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This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms.
Oral squamous cell carcinoma (SCC) is one of the most common malignancies in oral cavity. Hence, presenting methods for early diagnosis and find the etiologic factors of oral SCC are important. Saliva analysis can be used to discover various conditions because of its noninvasive methods. Copper as a useful metal has been used by men since ancient times. The level of copper increases when the cancerous changes occur in addition to biopsy, an alternative method for examining oral lesions is exfoliative cytology. The primary objective of this study was to determine the salivary copper level and cytomorphologic changes of oral mucosa among three study groups.
This cross-sectional study included 15 individuals with oral SCC, 15 workers exposed to copper, and 15 healthy individuals. Saliva samples were collected and analyzed by atomic absorption spectrophotometer. The exfoliative smears were prepared by brush biopsy and stained by Papanicolaou and argyrophilic nucleolar organizer region (AgNOR) staining methods. Data analysis using one-way ANOVA and Kruskal–Wallis test. P < 0.05 was considered significant.
There was a significant difference in mean salivary copper (P = 0.008), cytomorphology of oral mucosa, and AgNOR among the three groups (P < 0.001).
The results suggested that occupational exposure to copper increases the salivary levels of this element and causes changes in mucosal cells. Since this increase was very high and evidence of nuclear activity was seen in this group and in oral SCC patients, exposure to copper should be considered an important risk factor for oral mucosal changes.
Head-and-neck squamous cell carcinoma (SCC), which involves oral cavity, oropharynx, and hypopharynx, is the fifth most commonly occurring cancer in the world and is one of the most frequent causes of death in humans.
Copper is one of the most useful metals, mined and worked by men since ancient times, because of its essential use in various spheres of life.
Recently, saliva analysis had been adverted due to its simple, noninvasive collection, and low-cost storage methods. Oral fluid sampling is safe for the operator and patient.
These days, exfoliative cytology as an alternative method for oral lesions examination is simple, noninvasive, and inexpensive and can be used to conveniently identify biomarkers in laboratories. It is routinely done in medical and dental offices, which may increase the early diagnosis of oral mucosal malignancies in clinical trials.
Papanicolaou (PAP) staining is a common method used for cytology analysis and allows it to identify primary inflammation, dysplastic, or malignant changes.
Over time, the ability of other staining methods and markers to enhance the reliability of exfoliative cytology has been investigated, one of which is silver nitrate staining (argyrophilic nucleolar organizer region [AgNOR]).
The aim of this study was to determine the level of salivary copper and cytomorphologic changes of oral mucosa in patients with oral SCC, healthy people, and people with occupational exposure to copper.
In this cross-sectional study, 15 individuals with oral cancer, 15 workers exposed to copper (workers of the copper melting factory who had at least 5 years of work experience), and 15 healthy volunteers in dental school (control group) were included in the study. The participants were asked to wash their mouths with a physiological serum before starting saliva collection. Then, each individual sat in a comfortable position starting the saliva collection with open eyes, bending slightly forward, and evacuating 5 ml of saliva in the test tube. The specimens were then stored at −20°C and sent to the laboratory for evaluation of salivary copper. After the oral examination, using a disposable cytobrush on the mucosa (in the healthy group and workers, from the buccal mucosa and oral SCC from the lesion area), the cell samples were collected and transferred to a clean and dry slide. Before drying, the specimens were fixed immediately by Pathofix spray (Iran's antibacterial medicine company), which contains 95% ethanol. Then, the slides were stained separately by PAP and AgNOR.
Saliva samples were analyzed by atomic absorption spectrophotometer, and the results were expressed as micrograms per liter (μg/L). The PAP-stained slides were analyzed under a microscope (Olympus BX41TF, Tokyo, Japan) at (×100) magnification, and the parameters mentioned in the smears were analyzed and grouped. The parameters analyzed in the smears by PAP stain included large nuclei, nuclear changing in size and shape, pleomorphism, nuclear borders, nucleus-to-cytoplasm ratio, number of nuclei, and hyperchromatism. Dysplastic changes were registered and grouped in sequence
Class I: Normal Class II: Atypical Class III: Indeterminate Class IV: Suggestive of cancer Class V: Positive for cancer. Then, the AgNOR-stained slides were evaluated for NOR count. In each slide, several cells were explored at (×100) magnification, and one cell was randomly selected. The brown–black spots in the nucleus were counted. The interconnected and nucleolus points were considered one point. The areas of necrosis, severe inflammation, and artifacts were not calculated.
Ethics
This study has been approved by the Research and Ethics Committee of Isfahan University of Medical Sciences by No: 396089.
One-way ANOVA showed a significant difference in mean salivary copper (P = 0.008) and AgNOR (P < 0.001) among the three groups
Argyrophilic nucleolar organiser region stain (×100) (a) Control group, (b) Worker, (c) Oral squamous cell carcinoma patient.
Kruskal–Wallis test showed that the cytomorphology of oral mucosa was significantly different between the three groups (P < 0.001). To compare the cytomorphology of oral mucosa between two groups, Mann–Whitney test was used. This test showed that oral mucosal cytomorphology classes were significantly higher in people with oral SCC than in workers (P < 0.001) and in workers was significantly higher than in the control group [P < 0.001,
Papanicolaou stain (×100) (a) Normal cells (Class I), (b) Atypical cells (Class II), (c) Indeterminate cells (Class III), (d) Positive for cancer (Class V).
Oral SCC involves half of the oral cavity malignancies and is one of the major health problems in developing countries that leads to death.
Several factors are involved in the development of oral SCCs, including lifestyle and environmental factors. However, efforts have been made by clinical epidemiologists to illustrate the link between malignancies and scarcity or excessive presence of rare elements.
Some evidence and studies have shown that long-term exposure to an average amount of copper, as in various occupational environments, affects people's health. Moreover, some studies have reported a positive relationship between copper levels in the serum, saliva, and malignancy outbreak.
In this study, the copper level in saliva and cytomorphological changes in the oral mucosa of subjects with occupational exposure to copper in comparison with healthy people and those with oral cancer were evaluated using exfoliative cytology and AgNOR staining.
The salivary copper level in this study showed a significant difference between the three groups. It was 1215.5 in the workers, which was higher than normal and oral SCC patients. However, it was higher in people with SCC 143.3 than in healthy individuals 31.5.
Several studies have investigated the serum copper level in various oral, head, and neck lesions, but few studies have examined the salivary copper level in patients.
Al-Rawi and Talabani reported that the salivary levels of copper and iron were higher in malignant individuals than in healthy people.
The results of this study were along with those of the above studies, indicating that the amount of salivary copper increased when malignancy occurred. Although it cannot be argued with certainty that this increase is a secondary response to malignant changes or the patients; for some other reasons, had increased levels of salivary copper before malignancy occurrence. This increase stimulates the proliferation of endothelial cells and plays an important role in angiogenesis, which can be effective in the process of malignancy. Hence, increasing salivary copper content in the workers exposed to this element needs special consideration. Although saliva was easily accessible and could reflect the presence of rare elements in the body, in this study, salivary levels of copper were investigated using a precise method, atomic absorption.
The results of the exfoliative cytology evaluation showed a significant difference among the three groups. The exfoliated cells in patients with SCC were classified as Class V, which were positive for malignancy and showed malignant diagnostic criteria. Cellular changes in the oral mucosa of workers were significantly different from those in normal people. In this group, the cells showed atypical changes (Class II) and intermediate changes (Class III), while in the healthy people group, all cells were normal (Class I) without any changes.
In 2014, Gonzalez Segura et al. examined the relationship between PAP staining and cytomorphometric analysis in exfoliative cytology of oral mucosa and observed that these methods had high accuracy in the diagnosis of malignancy.
Verma et al. (2015) conducted a study to evaluate the usefulness of this method in the diagnosis of oral malignant lesions and observed that assessment of changes in the nucleus and cytoplasm by cytomorphometry could be utilized as a very useful complement in the diagnosis and prognosis of dysplastic lesions which could be transformed to malignancy.
The results of this study by this method were in agreement with those of previous studies. Cellular changes that have been made and their transformation from normal to atypical and intermediate cells can indicate the effects of copper on these cells, which requires a special follow-up despite the absence of clear tissue changes.
The mean number of AgNOR showed a significant difference between the three groups. The mean number of AgNORs in oral malignant lesions was 8.8, which was significantly higher than that of the workers with a mean of 4. In the normal group, the mean number of AgNOR was 1.9, which was significantly lower than those of the other two groups.
In a similar study in 2014, Mansoor Samadi et al. showed the mean staining dots by AgNOR in specimens of normal mucosa, leukoplakia, and SCC increased, respectively.
Since the diagnosis of dysplastic changes and suspected malignant lesions is difficult, and sometimes, there is no strong evidence to proof malignancy, evaluation of cytomorphological changes, especially the use of AgNOR staining can be helpful.
The results of this study and other similar studies showed that salivary levels of copper increased in the people exposed to various levels of copper due to the occupational environment, and despite the absence of any apparent changes in the tissue, the mucosal cells exhibited cellular changes. These changes consisted of Class II and Class III of cytomorphology classes, which were confirmed by counting AgNOR-positive points and were higher than the control group. An interesting point in this study was that having longer working experience and more exposure period to copper, increased the amount of salivary copper, and also the changes in epithelial cells. The cytology changes and the number of colored points by AgNOR were higher in these workers than in the others with less work experience. This could indicate that the harmful effects of copper would increase due to the increasing duration and amount. Therefore, populations that are exposed to the particles and dust of this element require special attention.
According to the results of the study, it can be concluded that occupational exposure to copper increases the salivary levels of this element and causes some changes in mucosal cells. These changes consist of nuclear activity enhancement and an increase in the number of NOR in the nucleus. Since this increase is very high in salivary levels of copper and nuclear activity in oral SCC patients, exposure to copper should be considered an important risk factor for oral mucosal changes.
Acknowledgments
This study has been approved by the Research and Ethics Committee of Isfahan University of Medical Sciences and has been supported financially by grant No: 396089.
The authors also would like thanks from Mr. Nasr for his kind cooperation in staining the slides.
Author's contribution
NT contributed in the conception of the work, conducting the study, definition of intellectual content, interpretation of data for the work, manuscript preparation, editing and review, approval of the final version of the manuscript, and agreed for all aspects of the work. AA contributed in the design of the study, definition of intellectual content, literature search, clinical and experimental, data acquisition, data analysis, statistical analysis, manuscript preparation, editing and review, approval of the final version of the manuscript, and agreed for all aspects of the work. NK contributed in the conducting the study, clinical and experimental and data acquisition, manuscript preparation, editing and review, approval of the final version of the manuscript, and agreed for all aspects of the work. AM contributed in the in the conducting the study, clinical, experimental and data acquisition, revising the draft, approval of the final version of the manuscript, and agreed for all aspects of the work.
Financial support and sponsorship
This study has been approved by the research and ethics committee of Isfahan University of medical sciences and has been supported financially by Grant No: 396089.
Conflicts of interest
The authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or non-financial in this article.