Changing the structure of PFOA and PFOS: a chemical industry strategy or a solution to avoid thyroid-disrupting effects?

BACKGROUND: The family of perfluoroalkyl and polyfluoroalkyl substances (PFAS) raised concern for their proven bioaccumulation and persistence in the environment and animals as well as for their hazardous health effects. As a result, new congeners of PFAS have rapidly replaced the so-called "old long-chain PFAS" (mainly PFOA and PFOS), currently out-of-law and banned by most countries. These compounds derive from the original structure of "old long-chain PFAS", by cutting or making little conformational changes to their structure, thus obtaining new molecules with similar industrial applications. The new congeners were designed to obtain "safer" compounds. Indeed, old-long-chain PFAS were reported to exert thyroid disruptive effects in vitro, and in vivo in animals and humans. However, shreds of evidence accumulated so far indicate that the "restyling" of the old PFAS leads to the production of compounds, not only functionally similar to the previous ones but also potentially not free of adverse health effects and bioaccumulation. Studies aimed at characterizing the effects of new-PFAS congeners on thyroid function indicate that some of these new-PFAS congeners showed similar effects. PURPOSE: The present review is aimed at providing an overview of recent data regarding the effects of novel PFAS alternatives on thyroid function. RESULTS AND CONCLUSIONS: An extensive review of current legislation and of the shreds of evidence obtained from in vitro and in vivo studies evaluating the effects of the exposure to novel PFOA and PFOS alternatives, as well as of PFAS mixture on thyroid function will be provided.

Cardiovascular and metabolic comorbidities in patients with thyroid nodules: the impact of incidental diagnosis.

PURPOSE: The prevalence of thyroid nodules (TN) in the general population has increased as screening procedures are implemented and an association with metabolic and cardiovascular disorders has been reported. The aim of this study was to investigate the reason leading to the diagnosis of TN and to compare the clinical characteristics of patients diagnosed incidentally with those of patients diagnosed for thyroid-related reasons. METHODS: We designed a retrospective cross-sectional study including consecutive patients with TN from two high-volume hospital-based centers for thyroid diseases (Pavia and Messina) in Italy. Data regarding reason leading to TN diagnosis, age, sex, BMI, presence of cardio-metabolic comorbidities were collected. RESULTS: Among the 623 enrolled subjects, the US diagnosis of TN was prompted by thyroid-related reasons in 421 (67.6%, TD group) and incidental in 202 (32.4%, ID group) with a similar distribution in the two centers (p = 0.960). The ID group patients were more frequently males (38.6% vs 22.1%, p < 0.001) and significantly older (58.9 ± 13.7 vs 50.6 ± 15.5 years, p < 0.001) than the TD group ones, and had a higher rate of cardiovascular comorbidities (73.8% vs 47.5%, p < 0.001), despite having a similar BMI (27.9 ± 5.2 vs 27.8 ± 13.5, p = 0.893). CONCLUSIONS: Stratification of patients with TN according to the diagnostic procedure leading to diagnosis allows a better epidemiological characterization of this inhomogeneous and large population.

A first-trimester serum TSH in the 4-10 mIU/L range is associated with obstetric complications in thyroid peroxidase antibody-negative women.

PURPOSE: The impact of mild subclinical hypothyroidism on pregnancy outcomes in TPOAb-negative women is poorly explored. The aim of the present study was the evaluation in a wide cohort of TPOAb-negative pregnant women the role of subclinical hypothyroidism (SCH) on several pregnancy outcomes. METHODS: The study included women aged ≥ 18 years with a singleton pregnancy without known thyroid disease with serum TSH concentration between 0.4 and 10 mIU/L and TPOAb negative. Data about clinical and demographic features were collected. A blood sample was drown to test TSH, TPOAb, ANA and ENA concentration. The mean uterine artery pulsatility index was measured. Risk of adverse obstetric and fetal outcomes was collected. RESULTS: The cohort included 2135 pregnant women. Pregnant women with TSH 4-10 mUI/L had a significantly higher frequency of family history of thyroid diseases, and personal history of celiac disease diseases, type 1 diabetes mellitus, rheumatic disease, antinuclear antibody (ANA) and anti-extractable nuclear antigen (ENA) positive tests. The risk for pre-eclampsia and small for gestational age (SGA) was significantly higher in pregnant women with first-trimester TSH 4-10 mIU/L. A first-trimester TSH serum level greater than 4 mIU/L was associated with a significant increase in the occurrence of abnormal uterine artery pulsatility index, with a more than threefold increase in the risk of developing pre-eclampsia and with the risk of SGA. CONCLUSIONS: In TPOAb-negative pregnant women, a first-trimester serum TSH level ranging from 4 to 10 mIU/L is significantly and independently linked to an increased uterine artery pulsatility index as well as to negative pregnancy outcomes such as pre-eclampsia, SGA and gestational diabetes.

Vitamin D and interferon-γ co-operate to increase the ACE-2 receptor expression in primary cultures of human thyroid cells.

BACKGROUND: A more severe course of COVID-19 was associated with low levels of Vitamin D (VitD). Moreover in vitro data showed that VitD up-regulates the mRNA of the Angiotensin Converting Enzyme 2 (ACE-2), the SARS-COV-2 receptor in different type of cells. ACE-2 is expressed in several type of tissues including thyroid cells, on which its mRNA was shown to be up-regulated by interferon-gamma (IFN-γ). The aim of the present study was to investigate if treatment with VitD alone or in combination with IFN-γ would increase ACE-2 both at mRNA and protein levels in primary cultures of human thyrocytes. MATERIALS AND METHODS: Primary thyroid cell cultures were treated with VitD and IFN-γ alone or in combination for 24 h. ACE-2 mRNA levels were measured by Real-time Polymerase Chain Reaction (RT-PCR). The presence of ACE-2 on thyroid cell membrane was assessed by immunocytochemistry basally and after the previous mentioned treatments. RESULTS: ACE-2 mRNA levels increased after treatment with VitD and IFN-γ alone. The combination treatment (VitD + IFN-γ) showed an additive increase of ACE-2-mRNA. Immunocytochemistry experiments showed ACE-2 protein on thyroid cells membrane. ACE-2 expression increased after treatment with VitD and IFN-γ alone and further increased by the combination treatment with VitD + IFN-γ. CONCLUSIONS: VitD would defend the body by SARS-COV2 both by regulating the host immune defense and by up-regulating of the expression of the ACE-2 receptor. The existence of a co-operation between VitD and IFN-γ demonstrated in other systems is supported also for ACE-2 up-regulation. These observations lead to an increased interest for the potential therapeutic benefits of VitD supplementation in COVID-19.

Detection of SARS-COV-2 receptor ACE-2 mRNA in thyroid cells: a clue for COVID-19-related subacute thyroiditis.

PURPOSE: SARS-COV-2 is a pathogenic agent belonging to the coronavirus family, responsible for the current global world pandemic. Angiotensin-converting enzyme 2 (ACE-2) is the receptor for cellular entry of SARS-CoV-2. ACE-2 is a type I transmembrane metallo-carboxypeptidase involved in the Renin-Angiotensin pathway. By analyzing two independent databases, ACE-2 was identified in several human tissues including the thyroid. Although some cases of COVID-19-related subacute thyroiditis were recently described, direct proof for the expression of the ACE-2 mRNA in thyroid cells is still lacking. Aim of the present study was to investigate by RT-PCR whether the mRNA encoding for ACE-2 is present in human thyroid cells. METHODS: RT-PCR was performed on in vitro ex vivo study on thyroid tissue samples (15 patients undergoing thyroidectomy for benign thyroid nodules) and primary thyroid cell cultures. RESULTS: The ACE-2 mRNA was detected in all surgical thyroid tissue samples (n = 15). Compared with two reporter genes (GAPDH: 0.052 ± 0.0026 Cycles-1; ß-actin: 0.044 ± 0.0025 Cycles-1; ACE-2: 0.035 ± 0.0024 Cycles-1), the mean level of transcript expression for ACE-2 mRNA was abundant. The expression of ACE-2 mRNA in follicular cells was confirmed by analyzing primary cultures of thyroid cells, which expressed the ACE-2 mRNA at levels similar to tissues. CONCLUSIONS: The results of the present study demonstrate that the mRNA encoding for the ACE-2 receptor is expressed in thyroid follicular cells, making them a potential target for SARS-COV-2 entry. Future clinical studies in patients with COVID-19 will be required for increase our understanding of the thyroid repercussions of SARS-CoV-2 infection.

The new generation PFAS C6O4 does not produce adverse effects on thyroid cells in vitro.

PURPOSE: Per- and poly-fluoroalkyl-substances (PFASs) are synthetic compounds that raised concern due to their potential adverse effects on human health. Long-chain PFAS were banned by government rules in many states, and thus, new emerging PFAS were recently introduced as substitutes. Among these, Perfluoro{acetic acid, 2-[(5-methoxy-1,3-dioxolan-4-yl)oxy]}, ammonium salt (C6O4) was recently introduced to produce a range of food contact articles and literature data about this compound are scanty. The aim of this study was to evaluate the in vitro effects of exposure to C6O4, compared with PFOA and PFOS on thyroid cells. METHODS: FRTL5 rat-thyroid cell lines and normal human thyroid cells (NHT) were incubated with increasing concentrations of C6O4 for 24, 48, 72, and 144 h to assess cell viability by WST-1. Cell viability was confirmed by AnnexinV/PI staining. Long-chain PFAS (PFOA and PFOS) were used at same concentrations as positive controls. The proliferation of cells exposed to C6O4, PFOA, and PFOS was measured by staining with crystal violet and evaluation of optical density after incubation with SDS. Changes in ROS production by FRTL5 and NHT after exposure to C6O4 at short (10, 20, and 30 min) and long-time points (24 h) were evaluated by cytofluorimetry. RESULTS: C6O4 exposure did not modify FRTL5 and NHT cell viability at any concentration and/or time points with no induction of necrosis/apoptosis. At difference, PFOS exposure reduced cell viability of FRTL5 while and NHT, while PFOA only in FRTL5. FRTL5 and NHT cell proliferation was reduced by incubation with by PFOA and PFOS, but not with C6O4. ROS production by NHT and FRTL5 cells was not modified after C6O4 exposure, at any time/concentration tested. CONCLUSIONS: The present in vitro study constitutes the first evaluation of the potential adverse effects of the new emerging PFAS C6O4 in cultured rat and human thyroid cells, suggesting its safety for thyroid cells in vitro.

Compared with classic Hashimoto's thyroiditis, chronic autoimmune serum-negative thyroiditis requires a lower substitution dose of L-thyroxine to correct hypothyroidism.

PURPOSE: Serum-negative-chronic-autoimmune-thyroiditis (SN-CAT) is considered a milder variant of classic Hashimoto's thyroiditis (CHT). However, its prevalence remains unknown and it is still unclear whether SN-CAT behaves differently in terms of L-thyroxine (LT4) substitution treatment of hypothyroidism. Aims of this study were to estimate the prevalence of SN-CAT in a large series of hypothyroid patients and to compare LT4 requirements in hypothyroid patients with SN-CAT and CHT. METHODS: Five-hundred-eighty-one consecutive patients with primary-autoimmune-hypothyroidism were enrolled in a cross-sectional study. LT4 requirements and thyroid-volume changes were longitudinally evaluated in 49 hypothyroid patients with SN-CAT and in 98 sex and age-matched hypothyroid patients with CHT. RESULTS: In our series the prevalence of SN-CAT was 20.8%. At diagnosis, patients in the CHT and SN-CAT groups had similar male/female ratio, age and BMI, while serum TSH and thyroid-volume were significantly greater in the CHT group. In the longitudinal study, during a follow-up of 8.9 ± 4.6 years, 8 out of 49 (16.3%) SN-CAT patients developed positive tests for of circulating TPO-Ab and/or Tg-Ab. Thyroid-volume significantly decreased in CHT patients, but not in those with SN-CAT. The maximum daily substitution dose of LT4 was smaller in SN-CAT patients as compared with the CHT ones. Multivariate analysis showed that age, BMI, basal TSH and thyroid antibody status independently and significantly predicted the maximum daily substitution dose of LT4. CONCLUSIONS: SN-CAT accounts for a significant proportion of patients with autoimmune hypothyroidism. Compared with hypothyroid patients diagnosed with CHT, the SN-CAT ones require smaller doses of LT4 to correct their hypothyroidism.

Laser photocoagulation therapy for thyroid nodules: long-term outcome and predictors of efficacy.

PURPOSE: The aim of the present study was to retrospectively evaluate the efficacy of interstitial laser photocoagulation (ILP) ablation of thyroid nodules during a 6-year follow-up period and to identify possible predictors of the final outcome. METHODS: Forty-three outpatients (38 women) were assigned to ILP therapy. The study group included euthyroid patients with benign thyroid nodules. Thyroid size, nodule volume and features, and autoimmune test were collected at baseline. Patients underwent US control after the ILP procedure and 1 month, 6 months, 12 months later and then annually. RESULTS: During the follow-up, two distinct groups of patients emerged: the responders (N = 33) and the non-responder (N = 10) ones to ILP. In the responder group, the nodule volume significantly decreased during the follow-up, but a trend toward a slight increase in nodule volume was recorded up to the end of follow-up. No significant decrease in nodule volume was observed in the non-responder group. Neither baseline clinical nor demographic features were significantly different between responders and non-responders groups. In the whole group of patients, the energy delivered per mL of nodule tissue was significantly correlated with the percent volume decrease at the end of follow-up. CONCLUSIONS: Interstitial laser photocoagulation is a safe technique able to reduce byabout 50% the volume of benign thyroid nodules in the majority of treated patients. However, due to the great variability of results, an active follow-up is required. The only independent predictor of ILP outcome is the energy delivered per mL of nodule tissue.

The clinical phenotype of Graves' disease occurring as an isolated condition or in association with other autoimmune diseases.

PURPOSE: Graves' disease (GD) can present as an isolated disease (iGD) or in association with other autoimmune diseases (aGD). The aim of this study, performed in two Endocrine referral centers settled in different geographical areas of Italy, was to compare the anthropometric, clinical, and biochemical phenotype of iGD patients with that of the aGD ones. METHODS: Clinical history, physical examination data, serum levels of TSH, FT4, FT3, thyroglobulin (TgAb), thyroid-peroxidase (TPOAb) and TSH-receptor (TRAb) antibody, presence of Graves' orbitopathy (GO), and thyroid ultrasound examination at disease diagnosis were recorded. RESULTS: 68 aGD and 136 iGD patients were consecutively recruited. At diagnosis, aGD and iGD patients did not differ for F/M ratio, age at presentation, thyroid function parameters, serum levels of TRAb, TgAb, TPOAb, presence of GO, and thyroid volume. The serum levels of TRAb were strongly correlated with the circulating concentrations of both FT3 (ρ = 0.667; p < 0.0001) and FT4 (ρ = 0.628; p < 0.001) in iGD patient, but not in the aGD ones (FT3: ρ = 0.231; p = 0.058; FT4: ρ = 0.096; p = 0.435). Compared with iGD patients, the aGD ones displayed a higher rate of transition from the previous hypothyroidism to hyperthyroidism (χ2 = 6.375; p = 0.012). CONCLUSION: Despite similar anthropometric, clinical, and biochemical features at diagnosis, aGD patients display a higher rate of transition from a thyroid functional status to the other as compared with iGD patients.

Effect of long- and short-chain perfluorinated compounds on cultured thyroid cells viability and response to TSH.

PURPOSE: Perfluorinated chemicals are widespread pollutants persistent in the environment with links to some major health issues. The two main compounds, perfluoro-octanoic acid (PFOA) and perfluoro-alkyl sulphonate (PFOS), were recently classified as carcinogenetic and thus their use has been restricted. Short-chain PFCs were recently developed as an alternative, but no data regarding the possible endocrine toxicities of these compounds are available. Aim of this study was to investigate whether short-chain PFCs could jeopardize thyroid cell viability and/or interfere with the functional effect TSH. METHODS: Fisher rat thyroid line-5 (FRTL-5) was treated with increasing concentrations of PFOA, PFOS, perfluorobutanesulfonic acid (PFBS), perfluorobutanoic acid (PFBA), pentafluoropropionic anhydride (PFPA), perfluoropentanoic acid (PFPeA) to evaluate modifications in cell viability and TSH-stimulated cAMP production. RESULTS: Neither long nor short-chain PFCs affected cell viability (apart from PFOS 100 µM), or interfered with cAMP production. CONCLUSIONS: The results of the present study demonstrate for the first time that short-chain PFCs have no acute cytotoxic effect on thyroid cells in vitro and that cAMP production is not modulated by any of the tested PFCs.

The AMPK-activator AICAR in thyroid cancer: effects on CXCL8 secretion and on CXCL8-induced neoplastic cell migration.

PURPOSE: The AMPK-activator AICAR recently raised great interest for its anti-cancer properties. With specific regard to thyroid cancer, AICAR reduces cancer cell growth, invasion and metastasis. CXCL8, a chemokine with several recognized tumorigenic effects, is abundantly secreted in thyroid cancer microenvironment. The aim of this study was to investigate if AICAR could inhibit the basal and the TNFα-induced CXCL8 secretion in normal human thyroid cells (NHT) and in thyroid cancer cell lines TPC-1 and BCPAP (RET/PTC and BRAFV600e mutated, respectively). METHODS: The effect of AICAR on basal and CXCL8-induced cell migration was assessed. Cells were incubated with AICAR (0.05, 0.5, 1, 2 mM) alone or in combination with TNF-α (10 ng/ml) for 24 h. CXCL8 concentrations were measured in cell supernatants. Transwell migration assays were performed in NHT, TPC-1 and BCPAP, basally and after treatment with AICAR (2 mM) and rh-CXCL8 (50 ng/ml) alone or in combination. RESULTS: AICAR dose dependently inhibited the basal secretion of CXCL8 in TPC-1 (F = 4.26; p < 0.007) and BCPAP (F = 6.75; p < 0.0001) but not in NHT. TNFα-induced CXCL8 secretion was dose dependently reduced by AICAR in NHT (F = 9.99; p < 0.0001), TPC-1 (F = 9.25; p < 0.0001) and BCPAP (F = 6.82; p < 0.0001). AICAR significantly reduced the basal migration of TPC-1 and BCPAP but not of NHT. CONCLUSIONS: CXCL8-induced cell migration was inhibited in NHT, TPC-1 and BCPAP. This is the first demonstration of the inhibition of CXCL8 secretion exerted by AICAR in TPC-1 and BCPAP indicating that the anti-cancer properties of AICAR are, at least in part, mediated by its ability to reduce the pro-tumorigenic effects of CXCL8.

Early spermatogenesis changes in traumatic complete spinal cord-injured adult patients.

STUDY DESIGN: Prospective longitudinal study. OBJECTIVES: To assess the impact of spinal cord injury (SCI) on the spermatogenesis of patients studied in the early subacute stage and ensuing months. SETTING: National hospital for SCI patients. METHODS: A prospective follow-up study was conducted on 28 male patients with complete SCI who were evaluated in the early subacute phase (~1 month), and 3 and 6 months after the injury. At each time point, fine needle aspiration biopsy samples were taken from the testes for cytological assessment, and serum levels of relevant hormones were measured. At the end of the study period, ejaculation was attempted for standard semen analyses. RESULTS: Cytological patterns indicative of defective spermatogenesis were found in 61%, 52% and 20% of the patients at 1, 3 and 6 months after SCI, respectively, suggesting an improvement over time. Serum hormone analyses showed a steady elevation of estradiol levels above the reference range, and increasing levels of testosterone, inhibin B and prolactin throughout the study period. Prolactin levels were above the reference range at all time points. Only 2 out of the 10 patients who were able to ejaculate at 6 months post injury showed normal sperm parameters. CONCLUSION: A majority of the patients showed impaired spermatogenesis soon after the injury, which in most cases recovered over time. That was accompanied by parallel increases in serum levels of inhibin B, testosterone and prolactin, possibly driving or reflecting the spermatogenesis recovery. Further studies are needed to elucidate the biological mechanisms underlying these changes.

Thyroid disruption by perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA).

BACKGROUND: Perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA) are two fluorinated compounds widely used in industry because of their useful chemical characteristics. They were identified as endocrine disruptors due to their ability to interfere with thyroid function. The resistance of PFOA and PFOS to environmental degradation, their bio-accumulation in food chains, and their long half-life raised concern in the scientific community, and several studies were performed with the aim to establish the real dangerousness of these compounds for the human health. PURPOSE: The present review will focus on the effects of PFOA and PFOS on the thyroid gland taking into account in vitro experiments, animal studies, and human data. PFOS and PFOA reduce the circulating levels of thyroid hormones in diet-exposed animals, mainly by increasing their metabolic clearance rate. CONCLUSIONS: An accumulation of PFOS and PFOA was documented in thyroid cells, and a cytotoxic effect was observed after exposure to extremely high concentrations of these compounds. In environmentally exposed communities and in the general population, the most consistent effect of exposure to PFOA, and to a less extent to PFOS, is the occurrence of hypothyroidism. Women and children appear to be more at risk of developing mild thyroid failure. Pregnant women with circulating thyroid antibodies might be at risk of developing subclinical hypothyroidism, mainly when exposed at high doses of PFOS. The relative risks for thyroid cancer in people exposed to PFOA and PFOS were low and based on a few cases. Moreover, there was no consistent finding across all or even most studies.

Expanding the therapeutic spectrum of metformin: from diabetes to cancer.

INTRODUCTION: Metformin, an oral hypoglycemic agent, was introduced in the clinical practice for the treatment of type 2 diabetes mellitus more than a half-century ago. Over the years, several studies demonstrated that diabetic patients treated with metformin have a lower incidence of cancer, raising the hypothesis that the spectrum of clinical applications of the drug could be expanded also to cancer therapy. Following these initial findings, a large number of studies were performed aimed at elucidating the effects of metformin on different types of tumor, at explaining its direct and indirect anti-cancer mechanisms and at identifying the molecular pathways targeted by the drug. Several clinical trials were also performed aimed at evaluating the potential anti-cancer effect of metformin among diabetic and non-diabetic patients affected by different types of cancer. While the results of several clinical studies are encouraging, a considerable number of other investigations do not support a role of metformin as an anti-cancer agent, and highlight variables possibly accounting for discrepancies. AIM: We hereby review the results of in vitro and in vivo studies addressing the issue of the anti-cancer effects of metformin. CONCLUSIONS: If in vitro data appear solid, the results provided by in vivo studies are somehow controversial. In this view, larger studies are needed to fully elucidate the role of metformin on cancer development and progression, as well as the specific clinical settings in which metformin could become an anti-cancer drug.