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Effect of areca nut on rabbit oral mucosa: evidence of oral precancerous condition by protein expression and genotoxic analysis
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Original Article
Effect of areca nut on rabbit oral mucosa: evidence of oral precancerous condition by protein expression and genotoxic analysis Tapasi Das a,∗ , Basudev Mahato b,c , Keya Chaudhuri a a
Molecular Genetics Division, CSIR- Indian Institute of Chemical Biology, Kolkata 700 032, India Department of Oral Pathology, Dr. R Ahmed Dental College & Hospital, Kolkata 700014, India c Multidisciplinary Organization of Technical and Education and Research, Kolkata 700047, India b
a r t i c l e
i n f o
Article history: Received 21 December 2016 Received in revised form 27 April 2017 Accepted 16 May 2017 Available online xxx Keywords: Areca nut collagen rabbit model oral submucous fibrosis MALDI-TOF
a b s t r a c t Background: In humans, areca nut induces oral submucous fibrosis (OSMF), a potentially malignant disorder, characterised by the deposition of collagen in the lamina propria. This study aimed to determine whether OSMF-like characteristics develop in a rabbit model following treatment with areca nut and lime. Results: The oral epithelial tissue upon treatment with areca nut extract at 6-day intervals for up to 6 months showed progressive changes in thickness from 3 months onwards, leading to blanching, ulceration, irregular growth and, finally, restricted mouth opening. The protein expression pattern of OSMF-like tissues of rabbit buccal mucosa was determined by 2-DE gel and MALDI-TOF and compared with normal buccal mucosa of the control group of rabbits. Three major proteins, namely tropomyosin beta chain (in the skeletal muscle), actin and collagen alpha-1(I) chain, have been identified in areca nut-treated rabbit tissues as compared to control. The genotoxic effect of areca nut was evaluated in the rabbit model by comet assay in the blood. A significant (p < 0.0001) DNA damage in areca nut-treated rabbits was observed as compared to the control group. Conclusion: Histological characteristics, comet assay and protein profile show the development of OSMFlike features in the mucosal tissue of rabbit followed by areca nut treatment. The increased expression of collagen alpha-1(I) chain in areca nut-treated rabbit correlated with the progressive development of the OSMF symptoms in the rabbit buccal mucosa, which might serve as a potential biological marker in the pathological development of OSMF. © 2017 Published by Elsevier Ltd on behalf of Japanese Stomatological Society.
1. Introduction Oral submucous fibrosis (OSMF), regarded as a pre-cancerous condition of the oral cavity, is a well-known chronic, progressive, virtually irreversible, and juxtaepithelial inflammatory disease of the oral mucosa that is more common in the Indian subcontinent and Southeast Asia, with sporadic occurrences in other parts of the world as well. The development and progression of OSMF are
Abbreviations: 2-DE, Two-dimension electrophoresis; MALDI-TOF, Matrixassisted laser desorption ionization-time of flight; MS/MS, Tandem mass spectrometry; IPGs, Immobilised pH gradient strips. ∗ Corresponding author at: Molecular Genetics Division, Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Kolkata 700 032, India. Tel: +91 33 2473 3491, Fax: +91 33 2473 5197. E-mail addresses: tapasi [email protected], [email protected] (T. Das), [email protected] (B. Mahato), [email protected] (K. Chaudhuri).
closely associated with adverse habits such as chewing areca nut and its commercially popular forms such as pan masala and gutkha. The strongest risk factor for OSMF is the chewing of betel quid containing areca nut. The amount of areca nut in betel quid and the frequency and duration of chewing betel quid are clearly related to the development of OSMF [1]. The common signs and symptoms of OSMF are burning sensation, dry mouth, blanching of the oral mucosa and ulceration. Blanching of the oral mucosa is caused by the impairment of local vascularity and increased fibrosis, and a lesion appears. In the advanced stage of the disease, the important characteristic is a fibrous band that restricts mouth opening and causes difficulty in mastication, speech, swallowing and maintaining oral hygiene. Development of fibrous bands in the lip and cheeks makes them thick, rubbery and rigid and makes the mucosa difficult to retract. In the more advanced stages of the disease, OSMF is characterised by the formation of thick bands of collagen and hyalinisation extending into the submucosal tissues and decreased vascularity.
http://dx.doi.org/10.1016/S1348-8643(17)30021-6 1348-8643/© 2017 Published by Elsevier Ltd on behalf of Japanese Stomatological Society.
Please cite this article in press as: Das T, et al. Effect of areca nut on rabbit oral mucosa: evidence of oral precancerous condition by protein expression and genotoxic analysis. Oral Sci Int (2017), http://dx.doi.org/10.1016/S1348-8643(17)30021-6
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Pathogenesis is believed to involve juxtaepithelial inflammatory reaction and fibrosis in the oral mucosa due to increased crosslinking of collagen through the up-regulation of lysyl oxidase activity [2]. OSMF is strongly associated with a risk of oral cancer, although the biology underlying this association is unresolved [3]. There are a few previous reports on the development of an animal model for OSMF. The oral epithelium of female BALB/c mice treated with areca nut for 300–600 days showed increased cellularity of fibroblasts and fibrosis of the connective tissue [4]. So far, the animal models included mice and rats, where the monitoring of clinical changes was difficult because of the small size of the oral cavity. This prompted us to assess rabbit as a model to have a better hold on the monitoring of the disease. The availability of a suitable animal model will enable a better understanding of the pathogenesis of OSMF. Considering this, the objective of the study was to (1) determine whether the features of OSMF appear in the rabbit model after treatment with areca nut and, if so, further confirm the disease genotoxically (2) identify differentially expressed proteins in the buccal mucosa of rabbits with OSMF features compared to the control group by two-dimension (2-DE) gel electrophoresis followed by in-gel digestion and MALDI-TOF MS. This is the first study to use a proteomic approach in oral OSMF tissues in an animal model. 2. Materials and methods 2.1. Animals and reagents For the development of OSMF in animal model, adult New Zealand white rabbits weighing between 1 and 1.5 kg each were segregated into three groups, namely control (negative), positive control (phenol), and areca nut-treated animals, each group containing three animals. For preparing the areca nut extract, 25 g dried areca nut was dissolved in deionised water, mixed using a cyclomixer and air dried. From this, 2 g power was mixed with slaked slime and 5 ml water and filtered, and the filtrate was used for the treatment of rabbits. Rabbits from each group received 1 ml submucous injection of the specified agents at 6 days interval: first group received 1X PBS (control), second group received phenol solution (4%) and third group received the filtered areca extract with lime. The doses were continued for the next 6 months. The oral mucosa was checked regularly. The study was duly approved by CPCSEA (New Delhi) and Institutional Animal Ethics Committee (Registration number 147/1999/CPCSEA). Housing conditions of the animals were maintained under standard conditions of 12 h/day/night cycle with water and food. The temperature of the animal houses was 22–23◦ C and humidity was 50–70%. All animals were deeply anaesthesitised by peptobartitine sodium solution before biopsy. 2.2. Histological staining of OSMF The biopsy specimens of buccal mucosa from each group were obtained by punch biopsy and fixed in a buffered formalin solution at neutral pH. The tissue sections were paraffinised, sectioned, and stained with haematoxylin and eosin. 2.3. Protein extraction by tissue lyser and fractionation of total proteins The biopsy samples from each rabbit were retained in 20 mM Tris–buffer (pH 7.5), washed with 5 mM MgCl2 , and homogenised using a tissue lyser (Qiagen, Hilden, Germany), and a protease inhibitor cocktail (Sigma, USA) was added. The homogenates were cleared to remove the cell debris by centrifugation at 14,000 rpm for 20 min at 4◦ C, and the supernatant was collected. The protein concentration of the resulting supernatant was determined using
the Bradford method. The supernatant was treated with DNase/N nuclease at 37◦ C for 30 min and again centrifuged. The precipitate was kept in Trichloroacetic acid: Acetone (1:9) at −20◦ C overnight and then washed with 80% cold acetone. The precipitate was air dried and solubilised in rehydration buffer [5 M urea, 2 M thiourea, 2% CHAPS, 40 mM Tris, 2% SB3-10, 0.2% Bio-lyte (working pH range 3–10), 10 l tributyl phosphine]. 2.4. 2-DE PAGE The first-dimension isoelectric focusing and second-dimension SDS-PAGE were performed according to the BIO-RAD manual provided with BIO-RAD 2-DE electrophoresis system. The protein (approximately, 200 g in 125 l) was adsorbed on to 7 cm IPG strips, pH 3–10, for 16 h and then isofocussed on an isoelectric focusing cell (PROTEAN IEF CELL, Bio-Rad) for 8000 Vh at 20 ◦ C. Strips were rehydrated for 12–16 h by passive rehydration in the presence of samples. The total volume for the rehydration was always 125 l for the total protein sample and was prepared in lysis buffer (BIO-RAD). Then reduction and alkylation of the focussed proteins was performed by equilibrating these strips in equilibration buffers I and II containing dithiothreitol (DTT) and iodoacetamide (IAA), respectively [375 mM Tris/HCl, pH 8.8, containing 6 M urea, 2% (w/v) SDS, 20% (v/v) glycerol containing either 2% (w/v) DTT or 2.5% (w/v) IAA], processed according to Bio-Rad manual. The equilibrated strips were placed on 12% SDS-PAGE separated in BIO-RAD gel apparatus stem (PROREAN II XI CELL). 2.5. In-gel digestion and identification of proteins by peptide mass fingerprinting The gels were stained using colloidal Coomassie blue (R-250), and desired proteins were excised manually from the stained gel. The excised gel pieces were digested using a gel digestion kit (PIERCE) according to the standard manufacturer’s protocol. The excised gel spots were destained by a destaining solution (100 mmol/L NH4 HCO3 in 50% acetonitrile) and reduced by a reduction buffer (25 mmol/L NH4 HCO3 and 10 mmol/L DTT) for 10 min at 60 ◦ C. Alkynation was done by an alkylation buffer (25 mmol/L NH4 HCO3 and 55 mmol/L iodoacetamide) in the dark for 1 h at room temperature at until the gel was colourless. After drying, the gel was treated with trypsin solution (100 g/mL) for overnight at 30 ◦ C. After digestion, these tryptic digestive peptides were centrifuged, and the supernatant was dried on a Speedvac concentrator and desalted using ZIPTIP (C18 column-Millipore, USA). The elution of peptides was performed in 50% acetonitrile containing 0.1% trifluroacetic acid (TFA). 2.6. Identification of proteins by (peptide mass fingerprinting) MALDI-TOF-TOF mass spectrometry The eluted peptides (0.5 l) from the gel were mixed with ␣-cyano-4-hydroxycinnamic acid (Sigma) in 0.1% TFA/50% acetonitrile as the matrix (1:1), spotted on MALDI plate by using the matrix–sample–matrix sandwich method at room temperature and analysed on a 4800 MALDI-TOF-TOF mass spectrometer (Applied Biosystems, USA). All the data were analysed using the GPS explore software. To identify the proteins, a search was performed of the combined MS and MS/MS by NCBI and SWISS-PROT using the MASCOT software v2.1. 2.7. Comet assay From each group of rabbits, blood was collected from the ear vein and suspended in PBS. The blood cells were embedded in thin
Please cite this article in press as: Das T, et al. Effect of areca nut on rabbit oral mucosa: evidence of oral precancerous condition by protein expression and genotoxic analysis. Oral Sci Int (2017), http://dx.doi.org/10.1016/S1348-8643(17)30021-6
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Fig. 1. A) Development of OSMF in the animal model (Rabbit). I) PBS-treated control rabbit. II) Ulceration involving the jaw angle of areca nut-treated rabbit. III) Epithelial crust in phenol-treated rabbit. B) Tissues collected from three differently treated animals. C) H/E staining of the normal PBS-treated (I), areca nut-treated (II) and phenol-treated (III) rabbits’ oral tissues. Representative H/E staining of areca nut-treated tissues showing the OSMF characteristics (IV, V and VI) such as hyalinisation of collagen and atropic stratified squamous epithelium of variable thicknesses, with superficially thick orthokeratinisation (IV), reduced thickness of oral epithelium (V), and reduced vascularity and hyperchromatic nuclei (VI).
agarose gel (1% low melting) on a slide, and then these blood cells were lysed by lysis buffer (2.5 M NaCl, 0.1 M EDTA, 10 mM Tris base, 1% triton, and 10% DMSO) and incubated overnight. Electrophoresis was performed for 20 min in an alkaline solution (300 mM NaOH, 0.1 mM EDTA, PH > 13 at 280 mA and 24 V). The damaged DNA migrated away from the nucleus; the slides were then neutralised by Tris buffer pH 7.5 and stained with ETBR. The comet of DNA was visualised by fluorescent microscope (Leica DM 3000) and measured by the comet Score software. 2.8. Immunohistochemistry Immunohistochemistry was performed using the Novolink polymer detection system (Novolink castra, UK). Briefly, biopsy tissues sections from buccal mucosa of different rabbits were washed with cold PBS and then fixed in formalin. Tissues were then embedded in paraffin at 56–60◦ C. Tissue sections of 4–5 mm thickness were embedded in poly-L-lysine-coated slides. Paraffin-embedded tissues were de-waxed by xylene and rehydrated through graded alcohol; the slides were treated with citrate buffer for unmasking the protein. Further immunostaining was performed using the Novolink polymer detection system and endogenous peroxidase, and the protein was blocked using the supplied blockers. Collagen I was detected using monoclonal anti-collagen I antibody (1:50, ab90395, Abcam, Cambridge, USA). After post-primary blocking, the sections were incubated with the Novolink polymer and were then developed with 3,3 -diaminobenzidine. The sections were then counterstained with haematoxylin and were observed under LEICA DM3000 microscope (Leica Microsystems, Switzerland). 3. Results The three groups of animals were injected with PBS, phenol, and areca nut into their oral epithelial tissue for 3 months at 6day intervals. After 3 months, strong fibrosis was developed in the oral mucosal layer with phenol and areca nut treatments. OSMF developed in areca nut-treated rabbits and oral cancer developed
in phenol-treated rabbits were confirmed by histological section (Fig. 1A–C). Areca nut-treated rabbits showed significant reduction in the mouth opening and oral mucosal blanching, suggesting OSMF-like characteristic development. From the above results, it could easily be inferred that the areca nut is fibrosis promoting and phenol is carcinogenic. The oral lesion was further characterised by staining with haematoxylin and eosin (H/E stating). Epithelial tissue sections from areca nut-treated rabbits have shown thinning of the epithelial layer compared to PBS-treated rabbit along with the fibrous bands (Fig. 1C). H/E-stained histological sections revealed atropic stratified squamous epithelium of variable thickness, with superficially thick orthokeratinisation (Fig. 1C-IV-V). Epithelium was hyperchromatic, showing individual cell keratinisation. Atypia characters such as atrophy, cellular pleomorphism and hyperchromatic nuclei are shown in Fig. (1C-IV). Reduced vascularity was found in the submucosal tissues (Fig. 1C-VI). The connective tissue was fibrocollagenous with minimum infiltration of chronic inflammatory cells and few capillaries present in the juxtaepithelial region. Hyalinisation and homogenisation of collagen fibres were also noted in the juxtaepithelial connective tissues (Fig. 1C-IV). Degeneration or loss of epithelial layer was observed in phenoltreated rabbits, which is the major characteristic of oral cancer. To monitor the DNA damage, blood samples were collected from the ear vein and analysed using comet assay (Fig. 2). Phenol-treated rabbits showed significantly (p < 0.0001) higher DNA damage than PBS-treated rabbit. Areca nut-treated rabbits showed moderate but significantly (<0.0001) higher DNA damage than PBS-treated rabbit (Fig. 2). Oral lesion biopsy tissue samples were collected from each group (Fig. 1B). Protein profiling was performed by SDS, 2-DE, and MALDI-TOF analysis. The overexpressed proteins in the control and OSMF tissues were compared in 12% 2-DE-PAGE. One part of the proteins was resolved in SDS-PAGE, and another part of the protein was solubilised in 2-DE rehydration buffer, consisting of urea, thiourea, CHAPS, 40 mM Tris, and ampholytes, pH 3–10. The proteins were loaded in gel rehydration for pH 3–10 IPG strips and 2-DE electrophoresis was conducted using Protein IIXi system (Biorad). Fig. 3 shows that approximately 100
Please cite this article in press as: Das T, et al. Effect of areca nut on rabbit oral mucosa: evidence of oral precancerous condition by protein expression and genotoxic analysis. Oral Sci Int (2017), http://dx.doi.org/10.1016/S1348-8643(17)30021-6
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Fig. 2. Comet assay on the blood of rabbit treated with PBS (A), areca nut (B) and phenol (C) and (D) graphical presentation of DNA damage. *** represents statistical significance at level p value <0.0001.
Fig. 3. Representative 2-DE PAGE gel images of proteins in normal and areca nut-treated rabbit tissues. The normal buccal mucosa (A) and oral submucous fibrosis-featuring rabbit buccal mucosa. (B) Proteins were separated by 2-DE PAGE using IPG strips (pH 3–10) in the first dimension and 12% SDS-PAGE in the second dimension. Table 1 Proteins showing increased expression in areca nut-treated rabbit tissue. Protein Accession Number
Protein Name
Molecular Weight and pI
Peptide matched
Sequence coverage
A24904 OCU18344 NID: – Oryctolagus cuniculus TMRBB A23022 I56246 Q9R168 AAB88871 S42634 CO1A1
actin alpha, skeletal muscle Oryctolagus cuniculus serum albumin precursor, mRNA, tropomyosin beta chain skeletal muscle [validated] – rabbit actin, cardiac muscle Lipopolysaccharide-binding protein Secreted frizzled-related protein sFRP-1 (Fragment)) AF036941 NID: – Rattus norvegicus homeoprotein collagen alpha-1(I) chain
42366; 5.23 70861; 5.85 32931; 4.66 42334; 5.23 53737; 8.99 18517; 9.22 14937; 6.37 31046; 7.10 45134; 5.23
14 12 11 15 18 23 14 14 12
36 48 39 24 16 29 49 19 24
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Fig. 4. Immunohistochemical staining of collagen I in buccal mucosa in (A) PBS-treated, (B) areca nut-treated and (C) phenol-treated rabbits. Buccal tissues were stained with anti-collagen antibodies and observed under microscope (Leica). Magnification 20X.
proteins appeared on the 2-DE gel. The concentrations of the total extracted protein in the control and OSMF tissues were almost similar. The protein spots excised from SDS-PAGE (data not shown) and 2-DE gel were then analysed by MALDI-TOF spectrometry. To identify the proteins, database search was performed by SWISS-PROT. Table 1 summarises matched overexpressed proteins (score > 60) with high sequence coverage, accession number, theoretical molecular weight (kDa)/PI, and protein scores. The identified proteins included tropomyosin beta, actin alpha, Oryctolagus cuniculus serum albumin precursor and collagen alpha-1(I). The MALDI-TOF peptide mass fingerprint of the tryptic digest of Collagen I and the matched peptide sequences are shown in SupplyFig1. For further confirmation, immunohistochemical staining of collagen I of buccal mucosa in (A) PBS-treated, (B) areca nut-treated and (C) phenol-treated rabbits was performed (Fig. 4). A diffused and weak localisation of collagen I in the squamous epithelium was observed. Collagen I was faintly immunostained in the lamina propria and interstium of any layers in control rabbits. A strong staining intensity of collagen I was observed in the lamina propria in areca nut-treated rabbits, while the staining was localised in phenol-treated rabbits. The increased collagen type I expression and its immunolocalisation in the lamina propria and comet results suggest that OSMF-like features are present in areca nut-treated rabbits. 4. Discussion Epidemiological studies have established a strong relationship between areca nut use and the development of OSMF in humans [5,6]. In the present study, rabbits were treated with areca nut extract, which culminated in OSMF-like features from 3 months onwards. The clinical changes in the rabbit oral mucosa were monitored by an expert clinician in this field when blanching of the oral mucosa along with ulceration and reduction in mouth opening could be observed. The histological evaluation showed a significant amount of fibrosis, thinning of epithelial layer and loss of rete ridges (Fig. 1-C). The degeneration or loss of epithelial layer was observed in phenol-treated rabbit, which is the major characteristic of oral cancer, which was further confirmed by comet assay where significant DNA damage was observed in phenol-treated rabbits (Fig. 2). Reports suggest that approximately 7–14% of OSMF cases turn to malignancy, and our previous studies have shown significantly higher mean comet tail length in OSMF than in control rabbits, but it was lower than OSCC cases [7]. Similar results are observed in the present study, and areca nut-treated rabbits showed significantly higher tail length than the control group but lower tail length than the phenol-treated group. These findings corroborated with the characteristic histological features of OSMF. Because OSMF is multifactorial in nature, the proteomic study is an important step in identifying the possible biomarkers of the disease and understanding the pathogenesis of OSMF. In the present study, we have identified a few proteins from MALDI-TOF analysis, which are shown in Table 1. Interestingly, increased expression
of the collagen alpha-1(I) chain in areca nut-treated rabbit tissue compared to control strongly suggested that the methodology we have used for the development of the fibrosis model in the rabbit was more precise and symptomatically correct as the accumulation of collagen type-I due to chewing areca nut is the hallmark of oral fibrosis. Moreover, overexpression of cytoskeleton proteins such as actin, tropomyosin and tubulin was also observed. These proteins have essential functional role and are critically involved in cell morphology. Actin has different isoforms; actin as a globular monomer is called G actin and as a filamentous polymer, it is called F actin. Actin is essential for the regulation of premalignant condition. The overexpression of this protein to counteract the collagen accumulation mediated reduction in the contractibility of the oral muscle. Tropomyosin plays an important role in the formation of cellular cytoskeleton. During bladder carcinogenesis, tropomysin levels decrease in T24 human cancer cells [8]. Tropomyosin 1 disappears in primary human breast tumours. This suggested that the overexpression of tropomyosin 1 in the rabbit model might impart protection against areca insult, which may further leads to oral cancer. For epithelial tissue differentiation, keratin plays an important role and acts as a biological marker of malignant formation [9]. In the present study, we observed that along with collagen expression, tropomyosin 1 may emerge as another marker for the OSMF model. To validate the collagen I expression from the proteomics study, areca nut-treated rabbit tissue was subjected to immunohistochemical analysis with collagen I antibody. Strongly positive collagen expression was observed throughout the thickness of the stratified squamous epithelium in areca nut-treated rabbits compared to controls, suggesting OSMF-like features. 5. Conclusions In the present study, three major proteins were identified in the progressive development of OSMF in the rabbit, namely tropomyosin beta chain (in the skeletal muscle), actin and collagen alpha-1(I) chain, which may serve as the biological marker in the pathological development of OSMF. Genotoxic effects of areca nut and significant (p < 0.0001) DNA damage were observed in the rabbit model as compared to the control group. The increased expression of the collagen alpha-1(I) chain in areca nut-treated rabbit tissue strongly suggested that the methodology we have used for the development of the fibrosis model in the rabbit is convenient. Moreover, it is the first study to use a proteomic approach to use oral OSMF tissues in an animal model. Further characterisation and functional analysis are required to gain more insight to understand the role of these proteins in the disease progression. Ethics approval and consent to participate The study was duly approved by CPCSEA (New Delhi) and Institutional Animal Ethics Committee (IAEC) (Registration number 147/1999/CPCSEA).
Please cite this article in press as: Das T, et al. Effect of areca nut on rabbit oral mucosa: evidence of oral precancerous condition by protein expression and genotoxic analysis. Oral Sci Int (2017), http://dx.doi.org/10.1016/S1348-8643(17)30021-6
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Funding
References
The study was supported by the Department of Science and Technology, Government of India and Council of Scientific and Industrial Research (CSIR), Govt. of India. Tapasi Das is the recipient of the DST (WOS-A).
[1] Rajalalitha P, Vali S. Molecular pathogenesis of oral submucous fibrosis–a collagen metabolic disorder. J Oral Pathol Med 2005;34:321–8. [2] Trivedy CR, Warnakulasuriya KA, Peters TJ, et al. Raised tissue copper levels in oral submucous fibrosis. J Oral Pathol Med 2000;29:241–8. [3] Mithani SK, Mydlarz WK, Grumbine FL, et al. Molecular genetics of premalignant oral lesions. Oral Dis 2007;13:126–33. [4] Sumeth Perera MW, Gunasinghe D, Perera PA, et al. Development of an in vivo mouse model to study oral submucous fibrosis. J Oral Pathol Med 2007;36:273–80. [5] Maher R, Lee AJ, Warnakulasuriya KA, et al. Role of areca nut in the causation of oral submucous fibrosis: a case-control study in Pakistan. J Oral Pathol Med 1994;23:65–9. [6] Gupta PC, Sinor PN, Bhonsle RB, et al. Oral submucous fibrosis in India: a new epidemic? Natl Med J India 1998;11:113–6. [7] Katarkar A, Mukherjee S, Khan MH, et al. Comparative evaluation of genotoxicity by micronucleus assay in the buccal mucosa over comet assay in peripheral blood in oral precancer and cancer patients. Mutagenesis 2014;29:325–34. [8] Pawlak G, McGarvey TW, Nguyen TB, et al. Alterations in tropomyosin isoform expression in human transitional cell carcinoma of the urinary bladder. Int J Cancer 2004;110:368–73. [9] Rao RS, Patil S, Ganavi BS. Oral cytokeratins in health and disease. J Contemp Dent Pract 2014;15:127–36.
Acknowledgements We are thankful to Dr Krishna Das Saha and Dr Rukhsana Chowdhury for helping with the instrument facility. We are thankful to Dr Sanjit Mukherjee and Dr Atul katarkar for helping with animal handling. We are also thankful to Dr Sandeep Chakroborty for MALDI-TOF program operation. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/S13488643(17)30021-6.
Please cite this article in press as: Das T, et al. Effect of areca nut on rabbit oral mucosa: evidence of oral precancerous condition by protein expression and genotoxic analysis. Oral Sci Int (2017), http://dx.doi.org/10.1016/S1348-8643(17)30021-6
ARTICLE IN PRESS Oral Science International xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Oral Science International journal homepage: www.elsevier.com/locate/osi
Original Article
Effect of areca nut on rabbit oral mucosa: evidence of oral precancerous condition by protein expression and genotoxic analysis Tapasi Das a,∗ , Basudev Mahato b,c , Keya Chaudhuri a a
Molecular Genetics Division, CSIR- Indian Institute of Chemical Biology, Kolkata 700 032, India Department of Oral Pathology, Dr. R Ahmed Dental College & Hospital, Kolkata 700014, India c Multidisciplinary Organization of Technical and Education and Research, Kolkata 700047, India b
a r t i c l e
i n f o
Article history: Received 21 December 2016 Received in revised form 27 April 2017 Accepted 16 May 2017 Available online xxx Keywords: Areca nut collagen rabbit model oral submucous fibrosis MALDI-TOF
a b s t r a c t Background: In humans, areca nut induces oral submucous fibrosis (OSMF), a potentially malignant disorder, characterised by the deposition of collagen in the lamina propria. This study aimed to determine whether OSMF-like characteristics develop in a rabbit model following treatment with areca nut and lime. Results: The oral epithelial tissue upon treatment with areca nut extract at 6-day intervals for up to 6 months showed progressive changes in thickness from 3 months onwards, leading to blanching, ulceration, irregular growth and, finally, restricted mouth opening. The protein expression pattern of OSMF-like tissues of rabbit buccal mucosa was determined by 2-DE gel and MALDI-TOF and compared with normal buccal mucosa of the control group of rabbits. Three major proteins, namely tropomyosin beta chain (in the skeletal muscle), actin and collagen alpha-1(I) chain, have been identified in areca nut-treated rabbit tissues as compared to control. The genotoxic effect of areca nut was evaluated in the rabbit model by comet assay in the blood. A significant (p < 0.0001) DNA damage in areca nut-treated rabbits was observed as compared to the control group. Conclusion: Histological characteristics, comet assay and protein profile show the development of OSMFlike features in the mucosal tissue of rabbit followed by areca nut treatment. The increased expression of collagen alpha-1(I) chain in areca nut-treated rabbit correlated with the progressive development of the OSMF symptoms in the rabbit buccal mucosa, which might serve as a potential biological marker in the pathological development of OSMF. © 2017 Published by Elsevier Ltd on behalf of Japanese Stomatological Society.
1. Introduction Oral submucous fibrosis (OSMF), regarded as a pre-cancerous condition of the oral cavity, is a well-known chronic, progressive, virtually irreversible, and juxtaepithelial inflammatory disease of the oral mucosa that is more common in the Indian subcontinent and Southeast Asia, with sporadic occurrences in other parts of the world as well. The development and progression of OSMF are
Abbreviations: 2-DE, Two-dimension electrophoresis; MALDI-TOF, Matrixassisted laser desorption ionization-time of flight; MS/MS, Tandem mass spectrometry; IPGs, Immobilised pH gradient strips. ∗ Corresponding author at: Molecular Genetics Division, Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Kolkata 700 032, India. Tel: +91 33 2473 3491, Fax: +91 33 2473 5197. E-mail addresses: tapasi [email protected], [email protected] (T. Das), [email protected] (B. Mahato), [email protected] (K. Chaudhuri).
closely associated with adverse habits such as chewing areca nut and its commercially popular forms such as pan masala and gutkha. The strongest risk factor for OSMF is the chewing of betel quid containing areca nut. The amount of areca nut in betel quid and the frequency and duration of chewing betel quid are clearly related to the development of OSMF [1]. The common signs and symptoms of OSMF are burning sensation, dry mouth, blanching of the oral mucosa and ulceration. Blanching of the oral mucosa is caused by the impairment of local vascularity and increased fibrosis, and a lesion appears. In the advanced stage of the disease, the important characteristic is a fibrous band that restricts mouth opening and causes difficulty in mastication, speech, swallowing and maintaining oral hygiene. Development of fibrous bands in the lip and cheeks makes them thick, rubbery and rigid and makes the mucosa difficult to retract. In the more advanced stages of the disease, OSMF is characterised by the formation of thick bands of collagen and hyalinisation extending into the submucosal tissues and decreased vascularity.
http://dx.doi.org/10.1016/S1348-8643(17)30021-6 1348-8643/© 2017 Published by Elsevier Ltd on behalf of Japanese Stomatological Society.
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Pathogenesis is believed to involve juxtaepithelial inflammatory reaction and fibrosis in the oral mucosa due to increased crosslinking of collagen through the up-regulation of lysyl oxidase activity [2]. OSMF is strongly associated with a risk of oral cancer, although the biology underlying this association is unresolved [3]. There are a few previous reports on the development of an animal model for OSMF. The oral epithelium of female BALB/c mice treated with areca nut for 300–600 days showed increased cellularity of fibroblasts and fibrosis of the connective tissue [4]. So far, the animal models included mice and rats, where the monitoring of clinical changes was difficult because of the small size of the oral cavity. This prompted us to assess rabbit as a model to have a better hold on the monitoring of the disease. The availability of a suitable animal model will enable a better understanding of the pathogenesis of OSMF. Considering this, the objective of the study was to (1) determine whether the features of OSMF appear in the rabbit model after treatment with areca nut and, if so, further confirm the disease genotoxically (2) identify differentially expressed proteins in the buccal mucosa of rabbits with OSMF features compared to the control group by two-dimension (2-DE) gel electrophoresis followed by in-gel digestion and MALDI-TOF MS. This is the first study to use a proteomic approach in oral OSMF tissues in an animal model. 2. Materials and methods 2.1. Animals and reagents For the development of OSMF in animal model, adult New Zealand white rabbits weighing between 1 and 1.5 kg each were segregated into three groups, namely control (negative), positive control (phenol), and areca nut-treated animals, each group containing three animals. For preparing the areca nut extract, 25 g dried areca nut was dissolved in deionised water, mixed using a cyclomixer and air dried. From this, 2 g power was mixed with slaked slime and 5 ml water and filtered, and the filtrate was used for the treatment of rabbits. Rabbits from each group received 1 ml submucous injection of the specified agents at 6 days interval: first group received 1X PBS (control), second group received phenol solution (4%) and third group received the filtered areca extract with lime. The doses were continued for the next 6 months. The oral mucosa was checked regularly. The study was duly approved by CPCSEA (New Delhi) and Institutional Animal Ethics Committee (Registration number 147/1999/CPCSEA). Housing conditions of the animals were maintained under standard conditions of 12 h/day/night cycle with water and food. The temperature of the animal houses was 22–23◦ C and humidity was 50–70%. All animals were deeply anaesthesitised by peptobartitine sodium solution before biopsy. 2.2. Histological staining of OSMF The biopsy specimens of buccal mucosa from each group were obtained by punch biopsy and fixed in a buffered formalin solution at neutral pH. The tissue sections were paraffinised, sectioned, and stained with haematoxylin and eosin. 2.3. Protein extraction by tissue lyser and fractionation of total proteins The biopsy samples from each rabbit were retained in 20 mM Tris–buffer (pH 7.5), washed with 5 mM MgCl2 , and homogenised using a tissue lyser (Qiagen, Hilden, Germany), and a protease inhibitor cocktail (Sigma, USA) was added. The homogenates were cleared to remove the cell debris by centrifugation at 14,000 rpm for 20 min at 4◦ C, and the supernatant was collected. The protein concentration of the resulting supernatant was determined using
the Bradford method. The supernatant was treated with DNase/N nuclease at 37◦ C for 30 min and again centrifuged. The precipitate was kept in Trichloroacetic acid: Acetone (1:9) at −20◦ C overnight and then washed with 80% cold acetone. The precipitate was air dried and solubilised in rehydration buffer [5 M urea, 2 M thiourea, 2% CHAPS, 40 mM Tris, 2% SB3-10, 0.2% Bio-lyte (working pH range 3–10), 10 l tributyl phosphine]. 2.4. 2-DE PAGE The first-dimension isoelectric focusing and second-dimension SDS-PAGE were performed according to the BIO-RAD manual provided with BIO-RAD 2-DE electrophoresis system. The protein (approximately, 200 g in 125 l) was adsorbed on to 7 cm IPG strips, pH 3–10, for 16 h and then isofocussed on an isoelectric focusing cell (PROTEAN IEF CELL, Bio-Rad) for 8000 Vh at 20 ◦ C. Strips were rehydrated for 12–16 h by passive rehydration in the presence of samples. The total volume for the rehydration was always 125 l for the total protein sample and was prepared in lysis buffer (BIO-RAD). Then reduction and alkylation of the focussed proteins was performed by equilibrating these strips in equilibration buffers I and II containing dithiothreitol (DTT) and iodoacetamide (IAA), respectively [375 mM Tris/HCl, pH 8.8, containing 6 M urea, 2% (w/v) SDS, 20% (v/v) glycerol containing either 2% (w/v) DTT or 2.5% (w/v) IAA], processed according to Bio-Rad manual. The equilibrated strips were placed on 12% SDS-PAGE separated in BIO-RAD gel apparatus stem (PROREAN II XI CELL). 2.5. In-gel digestion and identification of proteins by peptide mass fingerprinting The gels were stained using colloidal Coomassie blue (R-250), and desired proteins were excised manually from the stained gel. The excised gel pieces were digested using a gel digestion kit (PIERCE) according to the standard manufacturer’s protocol. The excised gel spots were destained by a destaining solution (100 mmol/L NH4 HCO3 in 50% acetonitrile) and reduced by a reduction buffer (25 mmol/L NH4 HCO3 and 10 mmol/L DTT) for 10 min at 60 ◦ C. Alkynation was done by an alkylation buffer (25 mmol/L NH4 HCO3 and 55 mmol/L iodoacetamide) in the dark for 1 h at room temperature at until the gel was colourless. After drying, the gel was treated with trypsin solution (100 g/mL) for overnight at 30 ◦ C. After digestion, these tryptic digestive peptides were centrifuged, and the supernatant was dried on a Speedvac concentrator and desalted using ZIPTIP (C18 column-Millipore, USA). The elution of peptides was performed in 50% acetonitrile containing 0.1% trifluroacetic acid (TFA). 2.6. Identification of proteins by (peptide mass fingerprinting) MALDI-TOF-TOF mass spectrometry The eluted peptides (0.5 l) from the gel were mixed with ␣-cyano-4-hydroxycinnamic acid (Sigma) in 0.1% TFA/50% acetonitrile as the matrix (1:1), spotted on MALDI plate by using the matrix–sample–matrix sandwich method at room temperature and analysed on a 4800 MALDI-TOF-TOF mass spectrometer (Applied Biosystems, USA). All the data were analysed using the GPS explore software. To identify the proteins, a search was performed of the combined MS and MS/MS by NCBI and SWISS-PROT using the MASCOT software v2.1. 2.7. Comet assay From each group of rabbits, blood was collected from the ear vein and suspended in PBS. The blood cells were embedded in thin
Please cite this article in press as: Das T, et al. Effect of areca nut on rabbit oral mucosa: evidence of oral precancerous condition by protein expression and genotoxic analysis. Oral Sci Int (2017), http://dx.doi.org/10.1016/S1348-8643(17)30021-6
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Fig. 1. A) Development of OSMF in the animal model (Rabbit). I) PBS-treated control rabbit. II) Ulceration involving the jaw angle of areca nut-treated rabbit. III) Epithelial crust in phenol-treated rabbit. B) Tissues collected from three differently treated animals. C) H/E staining of the normal PBS-treated (I), areca nut-treated (II) and phenol-treated (III) rabbits’ oral tissues. Representative H/E staining of areca nut-treated tissues showing the OSMF characteristics (IV, V and VI) such as hyalinisation of collagen and atropic stratified squamous epithelium of variable thicknesses, with superficially thick orthokeratinisation (IV), reduced thickness of oral epithelium (V), and reduced vascularity and hyperchromatic nuclei (VI).
agarose gel (1% low melting) on a slide, and then these blood cells were lysed by lysis buffer (2.5 M NaCl, 0.1 M EDTA, 10 mM Tris base, 1% triton, and 10% DMSO) and incubated overnight. Electrophoresis was performed for 20 min in an alkaline solution (300 mM NaOH, 0.1 mM EDTA, PH > 13 at 280 mA and 24 V). The damaged DNA migrated away from the nucleus; the slides were then neutralised by Tris buffer pH 7.5 and stained with ETBR. The comet of DNA was visualised by fluorescent microscope (Leica DM 3000) and measured by the comet Score software. 2.8. Immunohistochemistry Immunohistochemistry was performed using the Novolink polymer detection system (Novolink castra, UK). Briefly, biopsy tissues sections from buccal mucosa of different rabbits were washed with cold PBS and then fixed in formalin. Tissues were then embedded in paraffin at 56–60◦ C. Tissue sections of 4–5 mm thickness were embedded in poly-L-lysine-coated slides. Paraffin-embedded tissues were de-waxed by xylene and rehydrated through graded alcohol; the slides were treated with citrate buffer for unmasking the protein. Further immunostaining was performed using the Novolink polymer detection system and endogenous peroxidase, and the protein was blocked using the supplied blockers. Collagen I was detected using monoclonal anti-collagen I antibody (1:50, ab90395, Abcam, Cambridge, USA). After post-primary blocking, the sections were incubated with the Novolink polymer and were then developed with 3,3 -diaminobenzidine. The sections were then counterstained with haematoxylin and were observed under LEICA DM3000 microscope (Leica Microsystems, Switzerland). 3. Results The three groups of animals were injected with PBS, phenol, and areca nut into their oral epithelial tissue for 3 months at 6day intervals. After 3 months, strong fibrosis was developed in the oral mucosal layer with phenol and areca nut treatments. OSMF developed in areca nut-treated rabbits and oral cancer developed
in phenol-treated rabbits were confirmed by histological section (Fig. 1A–C). Areca nut-treated rabbits showed significant reduction in the mouth opening and oral mucosal blanching, suggesting OSMF-like characteristic development. From the above results, it could easily be inferred that the areca nut is fibrosis promoting and phenol is carcinogenic. The oral lesion was further characterised by staining with haematoxylin and eosin (H/E stating). Epithelial tissue sections from areca nut-treated rabbits have shown thinning of the epithelial layer compared to PBS-treated rabbit along with the fibrous bands (Fig. 1C). H/E-stained histological sections revealed atropic stratified squamous epithelium of variable thickness, with superficially thick orthokeratinisation (Fig. 1C-IV-V). Epithelium was hyperchromatic, showing individual cell keratinisation. Atypia characters such as atrophy, cellular pleomorphism and hyperchromatic nuclei are shown in Fig. (1C-IV). Reduced vascularity was found in the submucosal tissues (Fig. 1C-VI). The connective tissue was fibrocollagenous with minimum infiltration of chronic inflammatory cells and few capillaries present in the juxtaepithelial region. Hyalinisation and homogenisation of collagen fibres were also noted in the juxtaepithelial connective tissues (Fig. 1C-IV). Degeneration or loss of epithelial layer was observed in phenoltreated rabbits, which is the major characteristic of oral cancer. To monitor the DNA damage, blood samples were collected from the ear vein and analysed using comet assay (Fig. 2). Phenol-treated rabbits showed significantly (p < 0.0001) higher DNA damage than PBS-treated rabbit. Areca nut-treated rabbits showed moderate but significantly (<0.0001) higher DNA damage than PBS-treated rabbit (Fig. 2). Oral lesion biopsy tissue samples were collected from each group (Fig. 1B). Protein profiling was performed by SDS, 2-DE, and MALDI-TOF analysis. The overexpressed proteins in the control and OSMF tissues were compared in 12% 2-DE-PAGE. One part of the proteins was resolved in SDS-PAGE, and another part of the protein was solubilised in 2-DE rehydration buffer, consisting of urea, thiourea, CHAPS, 40 mM Tris, and ampholytes, pH 3–10. The proteins were loaded in gel rehydration for pH 3–10 IPG strips and 2-DE electrophoresis was conducted using Protein IIXi system (Biorad). Fig. 3 shows that approximately 100
Please cite this article in press as: Das T, et al. Effect of areca nut on rabbit oral mucosa: evidence of oral precancerous condition by protein expression and genotoxic analysis. Oral Sci Int (2017), http://dx.doi.org/10.1016/S1348-8643(17)30021-6
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Fig. 2. Comet assay on the blood of rabbit treated with PBS (A), areca nut (B) and phenol (C) and (D) graphical presentation of DNA damage. *** represents statistical significance at level p value <0.0001.
Fig. 3. Representative 2-DE PAGE gel images of proteins in normal and areca nut-treated rabbit tissues. The normal buccal mucosa (A) and oral submucous fibrosis-featuring rabbit buccal mucosa. (B) Proteins were separated by 2-DE PAGE using IPG strips (pH 3–10) in the first dimension and 12% SDS-PAGE in the second dimension. Table 1 Proteins showing increased expression in areca nut-treated rabbit tissue. Protein Accession Number
Protein Name
Molecular Weight and pI
Peptide matched
Sequence coverage
A24904 OCU18344 NID: – Oryctolagus cuniculus TMRBB A23022 I56246 Q9R168 AAB88871 S42634 CO1A1
actin alpha, skeletal muscle Oryctolagus cuniculus serum albumin precursor, mRNA, tropomyosin beta chain skeletal muscle [validated] – rabbit actin, cardiac muscle Lipopolysaccharide-binding protein Secreted frizzled-related protein sFRP-1 (Fragment)) AF036941 NID: – Rattus norvegicus homeoprotein collagen alpha-1(I) chain
42366; 5.23 70861; 5.85 32931; 4.66 42334; 5.23 53737; 8.99 18517; 9.22 14937; 6.37 31046; 7.10 45134; 5.23
14 12 11 15 18 23 14 14 12
36 48 39 24 16 29 49 19 24
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Fig. 4. Immunohistochemical staining of collagen I in buccal mucosa in (A) PBS-treated, (B) areca nut-treated and (C) phenol-treated rabbits. Buccal tissues were stained with anti-collagen antibodies and observed under microscope (Leica). Magnification 20X.
proteins appeared on the 2-DE gel. The concentrations of the total extracted protein in the control and OSMF tissues were almost similar. The protein spots excised from SDS-PAGE (data not shown) and 2-DE gel were then analysed by MALDI-TOF spectrometry. To identify the proteins, database search was performed by SWISS-PROT. Table 1 summarises matched overexpressed proteins (score > 60) with high sequence coverage, accession number, theoretical molecular weight (kDa)/PI, and protein scores. The identified proteins included tropomyosin beta, actin alpha, Oryctolagus cuniculus serum albumin precursor and collagen alpha-1(I). The MALDI-TOF peptide mass fingerprint of the tryptic digest of Collagen I and the matched peptide sequences are shown in SupplyFig1. For further confirmation, immunohistochemical staining of collagen I of buccal mucosa in (A) PBS-treated, (B) areca nut-treated and (C) phenol-treated rabbits was performed (Fig. 4). A diffused and weak localisation of collagen I in the squamous epithelium was observed. Collagen I was faintly immunostained in the lamina propria and interstium of any layers in control rabbits. A strong staining intensity of collagen I was observed in the lamina propria in areca nut-treated rabbits, while the staining was localised in phenol-treated rabbits. The increased collagen type I expression and its immunolocalisation in the lamina propria and comet results suggest that OSMF-like features are present in areca nut-treated rabbits. 4. Discussion Epidemiological studies have established a strong relationship between areca nut use and the development of OSMF in humans [5,6]. In the present study, rabbits were treated with areca nut extract, which culminated in OSMF-like features from 3 months onwards. The clinical changes in the rabbit oral mucosa were monitored by an expert clinician in this field when blanching of the oral mucosa along with ulceration and reduction in mouth opening could be observed. The histological evaluation showed a significant amount of fibrosis, thinning of epithelial layer and loss of rete ridges (Fig. 1-C). The degeneration or loss of epithelial layer was observed in phenol-treated rabbit, which is the major characteristic of oral cancer, which was further confirmed by comet assay where significant DNA damage was observed in phenol-treated rabbits (Fig. 2). Reports suggest that approximately 7–14% of OSMF cases turn to malignancy, and our previous studies have shown significantly higher mean comet tail length in OSMF than in control rabbits, but it was lower than OSCC cases [7]. Similar results are observed in the present study, and areca nut-treated rabbits showed significantly higher tail length than the control group but lower tail length than the phenol-treated group. These findings corroborated with the characteristic histological features of OSMF. Because OSMF is multifactorial in nature, the proteomic study is an important step in identifying the possible biomarkers of the disease and understanding the pathogenesis of OSMF. In the present study, we have identified a few proteins from MALDI-TOF analysis, which are shown in Table 1. Interestingly, increased expression
of the collagen alpha-1(I) chain in areca nut-treated rabbit tissue compared to control strongly suggested that the methodology we have used for the development of the fibrosis model in the rabbit was more precise and symptomatically correct as the accumulation of collagen type-I due to chewing areca nut is the hallmark of oral fibrosis. Moreover, overexpression of cytoskeleton proteins such as actin, tropomyosin and tubulin was also observed. These proteins have essential functional role and are critically involved in cell morphology. Actin has different isoforms; actin as a globular monomer is called G actin and as a filamentous polymer, it is called F actin. Actin is essential for the regulation of premalignant condition. The overexpression of this protein to counteract the collagen accumulation mediated reduction in the contractibility of the oral muscle. Tropomyosin plays an important role in the formation of cellular cytoskeleton. During bladder carcinogenesis, tropomysin levels decrease in T24 human cancer cells [8]. Tropomyosin 1 disappears in primary human breast tumours. This suggested that the overexpression of tropomyosin 1 in the rabbit model might impart protection against areca insult, which may further leads to oral cancer. For epithelial tissue differentiation, keratin plays an important role and acts as a biological marker of malignant formation [9]. In the present study, we observed that along with collagen expression, tropomyosin 1 may emerge as another marker for the OSMF model. To validate the collagen I expression from the proteomics study, areca nut-treated rabbit tissue was subjected to immunohistochemical analysis with collagen I antibody. Strongly positive collagen expression was observed throughout the thickness of the stratified squamous epithelium in areca nut-treated rabbits compared to controls, suggesting OSMF-like features. 5. Conclusions In the present study, three major proteins were identified in the progressive development of OSMF in the rabbit, namely tropomyosin beta chain (in the skeletal muscle), actin and collagen alpha-1(I) chain, which may serve as the biological marker in the pathological development of OSMF. Genotoxic effects of areca nut and significant (p < 0.0001) DNA damage were observed in the rabbit model as compared to the control group. The increased expression of the collagen alpha-1(I) chain in areca nut-treated rabbit tissue strongly suggested that the methodology we have used for the development of the fibrosis model in the rabbit is convenient. Moreover, it is the first study to use a proteomic approach to use oral OSMF tissues in an animal model. Further characterisation and functional analysis are required to gain more insight to understand the role of these proteins in the disease progression. Ethics approval and consent to participate The study was duly approved by CPCSEA (New Delhi) and Institutional Animal Ethics Committee (IAEC) (Registration number 147/1999/CPCSEA).
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Funding
References
The study was supported by the Department of Science and Technology, Government of India and Council of Scientific and Industrial Research (CSIR), Govt. of India. Tapasi Das is the recipient of the DST (WOS-A).
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Acknowledgements We are thankful to Dr Krishna Das Saha and Dr Rukhsana Chowdhury for helping with the instrument facility. We are thankful to Dr Sanjit Mukherjee and Dr Atul katarkar for helping with animal handling. We are also thankful to Dr Sandeep Chakroborty for MALDI-TOF program operation. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/S13488643(17)30021-6.
Please cite this article in press as: Das T, et al. Effect of areca nut on rabbit oral mucosa: evidence of oral precancerous condition by protein expression and genotoxic analysis. Oral Sci Int (2017), http://dx.doi.org/10.1016/S1348-8643(17)30021-6