Induction of epidermal proliferation and expressions of PKC and NF-κB by betel quid extracts in mouse: the role of lime-piper additives in betel quid

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Abstract

Components of betel quid (BQ) have been investigated for genotoxicity, mutagenicity, and animal toxicity. However, little information exists regarding their carcinogenic characteristics. Considerable attention has already been focused on tumor promoters that occur environmentally for human uptake. In this study, the promoting effects of BQ and lime-piper additives (LPA) in BQ on epidermal hyperplasia in CD-1 mouse skin are investigated. In the present study, we found that BQ and LPA at concentrations of 25, 50, 75 mg/ml caused significant induction of hyperplasia, but only LPA caused an increase of epidermal ornithine decarboxylase (ODC). Treatment of mouse skin with LPA caused remarkable increases in the production of H2O2 by 2.41-, 3.90-, and 3.76-fold (for the above-indicated concentrations respectively); as well as marked increases of myeloperoxidase (MPO) by 1.43-, 2.70-, and 2.29-fold. Application of LPA or BQ (50, 100, 150 mg/ml) also caused induction of protein kinase C-α (PKC-α) and NF-κB. LPA exhibited more significant effect than BQ. Thus, LPA might make a major contribution to the BQ-induced expression of PKC and NF-κB. These results indicated that BQ has the potential of being promoting agents, and that LPA should play a major role in increasing the effects of BQ-caused skin hyperplasia and inflammation. The promoting effects of BQ and LPA on mouse skin were associated with the induction of the expressions of PKC and NF-κB.

Introduction

Betel, known as Areca catechu, is a masticatory substance common to many oriental countries since a warm and humid climate is ideal for growing the Areca nut. In Taiwan, betel quid (BQ) is a natural masticatory. The most popular BQ is ‘red lime betel nut’ consisting of fresh nut of A. catechu, Piper betle flower and slacked lime paste which stains red from the addition of an extract of Chinese herb A. catechu. The other popular is ‘white lime betel nut’ composed of Areca nut, P. betle leaf and slacked lime [1]. Fresh areca nut contains lipids, fiber, alkaloids, polysaccharide and complex polyphenolic compounds, mainly hydroxychavicol and safrole [2]. The alkaloids in areca nut are arecoline (7.5 mg/g weight), arecaidine (1.5 mg/g weight), guvacoline (2.0 mg/g weight) and isoguvacine (2.9 mg/g weight) [3]. Several phenolic compounds are found in P. betle flower including safrole, hydroxychavicol, eugenol, methyl eugenol, isoeugenol, flavone and quercetin [4]. In P. betle leaf, a large amount of carotenes (80.5 mg/g weight) is found, as well as smaller amounts of phenolic compounds (21.9 mg/g weight) and ascorbic acid (1.9 mg/g weight) [2]. Acacia catechu contains apparently (+)-catechin and (−)-epicatechin [2]. In previous studies, different components existed in BQ were found to possess mutagenicity or genotoxicity [5], [6], [7], to induce chromosomal aberrations [8], and to increase the frequency of sister chromatid exchange of DNA in Chinese hamster ovary cells [9]. Some experiments on whether BQ or its components induced tumors or caused oral diseases showed positive results [10], [11], [12], [13], [14], whereas some did not, especially those that used betel leaf aqueous extract [15], [16], [17]. The different effects on tumorigenicity of BQ observed in previous study seemed to depend on the applied components, such as areca nut, arecoline, arecaidine or betel leaf. These controversial results led us to suspect that the mutagenic properties of BQ might be mainly caused by its additives. Actually, an epidemiological study has showed that a high frequency of oral cancers in humans can be linked to chewing BQ (4). Moreover, many investigations have demonstrated that chewing and smoking habits act synergistically, and that persons with mixed habits form a substantial fraction of the high-risk population. However, up to the present, there is no direct evidence to show that BQ is a carcinogen, even though BQ should play an important role in cancer progression. We found that most investigations on BQ were focused on its carcinogenic properties, but not on its tumor promoting properties. Furthermore, little research had clarified that it was BQ or its additives possessing the main ability to cause cancer. Therefore, a series of experiments were conducted in this study to evaluate the possible promoting properties of BQ and its additives. The kind of BQ we chose to evaluate the effect on carcinogenesis was ‘red lime betel nut’ consisting of the fresh nut of Areca catechu, P. betle flower and slacked lime paste. LPA means lime-piper additives consisted of P. betle flower and slacked lime paste. To investigate the promoting effect of BQ or LPA in CD-1 female mice, some short-term markers for promoting agent were observed, including morphological changes in mouse skin that represent skin inflammation and hyperplasia, induction of myeloperoxidase (MPO) activity and H2O2 formation, and induction of ornithine decarboxylase (ODC) activity.

Protein kinase C (PKC) is a Ca2+ and phospholipid-dependent serine/threonine protein kinase with fundamental importance in cellular growth control. PKC is activated by a wide variety of growth factors, hormones, and neurotransmitters; and it has been shown to be a high affinity receptor for the phorbol ester tumor promoters, as well as other agents possessing tumor promotion activity [18], [19], [20]. Since the alterations in PKC have been linked to the increased cell proliferation in response to tumor promotion, the effect of BQ and LPA on PKC expression in mouse skin was also investigated in this study. Additionally, an inappropriate regulation of NF-κB-mediated transcription has also been associated with cancer and inflammatory responses [21], [22], [23]. Therefore, we also determined the expression of NF-κB in the skin treated with BQ and LPA. The results of the present study could clarify the roles of BQ and LPA on carcinogenesis.

Section snippets

Materials and chemicals

Ethylenediaminetetraacetic acid (EDTA), sodium azide, phenol red, horseradish peroxidase, hexadecyltrimethyl ammonium bromide, 4-aminoantipyrine, dithiothreitol, Tris–HCl, sodium orthovanadate, sodium dodecyl sulfate (SDS), polyacrylamide, β-actin antibody were purchased from Sigma (St. Louis, MO). Nitrocellulose membrane was purchased from Millipore Corporation (USA). Enhanced chemiluminescence reagent (ECL) Kit, horseradish peroxidase (HRP)-labeled goat anti-mouse antibody, HRP-labeled goat

BQ and LPA induced mouse hyperplasia and inflammation

The effect of topical application of BQ- or LPA-induced alteration of cutaneous morphology were examined (Fig. 1 and Table 1). Topical application of BQ or LPA (50, 100 and 150 mg/ml) twice a day for 4 days to the dorsal surface of CD-1 mice resulted in increases of 1.5-–2.5-fold in the epidermal thickness. Inflammatory cell infiltration of the dermis was also observed. Topical application of BQ or LPA caused moderate or severe increase, respectively, in leukocyte infiltration and intercellular

Discussion

The components of Areca nut include phenolic compounds, which have been shown to posess mutagenicity or genotoxicity [5], [7], and to increase sister chromatid exchanges [9] in Chinese hamster ovary cells. At present, there is no direct evidence to relate BQ to oral cancer. Previous medical research reported that people over-chewing showed oral submucosa fibrosis (OSF) in oral cavity. Furthermore, people with a habit of chewing BQ only some show OSF at high levels with no further epidermal

Acknowledgments

The work was supported by the National Science Council Grant (NSC89-2320-B040-004) and the Chung Shan Medical and Dental College Research Fund (CSMC87-OM-B021).

References (38)

  • C.K. Wang et al.

    Study on the separation and hydrolysis of alkaloids and tumorigenicity of betel quid its ingredients

    J. Agric. Fd. Chem.

    (1996)
  • Y.C. Ko et al.

    Prevalence of betel quid chewing habit in Taiwan and related socio-demographic factors

    J. Oral Pathol. Med.

    (1992)
  • Y. Liu et al.

    6-Methylguanine-DNA methyltransferase activity in human buccal mucosal tissue and cell cultures. Complex mixtures related to habitual use of tobacco and betel quid inhibit the activity in vitro

    Carcinogenesis

    (1997)
  • S.F. Lee-Chen et al.

    Role of oxidative DNA damage in hydroxychavicol-induced genotoxicity

    Mutagenesis

    (1996)
  • D.S. Rupa et al.

    Chromosomal alterations affecting the 1cen-1q12 region in buccal mucosal cells of betel quid chewers detected using multicolor fluorescence in situ hybridization

    Carcinogenesis

    (1997)
  • B.J. Dave et al.

    In vitro genotoxic effects of areca nut extract and arecoline

    J. Cancer Res. Clin. Oncol.

    (1992)
  • S.V. Bhide et al.

    Carcinogenicity of betel quid ingredients: feeding mice with aqueous extract and the polyphenol fraction of betel nut

    Br. J. Cancer

    (1979)
  • K.J. Ranadive et al.

    Experimental studies on betel nut and tobacco carcinogenicity

    Int. J. Cancer

    (1976)
  • K.J. Ranadive et al.

    Betel quid chewing and oral cancer

    Int. J. Cancer

    (1979)
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