Free Radicals in Food - ACS Publications - American Chemical Society

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on February 1, 2017 | http://pubs.acs.org Publication Date: March 4, 2002 | doi: 10.1021/bk-2002-0807.ch022

Chapter 22

Wasabi: A Traditional Japanese Food That Contains an Exceedingly Potent Glutathione S-Transferase Inducer for RL34 Cells 1,3

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Yasujiro Morimitsu , Y. Nakamura , T. Osawa , and K. Uchida 1

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Laboratory of Food and Biodynamics, Nagoya University Graduate School of Bioagricultural Sciences, Nagoya 464-8601, Japan Laboratory of Organic Chemistry in Life Science, Graduate School of Agricultural, Kyoto University, Kyoto 606-8502, Japan Current address: Laboratory of Food Chemistry Ochanomizu University, Otsuka 2-1-1, Bunkyo-ku, Tokyo 112-8610, Japan Current address: Laboratory of Food and Biodynamics, Nagoya University Graduate School of Bioagricultural Sciences, Nagoya 464-8601, Japan

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In the course of our screening of edible plants on the induction of glutathione S-transferase (GST) activity in rat liver epithelial RL34 cells, 6-methylsulfinylhexyl isothiocyanate (MS-ITC) was isolated from wasabi (Wasabia japonica) as a potential inducer of GST. As a result from western blot analyses of GST expression induced by MS-ITC in RL34 cells, increasing of protein levels of both GST-Ya and GST-Υp were detected. And as a result from time dependent GSH level by MS-ITC, the GSH level in RL34 cells was initially decreased, then increased gradually to 1.8 times higher than its basal level after 12 hrs. By adding benzyl isothiocyanate (BITC) isolated from papaya instead of MS— ITC to the cells, the reactive oxygen intermediates (ROIs) detected by afluorescenceprobe were increased immediately From these data, we discuss the relation between levels of intracellular oxidation and the induction of GST.

© 2002 American Chemical Society Morello et al.; Free Radicals in Food ACS Symposium Series; American Chemical Society: Washington, DC, 2002.

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302 Wasabi (Wasabia japonica, syn. Eutrema wasabi) is one of traditional foods in Japan, and is now worldwide used in the pungent spice of the "sushi". Wasabi belong to the family Cruciferae such as horseradish, mustard, cabbage and broccoli. When Cruciferous vegetables are damaged, glucosinolates are hydrolyzed by myrosinase, and characteristic isothiocyanates are formed immediately. Most of these Crucifer isothiocyanates (cf. allyl isothiocyanate) are well known for having antimicrobial, fungicidal, and pesticidal activities (1,2). For example, a major wasabi flavor compound 6-methylthiohexyl isothiocyanate, has been shown the antimicrobial activity (5). On the other hand, epidemiological studies have demonstrated that consumption of cruciferous vegetables is associated with a lower incidence of cancers (4-6). Induction of phase II enzymes such as GST and/or quinone reductase (QR) has been demonstrated in broccoli (7,8% cabbage (9), and Brussel sprouts (10,11). Many natural isothiocyanates derived from cruciferous vegetables and somefruitshave been shown to induction of phase Π enzymes in cultured cells and rodents (12-14). We have studied on physiological, pharmaceutical or therapeutic effects of various edible plants, especially the sulfur-containing compoundsfromonion (15,16). Our continuous screening for the induction of GST activity in RL34 cells resulted in isolation of MS-ITCfromthe ethyl acetate (EtOAc) extract of wasabi. In this study, we describe the isolation of MS-ITCfromwasabi, and examined the protein levels of GST isozyme induced by MS-ITC. As a result from elucidation of the intracellular redox state of BITC instead of MS-ITC, the isothiocyanate moiety of these food factors plays an important role for the induction of phase II enzymes. Experimental Materials and Chemicals. Wasabi roots were kindly giftedfromKinjirushi Wasabi Co., Ltd (Aichi, Japan). All other vegetables andfruitswere purchased from the market in Nagoya, Japan. Authentic MS-ITC and its derivatives were synthesized in our laboratory. BITC and all other chemicals were purchased from Wako Pure Chemical Industries (Osaka, Japan). Horseradish peroxidaselinked anti-rabbit IgG immunoglobulin and enhanced chemiluminescence (ECL) Western blotting detection reagents were obtainedfromAmersham Pharmacia Biotech. The protein concentration was measured using the BCA protein assay reagent obtainedfromPierce. 2,7-Dichlorofluorescin diacetate (DCF-DA) was obtainedfromMolecular Probes (Leiden, the Netherlands). Cell Cultures and Enzyme Assay. The induction of GST was measured in RL34 cells by using 1 -chloro-2,4-dinitrobenzene as a substrate according to the

Morello et al.; Free Radicals in Food ACS Symposium Series; American Chemical Society: Washington, DC, 2002.


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reported method (17). The cells were grown as monolayer cultures in DMEM supplemented with 5% heat-inactivated fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, 0.3 mg/ml-glutamine, 0.11 mg/ml pyruvic acid, and 0.37% NaHCCfe at 37°C in an atmosphere of 95% air and 5% C02. Postconfluency cells were exposed to samples for 24 hours in the medium containing 5% fetal bovine serum in 24-well or 96-well microtiter plates (18J9). Usually, 25 μg/ml and 2.5 μg/ml of the samples (as final concentrations in the medium) were tested. Assessment of cytotoxicity after treatment of the sample was measured by protein concentration in an aliquot of the digitonin cell lysate with the BCA protein assay reagent. GSH Assay. Cellular GSH was measured by afluorometricassay (20). Typically, cell lysates were diluted with 0.1M potassium phosphate buffer, pH 8.0, and proteins were precipitated with trichloroacetic acid. A 100 μΐ sample then mixed with 100 μΐ of o-phthaldialdehyde (10 mg in 10 ml of methanol) and 1.8 ml of 0.1M potassium phosphate buffer (pH 8.0) containing 5 mM EDTA. The solution was incubated at room temperature for 15 min, and formation of GSH-phthalaldehyde conjugate was then measured by a fluorescence spectrometer (Hitachi Model F-2000). The conjugate was excited at a wavelength of 350 nm, and the fluorescence emission was measured at 420 nm. Western Blot Analysis. The sample treated and untreated cells were rinsed twice with PBS (pH 7.0) and lysed by incubation at 37°C for 10 min with a solution containing 0.8% digitonin and 2 mM EDTA (pH 7.8). Each whole-cell lysate was then treated with the Laemmuli sample buffer for 3 min at 100°C (21). The samples (20 μg) were run on a 12.5% SDS-PAGE slab gel. One gel was used for staining with Coomassie brilliant blue, and the other was transported on a nitrocellulose membrane with a semidiy blotting cell (TransBlot SD; Bio-Rad), incubated with Block Ace (40 mg/ml) for blocking, washed, and treated with the antibody. Intracellular Oxidative Products Determination. Intracellular oxidative products were detected by 2,7-dichlorofluorescin (DCF) as an intracellular fluorescence probe (22,23). The cells under confluency were treated with DCFDA (50 μΜ) for 30 min at 37°C. After washing twice with PBS, BITC was added to the complete medium and incubated for another 30 min. A flow cytometer (CytoACE 150; JASCO, Tokyo, Japan) was used to detect DCF formed by the reaction of H2DCF with the intracellular oxidative products.


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Results and Discussion Isolation of Inducers from Wasabi. Our screening for induction of GST activity in RL34 cells was undergone on many extracts of various vegetables and fruits. In the course of our screening, the EtOAc extracts of cruciferous vegetables were found to show potent induction activities, especially wasabi. The EtOAc extract of wasabi showed the potent induction activity even though the adding amount of extract was diminished (2.5 μg/ml). After continuous fractionations of the EtOAc extract of wasabi by conventional, a potential inducer of GST in RL34 cells was isolated with yield of 70mgfrom1.3 kg of wasabi root (Fig. 1A). And two homologous isothiocyanates were also isolated as minor inducible compounds. The major compound was identified as 6-methylsulfinylhexyl isothiocyanate (MS-ITC) and two minor compounds were also identified as 5-methylsulfinylpentyl isothiocyanate and 7-methylsulfinylheptyl isothiocyanate respectively by spectroscopic analyses (Fig. IB). MS-ITC is a homologous compound of sulforaphane, which was isolatedfrombroccoli as a potent inducer of phase Π enzymes (24,25). MS-ITC was previously reported to isolatefromcertain wild plants and termed hesperin (26). And MS-ITC has been identified and quantified in wasabi root as well as 6-methylthiohexyl isothiocyanate, a major wasabiflavorcompound (27). Induction of GST in RL34 Cells. Dose-dependent induction activity by MS-ITC in RL34 cells was examined (Fig. 2A). More than 10 μΜ, MS-ITC has begun to show cytotoxicity. As a resultfromwestern blot analyses of GST expression induced by MS-ITC in RL34 cells, increasing of protein levels of both GST-Ya and GST-Yp were detected (Fig. 2B). The GST-Yp isozyme could be one of the important determinants in cancer susceptibility, particularly in diseases where exposure to polycyclic aromatic hydrocarbons is involved. But a recent study using transgenic mice lacking the class π GST demonstrated that this class of GST was involved in the metabolism of carcinogens in mouse skin and had a profound effect on tumorigenicity (28). Additionally, the effect of MS-ITC on levels of phase II enzymes of ICR mice was also examined by gavage (Morimitsu et al., prepared for submitting). As a resultfromdetermination of phase II enzyme activities, MS-ITC was also found to be a potential inducer of GST in vivo, owing to increasing protein levels of GST-Ya. Intracellular Oxidation State Induced by Isothiocyanates. As a resultfromtime dependent GSH level by MS-ITC, the GSH level in RL34 cells was initially decreased, then increased gradually to 1.8 times higher



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Figure 1. Tim isolation scheme (A) mid the chemical structure β) of potent GST inducers in wasabi

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306

Figure 2 Dose-dependent effect of MS-ITC on the cellular GST (A), and timedependent effect of MS-ITC on the cellular GST-Ya and GST-Yp proteins β).



307 than its basal level after 12 hrs (Fig. 3A). In a recent study, we have isolated BITC from papaya as a potent GST inducer (29). In order to know intracellular oxidation degree, BITC in stead of MS-ITC was added to the cells. As shown in Fig. 3B, the reactive oxygen intermediates (ROIs; hydrogen peroxide, lipid peroxide, peroxinitrile, and so on) detected by afluorescenceprobe were increased immediately. The level of ROIs induced by different concentrations of BITC was increased in a dose-dependent manner. The level at 10 μΜ BITC was near 50-fold higher than that of the control. This ROIs generation was mainly caused by the isothiocyanate's highly reactivity with nucleophiles such as GSH, sulfhydryl residues of proteins and so on (25). Concerning about the nonenzymatically conjugation reaction between isothiocyanate group and GSH in RL34 cells at initial step after the addition of MS-ITC resulted in slight GSH depletion. The Michael addition acceptors were reported to be able to induce phase II enzyme activities in vitro and perhaps even in vivo (24,25). Isothiocyanates is right classed in this category. BITC was also decreased the GSH level, then was induced oxidative stress in RL34 cells. It is considered that this GSH depletion comes to the generation of ROIs in the cells. We assume that this slight oxidative stress may lead to increase the protein levels of GST, after induction of signal transductions and gene expressions in cells (30). Acknowledgements The authors thank Kinjirushi Wasabi Co. Ltd. for providing us wasabi research samples. This research project was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN). References 1. Soledade, M.; Pedras, C.; Sorensen, J. L. Phytochemistry 1998, 49, 1959. 2. Tajima, H.; Kimoto, H.; Taketo, Y.; Taketo, A. Biosci. Biotech. Biochem. 1998, 62, 491. 3. Ono, H.; Tesaki, S.; Tanabe, S.; Watanabe, M. Biosci. Biotech. Biochem. 1998, 62, 363. 4. Lee, H. P.; Gourley, L.; Duffy, S. W.; Esteve, J.; Lee, J.; Day, Ν. E. Int. J. Cancer 1989, 434, 1007. 5. Olsen, G. W.; Mandel, J. S.; Gibson, R. W.; Wattenberg, L. W.; Schuman, L. M. Cancer Causes Contr. 1991, 2, 291. 6. Mehta, B. G.; Liu, J.; Constantinou, Α.; Thomas, C. F.; Hawthorne, M.; You, M.; Gerhauser, C.; Pezzuto, J. M.; Moon, R. C.; Moriarty, R. M. Carcinogenesis 1995, 16, 399. 7. Aspry, K. E.; Bjeldanes L. F. Food Chem. Toxicol. 1983, 21, 133.



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Figure 3. Time-dependent effect of MS-ITC on the cellular GSH level (A), and changes in intracellular ROI levels after exposure ofRL34 cells to BTTC (B). For B, the DCFfluorescenceof >10,000 cells was monitored on a flow cytometer with excitation and emission wavelength at 488 nm and 510 nm, respectively. After BTTC stimulation for 30 min, the cells were washed with PBS and resuspended in PBS containing 10 mM EDTA: entry 1, untreated control; entry 2, BTTC 5 μΜ; enry 3, BTTC 10 μΜ; entry 4, BTTC 25 μΜ.

Time after treatment (h)


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12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.

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Free Radicals in Food - ACS Publications - American Chemical Society

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