Thyroid cancer and endocrine disruptive chemicals: a case-control study on per-fluoroalkyl substances and other persistent organic pollutants.

Objective: The aim was to evaluate the possible association between some endocrine disruptive chemicals and thyroid cancer (TC) in an Italian case-control cohort. Methods: We enrolled 112 TC patients and 112 sex- and age-matched controls without known thyroid diseases. Per- and poly-fluoroalkyl substances (PFAS), poly-chlorinated biphenyls (PCBs), and dichlorodiphenyltrichloroethane (4,4'-DDT and 4,4'-DDE) were measured in the serum by liquid or gas chromatography-mass spectrometry. Unconditional logistic regression, Bayesan kernel machine regression and weighted quantile sum models were used to estimate the association between TC and pollutants' levels, considered individually or as mixture. BRAFV600E mutation was assessed by standard methods. Results: The detection of perfluorodecanoic acid (PFDA) was positively correlated to TC (OR = 2.03, 95% CI: 1.10-3.75, P = 0.02), while a negative association was found with perfluorohexanesulfonic acid (PFHxS) levels (OR = 0.63, 95% CI: 0.41-0.98, P = 0.04). Moreover, perfluorononanoic acid (PFNA) was positively associated with the presence of thyroiditis, while PFHxS and perfluorooctane sulfonic acid (PFOS) with higher levels of presurgical thyroid-stimulating hormone (TSH). PFHxS, PFOS, PFNA, and PFDA were correlated with less aggressive TC, while poly-chlorinated biphenyls (PCB-105 and PCB-118) with larger and more aggressive tumors. Statistical models showed a negative association between pollutants' mixture and TC. BRAF V600E mutations were associated with PCB-153, PCB-138, and PCB-180. Conclusion: Our study suggests, for the first time in a case-control population, that exposure to some PFAS and PCBs associates with TC and some clinical and molecular features. On the contrary, an inverse correlation was found with both PFHxS and pollutants' mixture, likely due to a potential reverse causality.

Concise review on the combined use of immunocapture, mass spectrometry and liquid chromatography for clinical applications.

Immunocapture is now a well-established method for sample preparation prior to quantitation of peptides and proteins in complex matrices. This short review will give an overview of some clinical applications of immunocapture methods, as well as protocols with and without enzymatic digestion in a clinical context. The advantages and limitations of both approaches are discussed in detail. Challenges related to the choice of mass spectrometer are also discussed. Top-down, middle-down, and bottom-up approaches are discussed. Even though immunocapture has its limitations, its main advantage is that it provides an additional dimension of separation and/or isolation when working with peptides and proteins. Overall, this short review demonstrates the potential of such techniques in the field of proteomics-based clinical medicine and paves the way for better personalized medicine.

Associations between endocrine disruptor contamination and thyroid hormone homeostasis in Belgian type 1 diabetic children.

PURPOSE: Humans are daily exposed to many environmental pollutants, some of which being suspected to be thyroid disruptors. Some populations could be particularly susceptible to thyroid disruption, such like diabetics due to the well-known relation between the thyroid function and the control of carbohydrate homeostasis by pancreas. Therefore, the aim of this study was to investigate the associations between the exposure to several persistent and non-persistent chemicals and thyroid hormones levels in children with type 1 diabetes. METHODS: Blood and urine sample were collected from 54 children diagnosed for type 1 diabetes mellitus. The concentrations of 7 phthalate metabolites, 4 parabens, 7 bisphenols, benzophenone 3 and triclosan were measured in urine, while 15 organochlorine pesticides, 4 polychlorinated biphenyls (PCBs) and 7 perfluoroalkyl substances were analyzed in serum samples. In the same time, the blood levels of free thyroxine (fT4), thyroid stimulating hormone (TSH) and glycated hemoglobin (Hb1Ac) were determined. RESULTS: We highlighted positive associations between serum perfluorohexane sulfonate and urinary monoethylphthalate levels, and TSH level in blood. We also found that PCB 138 was positively associated to fT4 while urinary levels of bisphenol F were negatively correlated to this hormone. Finally, we observed positive associations between Hb1Ac levels and the contamination by PCB 153 and two urinary phthalate metabolites: mono-2-ethyl-5-hydroxyhexyl phthalate and mono-2-ethyl-5-oxoxyhexyl phthalate. CONCLUSION: Our results showed that our small cohort of children with type 1 diabetes mellitus is potentially susceptible to thyroid disruptions by some pollutants. Moreover, for these children, both di-(2-ethylhexyl) phthalate metabolites would potentially hamper the glucose homeostasis. Nevertheless, additional studies are mandatory to further explore these findings.

Inconsistencies in laboratory investigations of hypertension in a young woman taking ethinylestradiol/drospirenone association.

CASE PRESENTATION: A 34-year-old woman presented to the emergency department for arterial hypertension. Blood analysis requested by the endocrinologist showed very high level of aldosterone (1805 ng/L, normal values: <264 ng/L) and high level of renin activity (2.8 ng/mL/h, normal values: 0.1-2.0 ng/mL/h). The patient reported the use of Yasmin® (ethinylestradiol 30 µg/drospirenone 3 mg) continuously (without hormone-free week between cycles) as oral contraception. Medical imaging examinations revealed no anomaly in the kidneys and the adrenal glands. On the endocrinologist advice, patient stopped the intake of Yasmin®. Aldosterone and renin levels were measured several times after the discontinuation of the oral contraception and a diminution of these levels was observed with a complete normalization of both levels 26 days after the synthetic hormones discontinuation. DISCUSSION: The literature shows that ethynilestradiol/drospirenone association can interfere with the renin-angiotensin-aldosterone system and increase the levels of aldosterone and/or renin. We reported here a clinical case illustrating the significant impact of this medication on the renin-angiotensin-aldosterone axis of a young woman. However, this association is not listed among the drugs interfering with the aldosterone and renin-level measurements. CONCLUSION: Considering the data in the literature and our clinical case, we suggest adding drospirenone and the ethinylestradiol/drospirenone association in the list of drugs interfering with aldosterone and renin level determination.

Relevance of cycle threshold values in mass screening by reverse-transcription-PCR during COVID-19 pandemic in Belgium: a decision-making support?

Aim: The Belgium's strategy against COVID-19 was partly based on mass screening. Here, we reported the results observed in a Belgian mass screening center. Materials & methods: Between October 2020 and February 2021, 32,089 samples were collected analyzed with reverse-transcription PCR (Thermo Fisher Scientific kits and apparatus). Patients were categorized according to their contagiousness (extrapolated from the cycle threshold [Ct] values and the recommendation of Sciensano). Results: We observed association between Ct values and age, with higher Ct observed in extreme age groups (<6 years and >75 years). Conclusion: The analysis of the evolution of the contagiousness of these patients tested twice within a 7-day period showed the relevancy of the recommendation edited by Sciensano.

Investigation of discrepancies obtained during 15 years of non-invasive fetal RHD genotyping in apparent serologic RhD-negative pregnant women.

OBJECTIVES: In some European countries, non-invasive fetal RHD genotyping is the first step of anti-D allo-immunized pregnant women management but presence of RHD variant alleles may interfere with the results accuracy. We developed an algorithm allowing solving discordant results (due to the presence of RHD variant) in fetal RHD genotyping assay. METHOD: This study gathered the results of fetal RHD genotyping performed between 2006 and 2020 in the Medicine Laboratory of CHR Liège. Exons 4, 5 and 10 of the fetal RHD were profiled in maternal plasma using real time polymerase chain reaction (PCR). When the results were discrepant, maternal RHD variant was further explored by sequence-specific primer PCR on maternal buffy coat. RESULTS: A total of 11,630 pregnant women (mainly of both Caucasian and African origins) were tested during the study period and RHD variant alleles were detected in 247 women. The most frequent variant was RHD*08N.01 found in 66 women mainly of Black African origin. We identified 45 women with weak RHD variant type 1, 2 or 3. CONCLUSION: Women with weak RHD variant type 1, 2 or 3 can safely be considered as RhD positive in terms of RhIg prophylaxis and/or transfusion of blood components. Therefore, identification of RHD allele variants in women with discordant fetal RHD genotyping results contributes to save RhIg prophylaxis and RhD negative blood components.

Evolution of 17-ß-estradiol, estrone and estrone-sulfate concentrations in late pregnancy of different breeds of mares using Liquid Chromatography and Mass Spectrometry.

This study describes 17-ß-estradiol (E2), estrone (E1) and estrone-sulfate (E1S) concentrations between 4 and 11 months in healthy equine pregnancies of two different breeds using Liquid Chromatography coupled to Mass-Spectrometry (LC-MS). In 2 stud-farms including 15 Spanish PureBred (SPB) and 11 Showjumping (SJ) types mares, combined thickness of the uterus and the placenta (CTUP) was measured and blood was sampled monthly between 4 and 11 months of gestation. Concentrations of E2, E1 and E1S were assayed with LC-MS in mares with normal CTUP. Effects of breed, day of pregnancy and mare's parity and age on estrogens concentrations were investigated. Peak of E2 was observed at 5 months (median: 46.4 pg/mL; maximum: 201.5 pg/mL). A strong correlation was observed between E1 and E1S (p < 0.0001, r = 0.85). Peak of E1 (median: 571.0 pg/mL; maximum: 1641.9 pg/mL) and E1S (median: 573.6 ng/mL; maximum: 997.6 ng/mL) concentrations was observed at the 5th month and then E1S decreased quicker than E1 until the end of pregnancy. Higher E2 and E1 concentrations were observed in SJ than in SPB mares between the 6th and the 8th months. No difference between breeds was observed for E1S monthly evolution. Estrogen peak values were all observed at 5 months. Unlike recent LC-MS studies, E1S values observed here were in the same range than those previously established using immuno-assays. After the 6th month, E1S decreased quicker than E1. Effect of breed only observed on non-sulfonated estrogens should be further confirmed. These findings confirm that sulfonation activity of the allantochorion may be limited after the 6th month.

Bio-surveillance of environmental pollutants in the population of Kinshasa, Democratic Republic of Congo (DRC): a small pilot study.

BACKGROUND: Environmental pollutants are known to be ubiquitous and may present toxic effects (endocrine-disruption properties, carcinogenicity …) and represent a real threat to human health. The aim of the present pilot study was to assess the content of environmental pollutants (inorganic, persistent, and non-persistent pollutants) in biological samples (urine, serum, and whole blood), collected from volunteers in Kinshasa, capital of Democratic Republic of Congo, in order to identify pollutants of interest and to design a protocol for a larger scale study. METHODS: From randomly selected 15 volunteers living in Kinshasa, aged from 25 to 66 years, (mean age = 43.4 years), including 10 men and 5 women, urine, whole blood, and serum samples were used in this study to estimate the contents in these environmental pollutants, using inductively coupled plasma mass spectrometry, gas chromatography coupled to mass spectrometry, and liquid chromatography coupled to mass spectrometry. RESULTS: When compared to data nationally and internationally available, the preliminary outcomes of this study indicated a very high level of exposure to environmental pollutants in the population of Kinshasa, especially for heavy metals, parabens and triclosan. To a lesser extent, contamination measured for glyphosate, phthalates, organochlorine pesticides, pyrethroids and dialkylphosphate pesticides was also significant. In contrast, the investigated population of Kinshasa was found to be weakly exposed to other persistent organic pollutants like polychlorinated biphenyls, brominated flame retardants, phenolic organohalogens, and perfluoroalkyl substances. CONCLUSION: Although the biologic fluids were collected from a limited number of volunteers (n = 15), the results of the present report clearly indicate that the population of Kinshasa is not spared by the investigated environmental pollutants. Moreover, this study gives us important information to design a larger scale study protocol.

Development and validation of a liquid chromatography coupled to mass spectrometer (LC-MS) method for the simultaneous quantification of estrone-3-sulfate, progesterone, estrone and estradiol in serum of mares and American bisons.

Steroid concentrations in serum are fluctuating during pregnancy of many mammal species. The current knowledge about endocrinology of gestation is mainly based on immunoassays. However, the lack of specificity of these assays hampers the reliability of the results. In the present work, we developed and validated a methodology associating liquid chromatography (LC) and mass spectrometry (MS) to simultaneously quantify, with high specificity and accuracy, estrone-3-sulfate (E3S), progesterone (PRO), estrone (E1) and estradiol (E2) in serum of two different mammal species. The sample preparation procedure is based on a simple protein precipitation and a derivatization with dansyl chloride. After the chromatographical separation, compounds were analyzed with a triple-quadrupole mass spectrometer operating in multiple reaction monitoring. Mare and American bison serum samples were analyzed with the validated method and results were compared with concentrations measured with commercial radioimmunoassay (RIA), enzyme linked immunosorbent assay (ELISA) and chemiluminescent microparticle immunoassay (CMIA). Following these criterions: relative standard deviation <15% and relative bias <15%, lower limits of quantification of 0.5 ng/mL (E3S), 0.1 ng/mL (PRO) and 2 pg/mL (E1 and E2) were achieved. Most of the comparison between immunoassays and LC-MS showed poor correlation and proportional differences. Our LC-MS method is able to simultaneously quantify several steroid hormones with high specificity, accuracy and sensitivity in serum of two different mammal species. Our method constitutes a useful and performant tool for veterinary clinicians and LC-MS should thus be used to update and refine the current knowledge about the endocrinology of pregnancy in mammals.

Background contamination of perfluoralkyl substances in a Belgian general population.

The few Belgian studies on the human exposure to perfluoroalkyl substances (PFASs) have until now concerned the Northern part of Belgium (Flanders), while data related to Wallonia (South region) are missing. To fill this gap, 8 perfluorinated carboxylic acids and 3 perfluorinated alkyl sulfonates were measured in the serum of 242 adults (>18 years old) recruited in 2015 and living in the Province of Liege. Some multivariate regression models were also built with the PFAS levels and the participant's answers to a questionnaire about their diet and lifestyle habits in order to identify some predictors of exposure. The results obtained showed that although PFAS levels observed in our population seemed to be similar or lower than those reported in other countries, and especially lower than in the Northern part of Belgium, half of the population showed PFOS and PFOA serum levels above the health guidance values set by the German HBM Commission. As expected, age and gender were the main covariates explaining the different PFAS serum levels between participants, while breastfeeding (for women), consumption of fish and seafood, consumption of rice, and use of nail polish seemed also to impact the PFAS body burden of our population. Nevertheless, the statistical models were poorly predictive suggesting that the main sources of exposure were not taken into account.

The pathway through LC-MS method development: in-house or ready-to-use kit-based methods?

Historically, the determination of low concentration analytes was initially made possible by the development of rapid and easy-to-perform immunoassays (IAs). Unfortunately, typical problems inherent to IA technologies rapidly appeared (e.g. elevated cost, cross-reactivity, lot-to-lot variability, etc.). In turn, liquid chromatography tandem mass spectrometry (LC-MS/MS) methods are sensitive and specific enough for such analyses. Therefore, they would seem to be the most promising candidates to replace IAs. There are two main choices when implementing a new LC-MS/MS method in a clinical laboratory: (1) Developing an in-house method or (2) purchasing ready-to-use kits. In this paper, we discuss some of the respective advantages, disadvantages and mandatory requirements of each choice. Additionally, we also share our experiences when developing an in-house method for cortisol determination and the implementation of an "ready-to-use" (RTU) kit for steroids analysis.

Association between mixture of persistent organic pollutants and thyroid pathologies in a Belgian population.

Previous years, the incidence of autoimmune thyroid diseases has increased worldwide. The presence of many pollutants in the environment suspected to be thyroid disruptors may have contributed to the observed increase. Unfortunately, the results from epidemiological studies assessing the association between pollution and thyroid disorders remain inconsistent, maybe due to a nearly complete neglect of the mixture effect. The blood levels of 12 brominated flame retardants, 3 polychlorinated biphenyls, 16 organochlorine pesticides, 7 perfluoroalkyl substances and 16 phenolic organohalogens were measured in 35 hypothyroid and 44 hyperthyroid volunteers and in 160 individuals from the general population designed as controls. Weighted quantile sum (WQS) regressions were performed to compute indexes representing the mixture of POPs, and we assessed the relations with thyroid disorders. Nineteen pollutants were detected in more than 40% of the individuals and were thus included in the WQS indexes. The WQS index was statistically significantly associated with an increased odds of hypothyroidism (odds ratio (OR) = 98.1; 95% CI: 5.51-1747) with the highest weights attributed to PCB 138 (w = 0.210), 3-OH-CB 180 (w = 0.197), 4-OH-CB 146 (w = 0.188), 4',4-DDE (w = 0.156) while there were no evidence of a relation with increased odds of hyperthyroidism. Given the relative low number of individuals included in the present investigation, standard WQS methodology could not be used, this study should thus be considered as a preliminary, hypothesis-generating study. Nevertheless, these results highlighted the importance of considering the potential effect of chemical mixture when studying endocrine disruptors.

Association between organohalogenated pollutants in cord blood and thyroid function in newborns and mothers from Belgian population.

The last decades have seen the increasing prevalence of thyroid disorders. These augmentations could be the consequence of the increasing contamination of the environment by chemicals that may disrupt the thyroid function. Indeed, in vitro studies have shown that many chemicals contaminating our environment and highlighted in human serum, are able to interfere with the thyroid function. Given the crucial importance of thyroid hormones on neurodevelopment in fetus and newborns, the influence of these pollutants on newborn thyroid homeostasis is a major health concern. Unfortunately, the overall evidence for a deleterious influence of environmental pollutants on thyroid remains poorly studied. Therefore, we assessed the contamination by polychlorinated biphenyls (PCBs), organochlorine pesticides and perfluorinated compounds (PFC) in 221 cord blood samples collected in Belgium between 2013 and 2016. Our results showed that compared to previous studies performed on newborns recruited in Belgium during the two last decades, the present pollutant contamination is declining. Multivariate statistical analyses pointed out a decrease of thyroid stimulating hormone (TSH) level in male newborns with detectable level of 4,4'- dichlorodiphenyldichloroethylene (4,4'-DDE) in comparison with those with no detectable level (p = 0.025). We also highlighted a negative association between perfluorononanoic acid (PFNA) concentration and TSH in male newborns (p = 0.018). Logistic regression showed increased odds ratio for presentation of hypothyroid in mother for each one unit augmentation of log natural concentration of PFOA (OR = 2.30, [1.18-4.5]) and PFOS (OR = 2.03 [1.08-3.83]). Our findings showed that the residual contamination by PFCs and organochlorine pollutants in cord blood are correlated with thyroid hormone in the newborns and the risk of hypothyroid in mothers.

Determination of phenolic organohalogens in human serum from a Belgian population and assessment of parameters affecting the human contamination.

Many in vitro or in vivo studies highlighted the potential deleterious effects of phenolic organohalogenated compounds (POHs) on the health, particularly on the thyroid system homeostasis, however few large scale human epidemiological studies have been carried out, especially in Europe. Further studies monitoring the human contamination by POHs, the sources of exposure and the influence of these compounds on thyroid health are still needed. Therefore we determined the concentrations of 16 POHs (pentachlorophenol (PCP), tetrabromobisphenol A (TBBPA), 4 bromophenols (BPs), 3 hydroxy-polybromodiphenylethers (OH-PBDEs) and 7 hydroxy-polychlorobiphenyls (OH-PCBs)) in serum from 274 people aged from 18 to 76years old living in Liege (Belgium) and the surrounding area. A questionnaire about their alimentary habits, life style and home environment was also administered to the volunteers. The predominant compound measured in the population was PCP (median concentration of 593.0pgmL-1). 4-OH-CB 107, 4-OH-CB 146 and 4-OH-CB 187 were detected in all samples and contributed for 75% of the sum of OH-PCBs (ΣOH-PCBs). The median measured in our population for ΣOH-PCBs was 143.7pgmL-1. TBBPA and 2,4,6-tribromophenol were detected in 31% and 63.8% of the samples respectively while the detection frequency observed for the other BPs and the OH-PBDEs was close to zero. We computed multivariate regression models in order to assess the influence of demographic and lifestyle parameters on the PCP and ΣOH-PCBs contamination levels. Significant correlation was found between the PCP concentration and sex, smoker status, sea fish consumption and level of education, although the model seemed to be a poor (R2=0.14) predictor of the PCP concentration. The model computed for ΣOH-PCBs was more explanatory (R2=0.61) and involved age, BMI and sea fish consumption. Finally, we assessed the parameters affecting the ΣOH-PCBs/ΣPCBs ratio. The model proposed involved age, BMI, smoker status and parent PCB level, and explained 41% of the variability of the ΣOH-PCBs/ΣPCBs ratio.

Brominated flame retardant: environmental and exposed individuals' health impact.

Since Antiquity, men have used chemicals to protect their goods against fire. Effective and easy to use, brominated flame retardants are used since decades massively in plastic industry. Such like other organohalogenated compounds, brominated flame retardants are very persistent in the environment and able to accumulate along the food chain. Many authors highlight their presence in the environment, in many animal species and in the human serum. Worryingly, man is exposed as soon as the pregnancy and then by the breastfeeding. This exposition may have consequence on our health. Many studies (in vitro, in vivo or epidemiologic) highlight brominated flame retardant negative effects on the endocrine system, mainly on the thyroid function but also on the reproduction, the neurodevelopment in the children and on the metabolism with increasing diabetes risk. If authorities and some big enterprises are aware about the problematic, new studies are needed to confirm previous results, elucidate endocrine disrupting mechanisms and highlight hypothetical synergies with other pollutants such like PCBs.

Validation of a novel and rapid method for the simultaneous determination of some phenolic organohalogens in human serum by GC-MS.

Over the last decades, more and more studies focused on the impact of endocrine disruptors on the environment and human health. Among them, phenolic organohalogens (POHs) are a particular concern because of their structural resemblance with natural hormones. There are different methods that are known to quantify these compounds in human serum, however, the current extraction techniques are long, fastidious and using harmfull chemicals such as diazomethane and sulfuric acid. Consequently, we developed an alternative, sensitive and faster method to simultaneously quantify pentachlorophenol (PCP), tetrabromobisphenol A (TBBPA), 4 bromophenols, 7 hydroxypolychlorinated biphenyls (OH-PCBs) and 3 hydroxy-polybrominated diphenyl ether (OH-PBDEs) in human serum sample. The clean-up and the enrichment of the sample were performed in a single extraction step using strong anion-exchange solid phase cartridge. After a rapid liquid-liquid extraction step to remove acidic traces, the extract was derivatized using trimethylsilyldiazomethane (TMSD) and finally analyzed by a gas-chromatograph coupled with an electron negative capture chemical ionization source combined with a triple quadrupole mass spectrometer (GC-ENCI-MS) operating in single ion monitoring. The whole procedure was validated according to the total error approach. The inter and intra assay precision were demonstrated to be lower than 20% and the relative bias to be lower than 15% in the dosing range of concentrations. The limit of quantification (LOQ) ranged from 2pgmL-1 and 5pgmL-1, except for the PCP (44.6pgmL-1) and for the 2,4,6-tribromophenol (49.6pgmL-1). Finally, the method was successfully applied to measure the POH background contamination in serum samples collected from 20 Belgian blood donors recruited in CHU Mont-Godinne (Namur, Belgium) aged between 21 and 69 years old.