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ORIGINAL RESEARCH Table of Contents   
Year : 2006  |  Volume : 17  |  Issue : 3  |  Page : 104-10
Effect of glutathione on arecanut treated normal human buccal fibroblast culture.


Department of Oral Pathology, Ragas Dental College and Hospital, Chennai, India

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   Abstract 

BACKGROUND: Experimental studies have shown arecanut to be a cytotoxic substance with mutagenic and carcinogenic potential. OBJECTIVE: The present study was undertaken to evaluate the effect of glutathione on arecanut treated human buccal fibroblast culture and its potential as a chemopreventive agent. MATERIALS AND METHODS: Fibroblast culture was done in Dulbecco's Modified Eagle's Medium MEM) supplemented with 10% Fetal Calf Serum (FCS) and antibiotic at 370C degrees in an atmosphere of 5% carbon di-oxide and 95% air. The fibroblast cells were subjected to different concentrations of aqueous extracts of raw and boiled arecanut. Fibroblasts were plated in two 24-well culture plates and in each plate, cells were dividt,ednto 2 groups; 600gg microml of reduced glutathione was added to the first group of cells; subsequently, aqueous extracts of raw and boiled arecanut at least and highest concentrations i.e., 20j. microml and 100lg microml were added to the first group of cells in the respective plates whereas the second group served as a control. The morphological alterations and cell survival were assayed at 24, 48, 72, and 96 hours. Results Morphologically, the initial (10 hours) attached fibroblast cells were converted from spheroidal shape towards hexagonal and finally to a fully extended spindle shaped configuration. The three morphological types of fibroblasts at 48 hours were F-I, F-II and F-III. Aqueous extract of raw arecanut exhibited significant cytotoxicity (p < .0 001) at all time periods studied, when compared against the control values of untreated fibroblasts. Addition of reduced glutathione to cultures showed a significant (p < 0. 001) reduction in cytotoxicity, as indicated by higher optical density values and morphological reversion to the spindle-shaped configuration. CoCONCLUSION:Addition of glutathione reduced the cytotoxic and morphological alterations of the fibroblasts treated with aqueous extracts of both raw and boiled arecanut.

Keywords: buccal fibroblast culture oral submucous fibrosis, glutathione, arecanut

How to cite this article:
Saraswathi T R, Sheeba T, Nalinkumar S, Ranganathan K. Effect of glutathione on arecanut treated normal human buccal fibroblast culture. Indian J Dent Res 2006;17:104

How to cite this URL:
Saraswathi T R, Sheeba T, Nalinkumar S, Ranganathan K. Effect of glutathione on arecanut treated normal human buccal fibroblast culture. Indian J Dent Res [serial online] 2006 [cited 2019 Mar 22];17:104. Available from: http://www.ijdr.in/text.asp?2006/17/3/104/29881

   Introduction Top


Oral submucous fibrosis (OSF) is a crippling disease of the mouth with the potential for malignant transformation [1]. Caniff et al reported genetic predisposition of OSF, in individuals with the habit of arecanut chewing [2].Experimental studies have shown arecanut to be a cytotoxic substance with mutagenic and carcinogenic potential [3]. The alarming rise in the incidence of OSF in this part of the country [4] in spite of health education measures, necessiciates the identification of chemopreventive substances to reduce morbidity. Glutathione (GSH), a tripeptide, composed of cystine, glutamic acid and glycine, is involved in detoxification of a number of potentially toxic eletrophillic xenobiotics. This is conjugated to nucleophilic glutathione, catalized by the enzyme, Glutathione S-tranferase [5].In-vitro fibroblast culture studies have reported the effect of individual components of arecanut like arecoline, arecaidine, etc in the pathogenesis of OSF [6],[7]. However, there is paucity of in-vitro studies related to the use of chemopreventive substances along with arecanut extract. The present study is undertaken to evaluate the effect of glutathione on arecanut treated normal human buccal fibroblast culture and its potential as a chemopreventive agent. Aqueous extracts of different types of arecanut e.g. raw and boiled type as a whole were taken for the study to simulate the natural situation.


   Materials and methods Top


Biopsy specimens were obtained from the buccal mucosa of healthy non-arecanut chewers during the surgical removal of impacted mandibular third molars, with the consent of the patients who reported at the Tamilnadu Government Dental College and hospital, Chennai. The biopsied tissue was immediately placed in sterile test tube, containing Dulbecco's Modified Eagle's Medium (DMEM) and was transferred to the culture laboratory. It was washed thrice in phosphate buffer and the tissue was minced to small pieces in laminar flow cabinet. The minced tissue pieces were then incubated overnight with 0.005% collagenase in a sterile plastic tube containing DMEM supplemented with 10% fetal calf serum (FCS) and antibiotic at 37șC in an atmosphere of 5% carbon di-oxide and 95% air. It was centrifuged at 2000 rpm for 5 minutes. Existing medium was decanted and fresh DMEM supplemented with 10% FCS and antibiotics were added to the cell plates. Satisfactory attachment of the cells to the culture plates was observed in 24 - 48 hours. A monolayer of cells of about 2-3 x 106 subcells/mm were formed in 2 weeks. Subcultures were made from the monolayer. Fibroblasts between fourth and sixth passages were used for the present study.

Preparation of arecanut extract

The raw and boiled (cured) varieties of arecanut were ground into a fine powder. 5g of the powder of each type were suspended in 10ml of double distilled water. The suspension was filtered through glass wool filter and Whatman's filter paper No.1 and was boiled. A residue of 258mg and 326mg was obtained respectively for the raw and boiled arecanut. Both were dissolved in 1ml of double distilled water, each to yield stock solutions.

After trypsinisation, followed by overnight incubation, the cells [5] were subjected to 20, 40, 60 and 100 μg /ml of aqueous extracts of raw and boiled arecanut. The morphological alterations and cell survival were assayed at 24, 48, 72 and 96 hours. The cells were fixed with 70% methanol acetic acid and stained with Giemsa stain and observed under the inverted microscope.

MTT assay for cell viability

This calorimetric assay measured the reduction of 3-(4,5 dimethyl thiazol-2-41) 25-diphenyl tetrazolium bromide (MTT) by mitochondrial succinate dehydrogenase. MTT salt enters the cells and passes into the mitochondria where it is reduced to an insoluble purple formozon product upon cleavage of the tetrazolium by dehydrogenase enzymes. The cells are then solubilized with an organic solvent-isopropanol and the pink solubilized formozan reagent is measured spectrophotometrically. Reduction of MTT can occur only in metabolically active cells, the level of activity is a measure of viability of the cells.

Chemoprevention studies with glutathione became confluent and formed a monolayer in about 8 Fibroblasts were plated in two 24-well culture plates (24 days time after which they became indistinguishable. x103 subcells/mm3sub) and allowed to attach for 24 hours. For each plate, cells were divided into 2 groups; 600μg/ml of Aqueous extract of raw arecanut reduced glutathione was added to the first group of cells; Aqueous extract of raw arecanut exhibited significant subsequently, aqueous extracts of raw and boiled cytotoxicity (p<.0001) at all concentrations and time arecanut at least and highest concentrations i.e. 20μg/ml periods studied, when compared against the control and 100μg/ml were added to the first group of cells in the values of untreated fibroblasts, as determined by the respective plates; the second group served as a control. optical density units observed during the MTT assay. The morphological alterations and cell survival were A significant difference (p<0.05) was observed between assayed at 24, 48, 72 and 96 hours. various concentrations studied demonstrating a dose dependent increase in cytotoxicity of the aqueous extract of raw arecanut. Concomitant morphological study RESULTS showed significant alterations from control features at the high concentrations studied viz, at 20μg/ml and Normal fibroblast culture 100μg/ml. There was increased appearance of F-III The optical density units observed during the MTT assay fibroblasts and the cells affected by arecanut was found to increase with prolonged incubation in cytotoxicity appeared distorted [Figure - 4]. Some cells DMEM supplemented with 10% fetal calf. were detached from the dish surface and were found Morphologically, the initial attached cells were floating in the medium. converted from spheroidal shape towards hexagonal and finally to a fully extended spindle shaped configuration Aqueous extract of boiled arecanut by about 10 hours. The three morphological types of Aqueous extract of boiled arecanut showed decreased fibroblasts, F-I, F-II and F-III were appreciated at 48 cell survival of the cultured buccal fibroblasts hours of incubation. F-I cells were observed to be significantly (p<0.0001) at all concentrations studied, diploid, spindle-shape [Figure - 1]. F-II cells were diploid, when compared against the control values of untreated larger and epithelioid [Figure - 2]. F-III cells were larger fibroblast cultures. A dose dependent cytotoxicity was when compared to F-I and F-II cells and assumed a observed at all time points studied (p<0.05). stellate shape and were tetraploid [Figure - 3]. The cells Concomitant morphological study showed that at lower concentrations, up to 20μg/ml, there were no significant Extra-cellular addition of reduced glutathione alterations in morphology from the untreated cultures. At Raw arecanut extract treated cultures and boiled higher concentrations viz, at 100ug/ml, there was arecanut treated cultures to which glutathione was added increased appearance of F-III fibroblasts and the cells showed a significant (p<0.001) reduction in affected by arecanut cytotoxicity appeared distorted cytotoxicity, as indicated by higher optical density [Figure - 6]. Moreover, some attached cells had detached values at both concentrations of 20μg/ml and 100μg/ml from the cell surface, which was indicated by numerous, cells floating in the medium. A comparison of the cytotoxicity of aqueous extracts of raw and boiled, configuration [Figure 5, 7]. arecanut showed that there was a significant difference (p<0.001) between them. The aqueous extract of the boiled arecanut was found to be more cytotoxic than that of raw nut [Table - 1].


   Discussion Top


Oral submucous fibrosis is a cause for concern because it is at best, a crippling disease of the mouth and at worst, may give rise to oral cancer. Longitudinal studies in 66 cases over a period of 17 years in Kerala have shown malignant transformation in 5 patients (7.6%)[8]. Treatment available for oral submucous fibrosis helps in dissolution of inter-fibrillar substance, thereby reducing the stiffness of the oral mucosa, albeit not permanently. Use of arecanut has been pointed out as a possible etiological agent for the development of oral submucous fibrosis [1],[9]. In India, the younger generation practice chewing arecanut as a fashionable habit without any social stigma with the resultant is sudden rise of arecanut consumption [4]. Reducing the morbidity of oral submucous fibrosis therefore requires, either elimination of the habit in the society or alternatively identification of a chemo-preventive agent, to prevent the harmful effects of arecanut chewing.

The use of arecanut as a masticatory substance by humans has been known since the 4~ century A.D. in various parts of the world. It is estimated that over 600 million individuals consume arecanut in one form or the otherworldwide.

In India, arecanut is chewed in the form of various nut preparations such as scented supari, mawa, manipuri tobacco and pan masalaused in betel quid with or without tobacco [10]. Quid is a mixture of substances either pouched in the mouth or chewed and thus remains in contact with the mucosa Betel quid chewing has a long history among numerous Asian communities. It has been incorporated into many religious and cultural rituals of ethnic groups of the Indian sub-continent and has gained a degree of social acceptance [11].

Betel leaf has been found to contain phenolic compounds and beta-carotene, which are suggested to be anti­mutagenic agents giving the betel leaf a protective role against the harmful alkaloids of arecanut [3]. Chewers of betel nut (arecanut) alone, without betel leaf, stand a greater chance of mucosal damage. The absence of betel leaf and its carotenes in pan masala/gutkha could be responsible for the increased occurrence and earlier presentation of OSF in patients with this habit [12].

Various studies have been carried out with regard to the cy totoxic and genotoxic effects of individual components and extracts of arecanut, which include aqueous ethanolic and acetic acid extracts of the nut [6],[7],[13]. In this study we used a crude aqueous extract of raw and boiled varieties of arecanut to mimic the situation of human consumption of arecanut; saliva in which the betel nut is dissolved in the natural condition of human consumption of the nut is a water based solution. Hence, an aqueous extract of arecanut was chosen for this in-vitro study to simulate extraction of arecanut components by saliva in vivo.

Mollenhauer et al [14] described three types of fibroblasts F-I, F-II, F-III on morphological basis in rat skin and lung fibroblasts. According to their findings, F-1 cells

are diploid, spindle shaped and highly proliferative and they synthesized low levels of type I and type It collagen. F-11 cells are diploid, epitheloid and proliferate slowly, exhibiting elevated levels of collagen synthesis. F-III fibroblasts are large stellate, tetraploid cells that proliferate more slowly than the other types but synthesize large amounts of collagen. In the present study, the three types of normal fibroblasts were appreciated in untreated fibroblast culture with the predominance of F-I spindle shaped cells, at 48 hours of incubation. After 8 days the cells became confluent and indistinguishable.

Mollenhauer et al [14] suggested that fibroblasts differentiate sequentially i.e, F-I. F-11, F-111 in an irreversible manner and associated accumulation of F-III cells with physiological process of aging and some pathological states. The work of de Waal et al [15] on the fibroblast population of submucous fibrosis showed an increase in the F-III population in these tissues. They proposed that there is a permanent shift in the fibroblast population in oral submucous fibrosis toward a slower replicating population committed to producing larger quantities of collagen. Ma et al [16] also implied that a change in the synthesizing capabilities of the fibroblasts might take place with are canut chewing which could induce new or altered subpopulation of fibroblasts in the oral mucosa of OSF patients.

The present study did not extend to OSF tissues but it showed a shift towards RIII type when normal fibroblasts were treated with arecanut extract. It may be speculated that such a shift in the fibroblast subpopulation might be considered as a risk indicator for the development of fibrosis in apatientwith ahistory of arecanutchewing.

In the present study, aqueous extracts of raw and boiled arecanut were observed to decrease cell survival of the cultured buccal fibroblasts at concentrations ranging from 2pg/ml to 100pg/ml studied over 4 days incubation period in a concentration dependant rnanner. These results are in agreement with the work of Van Wyk et al [17] who observed that extracts of boiled, baked and raw arecanut inhibited buccal fibroblasts in a dose- dependent manner at concentrations ranging from 50pg/ml to 150pg/ml. The results of present study are also in concurrence with the observations of Jeng et al [3] who reported that arecanut extract decreased the cell survival of buccal fibroblasts in a dose dependant manner, concluding that arecanut extract contains cytotoxic agents. Arecanut extract has also been found to be cy totoxic to buccal epithelial cells and gingival keratinocy tes [12].

The cy totoxic effects of aqueous extract of raw and boiled arecanut extract differed significantly (p<0.001) from each other at comparable concentrations, at all time periods studied. The boiled arecanut extract exerted considerably more cy totoxicity than the raw type, which also correlated with morphological alterations. This variation could be attributed to the curing of the nut. Shivpurkar et al [18] reported that wide variations could occur in the composition of the 'cured' nut, showing a relative increase in the polyphenol and arecoline content in the cured nut. Glutathione is ubiquitous in eukaryotic cells and is implicated in many cellular functions. It is typically present in high concentrations (0.1-10mM) and is thus both the most prevalent cellular thiol and the most abundant low molecular weight peptide. Glutathione has adapted through evolution to perform diverse functions [19]. It acts as a reducing agent and anti-oxidant; participates in detoxification reactions of xenobiotics and metabolism of numerous cellular compounds. It is required for synthesis of some prostaglandins and is involved in cell cycle regulation and thermo-tolerance. This peptide plays a role in protection against tissue damage resulting from exposure to oxidizing environments such as hyperoxia, hyperbaric oxygen or ozone and can also protect against radiation, ultra violet light and photodynamic effects[20].

Glutathione is synthesized intracellularly and is exported from cells. Its breakdown is initiated by -glutamyl transpeptidase, an enzyme attached to the external surface of certain cell membranes. Export of glutathione functions in inter-organ and intra organ transfer of cystein moieties and in the protection of cell membranes [21]. Decreased glutathione has been associated with pathogenesis of specific diseases such as diabetes, alcoholic liver disease, AIDS and cataract. Glutathione deficiency is an important factor in the regulation of aging process. Depletion of glutathione has crucial effects on survival and low glutathione levels are associated with cell damage and increased susceptibility to toxic challenge.

Intra cellular depletion of glutathione can occur via the glutathione-S-transferase(GST) reaction, where glutathione after conjugation with a large number of foreign compounds with electrophillic centers is catalyzed by GST found in many tissues. Since the liver is such an important source of glutathione, metabolism of xenobiotics in the liver, which can drastically deplete liver glutathione, may also result in glutathione depletion in other tissues [20].

In the present study, addition of extra cellular glutathione did not significantly improve cell survival towards untreated controls. However, against comparable concentrations of arecanut extract treated controls, treatment with glutathione significantly (p<0.001) improved cell survival. These imply that addition of glutathione afforded a relative protection against arecanut extract induced toxicity, though not completely. These observations are similar to that of Jeng JH et al [12] who observed that addition of extracellular glutathione prevented only arecoline induced cytotoxicity but that it could not completely protect against arecanut extract induced toxicity. They concluded that mechanisms other than thiol depletion might be responsible for arecanut cytotoxicity. The work of Chan et al [22] also demonstrated that addition of extracellular glutathione prevented arecoline induced cytotoxicity.

An increase in the resistance of cells to oxidative damage and toxic compounds may be achieved by increasing intracellular glutathione levels. Glutathione is present in food and is one of the major antioxidants and antimutagens in the soluble fraction of cells. Its concentration may be influenced by dietary sulfur amino acids [23]. Dietary sources rich in glutathione include asparagus, avocado and walnuts, apart from fresh and frozen fruits and vegetables, fish and meat [19]. Chan JT et al [22] demonstrated that an antioxidant mixture which included glutathione significantly reduced the frequency of premalignant lesions and their subsequent development into tumours. Novi AM [24] reported that glutathione caused regression of tumour growth in rats bearing aflatoxin B1 induced liver tumours. Increasing dietary intake of glutathione rich foods or dietary supplement of glutathione may therefore, have chemopreventive potential to reduce arecanut quid associated lesions.


   Conclusion Top


This study revealed that aqueous extracts of raw and boiled arecanut were cytotoxic to normal human buccal mucosal fibroblasts in a dose dependent manner. Aqueous extract of boiled arecanut was more cytotoxic than that of raw arecanut at comparable concentrations. The cytotoxicity not only reduced cell survival but also induced morphological alterations towards more collagen producing F-III type of fibroblasts. Addition of glutathione reduced the cytotoxicity and morphological alterations of the fibroblasts to aqueous extracts of both raw and boiled arecanut, although not completely. The role of increased dietary intake of glutathione in reducing the cytotoxicity of arecanut is emphasized.



 
   References Top

1.Murti PR, Bhonsle RB, Gupta PC, Daftary DK, Pindborg JJ, Mehta FS. Etiology of Oral submucous fibrosis with special reference to the role of arecanut chewing. J Oral Pathol Med, 24:145-52, 1995.   Back to cited text no. 1    
2.Canniff JP, Harvey W: Etiology of oral submucous fibrosis: the stimulation of collagen synthesis by extracts of arecanut, Int J Oral Surg, 10 (S1): 163-7.   Back to cited text no. 2    
3.Jeng JH, Kuo ML, Hahn LJ, Kuo MYP: Genotoxic and non-genotoxic effects of betelquid ingredients on oral mucosal fibroblasts in vitro. J Dent Res, 73[5]:1043- 1049, 1994.   Back to cited text no. 3    
4.Gupta PC, Sinor PN, Bhonsle RB, Pawar VS, Mehta HC: Oral submucous fibrosis in India: A new epidemic? Natl Med J India, 11[3]: 113-116, 1998.   Back to cited text no. 4    
5.Murray RK: Metabolism of xenobiotics in Granner DK, Mayes PA, Rodwell VW (eds) Harper's th biochemistry, (24 ed) Appleton and Lange, pages: 752-755, 1996.   Back to cited text no. 5    
6.Chang YC, Tai KW, Cheng MH, Chou LSS, Chou MY: Cytotoxic and genotoxic effects of arecoline on human buccal fibroblasts in vitro, J Oral Pathol Med, 27: 68-71, 1998.   Back to cited text no. 6    
7.Sharan RN, Wary KK: Study of unscheduled DNA synthesis following exposure of human cells to arecoline and extracts of betel nut in vitro, Mutat Res, 27: 475-9, 1992.   Back to cited text no. 7    
8.Murti PR, Bhonsle RB, Pindborg JJ Daftary DK, Gupta PC, Mehta FS: Malignant transformation rate in oral submucous fibrosis over a 17 year period. Comm Dent Oral Epidemiol, 13: 340-1, 1985.   Back to cited text no. 8    
9.Van Wyk CW: Oral submucous fibrosis - The South African Experience, Int J Dent Res, 8[2]: 39-45, 1997.   Back to cited text no. 9    
10.Betel-quid and arecanut chewing. Lyon: International Agency for Research on Cancer. Monographs, 37: 141-202, 1986.   Back to cited text no. 10    
11.Bedi R: Betel quid-world wide use oral Dis, 3:284, 1997.   Back to cited text no. 11    
12.Jeng JH, Hahn LJ, Lin BR, Hsein EC, Chan CP, Chang MC: Effects of arecanut, inflorescence piper betel extracts and arecoline on cytotoxicity, total and unscheduled DNA synthesis in cultured gingival keratinocytes, J Oral Pathol Med, 28: 64-71, 1999.   Back to cited text no. 12    
13.Fang C, Liu S, Sheng Z, Liz, Tang F: Study on cytotoxic and DNA damaging effects in oral mucosal fibroblasts by arecanut extract, Hunan I,Ko Ta Hsuet Huest Pao 22[2]: 105-8, 1997.   Back to cited text no. 13    
14.Mollenhauer J, Bayreuther K et al : Donarage related changes in the morphology, growth potential and collagen biosynthesis in rat fibroblast subpopulations in vitro, Differentiation, 32: 165172, 1986.   Back to cited text no. 14    
15.Dewaal J, Oliver A, Van Wyk CW, Maritz JS, et al : The fibroblast population in oral submucous fibrosis, J Oral Pathol Med, 26: 69-74, 1997.   Back to cited text no. 15    
16.Ma RH, Tsai CC, Shieh TY: Increased lysyl oxidase activity in fibroblast cultured from oral submucous fibrosis associated with betel nut chewing in Taiwan, J Oral Pathol Med, 23: 65-69, 1994.   Back to cited text no. 16    
17.Van Wyk CW, Olivier A, De Miranda CM, Van der Bijl P, Grobler-Rabie AF: Observations on the effect of arecanut extracts on oral fibroblast proliferation, J Oral Pathol Med, 23: 145-148, 1994.   Back to cited text no. 17    
18.Shivpurkar NM, Bhide SV, Ranadive KJ: Biochemical studies of betel nut constituents, Ind J Pharmac, 10[3]: 191-200, 1978.   Back to cited text no. 18    
19.Jones DP, Coates RJ, Flagg EW et al : Glutathione in foods listed in the National Institute of Health Habits and History Food Frequency questionnaire, Nutr Cancer, 17: 57-75, 1995.   Back to cited text no. 19    
20.Deneke SM, Fanburg BL: Regulation of cellular glutathione, Am J Physiol, 257: 463-473, 1989.   Back to cited text no. 20    
21.Meister A: Glutathione metabolism and its modification. J Biol Chem, 263 (33): 17205-8, 1988.   Back to cited text no. 21    
22.Chan JT, Black HS: The mitigating effect of dietary anti-oxidants on chemically induced carcinogenesis, Experientia, 34:110-111, 1978.   Back to cited text no. 22    
23.Hunter EA, Grimble RF: Dietary sulfur amino acid adequacy influences glutathione synthesis and glutathione -dependent enzymes during inflammatory response to endotoxin and tumour necrosis in rats, Clinical Science, 92[3]:297-305, 1997.   Back to cited text no. 23    
24.Novi AM: Regression of aflatoxin B1 induced hepatocellular carcinoma by reduced glutathione, Science, 212: 541-542, 1981.   Back to cited text no. 24    

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Correspondence Address:
T R Saraswathi
Department of Oral Pathology, Ragas Dental College and Hospital, Chennai
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0970-9290.29881

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    Figures

[Figure - 1], [Figure - 2], [Figure - 3], [Figure - 4], [Figure - 5], [Figure - 6], [Figure - 7]

    Tables

[Table - 1], [Table - 2], [Table - 3]

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