Functional membrane androgen receptors in colon tumors trigger pro-apoptotic responses in vitro and reduce drastically tumor incidence in vivo

<p>Abstract</p> <p>Background</p> <p>Membrane androgen receptors (mAR) have been implicated in the regulation of cell growth, motility and apoptosis in prostate and breast cancer. Here we analyzed mAR expression and function in colon cancer.</p> <p>Results</p> <p>Using fluorescent mAR ligands we showed specific membrane staining in colon cell lines and mouse xenograft tumor tissues, while membrane staining was undetectable in healthy mouse colon tissues and non-transformed intestinal cells. Saturation/displacement assays revealed time- and concentration-dependent specific binding for testosterone with a K_Dof 2.9 nM. Stimulation of colon mAR by testosterone albumin conjugates induced rapid cytoskeleton reorganization and apoptotic responses, even in the presence of anti-androgens. The actin cytoskeleton drug cytochalasin B effectively inhibited the pro-apoptotic responses and caspase-3 activation. Interestingly, <it>in vivo </it>studies revealed that mAR activation resulted in a 65% reduction of tumor incidence in chemically induced Balb/c mice colon tumors.</p> <p>Conclusion</p> <p>Our results demonstrate for the first time that functional mARs are predominantly expressed in colon tumors and that their activation results in induction of anti-tumor responses <it>in vitro </it>and extensive reduction of tumor incidence <it>in vivo</it>.</p

Cadmium exposure in adults across Europe: Results from the HBM4EU Aligned Studies survey 2014-2020

ReviewThe objectives of the study were to estimate the current exposure to cadmium (Cd) in Europe, potential differences between the countries and geographic regions, determinants of exposure and to derive European exposure levels. The basis for this work was provided by the European Human Biomonitoring Initiative (HBM4EU) which established a framework for alignment of national or regional HBM studies. For the purpose of Cd exposure assessment, studies from 9 European countries (Iceland, Denmark, Poland, Czech Republic, Croatia, Portugal, Germany, France, Luxembourg) were included and urine of 20–39 years old adults sampled in the years 2014–2021 (n = 2510). The measurements in urine were quality assured by the HBM4EU quality assurance/quality control scheme, study participants' questionnaire data were post-harmonized. Spatially resolved external data, namely Cd concentrations in soil, agricultural areas, phosphate fertilizer application, traffic density and point source Cd release were collected for the respective statistical territorial unit (NUTS). There were no distinct geographic patterns observed in Cd levels in urine, although the data revealed some differences between the specific study sites. The levels of exposure were otherwise similar between two time periods within the last decade (DEMOCOPHES - 2011–2012 vs. HBM4EU Aligned Studies, 2014–2020). The age-dependent alert values for Cd in urine were exceeded by 16% of the study participants. Exceedances in the different studies and locations ranged from 1.4% up to 42%. The studies with largest extent of exceedance were from France and Poland. Association analysis with individual food consumption data available from participants’ questionnaires showed an important contribution of vegetarian diet to the overall exposure, with 35% higher levels in vegetarians as opposed to non-vegetarians. For comparison, increase in Cd levels due to smoking was 25%. Using NUTS2-level external data, positive associations between HBM data and percentage of cropland and consumption of Cd-containing mineral phosphate fertilizer were revealed, which indicates a significant contribution of mineral phosphate fertilizers to human Cd exposure through diet. In addition to diet, traffic and point source release were identified as significant sources of exposure in the study population. The findings of the study support the recommendation by EFSA to reduce Cd exposure as also the estimated mean dietary exposure of adults in the EU is close or slightly exceeding the tolerable weekly intake. It also indicates that regulations are not protecting the population sufficiently.The HBM4EU project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 733032. Co-funding for the HBM4EU Aligned Studies has been provided by the national programs: Sant´e Publique France and the French ministries of Health and the Environment (ESTEBAN, France); MEYS (No. LM2018121), and Cetocoen Plus project (CZ.02.1.01/0.0/ 0.0/15_003/0000469) (CELSPAC:YA, Czech Republic); the Ministry of Science and Higher Education of Poland (contract no.3764/H2020/ 2017/2) (POALES, Poland); Public Health Fund (Diet_HBM, Iceland); Croatian Institute of Public Health (HBM survey in Croatia); National Institute of Health Dr Ricardo Jorge (INSEF_ExpoQuim, Portugal); German Ministry for the Environment, Nature Conservation, Nuclear Safety and Consumer Protection (BMUV) (ESB, Germany); Luxembourg Institute of Health (LIH), the Laboratoire national de sant´e (human biomonitoring part), the Ministry of Higher Education and Research of Luxembourg and the Ministry of Health of Luxembourg (Oriscav-Lux2, Luxembourg); Candy Foundation (Nos. 2017–224 and 2020–344), Absalon Foundation (No. F-23653-01), The Danish Environmental Protection Agency (Miljøstyrelsen: MST-621-00012 Center on Endocrine Disrupters), The Research council of Capital Region of Denmark (No. E− 22717-11), Research council of Rigshospitalet (Nos. E− 22717-12, E− 22717-07, E− 22717-08), Aase og Ejnar Danielsens Fond (No. 10–001874), International Research and Research Training Centre for Male Reproduction and Child Health (EDMaRC, No. 1500321/1604357) (CPHMINIPUB (parents) and DYMS, Denmark). J.Kl. and L.A. thank the CETOCOEN EXCELLENCE project No. CZ.02.1.01/0.0/0.0/17_043/ 0009632 financed by MEYS for supportive background, and supported from the European Union’s Horizon 2020 research and innovation program under grant agreement No. 857560.info:eu-repo/semantics/publishedVersio

Concurrent Assessment of Phthalates/HEXAMOLL® DINCH Exposure and Wechsler Intelligence Scale for Children Performance in Three European Cohorts of the HBM4EU Aligned Studies

Information about the effects of phthalates and non-phthalate substitute cyclohexane-1,2-dicarboxylic acid diisononyl ester (HEXAMOLL® DINCH) on children's neurodevelopment is limited. The aim of the present research is to evaluate the association between phthalate/HEXAMOLL® DINCH exposure and child neurodevelopment in three European cohorts involved in HBM4EU Aligned Studies. Participating subjects were school-aged children belonging to the Northern Adriatic cohort II (NAC-II), Italy, Odense Child Cohort (OCC), Denmark, and PCB cohort, Slovakia. In each cohort, children's neurodevelopment was assessed through the Full-Scale Intelligence Quotient score (FSIQ) of the Wechsler Intelligence Scale of Children test using three different editions. The children's urine samples, collected for one point in time concurrently with the neurodevelopmental evaluation, were analyzed for several phthalates/HEXAMOLL® DINCH biomarkers. The relation between phthalates/HEXAMOLL® DINCH and FSIQ was explored by applying separate multiple linear regressions in each cohort. The means and standard deviations of FSIQ were 109 ± 11 (NAC-II), 98 ± 12 (OCC), and 81 ± 15 (PCB cohort). In NAC-II, direct associations between FSIQ and DEHP's biomarkers were found: 5OH-MEHP+5oxo-MEHP (β = 2.56; 95% CI 0.58-4.55; N = 270), 5OH-MEHP+5cx-MEPP (β = 2.48; 95% CI 0.47-4.49; N = 270) and 5OH-MEHP (β = 2.58; 95% CI 0.65-4.51; N = 270). On the contrary, in the OCC the relation between DEHP's biomarkers and FSIQ tended to be inverse but imprecise (p-value ≥ 0.10). No associations were found in the PCB cohort. FSIQ was not associated with HEXAMOLL® DINCH in any cohort. In conclusion, these results do not provide evidence of an association between concurrent phthalate/DINCHHEXAMOLLR DINCH exposure and IQ in children.This work received external funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No. 733032 [“European Human Biomonitoring Initiative” (HBM4EU)] and received co-funding from the author’s organizations. NAC-II: This research was funded by: the European Union through its Sixth Framework Program for RTD (contract “PHIME” No. FOOD-CT-2006-016253); the Institute for Maternal and Child Health IRCCS “Burlo Garofolo”, Trieste, Italy (RC 12/12 funded by Ministry of Health—Italy); CROME LIFE Project “Cross-Mediterranean Environment and Health Network” (LIFE12 ENV/GR/001040). OCC: The cohort was funded by the Odense University Hospital, Denmark; the Region of Southern Denmark, The Municipality of Odense, Denmark; The University of Southern Denmark; the Mental Health Service of the Region of Southern Denmark; Odense Patient data Exploratory Network (OPEN), Den mark; The Danish Center for Hormone Disrupting Chemicals (MST-611-00012); The Danish Research Council (4004-00352B_FSS); Novo Nordisk Foundation, Denmark (grant no. NNF19OC0058266 and NNF17OC0029404); Sygeforsikring Danmark (journalnr. 2021-0173); The Collaborative foundation between Odense University Hospital and Rigshospitalet, Helsefonden, Beckettfonden, the Danish Mental Health Fund, Health Insurance Denmark. The LS-MS/MS equipment was financially supported by the Velux Foundation. PCB: PCB cohort was funded by the Slovak Research and Development Agency, project no. APVV-0571-12 and the Ministry of Health of the Slovak Republic, project no. 2014/47-SZU-11. The APC was funded by the European Union’s Horizon 2020 research and innovation program under grant agreement No. 733032.S

Human biomonitoring in health risk assessment in Europe: Current practices and recommendations for the future.

Human biomonitoring (HBM) is an important tool to survey the internal exposure of humans which represents the real life chemical body burden to chemicals and/or their metabolites. It results from total exposure to chemical substances from different sources and via different routes. These substances may be regulated under different legislative frameworks on chemicals (e.g., environmental, occupational, food safety etc). In occupational health, HBM has long traditions to control the exposures at workplaces. By providing accurate data on internal exposure, HBM data can improve human health risk assessment (RA) for both the general population and workers. Although the past few years have shown good examples on the use of HBM in the RA of chemicals, there is still quite some work to be done to improve its use in a regulatory RA. Under the scope of the European Human Biomonitoring Initiative (project HBM4EU, 2017-2021), the current study reviews the state-of-the-art of HBM use in chemicals RA with a special focus in Europe, and attempts to identify hurdles and challenges faced by regulators. To gather information on the use of HBM, including the availability of guidance on how to use it in RA, the RA schemes applied by different European or international organizations were analysed. Examples of such use were identified for a few selected groups of chemicals of concern for human health. In addition, we present the results of a survey, aimed at collecting information from national regulatory risk assessors on their day-to-day RA practices, the use of HBM data, and the obstacles and challenges related to their use. The results evidenced and explained some of the current obstacles of using HBM data in RA. These included the lack of HBM guidance values or biomonitoring equivalents (BEs), limited toxicokinetic information to support the interpretation of HBM data and, in the occupational health and safety (OSH) field, the lack of legal enforcement. Therefore, to support the integration of HBM in regulatory RA, we recommend, on one hand, the elaboration of a EU level guidance on the use of HBM in RA and, on the other hand, the continuation of research efforts to integrate HBM with new RA approaches using in vitro/in silico data and Adverse Outcome Pathways (AOPs).This project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No 733032 HBM4EU, and received co-funding from the author's organizations. The authors thank to all other participants that contributed to this work.S

HBM4EU-MOM: Prenatal methylmercury-exposure control in five countries through suitable dietary advice for pregnancy – Study design and characteristics of participants

Background: Seafood is a major source of vital nutrients for optimal fetal growth, but at the same time is the main source of exposure to methylmercury (MeHg), an established neurodevelopmental toxicant. Pregnant women must be provided with dietary advice so as to include safely fish in their diet for nutrition and mercury control. The aim of this work is to present the design of a multicentre randomized control trial (RCT), which combines human biomonitoring (HBM) with dietary interventions using seafood consumption advice to pregnant women for MeHg control, and to collect information about other possible sources of exposure to mercury. It also presents the materials developed for the implementation of the study and the characteristics of the study participants, which were self-reported in the first trimester of pregnancy. Methods: The "HBM4EU-MOM" RCT was performed in the frame of the European Human Biomonitoring Initiative (HBM4EU) in five coastal, high fish-consuming European countries (Cyprus, Greece, Spain, Portugal and Iceland). According to the study design, pregnant women (≥120/country, ≤20 weeks gestational age) provided a hair sample for total mercury assessment (THg) and personal information relevant to the study (e.g., lifestyle, pregnancy status, diet before and during the pregnancy, information on seafood and factors related to possible non-dietary exposures to mercury) during the first trimester of pregnancy. After sampling, participants were randomly assigned to "control" (habitual practices) or "intervention" (received the harmonized HBM4EU-MOM dietary advice for fish consumption during the pregnancy and were encouraged to follow it). Around child delivery, participants provided a second hair sample and completed another tailored questionnaire. Results: A total of 654 women aged 18-45 years were recruited in 2021 in the five countries, primarily through their health-care providers. The pre-pregnancy BMI of the participants ranged from underweight to obese, but was on average within the healthy range. For 73% of the women, the pregnancy was planned. 26% of the women were active smokers before the pregnancy and 8% continued to smoke during the pregnancy, while 33% were passive smokers before pregnancy and 23% remained passively exposed during the pregnancy. 53% of the women self-reported making dietary changes for their pregnancy, with 74% of these women reporting making the changes upon learning of their pregnancy. Of the 43% who did not change their diet for the pregnancy, 74% reported that their diet was already balanced, 6% found it difficult to make changes and 2% were unsure of what changes to make. Seafood consumption did not change significantly before and during the first trimester of pregnancy (overall average ∼8 times per month), with the highest frequency reported in Portugal (≥15 times per month), followed by Spain (≥7 times per month). During the first-trimester of pregnancy, 89% of the Portuguese women, 85% of the Spanish women and 90%) were unaware of safe procedures for handling spillage from broken thermometers and energy-saving lamps, though >22% experienced such an incident (>1 year ago). 26% of the women had dental amalgams. ∼1% had amalgams placed and ∼2% had amalgams removed during peri-pregnancy. 28% had their hair dyed in the past 3 months and 40% had body tattoos. 8% engaged with gardening involving fertilizers/pesticides and 19% with hobbies involving paints/pigments/dyes. Conclusions: The study design materials were fit for the purposes of harmonization and quality-assurance. The harmonized information collected from pregnant women suggests that it is important to raise the awareness of women of reproductive age and pregnant women about how to safely include fish in their diet and to empower them to make proper decisions for nutrition and control of MeHg, as well as other chemical exposures.We acknowledge funding for HBM4EU from the European Union’s Horizon 2020 research and innovation program under grant agreement #733032 and the national governments of the participating countries. This publication reflects only the authors’ views, and the European Commission is not responsible for any use that may be made of the in formation it contains.info:eu-repo/semantics/publishedVersio

Trends of Exposure to Acrylamide as Measured by Urinary Biomarkers Levels within the HBM4EU Biomonitoring Aligned Studies (2000–2021)

This article belongs to the Special Issue Analysis of Human Biomonitoring Data and Risk Assessment of Human Exposure to Environmental Chemicals: What Do We Learn for Prevention?Acrylamide, a substance potentially carcinogenic in humans, represents a very prevalent contaminant in food and is also contained in tobacco smoke. Occupational exposure to higher concentrations of acrylamide was shown to induce neurotoxicity in humans. To minimize related risks for public health, it is vital to obtain data on the actual level of exposure in differently affected segments of the population. To achieve this aim, acrylamide has been added to the list of substances of concern to be investigated in the HBM4EU project, a European initiative to obtain biomonitoring data for a number of pollutants highly relevant for public health. This report summarizes the results obtained for acrylamide, with a focus on time-trends and recent exposure levels, obtained by HBM4EU as well as by associated studies in a total of seven European countries. Mean biomarker levels were compared by sampling year and time-trends were analyzed using linear regression models and an adequate statistical test. An increasing trend of acrylamide biomarker concentrations was found in children for the years 2014–2017, while in adults an overall increase in exposure was found to be not significant for the time period of observation (2000–2021). For smokers, represented by two studies and sampling for, over a total three years, no clear tendency was observed. In conclusion, samples from European countries indicate that average acrylamide exposure still exceeds suggested benchmark levels and may be of specific concern in children. More research is required to confirm trends of declining values observed in most recent years.This work received external funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No. 733032 and received co-funding from the author’s organizations. The Norwegian Institute of Public Health (NIPH) contributed to the funding of the Norwegian Environmental Biobank (NEB). The laboratory measurements were partly funded by the Research Council of Norway through research projects (275903 and 268465).info:eu-repo/semantics/publishedVersio

HBM4EU-MOM: Prenatal methylmercury-exposure control in five countries through suitable dietary advice for pregnancy - Study design and characteristics of participants

Background: Seafood is a major source of vital nutrients for optimal fetal growth, but at the same time is the main source of exposure to methylmercury (MeHg), an established neurodevelopmental toxicant. Pregnant women must be provided with dietary advice so as to include safely fish in their diet for nutrition and mercury control. The aim of this work is to present the design of a multicentre randomized control trial (RCT), which combines human biomonitoring (HBM) with dietary interventions using seafood consumption advice to pregnant women for MeHg control, and to collect information about other possible sources of exposure to mercury. It also presents the materials developed for the implementation of the study and the characteristics of the study participants, which were self-reported in the first trimester of pregnancy. Methods: The "HBM4EU-MOM" RCT was performed in the frame of the European Human Biomonitoring Initiative (HBM4EU) in five coastal, high fish-consuming European countries (Cyprus, Greece, Spain, Portugal and Iceland). According to the study design, pregnant women (≥120/country, ≤20 weeks gestational age) provided a hair sample for total mercury assessment (THg) and personal information relevant to the study (e.g., lifestyle, pregnancy status, diet before and during the pregnancy, information on seafood and factors related to possible non-dietary exposures to mercury) during the first trimester of pregnancy. After sampling, participants were randomly assigned to "control" (habitual practices) or "intervention" (received the harmonized HBM4EU-MOM dietary advice for fish consumption during the pregnancy and were encouraged to follow it). Around child delivery, participants provided a second hair sample and completed another tailored questionnaire. Results: A total of 654 women aged 18-45 years were recruited in 2021 in the five countries, primarily through their health-care providers. The pre-pregnancy BMI of the participants ranged from underweight to obese, but was on average within the healthy range. For 73% of the women, the pregnancy was planned. 26% of the women were active smokers before the pregnancy and 8% continued to smoke during the pregnancy, while 33% were passive smokers before pregnancy and 23% remained passively exposed during the pregnancy. 53% of the women self-reported making dietary changes for their pregnancy, with 74% of these women reporting making the changes upon learning of their pregnancy. Of the 43% who did not change their diet for the pregnancy, 74% reported that their diet was already balanced, 6% found it difficult to make changes and 2% were unsure of what changes to make. Seafood consumption did not change significantly before and during the first trimester of pregnancy (overall average ∼8 times per month), with the highest frequency reported in Portugal (≥15 times per month), followed by Spain (≥7 times per month). During the first-trimester of pregnancy, 89% of the Portuguese women, 85% of the Spanish women and 90%) were unaware of safe procedures for handling spillage from broken thermometers and energy-saving lamps, though >22% experienced such an incident (>1 year ago). 26% of the women had dental amalgams. ∼1% had amalgams placed and ∼2% had amalgams removed during peri-pregnancy. 28% had their hair dyed in the past 3 months and 40% had body tattoos. 8% engaged with gardening involving fertilizers/pesticides and 19% with hobbies involving paints/pigments/dyes. Conclusions: The study design materials were fit for the purposes of harmonization and quality-assurance. The harmonized information collected from pregnant women suggests that it is important to raise the awareness of women of reproductive age and pregnant women about how to safely include fish in their diet and to empower them to make proper decisions for nutrition and control of MeHg, as well as other chemical exposures.We acknowledge funding for HBM4EU from the European Union’s Horizon 2020 research and innovation program under grant agreement #733032 and the national governments of the participating countries. This publication reflects only the authors’ views, and the European Commission is not responsible for any use that may be made of the information it contains.S

Large-scale mapping of mutations affecting zebrafish development

BACKGROUND: Large-scale mutagenesis screens in the zebrafish employing the mutagen ENU have isolated several hundred mutant loci that represent putative developmental control genes. In order to realize the potential of such screens, systematic genetic mapping of the mutations is necessary. Here we report on a large-scale effort to map the mutations generated in mutagenesis screening at the Max Planck Institute for Developmental Biology by genome scanning with microsatellite markers. RESULTS: We have selected a set of microsatellite markers and developed methods and scoring criteria suitable for efficient, high-throughput genome scanning. We have used these methods to successfully obtain a rough map position for 319 mutant loci from the Tübingen I mutagenesis screen and subsequent screening of the mutant collection. For 277 of these the corresponding gene is not yet identified. Mapping was successful for 80 % of the tested loci. By comparing 21 mutation and gene positions of cloned mutations we have validated the correctness of our linkage group assignments and estimated the standard error of our map positions to be approximately 6 cM. CONCLUSION: By obtaining rough map positions for over 300 zebrafish loci with developmental phenotypes, we have generated a dataset that will be useful not only for cloning of the affected genes, but also to suggest allelism of mutations with similar phenotypes that will be identified in future screens. Furthermore this work validates the usefulness of our methodology for rapid, systematic and inexpensive microsatellite mapping of zebrafish mutations

It Pays to Prepare: Human Motor Preparation Depends on the Relative Value of Potential Response Options

Alternative motor responses can be prepared in parallel. Here, we used electroencephalography (EEG) to test whether the parallel preparation of alternative response options is modulated by their relative value. Participants performed a choice response task with three potential actions: isometric contraction of the left, the right, or both wrists. An imperative stimulus (IS) appeared after a warning cue, such that the initiation time of a required action was predictable, but the specific action was not. To encourage advanced preparation, the target was presented 200 ms prior to the IS, and only correct responses initiated within ±100 ms of the IS were rewarded. At baseline, all targets were equally rewarded and probable. Then, responses with one hand were made more valuable, either by increasing the probability that the left or right target would be required (Exp. 1; n = 31) or by increasing the reward magnitude of one target (Exp. 2, n = 36). We measured reaction times, movement vigor, and an EEG correlate of action preparation (value-based lateralized readiness potential) prior to target presentation. Participants responded earlier to more frequent and more highly rewarded targets, and movements to highly rewarded targets were more vigorous. The EEG was more negative over the hemisphere contralateral to the more repeated/rewarded hand, implying an increased neural preparation of more valuable actions. Thus, changing the value of alternative response options can lead to greater preparation of actions associated with more valuable outcomes. This preparation asymmetry likely contributes to behavioral biases that are typically observed toward repeated or rewarded targets