Effects of nanomolar methylmercury on developing human neural stem cells and zebrafish Embryo.

Exposure to mercury and its organic form methylmercury (MeHg), is of great concern for the developing nervous system. Despite available literature on MeHg neurotoxicity, there is still uncertainty about its mechanisms of action and the doses that trigger developmental effects. Our study combines two alternative methodologies, the human neural stem cells (NSC) and the zebrafish (ZF) embryo, to address the neurotoxic effects of early exposure to nanomolar concentrations of MeHg. Our results show linear or nonmonotonic (hormetic) responses depending on studied parameters. In ZF, we observed a hormetic response in locomotion and larval rotation, but a concentration-dependent response for sensory organ size and habituation. We also observed a possible delayed response as MeHg had greater effects on larval activity at 5 days than at 24 h. In NSC cells, some parameters show a clear dose dependence, such as increased apoptosis and differentiation to glial cells or decreased neuronal precursors; while others show a hormetic response: neuronal differentiation or cell proliferation. This study shows that the ZF model was more susceptible than NSC to MeHg neurotoxicity. The combination of different models has improved the understanding of the underlying mechanisms of toxicity and possible compensatory mechanisms at the cellular and organismal level.

Internalization and toxicity of polystyrene nanoplastics on inmortalized human neural stem cells.

Global plastic production has increased exponentially in recent decades, and a significant part of it persists in the environment, where it degrades into microplastics and nanoplastics (MPs and NPs). These can enter in humans by ingestion, inhalation, and dermal routes, and there is scientific evidence that they are able to reach the systemic circulation and penetrate and accumulate in various tissues and organs. Neurodevelopmental toxicity of NPs is one of the most worrying effects, as they can cross the blood-brain barrier. In the following study, we analyzed, by transmission electron microscopy, the in vitro uptake of 30-nm polystyrene nanoplastics (PS-NPs) into human neural stem cells (NSCs), their accumulation and subcellular localization within the cell. Furthermore, we studied the effects of different concentrations of PS-NPs on cell death, proliferation, and cell differentiation using immunocytochemistry and quantitative real time PCR for specific markers. This study demonstrated that PS-NPs were able to enter the cell, probably by endocytosis, accumulate, and aggregated in human NSCs, without being detected in the nucleus, causing cell death by apoptosis and decreased cell proliferation. This study provides new insights into the interaction and effects of PS-NPs in human NSC and supports the scientific evidence for the involvement of nanoplastic in neurodevelopmental disorders.

Molecular effects of polystyrene nanoplastics on human neural stem cells.

Nanoplastics (NPs) have been found in many ecological environments (aquatic, terrestrial, air). Currently, there is great concern about the exposition and impact on animal health, including humans, because of the effects of ingestion and accumulation of these nanomaterials (NMs) in aquatic organisms and their incorporation into the food chain. NPs´ mechanisms of action on humans are currently unknown. In this study, we evaluated the altered molecular mechanisms on human neural stem cell line (hNS1) after 4 days of exposure to 30 nm polystyrene (PS) NPs (0.5, 2.5 and 10 µg/mL). Our results showed that NPs can induce oxidative stress, cellular stress, DNA damage, alterations in inflammatory response, and apoptosis, which could lead to tissue damage and neurodevelopmental diseases.

DNA damage and molecular level effects induced by polystyrene (PS) nanoplastics (NPs) after Chironomus riparius (Diptera) larvae.

In this work, we analyzed the early molecular effects of polystyrene (PS) nanoplastics (NPs) on an aquatic primary consumer (larvae of Chironomus riparius, Diptera) to evaluate their potential DNA damage and the transcriptional response of different genes related to cellular and oxidative stress, endocrine response, developmental, oxygen transport, and immune response. After 24-h exposures of larvae to doses of PS NPs close to those currently found in the environment, the results revealed a large genotoxic effect. This end was evidenced after significant increases in DNA strand breaks of C. riparius larvae quantified by the comet assay, together with results obtained when analyzing the expression of four genes involved in DNA repair (xrrc1, ATM, DECAY and NLK) and which were reduced in the presence of these nanomaterials. Consequently, this reduction trend is likely to prevent the repair of DNA damage caused by PS NPs. In addition, the same tendency to reduce the expression of genes involved in cellular stress, oxidative stress, ecdysone pathway, development, and oxygen transport was observed. Taken together, these results suggest that PS NPs reduce the expression of hormonal target genes and a developmental gene. We show, for the first time, effects of PS NPs on the endocrine system of C. riparius and suggest a possible mechanism of blocking ecdysteroid hormones in insects. Moreover, the NPs were able to inhibit the expression of hemoglobin (Hb C), a protein involved in oxygen transport, and activate a gene of the humoral immune system. These data reveal for the first time the genomic effects of PS NPs in the aquatic invertebrate C. riparius, at the base of the food chain.

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 <50% of Greek, Cypriot and Icelandic women reported that they had consumed big oily fish. Relevant to non-dietary exposure sources, most participants (>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.

A simple method for direct mercury analysis in dried blood spots (DBS) samples for human biomonitoring studies.

Human exposure to mercury can have serious health effects, especially in vulnerable groups such as children and fetuses. The use of dried blood spot (DBS) samples to collect capillary blood greatly facilitates sample collection and fieldwork, being a less invasive alternative to blood collection by venipuncture, needing a small volume of sample, and does not require specialized medical staff. Moreover, DBS sampling reduces logistical and financial barriers related to transport and storage of blood samples. We propose here a novel method to analyze total mercury in DBS samples in a Direct Mercury Analyzer (DMA) that allow the control of the volume of the DBS samples. This method has shown good results in terms of precision (<6% error), accuracy (<10% coefficient of variation) and recovery (75-106%). The applicability of the method in human biomonitoring (HBM) was demonstrated in a pilot study involving 41 adults aged 18-65. Mercury concentrations of DBS samples from capillary blood collected by finger prick (real DBS samples) were determined in the DMA and compared with those determined in whole blood (venous blood) by ICP-MS, the method usually used in HBM. The sampling procedure was also validated by comparison of real DBS samples and DBS generated artificially in the laboratory by depositing venous samples in cellulose cards (laboratory DBS). There were no statistically significant differences in the results obtained using both methodologies (DMA: Geometric Mean (confidence interval 95%) = 3.87 (3.12-4.79) µg/L; ICP-MS: Geometric Mean (confidence interval 95%) = 3.46 (2.80-4.27) µg/L). The proposed method is an excellent alternative to be applied in clinical settings as screening methodology for assessing mercury exposure in vulnerable groups, such us pregnant woman, babies and children.

New approach methods to improve human health risk assessment of thyroid hormone system disruption-a PARC project.

Current test strategies to identify thyroid hormone (TH) system disruptors are inadequate for conducting robust chemical risk assessment required for regulation. The tests rely heavily on histopathological changes in rodent thyroid glands or measuring changes in systemic TH levels, but they lack specific new approach methodologies (NAMs) that can adequately detect TH-mediated effects. Such alternative test methods are needed to infer a causal relationship between molecular initiating events and adverse outcomes such as perturbed brain development. Although some NAMs that are relevant for TH system disruption are available-and are currently in the process of regulatory validation-there is still a need to develop more extensive alternative test batteries to cover the range of potential key events along the causal pathway between initial chemical disruption and adverse outcomes in humans. This project, funded under the Partnership for the Assessment of Risk from Chemicals (PARC) initiative, aims to facilitate the development of NAMs that are specific for TH system disruption by characterizing in vivo mechanisms of action that can be targeted by in embryo/in vitro/in silico/in chemico testing strategies. We will develop and improve human-relevant in vitro test systems to capture effects on important areas of the TH system. Furthermore, we will elaborate on important species differences in TH system disruption by incorporating non-mammalian vertebrate test species alongside classical laboratory rat species and human-derived in vitro assays.

How to use human biomonitoring in chemical risk assessment: Methodological aspects, recommendations, and lessons learned from HBM4EU.

One of the aims of the European Human Biomonitoring Initiative, HBM4EU, was to provide examples of and good practices for the effective use of human biomonitoring (HBM) data in human health risk assessment (RA). The need for such information is pressing, as previous research has indicated that regulatory risk assessors generally lack knowledge and experience of the use of HBM data in RA. By recognising this gap in expertise, as well as the added value of incorporating HBM data into RA, this paper aims to support the integration of HBM into regulatory RA. Based on the work of the HBM4EU, we provide examples of different approaches to including HBM in RA and in estimations of the environmental burden of disease (EBoD), the benefits and pitfalls involved, information on the important methodological aspects to consider, and recommendations on how to overcome obstacles. The examples are derived from RAs or EBoD estimations made under the HBM4EU for the following HBM4EU priority substances: acrylamide, o-toluidine of the aniline family, aprotic solvents, arsenic, bisphenols, cadmium, diisocyanates, flame retardants, hexavalent chromium [Cr(VI)], lead, mercury, mixture of per-/poly-fluorinated compounds, mixture of pesticides, mixture of phthalates, mycotoxins, polycyclic aromatic hydrocarbons (PAHs), and the UV-filter benzophenone-3. Although the RA and EBoD work presented here is not intended to have direct regulatory implications, the results can be useful for raising awareness of possibly needed policy actions, as newly generated HBM data from HBM4EU on the current exposure of the EU population has been used in many RAs and EBoD estimations.

Harmonized human biomonitoring in European children, teenagers and adults: EU-wide exposure data of 11 chemical substance groups from the HBM4EU Aligned Studies (2014-2021).

As one of the core elements of the European Human Biomonitoring Initiative (HBM4EU) a human biomonitoring (HBM) survey was conducted in 23 countries to generate EU-wide comparable HBM data. This survey has built on existing HBM capacity in Europe by aligning national or regional HBM studies, referred to as the HBM4EU Aligned Studies. The HBM4EU Aligned Studies included a total of 10,795 participants of three age groups: (i) 3,576 children aged 6-12 years, (ii) 3,117 teenagers aged 12-18 years and (iii) 4,102 young adults aged 20-39 years. The participants were recruited between 2014 and 2021 in 11-12 countries per age group, geographically distributed across Europe. Depending on the age group, internal exposure to phthalates and the substitute DINCH, halogenated and organophosphorus flame retardants, per- and polyfluoroalkyl substances (PFASs), cadmium, bisphenols, polycyclic aromatic hydrocarbons (PAHs), arsenic species, acrylamide, mycotoxins (deoxynivalenol (total DON)), benzophenones and selected pesticides was assessed by measuring substance specific biomarkers subjected to stringent quality control programs for chemical analysis. For substance groups analyzed in different age groups higher average exposure levels were observed in the youngest age group, i.e., phthalates/DINCH in children versus teenagers, acrylamide and pesticides in children versus adults, benzophenones in teenagers versus adults. Many biomarkers in teenagers and adults varied significantly according to educational attainment, with higher exposure levels of bisphenols, phthalates, benzophenones, PAHs and acrylamide in participants (from households) with lower educational attainment, while teenagers from households with higher educational attainment have higher exposure levels for PFASs and arsenic. In children, a social gradient was only observed for the non-specific pyrethroid metabolite 3-PBA and di-isodecyl phthalate (DiDP), with higher levels in children from households with higher educational attainment. Geographical variations were seen for all exposure biomarkers. For 15 biomarkers, the available health-based HBM guidance values were exceeded with highest exceedance rates for toxicologically relevant arsenic in teenagers (40%), 3-PBA in children (36%), and between 11 and 14% for total DON, Σ (PFOA + PFNA + PFHxS + PFOS), bisphenol S and cadmium. The infrastructure and harmonized approach succeeded in obtaining comparable European wide internal exposure data for a prioritized set of 11 chemical groups. These data serve as a reference for comparison at the global level, provide a baseline to compare the efficacy of the European Commission's chemical strategy for sustainability and will give leverage to national policy makers for the implementation of targeted measures.

Neurotoxicity and endocrine disruption caused by polystyrene nanoparticles in zebrafish embryo.

Nanoplastics (NP) are present in aquatic and terrestrial ecosystems. Humans can be exposed to them through contaminated water, food, air, or personal care products. Mechanisms of NP toxicity are largely unknown and the Zebrafish embryo poses an ideal model to investigate them due to its high homology with humans. Our objective in the present study was to combine a battery of behavioral assays with the study of endocrine related gene expression, to further explore potential NP neurotoxic effects on animal behavior. Polystyrene nanoplastics (PSNP) were used to evaluate NP toxicity. Our neurobehavioral profiles include a tail coiling assay, a light/dark activity assay, two thigmotaxis anxiety assays (auditory and visual stimuli), and a startle response - habituation assay in response to auditory stimuli. Results show PSNP accumulated in eyes, neuromasts, brain, and digestive system organs. PSNP inhibited acetylcholinesterase and altered endocrine-related gene expression profiles both in the thyroid and glucocorticoid axes. At the whole organism level, we observed altered behaviors such as increased activity and anxiety at lower doses and lethargy at a higher dose, which could be due to a variety of complex mechanisms ranging from sensory organ and central nervous system effects to others such as hormonal imbalances. In addition, we present a hypothetical adverse outcome pathway related to these effects. In conclusion, this study provides new understanding into NP toxic effects on zebrafish embryo, emphasizing a critical role of endocrine disruption in observed neurotoxic behavioral effects, and improving our understanding of their potential health risks to human populations.

Molecular effects of polystyrene nanoplastics toxicity in zebrafish embryos (Daniorerio).

Plastics pose a health hazard to living beings and the environment. Plastic degradation produces nano-sized plastic particles (NPs) that end up in terrestrial and aquatic ecosystems, including oceans, rivers, and lakes. Their presence in air, drinking water, sediments, food, and personal care products leads to a variety of exposure routes for living beings, including humans. The toxicity mechanisms of these nanomaterials (NMs) in living organisms and ecosystems are currently unknown, making it a priority to understand their effects at the molecular and cellular levels. The zebrafish (Zf) (Danio rerio) is a model organism which has a high homology with humans and has been widely used to assess the hazard of different xenobiotics. In this study, the expression changes of different genes in 120 hpf Zf embryos (Zfe) after exposure to polystyrene (PS) NPs (30 nm) at concentrations of 0.1, 0.5 and 3 ppm were investigated. The results showed that the gene encoding heat shock protein (hsp70) was down-regulated in a dose-dependent manner. The genes encoding superoxide dismutase (SOD 1 and SOD 2), apoptotic genes (cas 1 and cas 8) and interleukin 1-ß (il1ß) were activated at the concentration of 3 ppm PS NP, while the anti-apoptotic gene Bcl2α was inhibited at 0.5 and 3 ppm. In addition, the neurotransmitter-related gene Acetyl-Cholinesterase (ache) was significantly inhibited and the DNA repair genes (gadd45α and rad51) were also down-regulated. In contrast, the mitochondrial metabolism-related gene cox1 did not alter its expression in any of the treatments. Most of the changes in gene expression occurred at the highest concentration of NPs. Overall, the results indicated that NPs generated cellular stress that caused certain alterations in normal gene expression (oxidative stress, apoptotic and inflammatory processes, neurotoxicity and anti-apoptotic proteins), but did not cause any mortality after 120 hpf exposure at the three concentrations assayed. These results highlight the need for further studies investigating the effects, at the molecular level, of these materials in humans and other living organisms.

Methylmercury Risk Assessment Based on European Human Biomonitoring Data.

A risk assessment (RA) was conducted to estimate the risk associated with methylmercury (MeHg) exposure of vulnerable European populations, using Human Biomonitoring (HBM) data. This RA was performed integrating published data from European HBM surveys and earlier EFSA approaches (EFSA 2012). Children/adolescents (3 to 17 years old) and women of childbearing age (18 to 50 years old) were selected as relevant study population groups for this RA. Two types of HBM datasets were selected: HBM studies (n = 18) with mercury (Hg) levels (blood and hair, total Hg and/or MeHg) in the general population in different EU countries and the DEMOCOPHES harmonized study in child-mother pairs (hair, total Hg) in 17 EU countries as a reference. Two approaches were included in the RA strategy: the first one was based on estimations of the fraction of children/adolescents and women of childbearing age, respectively, from the EU general population exceeding the HBM-I value established by the German Human Biomonitoring Commission, measured as Hazard Quotients (HQ); and the second approach was based on estimations of the fraction of the two population groups exceeding the Tolerable Weekly Intake (TWI) (or their equivalent to Tolerable Daily Intake (TDI)) defined by EFSA in 2012. The HQ approach showed that for both groups, the risk varies across EU countries and that some EU areas are close to or exceeding the exposure guidance values. This is the case of Spain and Portugal, which showed the highest HQ (GM and/or P95), probably due to their higher fish consumption. Results from the EFSA approach show that hair values of children/adolescents and women of childbearing age (both in selected HBM studies and in DEMOCOPHES study) are below the TDI of 1.9 µg/g; therefore, in general, the European population does not exceed the daily average/intake dose for MeHg and/or Hg. A possible risk underestimation was identified in our assessment since for many studies no data on P95 were available, causing loss of relevant information for risk characterization on the upper bound. In addition, data from other European countries also with high seafood consumption, such as France, Greece or Iceland, were not available. For this reason, further RA refinement is needed with harmonized and more widespread HBM data to account for differences in European exposure and associated risks, so that interventions to protect vulnerable citizens, can be applied.

Toxicity of nanoplastics for zebrafish embryos, what we know and where to go next.

Nanoplastics (NP) are an emerging threat to human health and there is a need to understand their toxicity. Zebrafish (ZF) is extensively used as a toxicology model due to its power to com-bine genetic, cellular, and whole organism endpoints. The present review integrates results regarding polystyrene NP effects on ZF embryo development. Study design was evaluated against NP effects. NP size, concentration, and exposure time did not affect organism responses (mortality, development, heart rate, locomotion) or cellular responses (gene expression, enzymes, metabolites). However, NP accumulation depended on size. Smaller NP can reach internal organs (brain, eyes, liver, pancreas, heart) but larger (>200 nm) accumulate mainly in gut, gills and skin. Locomotion and heart rate were commonly affected with hypoactivity and bradycardia being more prevalent. Effects on genetic/enzymatic/metabolic pathways were thoroughly analyzed. Immunity genes were generally upregulated whereas oxidative stress response genes varied. Central nervous system genes and visual related genes were generally downregulated. Results of genetic and enzymatic analyses coincided only for some genes/enzyme pairs. Reviewed studies provide a basis for understanding NP toxicity but results are hard to integrate. We propose key recommendations and future directions with regard to experimental design that may allow greater comparability across future studies.