Thyroid benign nodules (TBNs) are the most common lesions of this endocrine gland that encountered globally and frequently discovered by palpation during a physical examination, or incidentally, during clinical imaging procedures. TBNs include non-neoplastic lesions, for example, colloid goiter (CG) and neoplastic lesion such as thyroid adenoma (TA) 1, 2, 3. An evaluation of the variant of TBNs is clinically important for subsequent therapeutic interventions. For this reason the finding of specific characteristics of CG and TA is the barest necessity for the differential diagnosis of these thyroid disorders.

For over 20th century, there was the dominant opinion that TBNs is the simple consequence of iodine deficiency. However, it was found that TBNs is a frequent disease even in those countries and regions where the population is never exposed to iodine shortage 4. Moreover, it was shown that iodine excess has severe consequences on human health and associated with the presence of TBNs 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 TBNs incidence 9, 10, 11. Among these factors a disturbance of evolutionary stable input of many chemical elements (ChE) in human body after industrial revolution plays a significant role in etiology of TBNs 12.

Besides iodine, many other ChE have also essential physiological functions 13. Essential or toxic (goitrogenic, mutagenic, carcinogenic) properties of ChE depend on tissue-specific need or tolerance, respectively 13.Excessive accumulation or an imbalance of the ChE may disturb the cell functions and may result in cellular 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 ChE 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 ChE content with age in the thyroid of males and females were studied and age- and gender-dependence of some ChE 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 ChE contents in CG and TA in comparison with normal thyroid was demonstrated 42, 43, 44.

To date, the etiology and pathogenesis of CG and TA has to be considered as multifactorial. The present study was performed to find differences in ChE contents between CG and TA group of samples, as well as to clarify the role of some ChE in the etiology of these thyroid lesions. Having this in mind, our aim was to assess the aluminum (Al), boron (B), barium (Ba), bromine (Br), calcium (Ca), chlorine (Cl), coper (Cu), iron (Fe), I, potassium (K), lithium (Li), magnesium (Mg), manganese (Mn), sodium (Na), phosphorus (P), sulfur (S), silicon (Si), strontium (Sr), vanadium (V), and zinc (Zn) contents in CG and TA tissue samples using a combination of non-destructive and destructive methods: instrumental neutron activation analysis with high resolution spectrometry of short-lived radionuclides (INAA-SLR) and inductively coupled plasma atomic emission spectrometry (ICP-AES), respectively. A further aim was to compare the levels of these ChE in CG and TA group of samples.

All studies were approved by the Ethical Committees of the Medical Radiological Research Centre (MRRC), Obninsk. 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.

All patients suffered from CG (n=46, mean age M±SD was 48±12 years, range 30-64) and TA (n=19, mean age M±SD was 41±11 years, range 22-55) were hospitalized in the Head and Neck Department of the Medical Radiological Research Centre. 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 TE contents. For all patients the diagnosis has been confirmed by clinical and morphological results obtained during studies of biopsy and resected materials (46 euthyroid CG, 4 toxic TA and 15 non-toxic TA). Histological conclusion for all thyroidal lesions was the CG (16 macro-follicular, 13 micro-follicular, and 17 macro-micro-follicular) and TA (4 macro-follicular, 4 micro-follicular, 11 macro-micro follicular).

All tissue samples were divided into two portions using a titanium scalpel 45. One was used for morphological study while the other was intended for TE analysis. After the samples intended for TE analysis were weighed, they were freeze-dried and homogenized 46.

The pounded samples weighing about 10 mg (for biopsy) and 100 mg (for resected materials) were used for ChE measurement by INAA-SLR. The content of Br, Ca, Cl, I, K, Mg, Mn, and Na were determined by INAA-SLR using a horizontal channel equipped with the pneumatic rabbit system of the WWR-c research nuclear reactor (Branch of Karpov Institute, Obninsk). After non-destructive INAA-SLR investigation the thyroid samples were used for ICP-AES. The samples were decomposed in autoclaves and aliquots of solutions were used to determine the Al, B, Ba, Ca, Cu, Fe, K, Li, Mg, Mn, Na, P, S, Si, Sr, V, and Zn mass fractions by ICP-AES using the Spectrometer ICAP-61 (Thermo Jarrell Ash, USA). Information detailing with the NAA-SLR and ICP-AES methods used and other details of the analysis were presented in our earlier publications concerning ChE contents in human thyroid, prostate, and scalp hair 33, 34, 47, 48, 49, 50, 51, 52.

To determine contents of the ChE by comparison with a known standard, biological synthetic standards (BSS) prepared from phenol-formaldehyde resins were used 53.In addition to BSS, aliquots of commercial, chemically pure compounds were also used as standards. Ten sub-samples of certified reference material (CRM) IAEA H-4 (animal muscle) and five sub-samples of CRM of the Institute of Nuclear Chemistry and Technology (INCT, Warszawa, Poland) INCT-SBF-4 Soya Bean Flour, INCT-TL-1 Tea Leaves, and INCT-MPH-2 Mixed Polish Herbs were treated and analyzed in the same conditions that thyroid samples to estimate the precision and accuracy of results

A dedicated computer program for INAA-SLR mode optimization was used 54. All thyroid samples were prepared in duplicate, and mean values of ChE contents were used in final calculation. Mean values of ChE contents were used in final calculation for the Ca, K, Mg, Mn, and Na mass fractions measured by two methods. Using Microsoft Office Excel software, a summary of the statistics, including, arithmetic mean, standard deviation, standard error of mean, and range (minimal - maximal value), was calculated for ChE contents in CG and TA tissue samples. The difference in the results between two groups of samples was evaluated by the parametric Student’s t-test and non-parametric Wilcoxon-Mann-Whitney U-test.

resents certain statistical parameters (arithmetic mean, standard deviation, standard error of mean, and range) of the Al, B, Ba, Br, Ca, Cl, Cu, Fe, I, K, Li, Mg, Mn, Na, P, S, Si, Sr, V, and Zn mass fraction in CG and TA tissue samples.

The ratios of means and the comparison of mean values of Al, B, Ba, Br, Ca, Cl, Cu, Fe, I, K, Li, Mg, Mn, Na, P, S, Si, Sr, V, and Zn mass fractions in CG and TA are presented in Table 2.

epicts the results of comparison the contents of Al, B, Ba, Br, Ca, Cl, Cu, Fe, I, K, Li, Mg,Mn, Na, P, S, Si, Sr, V, and Zn in CG and TA sample groups with those in normal thyroid (from data analysis of previous publications 43, 44), as well as comparison the contents of these ChE in CG and TA sample groups.

M – arithmetic mean, SD – standard deviation, SEM – standard error of mean.

M – arithmetic mean, SEM – standard error of mean, Significant values are in bold.

Table 3.

* From analysis of previous publications 43, 44, ↑ - content is higher, ↓ - content is lower, = - no difference.

As was shown before33, 34, 47, 48, 49, 50, 51, 52 good agreement of the Al, B, Ba, Br, Ca, Cl, Cu, Fe, I, K, Mg, Mn, Na, P, S, Sr, V, and Zn mass fractions in CRM IAEA H-4, INCT-SBF-4, INCT-TL-1, and INCT-MPH-2 samples determined by both INAA-SLR and ICP-AES methods with the certified data of these CRMs indicates acceptable accuracy of the results obtained in the study of CG and TA samples and presented in Tables 1–3.

In a general sense variations found for Al, B, Ba, Br, Ca, Cl, Cu, I, K, Na, Sr, and Zn contents in CG and TA tissue samples were similar in comparison with normal thyroid tissue (Table 3). In affected tissues contents of Al, B, Br, Cl, Cu, Na, and Zn increased, content of I decreased, whereas levels of Ba, Ca, K, and Sr did not changed. There was not found any differences between ChE contents of CG and TA, with the exception of Br and Mg (Tables 2 and 3). The Br level in TA tissue was almost 11 times higher, while the Mg content was 1.5 times lower than in CG tissue.

Published data on comparison of Al, B, Ba, Br, Ca, Cl, Cu, Fe, I, K, Li, Mg, Mn, Na, P, S, Si, Sr, V, and Zn levels in CG and TA were not found.

Thus, from obtained results it was possible to conclude that the common characteristics of CG and TA tissue samples were elevated level of Al, B, Br, Cl, Cu, Na, and Zn and reduced level of I in comparison with normal thyroid and, therefore, these ChE can be involved in etiology and pathogenesis of such thyroid disorders as CG and TA.

Aluminum

Al is the most widely distributed metal in the environment. Environmental media may be contaminated by Al from anthropogenic sources and through the weathering of rocks and minerals 55. The trace element Al is not described as essential, because no biochemical function has been directly connected to it. Toxic actions of Al induce oxidative stress, immunologic alterations, genotoxicity, and other disorders, including cell membrane perturbation, apoptosis, necrosis and dysplasia 55. Furthermore, it was shown in experimental and epidemiological studies that Al can affect thyroid iodide uptake and hormones secretion 56, 57.

Boron

Trace element B is known to influence the activity of many enzymes 58. Numerous studies have demonstrated beneficial effects of B on human health, including anti-inflammatory stimulus - reduces levels of inflammatory biomarkers, such as high-sensitivity C-reactive protein (hs-CRP) and tumor necrosis factor α (TNF-α); as well as raises levels of antioxidant enzymes, such as superoxide dismutase (SOD), catalase, and glutathione peroxidase 59. Why B content in adenomatous thyroid is higher than normal level and how an excess of B acts on thyroid are still to be cleared.

Bromine

Bris one of the most abundant and ubiquitous of the recognized ChE in the biosphere. Inorganic bromide is the ionic form of bromine which exerts therapeutic as well as toxic effects. An enhanced intake of bromide could interfere with the metabolism of iodine at the whole-body level. In the thyroid gland the biological behavior of bromide is more similar to the biological behavior of iodide 60. A significant age-related increase of Br content in human thyroid 25 26, 27, 28 correlated well with age-related prevalence of CG and TA 61, 62 63 . The main source of natural Br for human body is food. Food and environment media polluted by artificial Br-contained compounds, for example such as polybrominated biphenyls (PBBs) and diphenyl ethers (PBDEs), is other source. PBBs and PBDEs impact on thyroid function and thyroid hormones metabolism64 Thus, on the one hand, the accumulated data suggest that Br level in thyroid tissue might be responsible for CG, and TA development. But, on the other hand, Br compounds, especially potassium bromide (KBr), sodium bromide (NaBr), and ammonium bromide (NH₄Br), are frequently used as sedatives in Russia 65. It may be the reason for elevated levels of Br in specimens of patients with CG, and TA in comparison with normal thyroid. A nonuniform level of this TE in tissue of thyroid legions TA Br > CG Br may be explained by the different strength of emotional reactions of persons on the diagnosis TA and CG, and, as consiquence, different dozes of Br-conteined sedatives, which were used.

Chlorine and Sodium

Cl and Na are ubiquitous, extracellular electrolytes essential to more than one metabolic pathway. In the body, Cl and Na mostly present as sodium chloride. Therefore, as usual, there is a correlation between Na and Cl contents in tissues and fluids of human body. Because Cl is halogen like I and Br, in the thyroid gland the biological behavior of chloride has to be similar to the biological behavior of iodide. The main source of natural Cl for human body is salt in food and chlorinated drinking water. Environment (air, water and food) polluted by artificial nonorganic Cl-contained compounds, for example such as sodium chlorate (NaClO₃), and organic Cl-contained compounds, for example such as polychlorinated biphenyls (PCBs) and dioxin, is other source. There is a clear association between using chlorinated drinking water, levels NaClO₃, PCBs and dioxin in environment and thyroid disorders, including cancer64 66 67 68 69. Thus, on the one hand, the accumulated data suggest that Cl level in thyroid tissue might be responsible for CG and TA development. However, on the other hand, it is well known that Cl and Na mass fractions in human tissue samples depend mainly on the extracellular water volume 70 . Goitrous and adenomatous tissues can contain more colloid that normal thyroid. Because colloid is extracellular liquid, it is possible to speculate that CG and TA are characterized by an increase of the mean value of the Cl and Na mass fractions because the relative content of colloid in these thyroid lesions is higher than that in normal thyroid tissue. Overall, the elevated levels of Cl in goutrous and adenomatous thyroids could possibly be explored for diagnosis of CG and TA.

Coper

Cu is a ubiquitous trace metal in the human body which plays many roles at different levels. Various Cu-enzymes (such as amine oxidase, ceruloplasmin, cytochrome-c oxidase, dopamine-monooxygenase, extracellular superoxide dismutase, lysyl oxidase, peptidylglycineamidating monoxygenase, Cu/Zn superoxide dismutase, and tyrosinase) mediate the effects of Cu deficiency or excess. Cu excess can have severe negative impacts. Cu generates oxygen radicals and many investigators have hypothesized that excess copper might cause cellular injury via an oxidative pathway, giving rise to enhanced lipid peroxidation, thiol oxidation, and, ultimately, DNA damage71 72 73. Thus, Cu accumulation in thyroid parenchyma with age 25 26, as well as excessive Cu intake with diet or other environmental sources, may be involved in oxidative stress, dwindling gland function, and increasing risk of thyroid nodules, including CG and TA. The significantly elevated level of Cu in CG and TA tissue, observed in the present study, supports this speculation. The difference between Cu levels in CG and TA was not found (Table 2 and 3). Therefore, it is reasonable to conclude that role of Cu excess in etiology and pathogenesis of CG and TA is similar. However, an overall comprehension of Cu homeostasis and physiology, which is not yet acquired, is mandatory to establish Cu exact role in the CG and TA etiology and metabolism.

Iodine

Compared to other soft tissues, the human thyroid gland has higher levels of I, because this element plays an important role in its normal functions, through the production of thyroid hormones (thyroxin and triiodothyronine) which are essential for cellular oxidation, growth, reproduction, and the activity of the central and autonomic nervous system. Goitrous and adenomatous transformation are probably accompanied by a partial loss of tissue-specific functional features, which leads to a modest reduction in I content associated with functional characteristics of the human thyroid tissue.

Zinc

Zn as a trace metal has structural, catalytic and regulatory roles in normal and pathophysiology. This ChE is a constituent of more than 3000 proteins and is a cofactor for over 300 enzymes 74. Zn is an essential mediator of cell proliferation and differentiation through the regulation of DNA synthesis and mitosis. Zn also affects DNA repair pathways by regulating multiple intracellular signaling pathways and altering proteins involved in DNA maintenance 75. This metal also maintenance the balance of a cellular redox 76. Thus, Zn is important cofactors in diverse cellular processes. Concern the thyroid function, Zn is involved in the synthesis of TSH and important for the proper functioning of T3 because T3 nuclear receptors contain Zn ions 64. However, high Zn concentrations are toxic to the cells and the elevated level of Zn mass fractions in thyroid tissue may contribute to harmful effects on the gland. There are good reasons for such speculations since. experimental and epidemiological data support the hypothesis that Zn overload is a risk factor for benign and malignant tumors75 77 78 79.

Characteristically, elevated or reduced levels of ChE 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 ChE in affected thyroid tissues researchers try to determine the role of the deficiency or excess of each ChE in the etiology and pathogenesis of thyroid diseases. In our opinion, abnormal levels of many ChE in TBNs 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 ChE level in pathologically altered tissue is the reason for alterations or vice versa.

This study has several limitations. Firstly, analytical techniques employed in this study measure only twenty ChE (Al, B, Ba, Br, Ca, Cl, Cu, Fe, I, K, Li, Mg, Mn, Na, P, S, Si, Sr, V, and Zn) mass fractions. Future studies should be directed toward using other analytical methods which will extend the list of ChE investigated in normal thyroid and in pathologically altered tissue. Secondly, the sample size of CG group and, particularly, of TA group was relatively small and prevented investigations of ChE contents in these groups using differentials like gender, histological types of CG and TA, nodules functional activity, stage of disease, dietary habits of patients with CG and TA. Lastly, generalization of our results may be limited to Russian population. Despite these limitations, this study provides evidence on TBNs-specific tissue Al, B, Br, Cl, Cu, I, Na, and Zn level alteration and shows the necessity to continue ChE research of TBNs.

In this work, ChE analysis was carried out in the tissue samples of CG and TA using non-destructive analytical method INAA-SLR and destructive analytical method ICP-AES. It was shown that combination of these methods is an adequate analytical tool for the determination of twenty ChE (Al, B, Ba, Br, Ca, Cl, Cu, Fe, I, K, Li, Mg, Mn, Na, P, S, Si, Sr, V, and Zn) content in the tissue samples of human thyroid in norm and pathology, including needle-biopsy specimens. It was observed that in both CG and TA tissues tissues contents of Al, B, Br, Cl, Cu, Na, and Zn increased, content of I decreased, whereas levels of Ba, Ca, K, and Sr did not changed in comparison with normal thyroid tissue. It was not found any differences between ChE contents of CG and TA, with the exception of Br and Mg. The Br level in TA tissue was almost 11 times higher, while the Mg content was 1.5 times lower than in CG tissue.

From obtained results it was possible to conclude that the common characteristics of CG and TA tissue samples were elevated level of Al, B, Br, Cl, Cu, Na, and Zn and reduced level of I in comparison with normal thyroid and, therefore, these ChE can be involved in etiology and pathogenesis of such thyroid disorders as CG and TA.

Declaration of Conflicting Interests

The author has not declared any conflict of interests.

The author received no financial support for this study and for publication of this article.