Thyroid malignant nodules (TMNs) are the most common endocrine cancer and the fifth most frequently occurring type of malignancies ^{1, 2, 3}. The incidence of TMNs has increased worldwide over the past four decades. TMNs are divided into three main histological types: differentiated (papillary and follicular thyroid cancer), undifferentiated (poorly differentiated and anaplastic thyroid cancer, and medullary thyroid cancer, arising from C cells of thyroid ³. During the 20th century, there was the dominant opinion that TMNs is the simple consequence of iodine deficiency ⁴. However, it was found that TMNs is a frequent disease even in those countries and regions where the population is never exposed to iodine shortage. Moreover, it was shown that iodine excess has severe consequences on human health and associated with the presence of TMNs ^{5, 6, 7, 8}. It was also demonstrated that besides the iodine deficiency and excess many other dietary, environmental, and occupational factors are associated with the TMNs incidence ^{9, 10, 11}. Among these factors a disturbance of evolutionary stable input of many trace elements (TEs) in human body after industrial revolution plays a significant role in etiology of TMNs ¹².

Besides iodine, many other TEs have also essential physiological functions ¹³. Essential or toxic (goitrogenic, mutagenic, carcinogenic) properties of TEs depend on tissue-specific need or tolerance, respectively ¹³.Excessive accumulation or an imbalance of the TEs may disturb the cell functions and may result in cellular proliferation, degeneration, death, benign or malignant transformation ^{13, 14, 15}.

In our previous studies the complex of in vivo and in vitro nuclear analytical and related methods was developed and used for the investigation of iodine and other TEs contents in the normal and pathological thyroid ^{16, 17, 18, 19, 20, 21, 22}. Iodine level in the normal thyroid was investigated in relation to age, gender and some non-thyroidal diseases ^{23, 24}. After that, variations of many TEs content with age in the thyroid of males and females were studied and age- and gender-dependence of some TEs was observed ^{25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41}. Furthermore, a significant difference between some TEs contents in colloid goiter, thyroiditis, and thyroid adenoma in comparison with normal thyroid was demonstrated ^{42, 43, 44, 45, 46}.

To date, the etiology and pathogenesis of TMNs must be considered as multifactorial. The present study was performed to find out differences in TEs contents between the group of cancerous tissues, thyroid tissue adjacent to tumor, and “normal” thyroid (TEs as thyroid cancer biomarkers), as well as to clarify the role of some TEs in the etiology of TMNs. Having this in mind, the aim of this exploratory study was to examine differences in the content of copper (Cu), iron (Fe), iodine (I), rubidium (Rb), strontium (Sr), and zinc (Zn) in tumors and adjacent to tumor tissues of thyroids with TMNs, using a combination of non-destructive ¹⁰⁹Cd and ²⁴¹Am radionuclide-induced energy-dispersive X-ray fluorescent analysis, and to compare the levels of these TEs in two groups (tumor and adjacent to tumor tissues) of the cohort of TMNs samples. Moreover, for understanding a possible role of TEs in etiology and pathogenesis of TMNs, as well as thyroid cancer biomarkers, results of the study were compared with previously obtained data for the same TEs in “normal” thyroid tissue ^{42, 43, 44, 45, 46}.

All patients with TMNs (n=41, mean age M±SD was 46±15 years, range 16-75) were hospitalized in the Head and Neck Department of the Medical Radiological Research Centre (MRRC), Obninsk.. Thick-needle puncture biopsy of suspicious nodules of the thyroid was performed for every patient, to permit morphological study of thyroid tissue at these sites and to estimate their trace element contents. In all cases the diagnosis has been confirmed by clinical and morphological results obtained during studies of biopsy and resected materials. Histological conclusions for malignant tumors were: 25 papillary adenocarcinomas, 8 follicular adenocarcinomas, 7 solid carcinomas, and 1 reticulosarcoma. Tissue samples of tumor and visually intact tissue adjacent to tumor were taken from resected materials.

“Normal” thyroids for the control group samples were removed at necropsy from 105 deceased (mean age 44±21 years, range 2-87), who had died suddenly. The majority of deaths were due to trauma. A histological examination in the control group was used to control the age norm conformity, as well as to confirm the absence of micro-nodules and latent cancer.

All studies were approved by the Ethical Committees of MRRC. All the procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments, or with comparable ethical standards. Informed consent was obtained from all individual participants included in the study

All tissue samples obtained from tumors and visually intact tissue adjacent to tumors were divided into two portions using a titanium scalpel to prevent contamination by TEs of stainless steel ⁴⁷. One was used for morphological study while the other was intended for TEs analysis. After the samples intended for TEs analysis were weighed, they were freeze-dried and homogenized ⁴⁸.

To determine the contents of the TEs by comparison with known data for standard, aliquots of commercial, chemically pure compounds and synthetic reference materials were used ⁴⁹. Ten subsamples of the Certified Reference Material (CRM) IAEA H-4 (animal muscle) were analyzed to estimate the precision and accuracy of results. The CRM IAEA H-4 subsamples were prepared in the same way as the samples of dry homogenized thyroid tissue.

Details of the relevant facility for EDXRF determination of Cu, Fe, Rb, Sr, and Zn contents with ¹⁰⁹Cd radionuclide source, methods of analysis and the quality control of results were presented in our earlier publications concerning the ¹⁰⁹Cd-EDXRF analysis of human thyroid and prostate tissue ^{25, 26, 50, 51}.

Details of the relevant facility for EDXRF determination of I contents with ²⁴¹Am radionuclide source, methods of analysis and the quality control of results were presented in our earlier publication concerning the ²⁴¹Am-EDXRF analysis of human thyroid in norm and pathology ²¹.

All thyroid samples for TEs analysis were prepared in duplicate, and mean values of TEs contents were used in final calculation. Using Microsoft Office Excel software, a summary of the statistics, including, arithmetic mean, standard deviation of mean, standard error of mean, minimum and maximum values, median, percentiles with 0.025 and 0.975 levels was calculated for TEs contents in nodular and adjacent tissue of thyroids with TMNs. Data for “normal” thyroid were taken from our previous publications ^{42, 43, 44, 45, 46}. The difference in the results between three groups of samples (“normal”, “tumor”, and “adjacent”) was evaluated by the parametric Student’s t-test and non-parametric Wilcoxon-Mann-Whitney U-test.

Table 1 presents certain statistical parameters (arithmetic mean, standard deviation, standard error of mean, minimal and maximal values, median, percentiles with 0.025 and 0.975 levels) of the Cu, Fe, I, Rb, Sr, and Zn mass fraction in “normal”, “tumor”, and “adjacent” groups of thyroid tissue samples.

The ratios of means and the comparison of mean values of Cu, Fe, I, Rb, Sr, and Zn mass fractions in pairs of sample groups such as “normal” and “tumor”, “normal” and “adjacent”, and also “adjacent” and “tumor” are presented in Table 2, Table 3, and Table 4, respectively.

As was shown before ^{21, 25, 26, 50, 51} good agreement of the Cu, Fe, I, Rb, Sr, and Zn contents in CRM IAEA H-4 samples analyzed by EDXRF with the certified data of this CRM indicates acceptable accuracy of the results obtained in the study of “normal”, “tumor”, and “adjacent” groups of thyroid tissue samples presented in Table 1, Table 2, Table 3, and Table 4

From Table 2, it is observed that in cancerous tissue the mass fraction of I and Zn are 23 times and 25%, respectively, lower whereas mass fractions of Cu and Rb are 3.4 and 1.4 times, respectively, higher than in normal tissues of the thyroid. Thus, if we accept the TEs contents in thyroid glands in the “normal” group as a norm, we have to conclude that with a malignant transformation the Cu, I, Rb, and Zn in thyroid tissue significantly changed. In a general sense Cu, Fe, and Zn contents found in the “normal” and “adjacent” groups of thyroid tissue samples were very similar (Table 3,). However, in the “adjacent” group mean mass fractions of I and Rb were 1.75 and 2.06 times, respectively, higher, whereas mean value of Sr content 4 times lower than in the “normal” group. Significant changes of tumor TEs contents in comparison with thyroid tissue adjacent to tumor were found only for I (decrease) and Sr (increase). In malignant tumor Sr contents were approximately 5.4 times higher, while I content 40 times lower than in “adjacent” group of tissue samples (Table 4). Thus, from obtained results it was possible to conclude that the common characteristics of TMNs in comparison with “normal” thyroid and visually “intact” thyroid tissue adjacent to malignant tumors were drastically reduced level of I (Table 2, Table 4).

Characteristically, elevated or reduced levels of TEs observed in thyroid nodules are discussed in terms of their potential role in the initiation and promotion of these thyroid lesions. In other words, using the low or high levels of the TEs in affected thyroid tissues researchers try to determine the role of the deficiency or excess of each TE in the etiology and pathogenesis of thyroid diseases. In our opinion, abnormal levels of many TEs in TMNs could be and cause, and also effect of thyroid tissue transformation. From the results of such kind studies, it is not always possible to decide whether the measured decrease or increase in TEs level in pathologically altered tissue is the reason for alterations or vice versa. According to our opinion, investigation of TEs contents in thyroid tissue adjacent to malignant nodules and comparison obtained results with TEs levels typical of “normal” thyroid gland may give additional useful information on the topic because these data show conditions of tissue in which TMNs were originated and developed.

This study has several limitations. Firstly, analytical techniques employed in this study measure only six TEs (Cu, Fe, I, Rb, Sr, and Zn) mass fractions. Future studies should be directed toward using other analytical methods which will extend the list of TEs investigated in “normal” thyroid and in pathologically altered tissue. Secondly, the sample size of TMNs group was relatively small and prevented investigations of TEs contents in this group using differentials like gender, histological types of TMNs, tumor functional activity, stage of disease, and dietary habits of patients with TMNs. Lastly, generalization of our results may be limited to Russian population. Despite these limitations, this study provides evidence on many TEs level alteration in malignant tumor and adjacent to tumor tissue and shows the necessity to continue TEs research of TMNs.

In this work, TEs analysis was carried out in the tissue samples of TMNs using EDXRF. It was shown that EDXRF with using ¹⁰⁹Cd and ²⁴¹Am radionuclide sources is an adequate analytical tool for the non-destructive determination of Cu, Fe, I, Rb, Sr, and Zn content in the tissue samples of human thyroid in norm and pathology, including needle-biopsy specimens. It was observed that in cancerous tissue the mass fraction of I and Zn were 23 times and 25%, respectively, lower whereas mass fractions of Cu and Rb were 3.4 and 1.4 times, respectively, higher than in normal tissues of the thyroid. In a general sense Cu, Fe, and Zn contents found in the “normal” and “adjacent” groups of thyroid tissue samples were very similar. However, in the “adjacent” group mean mass fractions of I and Rb were 1.75 and 2.06 times, respectively, higher, while mean value of Sr content was 4 times lower than in the “normal” group. In malignant tumor Sr contents were approximately 5.4 times higher, while I content 40 times lower than in “adjacent” group of tissue samples. Thus, from results obtained, it was possible to conclude that the common characteristics of TMNs in comparison with “normal” thyroid and visually “intact” thyroid tissue adjacent to nodules were drastically reduced level of I. It was supposed that the drastically reduced level of I content in cancerous tissue could possibly be explored for differential diagnosis of benign and malignant thyroid nodules.

There were no any sources of funding that have supported this work.

The author is extremely grateful to Profs. B.M. Vtyurin and V.S. Medvedev, Medical Radiological Research Center, Obninsk, as well as to Dr. Yu. Choporov, former Head of the Forensic Medicine Department of City Hospital, Obninsk, for supplying thyroid samples.