Eriodictyol and thymonin act as GPR35 agonists.

Although herbs and spices have been used in traditional medicine for more than a century owing to their health benefits, the associated underlying mechanism is still not clear. Since the G protein-coupled receptor 35 (GPR35) has been linked to exert various antioxidant and anti-inflammatory effects, we screened 19 different herbs and spices for possible GPR35 agonist(s) to understand the GPR35-dependent functions of herbs and spices. Among the screened extracts, the ethyl acetate extract of thyme exhibited a remarkable GPR35 agonistic activity. Activity-guided separations allowed us to identify 2 polyphenolic phytochemicals, eriodictyol and thymonin, acting as GPR35 agonists. Both eriodictyol and thymonin showed a potent and specific agonist activity toward GPR35 with half maximal effective concentration values of 5.48 and 8.41 µm, respectively. These findings indicate that these phytochemicals may have beneficial health effects upon GPR35 activation.

Effects of 5-aminolevulinic acid with iron supplementation on respiratory responses to graded cycling and interval walking training achievement in older women over 75 yrs.

BACKGROUND: Exercise training above a given intensity is necessary to prevent age-associated physical disability and diseases; however, the physical and psychological barriers posed by deteriorated physical fitness due to aging may hinder older people from performing daily exercise training. Because 5-aminolevulinic acid (ALA), a precursor of heme, reportedly improves mitochondrial function, we examined whether ALA, combined with sodium ferrous citrate (SFC) for enhancement, improved aerobic capacity and voluntary exercise training achievement in older women aged over 75 yrs. METHODS: The study was conducted using a placebo-controlled, double-blind crossover design. Fifteen women aged ~78 yrs. with no exercise habits underwent two trials for 7 days each where they performed interval walking training (IWT), repeating fast and slow speeds of walking for 3 min each, at >70% and at ~40% of peak aerobic capacity for walking, respectively, with ALA+SFC (100 and 115 mg/day, respectively) or placebo supplement intake (CNT), with a 12-day washout period. Before and after each trial, subjects underwent a graded cycling test while having their oxygen consumption rate (V·O2), carbon dioxide production rate (V·CO2), and plasma lactate concentration ([Lac-]p) measured. Furthermore, during the supplement intake period, exercise intensity for IWT was measured by accelerometry. RESULTS: In ALA+SFC, the increases in V·O2 and V·CO2 during the graded cycling test were attenuated (both, P < 0.01) with a 13% reduction in [Lac-]p (P = 0.012) while none of these attenuated responses occurred in CNT (all, P > 0.46). Furthermore, energy expenditure and time during fast walking for IWT were 25% (P = 0.032) and 21% (P = 0.022) higher in ALA+SFC than in CNT. CONCLUSION: Thus, ALA+SFC supplementation improved aerobic capacity and thus increased fast-walking training achievement in older women.

Diosgenin Supplementation Prevents Lipid Accumulation and Induces Skeletal Muscle-Fiber Hypertrophy in Rats.

Diosgenin (Dio) is a steroid sapogenin found in plants such as Dioscorea species, and is recognized as a phytochemical against various disorders as well as a natural precursor of steroidal drugs. The present study used rats fed high-cholesterol (Chol) diets supplemented with or without 0.5% Dio for 6 wk to investigate the effects of dietary Dio on lipid metabolism. Dio supplementation significantly increased serum high-density lipoprotein Chol concentrations and fecal Chol content, and significantly decreased fecal bile acid content compared rats fed a high-Chol diet alone, showing that dietary Dio may facilitate excretion of Chol rather than bile acids. A reduction in the liver triglyceride content and intra-abdominal visceral fat was observed in Dio-supplemented rats. Interestingly, dietary Dio also significantly increased the skeletal muscle-fiber diameter and area in the thigh muscles of the rats. Mouse myoblast-derived C2C12 cells were used to examine whether Dio directly affected skeletal muscle. Dio promoted fusion of myoblasts into multinucleated cells or myotubes. Furthermore, in myotube C2C12 cells, protein levels of phosphorylated AMP-activated protein kinase (AMPK) increased with Dio treatment in a dose-dependent manner. These results indicate that Dio may not only induce myoblast fusion and enhance skeletal muscle as an energy expenditure organ, but may also activate the catabolic pathway via AMPK in skeletal muscle cells. Thus, these effects of Dio on skeletal muscles may contribute to inhibition of visceral fat accumulation.

Interval Walking Training and Nutritional Intake to Increase Plasma Volume in Elderly.

PURPOSE: Aerobic training-induced plasma volume (PV) expansion improves thermoregulation, and carbohydrate (CHO) + whey protein supplementation enhanced the effects in older people; however, these were suggested by studies on gym-based cycling training but not on home-based interval walking training (IWT). Moreover, long-term walking training effects on PV remain unknown. METHODS: Seventeen male and 10 female subjects (~69 yr), having performed IWT for ≥24 months before the study, were used. After pre-intervention measurement (PRE) of PV, plasma albumin content (Albcont), fasting glucose concentration ([Glc]f), and HbA1c, the subjects were randomly divided into two groups: CHO and Pro-CHO, either consuming CHO (22.5 g) alone or CHO (15 g) + whey protein (10 g), respectively, during additional 5-month IWT from May to November, 2009. After the additional IWT, we measured the same variables again (postintervention measurement). RESULTS: The baseline PV and Albcont were significantly correlated with the number of IWT days for the 12 months preceding PRE (r = 0.716, P < 0.001 and r = 0.671, P < 0.001, respectively). In postintervention, PV and Albcont marginally decreased in CHO from the baselines (P = 0.081 and P = 0.130, respectively) with increased HbA1c (P < 0.001) after correction for the baseline [Glc]f by ANCOVA, but these values remained unchanged in Pro-CHO (both, P > 0.74), with significant differences in the changes between groups (P = 0.020, P = 0.041, and P = 0.018 respectively). CONCLUSIONS: PV was proportional to the number of IWT days for 12 months and a CHO + whey protein supplementation during the 5-month IWT prevented PV reduction for the period of no supplementation, which might be partially linked with blood glucose control mechanisms.

Toll-like receptors as a target of food-derived anti-inflammatory compounds.

Toll-like receptors (TLRs) play a key role in linking pathogen recognition with the induction of innate immunity. They have been implicated in the pathogenesis of chronic inflammatory diseases, representing potential targets for prevention/treatment. Vegetable-rich diets are associated with the reduced risk of several inflammatory disorders. In the present study, based on an extensive screening of vegetable extracts for TLR-inhibiting activity in HEK293 cells co-expressing TLR with the NF-κB reporter gene, we found cabbage and onion extracts to be the richest sources of a TLR signaling inhibitor. To identify the active substances, we performed activity-guiding separation of the principal inhibitors and identified 3-methylsulfinylpropyl isothiocyanate (iberin) from the cabbage and quercetin and quercetin 4'-O-ß-glucoside from the onion, among which iberin showed the most potent inhibitory effect. It was revealed that iberin specifically acted on the dimerization step of TLRs in the TLR signaling pathway. To gain insight into the inhibitory mechanism of TLR dimerization, we developed a novel probe combining an isothiocyanate-reactive group and an alkyne functionality for click chemistry and detected the probe bound to the TLRs in living cells, suggesting that iberin disrupts dimerization of the TLRs via covalent binding. Furthermore, we designed a variety of iberin analogues and found that the inhibition potency was influenced by the oxidation state of the sulfur. Modeling studies of the iberin analogues showed that the oxidation state of sulfur might influence the global shape of the isothiocyanates. These findings establish the TLR dimerization step as a target of food-derived anti-inflammatory compounds.

Effects of bioreactive acrolein from automotive exhaust gases on human cells in vitro.

Acrolein is a toxic unsaturated aldehyde and widespread environmental pollutant produced during lipid peroxidation and also by burning of tobacco or liquid fuels. Inhalation or dermal exposure to acrolein could be toxic to organisms. This very reactive aldehyde has a strong affinity for binding to proteins thus forming pathogenic protein-adducts. In the present study we have analyzed formation of bioreactive acrolein-protein adducts in bovine serum albumin solution exposed to exhaust gases of mineral diesel fuel and of mineral diesel fuel supplemented with different amounts of a novel diesel fuel additive denoted Ecodiesel (produced by a genuine procedure of recycling of plant oils used for food preparation). The effects of acrolein-protein adducts were tested on human microvascular endothelial cells and on human osteosarcoma cells that are sensitive to bioactivities of lipid peroxidation products. The results have shown a reduction of the bioreactive acrolein in exhaust gases when mineral diesel was supplemented with 5-20% Ecodiesel. Moreover, acrolein-protein adducts obtained from mineral diesel supplemented with Ecodiesel were less toxic than those obtained from mineral diesel alone. Thus, we assume that supplementing mineral diesel fuel with Ecodiesel would be of benefit for the use of renewable energy, for environment and for human health due to reduced environmental pollution with bioreactive acrolein.

Iron-mediated oxidative stress plays an essential role in ferritin-induced cell death.

Previously, we have demonstrated an apoptosis-inducing activity of an acidic, H-chain-rich isoferritin secreted from primary rat hepatocytes in vitro. Because this proapoptotic property may be responsible for the growth-inhibitory and immunosuppressive effects described for certain ferritin species, we aimed to address the mechanism by which ferritin can trigger cell death. Suggesting a pivotal role for iron, iron chelation by desferrioxamine significantly abrogates ferritin-mediated apoptosis and necrosis in primary rat hepatocytes and substantially lowers the extent of protein modification by 4-hydroxynonenal (HNE)-a major lipid peroxidation (LPO) product. Furthermore, supplementing the cultures with the radical-scavenging compound trolox also provided significant protection from ferritin-mediated apoptosis. Moreover, a significant increase in micronucleated cells upon exposure to ferritin indicates that ferritin also introduces damage to DNA. Based on these observations we therefore propose that endocytosis of extracellular ferritin increases the level of free ferrous iron in the lysosomal compartment, promoting Fenton chemistry-based oxidative stress involving LPO and increased lysosomal membrane permeability. Subsequently, the release of reactive lysosomal content leads to cellular damage, in particular modification of protein and DNA induced by HNE and other reactive aldehydic LPO products. Together, these effects will trigger apoptosis and necrosis based on the upregulation of p53, increased mitochondrial membrane permeability, and proapoptotic Fas signaling as described recently. In conclusion, based on their iron-storing ability, secreted acidic isoferritins may act as soluble mediators of oxidative stress under certain physiological and pathophysiological conditions.

Black tea polyphenols modulate xenobiotic-metabolizing enzymes, oxidative stress and adduct formation in a rat hepatocarcinogenesis model.

The present study was designed to investigate the modulatory effects of black tea polyphenols (Polyphenon-B) on phase I and phase II xenobiotic-metabolizing enzymes and oxidative stress in a rat model of hepatocellular carcinoma (HCC). Liver tumours induced in male Sprague-Dawley rats by dietary administration of rho-dimethylaminoazobenzene (DAB) increased cytochrome P450 (total and CYP1A1, 1A2 and 2B isoforms), cytochrome b(5), cytochrome b(5) reductase, glutathione S-transferase (GST total and GST-P isoform) and gamma-glutamyltranspeptidase (GGT) with decrease in quinone reductase (QR). This was accompanied by enhanced lipid and protein oxidation and compromised antioxidant defences associated with increased expression of the oxidative stress markers 4-hydroxynonenal (4-HNE), anti-hexanoyl lysine (HEL), dibromotyrosine (DiBrY) and 8-hydroxy 2-deoxyguanosine (8-OHdG). Dietary administration of Polyphenon-B effectively suppressed DAB-induced hepatocarcinogenesis, as evidenced by reduced preneoplastic and neoplastic lesions, modulation of xenobiotic-metabolizing enzymes and amelioration of oxidative stress. Thus, it can be concluded that Polyphenon-B acts as an effective chemopreventive agent by modulating xenobiotic-metabolizing enzymes and mitigating oxidative stress in an in vivo model of hepatocarcinogenesis.

Galloylated catechins as potent inhibitors of hypochlorous acid-induced DNA damage.

Hypochlorous acid (HOCl), a strong oxidant derived from myeloperoxidase in neutrophils and macrophages, can chlorinate DNA bases at the site of inflammation. Because little is known about the protective role of natural antioxidants, such as polyphenols, for the myeloperoxidase-derived DNA damage, we screened the inhibitory effects of various phenolic antioxidants on the chlorination of the 2'-deoxycytidine residue by HOCl in vitro and found that green tea catechins, especially (-)-epicatechin gallate (ECg) and (-)-epigallocatechin gallate (EGCg), significantly inhibited the chlorination. These catechins also reduced nucleoside- and taurine-chloramines, which can induce secondary oxidative damage, into their native forms. Mass spectrometric and nuclear magnetic resonance analyses showed that ECg and EGCg can effectively scavenge HOCl and/or chloramine species resulting in the formation of mono- and dichlorinated ECg and EGCg. Using the HL-60 human leukemia cell line, it was found that ECg could efficiently accumulate in the cells. Immunocytometric analyses using antihalogenated 2'-deoxycytidine antibody showed that pretreatment of cells with ECg inhibited the HOCl-induced immunofluorescence. In addition, the chlorinated ECg derivatives were detected in the HOCl-treated HL-60 cells. These results showed that green tea catechins, especially 3-galloylated catechins, may be the plausible candidate for the prevention of inflammation-derived DNA damage and perhaps carcinogenesis.

Protection of cerebellar granule cells by tocopherols and tocotrienols against methylmercury toxicity.

Excessive free radical formation has been implicated as one of the causative factors in neurotoxic damage associated with variety of metals, including methylmercury (MeHg). Although the mechanisms associated with MeHg-dependent neurotoxicity remains are unclear, it is known that MeHg leads to neurotoxicity in cerebellar granule cells (CGCs). In vitro exposure of murine CGC primary cultures to MeHg resulted in time- and concentration-dependent cell death. The present study was designed to assess the effect of fat-soluble antioxidant tocopherols and tocotrienols (unsaturated vitamin E) on MeHg-induced neurotoxicity using cultured CGCs. Significant protection from MeHg-induced neuronal cell death was observed with both tocopherols and tocotrienols. Moreover, we observed that tocotrienols are multi-fold more potent than tocopherols in protecting CGCs against MeHg neurotoxicity. At micromolar concentration, tocotrienols, but not tocopherols, showed complete protection by an antioxidant mechanism. Similarly, tocopherols and tocotrienols showed a protective effect on CGCs migration against MeHg-toxicity. These results suggested that oxidative events may contribute to MeHg toxicity in isolated cerebellar granule neurons, and that tocotrienols are potent supplements for pharmacological protection of the developing brain exposed to MeHg.

Maintaining the redox-balance intact: gosha-jinki-gan but not insulin activates retinal soluble guanylate cyclase in diabetic rats.

Strategies to prevent hyperglycemia-induced cytotoxic reactive oxygen species in the retina include the prevention of free radical production, activation of radical-scavenging capacities and inhibition of aldose reductase. This study examined the effect of the standardized Japanese herbal extract product gosha-jinki-gan (GJG) in comparison to insulin treatment in the rat retina. Diabetes was induced in male Wistar rats by single injection of streptozotocin (50 mg/kg i.p.). At 6 and 12 weeks, eye-cups were removed for immunohistochemistry. At 12 weeks, lipid peroxidation (tested with the antiacrolein antibody, Ab5F6) was enhanced significantly in the untreated diabetic group. This effect was absent in both treatment groups, notably in the outer retina. A similar result was obtained for nitrotyrosine overproduction. As an early treatment effect, GJG -- but not insulin -- enhanced soluble guanylate cyclase (sGC) activation (using the function-sensing antibody, MoAb 3221). GJG not only reduces nitroxidative stress and lipid peroxidation in the retina, it also ameliorates glucose metabolism within the cells. We propose that the high glucose turnover in the insulin-treated model disturbs the intracellular redox equilibrium, one result of which might be the impaired sGC activation.

Selective induction of the tumor marker glutathione S-transferase P1 by proteasome inhibitors.

Exposure of cells to a wide variety of chemoprotective compounds confers resistance to a broad set of carcinogens. For a subset of the chemoprotective compounds, protection is generated by an increase in the abundance of phase 2 detoxification enzymes such as glutathione S-transferases (GSTs). Transcription factor Nrf2, which is sequestered in the cytoplasm by Keap1 (Kelch-like ECH-associated protein-1) under unstimulated conditions, regulates the induction of phase 2 enzymes. In this study, to explore the role of the proteasome in the detoxification response, we tested the effect of proteasome inhibitors such as MG132, clasto-lactacystin beta-lactone, and lactacystin on the induction of GST isozymes and found that these inhibitors selectively induced the class Pi GST isozyme (GST P1). Down-regulation of the proteasome by antisense oligonucleotides or RNA interference indeed resulted in significant up-regulation of GST P1, suggesting that a decline in the proteasome activity could be directly or indirectly linked to the induction of GST P1. From the functional analysis of various deletion constructs of the upstream regulatory region of the GST P1 promoter, GST P1 enhancer I was identified as the response element for proteasome inhibition. Overexpression of the wild-type and dominant-negative forms of Nrf2 and Keap1 had little effect on the induction of GST P1 not only by the proteasome inhibitor, but also by phase 2-inducing isothiocyanate, suggesting that there may be a process of GST P1 induction distinct from other phase 2 gene induction mechanisms. Because GST P1 is highly and specifically induced during early hepatocarcinogenesis as well as in hepatocellular carcinoma cells, these data may provide a potential critical role for the proteasome in the induction of a cellular defense program associated with carcinogenesis.

Protein-bound 4-hydroxy-2-hexenal as a marker of oxidized n-3 polyunsaturated fatty acids.

In the present study, to investigate the contribution of n-3 PUFAs in the oxidative modification of protein in vivo, we characterize the covalent binding of 4-hydroxy-2-hexenal (HHE), a potent cytotoxic aldehyde originating from the peroxidation of n-3 PUFAs, to protein and describe the production of this aldehyde in oxidatively modified LDL and in human atherosclerotic lesions. Upon incubation with BSA, HHE was rapidly incorporated into the protein and generated the protein-linked carbonyl derivative, a potential marker of oxidatively modified proteins under oxidative stress. To detect the protein-bound HHE in vivo, we raised monoclonal antibody HHE53 (MAb HHE53) directed to the HHE-modified protein and identified the Michael addition-type HHE-histidine adduct as the major epitope. This antibody reacted with copper-oxidized LDL, suggesting that HHE was produced during the oxidative modification of LDL. In addition, we demonstrated that the materials immunoreactive to MAb HHE53 indeed constituted the atherosclerotic lesions, in which intense positivity was associated primarily with macrophage-derived foam cells. The results of this study suggest that the reaction between oxidized n-3 PUFAs and protein might represent a process common to the formation of degenerative proteins during aging and its related diseases.

Bilirubin rinse: A simple protectant against the rat liver graft injury mimicking heme oxygenase-1 preconditioning.

Heme oxygenase (HO)-1 preconditioning through genetic or pharmacologic interventions was shown experimentally to improve posttransplant outcome of liver grafts. However, its clinical application requires careful consideration because of the complexity and economic costs of the procedures. This study aimed to examine if graft preconditioning with HO-1 could be substituted by a simple treatment with heme-degrading products such as bilirubin. Rats were pretreated with or without hemin, an HO-1 inducer for preconditioning. Their livers were harvested as grafts in University of Wisconsin (UW) solution for 16 hours at 4 degrees C and followed by reperfusion ex vivo or by transplantation in vivo. The control grafts were also treated with a rinse buffer containing varied concentrations of unconjugated bilirubin with different time intervals. The HO-1-preconditioned grafts ex vivo exhibited a marked improvement of bile output and cell injury that was cancelled by blocking HO with zinc protoporphyrin-IX. The aggravation of the graft viability by the inhibitor was repressed by supplementation of bilirubin but not by that of carbon monoxide. Furthermore, a short-term rinse treatment with micromolar levels of bilirubin attenuated biliary dysfunction and cell injury of the grafts both ex vivo and in vivo even without HO-1 preconditioning. The protective effects of HO-1 preconditioning or bilirubin rinse appeared to involve its inhibitory effects on lipid peroxidation in hepatocytes. In conclusion, these results suggest that bilirubin rinse serves as a simple strategy to ameliorate hyperacute oxidative stress and hepatobiliary dysfunction of the transplanted grafts, mimicking effects of HO-1-mediated preconditioning.

A phase II detoxification enzyme inducer from lemongrass: identification of citral and involvement of electrophilic reaction in the enzyme induction.

We have developed a simple system for the sensitive detection and measurement of glutathione S-transferase (GST) activity that detoxifies polycyclic aromatic hydrocarbons using the cultured rat normal liver epithelial cell line, RL34 cells. Citral (3,7-dimethyl-2,6-octadienal) was isolated from the methanol extract of lemongrass (Cymbopogon citratus) and identified as a novel inducer of GST. Citral, a mixture of the two stereoisomers geranial and neral, dose- and time-dependently induced the total and pi-class-specific activities of GST. The structure-activity relationship study revealed that geranial, an E-isomer, was mainly responsible for the inducing activity of citral mixture and the aldehyde group conjugated with a trans-double bond is an essential structural factor. The data were consistent with the in vitro observation that both glutathione (GSH) and protein thiol quickly and specifically reacted with the active isomer geranial, but not neral. Pretreatment of the cells with diethyl maleate significantly enhanced not only the basal activity but also the citral-stimulated activity of GST, while pretreatment with N-acetyl-cysteine inhibited it. Moreover, the treatment of RL 34 cells with geranial for 30 min significantly attenuated the intracellular GSH level, while application for 18 h enhanced it. These results strongly suggested that the electrophilic property characterized by the reactivity with intracellular nucleophiles including protein thiol or glutathione (GSH) plays an important role in the induction of GST. The present study also implied the antioxidant role of GST induction by citral in mouse skin, providing a new insight into skin cancer prevention.

Protective effect of colored rice over white rice on Fenton reaction-based renal lipid peroxidation in rats.

Rice has been one of the most important grains. While polished white rice is favored, colored strains of rice, red, or black, have been maintained for religious purposes in Japan. We studied whether feeding of unpolished colored rice instead of white rice ameliorates oxidative renal tubular damage in rats induced by ferric nitrilotriacetate. Whereas renal lipid peroxidation was exacerbated in white rice-fed group in comparison with standard chow group, this exacerbation was not observed in red or black rice-fed groups. These changes were dependent on the proportion of colored rice to standard chow in the diet. Cyanidin 3-O-beta-D-glucoside was detectable neither in the serum nor kidney after one week of colored rice diet, but serum protocatechuic acid was significantly increased after black rice diet. There was a generalized decrease in the renal glutathione peroxidase activity in rice diet groups. Renal enzymatic activities of superoxide dismutase, glutathione S-transferase and NAD(P)H quinone reductase were not associated with the levels of lipid peroxidation. However, renal catalase activity was significantly increased in black rice-fed groups. These may partly explain the antioxidative effect. Furthermore, colored strains of rice are rich in proteins. Thus, our data warrants further investigation of the antioxidative effect of colored rice.

Black tea polyphenols, theaflavins, prevent cellular DNA damage by inhibiting oxidative stress and suppressing cytochrome P450 1A1 in cell cultures.

Tea polyphenols have been demonstrated as chemopreventive agents in a number of experimental models. However, less is known about the mechanism of chemoprevention by black tea compared with that of green tea. Some beneficial properties of theaflavins, the black tea polyphenols, were investigated in the present study. Theaflavins showed inhibitory effects on H(2)O(2)- and tert-butyl hydroperoxide (tBuOOH)-induced cytotoxicity (evaluated by tetrazolium bromide reduction), cellular oxidative stress (detected by oxidation of 2', 7'-dichlorofluorescin), and DNA damage (measured by amount of 8-OHdG and comet assay) in rat normal liver epithelium cell RL-34 cell lines. In addition, theaflavins also exhibited suppression of cytochrome P450 1A1 induced by omeprazole in the human hepatoma HepG2 cell line. Furthermore, when HepG2 cells were pretreated with omeprazole to induce CYP1A1, then exposed to benzo[a]pyrene (B[a]P), DNA damage was observed using the comet assay. However, theaflavins could inhibit this DNA damage. These results indicated that theaflavins could prevent cellular DNA damage by inhibiting oxidative stress and suppressing cytochrome P450 1A1 in cell cultures.

A sulforaphane analogue that potently activates the Nrf2-dependent detoxification pathway.

Exposure of cells to a wide variety of chemoprotective compounds confers resistance to a broad set of carcinogens. For a subset of the chemoprotective compounds, protection is generated by an increase in the abundance of the protective phase II detoxification enzymes, such as glutathione S-transferase (GST). We have recently developed a cell culture system, using rat liver epithelial RL 34 cells, that potently responds to the phenolic antioxidants resulting in the induction of GST activity (Kawamoto, Y., Nakamura, Y., Naito, Y., Torii, Y., Kumagai, T., Osawa, T., Ohigashi, H., Satoh, K., Imagawa, M., and Uchida, K. (2000) J. Biol. Chem. 275, 11291-11299.) In the present study, we investigated the phase II-inducing potency of an isothiocyanate compound in vitro and in vivo and examined a possible induction mechanism. Based on an extensive screening of vegetable extracts for GST inducer activity in RL34 cells, we found Japanese horseradish, wasabi (Wasabia japonica, syn. Eutrema wasabi), as the richest source and identified 6-methylsulfinylhexyl isothiocyanate (6-HITC), an analogue of sulforaphane (4-methylsulfinylbutyl isothiocyanate) isolated from broccoli, as the major GST inducer in wasabi. 6-HITC potently induced both class alpha GSTA1 and class pi GSTP1 isozymes in RL34 cells. In animal experiments, we found that 6-MSHI was rapidly absorbed into the body and induced hepatic phase II detoxification enzymes more potently than sulforaphane. The observations that (i) 6-HITC activated the antioxidant response element (ARE), (ii) 6-HITC induced nuclear localization of the transcription factor Nrf2 that binds to ARE, and (iii) the induction of phase II enzyme genes by 6-HITC was completely abrogated in the nrf2-deficient mice, suggest that 6-HITC is a potential activator of the Nrf2/ARE-dependent detoxification pathway.