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.

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.

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.

Applicability of Food Monitoring Data for Assessing Relative Exposure Contributions of Pyrethroids in Retrospective Human Biomonitoring Risk Estimations.

The use of pyrethroids is very broad and shows increasing trends. Human biomonitoring studies represent the best approach for realistic risk estimations, but their interpretation requires a tiered approach. A previous HBM4EU study indicated levels in European children groups just around the threshold for concern, requiring further refinement. The main difficulty is that several pyrethroids with different toxicity potencies generate the same urinary metabolites. As diet is the main pyrethroid source for the general population, EU food monitoring data reported by EFSA have been used to estimate the relative contribution of each pyrethroid. The main contributors were cypermethrin for DCCA and 3-PBA and lambda-cyhalothrin for CFMP. Urinary levels predicted from food concentration according to the EFSA diets were mostly within the range of measured levels, except 3-PBA and CFMP levels in children, both below measured levels. The predicted lower levels for 3-PBA can be explained by the very low Fue value, initially proposed as conservative, but that seems to be unrealistic. The discrepancies for CFMP are mostly for the highest percentiles and require further assessments. The refined assessments included the revision of the previously proposed human biomonitoring guidance values for the general population, HBM-GV Gen Pop, following recent toxicological reevaluations, and the estimation of hazard quotients (HQs) for each individual pyrethroid and for the combined exposure to all pyrethroids. All HQs were below 1, indicating no immediate concern, but attention is required, particularly for children, with HQs in the range of 0.2-0.3 for the highly exposed group. The application of probabilistic methods offers assessments at the population level, addressing the variability in exposure and risk and providing relevant information for Public Health impact assessments and risk management prioritization.

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.

A Tiered Approach for Assessing Individual and Combined Risk of Pyrethroids Using Human Biomonitoring Data.

Pyrethroids are a major insecticide class, suitable for biomonitoring in humans. Due to similarities in structure and metabolic pathways, urinary metabolites are common to various active substances. A tiered approach is proposed for risk assessment. Tier I was a conservative screening for overall pyrethroid exposure, based on phenoxybenzoic acid metabolites. Subsequently, probabilistic approaches and more specific metabolites were used for refining the risk estimates. Exposure was based on 95th percentiles from HBM4EU aligned studies (2014-2021) covering children in Belgium, Cyprus, France, Israel, Slovenia, and The Netherlands and adults in France, Germany, Israel, and Switzerland. In all children populations, the 95th percentiles for 3-phenoxybenzoic acid (3-PBA) exceeded the screening value. The probabilistic refinement quantified the risk level of the most exposed population (Belgium) at 2% or between 1-0.1% depending on the assumptions. In the substance specific assessments, the 95th percentiles of urinary concentrations in the aligned studies were well below the respective human biomonitoring guidance values (HBM-GVs). Both information sets were combined for refining the combined risk. Overall, the HBM data suggest a low health concern, at population level, related to pyrethroid exposure for the populations covered by the studies, even though a potential risk for highly exposed children cannot be completely excluded. The proposed tiered approach, including a screening step and several refinement options, seems to be a promising tool of scientific and regulatory value in future.

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.