An ortho dimer of butylated hydroxyanisole inhibits nuclear factor kappa B activation and gene expression of inflammatory cytokines in macrophages stimulated by Porphyromonas gingivalis fimbriae

Archives of Biochemistry and Biophysics 449 (2006) 171–177 www.elsevier.com/locate/yabbi

An ortho dimer of butylated hydroxyanisole inhibits nuclear factor kappa B activation and gene expression of inXammatory cytokines in macrophages stimulated by Porphyromonas gingivalis Wmbriae Yukio Murakami a, Masao Shoji a, Atsushi Hirata a, Shoji Tanaka a, Shigemasa Hanazawa b, Ichiro Yokoe c, Seiichiro Fujisawa a,¤ b

a Department of Diagnostic and Therapeutic Sciences, Meikai University School of Dentistry, 1-1 Keyakidai, Sakado City, Saitama 350-0283, Japan Department of Applied Biological Science, College of Bioresource Science, Nihon University, 1866 Kameino, Fujisawa City, Kanagawa 252-8510, Japan c Faculty of Pharmaceutical Sciences, Josai University, 1-1 Keyakidai, Sakado City, Saitama 350-0295, Japan

Received 16 December 2005, and in revised form 10 February 2006 Available online 3 March 2006

Abstract Butylated hydroxyanisole, BHA, is widely used as a potent antioxidant, but its adverse eVects such as carcinogenesis and proinXammatory activity have been reported, which are possibly due to the prooxidant property of this compound. We recently demonstrated that the dimer of 2-methoxyphenols exhibits cyclooxygenase-2 inhibition, because of lessening of its prooxidant property caused by the dimerization. In the present study, toward our goal of developing a chemopreventive agent for chronic periodontal diseases, we examined whether 2-BHA (2-tert-butyl-4-methoxyphenol) and its synthetic ortho dimer, bis-BHA (3,3⬘-di-tert-butyl-5,5⬘-dimethoxy-1,1⬘-biphenyl-2,2⬘-diol) could inhibit the Porphyromonas gingivalis Wmbria-stimulated inXammatory reaction. The Wmbria-induced expression of interleukin-1 and neutrophil chemoattractant KC genes in RAW264.7 murine macrophages was strongly inhibited by bis-BHA. In contrast, 2-BHA showed only slight inhibition. bis-BHA also signiWcantly inhibited the Wmbria-stimulated phosphorylation-dependent degradation of the alpha inhibitor of nuclear factor-B and the transcriptional activity of this factor in the cells. These Wndings suggest that bis-BHA possesses anti-inXammatory activity against chronic periodontal diseases. © 2006 Elsevier Inc. All rights reserved. Keywords: Butylated hydroxyanisole; bis-BHA; Porphyromonas gingivalis Wmbriae; Anti-inXammatory activity; NF-B; InXammatory cytokine; Macrophage

The oral anaerobe Porphyromonas gingivalis is a key organism causing chronic periodontal diseases characterized by serious gingival tissue inXammation and tooth loss due to alveolar bone resorption. Especially, P. gingivalis Wmbriae are an important bacterial component that regulates the adherence to host cells of the organisms involved in these diseases. Our previous studies [1–4] demonstrated that the Wmbriae actually induced the expression of inXammatory cytokines such as IL-11, IL-6, and *

Corresponding author. Fax: +81 49 287 6657. E-mail address: [email protected] (S. Fujisawa). 1 Abbreviations used: BHA, butylated hydroxyanisole; IL-1, interleukin-1 beta; P. gingivalis, Porphyromonas gingivalis; NF-B, nuclear factor kappa B; IB-, alpha inhibitor of NF-B. 0003-9861/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2006.02.005

neutrophil chemoattractant KC (murine CXC chemokine) in several kinds of host cells and stimulated bone resorption via these endogenous cytokines such as IL-1 or GM-CSF by murine embryonic calvarial bone cells in vitro. These Wndings suggest that Wmbriae function not only as a component for binding to host cells, but also play an important role as a key virulence factor by promoting the various biological actions of inXammatory cytokines in the initiation and development of chronic periodontal diseases. Recently, phytophenols have become a focus of attention for the prevention of various chronic diseases including oral diseases such as periodontitis or dental caries. Natural o-methoxyphenols such as eugenol (4-allyl-2methoxyphenol) and isoeugenol (4-propenyl-2-methoxy-

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phenol), which are components of clover oil, are commonly used as a Xavoring agent in cosmetics, and food products. These compounds show an anti-inXammatory eVect but have adverse eVects characterized by inXammatory and allergic reactions when used at higher concentrations, possibly due to their prooxidant activity and consequent formation of reactive quinone methide intermediates [5,6]. Recently, we demonstrated that two synthetic dimers of o-methoxyphenols, i.e., bis-eugenol (3,3⬘-dimethoxy-5,5⬘-di-2-propenyl-1,1⬘-biphenyl-2, 2⬘-diol) and dehydrodiisoeugenol (2(3-methoxy-4-hydroxyphenyl)-3-methyl-5(1-propenyl)-7-methoxy-2,3-dihydrobenzofuran), which were designed to lessen the prooxidant activity derived from the parent monomers, inhibited lipopolysaccharide (LPS)-stimulated nuclear factor kappa B (NF-B) activation and suppressed the expression of inXammatory cytokines and/or cyclooxygenase-2 (COX-2) in macrophages [7,8]. These Wndings suggest that dimer compounds derived from various phenolic compounds may possess potent anti-inXammatory activity. tert-Butylated hydroxyanisole (BHA: 2-t-butyl-4methoxyphenol), an artiWcial phenolic antioxidant, is widely used in the food industry as an eYcient antioxidant. Several investigators have demonstrated that BHA signiWcantly inhibits cytokine-induced inXammatory responses in human and mouse cells via its potent antioxidant activity [9,10]. BHA is a mixture of 2- and 3-BHA. 3-BHA is well known to have a higher antioxidant activity than 2-BHA. Although BHA (93% or more as 3BHA) is an eVective antioxidant, its adverse eVects of promoting carcinogenesis and inXammatory activity have been suggested [11,12], eVects possibly due to its prooxidant activity. To explore the possible use of BHA as a potent chemopreventive agent for chronic periodontal disease, in the present study we investigated whether 2-BHA and its synthetic dimer, bis-BHA (3,3⬘-di-t-butyl5,5⬘-dimethoxy-1,1⬘-biphenyl-2,2⬘-diol), and could inhibit Wmbria-stimulated inXammatory cytokine expression and NF-B activation in mouse macrophage-like cell line RAW264.7. Our Wndings reported herein show that bis-BHA was anti-inXammatory and suggest that its anti-inXammatory action was mediated via the inhibition of NF-B action in the cells. Materials and methods

HO (H3C)3C

HO

OH

(H3C)3C

OMe

C(CH3)3

OMe

2-BHA

OMe

bis-BHA

Fig. 1. Chemical structures of 2-BHA and bis-BHA.

mouse IL-1 mRNA-speciWc antisense oligonucleotides, which consists of equimolar mixture of each of three 30mer individual single-stranded DNAs, was purchased from Genzyme Techne Co. (Minneapolis, MN, USA); and a 25-mer -actin oligonucleotide (single-stranded DNA) probe, from GeneDetect.com Ltd. (Bradenton, FL, USA). Mouse neutrophil chemoattractant KC cDNA was provided by C.D.Stiles, Harvard University. Mouse COX-2 cDNA probes having the length of approximately 1.2 kbp were purchased from Cayman Chemical Co. (Ann Arbor, MI, USA). Phospho-speciWc anti-IB- antibody (recognizing phospho-serine 32), anti-IB-, phospho-speciWc anti-IKK antibody (recognizing phospho-serines 177– 181), anti-IKK, all rabbit polyclonal antibodies, as well as HRP-conjugated goat anti-rabbit IgG and a Phototope-HRP Western blot detection kit, came from Cell Signaling Technology, Inc. (Beverly, MA, USA). -Actin rabbit polyclonal antibody was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). RPMI 1640, OptiMEM, and Lipofectamine™ were purchased from Invitrogen Corp. (Carlsbad, CA, USA). FBS was from HyClone (Logan, UT, USA); and Escherichia coli O111 B4-derived LPS was from List Biological Laboratories, Inc. (Campbell, CA, USA). Phorbol 12-myristate 13-acetate was purchased from Wako Chemical USA Inc. (Richmond, VA, USA). Cell culture Cells of the murine macrophage-like cell line RAW 264.7, obtained from the Riken Cell Bank, were used. They were cultured to the subconXuent state in RPMI 1640 medium supplemented with 10% FBS at 37 °C and 5% CO2 in air, washed, and then incubated overnight in serum-free RPMI 1640. Thereafter they were washed further and treated with test samples.

Reagents Preparation of P.gingivalis Wmbriae 2-BHA was purchased from Tokyo Kasei Co. (Tokyo, Japan). bis-BHA was synthesized from 2-BHA monomers by the CuCl(OH)-catalyzed coupling reaction described previously [13]. The chemical structures of BHA and bisBHA are shown in Fig. 1. Megaprime DNA labeling system, 5⬘-end labeling system, 5⬘-[-32P]dCTP, and [32 P]ATP were purchased from Amersham Biosciences Co. (Piscataway, NJ, USA). A mouse IL-1 probe cocktail for

Porphyromonas gingivalis ATCC 33277 Wmbriae were prepared and puriWed from cell washings by the method of Yoshimura et al. [14]. PuriWed Wmbria-induced biological activities were not attributable to lipopolysaccharide contaminants in the preparation, as documented previously [1,3]. Viability of the cells after Wmbriae exposure at the concentrations used was over 90% by the MTT (3-[4,5-

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dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) test. The protein content of the Wmbriae was measured by the method of Bradford [15]. Northern blot analysis After cells (106 cells) in Falcon 5-cm-diameter dishes (Becton Dickinson Labware, Franklin Lakes, NJ, USA) had been treated with test samples, total cellular RNA was extracted from them by the acid guanidine phenol chloroform procedure [16]. As described earlier [17], the RNA was electrophoresed in 1% agarose gels with 0.2 M sodium phosphate as running buVer and blotted onto nylon membranes (Micron Separations, Inc., Westboro, MA, USA). The membranes were then hybridized with each oligonucleotide probe, which had been labeled with [-32P]ATP by use of the 5⬘-end labeling system (Amersham Biosciences Co.). After hybridization, the membranes were washed, dried, and exposed overnight to Kodak X-ray Wlm (Eastman Kodak Co., Rochester, NY, USA) at ¡70 °C. -Actin was used as an internal standard for quantiWcation of total RNA in each lane of the gel. Measurement of IL-1 Cells in 24-well microculture plates (Nalge Nunc International, Kamstrup, Denmark) were treated with test samples for 6 h, and then supernatants of their culture medium were prepared. IL-1 protein in these supernatants was measured with an enzyme-linked immunosorbent assay (ELISA) kit utilizing anti-mouse IL-1 antibody (BioSource International, Inc.), as described previously [7]. Gel mobility shift assay Cells in Falcon 15-cm-diameter dishes (107 cells per dish) were treated with test samples. Their nuclei were then isolated, and the extracts were prepared for the gel mobility shift assay described previously [17]. BrieXy, the binding reactions were performed for 20 min at room temperature with 10 g of the nuclear proteins in 2 mM Tris (pH 7.5) containing 8 mM NaCl, 0.2 mM EDTA, 0.8% (v/ v) glycerol, 0.2 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl Xuoride (PMSF), 1 g of poly(dI–dC), and 20,000 cpm of a 32P-labeled NF-B oligonucleotide in a Wnal volume of 20 l. Poly (dI–dC) and nuclear extract were incubated at 4 °C for 10 min before addition of the labeled oligonucleotide. The double-stranded oligonucleotide containing a tandem repeat of the consensus sequence for the binding site, -GGGGACTTTCCC-, for NF-B was end-labeled by the T4 polynucleotide kinase[-32P]ATP method. DNA-protein complexes were electrophoresed in native 6% polyacrylamide gel in 0.25£ TBE buVer (22 mM Tris [pH 8.0], 22 mM boric acid, and 0.6 mM EDTA). The gel was dried, and then exposed to Kodak X-ray Wlm at ¡70 °C.

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Western blot analysis Cells in Falcon 5-cm-diameter dishes (106 cells per dish) were treated with test samples. Then the cells were solubilized with lysis buVer (20 mM Tris–HCl [pH 7.4], 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% [vol/vol] Triton X-100, 2.5 mM sodium pyrophosphate, 2 mM Na3VO4, 10 mM NaF, 1 mM -glycerolphosphate, 1 g/ml aprotinin, and 1 mM PMSF). The protein concentrations were measured by the method of Smith et al. [18]. Each sample (10 g of protein) was subjected to SDS–PAGE in a 12.5% or 7.5% polyacrylamide gel, and the separated proteins were transferred to a polyvinylidene diXuoride membrane (Millipore Co., Bedford, MA, USA). Then, the blots were blocked with 5% skim milk, washed, and incubated with 2000£ diluted primary antibodies, such as anti-IB-, phospho-speciWc anti-IB-, antiIKK or phospho-speciWc anti-IKK antibody in the working solution (5% BSA, 1£ TBS [50 mM Tris–HCl, pH 7.4, containing 150 mM NaCl], and 0.1% Tween 20) at 4 °C. Actin antibody was used at 0.1 g/ml after dilution by the working solution. After incubation, the blots were treated with a 4000 times- diluted HRP-conjugated secondary antibody at room temperature. Proteins were detected with a Phototope-HRP Western blot detection kit (Cell Signaling Technology, Inc.), and the blots were exposed to Kodak Xray Wlm for 10 min. -Actin was used as an loading control in each lane of the gel. Plasmid construction and transient expression assay The plasmid pNF-B-Luc (Clontech Laboratories, Inc., Palo Alto, CA, USA) was constructed by inserting a synthetic oligonucleotide containing four tandem copies of the NF-B consensus sequence into the corresponding sites of pTA-Luc (Clontech), which contains the HSV thymidine kinase (HSV-TK) promoter enhancer region located upstream of the WreXy luciferase gene. Also used was pRLTK, which contains the HSV-TK promoter located in the region upstream of Renilla luciferase (Promega Co., Madison, WI, USA). Transient expression was assayed as described previously [19]. BrieXy, cells in Falcon 5-cmdiameter dishes (106 cells per dish) were incubated for 1 h in serum-free Opti-MEM. Then they were transfected with the reporter plasmid at 2 g and pRL-TK at 0.2 g by using Lipofectamine™ (Invitrogen). After incubation for 24 h, the transfected cells were treated with test samples in serum-free RPMI 1640. Then, the cellular extracts were prepared in reporter passive lysis buVer (Promega) and examined for WreXy luciferase activity after determination of Renilla luciferase activity (pRL-TK). The latter was used as an internal control to normalize for variations in transfection eYciency. Results and discussion 2-BHA is moderately low in cytotoxic activity and a potent antioxidant, but bis-BHA possesses 10-fold lower

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cytotoxicity than 2-BHA [20]. No reports on the carcinogenicity of bis-BHA have yet appeared. Recently, we demonstrated that several 2-methoxy dimer compounds, such as bis-eugenol and dehydrodiisoeugenol, possess a potent anti-inXammatory activity at lower, less cytotoxic concentrations [7,8]. Thus, it was of interest to us to examine whether bis-BHA, a methoxyphenol dimer, could inhibit the Wmbria-stimulated inXammatory reaction. First, we examined by using the Northern blot assay whether 2-BHA and bis-BHA could inhibit the Wmbria-induced expression of inXammatory cytokines in RAW cells. The Wmbriainduced expression of IL-1 and neutrophil chemoattractant KC genes was strongly inhibited by bis-BHA at the low concentrations of 1 M or greater; whereas it was only slightly inhibited by 2-BHA (Figs. 2A and B). Against IL1 gene expression, 2-BHA showed a moderate inhibition at the high concentration of 100 M. We next examined whether these compounds could indeed inhibit the production of IL-1 protein. Similarly, bis-BHA clearly inhibited Wmbria-induced production of IL-1 at the concentration of 71 M: whereas 2-BHA slightly, but signiWcantly, inhibited it at the high concentration of 100 M (Fig. 3). In addition, bis-BHA, but not 2-BHA, inhibited E. coli O111 B4derived LPS or phorbol 12-myristate 13-acetate -induced gene expression of IL-1, as well as Wmbria-induced expression of the cyclooxygenase-2 gene, in RAW cells (data not shown). These Wndings suggest bis-BHA to be an eVective inhibitor of inXammatory mediators induced by Wmbriae. NF-B, which is activated by phosphorylation-dependent proteolysis of inhibitor B- (IB-), is an important transcriptional factor involved in the expression of various inXammatory cytokines [21]. In addition, NF-B plays a functional role in osteoclast survival [22], suggesting that NF-B is an important transcription factor that regulates inXammatory bone remodeling mechanisms in chronic

Fig. 3. bis-BHA inhibits the Wmbria-stimulated IL-1 production in RAW 264.7 cells. The cells were pretreated or not for 30 min with the indicated dosages of 2-BHA or bis-BHA and then treated or not with the Wmbriae at 2 g/ml. Thereafter, their culture supernatant was collected at 6 h after the addition of the Wmbriae. IL-1 protein levels were measured by use of ELISA. The results are expressed as the mean § SD of three diVerent experiments. Statistic analysis was performed by Student’s t test. There is a signiWcant diVerence between the same concentrations of 2-BHA and bis-BHA (¤p < 0.01).

periodontal diseases. As bis-BHA might also act as an inhibitor of the Wmbria-stimulated NF-B activation, next we investigated the eVect of bis-BHA on Wmbria-stimulated NF-B binding to its consensus sequence. As shown in Figs. 4 A and B, bis-BHA inhibited Wmbria-stimulated NFB binding to its consensus sequence in RAW cells. In contrast, 2-BHA did not have any eVect on the binding. These results suggest that inhibitory eVects of bis-BHA on the Wmbria-stimulated NF-B binding in RAW cells may have been due to the suppression of IB- degradation, which would prevent NF-B activity. To clarify this point, we also examined the eVect of bis-BHA on the Wmbria-stimulated

Fig. 2. Regulatory eVect of 2-BHA and bis-BHA on the Wmbria-induced expression of inXammatory cytokine genes in RAW 264.7 cells. (A) The cells were pretreated or not for 30 min with the indicated dosages of 2-BHA or bis-BHA and then treated or not with the Wmbriae at 2 g/ml. Thereafter, their total RNA was prepared at 3 h after the addition of the Wmbriae. Northern blot analysis was performed with radio-labeled IL-1, -actin antisense oligonucleotide, and KC cDNA probes. An identical experiment independently performed 3 times gave similar results. (B) QuantiWcation of IL-1 and KC expression shown in “A” was done by densitometry; and the data are expressed as the relative signal intensity (percentage of the maximum). Bars (black for IL-1, striped for KC) present the mean for three independent experiments. Standard errors <15%. There is a signiWcant diVerence between 2-BHA and bis-BHA at 10 M and 1 M (p < 0.001) for both IL-1 and KC.

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Fig. 4. bis-BHA inhibits the Wmbria-stimulated NF-B binding in RAW 264.7 cells. (A) The cells were pretreated or not for 30 min with 2-BHA or bisBHA at 10 M and then treated or not with the Wmbriae at 2 g/ml for 1 h. Then the nuclear proteins were prepared for a gel mobility shift assay, which was performed with these nuclear proteins and 32P-labeled oligonucleotide containing the NF-B consensus sequence. An identical experiment independently performed 3 times gave similar results. (B) QuantiWcation of NF-B binding in “A” was done by densitometry, and the data are expressed as the relative signal intensity (percentage of the maximum). Bars present the mean for three independent experiments. Standard errors <15%. There is a signiWcant diVerence between 2-BHA and bis-BHA (p < 0.01).

Fig. 5. bis-BHA inhibits Wmbria-stimulated IB- phosphorylation and degradation in RAW 264.7 cells. (A) The cells were pretreated or not for 30 min with 2-BHA or bis-BHA at 10 M and then treated or not with the Wmbriae at 2 g/ml. Thereafter, their cell lysates were prepared at various times after the start of treatment. Equal amounts of cell lysates were analyzed by Western blotting after SDS–PAGE with phospho-speciWc anti-IB- antibody, antiIB- antibody or anti--actin antibody. An identical experiment independently performed 3 times gave similar results. (B and C) QuantiWcation of expression of IB- (B) and phospho-speciWc IB- (C), respectively, shown in “A” was done by densitometry; and the data are expressed as the relative signal intensity (percentage of the maximum). Bars (black for Wmbriae only, dotted for Wmbriae plus 2-BHA, and striped for Wmbriae plus bis-BHA) present the mean for three independent experiments. Standard errors <15%. There is a signiWcant diVerence between 2-BHA and bis-BHA at 30 min (p < 0.01) for both IB- and phospho-speciWc IB-.

phosphorylation-dependent proteolysis of IB- in RAW cells by conducting a Western blot assay. The Wmbriae markedly stimulated both phosphorylation and degrada-

tion of the inhibitor at 30 min after the start of treatment, and these activities were signiWcantly inhibited by bis-BHA treatment (Figs. 5A–C).

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Fig. 6. bis-BHA inhibits the Wmbria-stimulated IKK phosphorylation in RAW 264.7 cells. (A) The cells were pretreated or not for 30 min with 2-BHA or bis-BHA at 10 M and then treated or not with the Wmbriae at 2 g/ml for 30 min. Thereafter, their cell lysates were prepared. Equal amounts of cell lysates were analyzed by Western blotting after SDS–PAGE with phospho-speciWc anti-IKK antibody, anti-IKK antibody or anti--actin antibody. An identical experiment independently performed 3 times gave similar results. (B) QuantiWcation of IKK phosphorylation in “A” was done by densitometry, and the data are expressed as the relative signal intensity (percentage of the maximum). Bars present the mean for three independent experiments. Standard errors <15%. There is a signiWcant diVerence between 2-BHA and bis-BHA (p < 0.01).

For IB- phosphorylation and degradation, the activation of IKK (IB kinase) is necessary. Especially, phosphorylation at two sites, serines 177–181, in the activation loop of IKK elicited by proinXammatory stimuli is an essential step for IKK activation [23]. To clarify this point, we further investigated inhibitory eVects of bis-BHA on Wmbria-stimulated phosphorylation of IKK. Figs. 6A and B shows that the Wmbria-stimulated phosphorylation of 87 kDa IKK was inhibited by bis-BHA, but not by 2-BHA. However, the total amount of IKK protein was not changed by the Wmbrial treatment. These Wndings strongly suggest that bis-BHA inhibited the Wmbria-stimulated transcriptional activity of NF-B by suppressing the phosphorylation-dependent proteolysis of IB- in the cells. Finally, to clarify the inhibitory eVect of bis-BHA on NF-B, we transfected RAW cells with a luciferase reporter gene under the control of a promoter containing NF-B sites prior to stimulation of the cells, and then conducted the luciferase assay. As expected, bis-BHA, but not 2-BHA, clearly inhibited the Wmbria-stimulated transcriptional activity of NF-B in the cells (Fig. 7). Together with these Wndings, it is clear that bis-BHA functioned as an eVective inhibitor of the Wmbria-stimulated NF-B activation. BHA (2- and 3-BHA) is well known to have a number of pharmacological and toxicological properties. Several previously reported studies showed BHA to be toxic and to act in the carcinogenic process in animal models [11,12,24]. We previously examined the cytotoxicity of 2-BHA and bis-BHA and discovered that 2-BHA was much more cytotoxic than bis-BHA, possibly due to the formation of semiquinone radicals and, consequently, of quinone methide derived from BHA [20]. BHA (93% or more as 3-BHA) was earlier shown to stimulate NF-B and activator protein-1 (AP-1) activities [25] and mitogen-activated protein kinase activation [26], all of which are inhibited by thiol antioxidants such as N-acetyl cysteine and glutathione. These Wndings suggest that BHA possesses a potent prooxidant and proinXammatory activity. In the present study, 2-BHA did not inhibit the Wmbria-stim-

ulated NF-B activation, possibly due to its own prooxidative activity. The phenolic O—bond dissociation energy (BDE) was previously calculated by a PM3 semiempirical method; and the BDE of 2-BHA (82.16 kcal/mol) was found to be lower than that of bis-BHA (83.19 kcal/mol), suggesting that 2-BHA is more oxidizable than bis-BHA. Possibly, the Wmbriae would stimulate living cells and generate intracellular ROS such as the .OH radical. Consequently, inXammatory cytokine expression might be induced via activation of redox-sensitive transcription factors such as NF-B or AP-1 in RAW cells. 2-BHA preferably scavenges much more O2¡ than bis-BHA [27]. This suggests that 2-BHA would be easy converted to reactive quinone intermediates. The quinone intermediates derived from 2-BHA are more active, when compared with those derived from 3-BHA [28]. In contrast, bis-BHA possess low cytotoxicity and moderate anti-radical activity [20]. Thus, bis-BHA may act as a non-steroidal antiinXammatory drug (NSAID)-like compound like aspirin and salicylic acid [29], leading to a reduction in the oxidative stress caused by Wmbrial treatment [30]. We previously demonstrated that transcription factor AP1 plays a functional role in the Wmbria-stimulated bone resorption via endogenous IL-1 in the mouse calvarial system [31]. These Wndings suggest that AP-1 inhibition may be connected with the prevention of stimulated bone resorption. Thus, we tested also whether bis-BHA could inhibit the Wmbria-stimulated AP-1 activity in RAW cells. bis-BHA strongly inhibited the binding of Wmbria-stimulated AP-1 to its consensus sequence, whereas 2-BHA did so only slightly (data not shown). This observation suggests that bis-BHA may function to prevent bone resorption in chronic periodontal diseases. In a further study, we plan to explore in greater detail the molecular mechanism of transcription factor inhibition by bis-BHA. In conclusion, we proposed herein a possible mechanism for the anti-inXammatory action of bis-BHA, suggesting that bis-BHA might be applicable to the chemoprevention

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Fig. 7. bis-BHA inhibits Wmbria-stimulated activation of NF-B in RAW 264.7 cells. The cells were co-transfected with pRL-TK and reporter plasmid (pNFB-Luc) and then washed three times. Next, the transfected cells were pretreated or not for 30 min with 2-BHA or bis-BHA at 10 M in serumfree RPMI 1640 and then treated or not with the Wmbriae at 2 g/ml. The cellular extracts were prepared 6 h later and subsequently subjected to the luciferase assay. The pRL-TK plasmid was used as an internal control to normalize for variations in transfection eYciency. The luciferase activity (relative signal intensity) is expressed as a percentage of the maximum obtained with the Wmbriae alone. The luciferase activity was determined in triplicate, and bars present the mean. Error bars show the standard deviation. Statistic analysis was performed by using Student’s t test. There is a signiWcant diVerence between 2-BHA and bis-BHA (¤p < 0.01).

of oral diseases, particularly chronic periodontal diseases; because this compound exhibited an inhibitory eVect on NF-B and inXammatory responses. Acknowledgment This work was supported by a Grant-in-Aid for scientiWc research (No. 15592133) from the Japanese Ministry of Education, Science Sports, and Culture. References [1] S. Hanazawa, Y. Murakami, K. Hirose, S. Amano, Y. Ohmori, H. Higuchi, S. Kitano, Infect. Immun. 59 (1991) 1972–1977.

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[2] S. Hanazawa, Y. Murakami, A. Takeshita, H. Kitami, K. Ohta, S. Amano, S. Kitano, Infect. Immun. 60 (1992) 1544–1549. [3] Y. Kawata, S. Hanazawa, S. Amano, Y. Murakami, T. Matsumoto, K. Nishida, S. Kitano, Infect. Immun. 62 (1994) 3012–3016. [4] Y. Murakami, M. Shoji, A. Hirata, S. Tanaka, S. Hanazawa, S. Fujisawa, FEMS Immunol. Med. Microbiol. 43 (2005) 205–211. [5] M.D. Baratt, D.A. Basketter, D.W. Roberts, J.-P. Lepoittevin, Chem. Res. Toxicol. 10 (1997) 335–343. [6] Y. Suzuki, K. Sugiyama, H. Furuta, Jpn. J. Pharmacol. 39 (1985) 381–386. [7] Y. Murakami, M. Shoji, S. Hanazawa, S. Tanaka, S. Fujisawa, Biochem. Pharmacol. 66 (2003) 1061–1066. [8] Y. Murakami, M. Shoji, A. Hirata, S. Tanaka, I. Yokoe, S. Fujisawa, Arch. Biochem. Biophys. 434 (2005) 326–332. [9] O.L. Brekke, T. Espevic, K.S. Bjeve, Lipids 29 (1994) 91–102. [10] A. Shrivastava, B.B. Aggarwal, Antioxid. Redox Signal. 1 (1999) 181–191. [11] N. Ito, S. Fukushima, A. Hagiwara, M. Shibata, T. Ogiso, J. Natl. Cancer Inst. 70 (1983) 343–352. [12] O. Takahashi, Y. Sakamoto, K. Hiraga, Toxicol. Lett. 27 (1985) 15–25. [13] K. Satoh, T. Atsumi, H. Sakagami, Y. Kashiwagi, Y. Ida, T. Ueha, Y. Sugita, I. Yokoe, S. Fujisawa, Anticancer Res. 19 (1999) 3947–3952. [14] F. Yoshimura, K. Takahashi, Y. Nodasaka, T. Suzuki, J. Bacteriol. 160 (1984) 949–954. [15] M.M. Bradford, Anal. Biochem. 72 (1976) 248–254. [16] P. Chomozynski, N. Sacci, Anal. Biochem. 162 (1987) 156–159. [17] S. Hanazawa, A. Takeshita, S. Kitano, J. Biol. Chem. 269 (1994) 21379–21384. [18] P.M. Smith, R.I. Krohn, G.T. Hermanson, A.K. Mallia, F.H. Gartner, M.D. Provenzano, E.K. Fujimoto, N.M. Goeke, B.J. Olson, D.C. Klenk, Anal. Biochem. 150 (1985) 76–85. [19] H. Iwahashi, A. Takeshita, S. Hanazawa, J. Immunol. 164 (2000) 5403–5408. [20] S. Fujisawa, T. Atsumi, Y. Kadoma, M. Ishihara, S. Ito, I. Yokoe, Anticancer Res. 24 (2004) 3019–3026. [21] S. Ghosh, M.J. May, E.B. Koop, Annu. Rev. Immunol. 16 (1998) 225–260. [22] K. Ozaki, H. Takeda, H. Iwahashi, S. Kitano, S. Hanazawa, FEBS Lett. 410 (1997) 297–300. [23] M. Delhase, M. Hayakawa, Y. Chen, M. Karin, Science 284 (1999) 309–313. [24] L. Della-Corte, G. Sgaragli, Pharmacol. Res. Commun. 16 (1984) 1041–1047. [25] R. Pinkus, L.M. Weiner, V. Daniel, J. Biol. Chem. 271 (1996) 13422–13429. [26] R. Yu, T.-H. Tan, A.-N.T. Kong, J. Biol. Chem. 272 (1997) 28962–28970. [27] K. Satoh, H. Sakagami, I. Yokoe, M. Kochi, S. Fujisawa, Anticancer Res. 18 (1998) 425–428. [28] L.K. Lam, P.K. Garg, S.M. Swanson, J.M. Pezzuto, J. Pharm. Sci. 77 (1988) 393–395. [29] M.-J. Yin, Y. Yamamoto, R.B. Gaynor, Nature 396 (1998) 77–80. [30] X. Shi, Z. Dong, C. Huang, W. Ma, K. Liu, J. Ye, F. Chen, S.S. Leonard, M. Ding, V. Castranova, V. Vallyathan, Mol. Cell. Biochem. 194 (1999) 63–70. [31] K. Naganuma, S. Amano, H. Takeda, S. Kitano, S. Hanazawa, Oral Microbiol. Immunol. 15 (2000) 53–57.