Serum Levels of Zinc, Copper and Ferritin in Patients with Salivary Gland Tumors

Background: Variation in serum levels of trace elements including zinc, copper and ferritin has been reported in cancer patients. The aim of this study was to evaluate these trace elements in the patients’ sera with benign and malignant salivary gland tumors (SGTs) and compare them with normal individuals. Methods: In this cross-sectional study, 60 patients with SGTs including 16 pleomorphic adenoma and 44 malignant SGTs, as well as 28 healthy controls, were enrolled. Serum levels of zinc, copper and ferritin were determined by atomic absorption and ELISA methods. Data were analyzed using one way ANOVA, Chi-square, Kruskal-Wallis and Mann- Whitney tests. Results: The mean concentration of zinc, copper, ferritin was1.5± 2 ppm, 1.2± 0.5 ppm, and 96.7± 65.7 ng/ml in PA, 1.5± 1.4,1.3± 0.4, and 111.2± 112 in malignant SGTs, and1.1±0.3, 1.2± 0.23 and 124±135.8 in normal control groups. There was no statistically significant difference between the patients and control groups, and between benign and malignant SGTs (P>0.05). Conclusion: The serum levels of trace elements in SGTs were not different from normal individuals. The results might have been affected by some interventional factors. Therefore, designing cohort complementary studies might result in obtaining more accurate data.


Introduction
Salivary gland tumors (SGTs) account for 3-6% of head and neck cancers. The most common benign SGT is pleomorphic adenoma (PA) and the malignant ones are adenoid cystic carcinoma (AdCC) and mucoepidermoid carcinoma (MEC). Irradiation, tobacco and nutritional factors have been reported as etiologic factors, but the exact mechanism is still unknown (Guzzo et al., 2010;Su et al., 2015). Some trace elements, such as iron, zinc (Zn) and copper (Cu), play a critical role in some pathological processes due to their presence in the structure of many enzymes, such as antioxidant enzymes (Zablocka-Slowinska et al., 2018). The reduced function of antioxidant enzymes due to imbalance of trace elements often results in oxidative damages (Huang et al., 2018). Oxidative stress might contribute to cancer development due to production of toxic and mutagenic metabolites (Martinez-Useros et al., 2017).
Zn is an important element which plays a role in a large number of metaloenzymes (Andreini and Bertini, 2012). In addition, it is considered as antioxidant component of metaloproteinases (Sharif et al., 2012). It is reported that serum Zn levels decreased or increased in some RESEARCH ARTICLE
Cu is an essential element and its concentration is elevated in cancer tissues, which promotes cancer development via some mechanisms, such as increasing angiogenesis (Wang et al., 2010).Tumor progression and metastasis initiated along with the increasing Cu serum levels in patients with lymphoma and breast cancer (Jamakovic and Baljic, 2013;Pavithra et al., 2015). Also, Cu/Zn ratio is stated to be effective in diagnosis of some cancers. In some studies, Zn/Cu ratio has been used to evaluate the prognosis of cancer patients (Golabek et al., 2012;Malavolta et al., 2015).
Ferritin, iron storage protein, stored in the human liver, spleen and bone marrow, reflects the amount of iron in the body. Ferritin makes iron available for critical cellular processes, while protecting lipids, DNA, and proteins from the potentially toxic effects of iron serum Baharvand et al., 2014b). Ferritin levels change in the inflammatory diseases as well as in some malignant tumors .
Since the role of micronutrients in SGTs is relatively unknown, this study aimed to evaluate Zn, Cu and Ferritin serum levels in patients with benign and malignant salivary gland tumors.

Materials and Methods
This cross-sectional study was approved by the Ethics Committee of Shiraz University of Medical Sciences (IR.sums.rec.1395.S1016). Sixty patients were enrolled in this study, including16 patients with PA, 44 patients with malignant SGTs (18 cases of MEC, 10 ADCC, 4 acinic cell carcinoma (ACC), 4 squamous cell carcinoma (SCC), 8 malignant mixed tumor), as well as 28 normal subjects. The diagnosis was confirmed for all patients histopathologically. The patients with any anti-neoplastic treatment or a history of other malignancies were excluded. The patients and normal controls did not have any history of other systemic diseases and complementary drug consumption. All subjects gave their written informed consent. Baseline data including the patients' age, gender and type of tumor were obtained from their medical files.
Before any therapeutic intervention, 5 ml blood samples were collected in tubes containing EDTA and centrifuged. The serum was separated and transferred to acid wash tubes and kept at -80ºC for further analysis.
The metal concentrations (Zn and Cu) were assessed by atomic absorption spectrophotometry (spectrophotometer type AAS1, Karl Zeiss, Jena, Germany).
Ferritin level was measured using enzyme linked immunosorbent assay (ELISA) kit (Monobind, USA). The mean Zn, Cu and ferritin serum levels, as well as Cu/Zn, in patients and control groups were analyzed and compared through SPSS (version 11), using Chi-square, one-way ANOVA, Kruskal-Wallis and Mann-Whitney tests.

Results
Baseline data of all groups are shown in Table 1. As shown, the mean serum Zn, Cu and ferritin levels, as well as Cu/ Zn, in all groups are shown in Tables2, 3.Moreover, normal or abnormal levels of trace elements in all study groups are illustrated in Table 4.
The mean serum Zn levels were 1.5± 2, 1.5± 1.4, and 1.1±0.3 ppm in the PA and malignant tumors and control group, respectively. Cu and ferritin levels were 1.2± 0.5 ppm and 96.7± 65.7 ng/ml in the PA patients; 1.3± 0.4 and 111.2± 112 in malignant tumors; and 1.2± 0.23 and 124±135.8 in normal individuals. Statistical analysis using one-way ANOVA test showed that the mean serum Zn, Cu and ferritin levels were not statistically different among the study groups (P> 0. 05).
As shown in Table 4, most of the patients and normal individuals exhibited normal Zn level. Although the percentage of normal Zn level in the control group (82%) and benign tumors (75%) was more than that in malignant ones (61.4%), Chi-square test showed no significant difference among the groups. Cu and ferritin levels presented a similar pattern and most of the subjects revealed a normal level of these elements. Chi-square test showed no difference among the groups regarding to the status of Cu and ferritin levels. (All p-values >0.05) Cu/Zn ratio was 1± 0.3, 1.1± 0.5 and 1.1± 0.3 in PA, malignant tumors and control groups, respectively. Kruskal-Wallis test show no difference among the study groups (p> 0.05).

Discussion
Researchers have revealed some relationships between the levels of trace elements and the tumor type, stage or activity. In the present study, we evaluated the serum levels of Zn, Cu and ferritin in patients with benign and malignant SGTs. The findings showed that the majority of patients expressed normal concentrations of these elements, which was not different from the normal individuals.
Zn is recognized as an antioxidant agent and plays an  Table 3. Cu/ Zn Ratio in All Study Groups concentration of these elements in a total sample group, while the normal range of these elements are too wide and the patients might be diagnosed in the normal range even in a different mean levels.
The present study did not find any change in the ferritin serum levels in the patients compared to the control group. Although the role of intracellular ferritin has been well recognized, the significance of its serum level is poorly understood in cancer prevention . A higher serum level has been detected in some human cancers including breast and oral carcinomas Baharvand et al., 2014a). It has been found that tumor-associated macrophages have expressed high levels of ferritin. Moreover, some cancer cells can secret ferritin. These elevated levels in the tumor environment and serum have correlated with more aggressive diseases and poor outcome. The elevation of ferritin level in cancer patients has been attributed to chronic inflammation . However, it has been shown that 90% of patients with non-small lung cancer, as well as patients with bladder cancer, showed lower serum ferritin levels (Kukulj et al., 2010;Mazdak et al., 2010). Ferritin might decrease as a result of anemia or tumor resection in cancer patients. Ferritin levels might be increased in anemic patients due to chronic inflammation (Rambod et al., 2008). Some studies reported the mean ferritin serum levels. However, this element has a wide and different normal range regarding the age and gender. Moreover, anemia might be prevalent in a population, which could affect the result.
In conclusion, based on the present findings, Zn, Cu and ferritin serum levels in patients with SGT were similar to normal subjects, in contrast to many human neoplasms. The results may be affected by the patients' dietary habits, socioeconomic factors, sample size, measurements and study design. Therefore, designing cohort complementary studies might result in obtaining more accurate data. Moreover, various types of malignant SGTs were enrolled in this study which had different values of the evaluated elements. This variation could affect the results; therefore, further studies focusing on a specific type of malignant SGT with a greater sample size are suggested.

Statement conflict of interest
There is no conflict of interest.

Acknowledgments
The authors would like to thank the Vice-chancellor anticancer role by activating some transcription factors that control the cell proliferation and apoptosis as well as DNA, RNA and ribosome stability. It is also effective in DNA repair. Low Zn concentration has been reported in many cancers, such as lung, bladder, colorectal and renal carcinomas (Mao and Huang, 2013;Pirincci et al., 2013;Khoshdel et al., 2016;Zablocka-Slowinska et al., 2018).
Another study (Gradinaru et al., 2007) reported low Zn concentration in a preliminary study in 31 patients with stage II and III malignant SGTs of the parotid glands. In this study, the plasma Zn level in patients was similar to our results; however, the control group showed a higher level of Zn. While Zn deficiency was reported in Iranian population (Ghasemi et al., 2012), it seems that serum Zn levels are related to dietary habits. It is shown that Zn supplementation might reduce DNA damage and inflammation sources (Sun et al., 2016). In addition, some studies have shown increased Zn levels in oral and testicular cancer patients (Baharvand et al., 2014a;Kaba et al., 2015). Some researchers have proposed that evaluating Zn concentration in tissues is a more precise marker for cancer development. Hashemian et al., (2014) in a review study explained that the benefits of Zn supplementation in cancer incidence was not observed.
Although many researchers have observed increased Cu concentration in the sera of some cancer patients, such as testicular, oral, bladder (Mao and Huang, 2013;Baharvand et al., 2014a;Kaba et al., 2015), the present findings regarding SGTs did not reveal any difference in comparison to the normal subjects. One study reported lower Cu serum level in patients with colorectal cancer in comparison with normal individuals and found Cu deficiency in the population (Khoshdel et al., 2016). In Gardinaru et al., 's study (2007), Cu exhibited a higher concentration in patients with malignant SGTs in comparison with the control group. Cu is an essential trace element, which is essential in several steps of cancer progression, and it elevates in cancer cells (Blockhuys et al., 2017). It was shown that the interaction between vitamin D and Cu leads to severe oxidative stress, DNA fragmentation and cell death, especially in malignant cells (Rizvi et al., 2015). Also, Cu is required for angiogenesis. Several studies have shown the tumor inhibitor effects of Cu chelating compounds (Santini et al., 2014