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J. Biol. Chem., Vol. 277, Issue 4, 2687-2694, January 25, 2002

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Ebselen, a Glutathione Peroxidase Mimetic Seleno-organic Compound, as a Multifunctional Antioxidant

IMPLICATION FOR INFLAMMATION-ASSOCIATED CARCINOGENESIS^*

Yoshimasa Nakamura, Qing Feng, Takeshi Kumagai, Koji Torikai^¶, Hajime Ohigashi^¶, Toshihiko Osawa, Noriko Noguchi, Etsuo Niki, and Koji Uchida^§

From the  Laboratory of Food and Biodynamics, Nagoya University Graduate School of Bioagricultural Sciences, Nagoya 464-8601, the ^¶ Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, and the  Research Center for Advanced Science and Technology, University of Tokyo, Tokyo 153-8904, Japan

Received for publication, October 5, 2001, and in revised form, November 9, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Ebselen, a seleno-organic compound showing glutathione peroxidase-like activity, is one of the promising synthetic antioxidants. In the present study, we investigated the antioxidant activities of ebselen using a 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated mouse skin model. Double pretreatments of mouse skin with ebselen significantly inhibited TPA-induced formation of thiobarbituric acid-reacting substance, known as an overall oxidative damage biomarker, in mouse epidermis, suggesting that ebselen indeed acts as an antioxidant in mouse skin. The antioxidative effect of ebselen is attributed to its selective blockade of leukocyte infiltration and activation leading to attenuation of the H₂O₂ level. In in vitro studies, ebselen inhibited TPA-induced superoxide generation in differentiated HL-60 cells and lipopolysaccharide-induced cyclooxygenase-2 protein expression in RAW 264.7 cells. In addition, we demonstrated for the first time that ebselen potentiated phase II enzyme activities, including NAD(P)H:(quinone-acceptor) oxidoreductase1 and glutathione S-transferase in cultured hepatocytes and in mouse skin. These results strongly suggest that ebselen, a multifunctional antioxidant, is a potential chemopreventive agent in inflammation-associated carcinogenesis.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Acute and chronic inflammatory states have been implicated as mediators of a number of pathological disorders, including cancer. Chronic inflammation appears to be linked to epithelial tumorigenesis in the lung, the bowel, the bladder, the colon, and the skin. Although the mechanisms by which inflammatory cells show their carcinogenic effects remain unclear, some potential pathways have been proposed (1). Treatment with UV light, endotoxins, or chemical tumor promoters are known to lead to chemotaxis, differentiation, and infiltration of inflammatory leukocytes, including granulocytes and monocytes, which produce reactive oxygen species (ROS),¹ prostaglandins (PGs), and cytokines. In fact, there is mounting evidence showing treatments of mouse skin with single or multiple doses of 12-O-tetradecanoylphorbol-13-acetate (TPA) induce production of superoxide anion (O₂) (2), hydrogen peroxide (H₂O₂) (3), PGE₂ and PGF₂ (4), cyclooxygenase (COX)-2 expression (5) as well as the release of tumor necrosis factor  (6). Thus, leukocyte activation resulting in ROS generation coupled to excessive production of chemotactic factors may play an important role in chronic inflammation and hyperplasia in mouse skin (7, 8). In addition, ROS production by double or multiple TPA treatments is closely associated with the metabolic activation of proximate carcinogens (9, 10) and the increased levels of oxidized DNA bases (11-13). These findings strongly suggest that activated inflammatory leukocytes play an important role in carcinogenesis.

We have recently developed a short term assay for skin oxidative stress, namely, double TPA application system (7, 8, 14-19). Double applications of phorbol esters was required for triggering production of ROS, including H₂O₂, lipid hydroperoxide, and peroxy radicals in mouse skin (9, 10, 13, 20). The available data reported previously suggested that each application induces two distinguishable biochemical events, namely, "priming" and "activation" (10). The former is characterized as recruitment of inflammatory cells such as polymorphonuclear leukocytes by chemotactic factors to inflammatory regions and edema formation. The latter is the process of activation of neutrophil- or other oxidant-producing cells, including keratinocytes, in which the second TPA application of phorbol ester induces oxidative stress. In addition, Sisskin et al. (21) pointed out that the result of a single topical application of TPA might not predict the response with multiple treatments for tumor promotion. Conversely, our recent studies have suggested that the double TPA application experiment is appropriate to predict the inhibitory potential of test compounds for chronic inflammation or tumor promotion in epithelial tissues, including not only skin but also digestive tract (22-24).

Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one; see Fig. 1 below) is a non-toxic seleno-organic drug that has been extensively studied during the last decade. It is now under active investigation as a neuroprotective agent in clinical trial (25). A substantial part of the pharmacological profile of ebselen appears to be due to its action as an antioxidant with a unique mode of action. A number of other antioxidants such as -tocopherol and probucol act as scavengers of free radicals and thus prevent lipid peroxidation, including secondary reaction triggered by them. In contrast, ebselen is a poor radical scavenger, if at all, but it is an effective scavenger of organic hydroperoxides, in particular, of lipid hydroperoxides. Thus, the particular interest in this drug is that ebselen mimics glutathione peroxidase (GPx) activities in particular that of phospholipid hydroperoxide GPx (26). There is some indication that the pseudoenzymatic catalytic activity may contribute in part to the pharmacodynamic profile of ebselen. However, it becomes plausible that ebselen displays a unique pattern of chemical reactions, which is not restricted to the GPx reaction (for review, see Ref. 27). Ebselen actually inhibits at low concentrations a number of enzymes involved in inflammation such as lipoxygenases, nitric-oxide synthases, NADPH oxidase, protein kinase C, and H⁺/K⁺-ATPase (26). Ebselen also suppresses inflammation in a variety of experimental animal models. Although its antioxidant capacities have been extensively investigated, relatively little is known about its anticarcinogenic activity. A previous study showed that ebselen inhibited TPA-induced down-regulation in gap-junctional intercellular communication (28). Reduction of gap-junctional intercellular communication has been proposed to be involved in the development of a number of pathogeneses, especially carcinogenesis. More recently, inhibition of ebselen on aflatoxin B₁-induced hepatocarcinogenesis in Fischer 344 rats and involvement of its antioxidative property in the anticarcinogenic effect have been reported (29). However, the mechanistic aspect of ebselen as an antioxidant in inflammatory oxidative events responsible for tumor promotion remains to be fully elucidated and is the topic of this report.

In the present study, we attempted to determine whether ebselen inhibits the priming and/or activation stages in the double TPA application model of mouse skin. To discuss the mode of action mechanism of anti-inflammatory activity of ebselen, the suppressive effect of ebselen on COX-2 expression was examined. Our studies also demonstrated for the first time that addition of ebselen to cultured rat liver epithelial cells and mouse skin results in induction of phase II enzyme, including glutathione S-transferase (GST) and NAD(P)H:(quinone-acceptor) oxidoreductase (NQO1), which may also participate in in vivo antioxidative effect of ebselen.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Chemicals and Animals-- Ebselen was kindly provided from Daiichi Pharmaceutical Co., Ltd., Tokyo, Japan. TPA was obtained from Research Biochemicals International, Natick, MA. RPMI 1640 medium was purchased from Invitrogen, Rockville, MD. All other chemicals were purchased from Wako Pure Chemical Industries, Osaka, Japan. Female ICR mice (7 weeks old) were obtained from Japan SLC, Shizuoka, Japan. Until the day of the experiment, mice were allowed free access to drink water and a commercial laboratory diet (MF; Oriental Yeast Co., Kyoto, Japan). Animals were treated in accordance with the Guidelines for Animal Experimentation of Kyoto University and Nagoya University. Animals were maintained in a room controlled at 24 ± 2 °C with a relative humidity of 60 ± 5% and a 12-h light/dark cycle (6:00 a.m. to 6:00 p.m.). The back of each mouse was shaved with surgical clippers 2 days before each experiment.

Double TPA Treatment Protocol-- One group was composed of five female ICR mice housed five per cage. Ebselen was topically applied to the shaved area of dorsal skin at various times before application of TPA solution (8.1 nmol/0.1 ml in acetone). This TPA dose (8.1 nmol) was used for the potentiation of oxidative responses compared with the dose for tumor promotion (1.6 nmol). The same doses of ebselen and TPA or acetone were applied twice at an interval of 24 h for H₂O₂ determination. Although the timing (24 h apart) of the double TPA application was different from tumor promotion protocol, the levels of oxidative stress were nearly the same when the time between the two TPA treatments was 24-72 h (data not shown).

Determination of Oxidative Stress Parameters-- Mice treated by the double-treatment protocol were sacrificed 1 h after the last TPA treatment. The H₂O₂ content was determined by the phenol red-horseradish peroxidase (HRPO) method (3, 7). The final results are expressed as equivalents of nanomoles of H₂O₂ per skin punch on the basis of a standard curve of HRPO-mediated oxidation of phenol red by H₂O₂. The level of thiobarbituric acid-reacting substances (TBARS) in mouse epidermis was determined by our previously reported method (7, 19). The final results are expressed as equivalents of nanomoles of malondialdehyde per square centimeters on the basis of a standard line of TBARS formation using the authentic malondialdehyde.

Inflammatory Biomarker Determination-- The modifying effect for single TPA application-induced inflammation was determined by two biomarkers, edema formation and myeloperoxidase (MPO) activity, as previously reported (3, 7). Mice were sacrificed by cervical dislocation 18 h after a single application of TPA. Mouse skin punches were obtained with an 8-mm-diameter cork borer and weighed using an analytical balance. The IE were expressed by the relative increasing ratio of the weight of a treated punch to that of a control punch; inhibitory effect (IE) (%) = [(TPA alone)  (test compound plus TPA)]/[(TPA)  (vehicle)] × 100. The MPO activity was calculated from the linear portion of the curve and expressed as units of MPO per skin punch. One unit of MPO activity was defined as the activity that degraded 1 µmol of H₂O₂ per min at 25 °C.

Cell Cultures-- Rat liver epithelial RL34 and human promyelocytoid HL-60 cells were obtained from the Health Science Research Resources Bank, Osaka, Japan (30, 31). Murine macrophage RAW 264.7 cells were kindly donated by Ohtsuka Pharmaceutical Co., Ltd. (Ohtsu, Japan). RL34 cells were grown as monolayer cultures in Dulbecco's modified Eagle's medium supplemented with 5% heat-inactivated fetal bovine serum, penicillin (100 units/ml), streptomycin (100 µg/ml), L-glutamine (0.3 mg/ml), pyruvic acid (0.11 mg/ml), and 0.37% NaHCO₃ at 37 °C in an atmosphere of 95% air and 5% CO₂. HL-60 cells were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum. RL34 cells were grown as monolayer cultures in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum. The cells were exposed to the test compound dimethyl sulfoxide (Me₂SO) solution (final concentration 0.5%) in the growing medium as mentioned above.

COX-2 Determination-- Confluent RAW 264.7 cells were treated with lipopolysaccharide (LPS, 100 ng/ml) and ebselen. After 20 h, the cells were washed twice with phosphate-buffered saline (pH 7.0) and lysed with lysis buffer (50 mM Tris-HCl (pH 7.5)/150 mM NaCl/1% Triton X-100/0.5% sodium deoxycholate/0.1% SDS/100 µg/ml phenylmethylsulfonyl fluoride) by incubation at 37 °C for 10 min with a solution containing 0.8% digitonin and 2 mM EDTA (pH 7.8). Each cell lysate was then treated with Laemmli sample buffer for 5 min at 100 °C (32). The samples were run on 10% SDS-PAGE slab gels. One gel was used for staining with Coomassie Brilliant Blue, and the other was transblotted on a nitrocellulose membrane (Hybond ECL) (Amersham Biosciences, Inc.), incubated with Block Ace (40 mg/ml) for blocking, washed, and then treated with the antibody. This procedure was followed by the addition of horseradish peroxidase conjugated to IgG and ECL reagents. The bands were visualized by exposure of the membrane to autoradiography film.

Superoxide Generation Test-- Inhibitory tests of TPA-induced O₂ generation in Me₂SO-differentiated HL-60 cells were done as previously reported (7, 8). Briefly, to determine the inhibitory effect of O₂ generation, a test compound dissolved in 5 µl of Me₂SO was added to Me₂SO-induced differentiated HL-60 cell suspension and incubated at 37 °C for 15 min. The cells were washed with PBS twice for removal of the extracellular test compound to eliminate O₂ scavenging effect. TPA (100 nM) and cytochrome c solution (1 µg/ml) were added to the reaction mixture, which was incubated for another 15 min. The reaction was terminated by placing it on ice. After centrifugation at 250 × g, the visible absorption at 550 nm was measured. The inhibitory effect was expressed by the relative decreasing ratio of absorbance of a test compound to the control experiment.

GST and NQO1 Assay-- GST activity was measured using 1-chloro-2,4-dinitrobenzene as substrate according to the method of Habig et al. (33). NQO1 activity was determined by the Prochaska test (34) with slight modifications. RL34 cells were grown for 24 h in 96-well plates (10,000 per well) and then exposed to serial dilutions of the chemicals.

Western Blot Analysis of GSTP1-- The ebselen-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 Laemmli sample buffer for 3 min at 100 °C (32). The samples (20 µg) were run on 12.5% SDS-PAGE slab gels. One gel was used for staining with Coomassie Brilliant Blue and the other was transblotted onto a nitrocellulose membrane with a semi-dry blotting cell (Trans-Blot SD, Bio-Rad), incubated with Block Ace (40 mg/ml) for blocking, washed, and then treated with the antibody.

CAT Assay-- A 3.0-kb fragment between 2.9 kb (EcoRI) and +59 bp (AccI) of GSTP1 was inserted into the HindIII site of pSV0CAT and designated as ECAT (35). 1CAT was constructed from the ECAT by using appropriate restriction enzymes (35). RL34 cells were transfected with 10 µg of plasmid construct by the calcium phosphate co-precipitation procedure described by Chen and Okayama (36). The cells were harvested 48 h after transfection. Cell lysates were obtained after five freeze-thaw cycles in 0.25 M Tris-HCl (pH 7.5). The protein was equalized and used for CAT assay. The degree of was determined by reading the intensity of the spots using the Fuji-BAS 2000 system (Fuji Photo, Tokyo, Japan).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Ebselen Inhibited Double TPA Application-induced Oxidative Stress in Mouse Skin-- Double applications of phorbol esters trigger ROS production in mouse skin (9, 10, 13, 20). The available data reported previously suggest that each application induces two distinguishable biochemical events, namely, priming and activation (10). The former is characterized as recruitment of inflammatory cells such as neutrophils by chemotactic factors to inflammatory regions and edema formation. The latter is the process of activation of neutrophils or other oxidant-producing cells, including keratinocytes, in which the second TPA application of phorbol esters induces oxidative stress. Importantly, the TPA dose used in the present experiment is in the tumor promotion range (1-10 nmol). First, we investigated whether ebselen showed antioxidative activity against double TPA application-induced oxidative damage in mouse skin (Fig. 1). The oxidative damage was evaluated by the TBARS level, a well-known biomarker of overall oxidative damage to cellular constituents such as membrane lipids. The quantitative data for the levels of TBARS formation in mouse epidermis homogenate are shown in Fig. 2. The increased level of TBARS caused by the double TPA application was significantly higher than that of the control (0.35 ± 0.06 versus 0.13 ± 0.02 nmol/cm², p < 0.001). The double pretreatment of ebselen (810 nmol) 30 min before each TPA treatment inhibited the increase in the TBARS level (0.23 ± 0.05 nmol/cm², p < 0.05 versus TPA). Reducing effect of ebselen on the TBARS level was dose-dependent. Thus, topically applied ebselen actually acts as an antioxidant in mouse skin.

View larger version (8K): [in this window] [in a new window]   Fig. 1.   Chemical structure of ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one).

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Antioxidant effects of ebselen on TBARS formation in the mouse epidermis. ICR mice (five mice in each group) were treated by the double TPA treatment protocol as described under "Experimental Procedures." Mouse skin was treated with ebselen or acetone 30 min prior to each TPA treatment. The mice were sacrificed 1 h after the second TPA application, and their epidermis was removed for TBARS assays. Significance determined by the Student's t test is expressed as: a, versus acetone (without TPA), p < 0.05; b, versus TPA, p < 0.05.

Although TBARS formation in vivo is considered not to reflect a single particular phenomenon but to indicate widespread oxidative damage, TBARS is known to be formed downstream of H₂O₂ generation in the presence of a metal ion as a catalyst. Therefore, we examined whether ebselen inhibits dermal oxidative damage via suppression of the ROS generating system, including leukocytes in inflamed regions. As shown in Fig. 3A, double applications of 8.1 nmol of TPA at 24-h interval increased the level of H₂O₂ by about 5-fold (1.07 ± 0.20 versus 5.02 ± 0.78 nmol/skin punch, p < 0.001) to that in the control mice treated twice with acetone instead of TPA. Pretreatment of ebselen (810 nmol; 100-fold dose of TPA) before each TPA treatment strongly inhibited H₂O₂ formation (2.56 ± 0.74 nmol/skin punch, inhibitory effect (IE) = 62%). Reducing effect of ebselen on the H₂O₂ level was dose-dependent (data not shown). To distinguish whether ebselen inhibits the priming or activation phase in a double TPA application model, ebselen was co-administered with either the first (priming) or second (activation) dose of TPA. Fig. 3A also shows the inhibitory effects of ebselen applied prior to either the first or second TPA treatment on H₂O₂ generation in mouse skin. A greater decrease in the H₂O₂ level was observed in the mice to which ebselen was co-administered in the priming phase (1.94 ± 0.39 nmol/skin punch, IE = 78%) than in the activation phase (3.63 ± 0.31 nmol/skin punch, IE = 35%).

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Inhibition of TPA-induced H2O2 generation and chemotactic action by ebselen. A, inhibitory effects of ebselen applied in the priming and/or activation phase on the H2O2 formation in mouse skin. Mice were treated with ebselen (810 nmol) or acetone 30 min before either the first and/or second TPA treatment. The mice were sacrificed 1 h after the second TPA application, and their dermis and epidermis was removed for determination of H2O2 level using a HRPO-phenol red method. B, inhibitory effect of ebselen on single TPA treatment-induced edema formation and leukocyte infiltration. Mice were sacrificed 18 h after a single application of TPA. Mouse skin punches were obtained with an 8-mm-diameter cork borer and weighed in an analytical balance. The MPO activity of homogenates of skin sample was determined by a 4-aminoantipyrine method (3). The MPO activity was calculated from the linear portion of the curve and expressed as units of MPO per skin punch. Statistical significance was determined by the Student's t test and is expressed as: a, versus acetone, p < 0.001; b, versus acetone, p < 0.05; c, versus TPA, p < 0.001; d, versus TPA, p < 0.01; e, versus TPA, p < 0.05.

Ebselen Attenuated Single TPA Application-induced Edema Formation and Leukocyte Infiltration-- The result that ebselen potently suppressed the priming phase of the double TPA application-induced H₂O₂ generation led us to determine whether ebselen influenced the chemotactic action of TPA in mouse skin. The effects of ebselen on the single application of TPA-induced skin edema formation and infiltration of polymorphonuclear leukocytes were thus examined. A single TPA application (8.1 nmol) resulted in edema formation (as measured by the weight of skin punch) by 1.9-fold (64.7 ± 17.1 versus 34.0 ± 6.1 mg/skin punch, p < 0.001) and increased the infiltration of polymorphonuclear leukocytes (as measured by MPO activity) by 25-fold (12.04 ± 3.22 versus 0.47 ± 0.23 unit/skin punch, p < 0.001) as compared with the control (Fig. 3B). The pretreatment of ebselen at a 100-fold molar dose to TPA (810 nmol) potently reduced the skin edema formation and infiltration of polymorphonuclear leukocytes (65 and 82%, respectively). These data are consistent with the inhibitory effect of ebselen on TPA-induced H₂O₂ generation and suggested that protection against the infiltration of polymorphonuclear leukocyte by TPA may be responsible at least in part for such an effect.

Ebselen Suppressed COX-2 Expression and NAD(P)H Oxidase Activity in Cultured Cells-- One of the most common events after topical application of TPA onto mouse skin is an inflammatory response mediated by the increased metabolism of arachidonic acid by induced levels of the enzymes such as cyclooxygenase (COX). The COX family of genes consists of COX-1 (constitutive COX) and COX-2 (inducible COX). An increased level of PGE₂, one of the major products of COX, has been linked with the increased expression of COX-2, which is expressed in mouse epidermis after topical treatment with TPA with no change in COX-1 expression (37). Consistent with these studies, we assessed the effect of ebselen treatment on COX-2 expression using LPS-stimulated RAW 264.7 cells in terms of COX-2 protein level. As shown in Fig. 4A, compared with the negative control, LPS treatment resulted in a strong increase in COX-2 protein. However, ebselen treatment resulted in significant inhibition of the COX-2 protein expression in a dose-dependent manner. The 50 µM ebselen completely attenuated COX-2 protein to the negative control level. Moreover, 100 µM ebselen reduced the COX-2 level to 50% of the control level, indicating that ebselen attenuated the basal induction of COX-2. The treatment of RAW 264.7 cells with TPA or ebselen did not produce any change in the expression of COX-1 protein (data not shown).

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Inhibition of COX-2 expression and NADPH oxidase-derived O2 generation in leukocytes in vitro by ebselen. A, dose-dependent effect of ebselen on COX expression in RAW 264.7 cells. Confluent RAW 264.7 cells were treated with lipopolysaccharide (LPS, 100 ng/ml) and ebselen. After 20 h, the cells were washed twice with phosphate-buffered saline and lysed with lysis buffer. COX-2 level was examined by an immunoblot analysis. The maximal standard deviation for the duplicated experiments was 5%. B, dose-dependent effect of ebselen and quercetin on TPA-induced O2 generation in differentiated HL-60 cells. HL-60 cells were preincubated with 1.25% Me2SO at 37 °C for 6 days, differentiating them into granulocyte-like cells. Ebselen or quercetin solution was added to the cell suspension, and the mixture was incubated at 37 °C for 15 min, followed by washing with PBS twice. Ninety seconds after stimulation with TPA (100 nM), cytochrome c solution was added to the reaction mixture. After incubation for another 15 min followed by centrifugation, visible absorption at 550 nm was measured.

Ebselen, applied in the activation phase when ROS are generated by inflammatory cells, significantly inhibited H₂O₂ generation in mouse skin (Fig. 3A). We, therefore, examined the inhibitory effects of ebselen on ROS generation in cultured leukocytes. The differentiated HL-60 cell system was chosen as a model of the NADPH oxidase system (38). To directly confirm whether or not ebselen inhibits NADPH oxidase, TPA stimulation was carried out after removing ebselen from the reaction mixture by washing with PBS. As shown in Fig. 4B, ebselen at a concentration of 10 µM strongly inhibited the O₂ generation by 62%. The suppressing effect of ebselen on TPA-stimulated O₂ generation was dose-dependent and much stronger than that of quercetin, a well-known antioxidative plant flavonol. These results suggested that the suppression of neutrophilic NADPH oxidase activation by TPA and the generation of O₂, one of the potential precursors of several ROS, may be responsible for at least in part the antioxidative action of ebselen in mouse skin.

Ebselen Induces Phase II Drug Metabolizing Enzymes in the Cultured Cells and Mouse Skin-- To protect against excessive ROS, aerobic organisms have developed a number of cellular defenses composed of non-enzymatic and enzymatic components. However, the treatment of mouse skin with TPA has been reported to result in strong depletion of antioxidant enzyme activities (39). Among the antioxidant enzymes, phase II drug metabolizing enzymes, including, GST and NQO1, play a major role in protecting cells against toxic and neoplastic chemicals, including polycyclic aryl hydrocarbons and their metabolites. In addition, some GST isozymes, being able to utilize the major products of lipid peroxidation, including fatty acid hydroperoxide and 4-hydroxy-2-nonenal, as substrates, play a physiological role in protection against oxidative stress (40, 41). Therefore, we examined whether ebselen induces phase II drug metabolizing enzymes in the cultured cells. To monitor the inducing potency of ebselen, we measured NQO1, which is induced to high levels in many animal tissues and cells by a variety of dietary and synthetic chemopreventive agents (42). As shown in Fig. 5 (A and B), the treatment of rat hepatocyte RL34 cells with ebselen resulted in significant induction of NQO1 activity. NQO activity was induced 3-fold when cells were exposed to 25 µM ebselen for 24 h. Inducing activity of ebselen was dose- and time-dependent and comparable to that of tBHQ, a well-known phase II inducer. As for GST activity, the GST activity also increased in a dose-dependent manner (Fig. 6A). GST activity was induced 3-fold when cells were exposed to 25 µM ebselen. Because ebselen showed a significant attenuation of the H₂O₂ level, we examined the possibility of GST induction to participate in the antioxidative action of ebselen in mouse skin. Mice were topically treated with different doses of ebselen for 24 h, and the GST activity was measured. As shown in Fig. 6B, the GST activity was significantly induced (1.6-fold compared with mice treated only with acetone) when the mice were exposed to 810 nmol of ebselen, which was able to reduce the H₂O₂ level in mouse skin (Fig. 3A). The NQO activity was also significantly induced in mouse skin (data not shown).

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Induction of NQO1 activity by ebselen in rat liver epothelial RL34 cells. A, dose-dependent effect of ebselen and tBHQ on cellular NQO1 activity. Cells post-confluency were exposed to the test compounds in the medium containing 5% fetal bovine serum. Cells were exposed to the test compounds for 24 h. We established the incubation time, because, for these compounds, increases in enzyme activity continued linearly with time for the first 24 h (see panel B). The cellular QR activity was evaluated by the Prochaska test (34). B, time-dependent effect of ebselen (25 µM) and tBHQ (25 µM) on cellular NQO1 activity.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Induction of GST activity by ebselen in RL34 cells and mouse skin. A, dose-dependent effect of ebselen on cellular GST activity. Cells post-confluency were exposed to the test compounds in the medium containing 5% fetal bovine serum for 24 h. GST activity was measured using 1-chloro-2,4-dinitrobenzene as substrate according to the method of Habig et al. (33). B, induction of GST activity by ebselen in mouse skin. Mice were treated with ebselen or acetone for 24 h, and their dermis and epidermis was removed for determination of GST activity. Statistical significance was determined by the Student's t test and is expressed as: a, versus acetone, p < 0.001; b, versus acetone, p < 0.05.

GPE1 Is an Ebselen Response Element-- To examine the GST isozyme responsible for the increase in the GST activity of ebselen-treated RL34 cells, an immunoblot analysis was carried out using the GST class-specific antibodies to confirm the apparent induction of GST proteins. The immunoblot analysis demonstrated a significant increase in the level of the Class  GST isozyme (GSTP1) by treatment with ebselen (Fig. 7A), whereas the amounts of the Class  GST isozyme (GSTA1) and Class µ isozyme (GST M1) were nearly unchanged (data not shown). The increase in the GSTP1 protein, therefore, coincided with a substantial rise in the GST activity.

View larger version (21K): [in this window] [in a new window]   Fig. 7.   Induction of GSTP1 protein expression and transcription of an GSTP1 gene 5'-flanking region-CAT reporter gene by ebselen. A, dose-dependent effect of ebselen on GSTP1 protein. Cells post-confluency were exposed to ebselen in the medium containing 5% fetal bovine serum for 24 h. GSTP1 level was examined by an immunoblot analysis. B, structures of constructs of ECAT and 1CAT used for the transfection experiments; C, effects of ebselen on ECAT or 1CAT activity. The cells were treated with ebselen (10 µM) or Me2SO for 24 h, and harvested for CAT activity assay.

We have previously reported that the 5'-flanking region of GSTP1 gene contains an element responsible for a cancer chemopreventer such as benzylisothiocyanate as previously reported (43). To examine how ebselen enhanced the expression of the GSTP1 gene and to confirm whether GSTP1 enhancer I (GPEI), a strong enhancer that is located about 2.5 kilobases upstream from the transcriptional initiation site of this gene (35), we utilized the construct, including 5'-flanking region of GSTP1 gene, namely ECAT and a GPEI-deletion mutant 1CAT (Fig. 7B), and tested the effect of ebselen on ECAT and 1CAT gene expression. As shown in Fig. 7C, ebselen (10 µM) could enhance the activity of ECAT gene 5-fold of the control in these cells, whereas ebselen could not activate a GPEI deletion mutant 1CAT, basal activity of which was low (0.7-fold). These results suggested that GPEI is the ebselen response element.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The results of the present study demonstrate that a synthetic seleno-organic compound, ebselen, had potent inhibitory activity against TPA-caused inflammatory biochemical events in mouse skin that are primarily associated with skin tumor promotion. Inflammatory cells produce a highly complicated mixture of growth and differentiation cytokines as well as biologically active arachidonate metabolites. In addition, they possess the ability to generate and release a spectrum of reactive oxygen species (ROS) and free radicals during oxidative burst. Among the inflammatory cells, polymorphonuclear leukocytes such as neutrophils are particularly adept at generating and releasing ROS, including O₂, H₂O₂, hypochlorous acid (HOCl), singlet oxygen, and hydroxyl radical (44-46). The generation of O₂ by polymorphonuclear leukocytes is attributed to the activation of a plasma membrane NADPH oxidase. Utilization of O₂-derived H₂O₂ by myeloperoxidase (MPO) results in the formation of HOCl. Further reaction of HOCl with H₂O₂ generates singlet oxygen. In addition, hydroxyl radical has been demonstrated to be generated from the interaction of HOCl with O₂ (45). Current evidence suggests that these ROS-derived inflammatory cells may be important in tumorigenesis. Recently, we have reported that O₂ from leukocytes plays an important role for the continuous and excessive production of chemotactic factors, leading to chronic inflammation and hyperplasia in mouse skin (7, 8). ROS production by double or multiple TPA treatments is closely associated with the metabolic activation of proximate carcinogens (9, 10) and the increased levels of oxidized DNA bases (11-13). In the continuous studies on the antioxidative behavior of chemopreventive food factors in mouse skin (7, 8, 14-18), we have recently demonstrated that a potent inhibitor of leukocyte-derived ROS generation (7, 8, 15, 16) effectively suppressed inflammation-related carcinogenesis (8, 15, 22-24). Thus, the regulation of ROS from activated leukocytes is proposed to be one of the most promising strategies for cancer control (17). Consistent with these results, we found that the pretreatment of mouse skin with ebselen results in the strong inhibition of TBARS formation and H₂O₂ generation (Figs. 2 and 3A). These potent antioxidant effects of ebselen in the mouse skin in vivo model are in accordance with those earlier reported in cultured cells and other in vitro systems (27) and confirmed in the present study as well.

One of the early cellular events after TPA treatment or UV irradiation is infiltration of polymorphonuclear leukocyte, and the accumulation of polymorphonuclear leukocytes is a characteristic feature of skin inflammation that is also used as a measure of the inflammation intensity. Studies have also indicated that infiltration of polymorphonuclear leukocyte is responsible in part for the oxidative burst in skin. As a component of polymorphonuclear leukocytes, MPO is a marker for tissue content of polymorphonuclear leukocytes, because MPO activity is well correlated with the number of infiltrated leukocytes in inflamed regions as confirmed by histological studies (7, 15, 16, 18). Normal skin possesses a low background of MPO, whereas skin inflamed by an infection, wounding, or the application of chemicals having a tumor promoting ability exhibits an accumulation of MPO. The present results demonstrate that ebselen inhibit both TPA-induced edema formation and enhancement of MPO activity in mouse skin and the co-administration of ebselen with TPA treatment in the priming phase significantly inhibited H₂O₂ formation (Fig. 3, A and B). These results indicated that the inhibitory effect of ebselen on chemotactic action of TPA, which regulates the infiltration of ROS-producing leukocytes, is a possible action mechanism for the inhibitory effects of TPA-induced H₂O₂ formation in mouse skin. MPO catalyzes the formation of HOCl using H₂O₂ as a substrate, as already mentioned. HOCl is highly toxic, mutagenic (47), and also activates carcinogens (48). The observation that ebselen potently inhibited not only generation of a substrate, H₂O₂, but also the H₂O₂ metabolizing activity of MPO strongly suggests that ebselen may effectively suppress the generation of HOCl in an inflamed region, and this may also contribute to significant inhibition of ebselen against TBARS formation, a representative biomarker of lipid peroxidation.

In vitro, ebselen was reported to exhibit potent inhibition of O₂ generation (49, 50). The present study also confirmed the inhibitory activity against TPA-induced, NADPH oxidase-dependent O₂ generation in differentiated HL-60 cells used as a model for neutrophils. Moreover, the co-administration of ebselen only with the second TPA treatment effectively inhibited H₂O₂ in mouse skin (Fig. 3A). Our previous studies indicated that the NADPH oxidase system of neutrophils rather than the epithelial xanthine oxidase system is implicated in the O₂- generating system in multiple TPA-treated mouse skin, because a xanthine oxidase inhibitor, allopurinol, showed no effect in this system (7). These findings clearly indicate that ebselen is an in vivo ROS generation inhibitor, which potentially inhibits the activation of leukocytes possibly through the inhibition of leukocytic NADPH oxidase (7). Thus we concluded that in vivo antioxidant activity of ebselen in mouse skin is exhibited through three potential pathways other than the directly scavenging action toward organic hydroperoxides: 1) inhibition of chemotactic action leading to accumulation of ROS-generating leukocytes, 2) inhibition of leukocytic NADPH oxidase, and 3) inhibition of MPO activity and reduction of HOCl formation.

Arachidonic acid metabolites, including hydroperoxyeicosatetraenoic acids and PGs have been implicated in TPA-caused skin inflammation (51). Among the arachidonate-metabolizing enzymes, COX, leading to the formation of PGs such as PGE₂, PGF₂, and PGD₂, also plays an important role in skin tumor promotion as well as cell proliferation, potentially responsible for skin inflammation. The role of the COX pathway in tumor promotion in mouse skin has been extensively studied by several laboratories. More recently, it was shown that the cause of the induction of COX activity in mouse epidermis is due to the expression of the inducible form of COX known as COX-2 and that COX-2 is constitutively overexpressed without any significant change in the expression of COX-1 in epidermal tumors obtained from the two-stage skin carcinogenesis protocol (52, 53). PGs has have been implicated to the induction of hyperplasia by TPA. Inhibitory effects of ebselen on COX-2 induction has been recently reported and also confirmed in the present study (Fig. 4A). This finding also supports the idea that ebselen could be explored as a cancer chemopreventive agent targeted toward inflammation-related carcinogenesis such as skin and colon cancer.

There is substantial and mounting evidence that phase II drug-metabolizing enzymes, e.g. GST, NQO1, epoxide hydrolase, heme oxygenase, and UDP-glucuronosyl-transferase, play important roles in the detoxification of electrophilic toxicants and their induction protects against carcinogenesis and mutagenesis (54). Dinkova-Kostova and Talalay (42) have identified many kinds of inducers of NQO1 activity using murine hepatoma cell lines. Some of them have proven to induce other phase II enzymes as described above in coordination with NQO1 gene expressions. An antioxidant/electrophile response element (ARE/EpRE; consensus sequence TGACNNNGC) or the related element, regulating both its basal and inducible expression, was mostly found in the 5'-flanking region of genes of phase II enzymes and may be recognized by a similar series of transcriptional factors (55). Thus, an inducer of NQO1 is regarded as a common inducer of phase II enzymes. Although NQO1 is not directly involved in the metabolic pathways of environmental carcinogens, the induced expression of this enzyme, therefore, has been found to correlate with the chemopreventive potential. The present study demonstrates for the first time that ebselen substantially induces NQO1 activity in cultured hepatocytes. It should be noteworthy that this activity is almost equivalent to that of tBHQ, a well-known strong inducer, suggesting that ebselen is a potential inducer of phase II enzymes. Recent evidence indicates that NQO1 may play an important role in supporting the function of two naturally occurring quinones: coenzyme Q (ubiquinone) (56) and -tocopherolquinone (57). Beyer et al. (56) demonstrated that NQO1 maintains various types of quinones in the reduced form, which is required for the protection of membranes against peroxidation. This protective function to internal antioxidants may also contribute to the in vivo antioxidative activity of ebselen in mouse skin.

In addition to NQO1 induction, ebselen expectedly induces GST activity in the cultured cell system and in mouse skin coincident with the GST protein expression and GST gene expression. Recently, two transgenic rodent studies clearly demonstrated that Class  GST (GSTP1), one of the GST isozymes, can profoundly alter susceptibility to chemical carcinogenesis in mouse skin (58) and rat liver (59). The Class  rat and human GST isozymes have been shown to be highly efficient in the glutathione (GSH) conjugation of carcinogenic benz[a]pyrene derivatives (60), and widespread environmental pollutants in cigarette smoke and automobile exhaust. In addition, GSTP1 is more effective for the detoxification of electrophilic ,-unsaturated carbonyl compounds produced by radical reactions, lipid peroxidation, ionizing radiation, and metabolism of drugs than other GSTs (61). Thus, the induction of GST is regarded as one of the important determinants in the cancer chemoprotection potential. In addition to GSH-conjugating activity, GST  class isozymes (GSTA1 and 2) show high GPx activity toward phospholipid hydroperoxides and they can catalyze GSH-dependent reduction of hydroperoxide in situ in biological membranes (40). Overexpression of human GSTA2 in human erythroleukemia K562 cells attenuates lipid peroxidation and confers resistance to these cells from H₂O₂ cytotoxicity. In addition to GPx-like activity, inducing ability of GST, being able to catalyze GSH-dependent reduction of hydroperoxide, may partly participate in its in vivo antioxidant effect in mouse skin.

We showed herein that ebselen stimulated the promoter activity of the 5'-flanking region of the GSTP1 gene and then induced GST protein in RL34 cells (Fig. 7, A and B). It appeared that this stimulation required the specific region containing the GPE1, ARE/EpRE of GSTP1. Venugopal and Jaiswal (62) have reported that the transcription factor NF-E2-related factor 2 regulates the ARE-mediated expression of Phase II detoxification enzyme genes. Itoh et al. (63) have also shown by gene-targeted disruption in mice that Nrf2 is a general regulator of the Phase II enzyme genes in response to electrophiles and reactive oxygens. More recently, the general regulatory mechanism underlying the electrophile counterattack response has been demonstrated in which electrophilic agents alter the interaction of Nrf2 with its repressor protein (Keap1), thereby liberating Nrf2 activity from repression by Keap1, culminating in the induction of the Phase II enzyme genes and antioxidative stress protein genes via ARE/EpREs (64). Keap1 contains 25 cysteine residues, 9 of which are expected to have highly reactive sulfhydryl groups (65). Because most of the inducers are redox-active, the Keap1·Nrf2 complex is a plausible candidate for the cytoplasmic sensor system that recognizes inducers, including ebselen. Further studies of factors interacting with GPEI and other functional cis-acting elements should be required for elucidation of the specific expression of the gene in association with the possible chemoprotective response of ebselen.

In conclusion, the results in the present study provide biological evidence that ebselen has a significant ability to suppress TPA-induced oxidative stress and inflammation through attenuation of the responsiveness of activated leukocytes and induction of the endogenous antioxidant protein such as phase II xenobiotic-metabolizing enzymes. Considering the importance of oxidative damage in carcinogenesis, the antioxidant effect of ebselen can be explored as a cancer chemopreventive agent targeted toward inflammation-related carcinogenesis such as skin and colon cancer.

	FOOTNOTES

^* This work was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence should be addressed: Tel.: 81-52-789-4127; Fax: 81-52-789-5741; E-mail: uchidak@agr.nagoya-u.ac.jp.

Published, JBC Papers in Press, November 19, 2001, DOI 10.1074/jbc.M109641200

	ABBREVIATIONS

The abbreviations used are: ROS, reactive oxygen species; PG, prostaglandin; TPA, 12-O-tetradecanoylphorbol-13-acetate; O₂, superoxide anion; H₂O₂, hydrogen peroxide; COX, cyclooxygenase; GPx, glutathione peroxidase; GST, glutathione S-transferase; NQO1, NAD(P)H:(quinone-acceptor) oxidoreductase; HRPO, horseradish peroxidase; TBARS, thiobarbituric acid-reacting substances; MPO, myeloperoxidase, IE, inhibitory effect; GPEI, GSTP1 enhancer I; HOCl, hypochlorous acid; ARE/EpRE, antioxidant/electrophile response element; CAT, chloramphenicol acetyltransferase; LPS, lipopolysaccharide; PBS, phosphate-buffered saline; GSH, reduced glutathione; tBHQ, t-butylhydroquinone.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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