Inhibitory Effects of 6-Methylsulfinylhexyl Isothiocyanate on Superoxide Anion Generation from Differentiated HL-60 Human Promyelocytic Leukemia Cells

Abstract

Wasabi is a plant of Japanese origin. It belongs to the Brassicaceae family and produces various isothiocyanates (ITCs). Because 6-methylsulfinylhexyl isothiocyanate (6-MSITC) is particularly stable among wasabi ITCs, it has potential as a functional compound. To clarify the antioxidative activity of 6-MSITC, we investigated its inhibitory effect on superoxide anion (O₂•⁻) generation from phorbol 12-myristate 13-acetate (PMA)-stimulated differentiated HL-60 human promyelocytic leukemia cells. Our results showed that 6-MSITC inhibited O₂•⁻ generation in these cells (IC₅₀ value: 28.9 µM). 6-MSITC at 0.8, 4, and 20 µM significantly inhibited the consumption of exogenously added NADH compared with the control. On the other hand, < 263 µM 6-MSITC did not scavenge O₂•⁻ in cell-free experiments. Therefore, we suggest that 6-MSITC inhibited O₂•⁻ generation by decreasing an enzymatic reaction with NADPH oxidase. Thus, 6-MSITC may be useful as a functional compound.

Introduction

When excess generation of reactive oxygen species (ROS) occurs in the body, biological compounds such as proteins, lipids, and DNA are oxidized. This oxidation promotes aging and causes diseases such as Alzheimer's, arteriosclerosis, and cancer (Yoshikawa, 2011). Therefore, functional foods and their constituents that exhibit antioxidative activity have been studied (Chow et al., 2007; Mizuno et al., 2011; Nakamura et al., 1998) and developed.

Neutrophils, a type of leukocyte, are the main source of ROS generation in the body. When microbes invade the body, neutrophils generate superoxide anions (O₂•⁻). O₂•⁻ and associated ROS play an important defensive role against microbes. However, the excess ROS generated by neutrophils promotes inflammation and is implicated in various diseases (Yoshikawa, 2011).

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, known to generate O₂•⁻ in neutrophils, is composed of membrane-bound cytochrome b₅₅₈ [gp91^phox (also called Nox2) and p22^phox] and cytosolic proteins (p47^phox, p67^phox, Rac, and p40^phox) (Chanock et al., 1994; Miyano and Sumimoto, 2009). When protein kinase C (PKC) is activated, PKC phosphorylates p47^phox (Benna et al., 1996). The phosphorylation of p47^phox causes the translocation of cytosolic proteins to the membrane (Chanock et al., 1994; Miyano and Sumimoto, 2009). When the NADPH oxidase complex is assembled, gp91^phox generates O₂•⁻ as a substrate in NADPH and/or nicotinamide adenine dinucleotide (reduced form) (NADH) (Chanock et al., 1994; Miyano and Sumimoto, 2009).

A human cell line derived from promyelocytic leukemia cells (HL-60 cells) can differentiate into granulocytes consisting of neutrophils, myelocytes, and metamyelocytes upon stimulation with retinoic acid (Breitman et al., 1980). Phorbol 12-myristate 13-acetate (PMA) translocates PKC-β, an isotype of PKC, to the membrane and activates NADPH oxidase in neutrophils (Dekker et al., 2000). Therefore, if the O₂•⁻ generation from PMA-stimulated differentiated HL-60 cells can be suppressed by certain compounds, such compounds may be useful as antioxidants.

Wasabi [Wasabia japonica (Miq.) Matsum. syn. Eutrema japonicum (Sieb.) Maxim.)] is a plant of Japanese origin (Yamane et al., 2016) that belongs to the Brassicaceae family. Grated wasabi rhizome, which contains various isothiocyanates (ITCs) (Etoh et al., 1990; Kumagai et al., 1994), is a popular condiment in Japan. Most ITCs are pungent and flavorful compounds. Because 6-methylsulfinylhexyl isothiocyanate (6-MSITC) has a weak wasabi flavor (Etoh et al., 1990), it is thought to be particularly stable among the ITCs in wasabi. In addition, 6-MSITC is the most abundant ITC in wasabi after allyl isothiocyanate (Etoh et al., 1990). Therefore, 6-MSITC may be useful as a functional compound.

6-MSITC has been studied for its physiological function, including antiplatelet (Morimitsu et al., 2000), anticancer (Morimitsu et al., 2000; Watanabe et al., 2003), detoxifying (Morimitsu et al., 2000; Toyama et al., 2011), anti-inflammatory (Uto et al., 2005), antidiabetic (Fukuchi et al., 2004; Yoshida et al., 2011), and antiallergic activities (Nagai and Okunishi, 2009; Yamada-Kato et al., 2012).

The antioxidative effects of 6-MSITC have also been studied. Kinae et al. (2000) reported that 6-MSITC may act as an antioxidant and/or radical scavenger in the formation of heterocyclic amines. The NF-E2-related factor 2/kelchlike ECH-associating protein 1 (Nrf2/Keap1) system is well known to play an important role in the expression of antioxidant enzymes. When the Keap1-Nrf2 complex is disrupted, Nrf2 translocates to the nucleus and activates the antioxidant response element (ARE). Morimitsu et al. (2002) reported that 6-MSITC induced the nuclear localization of Nrf2 and activated the ARE receptor genes in RL34 cells. Furthermore, Hou et al. (2011) revealed the molecular mechanisms behind the activation of the Nrf2/Keap1 system by 6-MSITC in human hepatoblastoma HepG2 cells. Additionally, Mizuno et al. (2011) reported that 6-MSITC significantly protected against hydrogen peroxide (H₂O₂)- and paraquat-induced cytotoxicity in primary neuronal cultures of rat striatum. Regarding the mechanism involved, 6-MSITC was suggested to increase the intracellular glutathione content via an increase in γ-glutamylcysteine synthetase expression induced by activation of the Nrf2/Keap1 system.

Benzyl isothiocyanate (BITC), which is contained in members of the Brassicaceae family such as papaya (Tang et al., 1983), has also been studied for its antioxidative effects. BITC inhibited PMA-induced O₂•⁻ generation from differentiated HL-60 cells (Miyoshi et al., 2004). In addition, it significantly inhibited the consumption of exogenously added NADH. However, BITC did not inhibit the translocation of PKC-β, although PKC-β was translocated to the membrane from the cytosol by PMA stimulation. Furthermore, it did not inhibit the translocation of p47^phox to the membrane and the assembly of the active NADPH oxidase complex, consisting of cytochrome b₅₅₈ and cytosolic proteins. BITC modified the residual protein thiols and decreased the enzymatic activity of glyceraldehyde-3-phosphate dehydrogenase. BITC might react with Cys-149 and/or Cys-153 in this enzyme, which are important for its activity. Therefore, BITC might directly modify gp91^phox by covalent cysteine modification in the substrate binding site and/or the oxidase catalytic site (Miyoshi et al., 2004; Nakamura, 2004).

In a previous study, other ITCs such as AITC, phenethyl isothiocyanate (PEITC), and methyl isothiocyanate (MITC) inhibited PMA-induced O₂•⁻ generation, while a BITC analog, O-methylbenzyl thiocarbamate [C₆H₅-CH2-NH-C(=S)-OCH₃; BITC-OMe], showed no such ability (Miyoshi et al., 2004). 6-MSITC was reported to conjugate with a thiol group of N-acetyl-l-cysteine (NAC) (Yamaguchi et al., 2008) and a cysteine residue of heat shock protein 90β (HSP90β) (Shibata et al., 2011). Hou et al. (2011) reported that 6-MSITC modified Keap1 protein probably by reacting with thiols in HepG2 cells. Therefore, 6-MSITC may exert an inhibitory effect on PMA-induced O₂•⁻ generation by the modification of gp91^phox.

In this study, to utilize 6-MSITC as a functional compound, we investigated its inhibitory effects on O₂•⁻ generation from differentiated HL-60 cells.

Materials and Methods

Reagents    6-MSITC was synthesized by the oxidation of 6-methylthiohexyl isothiocyanate (Ogawa & Co., Ltd., Tokyo, Japan) in our laboratory (Murata et al., 2004). BITC was purchased from Sigma (MO, USA) and used as a positive control. The chemical structures of these ITCs are shown in Fig. 1. 6-MSITC and BITC dissolved in ethanol were used as test samples.

Fig. 1.

Chemical structures of isothiocyanates.

Fetal bovine serum (FBS) was purchased from Nichirei Biosciences Inc. (Tokyo, Japan). Roswell Park Memorial Institute 1640 (RPMI-1640) medium, retinoic acid, and lucigenin were purchased from Sigma. PMA and β-NADH-2Na were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). L(+)-Ascorbic acid was purchased from Kishida Chemical Co., Ltd. (Osaka, Japan). Cell Count Reagent SF was purchased from Nacalai Tesque, Inc. (Kyoto, Japan).

Cell culture and differentiation    The HL-60 human cell line derived from promyelocytic leukemia cells (RCB3683) was purchased from Riken BioResource Center (Ibaraki, Japan). Cells were cultured in RPMI-1640 medium with 10% FBS at 37°C in a 5% CO₂ incubator.

HL-60 cells were plated at 1.0 × 10⁶ cells/mL in six-well culture plates (3.0 mL/well) with RPMI-1640 medium containing 10% FBS and 1 µM retinoic acid. They were cultured at 37°C in a 5% CO₂ incubator for 9 days to differentiate to neutrophils. After the medium was removed by centrifugation, the differentiated HL-60 cells were suspended at 1.0 × 10⁶ cells/mL in RPMI-1640 (serum-free medium).

Nitroblue tetrazolium (NBT) is known to be reduced by O₂•⁻, and cells are stained by this reduction. The differentiation of HL-60 cells to neutrophils was estimated using NBT staining. As a result of counting the number of stained cells, approximately 70% of the cells differentiated to neutrophils upon treatment with retinoic acid for 9 days (data not shown).

Cell viability    Cell viability was determined by the WST assay using 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonyl)-2H-tetrazolium (WST-8). The differentiated HL-60 cells were suspended at 1.0 × 10⁶ cells/mL in RPMI-1640 (serum-free medium). The cell suspension was transferred to 96-well plates (90 µL/well), and 10 µL of RPMI-1640 (serum-free medium) with the test samples was added and incubated at 37°C for 10 min in a 5% CO₂ incubator. After adding 100 µL of phosphate-buffered saline (PBS) with 20% FBS and 100 nM PMA, the cell suspension was incubated at 37°C for 30 min in a 5% CO₂ incubator. After adding 50 µL of RPMI-1640 medium with 10% FBS and Cell Count Reagent SF (final concentration 10%), the cell suspension was incubated at 37°C in a 5% CO₂ incubator. After 30 and 90 min, absorption at 450 nm was measured with the reference wavelength at 590 nm using a plate reader (Precision microplate reader; Molecular Devices Corporation, CA, USA). The absorption change per time unit was calculated to determinate the cell viability.

O₂•⁻ generation assay in differentiated HL-60 cells    The differentiated HL-60 cells were suspended at 1.0 × 10⁶ cells/mL in RPMI-1640 (serum-free medium). The cell suspension was then transferred to 96-well plates (90 µL/well), and 10 µL of RPMI-1640 (serum-free medium) with the test samples was added and incubated at 37°C for 10 min. After adding 100 µL of PBS with 20% FBS, 100 nM PMA, and 1 mM lucigenin, the cell suspension was incubated at 37°C for 5 min in a 5% CO₂ incubator. Luminescence was measured using a plate reader (Precision microplate reader; Molecular Devices Corporation).

NADH consumption    The differentiated HL-60 cells were suspended at 5.55 × 10⁶ cells/5 mL of RPMI-1640 with 10% FBS. After 1 µL of the test samples was added, the cell suspension was incubated at 37°C for 15 min in a 5% CO₂ incubator. After the medium was removed by centrifugation, the cells were suspended at 1.0 × 10⁶ cells/90 µL in PBS and the cell suspension was then transferred to 96-well plates (90 µL/well). After 10 µL of 10 mM NADH was added, the cell suspension was incubated at 37°C for 10 min in a 5% CO₂ incubator. Then, 100 µL of PBS with 200 nM PMA was added. After the cell suspension was incubated at 37°C for 15 min in a 5% CO₂ incubator, absorption at 340 nm was measured using a plate reader (Precision microplate reader; Molecular Devices Corporation).

Superoxide dismutase (SOD)-like activity    SOD-like activity was assessed using the SOD Assay Kit-WST in accordance with the manufacturer's instructions (Dojindo Laboratories, Kumamoto, Japan). The test samples were added to a 96-well plate (20 µL/well). After supplementation with 200 µL of WST working solution (Dojindo Laboratories), samples were mixed with a plate mixer. Enzyme working solution (Dojindo Laboratories) was then added (20 µL/well), followed by incubation at 37°C for 20 min. Absorption at 450 nm was measured using a plate reader (Precision microplate reader; Molecular Devices Corporation). The final concentration of ethanol in the test samples was 8.3%. The control containing ethanol at 8.3% did not have SOD-like activity (data not shown).

Statistical analysis    Results are expressed as mean ± standard deviation. Statistical comparisons were performed using a Student's t-test for paired values. Results with p < 0.05 were considered statistically significant.

Results and Discussion

Cell viability was determined by the WST assay. Cell viability decreased remarkably at more than 100 µM 6-MSITC and BITC, indicating cytotoxic effects had been detected (Fig. 2). To clarify that the antioxidative activity was not influenced by the decrease in cell viability, the treatment time of test samples was kept equal in the cell viability test and the O₂•⁻ generation assay.

Fig. 2.

Effects of 6-MSITC on HL-60 cell viability. The differentiated HL-60 cells were preincubated with test samples at 37°C for 10 min before adding PMA for 30 min. Then, the cell suspension was supplemented with Cell Count Reagent and incubated for 30 and 90 min. The absorption change per time unit was calculated to determinate the cell viability. Data are expressed as mean ± SD (n = 5).

Lucigenin is a chemiluminescent reagent and detects O₂•⁻ specifically among ROS. Myhre et al. (2003) reported that lucigenin detected O₂•⁻, but not nitric oxide, peroxynitrite, H₂O₂, or hydroxyl radical in neutrophils, while Akiyama et al. (2012) reported that lucigenin detects O₂•⁻ and hypochlorous acid in hypoxanthine-xanthine oxidase and myeloperoxidase-H₂O₂-chloride systems, respectively. In this study, when ROS were generated in the PMA-stimulated differentiated HL-60 cells, SOD dramatically reduced the luminescence (data not shown). Therefore, O₂•⁻ generation from NADPH oxidase in the differentiated HL-60 cells was specifically detected.

The O₂•⁻ generation from PMA-stimulated differentiated HL-60 cells was determined by luminescence, after the cells were supplemented with lucigenin. The luminescence of PMA-stimulated differentiated HL-60 cells in the control was 30,000–50,000 relative light units (RLU) (data not shown). As shown in Fig. 3, 6-MSITC and BITC inhibited O₂•⁻ generation from the differentiated HL-60 cells. The IC₅₀ values of 6-MSITC and BITC were estimated to be 28.9 µM and 33.9 µM, respectively. More than 100 µM 6-MSITC and BITC decreased cell viability (Fig. 2) and inhibited O₂•⁻ generation from PMA-stimulated differentiated HL-60 cells (Fig. 3). O₂•⁻ generation might have decreased because of cell death.

Fig. 3.

Inhibitory effect of 6-MSITC on O₂•⁻ generation from HL-60 cells. The differentiated HL-60 cells were preincubated with test samples at 37°C for 10 min before adding PMA for 5 min. Data are expressed as mean ± SD (n = 5).

To clarify the mechanism of inhibition of O₂•⁻ generation by 6-MSITC, the consumption of exogenously added NADH was determined. As a result, 6-MSITC at 0.8 µM, 4 µM, and 20 µM and BITC at 20 µM significantly inhibited NADH consumption compared with the control (Fig. 4).

Fig. 4.

Inhibitory effect of 6-MSITC on the consumption of exogenously added NADH. The differentiated HL-60 cells were preincubated with test samples at 37°C for 15 min before adding NADH for 10 min. The cell suspension was then stimulated by PMA for 15 min. Data are expressed as mean ± SD (n = 5). Statistically significant differences (Student's t-test) on comparing with the control value are indicated as *p < 0.05.

On the other hand, it is also possible that 6-MSITC scavenged the generated O₂•⁻. Therefore, the SOD-like activity of 6-MSITC was determined. If test samples have SOD-like activity, they may scavenge O₂•⁻ directly and/or reduce xanthine oxidase activity, although these possibilities are not distinguished in our experimental condition. The luminescence of lucigenin with a xanthine/xanthine oxidase system in the SOD Assay Kit-WST was approximately 150,000 RLU (data not shown). Thus, the amount of O₂•⁻ generated from this xanthine/xanthine oxidase system was higher than that from PMA-stimulated differentiated HL-60 cells. Ascorbic acid, an antioxidant with O₂•⁻ scavenging activity, was used as a positive control. As shown in Fig. 5, 6-MSITC, BITC, and ascorbic acid showed SOD-like activity in a concentration-dependent manner. The IC₅₀ values of 6-MSITC and ascorbic acid were estimated to be 4.4 mM and 14.4 µM, respectively. Although BITC at 2.63 mM scavenged 10% of O₂•⁻, its IC₅₀ value could not be estimated. The SOD-like activity was ineffective at less than 263 µM 6-MSITC.

Fig. 5.

SOD-like activity of 6-MSITC. The test samples were incubated with WST working solution and enzyme working solution. Data are expressed as mean ± SD (n = 3).

6-MSITC inhibited O₂•⁻ generation from the differentiated HL-60 cells, with an estimated IC₅₀ value of 28.9 µM. 6-MSITC at 0.8 µM, 4 µM, and 20 µM significantly inhibited NADH consumption compared with the control. On the other hand, the SOD-like activity was ineffective at <263 µM 6-MSITC, i.e., <263 µM 6-MSITC did not scavenge O₂•⁻. The concentration at which 6-MSITC scavenged O₂•⁻ is much higher than the concentration at which O₂•⁻ generation from differentiated HL-60 cells was inhibited by 6-MSITC. Therefore, the O₂•⁻ scavenging activity is not attributed to inhibitory effects on O₂•⁻ generation from the differentiated HL-60 cells. We suggest that 6-MSITC reduced O₂•⁻ generation by decreasing enzymatic reaction with NADPH oxidase.

Miyoshi et al. (2004) reported that BITC significantly inhibited PMA-induced O₂•⁻ generation from differentiated HL-60 cells. Because BITC modified the residual protein thiols, BITC might directly modify the gp91^phox by covalent cysteine modification (Miyoshi et al., 2004; Nakamura, 2004). On the other hand, 6-MSITC was also reported to conjugate with a thiol group of NAC (Yamaguchi et al., 2008) and a cysteine residue of HSP90β (Shibata et al., 2011). Therefore, 6-MSITC might directly modify the gp91^phox similarly to BITC. AITC, PEITC, and MITC were reported to inhibit the PMA-induced O₂•⁻ generation from differentiated HL-60 cells (Miyoshi et al., 2004). Because ITCs were reported to react with cysteine thiol groups of proteins (Zhang, 2011), ITCs may have an inhibitory effect on PMA-induced O₂•⁻ generation from differentiated HL-60 cells.

On the other hand, 6-MSITC might inhibit the upstream mechanism; it might inhibit the translocation of PKC-β by PMA and that of cytosolic protein to the membrane, although BITC reportedly did not inhibit the translocation of PKC-β (Miyoshi et al., 2004). Furthermore, 6-MSITC was reported to activate the Nrf2/Keap1 system, which is known to express antioxidant enzymes (Hou et al., 2011; Morimitsu et al., 2002). In this study, the inhibitory effect on PMA-induced O₂•⁻ generation from differentiated HL-60 cells might be influenced by the expression of antioxidant enzymes.

We reported that 6-MSITC inhibits O₂•⁻ generation from differentiated HL-60 cells, due to decreased enzymatic reaction with NADPH oxidase. These results indicate that 6-MSITC is a potential functional compound for antioxidative activity. Future research and development of 6-MSITC should be performed.

Acknowledgments The authors thank Enago (www.enago.jp) for the English language review.

References

•  Akiyama,  K.,  Ohota,  Y., and  Tokunaga,  K. (2012). Evaluation of reactive oxygenproduction using whole blood samples by chemiluminescence. Journal of Analytical Bio-Science (Seibutsu Shiryo Bunseki), 35, 140-145 (in Japanese).
•  Benna,  J.E.,  Faust,  R.P.,  Johnson,  J.L., and  Babior,  B.M. (1996). Phosphorylation of the respiratory burst oxidase subunit p47^phox as determined by two-dimensional phosphopeptide mapping. J. Biol. Chem., 271, 6374-6378.
•  Breitman,  T.R.,  Selonick,  S.E., and  Collins,  S.J. (1980). Induction of differentiation of the human promyelocytic leukemia cell line (HL-60) by retinoic acid. Proc. Natl. Acad. Sci. USA., 77, 2936-2940.
•  Chanock,  S.J.,  Benna,  J.E.,  Smith,  R.M., and  Babior,  B.M. (1994). The respiratory burst oxidase. J. Biol. Chem., 269, 24519-24522.
•  Chow,  S.E.,  Hshu,  Y.C.,  Wang,  J.S., and  Chen,  J.K. (2007). Resveratrol attenuates oxLDL-stimulated NADPH oxidase activity and protects endothelial cells from oxidative functional damages. J. Appl. Physiol., 102, 1520-1527.
•  Dekker,  L.V.,  Leitges,  M.,  Altschuler,  G.,  Mistry,  N.,  McDermott,  A.,  Roes,  J., and  Segal,  A.W. (2000). Protein kinase C-β contributes to NADPH oxidase activation in neutrophils. Biochem. J., 347, 285-289.
•  Etoh,  H.,  Nishimura,  A.,  Takasawa,  R.,  Yagi,  A.,  Saito,  K.,  Sakuta,  K.,  Kishima,  I., and  Ina,  K. (1990). ω-Methylsulfinylalkyl isothiocyanates in wasabi, Wasabia japonica Matsum. Agric. Biol. Chem., 54, 1587-1589.
•  Fukuchi,  Y.,  Kato,  Y.,  Okunishi,  I.,  Matsutani,  Y., and  Osawa,  T. (2004). 6-Methylsulfinylhexyl isothiocyanate, an antioxidant derived from Wasabia japonica MATUM, ameliorates diabetic nephropathy in type 2 diabetic mice. Food Sci. Technol. Res., 10, 290-295.
•  Hou,  D.X.,  Korenori,  Y.,  Tanigawa,  S.,  Yamada-Kato,  T.,  Nagai,  M.,  He,  X., and  He,  J. (2011). Dynamics of Nrf2 and Keap1 in ARE-mediated NQO1 expression by wasabi 6-(methylsulfinyl)hexyl isothiocyanate. J. Agric. Food Chem., 59, 11975-11982.
•  Kinae,  N.,  Masuda,  H.,  Shin  I.S.,  Furugori.,  M., and  Simoi,  K. (2000). Functional properties of wasabi and horseradish. BioFactors, 13, 265-269.
•  Kumagai,  H.,  Kashima,  N.,  Seki,  T.,  Sakurai,  H.,  Ishii,  K., and  Ariga,  T. (1994). Analysis of volatile components in essential oil of upland Wasabi and their inhibitory effects on platelet aggregation. Biasci. Biotech. Biochem., 58, 2131-2135.
•  Miyano,  K. and  Sumimoto,  H. (2009). Oxidative stress and the Nox family. Journal of Analytical Bio-Science (Seibutsu Shiryo Bunseki), 32, 289-296 (in Japanese).
•  Miyoshi,  N.,  Takabayashi,  S.,  Osawa,  T., and  Nakamura,  Y. (2004). Benzyl isothiocyanate inhibits excessive superoxide generation in inflammatory leukocytes: implication for prevention against inflammation-related carcinogenesis. Carcinogenesis, 25, 567-575.
•  Mizuno,  K.,  Kume,  T.,  Muto,  C.,  Takada-Takatori,  Y.,  Izumi,  Y.,  Sugimoto,  H., and  Akaike,  A. (2011). Glutathione biosynthesis via activation of the nuclear factor E2-related factor 2 (Nrf2) — antioxidant-response element (ARE) pathway is essential for neuroprotective effects of sulforaphane and 6-(methylsulfinyl) hexyl isothiocyanate. J. Pharmacol. Sci., 115, 320-328.
•  Morimitsu,  Y.,  Hayashi,  K.,  Nakagawa,  Y.,  Fujii,  H.,  Horio,  F.,  Uchida,  K., and  Osawa,  T. (2000). Antiplatelet and anticancer isothiocyanates in Japanese domestic horseradish, wasabi. Mech. Ageing Dev., 116, 125-134.
•  Morimitsu,  Y.,  Nakagawa,  Y.,  Hayashi,  K.,  Fujii,  H.,  Kumagai,  T.,  Nakamura,  Y.,  Osawa,  T.,  Horio,  F.,  Itoh,  K.,  Iida,  K.,  Yamamoto,  M., and  Uchida,  K. (2002). A sulforaphane analogue that potently activates the nrf2-dependent detoxification pathway. J. Biol. Chem., 277, 3456-3463.
•  Murata,  M.,  Uno,  M.,  Nagai,  Y.,  Nakagawa,  K., and  Okunishi,  I. (2004). Content of 6-methylsulfinylhexyl isothiocyanate in wasabi and processed wasabi products. J. Jpn. Soc. Food Sci. Technol. (Nippon Shokuhin kagaku Kogaku Kaishi), 51, 477-482 (in Japanese).
•  Myhre,  O.,  Andersen,  J.M.,  Aarnes,  H., and  Fonnum,  F. (2003). Evaluation of the probes 2′,7′-dichlorofluorescin diacetate, luminol, and lucigenin as indicators of reactive species formation. Biochem. Pharmacol., 65, 1575-1582.
•  Nagai,  M. and  Okunishi,  I. (2009). The effect of wasabi rhizome extract on atopic dermatitis-like symptoms in HR-1 hairless mice. J. Nutr. Sci. Vitaminol., 55, 195-200.
•  Nakamura,  Y.,  Ohto,  Y,  Murakami,  A.,  Osawa,  T., and  Ohigashi,  H. (1998). Inhibitory effects of curcumin and tetrahydrocurcuminoids on the tumor promoter-induced reactive oxygen species generation in leukocytes in vitro and in vivo. J. Cancer Res., 89, 361-370.
•  Nakamura,  Y. (2004). Cancer chemoprevention by isothiocyanates — Cancerous cell-specific control of cell proliferation and its molecular mechanism —. Environmental mutagen research communication (Kankyo Henigen Kenkyu), 26, 253-258 (in Japanese).
•  Shibata,  T.,  Kimura,  Y.,  Mukai,  A.,  Mori,  H.,  Ito,  S.,  Asaka,  Y.,  Oe,  S.,  Tanaka,  H.,  Takahashi,  T., and  Uchida,  K. (2011). Transthiocarbamoylation of proteins by thiolated isothiocyanates. J. Biol. Chem., 286, 42150-42161.
•  Tang,  C.S. and  Takenaka,  T. (1983). Quantitation of a bioactive metabolite in undisturbed rhizosphere ? Benzyl isothiocyanate from Carica papaya L. J. Chem. Ecol., 9, 1247-1253.
•  Toyama,  T.,  Shinkai,  Y.,  Yasutake,  A.,  Uchida,  K.,  Yamamoto,  M., and  Kumagai,  Y. (2011). Isothiocyanates reduce mercury accumulation via an Nrf2-dependent mechanism during exposure of mice to methylmercury. Environ. Health Perspec., 119, 1117-1122.
•  Uto,  T.,  Fujii,  M., and  Hou,  D.X. (2005). Inhibition of lipopolysaccharaide-induced cyclooxygenase-2 transcription by 6-(methylsulfinyl) hexyl isothiocyanate, a chemopreventive compound from Wasabia japonica (Miq.) Matsumura, in mouse macrophages. Biochem. Pharmacol., 70, 1772-1784.
•  Watanabe,  M.,  Ohata,  M.,  Hayakawa,  S.,  Isemura,  M.,  Kumazawa,  S.,  Nakayama,  T.,  Furugori,  M., and  Kinae,  N. (2003). Identification of 6-methylsulfinylhexyl isothiocyanate as an apoptosis-inducing component in wasabi. Phytochemistry, 62, 733-739.
•  Yamada-Kato,  T.,  Nagai,  M.,  Ohnishi,  M., and  Yoshida,  K. (2012). Inhibitory effects of wasabi isothiocyanates on chemical mediator release in RBL-2H3 rat basophilic leukemia cells. J. Nutr. Sci. Vitaminol., 58, 303-307.
•  Yamaguchi,  H.,  Noshita,  T.,  Kidachi,  Y.,  Umetsu,  H.,  Fuke,  Y., and  Ryoyama,  K. (2008). Detection of 6-(methylsulfinyl)hexyl isothiocyanate (6-MITC) and its conjugate with N-acetyl-l-cysteine (NAC) by high performance liquid chromatograpy-atmospheric pressure chemical ionization mass spectrometry (HPLC-MS/APCI). Chem. Pharm. Bull., 56, 715-719.
•  Yamane,  K.,  Sugiyama,  Y.,  Lu,  Y.X.,  Lü,  Na.,  Tanno,  K.,  Kimura,  E., and  Yamaguchi,  H. (2016). Genetic differentiation, molecular phylogenetic analysis, and ethnobotanical study of Eutrema japonicum and E. tenue in Japan and E. yunnanense in China. The Horticulture Journal, 85, 46-54.
•  Yoshida,  J.,  Nomura,  S.,  Nishizawa,  N,  Ito,  Y., and  Kimura,  K. (2011). Glycogen synthase kinase-3β inhibition of 6-(methylsulfinyl)hexyl isothiocyanate derived from wasabi (Wasabia japonica Matsum). Biosci. Biotechnol. Biochem., 75, 136-139.
•  Yoshikawa,  T. (2011). Free radicals and medicine. Journal of Kyoto Prefectural University of Medicine (Kyoto Furitsu Ikadaigaku Zasshi), 120, 381-391 (in Japanese).
•  Zhang,  Y. (2012). The molecular basis that unifies the metabolism, cellular uptake and chemopreventiveactivities of dietary isothiocyanates. Carcinogenesis, 33, 2-9.