fixed with paraformaldehyde was embedded in an OCT (optimal cutting temperature) compound (Sakura Finetech, Tokyo, Japan) and sectioned into 40 jam by acryostat. The sections were treated 3 times for 30 min with 0.6% hydrogen peroxide in 0.1 M PBS containing 0.1% Triton X-100 (PBST, pH 7.5) and then washed with PBST. Nonspecific protein binding was blocked by 5% normal goat serum (NGS; Chemicon International, Temecula, CA, USA) in PBST for lh. The sections were incubated with rabbit anti-rat GST-P primary antibody (Medical and Biological Laboratories, Tokyo, Japan) diluted to 1:50 in PBST containing 5% NGS at 4°C overnight. They were incubated with goat anti-rabbit immunoglobulins conjugated peroxidase labelled-dextran polymer in Tris-HCl buffer (EnVision Plus, Dako, Kyoto, Japan) for 30 min, washed with 0.1 M Tris-HCl (pH 7.5) and then reacted with 0.05% 3,3'-diaminobenzidine (DAB), 0.01% hydrogen peroxide, and 0.08% ammonium nickel sulfate in 0.1 M Tris-HCl (pH 7.5) for 4 min. The immunostained sections mounted on slides were dehydrated through a graded series of ethanol, cleared by xylene, and then coverslipped with an embedding compound. The DAB-stained GST-P positive foci were observed by light microscopy. Quantitative analysis of the GST-P positive foci was performed using NIH image version 1.61. The number and areas of GST-P positive foci >0.2 mm in diameter were measured. 2


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Measurement of p-Glucuronidase Activity The P-glucuronidase activity was measured as described previously (75). Briefly, the liver, kidney, lung, heart and thymus were homogenized in 20 mM Tris-HCl (pH 7.4) at 4°C. The protein contents in the homogenates were determined according to the Lowry method (14). The homogenates or plasma were incubated with 50 juL of assay buffer (200 mM sodium acetate, pH 5.0 containing 10 mM EDTA, 0.01% bovine serum albumin, 0.1% Triton X-100 and 0.5 mM 4-mathylumbelliferyl-P-D-glucuronide (MUG) as a substrate) at 37°C. The enzymatic reaction was stopped by adding 150 juL of 200 mM sodium carbonate. The incubation mixture was subsequently added to 5 JIL of 0.5 mM 9-chloromethylanthracecene (Tokyo Kasei Kogyo, Tokyo, Japan) as the internal standard, and centrifuged for 5 min at 15000 rpm. 4-Methylunbelliferone (MU), a hydrolyzed product, in the supernatants was analyzed by HPLC with a fluorescence detector (excitation at 355 nm, emission at 460 nm). The activity was expressed as MU nmols releasedfromMUG.

Results and Discussion The expression of GST-P, a liver-specific pre-cancer lesion, was measured by immunohistochemistry (Figure 2). The GST-P positive foci were not observed in the control group (Figure 2A). In contrast, the foci were significantly observed in the DEN/PB group; i.e., approximately 622 foci per cm were detected, and the area was approximately 2.11 mm /cm in the tissues (Figure 2B). This result indicates that this experimental model obviously induced the early stage of hepatocarcinogenesis in the DEN/PB group rats. To investigate whether the P-glucuronidase specifically increased in cancerous tissues, the activity of P-glucuronidase was compared between the livers of the Control and DEN/PB groups (Table I). The P-glucronidase in the GST-P-expressed liver (the DEN/PB group) significantly increased by 1.5-fold compared to the Control group; i.e., 640 ± 105 (MU nmols/mg protein/h) in the DEN/PB group, and 430 ± 29 (MU nmols/mg protein/h) in the Control group. On the other hand, the enzymatic activity of the other tissues, kidney, lung, heart, thymus and plasma was no significant differences between both groups. These results indicated that p-glucuronidase would be specifically activated in the early stage of carcinogenic tissues. There is a possibility that flavonoid aglycons increase by de-conjugation with activated P-glucuronidase in the carcinogenic tissues and prevent cancer development. To elucidate the contradiction between the metabolism and bioavailability of flavonoids and the epidemiologic studies, further studies are required; e.g., a comparative study of 2

2

2



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Figure 2. Expression of GST-P in liver. Fisher 344 rats were treated according to the experimental schedule shown in Figure 1. The GST-P positive foci in the liver were stained with DAB as described in the Materials and Methods. The back blots were DAB-stained GST-P positive foci. The data are representativ Original magnification *210.

Table I. Comparison of ^-Glucuronidase Activity in Tissues and Plasma P-Glucuronidase Group

Liver

a)

Kidney

a)

149 ± 14 Control 430 ± 2 9 DEN/P 640 ±105* 158 ± 5 a) b)

Lung 118 ± 9 121 ± 7

0

Heart*

14.3 ±1.1 15.8 ±1.9

Thymus

a)

Plasma

10

141 ±20 30.6 ±5.8 134 ± 9 31.3 ±6.9

The values are mean±SD. (MU nmols/mg protein/h) The values are mean±SD. (MU nmols/ml) Significantly different from the Control group (PO.05) as determined by the Student's t test.


107 the concentration of the aglycons form in these tissues as well as the kinetic study of the activation of β-glucronidase, among others.

References 1.


2. 3. 4. 5. 6.

7. 8. 9. 10. 11.

12. 13. 14.

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A Possible Mechanism That Flavonoids Exert Anticarcinogenesis with

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