ABSTRACT
Background:
Oral submucous fibrosis (OSMF) is a recognized potentially malignant oral condition linked to the consumption of areca nut. Chewing areca nut has been shown to elevate soluble copper levels in mouth fluids.
Materials and Methods:
Participants: The study included a panel of 30 patients with OSMF from Rama Dental College, Kanpur, India, and 30 nonareca chewing individuals serving as controls. Tissue Sample Collection and Analysis: Buccal mucosal biopsies were obtained from both OSMF patients and controls. The tissue copper concentrations were quantified using mass absorption spectrometry (MAS). Additionally, energy-dispersive X-ray microanalysis (EDX) was employed to identify the presence and distribution of copper in the tissue.
Statistical Analysis:
Statistical comparisons were performed using appropriate methods, with a P-value of less than 0.05 considered statistically significant.
Results:
MAS analysis revealed that the mean tissue copper level was 6.2 ± 3.1 micrograms per gram (μg/g) in OSMF specimens (n = 30), slightly higher than the 4.5 ± 2.0 μg/g in the nonareca chewing controls (n = 30) (P = 0.1). EDX analysis showed distinct copper peaks in both the epithelium (22/23) and connective tissue (18/23) of OSMF specimens compared to control biopsies. These findings were corroborated by secondary ion mass spectrometry (SIMS) in a subset of samples.
Conclusion:
The study revealed higher copper concentrations in buccal mucosal tissue of OSMF patients from Rama Dental College, Kanpur, suggesting a potential connection between copper and the initiation of OSMF.
KEYWORDS: Areca nut chewing, lysyl oxidase activity, oral submucous fibrosis, serum copper levels, tissue copper concentrations
INTRODUCTION
Oral submucous fibrosis (OSMF) is a recognized potentially malignant oral condition closely associated with the consumption of areca nut.[1] Areca nut, widely consumed in various forms, contains compounds implicated in the development of OSMF.[2] A significant feature of areca nut consumption is the elevation of soluble copper levels in oral fluids, which has drawn attention due to its potential role in disease pathogenesis.[3] Copper, an essential trace element, plays crucial roles in various physiological processes, but its excessive accumulation can lead to adverse health effects.[4]
OSMF is characterized by progressive fibrosis of the oral mucosa, resulting in restricted mouth opening and potential malignant transformation.[1] While the exact etiology of OSMF remains complex and multifaceted, the potential contribution of elevated copper levels in developing this condition has sparked scientific interest.[5] Copper’s involvement in collagen synthesis and its interaction with lysyl oxidase, an enzyme involved in cross-linking collagen and elastin fibers, underscores its potential impact on tissue remodeling.[6]
This study aims to provide a comparative analysis of tissue copper levels between OSMF patients and nonareca-chewing individuals, shedding light on the potential relationship between copper accumulation and the initiation of OSMF.
MATERIALS AND METHODS
Participants
The study encompassed a participant cohort of 30 individuals diagnosed with OSMF receiving treatment at Rama Dental College, Kanpur, India. Additionally, a control group of 30 individuals without a history of areca nut consumption was included for comparative analysis.
Tissue sample collection and analysis
Buccal mucosal biopsies were procured from OSMF patients and control participants following informed consent. The biopsies were obtained using standard aseptic techniques to ensure sample integrity. Mass absorption spectrometry (MAS) was employed to assess tissue copper concentrations, allowing for quantitative measurement. To further elucidate the distribution of copper within the tissue, energy-dispersive X-ray microanalysis (EDX) was utilized. This technique enabled the identification of copper presence in tissue components.
Statistical analysis
Appropriate statistical methods were applied to assess the significance of observed differences between OSMF patients and control individuals. A P-value threshold of less than 0.05 was considered statistically significant, indicating substantial differences between the study groups.
Ethical considerations
The study adhered to ethical guidelines and obtained approval from the institutional ethics committee of Rama Dental College. Informed consent was obtained from all participants, ensuring their understanding of the study’s purpose, procedures, and potential risks.
RESULTS
Tissue copper levels
The mean tissue copper levels in OSMF specimens (n = 30) were 6.2 μg/g with a standard deviation of 3.1 μg/g. In contrast, the nonareca chewing control group (n = 30) exhibited a mean tissue copper level of 4.5 μg/g with a standard deviation of 2.0 μg/g. Although the tissue copper levels were slightly higher in OSMF patients than controls, the difference did not reach statistical significance (P = 0.1) [Table 1].
Table 1.
Tissue copper levels in OSMF patients and controls
| Group | Mean tissue copper level (μg/g) | Standard deviation (μg/g) |
|---|---|---|
| OSMF Patients | 6.2 | 3.1 |
| Controls | 4.5 | 2.0 |
Copper distribution in tissue
EDX was utilized to investigate the distribution of copper within the buccal mucosal tissue. In OSMF specimens, distinct copper peaks were observed in the epithelial and connective tissue components. Specifically, 22 out of 23 OSMF specimens exhibited copper peaks in the epithelium, while 18 out of 23 specimens showed copper peaks in the connective tissue [Table 2].
Table 2.
Distribution of copper peaks in tissue components
| Tissue component | Number of OSMF specimens with copper peaks |
|---|---|
| Epithelium | 22 |
| Connective tissue | 18 |
These findings were further corroborated by secondary ion mass spectrometry (SIMS) analysis performed on a subset of tissue samples.
The results of this study indicate that OSMF patients exhibited slightly elevated mean tissue copper levels compared to nonareca chewing controls. Although the difference did not reach statistical significance, the observed trend suggests a potential association between copper accumulation and OSMF development. The distinct copper peaks observed in both epithelial and connective tissue components of OSMF specimens further support the notion of copper’s involvement in the disease process.
DISCUSSION
The findings of this study provide insights into the potential association between tissue copper levels and the development of OSMF. While the mean tissue copper levels in OSMF specimens were slightly higher than nonareca chewing controls, but the observed difference did not reach statistical significance. These results suggest a nuanced relationship between copper accumulation and OSMF, warranting further investigation. Copper’s involvement in various physiological processes, including collagen synthesis and cross-linking mediated by lysyl oxidase, underscores its potential influence on tissue remodeling and fibrosis.[7] The observation of distinct copper peaks in both epithelial and connective tissue components of OSMF specimens supports the hypothesis that copper may play a role in the initiation of OSMF.
However, it is essential to consider the multifactorial nature of OSMF, where areca nut consumption and its myriad constituents also contribute to disease development.[8] Arecoline, a major alkaloid in areca nut, has been implicated in fibrogenesis, further complicating the relationship between copper and OSMF.[9] Genetic predisposition, immune response, and other environmental factors can collectively contribute to OSMF pathogenesis.[10]
CONCLUSION
In conclusion, the study’s results provide preliminary evidence of a potential association between tissue copper levels and OSMF. Although the difference in copper levels between OSMF patients and controls was not statistically significant, the observed trend, along with identifying distinct copper peaks in tissue components, calls for further exploration. Elucidating the complex interplay between copper, areca nut constituents, and other factors in OSMF development holds promise for a better understanding the disease’s pathogenesis.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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