Methylglyoxal-induced neurotoxic effects in primary neuronal-like cells transdifferentiated from human mesenchymal stem cells: Impact of low concentrations.

In the last decades, advanced glycation end-products (AGEs) have aroused the interest of the scientific community due to the increasing evidence of their involvement in many pathophysiological processes including various neurological disorders and cognitive decline age related. Methylglyoxal (MG) is one of the reactive dicarbonyl precursors of AGEs, mainly generated as a by-product of glycolysis, whose accumulation induces neurotoxicity. In our study, MG cytotoxicity was evaluated employing a human stem cell-derived model, namely, neuron-like cells (hNLCs) transdifferentiated from mesenchymal stem/stromal cells, which served as a source of human based species-specific "healthy" cells. MG increased ROS production and induced the first characteristic apoptotic hallmarks already at low concentrations (≥10 µM), decreased the cell growth (≥5-10 µM) and viability (≥25 µM), altered Glo-1 and Glo-2 enzymes (≥25 µM), and markedly affected the neuronal markers MAP-2 and NSE causing their loss at low MG concentrations (≥10 µM). Morphological alterations started at 100 µM, followed by even more marked effects and cell death after few hours (5 h) from 200 µM MG addition. Substantially, most effects occurred as low as 10 µM, concentration much lower than that reported from previous observations using different in vitro cell-based models (e.g., human neuroblastoma cell lines, primary animal cells, and human iPSCs). Remarkably, this low effective concentration approaches the level range measured in biological samples of pathological subjects. The use of a suitable cellular model, that is, human primary neurons, can provide an additional valuable tool, mimicking better the physiological and biochemical properties of brain cells, in order to evaluate the mechanistic basis of molecular and cellular alterations in CNS.

Bisdemethoxycurcumin (BDC)-Loaded H-Ferritin-Nanocages Mediate the Regulation of Inflammation in Alzheimer's Disease Patients.

BACKGROUND: Bisdemethoxycurcumin (BDC) might be an inflammation inhibitor in Alzheimer's Disease (AD). However, BDC is almost insoluble in water, poorly absorbed by the organism, and degrades rapidly. We thus developed a new nanoformulation of BDC based on H-Ferritin nanocages (BDC-HFn). METHODS: We tested the BDC-HFn solubility, stability, and ability to cross a blood-brain barrier (BBB) model. We tested the effect of BDC-HFn on AD and control (CTR) PBMCs to evaluate the transcriptomic profile by RNA-seq. RESULTS: We developed a nanoformulation with a diameter of 12 nm to improve the solubility and stability. The comparison of the transcriptomics analyses between AD patients before and after BDC-HFn treatment showed a major number of DEG (2517). The pathway analysis showed that chemokines and macrophages activation differed between AD patients and controls after BDC-HFn treatment. BDC-HFn binds endothelial cells from the cerebral cortex and crosses through a BBB in vitro model. CONCLUSIONS: Our data showed how BDC-Hfn could improve the stability of BDC. Significant differences in genes associated with inflammation between the same patients before and after BDC-Hfn treatment have been found. Inflammatory genes that are upregulated between AD and CTR after BDC-HFn treatment are converted and downregulated, suggesting a possible therapeutic approach.

Occupational Exposure Assessment to Antineoplastic Drugs in Nine Italian Hospital Centers over a 5-Year Survey Program.

In the present study, surface contamination where antineoplastic drugs (ADs) are present was investigated, as occupational exposure risk is still an open debate. Despite recommendations and safety standard procedures being in place in health care settings, quantifiable levels of ADs are being reported in the recent literature. Thus, a survey monitoring program was conducted over five years (2016-2021) in nine Italian hospitals. The repeated surveys produced 8288 data points that have been grouped according to the main hospital settings, such as pharmacy areas and patient care units. Based on the most often prepared ADs, the investigated drugs were cyclophosphamide (CP), gemcitabine (GEM), 5-fluorouracil (5-FU), and platinum compounds (Pt). Patient care units had a frequency of positive wipe samples (59%) higher than pharmacies (44%). Conversely, pharmacies had a frequency of positive pad samples higher (24%) than patient care units (10%). Moreover, by statistical analysis, pad samples had a significantly higher risk of contamination in pharmacy areas than in patient care units. In this study, the 75th and the 90th percentiles of the contamination levels were obtained. The 90th percentile was chosen to describe a suitable benchmark that compares results obtained by the present research with those previously reported in the literature. Based upon surface contamination loads, our data showed that 5-FU had the highest concentration values, but the lowest frequency of positive samples. In pharmacy areas, the 90th percentile of 5-FU data distribution was less than 0.346 ng/cm2 and less than 0.443 ng/cm2 in patient care units. AD levels are higher than those reported for health care settings in other European countries yet trends of contamination in Italy have shown to decrease over time.

Long read sequencing overcomes challenges in the diagnosis of SORD neuropathy.

Biallelic mutations in sorbitol dehydrogenase (SORD) have been recently identified as a common cause of recessive axonal Charcot-Marie-Tooth neuropathy (CMT2). We aimed to assess a novel long-read sequencing approach to overcome current limitations in SORD neuropathy diagnostics due to the SORD2P pseudogene and the phasing of biallelic mutations in recessive disease. We conducted a screen of our Australian whole exome sequencing (WES) CMT cohort to identify individuals with homozygous or compound heterozygous SORD variants. Individuals detected with SORD mutations then underwent long-read sequencing, clinical assessment, and serum sorbitol analysis. An individual was detected with compound heterozygous truncating mutations in SORD exon 7, NM_003104.5:c.625C>T (p.Arg209Ter) and NM_003104.5:c.757del (p.Ala253GlnfsTer27). Subsequent Oxford Nanopore Tech (ONT) long-read sequencing was used to successfully differentiate SORD from the highly homologous non-functional SORD2P pseudogene and confirmed that the mutations were biallelic through haplotype-resolved analysis. The patient presented with axonal sensorimotor polyneuropathy (CMT2) and ulnar neuropathy without compression at the elbow. Burning neuropathic pain in the forearms and feet was also reported and was exacerbated by alcohol consumption and improved with alcohol cessation. UPLC-tandem mass spectrometry confirmed that the patient had elevated serum sorbitol levels (12.0 mg/L) consistent with levels previously observed in patients with biallelic SORD mutations. This represents a novel clinical presentation and expands the phenotype associated with biallelic SORD mutations causing CMT2. Our study is the first report of long-read sequencing for an individual with CMT and demonstrates the utility of this approach for clinical genomics.

Selective Targeting of Cancer-Associated Fibroblasts by Engineered H-Ferritin Nanocages Loaded with Navitoclax.

Cancer-associated fibroblasts (CAFs) are key actors in regulating cancer progression. They promote tumor growth, metastasis formation, and induce drug resistance. For these reasons, they are emerging as potential therapeutic targets. Here, with the aim of developing CAF-targeted drug delivery agents, we functionalized H-ferritin (HFn) nanocages with fibroblast activation protein (FAP) antibody fragments. Functionalized nanocages (HFn-FAP) have significantly higher binding with FAP+ CAFs than with FAP- cancer cells. We loaded HFn-FAP with navitoclax (Nav), an experimental Bcl-2 inhibitor pro-apoptotic drug, whose clinical development is limited by its strong hydrophobicity and toxicity. We showed that Nav is efficiently loaded into HFn (HNav), maintaining its mechanism of action. Incubating Nav-loaded functionalized nanocages (HNav-FAP) with FAP+ cells, we found significantly higher cytotoxicity as compared to non-functionalized HNav. This was correlated with a significantly higher drug release only in FAP+ cells, confirming the specific targeting ability of functionalized HFn. Finally, we showed that HFn-FAP is able to reach the tumor and to target CAFs in a mouse syngeneic model of triple negative breast cancer after intravenous administration. Our data show that HNav-FAP could be a promising tool to enhance specific drug delivery into CAFs, thus opening new therapeutic possibilities focused on tumor microenvironment.

Development and validation of a simple and versatile method for the quantification of everolimus loaded in H-ferritin nanocages using UHPLC-MS/MS.

Everolimus (Eve) is an immunosuppressive macrolide that is being analyzed in various biological matrices and fluids. Its antitumor activity makes this drug suitable not only for organ transplantation but also for breast cancer treatments. In the attempt to reduce the incidence and severity of its side effects, Eve was loaded in H-ferritin (HFn), a natural biomolecule that is involved in specific cellular uptake pathways. Thus, Eve pre-complexed with Cu(II) and encapsulated in HFn resulted in an Eve nanoformulation, named HEve. The quantification of HEve was performed using a tailored pH-induced procedure to precipitate H-ferritin. This sample preparation was effective enough to reduce the ion suppression effect on the mass spectrometric responses of Eve in electrospray ionization (ESI). The ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-ESI-MS/MS) system operating in positive ionization mode showed to be a versatile technique in achieving more than 77 % recovery of Eve from the cytoplasmic compartment. This simple, selective and sensitive method enabled the quantification of Eve within the linear range of 2.5-100 ng/mL in matrix spiked with the isotope-labeled internal standard, EveD4. This method was validated according to FDA Guidance. The intracellular distribution of HEve and its accumulation at a cytoplasmic level were studied in breast cancer cell lines. As expected, HEve was more effective than free Eve on sensitive (i.e. BT474) and resistant cell lines, as a result of a better penetration into the target subcellular compartment.

Biallelic mutations in SORD cause a common and potentially treatable hereditary neuropathy with implications for diabetes.

Here we report biallelic mutations in the sorbitol dehydrogenase gene (SORD) as the most frequent recessive form of hereditary neuropathy. We identified 45 individuals from 38 families across multiple ancestries carrying the nonsense c.757delG (p.Ala253GlnfsTer27) variant in SORD, in either a homozygous or compound heterozygous state. SORD is an enzyme that converts sorbitol into fructose in the two-step polyol pathway previously implicated in diabetic neuropathy. In patient-derived fibroblasts, we found a complete loss of SORD protein and increased intracellular sorbitol. Furthermore, the serum fasting sorbitol levels in patients were dramatically increased. In Drosophila, loss of SORD orthologs caused synaptic degeneration and progressive motor impairment. Reducing the polyol influx by treatment with aldose reductase inhibitors normalized intracellular sorbitol levels in patient-derived fibroblasts and in Drosophila, and also dramatically ameliorated motor and eye phenotypes. Together, these findings establish a novel and potentially treatable cause of neuropathy and may contribute to a better understanding of the pathophysiology of diabetes.

Author Correction: Biallelic mutations in SORD cause a common and potentially treatable hereditary neuropathy with implications for diabetes.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Everolimus Nanoformulation in Biological Nanoparticles Increases Drug Responsiveness in Resistant and Low-Responsive Breast Cancer Cell Lines.

Everolimus (Eve) is an FDA approved drug that inhibits mammalian target of rapamycin (mTOR). It is employed in breast cancer treatment even if its responsiveness is controversial. In an attempt to increase Eve effectiveness, we have developed a novel Eve nanoformulation exploiting H-ferritin nanocages (HEve) to improve its subcellular delivery. We took advantage of the natural tumor targeting of H-Ferritin, which is mediated by the transferrin receptor-1 (TfR1). Breast cancer cells overexpressing TfR-1 were successfully recognized by H-Ferritin, displaying quick nanocage internalization. HEve has been tested and compared to Eve for in vitro efficacy in sensitive and resistant breast cancer cells. Nanoformulated Eve induced remarkable antiproliferative activity in vitro, making even resistant cell lines sensitive to Eve. Moreover, the antiproliferative activity of HEve is fully in accordance with cytotoxicity observed by cell death assay. Furthermore, the significant increase in anticancer efficacy displayed in HEve-treated samples is due to the improved drug accumulation, as demonstrated by UHPLC-MS/MS quantifications. Our findings suggest that optimizing Eve subcellular delivery, thanks to nanoformulation, determines its improved antitumor activity in a panel of Eve-sensitive or resistant breast cancer cell lines.

Paclitaxel-Loaded Silk Fibroin Nanoparticles: Method Validation by UHPLC-MS/MS to Assess an Exogenous Approach to Load Cytotoxic Drugs.

The aim of this work was to load an anticancer drug, paclitaxel (PTX), on Silk Fibroin Nanoparticles (SFNs) by using an exogenous approach. SFNs were produced, freeze-dried and then loaded with PTX. An exogenous method allowed us to reduce both drug loss and environmental impact. In order to quantify PTX loaded in SFNs, a simple and reliable method using reversed phase liquid chromatography coupled to tandem mass spectrometry (rp-UHPLC-MS/MS) was developed. This methodology was validated by the determination of spiked QC samples in three consecutive days. Good accuracy and precision of the method were obtained, while the intra-day and inter-day precisions were less than 10.3%. For PTX, the limit of quantitation (LOQ) was 5.0 ng/mL. Recovery from the matrix (SFNs-PTX pellets) was calculated (81.2% at LOQ value) as PTX was entrapped in a new matrix like the polymer silk fibroin-based. This method was successfully applied to determine the encapsulation efficiency (1.00 ± 0.19%) and the nanoparticle loading (0.12 ± 0.02% w/w). The in vitro anticancer activity of SFNs-PTX was tested against CFPAC-1 cancer cells; results demonstrated a very high cytotoxic activity of SFNs-PTX, with a dose dependent inhibition of CFPAC-1 proliferation, confirmed by the IC50 value of 3450 ± 750 ng/mL.

From mathematical axioms to mathematical rules of proof: recent developments in proof analysis.

A short text in the hand of David Hilbert, discovered in Göttingen a century after it was written, shows that Hilbert had considered adding a 24th problem to his famous list of mathematical problems of the year 1900. The problem he had in mind was to find criteria for the simplicity of proofs and to develop a general theory of methods of proof in mathematics. In this paper, it is discussed to what extent proof theory has achieved the second of these aims. This article is part of the theme issue 'The notion of 'simple proof' - Hilbert's 24th problem'.

Validation of cleaning procedures used in an Italian Hospital Pharmacy for antineoplastic drug decontamination: a new tool for industrial hygiene.

BACKGROUND: Current Italian regulations and procedures for surface decontamination of antineoplastic drugs (ADs) are not clear. Therefore, most hospital pharmacies follow internal procedures as an interpretation of the recommended handling guidelines. OBJECTIVES: Our study compared 7 different cleaning procedures after controlled contamination of the work surface of a biological safety cabinet workbench in an Italian hospital oncology pharmacy (HOP) to determine which of them is more efficient and practical. Moreover, in order to approximate operative routine and improve risk awareness, cleaning procedures were carried out by the personnel that usually operate in the HOP. METHODS: Measured quantities, i.e. a drop (100 µL) of 5-FluoroUracil, IPhosfamide, CycloPhosphamide and Gemcitabine, were deposited on the work surface within precisely delimited areas. Following the wipe-test analysis using UPLC-MS/MS, the cleaning efficacy was calculated based on the ratio of the residual concentration of the AD, after the cleaning procedure, to the concentration of each AD before the procedure. RESULTS: Tested cleaning procedures were: 1) Hypo-Chlor®, hot water and Farmecol70®; 2) Hypo-Chlor® and hot water; 3) Farmecol70®; 4) Surfa'Safe SH® and hot water; 5) Amuchina® 10%, hot water and Farmecol70®; 6) Incidin® Oxyfoam and hot water; 7) liquid Marseille soap, hot water and Farmecol70®. Within the studied HOP, the Marseille soap was evaluated to be the optimal choice due to its efficacy, low cost, and the very short contact time needed before rinsing. DISCUSSION: The application of the protocol for procedure validation suggested here could be used in every HOP as a reliable industrial hygiene tool to demonstrate the validity of the chosen cleaning procedure.

Exposure duration and absorbed dose assessment in pesticide-exposed agricultural workers: Implications for risk assessment and modeling.

INTRODUCTION: Absorbed dose assessment from dermal exposure involves multiplying skin contamination by the dermal absorption coefficient, which is usually defined for the standard workday of 8 h. This strategy may suffer from limitations when the duration of exposure is extremely variable, such as in agricultural exposure to pesticides. OBJECTIVES: The aim of this study was to estimate the dose of mancozeb absorbed by agricultural pesticide applicators in a typical working day considering the real duration of exposure, to compare these estimates with those coming from the use of the Fixed Fractional Approach, and to assess the suitability of the dose estimates in the interpretation of biological monitoring results. METHODS: In a series of real-life field studies on 29 workers applying mancozeb in vineyards for 38 work days, three sets of data were collected: information regarding work activities for each work day, potential (on clothes) and actual skin exposure using the "patch" methodology, and excretion of ethylenethiourea (ETU) in the 24-h pre-exposure and 24-h post-exposure urine samples. The statistical analyses were done using the R Language and Environment for Statistical Computing. RESULTS: Accounting for the duration of exposure led to a substantial reduction in the absorbed dose estimates, compared to the estimates coming from the Fixed Fractional Approach. In particular, absorbed dose by the body, hands' and total absorbed dose were reduced by 50%, 81%, and 80% respectively. The body dose estimated considering both approaches still correlated better with post-exposure 24-h urine ETU levels than the hands' dose, although more than 90% of the estimated total absorbed dose comes from the hands. CONCLUSIONS: An accurate estimate of the absorbed dose, carried out considering the real duration of exposure, can result in a higher correlation with a biomarker of occupational exposure, such as urine ETU, or at least yield more accurate results. This can facilitate the interpretation of biological monitoring data in pesticide-exposed agricultural workers despite the absence of biological exposure limits. ETU should be evaluated as a potentially relevant source of exposure due to ethylenebisdithiocarbamates' (EBDCs) degradation in the formulated product or spray mixture.

A new, sensitive and versatile assay for quantitative determination of α-fluoro-ß-alanine (AFBA) in human urine by using the reversed-phase ultrahigh performance-tandem mass spectrometry (rp-UHPLC-MS/MS) system.

A method for the quantitation of α-fluoro-ß-alanine (AFBA), the main metabolite of capecitabine (Cape) and 5-fluoruracil (5-FU), is described. Among antineoplastic drugs (ADs), 5-FU and Cape (the new oral prodrug) are the most commonly applied drugs in cancer therapy. The main objective of this study was to develop a reliable method that would be easy to run on a reversed-phase UHPLC system coupled to tandem mass spectrometry. AFBA was derivatized with Sanger's reagent to ensure complete yield of a stable 2,4 dinitrophenil-α-fluoro-ß-alanine derivative. This method was based on the use of a mixed-mode anion exchange solid phase extraction enabling urinary extracts to be clear of endogenous interferences affecting quantitative results. The assay was validated in human urine according to FDA criteria with the use of a labeled internal standard (ß-alanine-d4) to minimize experimental error. Good accuracy and precision were demonstrated by determining spiked urine QC samples in four consecutive days. The recovery of AFBA was between 70.0 and 82.6%, with a matrix effect that was 12.8%-18.5%. The lower limit of quantitation (LOQ) was 0.5 ng/mL with a coefficient of variation of 5.3%. This assay was successfully applied to determine the levels of this metabolite in a large number of urine samples taken from personnel who were occupationally exposed to ADs.

Urinary levels of metal elements in the non-smoking general population in Italy: SIVR study 2012-2015.

The purpose of this study of the Italian Society of Reference Values (SIVR) was to provide the reference values for metals in the urine of the Italian general population. Thirteen sampling centres situated in the north, centre and south of Italy took part in this project. Each sampling centre selected 20 adult subjects. The sample was made up of 120 male and 140 female non-smokers or smokers who had abandoned the habit at least 5 years previously, aged between 18 and 60 years. Urine samples were obtained from the same subjects in June and again in November of the same year. During collection of these two samples, we administered an ad hoc questionnaire designed to assess factors that might influence exposure and the results of the study such as personal characteristics, occupational or extra-occupational activities, dietary habits, the presence of dental fillings containing amalgam, the frequency and mode of exposure to passive smoking and exhaust gases of motor vehicles (traffic). Therefore, the urinary levels of thirteen trace elements (Be, Cd, Co, Cr, Cu, In, Mn, Ni, Pb, Pt, Sb, Tl and V) were determined by Inductively Coupled Plasma Mass Spectrometry. The concentrations obtained (95th percentile) are in µg/L: 0.034, 0.900, 2.24, 0.600, 24.0, 0.013, 1.53, 4.44, 2.64, 0.022, 0.095, 0.759 and 0.855 for Be, Cd, Co, Cr, Cu, In, Mn, Ni, Pb, Pt, Sb, Tl and V, respectively. Further studies with a larger number of subjects are needed in order to continue the biomonitoring of the Italian general population and to monitor the modified levels over time.

Monitoring Surface Contamination by Antineoplastic Drugs in Italian Hospitals: Performance-Based Hygienic Guidance Values (HGVs) Project.

Antineoplastic drugs (ADs) will continue to represent a potential risk for personnel involved in the handling of these compounds and great concerns have been raised by the presence of ADs in many surveyed workplaces. Eight hospitals were investigated by means of wipe sampling for surface residue determination. Each wipe sample was tested for five ADs considered suitable exposure markers. Cyclophosphamide (CP), gemcitabine (GEM), 5-fluorouracil (5-FU), platinum-containing drugs (Pt), and epi-doxorubicin (EPI) contamination levels were measured in 85 per cent of the studied pharmacies and 93 per cent of outpatient care units (OpCUs). This study showed that 83 out of 349 samples were positive in Pharmacies, this proportion being statistically significant (χ2 = 42.9, p < 0.001). The positive samples provided evidence of at least one substance with levels greater than the limit of detection (LOD). The two most frequently detected substances were Pt (42%) and CP (30%). These accounted for 72 per cent of the whole dataset, followed by 5-FU and GEM. Based on the 90th percentile of wipe sampling data distribution, we suggest hygienic guidance values (HGVs) of 3.6, 1.0, 0.9, and 0.5 ng cm-2 for CP, 5-FU, GEM and Pt, respectively, as the best target levels of the surface contamination load in Italian pharmacies. The approach of proposing guidance values at the 90th percentile of results obtained from workplaces with good hygiene practice was found to be a simple and practical way of controlling occupational exposure. HGVs were challenged in this study as technical threshold limits to benchmark AD residual surface contamination at workplaces.

Methodology to define biological reference values in the environmental and occupational fields: the contribution of the Italian Society for Reference Values (SIVR).

BACKGROUND: Biological reference values (RVs) explore the relationships between humans and their environment and habits. RVs are fundamental in the environmental field for assessing illnesses possibly associated with environmental pollution, and also in the occupational field, especially in the absence of established biological or environmental limits. OBJECTIVES: The Italian Society for Reference Values (SIVR) determined to test criteria and procedures for the definition of RVs to be used in the environmental and occupational fields. METHODS: The paper describes the SIVR methodology for defining RVs of xenobiotics and their metabolites. Aspects regarding the choice of population sample, the quality of analytical data, statistical analysis and control of variability factors are considered. The simultaneous interlaboratory circuits involved can be expected to increasingly improve the quality of the analytical data. RESULTS: Examples of RVs produced by SIVR are presented. In particular, levels of chromium, mercury, ethylenethiourea, 3,5,6-trichloro-2-pyridinol, 2,5-hexanedione, 1-hydroxypyrene and t,t-muconic acid measured in urine and expressed in micrograms/g creatinine (µg/g creat) or micrograms/L (µg/L) are reported. CONCLUSIONS: With the proposed procedure, SIVR intends to make its activities known to the scientific community in order to increase the number of laboratories involved in the definition of RVs for the Italian population. More research is needed to obtain further RVs in different biological matrices, such as hair, nails and exhaled breath. It is also necessary to update and improve the present reference values and broaden the portfolio of chemicals for which RVs are available. In the near future, SIVR intends to expand its scientific activity by using a multivariate approach for xenobiotics that may have a common origin, and to define RVs separately for children who may be exposed more than adults and be more vulnerable.

Determination of Glucocorticoids in UPLC-MS in Environmental Samples from an Occupational Setting.

Occupational exposures to glucocorticoids are still a neglected issue in some work environments, including pharmaceutical plants. We developed an analytical method to quantify simultaneously 21 glucocorticoids using UPLC coupled with mass spectrometry to provide a basis to carry out environmental monitoring. Samples were taken from air, hand-washing tests, pad-tests and wipe-tests. This paper reports the contents of the analytical methodology, along with the results of this extensive environmental and personal monitoring of glucocorticoids. The method in UPLC-MS turned out to be suitable and effective for the aim of the study. Wipe-test and pad-test desorption was carried out using 50 mL syringes, a simple technique that saves time without adversely affecting analyte recovery. Results showed a widespread environmental pollution due to glucocorticoids. This is of particular concern. Evaluation of the dose absorbed by each worker and identification of a biomarker for occupational exposure will contribute to assessment and prevention of occupational exposure.

Exposure to perfluorinated compounds: in vitro study on thyroid cells.

Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) are widely used perfluorinated chemicals (PFCs). Previous studies detected PFOA and PFOS in human tissues including the thyroid gland. There are no studies on the in vitro effects of PFOA and PFOS on thyroid cells. Our study was aimed at evaluating the effect of the in vitro exposure to PFOA and PFOS on thyroid cell proliferation and viability. These objectives were investigated using Fisher rat thyroid line-5 (FRTL-5) cells. FRTL-5 cells cultured in the presence of PFOA and PFOS at concentrations up to 10(4) nM do not display changes in their viability and proliferation rate, while at a concentration of 10(5) nM of either PFCs, a significant inhibition of cell proliferation, mainly due to increased cell death, was found. PFOA and PFOS were detected in FRTL-5 cell pellets after 72 h of incubation with PFCs but not in control cultures. When FRTL-5 were incubated with PFCs then washed in PBS and re-cultured for 72 h without PFCs in the medium, no detectable concentrations of PFOA and PFOS were measured in the cell pellet. This indicates that PFOA and PFOS enter thyroid cells by a gradient-based passive diffusion mechanism. Future studies are required to evaluate the potential toxic effect resulting from prolonged in vivo exposure to even lower concentrations of PFCs.