Preventive Effect of Ortho Dimer of Butylated Hydroxyanisole on Activator Protein-1 Activation and Cyclooxygenase-2 Expression in Macrophages Stimulated by Fimbriae

Butylated hydroxyanisole (BHA; a mixture of 2and 3-BHA) is widely used as a potent antioxidant, but is reported to have adverse effects, such as carcinogenesis and pro-inflammatory activity, possibly due to the pro-oxidant property of this compound. 2-Methoxyphenol dimers derived from ferulic acid were recently demonstrated to inhibit the expression of lipopolysaccharide-stimulated cyclooxygenase-2 (COX-2) via redox-sensitive transcription factors such as nuclear factor kappa B or activator protein-1 (AP-1), due to a weakening of its pro-oxidant property by dimerization. To develop anti-inflammatory and/or anticancer drugs for the prevention of oral diseases, such as leukoplakia and destructive chronic periodontitis, whether 2-BHA (2-tert-butyl4-methoxyphenol) and its synthetic ortho dimer, bis-BHA (3,3’-di-tert-butyl-5,5’-dimethoxy-1,1’-biphenyl-2,2’-diol) can inhibit AP-1 transcriptional activity stimulated by Porphyromonas gingivalis fimbriae was examined. The fimbria-stimulated AP-1 activation of RAW 264.7 murine macrophages was markedly inhibited by bis-BHA. However, BHA showed slight inhibition. Furthermore, bis-BHA significantly inhibited fimbria-induced COX-2 gene expression, which is closely involved with inflammation and carcinogenesis. These findings suggest that bis-BHA may possess a potent anti-inflammatory effect against oral diseases. Butylated hydroxyanisole (BHA; 2-t-butyl-4-methoxyphenol), an artificial phenolic antioxidant, is widely used in the food industry as an efficient antioxidant. BHA (2and 3-BHA) has a number of pharmacological and toxicological properties. Several investigators have demonstrated that BHA significantly inhibits cytokine-induced inflammatory responses in human and mouse cells via its potent antioxidant activity (1, 2). Although BHA is an effective antioxidant, it has been reported to promote carcinogenesis and inflammatory activity in animals (3-5), possibly due to its pro-oxidant activity and consequent formation of reactive quinone methide intermediates (6, 7). Therefore, to weaken the potent oxidative activity of 2-BHA, bis-BHA (3,3’-di-tertbutyl-5,5’-dimethoxy-1,1’-biphenyl-2,2’-diol), an ortho dimer of 2-BHA, was recently synthesized and its antioxidant activities and cytotoxicity were investigated. Actuarially, bisBHA showed less cytotoxicity and less oxidation activity (8,9), suggesting that bis-BHA might act as a potent inhibitor of inflammatory and allergic responses. Porphyromonas gingivalis is an oral anaerobic bacterium causing chronic periodontal diseases characterized by serious gingival tissue inflammation and tooth loss due to alveolar bone resorption. P. gingivalis fimbriae are an especially important bacterial component that regulate adhesion to host cells of the organisms affected by these diseases. Our previous studies (10-12) demonstrated that the fimbriae actually induced the expression of inflammatory cytokines, such as IL-1 beta, IL-6 and neutrophil chemo-attractant KC (murine CXC chemokine) in several kinds of host cells, and stimulated bone resorption in murine embryonic calvarial bone cells. These findings suggest that the fimbria function not only as a component 2915 Abbreviations: AP-1, activator protein-1; COX-2, cyclooxygenase2; P. gingivalis, Porphyromonas gingivalis. Correspondence to: Seiichiro Fujisawa, Department of Diagnostic and Therapeutic Sciences, Meikai University School of Dentistry, 1-1 Keyakidai, Sakado City, Saitama 350-0283, Japan. Tel: 81-49285-5511, Fax: 81-49-287-6657, e-mail: [email protected] or [email protected]