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Year : 2015  |  Volume : 11  |  Issue : 1  |  Page : 204-210

In vitro analysis of particle penetration of smokeless tobacco forms using egg shell membrane as a substrate

Department of Oral Pathology, Saveetha University, Vellapanchavadi, Chennai, Tamil Nadu, India

Date of Web Publication16-Apr-2015

Correspondence Address:
Nithya Jagannathan
Department of Oral Pathology, Saveetha University, No. 162, Poonamalle High Road, Vellapanchavadi, Chennai - 600 077, Tamil Nadu
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0973-1482.138098

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 > Abstract 

Aims: The aim of the following study is to determine the particle sizes of smokeless tobacco forms and thereby evaluate the degree of diffusion of the products using an egg shell membrane as a natural substrate by scanning electron microscopy (SEM).
Subjects and Methods: The particle size of smokeless tobacco forms namely mawa, gutka, khaini, and tobacco leaves was determined by image analysis and the products were subjected on an egg shell membrane subjected to artificial saliva along with constant grinding force. The processed egg shell membrane was then examined by SEM to evaluate the morphology and the degree of diffusion of these particles.
Results: The morphometric image analysis revealed khaini to be smallest in size followed by mawa, gutka, and tobacco leaves. The control group (egg shell membrane not subjected to any products) under SEM demonstrated intricately woven collagen meshwork, which was regular in length, branched and anastamosed with each other. Khaini exposed membrane demonstrated maximum particle diffusion with disruption of collagen meshwork. Mawa exposed membranes demonstrated minimal particle penetration which were adherent to the collagen meshwork which was irregular with increased interfibrillar space. The egg shell membrane exposed to gutka demonstrated particle penetration at higher magnification with more or less a regular collagen meshwork. The tobacco leaf exposed egg shell membrane demonstrated minimal blebs on the meshwork surface.
Conclusions: The degree of diffusion gradually increased with a decrease in the particle size establishing an inverse relationship.

Keywords: Diffusion, malignancy, potentially malignant disorders, scanning electron microscope

How to cite this article:
Jagannathan N, manian AR, Ramani P, Premkumar P, Natesan A, Sherlin HJ. In vitro analysis of particle penetration of smokeless tobacco forms using egg shell membrane as a substrate. J Can Res Ther 2015;11:204-10

How to cite this URL:
Jagannathan N, manian AR, Ramani P, Premkumar P, Natesan A, Sherlin HJ. In vitro analysis of particle penetration of smokeless tobacco forms using egg shell membrane as a substrate. J Can Res Ther [serial online] 2015 [cited 2018 Oct 20];11:204-10. Available from: http://www.cancerjournal.net/text.asp?2015/11/1/204/138098

 > Introduction Top

Tobacco continues to be a major, although a preventable cause of potentially malignant and malignant disorders of the oral cavity and its use in children and adolescents is reaching pandemic levels. [1],[2] Tobacco may be used in the smoking and smokeless form, with the smokeless form showing an increasing usage. [3] Both these forms are associated with oral cancer, but smokeless tobacco appears to have the strongest correlation, which could be attributed to the direct contact of carcinogens with the oral epithelium. [2],[3],[4]

In a developing country like India, smokeless tobacco is frequently used in traditional forms and the most common forms are betel quid chewing, khaini, gutka, mawa, and tobacco leaves. Smokeless tobacco is not homogeneous as it is often combined with betel leaves, sliced areca nut and slaked lime and other additives that enhance the toxicity and psychotropic effects of tobacco. [5],[6],[7] While khaini is a mixture of roasted tobacco flakes and slaked lime, both gutka and mawa are mixtures of tobacco and areca nut. Mawa in addition also contains slaked lime. These forms of smokeless tobacco are placed in the buccal mucosa as they present a relatively smooth and immobile surface. [8] These chewable forms of tobacco are often mixed with fiberglass which creates microabrasions on the mucosa which helps in easier penetration of the particles to reach the circulation. Tobacco leaves are another form of chewable tobacco that are obtained primarily from the plants, which are air cured and shredded into flakes and often treated with sweet flavoring solutions. [9]

Tobacco exhibits a dose and time dependent effect on oral cancer, with the dose dependent effect having a stronger association. [10],[11],[12] Smokeless tobacco exposure in humans produces significant changes in the luminal environment of buccal mucosa. Further, the barrier and electrolyte transport function of epithelia are responsive to changes with the use of smokeless tobacco. [13]

There is an established association of certain tobacco products like gutka and khaini with oral submucous fibrosis (OSMF) and other forms like betel leaves with oral cancer. [14],[15],[16] There also exists a strong relationship between the tobacco particle size and tobacco related cancer and potentially malignant disorders. Over the past decades, numerous studies have been done to analyze the effect of chemical composition of these products on oral mucosa. However, the effects of particle size distribution of these smokeless tobacco forms on the oral mucosa are yet to be known.

Against this background, this study was a novel attempt which was aimed at determining the particle size distribution of different smokeless tobacco using computer assisted morphometric analysis and thereby to assess the degree of diffusion of these different tobacco forms using a scanning electron microscope (SEM).

 > Subjects and methods Top

Determination of particle size

The study included various types of tobacco products which comprised of khaini (Group 1), mawa (Group 2), gutka (Group 3) and tobacco leaves (Group 4). The products were photographed and 30 particles were chosen at random. The length and width of each particle was calculated and an average was taken using image analysis Image J software for Windows 95/WT/98 developed by the National Institute of Heath {Bethesada, Maryland, USA} (Wayne Rasband, NIH) after accurate calibration.

Analysis of degree of diffusion of tobacco products

The egg shell membrane test was used to study the degree of diffusion of the tobacco forms. The egg shell membrane, which is composed of ground substance and keratin, was used to simulate the buccal mucosa. [17] The eggshell membrane was obtained from fresh chicken egg, by opening the egg shell at pole opposite to the air chamber, and then emptying the albumen and yolk followed by a thorough rinse with distilled water. After emptying the contents, the external egg shell was removed by dipping the shell in 0.1N hydrochloric acid for about 1 h followed by manual removal of the shell. Thereafter, the underlying membrane was isolated, taking care to maintain the integrity of the membrane and it was washed and stored in distilled water at 4°C until use. [17],[18] Prior to the onset of the experiment, the membrane was brought to room temperature and cut in sheets of 1 cm 2 .

Subjecting the egg shell membrane to tobacco products

Prior to the exposure of the tobacco products, the membranes were rinsed with ringer solution to provide a uniform environment for the measurement of diffusion. About 0.5 g of tobacco products were measured and placed on the membrane for about 5 h/day. The membrane along with tobacco products were subjected to 5 ml of artificial saliva at equal intervals for 4 days. During this period, a constant grinding force was applied on the membrane to simulate the oral environment. Egg shell membrane not subjected to any product served as control.

Scanning electron microscope analysis

The eggs shell membrane which was exposed to various tobacco products was prepared for examination under the SEM by prefixation in 2.5% glutaraldehyde buffered with 0.1M cacodylate buffer overnight at 4°C, and rinsed with three changes of 0.1M phosphate-buffered saline (PBS) at pH 7.2 for about 15 min followed by postfixation in 1% osmium tetroxide for 2 h at room temperature. It was rinsed with two changes of 0.1M PBS for 15 min at pH 7 and dehydrated using two changes of increasing ethanol concentrations: 50%, 60%, 70%, 85%, 95%, and 100% for about 15 min.

The processed specimen was dried by the critical-point method for 3 h, mounted on aluminum stubs, and gold sputtered with colloidal gold of 100-300A° thickness. The undersurface of the specimen was then viewed with a JEOL JSM-6360 LV SEM, at an accelerating voltage of 3-30 kV. The specimen angle in the instrument was varied to give the best visual results for each specimen.

Penetration levels of tobacco products

The SEM images of various smokeless tobacco products were examined under Image analysis software for determination of the penetration levels. The particles were encircled on the image and the area of penetration with minimum and maximum particle size were calculated using Image J, Image analysis software for further comparative analysis.

 > Results Top

Particle size distribution

The dimensions of the various tobacco products were determined by image analysis [Table 1] and the particle size distribution was presented in length and width (in mm). Khaini was found to have a dimension of about 1 × 0.4 mm and was smallest in size. The dimensions of mawa and gutka were 2 × 1 mm and 4 × 3 mm and tobacco leaves with a dimension of 9 × 7 mm were found to be largest in size.
Table 1: Dimension of smokeless tobacco products as measured by image analysis

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Scanning electron microscope analysis

The egg shell membrane which was not subjected to any products (control group) demonstrated an intricately woven network composed of fibers which were randomly oriented and criss-crossing each other. There was presence of few fibers which anastomosed with each other giving a branched appearance. The fibers were parallel in the middle of the membrane, whereas the surface demonstrated a random arrangement. The fibers were regular in length and there were abundant branching among them [Figure 1].
Figure 1: Lower magnification (100) of normal egg shell membrane composed of intricately woven network with randomly oriented criss-crossing fibers

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Under ×500 magnification, the egg shell membrane exposed to khaini showed maximum particle distribution which were scattered amidst the collagen meshwork of the egg shell membrane [Figure 2]. At ×5000, the membrane demonstrated high particle penetration and disruption in the collagen meshwork. The collagen meshwork had a loose arrangement as compared to normal membrane [Figure 3].
Figure 2: Lower magnification (×500) of khaini exposed egg shell membrane showing aggregates of tobacco particles scattered among the meshwork

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Figure 3: Higher magnification (×5000) of khaini exposed egg shell membrane showing clumps of tobacco particles adherent to the meshwork along with disruption of the collagen meshwork

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The membrane exposed to mawa demonstrated a minimal particle penetration in the egg shell membrane in lower magnification (×500) [Figure 4]. At ×5000, adhesion of the particles to the meshwork was evident and the collagen meshwork demonstrated irregular arrangement with increase in the interfibrillar space [Figure 5].
Figure 4: Lower magnification (×500) of mawa exposed egg shell membrane showing tobacco particle penetration in the form of globules

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Figure 5: Higher magnification (×5000) of mawa exposed egg shell membrane showing tobacco particle adherent to the meshwork which are irregularly placed with increased distance between them

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The egg shell membrane exposed to gutka resembled that of the normal membrane under lower magnification [Figure 6]. However, the particle penetration was evident in higher magnification and the collagen meshwork was more or less regular in higher magnification. When the membrane was magnified under ×5000 objective, the collagen meshwork had an irregular surface with particle adherent to it [Figure 7].
Figure 6: Lower magnification (×500) of gutka exposed egg shell membrane resembling a normal membrane consisting of densely intricate collagen meshwork

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Figure 7: Higher magnification (×5000) of gutka exposed egg shell membrane demonstrating particle penetration with irregularly arranged meshwork of collagen fibers

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The tobacco leaf exposed egg shell membrane had minimal particle penetration in ×500 magnification [Figure 8]. However, higher magnification showed a normal collagen meshwork with minimal blebs on the surface [Figure 9].
Figure 8: Lower magnification (×500) of tobacco leaf exposed egg shell membrane demonstrating minimal particle penetration

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Figure 9: Higher magnification (×5000) of tobacco leaf exposed egg shell membrane demonstrating normal collagen meshwork with blebs on the surface of the meshwork

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Distribution of the particles on the egg shell membrane

Analysis on the rate of penetration of various tobacco products showed a large variation between the various forms with a greater penetration level for khaini particles. This was followed by mawa and gutka. The test of significance showed a P < 0.05 suggesting that the difference in areas among these three products were statistically significant. The average particle size of tobacco and gutka did not show a significant difference when the particles were analyzed at lower magnification. However, higher magnification demonstrated a statistical significance in the penetration of particles of tobacco and gutka [Table 2].
Table 2: Average area of penetration based on the particle size

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 > Discussion Top

The World Health Organization predicts that tobacco deaths in India may exceed 1.5 million annually by 2020 and this could be attributed to the use of smokeless tobacco, in various forms, which has been shown to be strongly associated with oral cancer and other potentially malignant disorders. [19] Oral cancer affects as many as 274,000 people worldwide annually, and the frequency is often indicative of the patterns of tobacco use. [20],[21] This disparity in cancer prevalence may be attributable to well-known risk habits of tobacco, paan, and alcohol abuse. [22] Most of the Indians (almost 75%) consume smokeless forms of tobacco that includes paan, gutka, pan masala, khaini, and mawa. [23] There is a rise in the incidence of oral cancers and potentially malignant disorders in recent years and the role of smokeless tobacco in oral cancer is of concern. [24] Though various factors have been identified to evaluate the association of smokeless tobacco with oral cancers and potentially malignant disorders, the direct association of particle size on these disorders has not been studied. Therefore, this study was first of its kind to assess the particle diffusion of various smokeless tobacco forms.

This study utilized the egg shell membrane, a natural substrate to simulate the buccal mucosa. The egg shell membrane, also called membrane putaminis, is a thin bilayered barrier membrane of about 150-250 μm which forms the inner portion of the egg shell and is present between egg white and shell. [25],[26] On its inner surface, the fibers are lined by a limiting membrane which is a continuous impervious layer separating the fibers from the albumen, whereas the outer layers confronting the air sac was not covered by this membrane. [27] This membrane is composed of an intricate matted lattice network of stable and water insoluble fibers with chemical properties that are intermediate between keratin and chitin. [28],[29] The organic matter of eggshell membranes contains proteins as major constituents with small amounts of carbohydrates and lipids. [30] It also contains Type I, V, and X collagens with variable amounts of proteoglycans and glycoasminoglycans namely chondroitin sulfate and hyaluronic acid. [31],[32],[33],[34],[35] Furthermore, it also represents a standard smooth and a well characterized surface, with a high surface area, which is easily available and convenient to handle than animal tissues or mucous membrane. [36] This is the first study to use the egg shell membrane to simulate the buccal mucosa for diffusion of tobacco products, and this method has been only previously used in pharmaceutical sciences to assess the drug penetration. [36]

The results of this study demonstrated a strong correlation between particle size and diffusion into the egg shell membrane. Nevertheless, all the study groups showed diffusion of particles across the membrane. Khaini (Group 1) which had the smallest dimension of about 1 × 0.5 mm demonstrated a numerous particles which were attached to the meshwork of collagen fibers. This correlated with the literature, that patients with increased usage of khaini were more prone to development of potentially malignant disorders. [37] There was also a progressive decrease in the penetration of the particles with an increase in particle size as seen in mawa (Group 2).

The particle penetration of egg shell membrane exposed to tobacco leaves (Group 4) showed tobacco particles in lower magnification which was not evident in gutka though tobacco leaves had a bigger dimension (Group 3). However, the leaves had tobacco dusts mixed with it and this explains the penetration of tobacco particles in leaves under lower magnification. Under higher magnification, egg shell membrane exposed to tobacco leaves showed more or less normal morphology, whereas the membrane exposed to gutka demonstrated particles adherent to the tubules with irregularity in the meshwork.

The results of this study can be correlated to several studies wherein the prevalence of potentially malignant disorders and oral cancer were assessed in smokeless tobacco users. A review of literature on various smokeless tobacco products in potentially malignant and malignant disorders demonstrates a greater prevalence of OSMF in gutka and mawa users and leukoplakia, erythroplakia and malignancies in tobacco leaf chewers [Table 3]. This could be due to the release of nitrosamines and other carcinogens from the tobacco leaf which could have a synergistic action on the particle size of the tobacco products. [24] Also, mawa and gutka have been the predominant forms of smokeless tobacco forms since 1970's. [38] Though khaini, having the smallest particle size has greatest association with OSMF compared with other forms, the number of studies done with khaini is comparatively less probably due to the late emergence of these products in 1990's. However, tobacco leaves which had the largest dimension have shown a strong correlation with oral cancer due to the differential release and absorption of toxic components of tobacco which are placed for a much longer time by the users. [3],[12]
Table 3: Prevalence of potentially malignant disorders/ carcinoma in patients using smokeless tobacco

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Hence, this study explains that all forms of smokeless tobacco created significant changes in the membrane irrespective of their size. However, there was a gradual increase in the rate of diffusion of particles with a decrease in particle size thus establishing an inverse relationship. Although egg shell membrane is easily available and easy to handle, it does not completely simulate the clinical scenario. Further in vitro animal studies are required to characterize the diffusion kinetics and to analyze exact changes in the buccal mucosa at the microscopic level.

This study, the first of its kind proves the association of smokeless tobacco and its forms in oral cancer and potentially malignant disorders and thus establishes a striking correlation with the particle size. The majority of oral carcinoma is preventable in India by making forceful public awareness. Identification of the high risk tobacco forms helps us to assess the magnitude of risk and thereby to decrease the incidence of precancerous lesions eventually resulting in a decrease the mortality rates and thereby being beneficial to the mankind.

 > Acknowledgment Top

The authors would like to thank the Department of Geological Sciences, Anna University for having helped us with the scanning electron microscope.[46]

 > References Top

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]

  [Table 1], [Table 2], [Table 3]


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