Concentration of Metals and Trace Elements in the Normal Human and Rat Thyroid: Comparison with Muscle and Adipose Tissue and Volcanic Versus Control Areas

Abstract

Background:

The concentration of trace elements and metals in the thyroid is the result of exposure, uptake, retention, and clearance. The specificity and selectivity of thyroid capacity to concentrate these elements relative to other tissues are not known. To obtain this information, we measured the tissue concentration of 26 elements in the thyroid, muscle, and fat of euthyroid human subjects and also in normal rats.

Methods:

At programmed surgery, small (<1 g) tissue fragments were collected in 77 euthyroid subjects. Macroscopically normal thyroid tissue, sternothyroid muscle, and neck subcutaneous fat samples were excised, and thyroid tissue was confirmed to be morphologically normal through microscopy. Tissue specimens (thyroid, hindlimb muscle, and abdominal fat) were also obtained from normal rats. Measurements of trace elements were performed on tissues using inductively coupled plasma mass spectrometry (DRC-ICP-MS).

Results:

Only 19 of the 26 investigated elements were measurable as 7 elements were below the limit of detection. The ranking concentration in human thyroid tissue, not considering iodide, indicated that Zn, Br, Cu, Cr, Se, and Mn represented over 95% of the measured elements. A similar ranking was observed in the rat thyroid. A comparison with other tissues indicated that in addition to I, also Br, Mn, Se, and Sn were significantly more concentrated in the thyroid, and this was also the case for the recognized carcinogens As, Cd, and Hg. As and Hg, but not Cd (which was not detectable in any of the rat tissues), were also more concentrated in the rat thyroid. Since human thyroid specimens were also obtained from residents of a volcanic area, where environmental pollution may cause human biocontamination, we compared the trace element concentration in specimens from the volcanic area with controls. Many trace elements were slightly, but not significantly, increased in the volcanic area specimens.

Conclusions:

In the normal human thyroid, many trace elements, including Br, Mn, Se, and Sn, and the recognized carcinogens, As, Cd, and Hg, are significantly more concentrated than in muscle and fat of the same individual. Similar data were observed in rats. The reason for the differential element accumulation in the thyroid is unclear; a better understanding may be useful to further clarify thyroid biology.

Introduction

Metals are chemical elements that are not biologically synthesized, but are present in the environment and acquired by living cells. Some metals (e.g., zinc, copper, iron, and selenium) are essential for life and are considered necessary micronutrients that must be available in a defined range to allow normal physiological processes of living cells. Other metals are called nonessential and may even be toxic in small amounts (e.g., arsenic, cadmium, and mercury). Excessive exposure to the metals in this latter group may interfere and damage normal biological processes.

In endocrinology, metals and trace elements have been investigated mainly as chemical agents that may interfere with hormone production and function (endocrine-disrupting chemicals or endocrine disruptors) (1). Much less studied is their potential role in endocrine cell proliferation, differentiation, apoptosis, and mutagenesis.

Metal homeostasis and concentration in tissues are dependent on metal uptake, compartmentalization, retention, and clearance. Appropriate metal availability in the environment and normal absorption and distribution to different tissues may not be sufficient to assure correct cell function when congenital or acquired abnormalities of mechanisms involved in metal cellular metabolism are present. These concepts are well known for the thyroid because of its peculiar activity regarding iodide uptake, retention, and compartmentalization and thyroid hormone synthesis and secretion. Diseases derived from an abnormality in these different steps of iodide uptake and metabolism have been widely studied. Because it is less relevant to thyroid physiopathology and clinical consequences, the capacity of the thyroid to uptake and metabolize other chemicals and metals is scarcely studied, even if some of them (i.e., selenium) are essential for the thyroid function. Reference concentration values in the thyroid are a necessary prerequisite to plan biological and molecular studies on the role of metals in thyroid physiology and pathology, but our present knowledge of the normal concentration range of these elements and metals in the thyroid is poor and is sometimes affected by preanalytical and analytical problems (2). Moreover, for some metals, the thyroid tissue concentration has never been investigated.

Recently, we have confirmed the previous observation that thyroid cancer incidence is greatly increased in volcanic areas (3 –6) and we have documented for the first time that this increase is associated with relevant, nonanthropogenic, multielemental environmental pollution concerning many metals and metalloids, with the consequent contamination of residents (7). These observations raise the possibility of a relationship between one or more of the increased metals and thyroid cancer, especially if some of the environmentally increased metals accumulate in the thyroid.

In the present study, we investigated the multielemental concentration of metals in normal thyroid tissue by measuring the content of iodide and 25 additional trace elements and metals in the thyroid, as well as the muscle and adipose tissues of the same euthyroid individual, to evaluate whether the thyroid has a tissue-specific capacity to differentially accumulate some metals or trace elements. We also measured some of these elements in the same tissues of normal rats to evaluate possible species differences in an experimental animal commonly used for thyroid research. Finally, we evaluated the possible differences in element concentration in the thyroid tissue of individuals exposed to volcanic environmental contamination relative to control subjects living in adjacent nonvolcanic areas.

Methods

Human subjects and tissues

Seventy-seven adult subjects with normal thyroid function (assessed by thyroid hormone and thyrotropin (TSH) serum levels in the reference range and negative antithyroid antibodies) undergoing thyroidectomy at the Garibaldi-Nesima Medical Center (a tertiary referral center for thyroid diseases) due to a discrete (maximum diameter ≤2 cm), single thyroid nodule cytologically evaluated as TIR3, TIR4, or TIR5 at fine-needle aspiration biopsy (8) signed informed consent to donate small tissue fragments (<1 g) collected at surgery and subsequently entered the study. On pathological examination, 42 nodules (55.5%) were benign and 35 were malignant (differentiated papillary thyroid cancer in all cases).

Donated tissues for each patient included macroscopically normal thyroid tissue (at least 1 cm distance from the nodule) and sternothyroid muscle tissue and neck subcutaneous adipose tissue.

Average patient age was 48.7 ± 16.7 years and there were 54 (70.1%) women and 23 men. Their residence for the last 10 years was either in the Mount Etna volcanic area (province of Catania, 43 cases) or in adjacent nonvolcanic areas in Sicily (34 cases). All patients were and had always been euthyroid, as evaluated by their medical records, clinical examination, and biochemical measurements of TSH, thyroid hormones, and negative antibodies before surgery. At enrollment time, serum TSH values ranged from 0.42 to 4.50 mIU/L (reference range 0.35–4.94), free triiodothyronine 3.25 to 5.21 pmol/L (reference range 2.63–5.70), and free thyroxine 10.30 to 17.85 pmol/L (reference range 9.0–19.05) (Supplementary Table S1).

The following exclusion criteria were adopted: previous clinical or laboratory evidence of abnormal thyroid function and/or treatment for thyroid diseases, history of previous head/neck irradiation, obesity (body–mass index >30 kg/m²), smoking, taking medications that can interfere with thyroid function, including iodide and nutritional supplements, and chronic disease with specific attention to gastrointestinal, liver, and kidney diseases.

Thyroid tissue specimens were collected at surgery using a titanium scalpel, divided into two fragments, and placed in conical tubes containing saline. One portion was used for morphological examination. The other was weighed and frozen in liquid nitrogen within one hour from excision and stored deep frozen until the experimental measurement.

This study was approved by the Institutional Ethics Committee of our Hospital (Catania-2).

Experimental animals and sample collection

Eight female Wistar rats (9 weeks old, 200–230 g) were kept under standard housing conditions (temperature 21–23°C, relative humidity 45–65%, and 12-h light:12-h dark cycle) and fed a standard pellet diet for rodents (Mucedola 4RF2) and tap water ad libitum. All animals were housed in plastic cages containing two animals/cage.

All animal experiments were approved by the ethics committee of the Catholic University of Rome, Italy (n. Q42), and were conducted in accordance with institutional guidelines, which are in compliance with national (D.L. No. 116, G.U., Suppl. 40, February 18, 1992; Circular No. 8,G.U., July 1994) and international laws (EEC Council Directive 86/609, OJ L 358. 1, December 12, 1987; Guide for the Care and Use of Laboratory Animals, United States National Research Council, 1996) on the ethical use of animals.

For specimen collection, rats were sacrificed by CO₂ inhalation. The thyroid gland, hindlimb muscle, and abdominal visceral fat were surgically removed with a sterile scalpel and immediately snap-frozen in liquid nitrogen for subsequent metal measurements.

Measurements of trace elements

Multielemental quantitative determination was performed in wet tissue specimens in the Laboratory of Experimental and Clinical Toxicology at the ICS Maugeri in Pavia. In most previous studies, element measurements were normalized on the basis of the weight of dry tissue specimens. Therefore, to better compare our data with previous studies, we converted our measured concentrations from wet to dry weight under the assumption that the water content in the thyroid tissue is 74.2% ± 3.4 (9).

The following 26 elements were measured: silver (Ag), arsenic (As), boron (B), barium (Ba), bromine (Br), cadmium (Cd), cobalt (Co), chromium (Cr), copper (Cu), mercury (Hg), iodide (I), lithium (Li), manganese (Mn), molybdenum (Mo), nickel (Ni), lead (Pb), palladium (Pd), antimony (Sb), selenium (Se), tin (Sn), strontium (Sr), thallium (Tl), vanadium (V), tungsten (W), zinc (Zn), and zirconium (Zr).

Measurements were performed using an inductively coupled plasma mass spectrometry (DRC-ICP-MS) equipment (ELAN 6100 DRCII ICP-MS PerkinElmer SCIEX Instruments, Canada) with a dynamic reaction cell (DRC) and a quadrupole mass filter, a cyclonic spray chamber with a concentric nebulizer, and an AS90 plus autosampler (PerkinElmer). DRC-ICP-MS is a very sensitive technique that is suitable for determination of trace and ultratrace elements. This multielemental technique measures different elements at the same time on the basis of their mass-to-charge ratio (m/z). The DRC allows reduced chemical interference (10) and allows the acquisition of background values <1 cps and detection limits on the order of ng/L. The mass spectrometer also allows working on complex matrices (biological and environmental samples) in a linear way in a wide dynamic range to analyze the various analytes at different concentrations with comparable precision and accuracy.

The following procedure was used to perform sample processing:

Before the analysis, tissue samples underwent pretreatment in a microwave CEM model MARS-Xpress (CEM Corp., Matthews, NC) to reduce interferences caused by the matrix. Temperature was recorded at one-second intervals with an infrared thermometer sensor. Microwave oven operations (including time, temperature, and power settings) were controlled by an in-built digital computer.

Tissue samples (∼100 mg) were added to the digestion vessels with 4 mL HNO₃ (65% m/v) and 0.5 mL H₂O₂ (30% m/v). After complete digestion and cooling, samples were filtered and transferred to 50-mL graduated polypropylene tubes and diluted to volume with deionized water.

The DRC was vented and pressurized with reactive gases (CH₄ and NH₃) under computer control, and online chemical modifications of the ion beam were provided to eliminate interferences. The specificity of interference rejection was obtained through selection of the reaction gas and the operating conditions. Ag, B, Ba, Br, Cd, Co, Cu, Hg, I, Li, Mn, Mo, Pb, Pd, Sb, Sn, Sr, Tl, W, Zn, and Zr were determined in standard mode, while As, Cr, Ni, Se, and V were determined in enhanced mode (CH₄ and NH₃).

To optimize the ICP-MS signal, a standard solution containing 1 μg/L of three elements that covered the entire mass range (²⁴Mg, ¹¹⁵In, and ²³⁸U) was used. A fourth mass (220) was used to monitor background noise in the system.

Calibration was performed by an external calibration method. Multielement standard solutions (calibration from 0.1 to 20 μg/L) were prepared from ICP-MS multielemental standard solutions, 10 mg/L (Standard 3, 4, MS3, MS1 CPI International, Amsterdam, NL); from 1000 mg/L I and Br standard solutions (CPAchem Ltd.); and from standard solution 1 μg/L ELAN 6100 DRC SETUP/STAB/MASSCAL SOLUTION (PerkinElmer Life and Analytical Sciences, Shelton). Standards were diluted with high purity water containing the same amount of acids as the samples. The working standard solutions were used for preparation of the quality control samples.

The stability of the calibration curve was assessed by generating linear calibration curves (from 0.1 to 20 μg/L) of all multielemental standard solutions. To evaluate the day-to-day stability of calibration curves, spiked aqueous solutions were analyzed on five consecutive days. No significant variations were observed and the analytes were therefore considered to be stable over this time period.

Statistical analyses

Chemical concentrations are expressed as median and range or either arithmetic mean (± standard deviation [SD]) or geometric mean (GM) with 95% confidence interval (CI). The GM was calculated to estimate the central tendency by taking the log of each concentration and then computing the mean and its 95% CI of the log-transformed values. Values below the limit of detection (LOD) were also included by assigning a value equal to the LOD divided by the square root of 2 (11). If the number of measurements below the LOD was greater than 40% of specimens examined, the GM was not calculated, and the element was classified as nondetectable in that tissue. To verify the difference in chemical concentrations between the different tissue specimens, linear regression analysis was used by including the log-transformed values of each chemical in the model. Age and sex were also included in the linear regression model to control the differences for these matching variables. p Values lower than 0.05 were considered statistically significant for a two-tailed test. All statistical analyses were performed using the STATA 13.1 statistical package (StataCorp LP, College Station, TX).

Results

Precision and trueness of results

The precision of the method was determined in terms of repeatability (intra-assay precision) and is expressed as relative SDs calculated from 10 replicate measurements on 6 samples at different concentrations. The CV% was 3–4% at concentration levels of 0.01 μg/L and 1–2% at 10 μg/L.

Trueness was evaluated by analyzing 10 replicates of 5 different samples spiked with three different concentrations (0.01, 10, and 20 μg/L) of all studied analytes. The elements were determined by DRC-ICP-MS according to previously reported experimental conditions. The recoveries, expressed as the percentage recovery mean ± SD (n = 5), were between 98.2% and 100.6% (±1.0–1.8%).

The detection limits (LOD, defined as three times the SD of five repeated readings of a blank sample prepared on five consecutive days) for all trace elements were in the range 0.0001–0.016 μg/L.

Under the same conditions, the limit of quantification (defined as 10 times the SD of 5 repeated readings of a blank sample prepared on 5 consecutive days) for all the trace elements was in the range 0.0003–0.048 μg/L.

Element concentration in normal human thyroid tissue and comparison with published data

Of the 26 investigated elements, data are presented for only 19 because the remaining 7 (B, Co, Li, Pd, Sb, Tl, and V) were at a concentration lower than the LODs for the assay in the thyroid tissue of more than 40% (31/77) of the examined specimens. For these elements, the analysis of the concentration in the thyroid was considered inadequate to establish accurate values.

For each element, we first examined whether patient sex influenced the results and found no significant difference between values in the specimens from 54 women and those from 23 men. Then, we also separately evaluated the concentration for each element in the normal thyroid tissue of patients affected by either benign (42 cases) or malignant (35 cases) thyroid nodules. Again, no significant difference was found between the two groups. As a consequence, all data were evaluated cumulatively to assess the element concentration values in normal human thyroid tissue.

Iodide, as expected, was the element at the highest concentration in thyroid tissue (433.7 μg/g of wet tissue or 1682.8 mg/g of dry tissue, see Table 1), followed by Zn, Br, and Cu in the microgram range, Cr, Se, Mn, Ni, Sr, Ba, and Cd in the nanogram range (decreasing average values between 300 and 140 ng/g of tissue), and then As, Hg, Zr, Ag, W, Sn, Mo, and Pb (decreasing average values between 70 and 0.17 ng/g of tissue).

Table 1.

Element Concentration (μg/g of Wet Tissue) in Human Thyroid Tissue

Element	GM (95%CI)	M ± SD	Median (range)	GM calculated in dry weight^a
I	433.7 (329.1–571.5)	757.5 ± 764.4	548.6 (25.3–3969.2)	1682.8
Zn	11.97 (10.49–13.67)	13.91 ± 7.31	12.41 (2.05–37.52)	46.4
Br	8.2 (7.6–8.9)	8.67 ± 2.9	8.37 (2.40–16.71)	31.8
Cu	0.95 (0.9–1.1)	1.06 ± 0.51	1.04 (0.32–3.40)	3.69
Cr	0.30 (0.20–0.40)	0.48 ± 0.45	0.30 (0.02–1.68)	1.12
Se	0.28 (0.24–0.33)	0.35 ± 0.23	0.28 (0.02–0.98)	1.08
Mn	0.22 (0.19–0.25)	0.26 ± 0.15	0.23 (0.06–0.7)	0.85
Ni	0.18 (0.11–0.24)	0.30 ± 0.28	0.21 (0.01–0.98)	0.70
Sr	0.18 (0.15–0.21)	0.22 ± 0.13	0.20 (0.04–0.81)	0.70
Ba	0.17 (0.14–0.2)	0.20 ± 0.19	0.14 (0.01–0.95)	0.66
Cd	0.14 (0.11–0.17)	0.21 ± 0.21	0.14 (0.01–1.31)	0.54
As	0.07 (0.05–0.09)	0.10 ± 0.07	0.08 (ND–0.28)	0.27
Hg	0.04 (0.03–0.05)	0.09 ± 0.14	0.03 (ND–0.8)	0.16
Zr	0.03 (0.03–0.04)	0.04 ± 0.02	0.02 (ND–0.14)	0.12
Ag	0.03 (0.01–0.05)	0.09 ± 0.16	0.02 (ND–0.8)	0.12
W	0.03 (0.02–0.03)	0.07 ± 0.22	0.03 (ND–1.96)	0.12
Sn	0.02 (0.02–0.03)	0.03 ± 0.02	0.03 (0.01–0.10)	0.08
Pb	0.02 (0.02–0.02)	0.04 ± 0.04	0.02 (ND–0.29)	0.08
Mo	0.02 (0.02–0.02)	0.02 ± 0.02	0.02 (ND–0.20)	0.08

B, Co, Li, Pd, Sb, Tl, and V are not included in the table because their concentration in thyroid tissue is inferior to the LOD in over 40% of specimens examined.

Calculated concentration in dry weight = concentration in wet weight × 3.88, assuming that dry weight is equal to 25.8% of wet weight (9).

GM, geometric mean; LOD, limit of detection; M, arithmetic mean; SD, standard deviation.

These values indicate the average element content in the thyroid tissue, including not only thyrocytes and the colloid but also all other cellular and stromal components of the thyroid gland.

When considering measurement values obtained in other studies over the last two decades (Table 2), in which the number of evaluated chemicals was much smaller than in our study, a similar concentration ranking was observed. After iodide, Zn was the highest concentrated element in the thyroid, followed by Br in studies where both elements were measured. The [Zn]: [Cu] ratio ranged from 13.6 to 26.6 (12 –15) (12.6 in our series). Selenium, an essential component of selenoenzymes that play an important role in thyroid hormone metabolism, was found in a large range of concentrations (0.49–3.69 μg/g of dry tissue) (Table 2).

Table 2.

Comparison of Element Concentration (μg/Dry Human Thyroid Tissue) as Measured with Different Methods in Different Series of Subjects of Different Ethnicities

Author Year Country Sampling N. specimens Reference	Katoh et al. 2002 Japan Postmortem 64 (males) (9)	Reddy et al. 2002 India Ex vivo 4(18)	Yaman et al. 2004 Turkey Ex vivo 13^a (19)	Al-Sayer et al. 2004 Kuwait Ex vivo 45 (15)	Zhu et al. 2010 China Postmortem 68 (males)^b (17)	Blazewicz et al. 2010 Poland Postmortem 50 (12)	Zaichick et al. 2018 Russia Postmortem 105 (14)	Present study 2019 Italy Ex vivo 77^a
As	0.87 ± 0.9	125.1 ± 12.0			0.053 (0.009–0.08)			0.39 ± 0.27
Br	18.1 ± 5.1				1.28 (0.7–1.7)		13.9 ± 12	33.6 ± 11.3
Cd	16.1 ± 22.5				0.59 (0.1–5.4)	ND		0.81 ± 0.81
Cu		54.9 ± 5.5	2.7 ± 0.8	1.2 ± 2.7	2.05 (0.7–3.1)	5.24 ± 0.51	4.23 ± 1.52	4.11 ± 1.98
Hg		98.5 ± 10.0			0.006 (0.001–1.1)			0.35 ± 0.54
Mn	1.34 ± 0.68	17.3 ± 1.8		0.31 ± 1.0	0.49 (0.13–0.7)	0.68 ± 0.10		1.0 ± 0.58
Pb		17.2 ± 1.7			0.067 (0.04–0.09)			0.15 ± 0.15
Se	3.69 ± 2.33			0.49 ± 1.6	0.7 (0.4–1.2)			1.36 ± 0.89
Zn	129 ± 67	149.8 ± 15	58.2 ± 23.3	32.0 ± 72.4	28.8 (14.4–31)	101 ± 10.9	112 ± 44	54.0 ± 28.4

Measured in tissue wet weight and converted to dry weight values.

All data are presented as arithmetic mean ± SD except data by Zhu et al. (median value and 90% CI).

Element concentration in the human thyroid compared with muscle and adipose tissue

Comparing the thyroid tissue concentrations of the studied elements with those measured in the sternothyroid muscle and the subcutaneous neck fat tissue of the same individual, we observed a significant difference in the tissue accumulation of different chemicals.

In addition to iodide, seven other elements were significantly more concentrated in the thyroid tissue than in muscle and adipose tissue (p < 0.01 for all) (Table 3); among them were the halogen Br, as well as Se, an indispensable component of selenoenzymes involved in thyroid hormone metabolism, and Mn and Sn, whose role in thyroid biology is not known. The bromine concentration was 2–3-fold higher than iodide in muscle and adipose tissue, in contrast to the thyroid where iodide is present at an over 50-fold higher concentration relative to bromine. However, Br is significantly more concentrated in the thyroid than in the other tissues. The specific ability of the thyroid to concentrate Br⁻ is probably related to its chemical similarity with iodide: the two halides are transported inside thyroid follicular cells through the NIS (sodium–iodide symporter), specifically expressed at a high level in those cells (16).

Table 3.

Concentration of Trace Elements and Metals (μg/g of Wet Tissue) in Human Thyroid, Muscle, and Adipose Tissue of the Same Individuals

	Thyroid		Muscle		Adipose tissue
	GM (95% CI)	No. of cases <LOD	GM (95% CI)	No. of cases <LOD	GM (95% CI)	No. of cases<LOD
A—Elements significantly more prevalent in the thyroid
I	433.7 (329.1–571.5)	0	0.37 (0.21–0.68)^**	4	1.05 (0.61–1.8)^**	4
As	0.07 (0.05–0.09)	7	ND^**	51	ND^**	40
Br	8.2 (7.6–8.9)	0	1.29 (0.95–1.76)^**	0	2.39 (1.83–3.12)^**	0
Cd	0.14 (0.11–0.17)	0	0.02 (0.02–0.03)^**	6	ND^**	40
Hg	0.04 (0.03–0.05)	7	ND^**	44	ND^**	58
Mn	0.22 (0.19–0.25)	0	0.08 (0.06–0.10)^**	2	0.09 (0.07–0.11)^**	2
Se	0.28 (0.24–0.33)	0	0.07 (0.06–0.08)^**	0	0.03 (0.03–0.04)^**	0
Sn	0.02 (0.02–0.03)	0	0.01 (0.01–0.02)^**	27	0.01 (0.01–0.02)^**	26
B—Elements significantly less prevalent in the thyroid
B	ND	53	0.47 (0.36–0.62)^**	2	0.53 (0.39–0.73)^**	3
Cr	0.30 (0.20–0.40)	0	0.55 (0.45–0.67)^**	1	0.37 (0.29–0.46)	0
Ni	0.18 (0.11–0.24)	0	0.40 (0.33–0.47)^**	0	0.29 (0.22–0.36)^*	0
C—Elements at similar concentration in the examined tissues
Ba	0.17 (0.14–0.2)	0	0.14 (0.11–0.17)	0	0.16 (0.13–0.20)	0
Sr	0.18 (0.15–0.21)	0	0.15 (0.11–0.19)	0	0.11 (0.03–0.19)	0
W	0.03 (0.02–0.03)	20	0.02 (0.02–0.03)	29	0.03 (0.02–0.04)	16
Zr	0.03 (0.03–0.04)	0	0.03 (0.03–0.04)	0	0.04 (0.03–0.05)	0
D—Other conditions
Ag	0.03 (0.01–0.05)	22	ND^**	63	0.05 (0.01–0.2)	26
Cu	0.95 (0.9–1.1)	0	1.07 (0.95–1.2)	0	0.56 (0.49–0.65)^**	0
Mo	0.02 (0.02–0.02)	2	0.02 (0.02–0.02)	3	0.01 (0.01–0.02)^**	5
Pb	0.02 (0.02–0.02)	19	0.03 (0.02–0.03)^*	12	0.03 (0.02–0.03)	10
Zn	11.97 (10.49–13.67)	0	19.5 (16.8–22.5)^**	0	3.3 (2.76–3.94) ^**	0

ND because values are <LOD (LODs) in over 40% specimens examined.

p < 0.05; ^** p < 0.01 relative to thyroid tissue.

GM, geometric mean; ND, nondetectable.

Of note, three recognized carcinogens (As, Cd, and Hg) were significantly more concentrated in the thyroid relative to the other tissues studied. Finally, the [Zn]: [Cu] ratio was 12.6 in the thyroid versus 5.9 in adipose tissue and 18.2 in the muscle. The relatively similar ratios suggest that the human thyroid has no specific mechanism for the uptake and accumulation of these two metals, both with a relevant role in cell enzyme function.

Element concentration in the rat thyroid

Given the relevance of rats as the most widely used experimental animals in thyroid studies, species differences between man and rat were evaluated. Eleven metals were measured in the thyroid, muscle, and adipose tissue of eight normal rats (Table 4). As in the human thyroid, after iodide, the highest concentrated element in the rat thyroid was Zn, followed by Br and Cu, all of which were in the μg/g tissue range and at a concentration similar to that measured in the human thyroid. In contrast, Mn was markedly more concentrated in the rat thyroid compared with the human thyroid (rat/man ratio ∼5:1).

Table 4.

Element Concentration (μg/g of Wet Tissue) in Rat Tissues

Element	Thyroid	Muscle	Adipose	Human thyroid^a
I	245.7 (129.0–506.1)	0.18 (0.12–0.26)^*	0.58 (0.22–0.81)^*	433.7 (329.1–571.5)
Zn	14.14 (13.47–14.81)	7.62 (6.39–8.84)^*	5.54 (3.34–7.75)^*	11.97 (10.49–13.67)
Br	8.95 (7.95–9.94)	6.41 (5.14–7.68)^*	5.80 (3.35–8.25)^*	8.2 (7.6–8.9)
Cu	1.79 (1.53–2.04)	1.09 (0.91–1.27)^*	0.79 (0.56–1.02)^*	0.95 (0.9–1.1)
Se	0.25 (0.19–0.32)	0.10 (0.09–0.12)^*	0.07 (0.03–0.10)^*	0.28 (0.24–0.33)
Mn	1.28 (0.79–1.79)	0.11 (0.09–0.12)^*	0.18 (0.10–0.27)^*	0.22 (0.19–0.25)
Ba	ND	ND	0.01 (0.01–0.03)	0.17 (0.14–0.2)
Cd	ND	ND	ND	0.14 (0.11–0.17)
As	0.12 (0.08–0.15)	0.05 (0.03–0.06)^*	0.08 (0.04–0.12)	0.07 (0.05–0.09)
Hg	0.01 (0.003–0.44)	ND	ND	0.04 (0.03–0.05)
W	ND	ND	ND	0.03 (0.02–0.03)
Mo	0.07 (0.05–0.08)	0.02 (0.02–0.03)^*	0.08 (0.05–0.10)	0.02 (0.02–0.02)

Eight female Wistar rats were used. Data are presented as GM and 95% CI (between brackets).

Values for human thyroid are indicated in the fourth column for comparison.

p < 0.01 relative to the thyroid tissue.

ND, nondetectable.

Of the remaining elements, Se, As, Mo, and Hg values were in the range of ng/g of tissue (250–10), as observed in human thyroid, while Ba, Cd, and W were neither detectable (lower than detection limits) in the rat thyroid nor in the muscle and adipose tissue of the examined animals. When comparing the rat thyroid concentration with that of muscle and adipose tissue, As, Br, Hg, Mn, and Se were all more concentrated in the thyroid, reflecting a similar behavior observed in humans. In contrast to human specimens, Zn, Cu, and Mo were more concentrated in the rat thyroid relative to the other tissues examined. As in humans, however, the [Zn]: [Cu] ratio was remarkably similar in the tissues evaluated: 7.9 for the thyroid and 7.0 in both muscle and adipose tissues. Moreover, as in man, both the carcinogenic metals As and Hg were found at a higher concentration in the thyroid, while no comparative evaluation was possible for Cd since this metal was present at a lower than measurable concentration in all rat tissues examined.

Element concentrations in the thyroid of residents in volcanic versus nonvolcanic areas

We previously observed than in the volcanic area of Mount Etna, where thyroid cancer incidence is double relative to adjacent nonvolcanic areas (6), significant environmental pollution is present. Urine values of many elements were significantly higher than values in the urine of residents of adjacent nonvolcanic areas (7), indicating biocontamination.

We comparatively examined the concentration of these elements in the thyroid of residents of the 2 areas, 43 from the volcanic area (mean age 45 ± 18 years, F = 33) and 34 from the control area (mean age 52.2 ± 14.6, F = 21). For many elements (As, Ba, Br, Cr, Cu, Hg, Mn, Ni, Se, Zr, and Zn), concentrations in the thyroid were slightly higher in individuals living in the volcanic area compared with residents of control areas. However, a large overlap of individual values was present and differences were not significant (Table 5). In addition, in the muscle and adipose tissues of residents of the volcanic area, the concentrations of most examined elements were also slightly higher, suggesting that the increase was the general consequence of the increased exposure for all tissues and that no specific accumulation occurs in the thyroid. Only As and Hg were increased in the thyroid, but not in muscle and adipose tissues, of residents of the volcanic area. In addition, for these carcinogenic metals, however, differences were small and nonsignificant relative to values found in thyroids of the control group.

Table 5.

Concentration of Trace Elements and Metals (μg/g of Wet Tissue) in the Thyroid of Residents of the Mount Etna Volcanic Area Versus Values in the Thyroid of Inhabitants of Adjacent Nonvolcanic Areas

	Ag	As	Ba	Br	Cd	Cr	Cu	Hg	Mn	Mo	Ni	Pb	Se	Sn	Sr	W	Zr	Zn
Volcanic area (no. of cases 43)
No. <LOD	11	4	0	0	1	0	0	4	0	1	0	10	0	0	0	9	0	0
GM	0.02	0.07	0.18	8.83	0.13	0.33	1.03	0.04	0.24	0.02	0.18	0.02	0.29	0.02	0.16	0.02	0.04	12.2
95% CI	0.01–0.05	0.05–0.1	0.15–0.23	7.4–9.7	0.09–0.19	0.19–0.48	0.9–1.2	0.03–0.07	0.2–0.3	0.01–0.02	0.09–0.27	0.01–0.03	0.23–0.36	0.02–0.03	0.13–0.20	0.02–0.03	0.03–0.04	10–14.8
Control area (no. of cases 34)
No. <LOD	11	3	0	0	0	0	0	3	0	1	0	9	0	0	0	11	0	0
GM	0.03	0.06	0.16	7.44	0.14	0.27	0.87	0.03	0.19	0.02	0.17	0.02	0.28	0.02	0.20	0.03	0.03	11.7
95% CI	0.01–0.06	0.04–0.1	0.12–0.21	6.7–8.25	0.1–0.2	0.13–0.40	0.8–1.0	0.02–0.05	0.15–0.23	0.02–0.03	0.09–0.25	0.02–0.03	0.22–0.36	0.01–0.03	0.16–0.25	0.02–0.04	0.02–0.04	9.8–14

Iodine concentration in the thyroid was 464.2 (GM, μg/g tissue) in the volcanic area versus 397.9 in the control area (nonsignificant).

Discussion

Using an advanced spectrochemical technique, we measured the concentration of 26 trace elements in the human thyroid gland, paying special attention not only to the quality of the analytical technique but also to preanalytical factors such as patient euthyroidism, normal tissue selection, sample handling and processing in a highly specialized laboratory, and postanalytical calculations.

In some previous studies, for instance, measurements were carried out in the thyroid of deceased individuals (9,12,14,17) with no information on postmortem period duration or on possible interfering factors such as previous thyroid disease, disorders in other organs, or intake of medications. In contrast, the use of strict inclusion criteria is one of the strengths of this study: we investigated thyroid tissue collected during planned surgery for a single nonfunctioning thyroid nodule in patients with confirmed past and present euthyroid status. The normal morphology of the excised thyroid tissue was further controlled at pathology, minimizing possible variability due to altered tissue architecture. The normal thyroid, in fact, has a large colloid component (∼20% of wet weight) and this component may be increased, reduced, or absent in different thyroid conditions such as iodide deficiency, hyperthyroidism, adenomas, and cancer. Since the relative concentration of metals in cells versus colloid has never been measured, the abnormal cellular/colloid ratio may influence the results. For this reason, the comparison of the chemical element concentration per gram of tissue in the normal and pathological thyroid may be of limited significance.

As far as the analytical procedure is concerned, the DRC-ICP-MS method ensures excellent detection limits that (for most elements) are one or two orders of magnitude below the μg/L (ng/L—ppt) concentration. Moreover, it allows the possibility of multielement analysis of isotope mixtures with extremely accurate results. For these reasons, DRC-ICP-MS is currently the most widely used method for detection of trace elements and metals.

Another point that deserves attention is data calculation. Most previous studies presented data as the arithmetic mean of measured values. These values, however, follow an asymmetric rather than normal (Gaussian) distribution for practically all substances, and nonparametric models such as the median with range values or the GM with 95% CI are preferable. Different calculations provide different values with the arithmetic mean levels often significantly higher (increased from 20% to 50% up to 2–3-fold) than values obtained with nonparametric analyses (Table 1). These differences are due to the influence of occasional exceptionally high values on the arithmetic mean and variation. Since data from most previous studies were presented as the mean ± SD, when comparing data from different series, we also present our data in this form in Table 2.

Considering these preanalytical, analytical, and postanalytical differences and considering the different ethnicities, diets, and environments of the subjects studied, the differences in observed values between the present and previous data can be easily explained (Table 2). Although different, however, the concentration values obtained in studies performed in the last 20 years are in most cases in the same range found in this study (Table 2) except for the study by Reddy et al. (18), which was performed in only 4 normal thyroid specimens and in a very different ethnic and environmental context. In that study, a surprisingly low iodide/zinc ratio has been reported (1.54 vs. 36.2 in our series) and concentration values of most elements are one or two orders of magnitude higher than in other studies (Table 2), suggesting severe overestimation because of possible problems for tissue selection, tissue specimen processing, including those related to the freeze-dry procedure of samples (19), or the analytical procedure (20).

Considering all the trace elements measured in our study and excluding iodide, nearly 90% of all trace elements in human and rat thyroid is represented by Zn and Br.

Bromine has no recognized specific physiological function in any organ and is minimally incorporated into organic compounds. Its elevated concentration in the thyroid was significantly higher than in muscle and fat in humans (Table 3) and was also higher in the rat thyroid (Table 4), likely the consequence of its chemical similarity and functional competition with iodide (both halogens) (16). In our series, the observed [I]: [Br] ratio was 52.9 in humans and 27.4 in rats.

In contrast to Br, Zn is an essential metal that is involved in many aspects of cell biology as a structural element and as a regulatory factor. The free intracellular (Zn⁺²) level is very low since most Zn is associated with proteins, predominantly metallothioneins and metalloenzymes (20,21). Zn, together with Cu, has an important role as a cofactor for superoxide dismutase (SOD), a key enzyme that protects cells from superoxide toxicity, a very relevant function in the biology of the thyroid. In the human thyroid, the [Zn]: [Cu] ratio is greater than 10 in all studies except the one by Reddy et al. (18). In rats, the two metals were more abundant in the thyroid relative to muscle and fat (Table 4), but this was not the case in humans, in which both metals were present at a higher level in muscle (Table 1). The difference between humans and rats may be not only due to species-specific differences in physiological mechanisms but also to the different environmental conditions of our experimental models, including the limited mobility and artificial standardized diet given to rats, as well as the different localization of excised muscle and fat in measured specimens from humans and rats. Zn and Cu also serve as signaling factors for regulation of cell proliferation, differentiation, and death (20,22,23) and have been suggested to play a role also in thyroid cancer etiology and progression (24 –26). However, until now, the chemical speciation, compartmentalization, and role in thyroid physiopathology of these two essential metals remain unknown and require future research.

The thyroid concentration of Se is remarkably similar in humans and rats; in both species, values in the thyroid are higher than in muscle and fat. Selenoenzymes (type I, II, and III deiodinases, as well as glutathione peroxidase and thioredoxin reductase) are known to play an essential role in thyroid hormone metabolism and redox processes. Whether the increased concentration of Se in the thyroid is due to facilitated uptake or increased retention due to incorporation into proteins, or both, is not known.

Another metal with a concentration in the human thyroid that is more than twice as high relative to muscle and fat is Mn. This metal is potentially toxic at high levels and is possibly carcinogenic (27), but at the same time, it is an essential constituent of many enzymes, including Mn-SOD, an SOD that is a major antioxidant for neutralizing the toxic effect of reactive oxygen species. In the rat thyroid, Mn was found at a much higher level than that in the human thyroid and at ∼10 times greater levels than in muscle and fat. Since Mn concentrations in muscle and fat tissues are similar to those found in humans, this observation suggests that for unknown reasons, the rat thyroid accumulates Mn more than the human thyroid.

Another interesting observation is the significantly higher concentration of three important carcinogens in the thyroid relative to the other tissues studied. The levels of As, Cd, and Hg in the human thyroid were significantly higher (p < 0.01) than those in muscle and fat (Table 3). This same increase was observed for As and Hg (but not for Cd) in the rat thyroid (Table 4). How the relative abundance of these toxic metals can influence the very frequent occurrence of benign and malignant nodules in the thyroid is not known. In middle and advanced age individuals, thyroid nodules occur in 50% or more cases (28,29), and the thyroid mutation rate is calculated to be 8–10-fold higher than in other organs (30). Mutations may accumulate because follicular cells are constantly exposed to the toxic effect of free radicals produced by the continuous generation of hydrogen peroxide necessary for oxidation of iodide (I⁻) to derivatives such as hypoiodite, hypoiodous acid, and iodinium (31). This unfavorable microenvironment promotes spontaneous mutagenesis (30) and the increased presence and activity of carcinogenic metals may favor cell transformation, leading to formation of benign and malignant nodules.

Thus, it is interesting that in the thyroid of residents of the Mount Etna volcanic area, where thyroid cancer incidence is doubled (6,32), many elements and metals are present at a higher concentration than in the thyroid of residents of adjacent nonvolcanic areas (7,33). The differences between the two groups are not statistically significant, and therefore, no conclusion can be drawn until more cases are studied. It is noteworthy that in residents of the volcanic area, the small increase of most elements observed in the thyroid also occurs in muscle and fat, which suggests generalized biocontamination from a polluted environment. This is not the case, however, for As and Hg, two carcinogens that are increased (+16.6% and +25%, respectively) in the thyroid, but not in the two other examined tissues. As already mentioned, in our limited series, the increases are small and not statistically significant, but a potential biological effect of a very low dose increase is now documented for various metals and chemicals such as As (34), Cd (35,36), Hg (36,37), W (38), and bisphenol A (39) due to a hormetic dose response following a biphasic pattern (40).

In conclusion, our study provides novel information on the concentration of numerous elements in the human thyroid relative to other tissues of the same individual and to similar tissues in the rat. This latter comparison has the weakness of different environment and dietary conditions between the two species.

The reference data provided by our study involve many trace elements that play an important role as determinants of thyroid function, such as the antioxidant defense system (metalloenzymes), hormone production and metabolism, and thyroid cell growth, differentiation, and death. Some of the studied elements, when in excess, may also have toxic effects through metal-mediated formation of free radicals, DNA damage, and enhanced lipid peroxidation (41), possibly influencing some genetic alterations predisposing to thyroid cancer (42 –44). It must be stressed, however, the biological meaning of data reported in this study, as well as in previous studies, is hampered by the consideration that the values observed represent the average of heterogeneous levels present in the different structural and functional compartments of the gland. Follicular cells and colloid, C cells, stromal components, and vessels might have a markedly different concentration of the studied chemical, as has been observed for iodide.

Thus, bridging interdisciplinary endocrine research is required to connect variations in the overall tissue concentration to the chemical, biochemical, and molecular aspects of trace element accumulation considering the different components of thyroid tissue. These studies will allow a better understanding of the role of trace elements and metals in thyroid physiology and pathology.

Footnotes

Acknowledgment

Ms. Rita Pennisi is gratefully acknowledged for her skillful assistance during manuscript and table preparation.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This study was supported, in part, by a grant from the AIRC Foundation, Italy (Grant No. 19897), to R.V.

Supplementary Material

Supplementary Table S1

References

Gore

, Chappell

, Fenton

, Flaws

, Nadal

, Prins

, Toppari

, Zoeller

. 2015. EDC-2: the endocrine society's second scientific statement on endocrine-disrupting chemicals. Endocr Rev, 36:E1–E150.

Minoia

, Pietra

, Sabbioni

, Ronchi

, Gatti

, Cavalleri

, Manzo

. 1992. Trace element reference values in tissues from inhabitants of the European Community. III. The control of preanalytical factors in the biomonitoring of trace elements in biological fluids. Sci Total Environ, 120:63–79.

Arnbjornsson

, Arnbjornsson

, Olafsson

. 1986. Thyroid cancer incidence in relation to volcanic activity. Arch Environ Health, 41:36–40.

Kolonel

, Hankin

, Wilkens

, Fukunaga

, Hinds

. 1990. An epidemiologic study of thyroid cancer in Hawaii. Cancer Causes Control, 1:223–234.

Kung

, Ng

, Gibson

. 1981. Volcanoes and carcinoma of the thyroid: a possible association. Arch Environ Health, 36:265–267.

Pellegriti

, De Vathaire

, Scollo

, Attard

, Giordano

, Arena

, Dardanoni

, Frasca

, Malandrino

, Vermiglio

, Previtera

, D'Azzo

, Trimarchi

, Vigneri

. 2009. Papillary thyroid cancer incidence in the volcanic area of Sicily. J Natl Cancer Inst, 101:1575–1583.

Malandrino

, Russo

, Ronchi

, Minoia

, Cataldo

, Regalbuto

, Giordano

, Attard

, Squatrito

, Trimarchi

, Vigneri

. 2016. Increased thyroid cancer incidence in a basaltic volcanic area is associated with non-anthropogenic pollution and biocontamination. Endocrine, 53:471–479.

Fadda

, Basolo

, Bondi

, Bussolati

, Crescenzi

, Nappi

, Nardi

, Papotti

, Taddei

, Palombini

, Group

S-IICW

. 2010. Cytological classification of thyroid nodules. Proposal of the SIAPEC-IAP Italian Consensus Working Group. Pathologica, 102:405–408.

Katoh

, Sato

, Yamamoto

. 2002. Determination of multielement concentrations in normal human organs from the Japanese. Biol Trace Elem Res, 90:57–70.

10.

Baranov

, Tanner

. 1999. A dynamic reaction cell for inductively coupled plasma mass spectrometry (ICP-DRC-MS). Part 1. The RF-field energy contribution in thermodynamics of ion-molecule reactions. J Anal Atom Spectrom, 14:1133–1142.

11.

Hornung

, Reed

. 1990. Estimation of average concentration in the presence of nondetectable values. Appl Occup Environ Hyg, 5:46–51.

12.

Blazewicz

, Dolliver

, Sivsammye

, Deol

, Randhawa

, Orlicz-Szczesna

, Blazewicz

. 2010. Determination of cadmium, cobalt, copper, iron, manganese, and zinc in thyroid glands of patients with diagnosed nodular goitre using ion chromatography. J Chromatogr B Analyt Technol Biomed Life Sci, 878:34–38.

13.

Kucharzewski

, Braziewicz

, Majewska

, Góźdź

. 2003. Copper, zinc, and selenium in whole blood and thyroid tissue of people with various thyroid diseases. Biol Trace Elem Res, 93:9–18.

14.

Zaichick

, Zaichick

. 2018. Associations between age and 50 trace element contents and relationships in intact thyroid of males. Aging Clin Exp Res, 30:1059–1070.

15.

Al-Sayer

, Mathew

, Asfar

, Khourshed

, Al-Bader

, Behbehani

, Dashti

. 2004. Serum changes in trace elements during thyroid cancers. Mol Cell Biochem, 260:1–5.

16.

Pavelka

. 2004. Metabolism of bromide and its interference with the metabolism of iodine. Physiol Res, 53:S81–S90.

17.

Zhu

, Wang

, Zhang

, Wu

, Chen

, Gao

, Chang

, Liu

, Fan

, Li

, Wang

. 2010. Element contents in organs and tissues of Chinese adult men. Health Phys, 98:61–73.

18.

Reddy

, Charles

, Kumar

, Reddy

, Anjaneyulu

, Raju

, Sundareswar

, Vijayan

. 2002. Trace elemental analysis of adenoma and carcinoma thyroid by PIXE method. Nucl Instr Methods Phys Res B Beam Interac Mater Atoms, 196:333–339.

19.

Yaman

, Akdeniz

. 2004. Sensitivity enhancement in flame atomic absorption spectrometry for determination of copper in human thyroid tissues. Anal Sci, 20:1363–1366.

20.

Beyersmann

, Hartwig

. 2008. Carcinogenic metal compounds: recent insight into molecular and cellular mechanisms. Arch Toxicol, 82:493–512.

21.

Boulyga

, Loreti

, Bettmer

, Heumann

. 2004. Application of SEC-ICP-MS for comparative analyses of metal-containing species in cancerous and healthy human thyroid samples. Anal Bioanal Chem, 380:198–203.

22.

Truong-Tran

, Carter

, Ruffin

, Zalewski

. 2001. The role of zinc in caspase activation and apoptotic cell death. In: Maret

(ed). Zinc Biochemistry, Physiology, and Homeostasis. Springer, Dordrecht, Netherlands, pp 129–144.

23.

Watjen

, Haase

, Biagioli

, Beyersmann

. 2002. Induction of apoptosis in mammalian cells by cadmium and zinc. Environ Health Perspect, 110 Suppl 5:865–867.

24.

Brady

, Crowe

, Turski

, Hobbs

, Yao

, Chaikuad

, Knapp

, Xiao

, Campbell

, Thiele

. 2014. Copper is required for oncogenic BRAF signalling and tumorigenesis. Nature, 509:492.

25.

Shen

, Cai

W-S

, Li

J-L

, Feng

, Cao

, Xu

. 2015. The association between serum levels of selenium, copper, and magnesium with thyroid cancer: a meta-analysis. Biol Trace Elem Res, 167:225–235.

26.

, Casio

, Range

, Sosa

, Counter

. 2018. Copper chelation as targeted therapy in a mouse model of oncogenic BRAF-driven papillary thyroid cancer. Clin Cancer Res, 24:4271–4281.

27.

Assem

, Holmes

, Levy

. 2011. The mutagenicity and carcinogenicity of inorganic manganese compounds: a synthesis of the evidence. J Toxicol Environ Health B Crit Rev, 14:537–570.

28.

Guo

, Sun

, He

, Chen

, Li

, Tang

, Lu

, Bi

, Ning

. 2014. The prevalence of thyroid nodules and its relationship with metabolic parameters in a Chinese community-based population aged over 40 years. Endocrine, 45:230–235.

29.

Jiang

, Tian

, Yan

, Kong

, Wang

, Dou

, Liang

, Mu

. 2016. The prevalence of thyroid nodules and an analysis of related lifestyle factors in Beijing communities. Int J Environ Res Public Health, 13:442.

30.

Maier

, Van Steeg

, Van Oostrom

, Karger

, Paschke

, Krohn

. 2006. Deoxyribonucleic acid damage and spontaneous mutagenesis in the thyroid gland of rats and mice. Endocrinology, 147:3391–3397.

31.

Kopp

. 2012. “Thyroid hormone synthesis” Werner and Ingbar's the Thyroid: A Fundamental and Clinical Text. 10th ed. Lippincott Williams & Wilkins, Philadelphia, pp 48–74.

32.

Vigneri

, Malandrino

, Vigneri

. 2015. The changing epidemiology of thyroid cancer: why is incidence increasing?. Curr Opin Oncol, 27:1–7.

33.

Vigneri

, Malandrino

, Gianì

, Russo

, Vigneri

. 2017. Heavy metals in the volcanic environment and thyroid cancer. Mol Cell Endocrinol, 457:73–80.

34.

Yang

, Chang

, Zheng

, Huang

, Sun

, Zhao

, Li

, Xiu

. 2017. Anti-angiogenic effect of arsenic trioxide in lung cancer via inhibition of endothelial cell migration, proliferation and tube formation. Oncol Lett, 14:3103–3109.

35.

Damelin

, Vokes

, Whitcutt

, Damelin

, Alexander

. 2000. Hormesis: a stress response in cells exposed to low levels of heavy metals. Hum Exp Toxicol, 19:420–430.

36.

Jiang

, Zhao

, Fang

, Xiao

. 2018. Physiological responses and metal uptake of Miscanthus under cadmium/arsenic stress. Environ Sci Pollut Res Int, 25:28275–28284.

37.

Hao

, Hao

, Wei

, Xing

, Jiang

, Shang

. 2009. The role of MAPK in the biphasic dose-response phenomenon induced by cadmium and mercury in HEK293 cells. Toxicol In Vitro, 23:660–666.

38.

Kelly

, Lemaire

, Young

, Eustache

, Guilbert

, Molina

, Mann

. 2013. In vivo tungsten exposure alters B-cell development and increases DNA damage in murine bone marrow. Toxicol Sci, 131:434–446.

39.

Prins

, Patisaul

, Belcher

, Vandenberg

. 2018. CLARITY-BPA academic laboratory studies identify consistent low-dose Bisphenol A effects on multiple organ systems. Basic Clin Pharmacol Toxicol, 125 Suppl 3:14–31.

40.

Calabrese

. 2004. Hormesis: a revolution in toxicology, risk assessment and medicine. EMBO Rep 5 Spec No:S37–S40.

41.

Valko

, Morris

, Cronin

. 2005. Metals, toxicity and oxidative stress. Curr Med Chem, 12:1161–1208.

42.

Patel

, Singh

. 2006. Genetic considerations in thyroid cancer. Cancer Control, 13:111–118.

43.

Petr

, Else

. 2016. Genetic predisposition to endocrine tumors: diagnosis, surveillance and challenges in care. Semin Oncol, 43:582–590.

44.

Vella

, Puppin

, Damante

, Vigneri

, Sanfilippo

, Vigneri

, Tell

, Frasca

. 2009. DeltaNp73alpha inhibits PTEN expression in thyroid cancer cells. Int J Cancer, 124:2539–2548.