Assessment of chemical mixtures using biomarkers of combined biological activity: A screening study in human placentas.

Humans are simultaneously exposed to complex mixtures of chemicals with limited knowledge on potential health effects, therefore improved tools for assessing these mixtures are needed. As part of the Human Biomonitoring for Europe (HBM4EU) Project, we aimed to examine the combined biological activity of chemical mixtures extracted from human placentas using one in vivo and four in vitro bioassays, also known as biomarkers of combined effect. Relevant endocrine activities (proliferative and/or reporter gene assays) and four endpoints were tested: the estrogen receptor (ER), androgen receptor (AR), and aryl hydrocarbon receptor (AhR) activities, as well as thyroid hormone (TH) signaling. Correlations among bioassays and their functional shapes were evaluated. Results showed that all placental extracts agonized or antagonized at least three of the abovementioned endpoints. Most placentas induced ER-mediated transactivation and ER-dependent cell proliferation, together with a strong inhibition of TH signaling and the AR transactivity; while the induction of the AhR was found in only one placental extract. The effects in the two estrogenic bioassays were positively and significantly correlated and the AR-antagonism activity showed a positive borderline-significant correlation with both estrogenic bioassay activities. However, the in vivo anti-thyroid activities of placental extracts were not correlated with any of the tested in vitro assays. Findings highlight the importance of comprehensively mapping the biological effects of "real-world" chemical mixtures present in human samples, through a battery of in vitro and in vivo bioassays. This approach should be a complementary tool for epidemiological studies to further elucidate the combined biological fingerprint triggered by chemical mixtures.

Is There Such a Thing as "Anti-Nutrients"? A Narrative Review of Perceived Problematic Plant Compounds.

Plant-based diets are associated with reduced risk of lifestyle-induced chronic diseases. The thousands of phytochemicals they contain are implicated in cellular-based mechanisms to promote antioxidant defense and reduce inflammation. While recommendations encourage the intake of fruits and vegetables, most people fall short of their target daily intake. Despite the need to increase plant-food consumption, there have been some concerns raised about whether they are beneficial because of the various 'anti-nutrient' compounds they contain. Some of these anti-nutrients that have been called into question included lectins, oxalates, goitrogens, phytoestrogens, phytates, and tannins. As a result, there may be select individuals with specific health conditions who elect to decrease their plant food intake despite potential benefits. The purpose of this narrative review is to examine the science of these 'anti-nutrients' and weigh the evidence of whether these compounds pose an actual health threat.

Rapid determination of propylthiouracil and methimazole by surface-enhanced Raman scattering based on sodium alginate-protected silver nanoparticles.

A rapid, ultrasensitive and selective method has been established to determine antithyroid drugs [propylthiouracil (PTU) and methimazole (MTZ)] by surface-enhanced Raman scattering (SERS). Silver nanoparticles (AgNPs) were synthesized by reduction of silver nitrate (AgNO3) with sodium alginate (SA) and were used as SERS substrates. Through optimization of the SERS conditions, including the volume of SA-protected AgNPs solution, pH of Britton-Robinson (BR) buffer solution and concentration of NaCl solution, linear responses were obtained for PTU and MTZ in the concentration ranges of 3.02 × 10-9-1.06 × 10-5 mol L-1 and 1.21 × 10-9-1.21 × 10-5 mol L-1, respectively. By the present method, the limits of detection (LODs) for the determination of PTU and MTZ were as low as 1.58 × 10-10 mol L-1 and 2.97 × 10-11 mol L-1. The method was successfully applied for the determination of PTU and MTZ in real samples. The recovery of PTU and MTZ from actual samples ranged between 97.20 and 104.80%, with relative standard deviations (RSDs) less than 3.63%.

Chromatographic methods development, validation and degradation characterization of the antithyroid drug Carbimazole.

The current paper reports the development and validation of stability-indicating HPLC and HPTLC methods for the separation and quantification of main impurity and degradation product of Carbimazole. The structures of the degradation products formed under stress degradation conditions, including hydrolytic and oxidative, photolytic and thermal conditions, were characterized and confirmed by MS and IR analyses. Based on the characterization data, the obtained degradation product from hydrolytic conditions was found to be methimazole-impurity A of Carbimazole as reported by the British Pharmacopeia and the European Pharmacopeia. A stability-indicating HPLC method was carried out using a Zorbax Eclipse Plus CN column (150 × 4.6 mm i.d, 5 µm particle size) and a mobile phase composed of acetonitrile-0.05 m KH2 PO4 (20: 80, v/v) in isocratic elution, at a flow rate of 1 mL/min. The method was proved to be sensitive for the determination down to 0.5% of Carbimazole impurity A. Additionally, a stability-indicating chromatographic HPTLC method was achieved using cyclohexane-ethanol (9:1, v/v) as a developing system on HPTLC plates F254 with UV detection at 225 nm. The proposed HPLC and HPTLC methods were successfully applied to Carbimazole® tablets with mean percentage recoveries of 100.12 and 99.73%, respectively.

Goitrogenic/antithyroidal potential of moringa leaves (Moringa oleifera) and spinach (Spinacia oleracea) of Indian origin on thyroid status in male albino rats

Moringa leaves and spinach used as vegetables containing cyanogens and polyphenols having suspected antithyroidal activity; however, detailed studies in this aspect found unavailable. Goitrogenic/antithyroid potentiality of those plant foods of Indian origin was evaluated. To explore the goitrogenic/antithyroid effect of those plant foods cyanogenic glucosides, glucosinolates, thiocyanate and polyphenol content were measured. These plant foods were fed with diet regularly to adult male albino rats for 30 and 60 days respectively, followed by evaluation of morphological and functional status of thyroid,as thyroid gland weight, the activity status of thyroid hormone synthesizing enzymes viz. TPO, Na+-K+-ATPase, deiodinase I, thyroid hormone profiles, thyroid histoarchitecture as well as urinary iodine and thiocyanate. In moringa leaves and spinach fed diet, there was a significant increase in urinary excretion of thiocyanate and iodine, enlargement of thyroid gland along with hypertrophy of thyroid follicles and altered in the activities of thyroid hormone synthesizing enzymes with concomitant changes of thyroid hormone profiles. Effects were more severe in moringa leaves treated group than that of spinach. Biochemical analysis moringa leaves and spinach reveal that those are rich sources of goitrogens and their prolonged consumption gradually developed a relative state of biochemical and morphological hypothyroidism.

Antinutritional factors in pearl millet grains: Phytate and goitrogens content variability and molecular characterization of genes involved in their pathways.

Pearl millet [Pennisetum glaucum (L.) R. Br.] is an important "orphan" cereal and the most widely grown of all the millet species worldwide. It is also the sixth most important cereal in the world after wheat, rice, maize, barley, and sorghum, being largely grown and used in West Africa as well as in India and Pakistan. The present study was carried out in the frame of a program designed to increase benefits and reduce potential health problems deriving from the consumption of pearl millet. The specific goal was to provide a database of information on the variability existing in pearl millet germplasm as to the amounts of phytate, the most relevant antinutrient compound, and the goitrogenic compounds C-glycosylflavones (C-GFs) accumulated in the grain.Results we obtained clearly show that, as indicated by the range in values, a substantial variability subsists across the investigated pearl millet inbred lines as regards the grain level of phytic acid phosphate, while the amount of C-GFs shows a very high variation. Suitable potential parents to be used in breeding programs can be therefore chosen from the surveyed material in order to create new germplasm with increased nutritional quality and food safety. Moreover, we report novel molecular data showing which genes are more relevant for phytic acid biosynthesis in the seeds as well as a preliminary analysis of a pearl millet orthologous gene for C-GFs biosynthesis. These results open the way to dissect the genetic determinants controlling key seed nutritional phenotypes and to the characterization of their impact on grain nutritional value in pearl millet.

[Determination of thyreostats in bovine urine using ultra-high performance liquid chromatography-tandem mass spectrometry].

Five thyreostats (TSs), namely tapazole, thiouracil, methylthiouracil, propylthiouracil, and phenylthiouracil, were determined in bovine urine using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) in positive electrospray ionization mode. Extraction and clean-up were achieved using a ChemElut cartridge with tert-butyl methyl ether, without a derivatization step. Separation was achieved on an Acquity UPLC SS T3 column. The mobile phase was acetonitrile and water containing 0.2% (v/v) formic acid. The mass spectrometer was operated in multiple reaction monitoring mode. Urine samples were spiked with TS solution at levels corresponding to 5, 10, 15, and 20 µg/L. The accuracy (internal standard corrected) ranged from 92% to 107%, with a repeatability precision (relative standard deviation, RSD) less than 15% for all five analytes. The RSDs within-laboratory reproducibility was less than 26%. The decision limits (CCα) and detection capabilities (CCß) were obtained from a calibration curve and were in the ranges of 3.1-6.1 µg/L and 4.0-7.4 µg/L, respectively. The CCα and CCß values were below the recommended concentration, which was set at 10 µg/L. The results show that the described method is suitable for the direct detection of TSs in bovine urine. This method can also be used to determine TSs in porcine urine.

Residue analysis of thyreostats in baby foods via matrix solid phase dispersion and liquid chromatography - dual-polarity electrospray - tandem mass spectrometry.

This paper describes a rapid method for confirming residues of thyreostats in meat-based baby foods by using liquid chromatography - dual polarity electrospray - tandem mass spectrometry (LC-ES(±)-MS/MS). Six thioureylenes, belonging to the group of thiouracil and imidazole, were selected for this work: thiouracil (TU), methylthiouracil (MTU), propylthiouracil (PTU), phenylthiouracil (PhTU), mercaptobenzimidazole (MBI) and tapazole (TAP). The amphoteric nature of these compounds allows their electrospray detection in both positive and negative ionisation. Nevertheless, MS detection is not favoured by their low molecular weights, while their chromatographic retention is also thwarted by their high polarity. A pentafluorophenyl (PFP) core-shell phase column was selected to avoid peak asymmetry or peak splitting, and a dual-polarity ionisation method was optimised to obtain a sensitivity as high as possible. The method was validated according to the Commission Decision 657/2002/EC. A simple and fast procedure based on matrix solid phase dispersion (MSPD) was optimised to extract analytes from baby foods with recoveries exceeding 82%. Limit of decision (CCα) and detection capability (CCß) were lower than the permissible maximum concentration (10 ng g-1). The validated method was then applied to assess the potential occurrence of the six selected thyreostats in nine commercial products. All the samples were found free of contamination.

Evaluation and characterization of thyroid-disrupting activities in soil samples along the Second Songhua River, China.

In this study, a recombinant thyroid receptor (TR) gene yeast assay combined with Monte Carlo simulation were used to evaluate and characterize soil samples collected from Jilin (China) along the Second Songhua River, for their ant/agonist effect on TR. No TR agonistic activity was found in soils, but many soil samples exhibited TR antagonistic activities, and the bioassay-derived amiodarone hydrochloride equivalents, which was calculated based on Monte Carlo simulation, ranged from not detected (N.D.) to 35.5µg/g. Hydrophilic substance fractions were determined to be the contributors to TR antagonistic activity in these soil samples. Our results indicate that the novel calculation method is effective for the quantification and characterization of TR antagonists in soil samples, and these data could provide useful information for future management and remediation efforts for contaminated soils.

Analysis of antithyroid drugs in surface water by using liquid chromatography-tandem mass spectrometry.

This paper describes development and validation of a new method for the simultaneous determination of six antithyroid drugs (ATDs) in surface waters by using liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS). Target compounds include two ATD classes: thiouracil derivatives (thiouracil (TU), methyl-thiouracil (MTU), propyl-thiouracil (PTU), phenyl-thiouracil (PhTU)) and imidazole derivatives (tapazole (TAP), and mercaptobenzimidazole (MBI)). Sensitivity and selectivity of the LC-multiple reaction monitoring (MRM) analysis allowed applying a simple pre-concentration procedure and "shooting" the concentrated sample into the LC-MS/MS system without any other treatment. Recoveries were higher than 75% for all analytes. Intra-day precision and inter-day precision, calculated as relative standard deviation (RSD), were below 19 and 22%, respectively. Limits of detection (LODs) ranged from 0.05 to 0.25 µg/L; limits of quantitation (LOQs) varied between 0.15 and 0.75 µg/L. The validated method was successfully applied to the analysis of ATD residues in surface water samples collected from the Tiber River basin and three lakes of Lazio (central Italy). The analytes were quantified based on matrix-matched calibration curves with mercaptobenzimidazole-d4 (MBI-d4) as the internal standard (IS). The most widespread compound was TAP, one of the most common ATDs used in human medicine, but also TU and MBI were often detected in the analysed samples.

A new Methimazole sensor based on nanocomposite of CdS NPs-RGO/IL-carbon paste electrode using differential FFT continuous linear sweep voltammetry.

A Methimazole sensor was designed and constructed based on nanocomposite of carbon, ionic liquid, reduced graphene oxide (RGO) and CdS nanoparticles. The sensor signal was obtained by Differential FFT continuous linear sweep voltammetry (DFFTCLSV) technique. The potential waveform contains two sections, preconcentration potential and potential ramp. In this detection technique, after subtracting the background current from noise, the electrode response was calculated, based on partial and total charge exchanges at the electrode surface. The combination of RGO and CdS nanoparticles can catalyze the electron transfer, which outcomes of the amplification of the sensor signal. The result showed that the sensor response was proportional to the concentrations of Methimazole in the range of 2.0 to 300 nM, with a detection limit of 5.5×10(-10) M. The sensor showed good reproducibility, long-term of usage stability and accuracy. The characterization of the sensor surface was studied by atomic force Microscopy and Electrochemical Impedance Spectroscopy. Moreover, the proposed sensor exhibited good accuracy, and R.S.D value of 2.82%, and the response time of less than 7 s.

Identification of thyroid-receptor antagonists in water from the Guanting Reservoir, Beijing, China.

Thyroid hormone (TH) has long been known to be essential for normal brain development in both humans and animals, and increasing evidence suggests that environmental components may disrupt TH signaling. In the present study, two-hybrid yeast bioassay and chemical analysis were used to evaluate and identify thyroid-receptor (TR) disruptors in water from the Guanting Reservoir, Beijing, China. Modified yeast bioassay showed that the water samples could affect TH signaling. The bioassay-derived amiodarone hydrochloride equivalents ranged from 33.8 ± 3.3 to 308.5 ± 31.8 µg/L. Solid-phase extraction was used to separate the organic extracts, which were subjected to bioassay and chemical analysis. The organic extracts significantly antagonized the TR, which accounted for >86.0 % of the total effects. Thus, organic extracts may play a major role in the TR-disruption activity of the water. Phenols, organochlorine pesticides, and phthalate esters were detected in the organic extracts. Chemical analysis and toxic-equivalent calculation showed that a major cause of the TR antagonism of the water was dibutyl phthalate (80.1 to 122.7 %).

A method for the determination of total and reduced methimazole in various biological samples.

A simple, rapid, reproducible and sensitive method based on HPLC with ultraviolet detection was developed for the determination of methimazole (MMI) in animal tissues and plasma samples. Under the optimum experimental conditions, the calibration curves for MMI were linear in the tested range 0.5-20 mg kg(-1) tissue sample (mg l(-1) plasma) with correlation coefficients better than 0.99. The performance of the proposed method was tested for the determination of MMI levels in brain, liver, thyroid gland and plasma of MMI-treated hens, as well as in their eggs and embryos. The proposed method reduces time and simplifies the sample preparation procedure.

Capillary electrophoresis coupled with electrochemiluminescence detection for the separation and determination of thyreostatic drugs in animal feed.

A rapid, simple, and practical method for the determination of four of the most used thyreostatic drugs (methimazole, 2-thiouracil, 6-methyl-2-thiouracil, and 6-propyl-2-thiouracil) using CE coupled to electrochemiluminescence detection has been established, based on the electrochemiluminescence enhancement of tris(2,2-bipyridyl)ruthenium(II) with these analytes. Parameters that affect separation and detection were optimized. Under the optimum experimental conditions, the four analytes could be well separated within 11 min at the separation voltage of 16 kV in a running solution containing 20 mM phosphate buffer (pH 9.0) and 1.0 × 10(-4) M Ru(bpy)(3)(2+), with a solution of 20 mM phosphate buffer (pH 12.0) containing 1.0 × 10(-4) M Ru(bpy)(3)(2+) in the electrochemiluminescence detection cell. The detection limits for methimazole, 6-methyl-2-thiouracil, 6-propyl-2-thiouracil, and 2-thiouracil were 0.1, 0.05, 0.05, and 0.01 µM, respectively. The proposed method was applied to analyze these drugs in spiked animal feed samples. The recoveries were 88.2∼99.0 and 86.4∼98.7% for the intraday and interday analyses, respectively. The RSDs were 2.7∼4.8 and 1.8∼5.0% for the intraday and interday analyses, respectively. The results demonstrate that the proposed method has promising applications in the detection of thyreostatic drugs in animal feeds.

Au-Ag-Au double shell nanoparticles-based localized surface plasmon resonance and surface-enhanced Raman scattering biosensor for sensitive detection of 2-mercapto-1-methylimidazole.

In this paper, Au-Ag-Au double shell nanoparticles were prepared based on the reduction of the metal salts HAuCl4 and AgNO3 at the surface of seed particles. Due to the synergistic effect between Au and Ag, the hybrid nanoparticles are particularly stable and show excellent performances on the detection of 2-mercapto-1-methylimidazole (methimazole). The binding of target molecule at the surface of Au-Ag-Au double shell nanoparticles was demonstrated based on both localized surface plasmon resonance (LSPR) and surface-enhanced Raman scattering (SERS) spectra. The LSPR intensity is directly proportional to the methimazole concentration in the range of 0.10-3.00×10(-7) mol L(-1). The SERS spectrum can be applied in identification of methimazole molecule. The LSPR coupled with SERS based on the Au-Ag-Au double shell nanoparticles would be very attractive for the quantitative determination and qualitative analysis of the analytes in medicines.

Method for the determination of thyreostats in milk samples using LC-MS/MS.

A sensitive and selective analytical method for the determination of four thyreostats (tapazol, thiouracil, methylthiouracil and propylthiouracil) in cow's milk, lamb's milk, and goat's milk was developed and validated according to 2002/657/EC criteria. Proteins in milk samples were precipitated by acetonitrile and analytes were derivatised with 3-iodobenzylbromide. Afterwards, derivatives were separated from the matrix by liquid-liquid extraction with ethyl acetate as an organic solvent and analysis was carried out using LC-MS/MS in a positive electrospray mode. The method provides, for all determined analytes, decision limits CCα below 1 ng ml(-1) and a detection capability CCß value below 1.5 ng ml(-1). The stability of analytes in sample extracts stored at various conditions was also tested and evaluated.

Determination of thyreostats in muscle and thyroid tissues by QuEChERS extraction and ultra-performance liquid chromatography tandem mass spectrometry.

The use of thyreostats as veterinary drugs is banned in the European Union since 1981 because of their carcinogenic and teratogenic properties. Controlling their illegal use in breeding animals is quite difficult because of their low molecular weight, high polarity and the presence of tautomeric forms. To harmonise the performance of analytical methods, the recommended concentration for thyreostats such as thiouracil, methylthiouracil, propylthiouracil and tapazole established by the Community Reference Laboratory in 2007 is 10 ng g(-1). The majority of the currently available analytical methods require a time-consuming derivatisation step and/or an SPE clean-up step. In this study, a rapid confirmatory method for the determination of six thyreostatic drugs - thiouracil, methylthiouracil, propylthiouracil, phenylthiouracil, tapazole and 2-mercaptobenzoimidazole - in thyroid and muscle at recommended concentration is presented. Quick sample extraction has been achieved by QuEChERS with ethyl acetate without further clean-up or derivatisation steps. Analysis has been carried out by using UPLC-ESI-MS/MS. Performance characteristics of the method have been determined in agreement with Commission Decision 2002/657/EC requirements for confirmatory methods and calculated decision limits (CCα) are below the recommended concentration (10 ng g(-1)).

[Determination of the thyreostats in animal muscle tissue by matrix solid-phase dispersion (MSPD) and liquid chromatography-tandem mass spectrometry]. / Zastosowanie metody ekstrakcji rozpraszania próbki w fazie stalej (MSPD) do oznaczania tyreostatyków w tkance miesniowej zwierzat metoda chromatografii cieczowej-tandem spektometrii mas.

BACKGROUND: The residues ofthyreostats must not be present in the edible animal tissues. The proposed in the EU minimum required performance limit (MPRL) in the animal tissues is 10 microg/kg. This implies the decision limit (CCalpha) and decision capability (CCbeta) of the analytical methods used for the determination of these compounds lower than 10 microg/kg. OBJECTIVE: This study aimed at the development, basing on the literature data and own studies the analytical method allowing for the identification and quantification of five thyreostats: tapazole (TAP), thiouracil (TU), methylotiouracil (MTU), propylothiouracil (PTU) and phenylotiouracil (FTU)) in the bovine muscle tissue, which would meet the criteria set in the Commission Decision No 2002/657/EC. MATERIAL AND METHODS: The developed method used liquid chromatography-tandem mass spectrometry (LC-MS/MS). The sample was extracted and cleaned using the matrix solid-phase dispersion (MSPD) method. The LC was equipped with column Luna C18 Phenomenex. Dimetylotiouracyl was used as internal standard. The samples were fortified at levels: 5, 10 and 20 microg/kg. The method was validated according to the criteria laid down in Commission Decision No. 2002/657/EC. RESULTS: At the levels, mean relative recoveries was in the range 90 - 109% and repeatability (CV %) was less than 10%. Decision limit (CCalpha) and detection capability (CCbeta) calculated for all thyreostats were below the recommended minimum required performance limit (MRPL) - 10 microg/kg. CONCLUSIONS: The developed and validated LC-ESI-MS/MS method allows for the identification and quantification of five thyreostats in the bovine muscle tissue in the quantities below 10 microg/kg. Analytical procedure meets the criteria of Commission Decision No 2002/657/EC.

Analysis of antithyroid drug residues in food animals.

In the late 1970s, concerns were raised regarding why antithyroid drugs were being administered to food animals to promote growth despite the fact that they had been implicated as being carcinogenic and teratogenic; the growth promotion process produced an inferior quality meat with increased water retention in the animal's gastrointestinal tract. An increased incidence of aplasia cutis (a characteristic scalp defect) in consumers in Spain was linked to an increased consumption of antithyroid-contaminated meat. Therefore, to protect human health, the EU banned the use of antithyroid drugs in food animal production in 1981. This article reviews the impact of this regulatory decision on the regulatory analysis of these compounds in foods of animal origin. It discusses the physiology of the thyroid gland, the chemistry of antithyroid drugs and critically evaluates the suitability of the analytical methods that have been developed and validated to support enforcement of the regulation.

Optimization of ion-pair based hollow fiber liquid phase microextraction combined with HPLC-UV for the determination of methimazole in biological samples and animal feed.

Ion-pair based hollow fiber liquid phase microextraction (IP-HFLPME) coupled with high performance liquid chromatography-ultraviolet detection was applied for the preconcentration and determination of methimazole in biological samples and animal feed. Optimization of the conditions for the high extraction efficiency was studied simultaneously using the experimental design. For the first step, the Plackett-Burman design was applied to screen the significant factors on the extraction efficiency. Central composite design (CCD) was then used for the optimization of important factors and the response surface equations were obtained. The optimum experimental conditions were donor phase pH, 12.2; extraction temperature, 45°C; extraction time, 50 min; sodium perchlorate concentration, 1.5 M; cetyltrimethylammonium bromide concentration, 0.65 mM, and without salt addition in donor phase. The limit of detection and the dynamic linear range were in the range of 0.1-0.7 µg L(-1) and 0.5-1000 µg L(-1), respectively. Preconcentration factors were obtained in the range of 93-155 in different matrices. Finally, the performance of the proposed method was tested for the determination of trace amounts of methimazole in plasma, urine, bovine milk, and animal feed samples, and satisfactory results were obtained (RSDs < 7.1%).