| Abstract|| |
Background: Oral submucous fibrosis (OSMF) commonly seen in the South Asian countries is mostly associated with the chewing of areca nut (AN). Copper content in the AN has been implicated to play a major role in the pathogenesis of OSMF. It was found that most of the AN plantations in South India commonly use a copper-based fungicide, Bordeaux mixture (BM). Objective: To evaluate the level of copper in the AN, root, and soil of the AN plantation with and without the application of BM and to correlate the role of copper in the pathogenesis of OSMF. Materials and Methods: ANs, roots, and soil were obtained from plantations located in Tanniadi, Kerala. Four areas were selected from the plantation with and without BM application. The samples were collected twice with the interval of 6 months during January 2015 and July 2015, respectively. Statistical Analysis: Arithmetic mean and standard deviation were calculated. The differences between means were calculated by paired sample t-test. Results: There was statistically significant difference in the copper content of ANs, soil, and roots from both groups (P < 0.05). Samples treated with BM showed significantly higher copper levels as compared to their counterparts in January and July 2015. Conclusions: External copper from BM and increased processing for the commercial products could collectively increase the total copper content of the commercial AN products, and this high copper concentration may be implicated to the pathogenesis and the increasing prevalence of OSMF.
Keywords: Areca nut, Bordeaux mixture, copper, oral submucous fibrosis
|How to cite this article:|
Alexander AJ, Ramani P, Sherlin HJ, Gheena S. Quantitative analysis of copper levels in areca nut plantation area – A role in increasing prevalence of oral submucous fibrosis: An in vitro study. Indian J Dent Res 2019;30:261-6
|How to cite this URL:|
Alexander AJ, Ramani P, Sherlin HJ, Gheena S. Quantitative analysis of copper levels in areca nut plantation area – A role in increasing prevalence of oral submucous fibrosis: An in vitro study. Indian J Dent Res [serial online] 2019 [cited 2020 Feb 21];30:261-6. Available from: http://www.ijdr.in/text.asp?2019/30/2/261/259226
| Introduction|| |
Oral submucous fibrosis (OSMF) is a potentially malignant disorder and crippling condition of the oral mucosa. In 1953, Joshi described this condition as “submucous fibrosis.”,, It was defined by Pindborg and Sirsat in 1966 as an insidious, chronic disease affecting any part of the oral mucosa, sometimes the pharynx. Although occasionally it is preceded by and/or it is associated with vesicle formation, it is always characterized by juxta epithelial inflammatory reaction followed by fibroelastic change in the lamina propria and epithelial atrophy, leading to stiffness of oral mucosa causing trismus and inability to eat.,
In an epidemiological study on oral cancer and precancerous lesions in a rural Indian population, the malignant transformation rate of OSMF was 7.6% (5 of 66) over a 17-year period (median observation, 10 years).
It is hypothesized that OSMF is associated with increased collagen production and reduced collagen degradation. The synthesis of collagen is influenced by a variety of mediators, including growth factors, hormones, cytokines, and lymphokines. A prominent mediator is transforming growth factor-beta (TGF-β); TGF-β1, in particular, seems to be the one that plays a major role in wound repair and fibrosis. This growth factor has also been implicated in the development of many fibrotic diseases. Although TGF-β is essential for healing, overproduction leads to scar tissue and fibrosis.
Among all the possible etiological factors, areca nut (AN) plays a major role in the pathogenesis of OSMF.
AN is the fourth commonly used psychoactive substance chewed as an aid to digestion and as stimulant, either used alone or added with different tobacco or nontobacco substances to make different combinations.
The proven carcinogenic contents of AN are the flavonoids, tannins, and most of the alkaloids. The alkaloids are the arecoline, arecadine, guvacine, and guvacoline. Arecoline is the main agent responsible for fibroblast proliferation. Under the influence of slaked lime (Ca (OH)2), arecoline gets hydrolyzed to arecadine, which has pronounced effects on fibroblasts. Areca flavonoids, tannins, and catechins can cause increased fibrosis by forming a more stable and nonsoluble collagen structure by inhibiting collagenase enzyme activity. Apart from this, AN also contains a significant amount of copper (302 nmol/g) that also plays a major role in the pathogenesis of OSMF.
It is suggested that substantial amounts of copper is released into saliva on chewing areca products and is absorbed into oral mucosa. The copper is either bound to protein metallothionein or transferred across the basolateral membrane. Glycyl-L-histidyl-L-lysine (GHK), a tripeptide that possesses a high affinity for copper ions, with which it forms a complex (GHK-Cu) is reported to stimulate collagen synthesis. This is released within the lamina propria zone of the AN chewers.
Copper acts by upregulating lysyl oxidase activity, which is the key enzyme in collagen metabolic pathway. Thereby, copper enhances collagen synthesis by fibroblasts, facilitates its crosslinking, and eventually inhibits its degradation., Copper may also bind to the protein product of p53 causing p53 aberrations in the oral keratinocytes. It is hypothesized that the levels of copper is increased in the AN due to the application of a fungicide (Bordeaux mixture [BM]) which is used to spray on the plantation during the monsoon season.
BM is a mixture of copper sulfate (CuSO4) and slaked lime (Ca(OH)2). It achieves its effect by means of the copper ions (Cu2+) of the mixture. These ions affect enzymes in the fungal spores in such a way as to prevent germination. It is hypothesized that the incorporation of copper into the AN is through the BM of which CuSO4 is an important constituent. The copper may get incorporated into the nut and leaves which is again absorbed by the plant as the leaves and nut shed off and get decomposed and absorbed within the soil.
Kerala accounts for 22.47% of the area under AN in India contributing to 13.70% of national production. According to Rajendran et al., the epidemiological assessment shows 0.4% prevalence for OSMF in Kerala, South India, which is among the highest recorded. Therefore, this study was designed to explore and quantify the levels of copper in the AN, root, and soil from the AN plantations in Kerala with and without the application of BM.
| Materials and Methods|| |
One AN plantation from Tanniadi, Kerala, with a 30-year history of BM application was selected in one group (Group A). Another plantation without BM usage for the last 20 years was identified in the same place and constituted the other group (Group B) in the study. ANs, roots, and soil were obtained from these plantations. Four areas from each plantation were selected. Five ANs, root, and soil were obtained from each area [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]. The samples were collected twice with the interval of 6 months during January 2015 and July 2015, respectively.
A total number of 24 samples were obtained in January 2015: 4 AN with BM application, 4 root with BM application, 4 soil with BM application, 4 AN without BM application, 4 root without BM application, and 4 soil without BM application. The same number of samples was obtained in July 2015 from the same areas from those AN plantations.
All ANs were dehusked, and the nut was grounded and homogenized using a mortar and pestle and was stored in clean polyethene bags. The roots were dried, scrapped, and homogenized using a mortar and pestle; soil was dried and stored in clean polyethene bags. Five milliliters of nitric acid was added to the samples to dissolve the copper in CEM microwave digester using MARSXpress (self-regulating microwave vessel). The digested solution was made up to 50 ml using deionized water, and it was thoroughly filtered using Whatmann 40 filter paper. The clear solution was analyzed by inductively coupled plasma optical emission spectroscopy (ICP-OES). A blank solution was also prepared in a similar manner and the intensity values were subtracted. This solution was subjected to copper analysis with ICP-OES by producing excited atoms and ions that emit electromagnetic radiation at wavelengths characteristic of a copper. The obtained results were expressed in mg/L.
The data obtained were subjected to a statistical analysis, and basic variation statistical values (arithmetic mean and standard deviation) were calculated. The differences between means were calculated using paired sample t-test.
| Results|| |
The study included a total of n = 24 samples which were divided into six groups. [Table 1] represents the levels of copper in AN, root, and soil between the two plantations with and without the application of BM in January 2015. [Table 2] represents the difference between the mean value of the samples with and without BM in January 2015. [Table 3] and [Table 4] represent the mean and standard deviation and the difference between the samples with and without Bordeaux mixture in July 2015, respectively. The comparison in copper content between AN, root, and soil from January and July 2015 was also calculated [Table 5]. The data show a significant increase in the copper content of the samples in July as compared to the samples in January.
|Table 2: Difference between the samples with and without Bordeaux mixture in January 2015|
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|Table 4: Difference between the samples with and without Bordeaux mixture in July 2015|
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|Table 5: Statistical analysis of copper levels among the variables in the January and July samples|
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| Discussion|| |
While the AN is fresh, the husk is green and the nut inside is very soft, whereas in a ripe fruit, the husk becomes yellow or orange and the fruit inside hardens to a wood-like consistency. As the nut matures, the moisture content decreases in the nut, thereby the copper content increases as it mineralizes. Jayalakshmi and Mathew have mentioned about the difference in moisture content in the unripe and ripe AN. These unripe nuts which are rich in moisture content showed the least copper levels for a given weight compared to the exfoliated mature nuts that showed higher amounts of copper and relatively less moisture. Similar results were obtained by Mathew et al. in which copper content increased significantly with maturity in raw ANs, showing that the copper content in commercial products was significantly higher than the raw AN. Studies by Shakya et al. and Trivedy et al. also revealed higher copper levels in the commercial AN products.,
Prevention of the diseases of the areca plantation during the monsoon season is done by spraying 1% BM as a prophylactic spray just before the onset of monsoon. BM is a fungicide which is prepared by adding milk of lime to a solution of CuSO4 until alkaline reaction is obtained. The spray gives a coating on the tender nut and prevents it from rotting and falling off. Thus, postmonsoon, the ripened nuts are picked. This copper incorporated in the nut and leaves is again absorbed by the plant as the nut and leaves shed off and get decomposed and absorbed within the soil. It is emphasized that the incorporation of copper into the AN is through the BM.
In our study, the samples from January 2015 and July 2015 showed increased amount of copper in the AN, root, and soil from the plantations with the application of BM compared to their counterparts in the other group, the copper being highest in the soil followed by root and the least in the AN.
Our findings correlate with a study by Mathew in 2015 in which the copper content in the ANs from plantations with BM application was significant higher than from the plantations without BM application.
Spraying of BM on the areca branches, leaves, and tender nuts may enable the soluble copper to penetrate the cuticle and gain access to the AN. The copper incorporated in the nut and leaves is again absorbed by the plant and the leaves and the nut shed off and get decomposed and absorbed within the soil. While spraying the BM onto the trees, certain amount of the BM also falls onto the soil in the plantation. Moreover, during heavy rainfall, the BM also is washed away from the nut and leaves and is incorporated into the soil which is further absorbed by the soil. Our study showed statistical significance in the difference in the levels of copper content of AN among the plantations with and without BM application during January (P < 0.001) and July 2015 (P < 0.003). Both the plantations were from the same locality that was exposed to similar natural geographical features and with not much difference in the mineral contents of the soil for the plantation.
The copper from the soil is mostly transported via the plant root and thereby is incorporated in the nut. Our study showed statistical significance in the difference of the levels of copper content of root of the plantations with and without BM application in January 2015 (P < 0.007) and July 2015 (P < 0.008). Furthermore, it showed statistical significance in the difference of the levels of copper content of soil of the plantations with and without BM application in January 2015 (P < 0.002) and July 2015 (P < 0.002). Hence, this shows that the copper levels were higher in the plantations with BM application and the copper from the soil is absorbed by the roots. Copper has low solubility, which enables it to persist in the soil for years.
According to Liu et al. in 2001, the plants transported and concentrated only a small amount of Cu to their shoots. Furthermore, Graham et al., in 1985, suggested that copper is accumulated in roots in proportion to the amount added to the soil. According to Badilla-Ohlbaum et al., copper contents of stems and leaves were significantly higher for plants growing in the high-Cu soils.
The comparison of the samples from January 2015 to July 2015 was done to check for absorption of copper over the period of 6 months after the application of BM.
It is clear from our study that BM plays an important role in the copper content of the AN. The plantations which have been subjected to the application of BM for years have developed an increase in the amount of copper content in the soil and the root. This leads to increased incorporation of the copper into the AN. It was observed that minute amounts of copper through the BM were distributed through all plant tissues. Others thought that copper might be an essential element in the metabolism of plants and animals.
Most of the plantations in South India use BM application. Understandably, the high concentration of copper in the commercial areca products can be attributed because of the mineralization of copper and further because of the BM.
The reason for rapid increase of the disease is reported to be due to an upsurge in the popularity of commercially prepared AN preparations in India and increased uptake of this habit by young people is because of addiction to this habits.,
Thus, external copper from BM and increased processing for the commercial products could collectively increase the total copper content of the commercial AN products, and this high copper concentration may be implicated to the pathogenesis and the increasing prevalence of OSMF. Our study has clearly quantified the copper levels, and it has given a good insight into the present trend of OSMF.
Although various campaigns and government initiatives including bans are in vogue, it is not in our power to diminish the usage of AN by population. However, elimination of the addition of BM which is rich in copper content that plays a crucial role in the pathogenesis of OSMF can be advocated and the farmers can be educated on the same. In addition, copper-free fungicidal alternatives can be utilized. The previous study done on this analyzed only the copper levels in the AN with and without BM application. This is the first study of its kind which has analyzed the copper levels in AN, root, and soil and gives an insight on the current trend and possible role of copper in the pathogenesis of OSMF.
| Conclusions|| |
The present study concludes that the difference in the levels of copper between AN, root, and soil from the plantations with the application of BM compared to the plantation without the application of BM in January and July 2015 showed statistical significance. The copper-based fungicide (BM) plays an important role in the copper content of the AN. It can be assumed that copper through the soil reaches the root and further the nuts and attributes to the increased copper content in it. External copper from BM and increased processing for the commercial products could collectively increase the total copper content of the commercial AN products, and this high copper concentration may be implicated to the pathogenesis and the increasing prevalence of OSMF. OSMF has now shown a change in the trend with increase in the occurrence among the younger age group including the 2nd decade of life due to the low cost, ease of availability of the products, attractive packages, and aggressive marketing.
We would like to thank Dr. Kritika Jangid and Dr. Zoha Abdullah for giving their timely help rendered in the statistical analysis of the study.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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Dr. Aurelian Jovita Alexander
Department of Oral and Maxillofacial Pathology, Saveetha Dental College, Chennai, Tamil Nadu
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]