Fibrotic Effects of Arecoline N-Oxide ... - ACS Publications

Jun 10, 2015 - Department of Pathology, Changhua Christian Hospital, Changhua, ... of Biochemistry, College of Medicine, Kaohsiung Medical University,...
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Fibrotic Effects of Arecoline N‑Oxide in Oral Potentially Malignant Disorders Tzer-Min Kuo,†,Θ Shun-Yuan Luo,‡ Shang-Lun Chiang,†,§ Kun-Tu Yeh,Δ Hui-Ting Hsu,Δ Cheng-Tien Wu,⊥ Chi-Yu Lu,# Ming-Hsui Tsai,Π Jan-Gowth Chang,⊗,Θ and Ying-Chin Ko*,†,Θ †

Environment-Omics-Diseases Research Centre, China Medical University Hospital, Taichung, Taiwan Department of Chemistry, National Chung Hsing University, Taichung, Taiwan § Department of Health Risk Management, College of Management, China Medical University, Taichung, Taiwan Δ Department of Pathology, Changhua Christian Hospital, Changhua, Taiwan ⊥ Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan # Department of Biochemistry, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan Π Department of Otorhinolaryngology, China Medical University Hospital, Taichung, Taiwan ⊗ Department of Laboratory Medicine, China Medical University Hospital, Taichung, Taiwan Θ Graduate Institute of Clinical Medical Science, China Medical University, Taichung, Taiwan ‡

S Supporting Information *

ABSTRACT: The metabolites of environmental chemicals play key roles in carcinogenesis. Areca nut is strongly associated with the development of oral potentially malignant disorders (OPMD) or cancer. The main alkaloid in the areca nut is arecoline, which is highly cytotoxic and genotoxic. Arecoline N-oxide, a metabolite of areca nut alkaloids, which has been identified in animal urine, has been shown to induce mutagenicity in bacteria. In this study, it was found that its protein adduct could be detected in oral keratinocytes treated with areca nut extract. Increased collagen expression and severity of squamous hyperplasia were observed in arecoline N-oxide treated mice. In cultured oral fibroblasts, arecoline N-oxide showed stronger effects on the increase of fibrotic related genes including TGF-beta1, S100A4, MMP-9, IL-6, and f ibronectin and a decrease of E-cadherin as compared with arecoline. Finally, arecoline N-oxide stimulation effectively increased the DNA damage marker, gamma-H2A.X, both in vitro and in vivo. Taken together, these results indicate that arecoline N-oxide shows a high potential for the induction of OPMD. KEYWORDS: areca nut, arecoline, arecoline N-oxide, collagen, fibrosis

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INTRODUCTION Betel palm (Areca catechu), a tropical Asian tree, is widely cultivated for its edible seeds. Chewing betel quid is a prevalent habit, with an estimated 600 million users worldwide, particularly in Asian and migrant populations.1 Oral potentially malignant disorders (OPMD), including oral leukoplakia (OL) and oral submucous fibrosis (OSF), are oral precancerous conditions or lesions with a high incidence of malignant transformation.2 Betel chewing is highly associated with OPMD and oral cancer.3 The areca (betel) nut has been recognized as a group I carcinogen by the International Agency for Research on Cancer (IARC) of the World Health Organization.4 Previous studies identified that arecoline, arecaidine, guvacoline, and guvacine are the four major alkaloids among the chemical constituents of the areca nut.5 Arecoline, the most abundant and active alkaloid, is suggested to be a potential carcinogen.4 Besides its reported cytotoxic effects on cultured oral cells, studies with bacteria, mammalian cells, and experimental animals have revealed arecoline’s mutagenic and genotoxic effects.6 Both the areca nut and arecoline affect fibrotic contraction, collagen metabolism, and the TGF-beta pathway in cultured cells, which are suggested to cause OSF.7 However, © XXXX American Chemical Society

neither oral pathologic lesions nor tumor formation, induced by areca nut or arecoline alone, was reported.8 IARC also recognizes the carcinogenicity of arecoline as supported only by “limited evidence in experimental animals”.4 The specific constituents of the areca nut, which potentially cause oral tumorigenesis, remain a controversial issue. The bioactivation of chemical carcinogens through enzymatic transformation plays a critical role in carcinogenesis.9 Chemical carcinogens can activate multiple steps in carcinogenesis through their DNA and protein adducts or through the generation of reactive oxygen species. Arecoline N-oxide (or arecoline 1-oxide) is a metabolite of arecoline identified in animal urine, and its metabolism has been studied.10 Although the mutagenicity of arecoline N-oxide on prokaryotic Salmonella has been demonstrated by Lin et al., neither eukaryotic cells nor animal studies were reported.6b In the present study, we found that the protein adduct of arecoline NReceived: March 16, 2015 Revised: May 13, 2015 Accepted: May 28, 2015

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DOI: 10.1021/acs.jafc.5b01351 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Carlsbad, CA, USA) according to the manufacturer’s protocol. The cDNA templates were produced by reverse transcription of the total RNA with random primer and a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA). The products were then subjected to RT-qPCR analysis with the specific primer pairs using the ABI StepOnePlus Real-Time PCR Systems (Applied Biosystems) with Power SYBR Green PCR Master Mix (Invitrogen Corp.). The primer sequences are listed in Table S1. Western Blotting. To detect H2A.X phosphorylation, the cells were lysed in lysis buffer A (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1 mM EDTA, 10 mM magnesium acetate, and 1% NP-40) containing a cocktail of protease inhibitors (Roche, Germany) on ice for 10 min. The extracts were centrifuged at 15600g for 5 min at 4 °C to sediment the insoluble fraction. The pellet was resuspended in lysis buffer A and mixed with an equal volume of 4× Laemmli’s sample buffer. Sample loading of the insoluble fraction was normalized by protein concentration of the soluble fraction. Protein samples from both the soluble (for GAPDH) and insoluble (for H2A.X phosphorylation) fractions were separated by SDS−polyacrylamide gel and electrotransferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes were incubated with primary antibodies as indicated and peroxidase-conjugated secondary antibodies, and protein signals were detected by enhanced chemiluminescence reagent (Millipore). For detection of collagen 1, cell lysates were prepared in lysis buffer B (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1 mM EDTA, 10 mM magnesium acetate, 1% Triton X-100, and protease inhibitors cocktail). Monomeric collagen 1 was detected using a denaturing polyacrylamide gel electrophoresis (PAGE). To ensure equal loading, Western blotting was performed by using a denatured PAGE with double layers (6 and 9%) for separation. For detection of polymeric collagen, total protein (120 μg) was separated by nonreducing PAGE. The primary antibodies used were antiphospho-histone H2A.X (Ser139; Cell Signaling Technology), anticollagen 1 (St. John’s Laboratories, United Kingdom), and anti-GAPDH (GeneTex Inc., Taiwan). Immunohistochemistry Analysis. Tongue tissue samples from all 20 treated mice of 21 weeks were fixed with 4% paraformaldehyde for 3 days, embedded in paraffin. The 3−5 μm sections were stained with hematoxylin and eosin (H&E), specific antibodies, and Masson’s trichrome for histological evaluation and scoring. Immunohistochemical detection of mouse H2A.X phosphorylation (gamma-H2A.X) was performed using the antiphospho-histone H2A.X (Ser139; Cell Signaling Technology) antibody. The protein signal was detected using the appropriate primary antibody amplifier, horseradish peroxidase (HRP)-conjugated polymer, and DAB chromogen/ substrate. The positive signals of gamma-H2A.X were counted in a high-power field (200× magnification) in five mice from each group. The results were averaged, the numbers from 60 cells per f5 randomly selected sublingual positions each mouse. A trichrome stain for assessment of fibrosis was performed by using a Trichrome Modified Masson’s Stain Kit (Scytek Laboratories, Logan, UT, USA) according to the manufacturer’s instructions. Tongue fibrosis sections were counted and scored under 50× magnification of five separated fields of three mice per group. The quantification was used by the Fovea Pro 3.0 and OptiPix 2.0 free Wide Histogram Photoshop plug-in software. In this study, results of all immunohistochemical were interpreted by two pathologists.

oxide could be detected in areca nut extract (ANE)-treated oral cells. We therefore chemically synthesized arecoline N-oxide and investigated its effects on normal human cells and immunodeficient NOD/SCID mice. Our study unearthed clues about how areca nut induces OMPD. This study also increases our understanding of the mechanism by which betel chewing may lead to tumorigenesis.

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MATERIALS AND METHODS

Cell Culture and Treatment. Normal human gingival fibroblast cells (HGF-1, CRL-2014) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA)/Bioresource Collection and Research Center (BCRC, Taipei, Taiwan). Cells were grown and maintained in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum, at 37 °C in a 5% CO2 incubator. Normal human oral keratinocyte HOK cells purchased from ScienCell Research Laboratories (Carlsbad, CA, USA) were cultured in the oral keratinocyte growth medium (ScienCell Research Laboratories). For identifying the protein adducts of the areca nut in HOK cells, equal amounts of cells were treated with 200 μg/mL ANE or saliva-contained ANE for 24 h. For determination of DNA damage or fibrotic related genes, equal amounts of HGF-1 cells were seeded and grown until confluent for chemical treatment, avoiding strong cytotoxicity via arecoline or arecoline N-oxide. Cells were then treated with arecoline or arecoline N-oxide for 7 days. The culture medium was refreshed every 2 days during the experiment. Chemicals. ANE or saliva-contained ANE were prepared as previously described.11 Arecoline hydrobromide was purchased from Sigma-Aldrich (St. Louis, MO, USA). Arecoline N-oxide was synthesized as previously described.10a In brief, a solution of arecoline in ether was treated with aqueous 3 M peroxyacetic acid (1.95 g in 13 mL of H2O) dropwise during 30 min at 0 °C. After 2 h, the oily lower layer was separated and then precipitated three times by the addition of ether, which concentrated under reduced pressure to afford arecoline N-oxide as a pale yellow viscous oil. The synthesized chemical has been confirmed by high-resolution mass spectrometry and NMR analysis. The used arecoline N-oxide was dissolved in 0.1% DMSO/deionized water. Identification of Protein Adducts in HOK Cells. Protein adducts from ANE-exposed cells were identified using the LC-MS/MS system (LTQ Orbitrap Discovery hybrid Fourier transform mass spectrometer; Thermo Fisher Scientific Inc., Germany) as previously described.12 The raw data files from each treated cell were processed with Proteome Discoverer 1.1 software (Thermo Scientific) to gain the file of peak lists. Protein identification was analyzed by an in-house Mascot server. Cytotoxicity Detection. To measure the cytotoxicity, 104 HGF-1 cells were seeded into 96-well culture plates and cultured for 24 h for arecoline or arecoline N-oxide treatment. The released lactate dehydrogenase (LDH) in the culture medium was assessed using a LDH assay (CytoTox-ONE Homogeneous Membrane Integrity Assay, Promega, Madison, WI, USA). Animals and Experimental Protocol. In this study, the experimental animals used were 10-week-old NOD.CB17-Prkdcscid/ NcrCrl (NOD SCID Mouse) animals purchased from LASCO Charles River Technology (Taiwan) and then maintained in a specific pathogen-free environment. In the present study, animal procedures conformed to the guidelines published by the National Institutes of Health (NIH Publication No. 85-23) and were approved by the Institutional Animal Care and Use Committee (IACUC) of China Medical University (CMU). The mice (each weighing approximately 28 g) were randomly divided into two groups. Group 1 (the control group, n = 8) was treated with 0.1% DMSO/deionized water by cotton swab smearing the oral cavity once daily, 5 days per week. Group 2 (n = 12) was treated with 500 μg/mL arecoline N-oxide dissolved in 0.1% DMSO/deionized water in the same manner. During treatment, a standard laboratory diet and water were offered to all mice. Real-Time Quantitative PCR (RT-qPCR). Total RNA was isolated from treated cells using TRIzol reagent (Invitrogen Corp.,

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RESULTS Detection of Arecoline N-Oxide Protein Adduct in Cultured Oral Keratinocytes. Diseases caused by chemical exposure have been demonstrated through the metabolism of chemically bound products, such as DNA or protein adducts.9a To confirm the possibility of arecoline N-oxide presence in oral cells exposed to areca nuts, normal HOK cells were stimulated with ANE or saliva-contained ANE, and protein samples from the cell lysate were assayed to identify protein adducts (Table 1). The protein adduct of arecoline N-oxide-bound annexin A1 B

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observed in the upper surface of the tongue in both groups. These results indicate that arecoline N-oxide increased the severity of squamous hyperplasia in the sublingual tongue. Arecoline N-Oxide Increased Expression of Collagen 1. Our mouse experiment indicated that arecoline N-oxide induced oral collagen deposition, which implies that arecoline N-oxide has fibrotic potential.7a Next, we tested the fibrotic related effects of arecoline N-oxide in cultured HGF-1 cells. During betel chewing, the concentration of arecoline in saliva can reach as much as 97.39 μg/mL (about 600 μM).14 First, we compared the cytotoxicity between arecoline and arecoline Noxide by determining the LDH release in cells treated with various concentrations of arecoline or arecoline N-oxide. Indeed, cytotoxicity was induced in both treatment groups (Figure 2A). Arecoline N-oxide induced higher LDH release than arecoline, with a significant effect observed at a concentration of 400 μM (1.44-fold, p < 0.05, vs arecoline). Taken together, these results indicate that arecoline N-oxide induced stronger cytotoxicity than arecoline. To ascertain if arecoline N-oxide stimulation increases collagen 1 production, confluent HGF-1 cells were treated with arecoline N-oxide for 7 days and then analyzed for levels of collagen 1 mRNA (Figure 2B) and protein (Figure 2C,D). Arecoline N-oxide significantly increased the expression of collagen 1 mRNA and protein, and significant induction was observed at both 200 and 400 μM concentrations, respectively. These results indicate that arecoline N-oxide increased collagen 1 production in HGF-1 cells, which corresponded to the observations of our mouse study. Arecoline N-Oxide Increases the Expression of Oral Fibrotic Related Genes. TGF-beta 1 is a well-known fibrotic signaling molecule that is increased in OSF tissues.15 The effects of arecoline N-oxide on TGF-beta1 induction in HGF-1 cells were estimated by RT-qPCR. Both arecoline and arecoline N-oxide significantly increased the mRNA of TGF-beta1 (Figure 3 A). The effect of arecoline N-oxide was significantly higher than that of arecoline (at both 200 and 400 μM, p < 0.05). A series of oral precancerous related gene transcripts were also examined. These genes included S100A4,16 matrix metalloproteinase-9 (MMP-9),17 interleukin-6 (IL-6),18 f ibronectin,19 E-cadherin,20 and alpha-smooth muscle actin (alphaSMA)21 (Figure 3B−F and Figure S3). Arecoline N-oxide significantly stimulated increased expression of S100A4, MMP9, IL-6, f ibronectin, and alpha-SMA. It also reduced the level of E-cadherin mRNA. These results correlate with previous observations of genetic regulation in OSF. Arecoline N-oxide showed higher activity than arecoline in its effects on the expression of these genes expect alpha-SMA (at 400 μM, p < 0.05, vs arecoline). Thus, arecoline N-oxide may be a causative agent of OSF. Arecoline N-Oxide Increased H2A.X Phosphorylation. DNA damage is triggered early in tumorigenesis. To compare the effect on DNA damage, phosphorylation of histone H2A.X at serine (Ser) 139 (also called gamma-H2A.X), a marker of DNA double-strand breaks,22 was determined in confluent cells after 7 days of treatment. Whereas a slight effect of arecoline was observed, arecoline N-oxide significantly induced gammaH2A.X (Figure 4A,B). At arecoline N-oxide concentrations of 200 and 400 μM, levels of gamma-H2A.X increased 4.32- and 5.13-fold, respectively, as compared with the control. Arecoline N-oxide showed higher activity than arecoline at an equal concentration (at both 200 and 400 μM, p < 0.05). Similar results were shown when we measured 8-OHdG, another

Table 1. Identification of Areca Nut Related Protein Adduct in HOK Cells by LTQ Orbitrap treatment ANE

salivaANE

protein name (accession no.)

peptide sequencea

myosin gi|531138

arecaidine IVEANPLLEAFGNAK

S100a6 gi|20664042

arecaidine N-oxide A EPLDQAIGLLVAIFHK

beta-actin gi|62897625

arecaidine N-oxide LCYVALDFEQEMATAASSSSLEKb

myosin gi|531138

arecaidine IVEANPLLEAFGNAK

S100a6 gi|20664042

arecaidine N-oxide AEPLDQAIGLLVAIFHK

annexin A1 gi|4502101

arecoline N-oxide DLAKDITSDTSGDFR

a

Chemically bound amino acids are shown in bold and underlined. Both arecaidine N-oxide bound leucine 215 and cysteine 216 of betaactin were identified. b

was identified in the saliva-contained ANE-treated HOK cells. Products of arecaidine and arecaidine N-oxide bound proteins were also detected. These results indicated the presence of arecoline N-oxide in oral cells exposed to areca nuts. Arecoline N-Oxide Increased Oral Collagen Deposition in Mice. Protein or DNA targets of arecoline N-oxide remain uncharacterized. Because the effects of arecoline Noxide might result from multiple DNA or protein adducts in vivo, we investigated its effects by treating it directly. Commercial arecoline N-oxide is rarely obtained. Hence, we produced arecoline N-oxide chemically. We tested whether arecoline N-oxide can cause oral pathological changes in NOD/ SCID mice. The immunodeficiency NOD/SCID mice were used as a model to avoid antitumor immune response. The average body weight of the mice, in both arecoline Noxide and control groups, slightly increased during the 21 weeks of treatment. The average increases in body weight in the control and arecoline N-oxide-treated mice were 1.08 and 1.1%, respectively. No tumor formation was observed in the exterior alimentary canal. An increase in collagen synthesis is a main cause of OSF.13 To assess whether arecoline N-oxide enhanced collagen expression, tongue specimens were examined, and collagen deposition was found to be significantly higher in arecoline N-oxide-treated mice than in control mice with obvious observation in sublingual tongue (Figure 1A,B and Figure S1). Quantitative analysis of staining intensity and percentage indicated that arecoline N-oxide-treated mice showed a 1.97-fold increase in collagen deposition on the tongue. These results suggest that oral fibrotic potential is shown in arecoline N-oxide-treated mice. Pathologic changes in all mouse tongues were observed by H&E staining (Figure 1C,D). The arecoline N-oxide-treated group showed significant pathologic hyperplastic lesions in the epithelium of the sublingual tongue (Figure 1D). In contrast, control groups showed mild pathologic change of hyperplasia (Figure 1C), which might be due to long-term exposure to a low concentration of DMSO. No pathologic changes were C

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Figure 1. Arecoline N-oxide increased collagen deposition and severity of squamous hyperplasia in mouse tongue. Mice were consecutively treated with arecoline N-oxide (500 μg/mL) for 21 weeks. Tongues were stained with Masson’s trichrome staining in control (A) and arecoline N-oxide groups (B). Sublingual tongues are shown (magnification, 50×; scale bar, 50 μm). Pathological changes of sublingual tongue samples from the control (C) and arecoline N-oxide groups (D) were visualized by H&E staining. Indicated areas in left panels (black arrowheads, magnification, 50×) were observed under higher magnification (200×). Scale bar, 50 μm.

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DISCUSSION Arecoline N-oxide was first identified as a metabolite of arecoline in rat urine in 1971.10a A later study also identified it in mouse urine, which indicates that it is a major metabolite of arecoline.23 The metabolic interconversion between arecoline, arecaidine, arecoline N-oxide, and arecaidine N-oxide has been suggested.10b,23 Previous study showed that arecoline was converted to arecoline N-oxide by the human flavin-containing monooxygenases FMO1 and FMO3.10b The FMO1 and FMO3 are predominantly expressed in kidney or liver.24 In the present study, we identified arecoline N-oxide in ANE-treated human

marker for oxidative stress and DNA damage (Figure S2). Our data indicated that arecoline N-oxide induced gamma-H2A.X in HGF-1 cells. Therefore, immunostaining of tongue sections from each mouse group using the anti-gamma-H2A.X antibody was performed (Figure 4C,D). Significant expression of gamma-H2A.X was detected in the cells of the sublingual tongue in the arecoline N-oxide group (10.93-fold, p < 0.05, vs the control group), indicating that DNA damage occurred in this region. Taken together, these data indicate that arecoline N-oxide induced DNA damage response in vitro and in vivo. D

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Figure 2. Arecoline N-oxide increased the expressions of collagen 1. (A) Arecoline N-oxide induced cytotoxicity in HGF-1 cells. HGF-1 cells (104) in a 96-well plate were exposed to arecoline (abbreviated A) or arecoline N-oxide (abbreviated ANO) for 24 h. Cytotoxicity was measured by LDH assay. (B−D) Arecoline N-oxide increased the expression of collagen 1. Confluent cells were treated with arecoline N-oxide for 7 days. (B) Transcripts of collagen 1 were measured by qRT-PCR. (C) Protein expression of collagen 1 was confirmed by Western blotting of collagen 1 in native (for polymer, upper panel) and denatured (for monomer, lower panel) PAGE gels. (D) An increased effect on collagen 1 is plotted on a bar graph. Signals of collagen 1 were quantified by densitometry analysis. For panels A, B, and D, the graphs represent mean (±SD) values of three or more independent experiments. (∗) p < 0.05 compared with cells without arecoline or arecoline N-oxide stimulation; (#) p < 0.05, arecoline N-oxide group compared to arecoline; (§) p < 0.05, compared to the last dose of the same chemical.

Figure 3. Arecoline N-oxide increased the levels of oral fibrotic related gene transcripts. Confluent HGF-1 cells were treated with arecoline (abbreviated A) or arecoline N-oxide (abbreviated ANO) for 7 days. Transcripts of TGF-beta1 (A), S100A4 (B), MMP-9 (C), IL-6 (D), fibronectin (E), and E-cadherin (F) were measured by qRT-PCR. The graphs represent mean (±SD) values, and each experiment was performed more than three times in triplicate. (∗) p < 0.05 compared with control cells; (#) p < 0.05, arecoline N-oxide group versus arecoline; (§) p < 0.05, arecoline Noxide 400 μM versus arecoline N-oxide 200 μM.

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Figure 4. Arecoline N-oxide induced gamma-H2A.X in HGF-1 cells and mice. (A) Gamma-H2A.X (γ-H2AX) protein expression was detected by Western blotting in confluent cells treated with arecoline or arecoline N-oxide for 7 days. (B) Induction effect on gamma-H2A.X is plotted on a bar graph. Signals of gamma-H2A.X were quantified by densitometry analysis and expressed as average percentage of respective control cells. Results depict mean (±SD) of three independent experiments. (∗) p < 0.05 compared with cells without arecoline or arecoline N-oxide stimulation; (#) p < 0.05, arecoline N-oxide group versus arecoline. Arecoline N-oxide induced expression of gamma-H2A.X in mice. Immunohistochemical expression of gamma-H2A.X in sublingual tongue samples from the control (C) and arecoline N-oxide groups (D). Magnification, 200×.

Differences affecting the metabolism of areca nut, such as oral condition or genetic background, might lead to different outcomes in humans and other experimental animals during areca nut exposure. Areca nut might show a higher potential for oral tumorigenesis in humans than in other experimental animals. It has been reported that N-oxide derivatives of various chemicals show DNA binding activity or reaction with cellular ion containing enzymes. Formation of DNA and protein adducts is central to tumorigenesis via environmental chemicals, such as tobacco or alcohol.29 Recently, we established a screening platform for identifying protein adducts in samples exposed to arecoline and arecaidine.12 In this study, protein adducts of ANE in culture keratinocytes were identified (Table 1). Myosin and beta-actin are abundantly expressed in most types of cells, which might be easy binding targets for chemicals. In contrast, they are important cell regulators. It has been shown that some adducts to abundantly expressed proteins, such as antibodies or tubulins, are involved in the immune response or drug sensitivity.30 Most importantly, the N-oxide derivatives from the areca nut, arecoline N-oxide and arecaidine N-oxide, exist in areca nut exposed oral cells. The influence of those metabolites on diseases requires further investigation. Both the areca nut and arecoline have been reported to induce DNA damage. Both of these molecules have been suggested as a cause of oral cell carcinogenesis.6c In this study, we used gamma-H2A.X as a marker to determine genotoxicity via arecoline N-oxide (Figure 4). When DNA damage occurs,

oral cells. Our data indicated effective cytotoxicity, genotoxicity, and fibrotic gene regulation and increased pathological changes induced by arecoline N-oxide. Because arecoline N-oxide showed a potential influence on tumorigenesis, it is reasonable to suspect that the toxicity of the areca nut on oral tumorigenesis might be dependent on the cellular amount of its metabolites. Hence, cellular conditions affecting the metabolism of areca nut might play a key role in the occurrence of oral carcinogenesis via betel chewing. To test whether the constituents of the areca nut enhance oral tumorigenesis or cause precancerous lesions in vivo, numerous studies have treated experimental animals with areca nut or arecoline through diet, drinking water, or intraperitoneal injection. Mice treated with areca nut or arecoline showed higher gene mutation frequency and mutations in BRCA1.25 Lung adenocarcinoma, stomach squamous cell carcinoma, testicular lymphoma, and liver hemeangioma induced in areca nut-stimulated animals have also been studied.26 In one study focused on the induction of oral lesions or tumor formation, cotreatment with arecoline and 4-NQO of tobacco-related carcinogen induced tongue tumors in mice; however, arecoline alone did not induce any oral pathological lesions.27 In addition, 3-(methymitrosamino)propionitriIe (MNPN) and N-(nitrosomethylamino)propionitrile (NMAP), lime-enhancing synthesized derivatives from the areca nut in saliva, showed lower effects on tumor induction in the oral cavity of rats.28 Although a strong association between oral precancer or cancer and betel chewing has been demonstrated,3 the results from multiple animal studies have not proven that there is a link. F

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the histone H2AX is rapidly phosphorylated at Ser 139 (gamma-H2A.X). This process is involved in the DNA doublestrand break repair pathway.22b Oxidative stress via the areca nut and arecoline is the main pathway causing DNA damage.6c Therefore, N-oxide derivatives of the areca nut might induce strong oxidative stress. Gamma-H2A.X was reported as an independent prognostic marker in oral squamous cell carcinoma, which is associated with reduced overall survival time.22a Here, we suggest that it might be a marker of the incidence of malignant transformation in OPMD. To avoid strong side effects via other solvents, we used a lower concentration (0.1%) of DMSO to dissolve the arecoline Noxide. However, our results suggested that arecoline N-oxide increased the severity of hyperplasia (Figure 1), which is based on the observation of mild hyperplasia in the 0.1% DMSO treated group (the control group). DMSO induced the proliferation of human gingival fibroblasts31 and sebaceous hyperplasia.32 Thus, the trace of hyperplasia in the control group is most likely due to DMSO. OSF is a precancerous condition with high collagen expression in oral carcinogenesis. It is a chronically insidious oral mucosal condition, with submucosal fibrosis affecting the oral cavity, pharynx, and esophagus.13 TGF-beta is well-known as a potent stimulator of the production and deposition of collagen.33 The areca nut increases collagen accumulation and activates the TGF-beta signaling pathway. Our data also showed an increase in collagen and TGF-beta production by arecoline N-oxide, which suggests that arecoline N-oxide may be an inducer of OSF. Interestingly, most of the effects of arecoline N-oxide were observed in the regions located in the sublingual space below the tongue, possibly due to longer chemical exposure time in that area than in other areas of mouth. In addition, these chemicals might also react with saliva for longer periods of time, which implies that there are more toxic derivatives being produced. The presence of arecoline N-oxide in saliva during betel chewing has not been studied to our knowledge. Arecoline concentration in the saliva can reach around 600 μM.14 Although the cellular concentration of arecoline N-oxide is unclear, our results indicate that fibrotic gene regulation was induced by 200 μM arecoline N-oxide (Figure 3). In addition to arecoline, other alkaloids of the areca nut might also metabolically convert to N-oxide products, which can also induce oral fibrosis or tumorigenesis. The inhibitory effects on mutagenicity of arecoline N-oxide by sulfhydryl compounds and titanium trichloride in bacteria were cited by Lin et al.6b Therefore, uptake of inhibitors against those chemicals during betel chewing might be a way to prevent related oral diseases. Although oral tumors were not induced by arecoline N-oxide in our study, this compound may still enhance tumorigenicity. Exposure to arecoline N-oxide in an increasing frequency for longer periods might be required for tumor formation. Because the immunodeficient mice are prone to spontaneous cancer and other diseases, further study using wild-type mice is required. Further investigation is also necessary to reveal arecoline Noxide-targeted DNA or protein adducts that lead to tumorigenicity. In conclusion, our study showed the oral fibrotic potential of arecoline N-oxide in vitro and in vivo. Our findings provide new insights into the mechanism of carcinogenesis of the areca nut.

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ASSOCIATED CONTENT

S Supporting Information *

Table S1 and Figures S1−S3. Experimental procedures for determination of 8-OHdG. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b01351.

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AUTHOR INFORMATION

Corresponding Author

*(Y.-C.K.) E-mail: [email protected], [email protected]. tw. Phone: 886-4-2205-2121, ext. 7621. Mail: China Medical University Hospital, 2 Yude Road, Taichung, Taiwan 40447, Taiwan. Funding

This study was supported by the Health and Welfare surcharge of tobacco products, the China Medical University Hospital Cancer Research Center of Excellence (MOHW104-TDU-B212-124-002), the National Science Council (NSC 101-2314B-037-051-MY3, MOST 103-2314-B-039-002), and research projects (DMR-103-113, DMR-103-114, DMR103-120), China Medical University Hospital, Taiwan. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED ANE, areca nut extract; ANO, arecoline N-oxide; HGF-1, human gingival fibroblast; HOK, human oral keratinocyte; IL-6, interleukin-6; OPMD, oral potentially malignant disorders; OSF, oral submucous fibrosis; TGF-beta1, transforming growth factor-beta 1

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REFERENCES

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DOI: 10.1021/acs.jafc.5b01351 J. Agric. Food Chem. XXXX, XXX, XXX−XXX