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 Table of Contents  
Year : 2019  |  Volume : 31  |  Issue : 4  |  Page : 293-297

Evaluation of the salivary zinc assay as a potential diagnostic tool in potential malignant and malignant lesions of the oral cavity

1 Department of Oral Medicine, Faculty of Dentistry, Tishreen University, Latakia, Syria
2 Department of Laboratory Medicine, Tishreen University Hospital, Latakia, Syria

Date of Submission01-Nov-2019
Date of Acceptance11-Jan-2020
Date of Web Publication03-Mar-2020

Correspondence Address:
Dr. Marwa Mohmmad Khalil
Department of Oral Medicine, Faculty of Dentistry, Tishreen University, Latakia
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jiaomr.jiaomr_180_19

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Introduction: Recently, trace elements are receiving much attention in the detection of oral cancer and potentially malignant disorders, as they are found to be significantly altered in the head and neck, lung, and breast carcinomas. Undoubtedly, by early diagnosis of potentially malignant lesions based on the values of micronutrients, the occurrence of cancers may be decreased. Aims and Objectives: The aim of the study was to evaluate the salivary levels of zinc (Zn) in patients with oral squamous cell carcinoma (OSCC) and oral leukoplakia (OL). Materials and Methods: The study subjects included histopathologically confirmed cases of OL and OSCC with age and sex-matched healthy controls, reporting to the Department of Oral Medicine, Faculty of Dentistry, Tishreen University from January 2017 to July 2019. The subjects were asked to spit in a graduated container every 1 min till 5 mL of saliva was obtained. Levels of Zn were estimated by standard spectrophotometric methods in accordance with the underlying validated principles using the kits from Medichem, Germany. Settings and Design: This study is a randomized clinical trial. Statistical Analysis Used: ANOVA and Chi-square'' test. Results: There was a highly significant decrease in the level of salivary Zn in patients with OSCC when compared to OL patients and controls (P < 0.05). Salivary Zn levels were decreased in OL patients when compared to controls. However, the difference was not statistically significant. Conclusion: Salivary Zn could be used as a biomarker for OSCC and OL.

Keywords: Leukoplakia, oral squamous cell carcinoma, saliva, zinc

How to cite this article:
Khalil MM, Zraiki S, Khayat MI. Evaluation of the salivary zinc assay as a potential diagnostic tool in potential malignant and malignant lesions of the oral cavity. J Indian Acad Oral Med Radiol 2019;31:293-7

How to cite this URL:
Khalil MM, Zraiki S, Khayat MI. Evaluation of the salivary zinc assay as a potential diagnostic tool in potential malignant and malignant lesions of the oral cavity. J Indian Acad Oral Med Radiol [serial online] 2019 [cited 2022 May 26];31:293-7. Available from: https://www.jiaomr.in/text.asp?2019/31/4/293/279860

   Introduction Top

Oral cancer is the most prevalent malignant neoplasms within the head and neck that accounts for more than 300,000 new cancer cases and 145,000 deaths per year worldwide, with higher rates in the developing countries.[1] The development of oral cancer is a multistep process arising from preexisting potentially malignant lesions. Leukoplakia is the most common precancer representing 85% of such lesions [Figure 1]. It has been suggested that a vast majority of oral squamous cell carcinomas (OSCC) arise from preexisting leukoplakia.[2] Histologically, over 95% of oral cancers are squamous cell carcinomas [Figure 2].[3] Alcohol, viruses, genetic mechanisms, Candida, chronic irritation, and diet deficiency states are also implicated in the etiology.[2]
Figure 1: 50-year-old patient with oral leukoplakia

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Figure 2: Patient with oral squamous cell carcinoma stage III

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At the cellular level, most carcinogens cause free-radical induction and the damage inflicted by it is implicated in the etiopathogenesis of cancer.[4],[5] The electrophilic reactive oxygen species [ROS] and reactive nitrogen species [RNS] generated damage to DNA and nucleophilic biomolecule. When the generation of free radicals supersedes the protective effects of antioxidants, it will consequentially cause genetic damage to initiate carcinogenesis. On the other hand, the length and the concentration of the exposure to carcinogens is the most pivotal extrinsic factor for initiation, propagation, and progression of cancer.[5]

In addition to the free radicals, metals such as iron, zinc, copper, molybdenum, and selenium are essential in the metabolism and cell defense process.

Many studies have shown that increased levels of iron and copper catalyze the formation of ROS through Fenton and Haber-Weiss reactions to generate highly reactive hydroxyl radicals.[6] On the contrary, when compared to iron and copper, zinc has a contrasting role and is an essential nutrient for wound healing, normal growth, response to infections, growth of epithelial cells, and tissue repair.[7],[8]

Zn is reported to have antioxidant properties and plays a key role in the regulation of the immune system and cell-mediated immunity of the human body.[9]

In addition, Zn has antiproliferative and proapoptotic properties useful in terming as a chemoprotective agent in carcinogenesis.[5] Zn also stimulates gene transcription and cell multiplication and is critical for the activation of DNA and RNA polymerase activity and is important in the multiplication of tumor cells.[10] Reduced serum Zn concentrations are well known as typical laboratory characteristics in advanced head and neck cancer.

Recently, saliva has been gaining importance as a valuable prognostic marker in head and neck ailments, mainly because its collection is noninvasive, it does not require phlebotomist, and has the least compliance problems when compared to blood.[11],[12]

Most of the molecules that are found in the body fluids like blood and urine are also found in the saliva, although in lesser concentration thus making saliva an important diagnostic tool.[13] Salivary research has shown that saliva represents a valid alternative diagnostic fluid for multivarious local or systemic diseases, making research in this field a top biomedical priority of the 21st century.[14] Saliva possesses an array of qualities which recommend it as an appropriate diagnosis approach such as effortless collection procedure, facile manipulation and sampling methods, favorable sensitivity, low quantity samples are required for detection, good cooperation with patients during collection, positive correlation between blood markers and the ones found in saliva, and the possibility of caring out dynamic studies.[15],[16],[17]

At present, early and accurate clinical non-invasive prediction of oral cancer and oral potentially malignant disorders remains challenging. Hence, the present study was undertaken to estimate the Zn levels in the unstimulated whole saliva of normal individuals and patients with OL and OSCC and to evaluate its usefulness so that it may be considered as a biomarker in the process and progress of carcinogenesis.

   Materials and Methods Top

This study is a randomised clinical trial. The study subjects included 15 histopathologically confirmed cases of OL and 15 histopathologically confirmed cases of OSCC with 15 healthy controls matched in age and gender, reporting to the Department of Oral Medicine, Faculty of Dentistry, Tishreen University from January 2017 to July 2019. The inclusion criteria consisted of freshly diagnosed cases of oral cancer and leukoplakia without having been incurred by any prior treatment and age group of 20 to 75 years. The exclusion criteria consisted of volunteers and patients who have had undergone any treatment for cancer, patients with previous history of malignancy, patients who were administered medication containing minerals, or medication which alter significantly the homeostasis of Zn like patients who were administered diuretic one month prior to participation to the study, and subjects who suffer from diseases chronic or acute which may interfere with the homeostasis of Zn such as renal failure, liver diseases, heart failure, diabetes mellitus, and bleedings.

A detailed case history with a habit index was taken from each subject in the study. Information on family history of cancer, smoking habits, alcohol consumption, body mass index (BMI), and oral health conditions were collected using a structured questionnaire for both cases and controls. Smokers were defined as those who had smoked at least 100 cigarettes during their lifetime. Subjects were considered alcohol drinkers if they had consumed at least 1 drink/week for at least 6 months continuously. The disease-free status was ascertained according to the results of physical examination was done for each of the study subjects. The informed consent was taken from all the participants included in the study. Unstimulated saliva was collected from the study subjects between 9 am and 12 am to avoid diurnal variation.[18] The salivary samples of cases that they were clinically diagnosed as OL or OSCC were collected before taking the biopsies to avoid any alterations that may occur. The subjects were requested not to eat, drink, perform oral hygiene activities, or chew 60 min prior to the saliva collection procedure. The subjects were then seated on the dental chair and asked to spit in a graduated container every 1 min till 5 mL of saliva was obtained. During saliva collection, subjects were instructed not to speak or swallow.

Levels of Zn were estimated by standard spectrophotometric methods in accordance with the underlying validated principles using the kits from Medichem, Germany. The assays were carried out in accordance with the instructions suggested.

Statistical analysis

The data of total patients were collected, evaluated and analyzed statistically using SPSS. Comparisons of demographic characteristic distributions between cases and controls were examined with Chi-square test. ANOVA was used to compare the mean values of salivary zinc among the three groups. The probability value less than [0.05] was considered significant.

   Results Top

The distributions of age, gender, alcohol drinking, BMI, and family history of cancer were similar between cases and control subjects (P > 0.05). However, significant differences were observed with regard to tobacco smoking, tobacco smoking with alcohol drinking, smoking pack-years, and duration of smoking (P < 0.05). With respect to the biochemical data, the mean salivary Zn levels in group OSCC, OL, and HC were (116 ± 8.45) μg/dL, (151.73 ± 11.37) μg/dL, (156.13 ± 10.12) μg/dL, respectively. There was a highly significant reduction in the salivary Zn levels in OSCC patients compared with controls and OL patients. A slight decrease in Zn levels was observed in OL patients compared with controls. It was statistically insignificant (P > 0.05) [Figure 3].
Figure 3: The mean salivary values of oral leukoplakia, oral squamous cell carcinoma, and healthy group

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This study confirmed that the most affected individuals are males with 60% in both OSCC and OL. The mean age of patients with OSCC and OL was 55.67 ± 2.89 and 51.2 ± 3 years, respectively.

Risk factors

It was noted that 53.3% of OL patients and 60% of OSCC patients reported alcohol use. A considerable majority of the patients 86.7% of both OSCC patients and OL patients admitted to being smokers.

Presentation and Characteristics: The lateral tongue was most frequently involved in 26.7% of OSCC cases while the majority of OL lesions occurring on the buccal mucosa. According to histopathology, 46.7%of OSCC patients had well-differentiated lesions [Figure 4]. 66.7%of OL cases in our study showed hyperplasia with no dysplasia [Figure 5]. Clinically, 80% of OL patients showed homogeneous leukoplakia [Figure 6].
Figure 4: A graphical representation of the percentage of patients presenting with OSCC as a function of differentiation (poorly differentiated, moderately differentiated, well-differentiated)

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Figure 5: A graphical representation of the percentage of patients presenting with OL as a function of histological appearance (hyperplasia, mild dysplasia, moderate dysplasia, severe dysplasia)

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Figure 6: A graphical representation of the percentage of patients presenting with OL as a function of clinical appearance (nonhomogenous, homogenous)

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   Discussion Top

Apart from the essential role of Zn in the functions of the human body, it seems that changes in the saliva levels of Zn may play a role in the pathogenesis of oral cancer.[19] Zn as anticarcinogen was concomitantly reduced, strongly suggests that the cycle of free-radical generation enhanced the process of carcinogenesis.[20] The role of Zn in carcinogenesis can be suggested by its role in DNA and RNA polymerase, inhibitory effect on phosphodiesterase, and activation of membrane-bound adenylyl cyclase. Deficiency of Zn also contributes to cancer initiation by the activation of nuclear factor-kappa B expression and consequent induction of tumorigenic signaling. Reduced levels of Zn were found in most studies and were attributed to the decreased immunity status of individuals.[21],[22] Firstly, Zn is a constituent of SOD, an enzyme that removes free radicals, and has the reverse effect and protects against carcinogenesis.[23] Secondly, Zn decreases the activity of Cu-containing lysyl oxidase enzyme, and thus causes the inhibition of cross-linkage of collagen peptides. It also plays a noteworthy role in promoting collagen degradation through collagenase and matrix metalloproteinase.[24]

From a practical application perspective, these observations have immense clinical significance because on a day-to-day basis for screening purpose, saliva collection will be better than blood and being a noninvasive process will not need the services of a phlebotomist. In addition, from the patient's viewpoint, the collection of saliva is always preferred over invasive blood and has better compliance. This will go a long way in establishing saliva as a diagnostic tool complementary to blood.[20]

Decreased sera levels of Zn have been reported in patients suffering from gastrointestinal cancers, gynecological tumors, lymphomas, and breast and lung carcinomas. An animal experiment has also shown that Zn deficiency significantly increased the risk of oral and esophageal cancer in p53-deficient mice.[25]

Previous studies have also pointed out that Zn deficiency in serum was positively correlated with oral cancer risk indicate that oxidative stress was more in cancer patients than in healthy individuals.[15],[26],[27] By contrast, some studies have shown increased serum Zn levels in oral and testicular cancer patients.[28],[29]

Some studies indicated that the salivary value of Zn in patients with OSCC had significantly decreased.[20] Our findings were in agreement with these studies. The possible mechanism can be that Zn deficiency leads to increased oxidative DNA-damaging by affecting many antioxidant defense proteins and transcription factors.[30]

It was reported in earlier studies that the reduction in salivary trace elements in oral cancer patients may be explained on the basis that tumor cells and tissue have increased metabolic requirement of them which result in an increased uptake from an adjacent structure such as glandular secretion. Nevertheless, Zn deficiency also causes immune dysfunctions and increased inflammation which may eventually promote the occurrence of oral cancer.

A review of the literature indicates that serum Zn levels have been observed to decrease in potentially premalignant disorders such as oral submucous fibrosis and OL.[31],[32],[33],[34] Some studies have revealed a lower Zn level in the saliva of patients with OL.[20]

In our study, the mean levels of Zn were slightly decreased in the OL group. However, the difference is not statistically significant which may be due to the majority of our OL cases were histologically hyperplasia with no dysplasia. We found that salivary Zn values were significantly lower in OL patients of our study samples which histologically showed dysplasia. Dysplastic cells and tissues have increased metabolic requirements of Zn, which results in an increased uptake from adjacent structures such as salivary secretion. This could be the reason for decreased Zn levels in OL salivary samples.

In spite of all these observations, it can be deduced that saliva is a useful body fluid to ascertain the levels of Zn and that further studies are required to refine the assay for better diagnostic and application in healthcare. Thus, the alteration in salivary Zn can be used as a potential biomarker in early detection of numerous oral precancerous lesions and conditions as well as their malignant transformation and turning into frank cancers at an early enough stage.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Financial support and sponsorship

Tishreen University.

Conflicts of interest

There are no conflicts of interest.

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


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