Nanoplastics (NPs) have emerged as a growing environmental and health concern due to their widespread presence in various environmental matrices, including food, water, and air. They are inadvertently ingested, inhaled, or absorbed through the skin, raising concerns about their potential to interact with cellular systems, including the nervous system. Despite increasing awareness, their neurotoxic potential, particularly the effects of functionalized NPs, remains poorly understood. This study provides novel insights into the neurotoxicity of four types of polystyrene nanoplastic (PS-NPs)—plain PS-NPs (50 nm and 100 nm) and amine- and carboxyl-functionalized PS-NPs (100 nm)—on human SH-SY5Y cells. Cells were exposed to NP concentrations ranging from 1 to 500 µg/mL for 24 and 48 hours, with NP characterization and stability assessments conducted in culture media prior to toxicity evaluations (cell viability, ROS/RNS production, NP internalization, morphological and ultrastructural changes). In-depth toxicity assessments revealed that functionalized NPs, particularly amine-modified ones, induced greater cytotoxicity than their plain counterparts. Cellular viability assays demonstrated a concentration- and time-dependent reduction, with significant cytotoxicity observed at 200-500 µg/mL. ROS/RNS production was markedly elevated in plain 100 nm and amine-functionalized NPs at concentrations of 200-500 µg/mL, with oxidative stress intensifying over time. Transmission electron microscopy uncovered distinct subcellular damage patterns, including endoplasmic reticulum dilation, mitochondrial impairment, and Golgi fragmentation, correlating with NP size, concentration and functionalization. Notably, functionalized NPs exhibited greater cellular uptake, with amine-modified NPs showing the highest internalization. Further mechanistic analyses revealed that PS-NPs induced apoptosis, autophagy, and lysosomal dysfunction, with functionalized variants exhibiting more pronounced effects. These findings underscore the critical influence of NP functionalization on neurotoxicity and highlight the urgent need for further investigation into their potential health implications, particularly concerning human exposure and neurodegenerative risk.

Acknowledgments: This work received support from PT national funds through the UID/50006 project and FCT/MCTES through the SALIVA+ project (DOI 705 10.54499/2022.08978.PTDC).

Microcystins (MCs), common toxins produced during cyanobacterial blooms, are frequently found in water sources used for irrigation, leading to their introduction into agricultural environments. While MCs are well-known for their toxic effects on plant growth and physiology, their influence on plant-associated microbial communities remains underexplored. To address this gap, we studied how exposure to 100 µg/L of MCs affects the bacterial microbiota inhabiting bulk soil (BS), root-adhering soil (RAS), and root tissue (RT) in Vicia faba using a greenhouse pot experiment. Our findings revealed that MC exposure significantly altered the microbial community structure, co-occurrence patterns, and assembly mechanisms, with the most pronounced changes observed in RT microbiota, followed by BS and then RAS. Notably, MCs caused a marked decline in several Actinobacteriota taxa within RT, particularly members of the genus Streptomyces, which are commonly associated with plant health. In contrast, the abundance of certain Proteobacteria, including Methylobacillus, Methylotenera, and Paucibacter, genera potentially involved in MC degradation, increased under MC treatment. Network analysis showed a reduction in microbial co-occurrence complexity in MC-exposed conditions compared to the control. Moreover, community assembly in RT appeared to be predominantly governed by deterministic processes, while both deterministic and stochastic processes influenced BS and RAS microbiota, with a stronger deterministic trend overall. These results suggest that MCs may reshape the microbiota structure in the soil-plant system by reducing bacterial taxa with potential phytobeneficial traits and increasing other taxa with a potential capacity to degrade MCs.

Introduction Titanium dioxide(TiO₂) is frequently used as a whitening agent(E171). Although historically classified as "generally recognized as safe", the European Food Safety Authority (EFSA) suspended its food additive approval due to unresolved concerns(EFSA Panel, 2021), while China and the United States maintain permissible limits. E171 ranges between 200 and 300 nm in diameter while smaller nanoparticles are more likely to be absorbed by intestinal epithelial cells. Based on bioinformatics, we studied the molecular mechanism of the TiO₂-induced intestinal damage process.

Methods Comparative Toxicogenomics Database (CTD) identified 160 colitis- and 258 colorectal cancer-associated genes modulated by TiO₂ and Cytoscape was used to make a PPI network diagram. Functional enrichment analysis (GO/KEGG) revealed key pathways, cross-validated with transcriptomic datasets from TiO₂-exposed enteritis (GSE92563) and colorectal tumor models (GSE109520). We screened the AOP-wiki database and constructed a new pathway network factors. Meanwhile, we used MECE and CT26 cells separately co-cultured with E171 and TiO₂-10nm for 48h for gene sequencing to validate our predictive model.

Results In the intestinal inflammation model, TiO₂ exposure initially activates the JNK and MAPK signaling pathways, causing changes in the cell membrane (invagination, endocytosis), cell death, inflammation, and hypoxia, leading to inflammatory bowel disease. For colorectal cancer, cytokines (Stat1, Pik3cd, Cxcr4, etc.) were activated, leading to extracellular matrix response and apoptosis, resulting in colorectal cancer and migration. Meanwhile, we analyzed the existing AOPs with high relevance to the intestinal damage mechanism of TiO₂ (AOP392, AOP451, and AOP303) and constructed a new AOP. In cell sequencing, we also enriched the relevant pathways, which pointed to different factors in the two types of cells.

Conclusion We introduced these regulatory cascades as new MIE and KE into the AOP framework, outlining the precise mechanism by which TiO₂ exposure induces colorectal damage and providing new evidence for food safety issues.

Introduction
Drug-induced liver injury (DILI) is a major global health concern and a leading cause of acute liver failure. While intrinsic DILI, such as that caused by acetaminophen, is dose-dependent and predictable, idiosyncratic DILI (iDILI) occurs unpredictably at therapeutic doses. The incidence of iDILI is rising due to polypharmacy, aging populations, and increased use of herbal products. Although host factors such as age, immune response, and genetic variants (e.g., HLA alleles) influence susceptibility, they are not fully predictive. Emerging evidence points to acquired conditions—such as exposure to persistent organic pollutants (POPs) and preexisting hepatic steatosis—as contributors to iDILI by altering liver susceptibility and enhancing drug toxicity.

Methods
We used Upcyte human hepatocytes, which retain key metabolic and transport functions of primary liver cells. Hepatocytes were exposed to the POPs Bisphenol A (BPA) and Perfluorooctanoic acid (PFOA), with or without co-treatment with azathioprine, valproic acid (VPA), or amoxicillin/clavulanic acid. Cell viability was assessed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. To model hepatic steatosis, cells were preincubated with 0.2 mM oleate/palmitate (2:1) for 44 hours. Triglyceride (TG) accumulation was measured using a fluorescence-based assay.

Results
A synergistic cytotoxic effect was observed in cells co-treated with azathioprine plus BPA, VPA plus BPA, and amoxicillin/clavulanic acid plus PFOA. Metabolic steatosis preconditioning enhanced TG accumulation when combined with azathioprine or VPA. Interestingly, BPA reduced the steatotic effect of VPA, possibly because BPA may operate as an antisteatotic polyphenol or due to modulation of estrogen receptor activity.

Conclusions
These preliminary findings suggest that environmental exposures and hepatic steatosis modulate drug toxicity and may increase susceptibility to iDILI. POPs like BPA and PFOA can exacerbate hepatotoxicity, highlighting the importance of including such factors in risk assessment and prevention strategies for drug-induced liver injury.

Introduction: Endometriosis is a chronic gynecological disease affecting up to 7 - 10% of menstruating individuals worldwide. To date, its etiopathogenesis remains largely unknown, with environmental factors such as potentially toxic elements (PTEs) being contributors.

Methods: Peritoneal fluid (PF) samples were collected from 13 patients with superficial peritoneal endometriosis (SUP), 13 with ovarian endometrioma (OMA), 4 with deeply infiltrative endometriosis (DEEP), and 13 non-endometriosis controls. The samples were collected between June 2020 and September 2022 from patients who underwent laparoscopic procedures within the Gynecology Service of a University Hospital in San Juan (Spain). The group classification was based on surgical findings and histopathological reports. After sample processing, the PF was stored in the dark at -20°C until analysis by inductively coupled plasma tandem mass spectrometry (Agilent 8900 ICP-MS/MS). Statistical analyses were performed using IBM SPSS Statistics version 28.0 (IBM, Armonk, NY, USA). Elemental ratios were calculated by normalizing the average concentration of each element to that of the control group.

Results: The concentration of Fe was significantly elevated in the OMA group compared to the control group (p=0.005), with mean concentrations of 8,172 ± 13,361 and 967 ± 1,524 µg L⁻¹, respectively. No statistically significant differences were found when comparing the elemental concentrations in SUP and DEEP with those in the control group. When examining the elemental ratios (threshold of >1.5), elevated levels were observed as follows: the levels of Fe and Co were above average in SUP, OMA, and DEEP; the level of Ba was above average in OMA and SUP; the level of Pb was above average in SUP and DEEP; the levels of Cd and Mn were above average in OMA; and finally, the levels of Cu and Zn were above average in DEEP. Distinct patterns of Spearman correlations between the elements were identified depending on the specific endometriosis entity.

Conclusions: These findings suggest that multielemental profiles in peritoneal fluid vary according to the specific endometriosis entity.

Structural firefighting exposes firefighters to intense physical and thermal stress, and emissions of health-burden compounds [e.g., particulate matter and polycyclic aromatic hydrocarbons (PAHs)]. The use of a self-contained breathing apparatus (SCBA) is mandatory during structure fires, although in the overhaul phase, firefighters often remove it. The characterization of inhaled PAHs during structure fires remains scarce, particularly for European firefighters. This study assessed, for the first time, the levels of PAH in the air of firefighters outside and inside the SCBA during training firefighting activities and at fire stations on regular working days. The control group included 13 male volunteer firefighters without active participation in firefighting in the previous 7 days, while the exposed group included 31 professional sapper firefighters. Outside PAHs were monitored with a pre-treated polyurethane foam (PUF), placed in the personal protective equipment, while a cotton disk was placed inside the SCBA. PAHs were quantified by high-performance liquid chromatography. Total PAH levels in PUFs of exposed firefighters were higher than PUFs from the control group (11.5–28.0 µg/m³ versus 1.41 µg/m³), and significantly higher than levels detected inside the SCBA (1.96-10.40 ng/m³). Regarding carcinogenic PAHs, levels quantified outside the SCBA were higher for exposed firefighters than the control group (5.80-17.2 µg/m³ versus 0.84 µg/m³), and superior to the inside SCBA’ levels (0.77-0.93 ng/m³) (p<0.005). International occupational guidelines were not exceeded. The inside–outside ratios (<1) suggested that ambient PAHs contributed to the levels quantified inside the SCBA. Extracts collected outside and inside the SCBA caused cytotoxicity in human alveolar and bronchial cell lines. Moreover, the collected samples were applied to an in vitro co-culture air–blood barrier model to assess permeability. Carcinogenic benzo(a)pyrene (100%) permeated the model. Overall, the negative effects of fire emissions on human respiratory cell lines were confirmed, suggesting that fire pollutants contribute to firefighters’ respiratory health burden.

Introduction Complete blood count (CBC) is one of the fundamental tests for assessing a patient's condition. Despite widespread automation in hematology, manual blood smear analysis remains essential. This study aimed to evaluate the use of manual blood smears, both unstained and May–Grünwald–Giemsa (MGG)-stained, as a novel alternative biological matrix for xenobiotic determination.

Methods Analytical standards of selected 2-phenylethylamine derivatives were added to K₂EDTA whole blood, followed by manual smear preparation. Half of the smears were stained using the MGG technique. The developed method involved transferring the matrix from the slide into a test tube, followed by extraction with an n-hexane/ethyl acetate mixture under alkaline conditions. Analyses were conducted using UHPLC-ESI-QqQ-MS/MS. In unstained smears, xenobiotics were monitored across concentrations ranging from pg/mL to µg/mL. Total hemoglobin was evaluated at low (critical), moderate, and normal levels. The method was further applied to evaluate xenobiotic stability in blood smears stored at room temperature, the typical storage condition for such samples.

Results The study demonstrated that unstained blood smears can serve as a valuable alternative matrix for xenobiotic determination, enabling detection not only in poisoning cases (toxic/lethal concentrations) but also in exposure assessments, with a limit of quantification down to the ng/mL range. Despite substantial differences in total hemoglobin concentration, effective xenobiotic detection was achieved. In MGG-stained smears, however, staining steps reduced detection capabilities, and a qualitative interpretation approach is recommended.

Conclusions The results indicate that small blood volumes are sufficient for both qualitative and quantitative toxicological analyses, particularly in unstained smears. Furthermore, the ability to detect substances at trace levels highlights the necessity for cautious interpretation due to contamination risk. The findings regarding unstained blood smears, including stability results, may provide valuable support for toxicological examinations of blood traces on other glass surfaces.

Introduction:

Traditional neurotoxicity (NT) testing relies mainly on rodent-based guidelines (OECD TG 424 and 426). Despite their relevance to many neurotoxic mechanisms, these tests are costly and ethically challenging, and they overlook behavioral endpoints related to anxiety. To address the urgent need for animal replacement in NT testing, the European Partnership for the Assessment of Risks from Chemicals (PARC), subtask 5.2.1e, focuses on the development of human-relevant New Approach Methodologies (NAMs). In this context, the zebrafish embryo (ZFe), up to 120 hours post fertilization (hpf), is an in vivo model that has high human homology, but is not included in animal welfare regulations and is not considered an NAM.

Methods:

We developed a high-throughput thigmotaxis assay based on 120 hpf ZFEs to evaluate anxiety-like behavior as a biomarker of NT. Thigmotaxis—the tendency to remain close to the edge of a container—is a robust indicator of altered neural function. We optimized the assay to include visual and acoustic stimuli and evaluated two plate formats (24-well round and 96-well square). A range of known anxiogenics, anxiolytics, negative controls and known neurotoxicants (e.g., nicotine, chlorpyriphos) were tested to establish baseline responses and assay reliability. Thigmotaxis response was compared to general locomotion to test for assay sensitivity.

Results:

The 96-well square plate format offered optimal throughput and reproducibility. Exposure to positive control compounds resulted in significantly elevated/decreased thigmotactic behavior, consistent with anxiogenic/anxiolytic responses. Negative controls showed no behavioral alteration, supporting assay specificity. Comparison with general locomotion showed that thigmotaxis evaluation increased assay sensitivity.

Conclusions:

This thigmotaxis assay is a high-throughput and ethically viable approach for screening chemicals for neurotoxic potential. It effectively captures anxiety-related behaviors and serves as a promising NAM for inclusion in alternative developmental and adult neurotoxicity test batteries. Further validation under PARC is underway to support regulatory adoption.

Pollution with lead (Pb) constitutes a primary route for the contamination of foods like cow's milk (CM) or soya beverages (SBs), which are frequently consumed on a daily basis [1]. Even at low exposure levels, Pb is toxic; no level is safe, with effects seen at blood Pb concentrations of just 3.5 µg/dL [2], which is the current Blood Lead Reference Value of the Centers for Disease Control and Prevention. In alignment with this value, the US Food and Drug Administration (FDA) establishes an Interim Reference Level (IRL) for dietary Pb of 8.8 μg/day for females of childbearing age (FCBAs), considering the potential for fetal Pb exposure [3]. The objective of this work was to assess whether the exposure to Pb of FCBAs through the consumption of CM or SB brands available on the Portuguese market was higher than the FDA’s IRL. The lead levels of 14 CM and 14 SB brands were determined by Atomic Absorption Spectrophotometry. Three CM or SB consumption scenarios were considered: the estimated adult CM consumption in Portugal [4], and the minimum and maximum recommended CM consumption (MinC and MaxC) [5]. For the estimated CM intake, totalling 0.1 L/day, all the analysed samples exceeded the FDA’s IRL. However, for MinC (0.5 L/day), 42 and 21% of the CM and SB brands, respectively, surpassed the IRL; in this scenario, the mean Pb exposure from the 14 analysed CM brands was 9.66 µg/day, and the mean Pb exposure from the 14 SB brands was 6.72 µg/day. Regarding MaxC (0.75 L/day), 64 and 42% of the analysed CM and SB brands, respectively, surpassed the FDA’s IRL; the mean Pb exposure from the CM brands was 14.49 µg/day, and from the SB brands, it was 10.08 µg/day. Although CM constitutes a higher contributor to dietary Pb exposure than SBs, greater efforts must be made to control the exposure of FCBAs to Pb through the consumption of both food products.

Plastic products, such as bisphenol A (BPA), are widely released into the environment, causing adverse effects in aquatic organisms. In response to regulatory restrictions on BPA, structurally similar bisphenol analogues have been introduced as alternatives. However, these compounds often lack comprehensive toxicological evaluation and may pose comparable or even greater environmental risks.

This study investigated the toxicity of BPA, two BPA analogues (BPAP and BPAF), and one BPA monomer derivative (BADGE·2HCl) using zebrafish liver (ZFL) cell spheroids. ZFL spheroids were exposed to 5 µM for acute exposure (24 hours) and 1–5 µM for chronic exposure (6 days). Afterwards, metabolic activity, changes in gene expression and alterations in the lipidome were evaluated.

For acute exposure, upregulation of cyp1a expression was detected after exposure to BADGE·2HCl, suggesting activation of xenobiotic metabolism pathways. For chronic exposure, ATP levels decreased at 5 µM for BPAP, BPAF, and BADGE·2HCl. Notably, chronic exposure to BADGE·2HCl (1–5 µM) and BPA (5 µM) caused significant alterations in membrane lipid composition, predominantly affecting phosphatidylcholines, ether-linked phosphatidylcholines, and phosphatidylethanolamines, lipids essential for maintaining membrane integrity. Additionally, exposure to 5 µM BADGE·2HCl led to a substantial upregulation of ceramides and sphingomyelins, which are associated with mitochondrial dysfunction and apoptotic pathways.

In conclusion, BADGE·2HCl elicited the most pronounced alterations in lipid metabolism, followed by BPA. The results highlight the need for more rigorous testing and stricter regulatory measures to mitigate the potential environmental and health risks posed by these compounds. Moreover, this study highlights the utility of ZFL spheroids as a robust in vitro model for evaluating the chronic toxicity of environmental contaminants, offering a physiologically relevant alternative to traditional monolayer cultures.