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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 14  |  Issue : 1  |  Page : 150-155

Determination of phenotypic alteration of arecoline-induced buccal mucosal fibroblasts: An in-vitro cell culture study


1 Department of Oral Pathology and Microbiology, KLE's VK Institute of Dental Sciences, KLE Academy of Higher Education and Research, Belgaum, Karnataka, India
2 Dr. Prabhakar Kore Basic Science Research Center, KLE Academy of Higher Education and Research, Belgaum, Karnataka, India

Date of Submission04-Sep-2020
Date of Acceptance26-Nov-2020
Date of Web Publication09-Feb-2021

Correspondence Address:
Dr. Ritiha Patil
Department of Oral Pathology and Microbiology, VK Institute of Dental Sciences, KLE Academy of Higher Education and Research, Belagavi, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/kleuhsj.kleuhsj_277_20

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  Abstract 

Introduction: Oral cancer is one of the worldwide health problems accounting as the 6th common of all malignancies. Majority of the oral cancer develop from premalignant conditions of the oral cavity due to the chronic habit of tobacco chewing and smoking. The prominent cells of the oral mucosa are fibroblasts playing a major role in synthesis of extracellular matrix, wound healing, and wound repair. Arecoline, one of main ingredients of tobacco is considered as a risk factor for the development of oral premalignant lesions and cancer. The arecoline is reported to have both genotoxic and morphological alteration of oral fibroblasts leading to Oral submucous fibrosis. Thus in our study a dose dependent effect of arecoline was assessed on the morphology of cultured Human Buccal Mucosal Fibroblasts.
Aim: The aim of our experiment is to assess the effect of different concentration of arecoline on the morphological variation of primary cell lines of human buccal mucosal fibroblasts to develop a model of altered fibroblasts.
Materials and Methods: The primary cell lines of human buccal mucosa were established in BSRC KAHER Belagavi, and authenticated by STR profiling from DNA forensics Lab New Delhi. The cells were further cultured and assessed after treating with different concentration of arecoline hydrobromide. The treated cells were then observed for the phenotypic changes and recorded. The morphological alterations were compared to the untreated fibroblasts.
Results and Conclusion: In our study, a dose-dependent effect of arecoline was assessed on phenotypic or morphological alteration of buccal mucosal fibroblasts. The results justified that concentration of arecoline lower than 125 μg/ml did not show change in the morphology of buccal mucosal fibroblasts, whereas the concentration of arecoline >250 μg/ml showed altered fibroblasts. Hence, it can be concluded that the levels of arecoline in arecanut chewers if it is >250 μg/ml, the mucosal fibroblasts may undergo changes to cause fibrosis of collagen. The future scope of our study is to determine the genotoxic effects of arecoline on buccal mucosal fibroblasts and also to develop the therapeutic effects.

Keywords: Arecoline hydrobromide, cytotoxicity, human buccal mucosal fibroblasts, oral submucous fibrosis


How to cite this article:
Patil R, Kale AD, Mane DR, Patil D. Determination of phenotypic alteration of arecoline-induced buccal mucosal fibroblasts: An in-vitro cell culture study. Indian J Health Sci Biomed Res 2021;14:150-5

How to cite this URL:
Patil R, Kale AD, Mane DR, Patil D. Determination of phenotypic alteration of arecoline-induced buccal mucosal fibroblasts: An in-vitro cell culture study. Indian J Health Sci Biomed Res [serial online] 2021 [cited 2021 Feb 27];14:150-5. Available from: https://www.ijournalhs.org/text.asp?2021/14/1/150/308963




  Introduction Top


Oral cancer is one of the worldwide health problems accounting as the 6th common of all malignancies. The most common oral malignancy is oral squamous cell carcinomas (SCCs). The oral cancer develop from premalignant conditions of the oral cavity such as leukoplakia, erythroplakia, palatal lesion cigar smoking, oral lichen planus, oral submucous fibrosis, discoid lupus erythematosus, and epidermolysis bullosa. Although there are many advancements in the treatment modalities of oral cancer and precancerous conditions, there is no significant improvement in over the past several decades.[1],[2]

Oral mucosa is the lining of oral cavity, located anatomically between the skin and gastrointestinal tract. It protects the deeper tissues from mechanical forces resulting from mastication and from abrasive nature of foodstuffs. The cells of the oral mucosa comprise the epithelial cells, keratinocytes, and connective tissue cells mainly the fibroblasts. Fibroblasts play an important role in the synthesis of extracellular matrix in connective tissue and in wound healing. Human oral fibroblasts located in the oral cavity contrast to skin fibroblasts, have the ability to rapidly repair defects in the oral cavity more quickly, reorganize the extracellular matrix and migrate for wound repair. Abnormal proliferation or any morphological or genetic alteration of human oral fibroblasts can lead to the development of oral SCC, oral submucous fibrosis. Human oral fibroblasts are a useful model for elucidating the mechanisms of fibrosis and developing treatments for oral cancers. One of the common conditions of oral cavity where altered fibroblasts are involved is oral submucous fibrosis. It is a potentially malignant condition characterized by changes in the connective tissue fibers of the lamina propria and deeper parts, leading to stiffness of the mucosa and restricted mouth opening seen predominantly in people of Asian descent. Oral submucous fibrosis predominantly affects the buccal mucosa and other parts of oral cavity, pharynx, and upper third of the esophagus.[3],[4]

In 2003, the International Agency for Research and Cancer (IARC), a World Health Organization sponsored group, found sufficient evidence that the habit of chewing betel quid, with or without tobacco, causes cancer and has considered as an important environmental risk factor for the development of oral premalignant lesions and cancer. Arecanut is now considered to be Group I carcinogen by IARC, International Agency of Research Cancer by the World Health Organization.[5]

Arecoline consists of alkaloids such as arecoline, arecadine, guvacoline, and guvacine. Arecoline is the major alkaloid of areca nut, causes cytotoxicity and genotoxicity in mammalian cells causing carcinogenicity.[6],[7]

Arecoline has been documented as the main agent responsible for fibroblast proliferation, in presence of slaked lime (Ca (OH) 2), arecoline get hydrolyzed to arecadine, thus affecting the fibroblast proliferation. In a study done by Harvey et al. they found that showed that exposures to 0.1–10 μg/ml arecoline stimulates fibroblasts and concentrations >25 μg/ml, inhibits fibroblast growth and collagen synthesis. Jeng et al. in their study found that depletion of cellular glutathione levels by arecoline predisposes the oral mucosal fibroblasts to various genotoxic and cytotoxic stimulation. Hence, based on the literature survey, the effect of arecoline on the oral fibroblasts is concentration dependent, frequency of consuming the arecanut and geographical variation.[8],[9]

In OSMF, the oral mucosal fibroblasts are affected because of the effect of alkaloids present in arecanut. The alkaloids cause increased formation of collagen, leading to stabilization and cross linking of collagen fibers, which further causes accumulation of collagen resulting in disease – OSF.[10],[11] Evidence from the literature suggest that oral mucosal fibroblasts undergo morphological and biological changes due to alkaloids present in arecanut based on the consuming habit of arecanut. Thus, the present study aims at assessing the effect of different concentration of arecoline on the morphological variation of oral fibroblasts using HBMF cell lines.[10],[11]


  Materials and Methods Top


The study comprised the primary cell lines of human buccal mucosal fibroblasts derived from the tissue samples of healthy individuals undergoing extraction of impacted third molars with the informed consent of the patients from Department of Oral surgery of KLE V K Institute Dental Sciences. The culture of primary cell lines was carried out using standard protocols in Tissue Culture Laborotary of Dr. Prabahakar Kore Basic Science Research Centre KAHER, Belagavi. The cell lines was characterized and authenticated from DNA Forensics Lab, New Delhi.[2]

Ethical clearance for the study was obtained from the Ethics Committee of KLE Academy of Higher Education and Research with the reference number /ethical number: KLEU/Ethic/2016-2017/D-227. Informed consent was also obtained from all the patients involved in our study.

Cell growth and culture

The authenticated cell lines were cultured in T-25 flask with complete Dulbeccos Modified Eagles Media (DMEM) media and on reaching a confluency of 80%, the cell lines were sub-cultured and passage number was given as P1-2-3 and P4. During the initial days of culture, fibroblasts showed spindle (F1) epithelioid (F2) and stellate shaped (F3) fibroblasts and mixture of round-to-spherical-shaped cells.[2],[12]

Arecoline hydrobromide was procured from Sigma Aldrich in the powder form and different concentration was prepared by dissolving in DMEM Media. The stock solution was prepared as 1 mg/2 ml of DMEM and further concentration were prepared using serial dilution method.

Morphological assessment

Day 1: The cell counting and seeding

The cells were cultured till they reached the confluency and the cells of the 4th passage were selected for the study which showed all the shapes of fibroblast cells (F1, F2, and F3). The cells were incubated for 24 h in CO2 incubator supplemented with DMEM Media, fetal bovine serum, and antibiotics. The adherent cells were detached from the flask by trypsinization and centrifuged to form a cell pellet. The pellet is then mixed with fresh media and cell counting is done. Approximately 6000 cells were seeded in a 12 well culture plate. The cells were allowed for attachment in a CO2 incubator for 24 h.

Day 2: Addition of arecoline hydrobromide on the cell lines

Arecoline hydrobromide was incorporated on the cell lines with different concentrations by using serial dilution method [Table 1]. DMEM media was used as a dissolving media and around 200 μl of the compound was added in each well. The cells were incubated for 24 h in CO2 incubator and were assessed for the morphological changes.
Table 1: Concentration of arecoline hydrobromide

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Assessment of morphology of fibroblast cell lines

The morphological changes were categorized as shape of the cell, outline of the cells, nucleus, and cytoplasm for the assessment of the effect of AH on cell lines as seen in [Table 2].
Table 2: Morphological assessment of arecoline induced primary human buccal mucosal fibroblasts

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  1. 500 μg/ml: The cells treated with this concentration of AH were morphologically altered showing mainly F2 (epithelioid)-shaped cell lines. When observed under inverted microscope the outline of the cells were dead cells with majority of them being round cells, closed nucleus-containing dense granular scanty cytoplasm [Figure 2]b and [Figure 2]c
  2. 250 μg/ml: The culture wells treated with 250 μg/ml showed epithelioid (F2)-shaped cells. The fibroblasts had a plump-shaped outline with the absence of spindle-shaped fibroblasts [Figure 3]b and [Figure 3]c. The cells possessed closed nucleus with scanty dense granular cytoplasm indicating granular cells [Figure 3]c
  3. 125 μg/ml: The culture wells showed cells of mixed population with F1, F2, and F3-shaped fibroblasts. The majority of the cells were spindle shaped and few plump cells. The nucleus was open with scanty clear cytoplasm which is feature of granular cells
  4. 62.5 μg/ml: The cells treated with this concentration of AH were not much morphologically altered as they showed majority of the cell lines possessing F1 cells and remaining of the well with both F2 and F3 cells. The cells were spindle shaped, round and plump fibroblasts with open nucleus showing abundant clear cytoplasm
  5. 31.25–3.90 μg/ml: There were no much morphological changes seen with the cells treated with 31.25–3.90 μg/ml of arecoline hydrobromide. Majority of the cells were spindle-shaped, and stellate-shaped cells. The culture wells also showed cells with open nucleus and abundant clear cytoplasm.
Figure 1: (a and b) Primary culture of human buccal mucosal fibroblasts

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Figure 2: (a-c) :Photomicrogrph of fibroblast cell lines treated with 500 μg/ml arecoline hydrobromide

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Figure 3: (a-c) Photomicrograph of fibroblast cell lines treated with 250 μg/ml of arecoline hydrobromide (a: ×10, b: ×20, c: ×40)

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  Results and Observation Top


The primary cell lines were established in BSRC KAHER Belagavi, and authenticated by STR profiling from DNA forensics Lab New Delhi and published.[12] The cultures had long spindle shaped cells with round nucleus and abundant cytoplasm as seen in [Figure 1]a and [Figure 1]b. The cells were further cultured and assessed after treating with different concentration of Arecoline Hydrobromide. The results showed that the concentration of 500 μg/ml [Figure 2]a,[Figure 2]b,[Figure 2]c was cytotoxic to the cell lines where the majority of the cells were round with granular nucleus and scanty cytoplasm indicating dead cells. [Table 3] shows the effect of different concentration of Arecoline Hydrobromide on the morphology of buccal mucosal fibroblasts. The concentration from 250 to 125 μg/ml [Figure 3]a,[Figure 3]b,[Figure 3]c showed mixture of cells with majority of them being granular cells indicating the cells attaining death and very few being spindle/plump-shaped cells. The concentration from 125 to 7.81 μg/ml [Figure 4]a,[Figure 4]b,[Figure 4]c and [Figure 5]a,[Figure 5]b,[Figure 5]c had no much cytotoxic effect with F1, F2, and F3 types [Table 3] with clear abundant cytoplasm. The lower concentration of the Arecoline hydrobromide showed less toxicity to the cells and could not exhibit altered shapes of fibroblasts with closed nucleus and abundant cytoplasm.
Table 3: Shapes of fibroblast

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Figure 4: (a-c) Fibroblast cell lines treated with 125 μg/ml of arecoline hydrobromide. (a: ×10, b: ×20, c: ×40)

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Figure 5: (a and b) Fibroblast cell lines treated with 62.5 μg/ml of arecoline hydrobromide. (a: ×10, b: ×20)

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  Discussion Top


Oral cavity is lined by oral mucosa consisting of epithelium and connective tissue. The major cells of connective tissue in oral cavity are fibroblasts. The fibroblasts play an important role in the synthesis of collagen fibers maintaining the architecture of tissue microenvironments by depositing and remodeling of extracellular matrix components. The cells actively play role in the production of collagen and the maintenance of extracellular matrix, thus regulating the wound healing.[1],[2] Hence, any damage or alteration in the shape or function of fibroblasts may lead to pathologies of oral mucous membrane such as oral submucous fibrosis and oral SCC. The etiology of the precancerous condition in India is mainly the consumption of tobacco either in smoke and smokeless form. Tobacco is the leading cause for the precancerous condition in India. The main ingredient of tobacco is arecanut which consists of Arecoline and Arecadine. The increased concentration of Arecoline and the constant habit of chewing tobacco results in the fibrosis of oral mucous membrane mostly resulting in oral submucous fibrosis. The arecoline will be absorbed by buccal mucosa harming the connective tissue and hence is considered as the initial risk factor of OSF in betel chewers. The increased gingivitis, periodontitis and the changes in the buccal mucosa in betel chewers might implicate that there exist some possible morphological and molecular damage.[2],[3]

In previous studies done on fibroblasts cell culture, three basic morphological forms of fibroblasts were appreciated. Based on the animal model and cell culture experiments, they were classified as F1 (spindle shaped), F2 (epithelioid shaped), and F3 (stellate shaped). F1 produces very low levels of collagen (type 1 and 3), are more proliferative in nature. F2 is less proliferative and secretes more collagen (type 1 and 2). F3 cells produces high levels of collagen (type 1 and 3) with proliferative activity indicating the terminal fibroblasts [Table 3].[1]

The isolation and culture of primary cells from the explant tissue have been documented since decades. The primary culture is an essential component of animal tissue and cell culture technology.[2] The advantage of culturing the primary cells over the cancer cells is that as they are the reliable source in understanding the normal physiological, morphological, and molecular process of the human cells. Our study mainly aims to study the morphological variation of the cultured primary cell lines of human buccal mucosal fibroblasts taken from healthy individuals. The alteration in the morphology, genetic, and their function help in understanding the pathogenesis of many pathological conditions. Culture of oral fibroblast cells helps the oral biologists and researchers to study the morphological and molecular process in the oral diseases.[11],[12]

In continuation to our previous research of developing primary cell line, we conducted this research to determine the dose-dependent effect of Arecoline on morphology of primary buccal mucosal fibroblasts. The primary cell lines of buccal mucosal fibroblasts were cultured, treated with different concentration of Arecoline for 24 h and the morphological changes were observed under inverted microscope. On observation, we found that the concentration of 500 μg/ml was cytotoxic as the majority of the cells showed the features of granular cells or dead cells. The remaining cells which (10-15) showed alteration from spindle shape to round or spherical with scanty granular cytoplasm thus attaining cell death. At concentration of 250 μg/ml to 150 μg/ml in our study, we had a mixture of cells from spindle cells to plump-shaped fibroblast. Along with plump-shaped cells, round dead cells were also present. Hence in our study, we observed that the concentration of 500 μg/ml was completely cytotoxic to the cells, whereas the concentration ranging from 250 to 150 μm/ml was partially cytotoxic showing mixture of dead and viable cells.

A study conducted by Chang Y C et al. in the year 2001 assessed the cytotoxic effects of Arecoline on the PDLF in Taiwan in patients with the habit of smoking. Their study showed that the concentration >200 μg/ml arecoline inhibits cell growth, proliferation, and protein synthesis on human PDLF indicating that betel nut chewing might be another risk factor in the pathogenesis of periodontal diseases. The above observations are similar to our findings.[2],[3]

Shang Lu Chiang in the year 2007 conducted a study to assess the dose-dependent effect of Arecoline on Human Gingival Fibroblasts (HGF) of normal individuals. The cells were treated with 100 μg/ml (or >100 μg/ml) of arecoline for 24 h, the cells appeared less dense than control and cell retraction was observed. Whereas the concentration >400 μg/ml resulted in granular and dead cells indicating cyto-toxicity of Arecoline to normal fibroblasts. This was in accordance to our observation.[4]

A similar experiment was conducted by Deepu George Mathew et al. in 2011 to compare the arecoline effect on fibroblasts of chewers with or without OSMF. They divided their study groups as Arecanut chewers with normal mucosa, arecanut chewers with OSMF and a control group with no chewing habit and normal mucosa. Their study concluded that the primary culture of fibroblasts revealed not only the basic F1, F2, F3 morphological variants, but in addition, they also found f1, f2, f3 (mitotic) and f4, f5, f6 (postmitotic) shapes of fibroblasts in arecanut chewers with OSMF. This showed that there are morphological variations when fibroblasts in the buccal mucosa are subjected to betel nut chewing. However, this study did not highlight the dose-dependent effect of arecoline on the morphology of buccal fibroblasts.[5]

A recent study was carried out by Abhishek Banerjee et al. in the year 2017 to identify the various morphological forms of fibroblasts and to understand and assess the response of the fibroblast cell lines to different concentrations of Arecoline. The primary cell lines were cultured and treated with different concentration ranging from 50/100/150/300/500 ug/ml of Arecoline. The cell lines were then observed for 8 days to assess the morphological variation and cell counts. Our results were concomitant to the above findings when less concentration of arecoline was stimulative and showed toxic effect at higher concentration.[10]


  Conclusion Top


The adverse effects of arecanut chewing and its constituents on buccal mucosa have been studied from the past decades. The arecoline and arecadine being the main constituent of arecanut have both phenotypic and genotoxic effect on the cells of buccal mucosa that are predominantly fibroblasts. Hence in our study, the dose-dependent effect of Arecoline was assessed on phenotypic or morphological alteration of buccal mucosal fibroblasts. The results justified that concentration of arecoline lower than 125 μg/ml did not show change in the morphology of buccal mucosal fibroblasts in Indian healthy individuals. Whereas the concentration of arecoline >250 μg/ml showed altered fibroblasts. Hence, it can be concluded that the levels of arecoline in arecanut chewers if it is >250 μg/ml, the mucosal fibroblasts may undergo changes to cause fibrosis of collagen. The future scope of our study is to determine the genotoxic effects of arecoline on buccal mucosal fibroblasts and also to develop the therapeutic effects.

Acknowledgment

We would like to acknowledge and thank Dr. Prabhakar Kore Basic Science Research Centre, KLE Academy of Higher Education and Research, Belgaum, for providing the facilities for successful carrying out our experiment in the tissue culture lab.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Waal J, Olivier A, van Wyk CW, Maritz JS. The fibroblast population in oral submucous fibrosis. J Oral Pathol Med 1997;26:69 74.  Back to cited text no. 1
    
2.
Chang YC, Lii CK, Tai KW, Chou MY. Adverse effects of arecoline and nicotine on human periodontal ligament fibroblasts in vitro. J Clin Periodontol 2001;28:277-82.   Back to cited text no. 2
    
3.
Chiang SL, Jiang SS, Wang YJ, Chiang HC, Chen PH, Tu HP. Characterization of arecoline-induced effects on cytotoxicity in normal human gingival fibroblasts by global gene expression profiling. J Toxicological Sci 2007;100:66-74.   Back to cited text no. 3
    
4.
Mathew DG, Skariah KS, Ranganathan K. Proliferative and morphologic characterization of buccal mucosal fibroblasts in areca nut chewers: A cell culture study. Indian J Dental Res 2011;22:6.  Back to cited text no. 4
    
5.
Gunjan S, Pankaj C, Sagar V. Arecanut as an emerging etiology of oral cancers in India Indian Journal of Medical and Paediatric Oncology 2012;33:2.  Back to cited text no. 5
    
6.
Chang MC, Lin LD, Wu HL, Ho YS, Hsien HC, Wang TM, et al. Areca nut-induced buccal mucosa fibroblast contraction and its signaling: A potential role in oral submucous fibrosis: A precancer condition. J Crcinogenesis 2013;34:1096-104.  Back to cited text no. 6
    
7.
Rachana VP, Vishnudas P, Laxmikanth C, Shenai P, Nithin S, Dandekeri S, et al. Areca nut and its role in oral submucous fibrosis. J Clin Exp Dent 2014;6:569-75.   Back to cited text no. 7
    
8.
Chang YC, Tsai CH, Lai YL, Yu CC, Chi WY, Li JJ, et al. Arecoline induced myofibroblast transdifferentiation from human buccal mucosal fibroblasts is mediated by ZEB1. J Cell Mol Med 2014;18:698-708.   Back to cited text no. 8
    
9.
Shwetha HR, Chaitanya B, Saad A, Anuj G. Effect of arecanut on oral epithelium: A review of literature. University J Dent Science 2015;1:38-4.   Back to cited text no. 9
    
10.
Banerjee A, Kamath VV, Kotrashetti V, Bhatt K. Fibroblastic phenotype in oral submucous fibrosis- A cell culture analysis. Saudi J Pathol Microbiol 2017;2:36-47.   Back to cited text no. 10
    
11.
Wen QT, Tao W, Yu DH, Wang ZR, Sun Y, Liang CW. Development of a mouse model of arecoline-induced oral mucosal fibrosis Asian Pacific. J Tropical Med 2017;10:1177-84.   Back to cited text no. 11
    
12.
Patil R, Kale AD, Mane DR, Patil D. Isolation, culture and characterization of primary cell lines of human buccal mucosal fibroblasts: A combination of explant enzamytic technique. J Oral Maxillofac Pathol 2020;24:68-75.  Back to cited text no. 12
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    Figures

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