Effects of exposure to nano-plastic drinking during pregnancy on cognitive related proteins in offspring of SD rats

ABSTRACT

Nanoplastics, an emerging contaminant, pose risks to fetal neural development, transferring from mother to offspring via placenta and breast milk. Assessing these risks, hippocampal CA3 samples from nanopolystyrene-exposed rat offspring were proteomically analyzed. Findings revealed reduced expression of neural developmental proteins (KIF21A, STMN2, DMTN, DLG1) and increased inhibitory proteins (PZP, α-2M, FN1, SERPINA1, ALOX15) in the hippocampus. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis highlighted ferroptosis enrichment, validated by significantly expressed ALOX15 and TF proteins. These alterations suggest nanopolystyrene’s potential contribution to cognitive deficits and neurodevelopmental disorders, emphasizing its hazardous impact on neural development. This study provides novel insights into nanopolystyrene’s risks.

KEYWORDS:

• Nanopolystyrene
• proteomic analysis
• transgenerational neurotoxicity
• hippocampal

Introduction

Nanoplastics are of increasing concern worldwide as emerging pollutants because they are more easily ingested by living organisms [1]. Long-term and continuous exposure to these particles, occurring through fruits, vegetables, and drinking water, is a part of everyday life for humans [2,3]. However, the health effects of Nanoplastics on humans still remain largely unclear [4].

Previous studies have demonstrated accumulation of nanoplastics in the brain of aquatic organisms produces oxidative stress and inhibits acetylcholinesterase activity leading to neurotoxicity to aquatic organisms [5–9]. A recent in vivo experiment revealed the bioaccumulation of polystyrene micro-nano plastics in rodent organs such as the liver, spleen, kidneys, brain, lungs, and intestines, exhibiting a range of adverse effects such as reproductive toxicity and transgenerational toxicity [10]. However, fewer studies reported the transgenerational neurotoxicity of Nanoplastics in mammals. It has been previously established that the neurotoxic effects of these particles can be transmitted from parent to offspring in Caenorhabditis elegans and can also be transferred directly to mammalian fetal organs such as the liver and brain via the placenta and breast milk [6,11,12]. This transfer has been associated with cognitive impairments and other neurodevelopmental disorders in offspring.

The hippocampus of the brain, which includes areas CA1-to-CA4, is integral to learning and memory processes [13]. The CA3 area, consisting of pyramidal neurons, regulates cognitive functions such as learning and memory [14]. The hippocampal CA3 region has a distinctive role in memory processing, epilepsy susceptibility, and neurodegenerative changes, thereby significantly contributing to overall hippocampal function [15,16]. Studies have revealed that nanopolystyrene can decreased expression of neurotransmitter-related genes and reduce superoxide dismutase enzyme levels in the rat hippocampus, leading to neuronal damage and subsequent learning and memory impairments [17]. However, there is limited literature discussing the damage mechanisms of Nanoplastics on the mammalian hippocampus. Fetal development is a sensitive period for neurodevelopment, and exposure to environmental pollutants can potentially result in irreversible damage on the nervous system during this time.

Proteomics is an important tool because it is capable of reflecting the interactions between different proteins and the roles they play in an organism [18]. Thus, proteomics is a large-scale technique for the study of proteins in health and disease from a given biological samples; therefore, label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) with high sensitivity and specificity are appropriate tools for studying protein changes in biological samples [19,20].

The aim of this study was to explore whether nanoplastics can be passed from mother to offspring in mammals, leading to neurodevelopmental toxicity in the offspring and providing new insights into the prevention of neurological disorders in children. Thus, the samples of CA3 region in the hippocampus of the offspring rats were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS), followed by statistical analyses of the differential proteins, using 4D-Label-free quantitative proteomics technique.

Materials and methods

Construction of the animal model and sample collection

Research and animal care procedures were approved by the animal and human use in research committee of the Tianjin Institute of Enviormental and Occupational Medicine (IACUC of AMMS-04-2022-015), and all animal experiments were performed in accordance with relevant guidelines and regulations. Healthy pregnant Sprague-Dawley (SD) rats around 350 g at 5 months of age were procured from SiPeiFu (BeiJing) Biotechnology Co., Ltd. The rats were housed for general feeding that were accommodated in a specific pathogen-free (SPF) animal room maintained at 23 ± 2°C with a 12-h light/dark cycle and a relative humidity between 40% and 60%.

Eight healthy pregnant SD rats were randomly divided into experimental and control groups. From the initial day of pregnancy, rats in the experimental group were exposed to 2.5 mg/kg/day of nanopolystyrene (PS-NPs) (50 nm, Beijing Zhongkeleiming Technology Co., Ltd) through gavage, continuing until weaning on the 21st day after birth，altogether 43 days. The control group received ultra-pure water. Each group produced 9 to 13 offspring per female rat, and daily observations revealed no deaths of offspring in rearing. Two days post-weaning, the pups were euthanized via cervical dislocation, and the hippocampal CA3 region was harvested for subsequent label-free proteomics analysis (Figure 1).

Figure 1. Schematic diagram of the animal modeling for the proteomics experiment on the hippocampal CA3 region.

Note: P1 refers to the maternal rat; F1 refers to the offspring.

Protein extraction procedures and quantification methods

Hippocampal CA3 brain tissue was combined with an adequate amount of SDT lysis buffer (4% Sodium dodecyl sulfate, 100 mM Tris-HCl, pH 7.6) in a 2 mL centrifuge tube pre-filled with an appropriate amount of quartz sand (extra ¼-inch ceramic bead MP6540–424 was included for tissue samples). Homogenization was performed using an MP homogenizer (24 × 2, 6.0 M/S, 60s, twice), followed by ultrasonication (2,000 W, working for 30 s, resting for 30 s, for 10 cycles) and treatment in a boiling water bath for 8 min. After centrifugation at 12,600 × g for 20 min, the supernatant was filtered through a 0.22 µm filter, and the filtrate was collected. Protein quantification was performed using the Bicinchoninic Acid Assay (BCA) method. The samples were aliquoted and stored at −80°C.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) separation

The samples (20 µg of protein) were combined with 5× loading buffer, heated for 5 min, and then separated on a 4% to 20% SDS-PAGE gel at a constant current of 180 V for 45 min. Protein bands were visualized by staining with Coomassie Blue R-250.

Filter-assisted sample preparation (FASP) digestion

Dithiothreitol (DTT, final concentration of 40 mM) was added to each sample and mixed at 37°C and 600 rpm for 1.5 h. The samples were cooled to room temperature, followed by the addition of iodoacetamide (IAA) to a final concentration of 20 mM for the reduction and blocking of cysteine residues, and subsequent incubation in darkness for 30 min. Samples were then transferred to a filter, which was rinsed thrice with 100 μL of Urea lysis buffer (8 M urea，150 mM Tris‐HCl，pH 8.0) buffer, and then twice with 100 μL of 25 mM NH₄HCO₃ buffer. Finally, trypsin was added to the samples (trypsin: protein (wt/wt) ratio of 1:50), the protein suspension was digested overnight at 37°C, and peptides were collected as the filtrate. The peptides were desalted on a C18 column (Empore™ SPE Cartridges C18 (standard density), bed I.D. 7 mm, volume 3 mL, Sigma), concentrated by vacuum centrifugal evaporation, and re-dissolved in 40 µL of 0.1% (v/v) formic acid. The peptide content was determined using an Ultraviolet spectrophotometer by measuring the spectral density at 280 nm using an extinction coefficient of 1.1 for a solution of 0.1% (g/L) (calculated based on the frequencies of tryptophan and tyrosine in vertebrate proteins).

Mass spectrometry

The peptide fractions were analyzed using a timsTOF Pro mass spectrometry (Bruker) coupled to Nanoelute (Bruker). Peptides were loaded onto a C18 reversed-phase analytical column (Thermo Scientific Easy-Column, Length: 25 cm, inner diameter: 75 μm, C18 resin: 1.9 μm) in 95% Buffer A (0.1% formic acid aqueous solution), and separated using a linear gradient of Buffer B (99.9% acetonitrile and 0.1% formic acid) at a flow rate of 300 nL/min. The mass spectrometer was operated in the positive ion mode with an applied electrospray voltage of 1.5 kV. Precursors and fragments were analyzed in the m/z 100–1,700 mass range on the Time-of-Flight (TOF)detector, with a dynamic exclusion duration of 24 s, and the timsTOF Pro operated in the parallel accumulation-serial fragmentation (PASEF) mode. The PASEF mode data acquisition was based on the following parameters: Ion mobility coefficients (1/K0) of 0.6–1.6 Vscm²; 1 MS and 10 MS/MS PASEF scans. The instrument was set to operate in peptide identification mode.

Statistical analysis for proteomics approach

The CA3 region of the hippocampus of the offspring rats was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The LC-MS/MS raw data acquired for samples from hippocampus CA3 region from the offspring rats were combined and searched separately using The MaxQuant software version 1.6.14 (http://coxdocs.org/doku.php?id=maxquant:common:download_and_installation) for identification and quantitation analysis. Related parameters and instructions are as follows: Max missed cleavages = 2; Main search= ± 6 ppm; First search= ± 20ppm; MS/MS tolerance = 20ppm.

The resulting data processing was submitted to bioinformatic analysis. Initially, Cluster 3.0 (http://bonsai.hgc.jp/~mdehoon/software/cluster/software.htm) and Java Treeview software (http://jtreeview.sourceforge.net) were used to performing hierarchical clustering analysis for samples. Euclidean distance algorithm for similarity measure and average linkage clustering algorithm (clustering uses the centroids of the observations) for clustering were selected when performing hierarchical clustering. Next, Protein sequences of samples are searched using the InterProScan software to identify protein domain signatures from the InterPro member database Pfam. Subsequently, the protein sequences of the selected differentially expressed proteins were locally searched using the NCBI BLAST+client software (ncbi-blast-2.2.28±win32.exe) and InterProScan to find homologue sequences, then gene ontology (GO) terms were mapped and sequences were annotated using the software program Blast2GO. Following annotation steps, the studied proteins were blasted against the online Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://geneontology.org/) to retrieve their KEGG orthology identifications and were subsequently mapped to pathways in KEGG.

Moreover, enrichment analysis of differential proteins in samples were applied based on the Fisher’ exact test, considering the whole quantified proteins as background dataset. Benjamini- Hochberg correction for multiple testing was further applied to adjust derived p-values. And only functional categories and pathways with p-values under a threshold of 0.05 were considered as significant. Finally, we processed to the protein – protein interaction (PPI) information of the studied proteins also in IntAct molecular interaction database (http://www.ebi.ac.uk/intact/) by their gene symbols or STRING software (http://string-db.org/). Protein validation was analyzed using Graph Pad Prism 9.5.1 (GraphPad Software, U.S.A.). Comparisons between two experimental groups were performed using unpaired twotailed t-tests and the standard for determining statistical significance was set at *p < 0.05 or **p < 0.01.

Results and conclusion

Effects of nanopolystyrene exposure on the protein expression profile in hippocampal CA3 of offspring rats

To clarify the impact of nanopolystyrene on the CA3 region of the hippocampus of offspring rats, a label-free proteomics study was performed. The mass spectrometry generated 349,156 spectra, identifying 61,440 peptides, among which 53,263 were unique. A total of 6,451 proteins were identified and quantified (quantified proteins were defined as those presenting quantitative information in at least one comparison group) (Figures 2). The experimental and control groups exhibited a significant difference in 170 proteins (Fold Change > 1.5, P < 0.05), with 128 upregulated and 42 downregulated proteins (Figure 3). The heatmap results showed higher expression of proteins inhibiting neural development and lower expression of proteins affecting neural function in the nanopolystyrene intervention than in the control group (Figures 4).

Figure 2. Proteins were identified and quantified. a. Histogram of proteomic identification and quantitative results of the hippocampal CA3 region. Note: total spectrum: total number of MS/MS spectra; matched spectrum (PSM, peptide spectrum match): total number of spectra matching the database; peptides: total number of peptides; unique peptides: total number of unique peptides; identified proteins: total number of identified proteins, also known as protein groups; quantified proteins: quantifiable proteins, specifically those for which intensity values are available in more than half of the biological replicates in at least one comparison group. b. The Venn diagram of proteins identified in all replicates of the control group. c. Venn diagram of proteins identified in all replicates of the experimental group. d. The Venn diagram of proteins identified between groups.

Figure 3. Volcano plot of significantly differentially expressed proteins in the hippocampal CA3 region.

Note: The horizontal axis represents the fold change (log2 transformed), and the vertical axis represents the P value which reflects the significance of the differences (log10 transformed). The red dots in the figure represent significantly upregulated proteins; green dots represent significantly downregulated proteins, and grey dots represent proteins that did not exhibit differential changes.

Figure 4. Cluster analysis of significantly differentially-expressed proteins. a. Cluster analysis of significantly differentially-expressed proteins in the hippocampal CA3 region. Note: the results of hierarchical clustering are depicted in a heatmap where each column represents a sample group (the horizontal axis represents sample information), and each row corresponds to a protein (the vertical axis represents significantly differentially-expressed proteins). Expression levels of these proteins across different samples are standardized by the z-score method and shown in various colors on the heatmap. Red indicates upregulated proteins, blue indicates downregulated proteins, and the gray sections denote an absence of protein quantification information. b. Protein-protein interaction network of differentially-expressed proteins in the hippocampal CA3 region. Note: the circles in the figure represent differentially-expressed proteins, while the lines represent protein-protein interactions. The circle color represents the differential expression of proteins, with upregulation shown in blue and downregulation shown in yellow. The circle size indicates the degree of interaction, i.e. the number of proteins directly interacting with a particular protein.

Gene ontology (GO) analysis of differentially-expressed proteins in the hippocampal CA3 region of offspring rats exposed to nanopolystyrene

The GO analysis results exhibited that significantly upregulated proteins were primarily involved in cellular processes and biological regulation responses. They were mainly expressed within cells, with secondary expression in cell regions and organelles, and potentially participated in adhesive functions and catalytic activities (Figure 5). The main biological processes enriched by the differentially-expressed proteins are defense responses, regulation of protein hydrolysis, and inflammatory responses (Figure 6).

Figure 5. GO annotation statistics of differentially-expressed proteins in the hippocampal CA3 region.

Note: The x-axis in the figure represents the second-level GO functional annotation information, which includes biological process, cellular component, and molecular function, distinguished by red, yellow, and blue, respectively. The y-axis indicates the number of differentially-expressed proteins under each functional category.

Figure 6. Bubble plot of GO functional enrichment under the classification of the biological process of enrichment of differentially-expressed proteins in the hippocampal CA3 region.

Note: The x-axis represents the enrichment factor (rich factor ≤ 1), reflecting the ratio of differentially-expressed proteins annotated to a particular GO functional category to all identified proteins within that category. The y-axis indicates the statistical analysis results of differentially-expressed proteins under each GO functional category. In the diagram, the color of the bubble indicates the significance of the enrichment of the GO functional category, i.e. the P-value calculated via Fisher’s exact test. The color gradient represents the P-value (expressed as -log10), with a color closer to red denoting a smaller P-value and a higher level of significance for the enrichment of the corresponding GO functional category. a. The enrichment analysis of the biological processes of differentially-expressed proteins in the hippocampal CA3 region; b. The enrichment analysis of biological processes of differentially-expressed proteins that may impact neural development.

Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of differentially-expressed proteins in the hippocampal CA3 region of offspring rats exposed to nanopolystyrene

The KEGG pathway analysis revealed that the differentially-expressed proteins were primarily involved in processes such as neuroactive ligand-receptor interaction, arachidonic acid metabolism, ferroptosis, and regulation of the actin cytoskeleton (Figure 7).

Figure 7. Bubble plot of the KEGG pathway enrichment analysis for differentially-expressed proteins in the hippocampal CA3 region.

Note: The x-axis represents the enrichment factor (rich factor ≤ 1), reflecting the ratio of differentially-expressed proteins annotated to a particular KEGG pathway category to all identified proteins within that category. The y-axis indicates the statistical analysis results of differentially-expressed proteins under each KEGG pathway category. In the diagram, the color of the bubble indicates the significance of the enrichment of the KEGG pathway category, i.e. the P-value calculated via Fisher’s exact test. The color gradient represents the P-value (expressed as -log10), with a color closer to red denoting a smaller P-value and a higher level of significance for the enrichment of the corresponding KEGG metabolic pathway category. a. The KEGG pathway analysis of differentially-expressed proteins in the hippocampal CA3 region; b. The KEGG pathway analysis of differentially-expressed proteins that may impact neural development.

Analysis of the functions of significantly differentially-expressed proteins

Nanopolystyrene intervention resulted in an upregulation of pregnancy-zone protein (PZP), alpha-2-macroglobulin (α-2 M), and fibronectin 1 (FN1) in the hippocampal CA3 region of offspring rats. This change may be associated with biological processes related to neural cell growth and differentiation. PZP and α-2 M, members of the α-2 M gene family, can produce monoamine-activated (MA) macroglobulins. The MA forms of human α-2 M and PZP and rat α-2 M have been shown to inhibit neuron activity promoted by various neurotrophic proteins [21]. PZP, a multifunctional carrier of neurotrophic factors, along with MA-PZP and MA-α-2 M, can inhibit neurite outgrowth and Trk-mediated signal transduction [22]. Another study has found high PZP expression near senile plaques in the brains of Alzheimer’s patients, suggesting a potential link between PZP and plaque formation [23]. Fibronectin 1 (FN1) is a glycoprotein involved in the processes of cell adhesion and migration, and the FN1 axis is shown to promote nerve cell regeneration [24]. The elevated expression of these proteins indicates potential neural damage in the experimental group.

In the experimental group, the hippocampal expression of serpin family A member 1 (SERPINA1) increased, which is potentially associated with the biological processes of synaptic transmission in the nervous system. SERPINA1 inhibits agrin cleavage by suppressing neurotrypsin activity, thereby reducing the production of agrin-22 necessary for synaptic stability and indirectly disrupting synaptic homeostasis [25].

An increase in arachidonate 15-lipoxygenase (ALOX15) expression may be associated with the biological process of cell ferroptosis. ALOX15 plays a key role in oxidative stress and the pathologic process of neuronal death [26]. ALOX15, which is highly expressed in the cerebral cortex, hippocampus, prefrontal cortex, and olfactory bulb, is a vital enzyme in the arachidonic acid cascade reaction, participating in neuronal cell death [27]. ALOX15 can oxidize unsaturated fatty acids, both membrane-bound and in the form of phospholipids, thus damaging the cell membrane and organelles [28].

The nanopolystyrene intervention resulted in the downregulation of kinesin family member 21A (KIF21A) and stathmin 2 (STMN2) in the hippocampus of offspring rats, potentially related to the biological process of synaptic development. KIF21A, a neuronal kinesin family member, is essential for the neuronal transport of cellular components along axonal and dendritic microtubules via kinesins and dyneins [29]. Downregulation of KIF21A may impact synaptic functions. Stathmin family proteins regulate tubulin assembly and breakdown, thereby affecting microtubule dynamics. STMN2, which is highly expressed in motor neurons, plays a key role in axon maintenance and is significant in neurodegenerative diseases [30]. STMN2 reduction can lead to neuronal axon injury, which is a marker of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) [31].

The hippocampus of offspring rats showed a decrease in dematin actin-binding protein (DMTN) expression, which may be associated with the biological process of cell proliferation. DMTN can suppress glioblastoma multiforme (GBM) proliferation and invasion by influencing cell cycle regulation and actin remodeling. Studies have demonstrated that DMTN expression in gliomas is significantly lower than in normal brain tissue, suggesting its inhibitory effect on tumor proliferation and migration [32]. The downregulation of DMTN suggests a higher potential for brain tumor occurrence in the experimental than in the control group.

The experimental group showed a downregulation of the MAGUK scaffold protein discs large (DLG1), which may be associated with biological processes of neuronal development and synaptic regulation. DLG1 serves as a molecular scaffold in the development and regulation of neuronal and immune synapses and is expressed in neurons, lymphocytes, and epithelial cells [33]. In neuronal synapses, DLG1 binds to subtypes of glutamate receptors and is responsible for their transportation to the plasma membrane [34].

Discussion

Previous studies have suggested that micro-nano plastics alter physiological indicators associated with glucose and lipid metabolism in the serum and liver of ICR mice, as well as in their F1 and F2 offspring [35]. However, studies exploring the effect of nanoplastics on the neuronal development of offspring rats are limited. Thus, to explore the potential association between nanoplastics and neurodevelopmental disorders, a model was established in which pregnant and lactating rats were exposed to nanopolystyrene. In the study, differential protein expression in the hippocampal CA3 region due to Nano polystyrene exposure in offspring rats was analyzed using label-free LC-MS/MS. Yudhiakuari Sincihu et al. have shown that nanoplastics elevate the expression of oxidative stress-related proteins in rat hippocampal neurons [17]. However, the work reveals a significant reduction in the expression of proteins related to neuronal development (DMTN， DLG1 and MAPT) and function (KIF21A, ADCY8, STMN2, HTR1B, SHISA9, NTNG1, SYBU, and CAMK2G) in the hippocampal CA3 region of offspring rats post-intervention (Figure 8). Among these, MAPT and TAU represent two critical microtubule-associated proteins in the vertebrate nervous system, promoting microtubule assembly and stability, and potentially contributing to the establishment and maintenance of neuronal polarity [36]. The AMPA receptor is an essential medium for neurotransmission in the central nervous system, and SHISA9, a transmembrane protein in the AMPA receptor-associated protein complex, plays a crucial role in regulating central neuronal synaptic plasticity [37]. Cognitive impairment and decline in rats have been linked to reduced SYBU expression, suggesting its significant predictive value in dementia diseases such as Alzheimer’s [38]. These findings are consistent with those of a previous study that micro-nano plastics cause brain developmental abnormalities, leading to neuronal dysfunction and cognitive deficits [12]. In this study, neuronal developmental abnormalities were predicted by differences in the protein level. The work also observed that nanopolystyrene intervention led to significant expression of PZP and α-2 M. A previous study has identified PZP and α-2 M as multifunctional binding carriers of neurotrophic factors with neuroinhibitory effects [21], which is consistent with the present study findings.

Figure 8. Schematic representation of the findings of nanopolystyrene action on hippocampal CA3.

Cell death is critical in mammalian development and is closely linked to other biological processes [39]. Ferroptosis is a distinctive form of iron-dependent non-apoptotic cell death associated with neuronal damage [40]. Inflammation serves as a primary indicator of ferroptosis [41], involving inflammatory factors related to immune responses and neuroinflammation. In the enrichment data of our differentially-expressed proteins across diverse biological processes, the proteins differing between the experimental and control groups are predominantly associated with processes such as agrin-dependent defense, regulation of protein hydrolysis, and inflammatory responses. However, emerging evidence suggests that Ferroptosis is caused by polyunsaturated fatty acids attacking the cell membrane [39]. Our KEGG pathway analysis revealed that the differentially-expressed proteins were primarily enriched in neuroactive ligand-receptor interactions, arachidonic acid metabolism, ferroptosis, and the regulation of the actin cytoskeleton. Among them, enrichment of the ferroptosis pathway due to the upregulation of two proteins, ALOX15and transferrin (TF). Validation by western bolt was found to be consistent with the study and ALOX15 and TF were significantly expressed in the experimental group compared to the control group (Figure 9). This suggests that nanopolystyrene may lead to the discovery of ferroptosis. These findings are consistent with the study result of Liu Xiu et al., which suggests that exposure to micro-nano plastics can trigger cognition-related brain iron metabolism and exacerbate cognitive impairment [42].

Figure 9. Immunoblotting and quantification of ferroptosis-related proteins. *p < 0.05, **p < 0.01 vs. The control group.

Although the differentially-expressed proteins in the study exert an effect on neural system development, it is important to note that proteins can exhibit species variability. While these findings suggest that nanopolystyrene may potentially cause cognitive deficits in offspring rats, the study still lacks direct behavioral and testing evidence to substantiate this claim, necessitating further investigation.

Conclusion

In summary, the study firstly provides the proteomic analysis of transgenerational neurotoxicity in rat hippocampus due to exposure to nanopolystyrene. First, nanopolystyrene could influence the growth and development of neurons in the hippocampal CA3 region, impair neuronal function, and potentially induce neuronal cell death using by differentially-expressed proteins analysis. Secondly, the work discovered that nanopolystyrene could potentially promote neuronal ferroptosis, thereby impacting neurodevelopment. These findings shed light on the transgenerational neurotoxicity of nanopolystyrene at the protein level, which provide a new insight for investigating the neurodevelopmental toxicity of nanopolystyrene.

Author Contribution

J.C. carried out data analysis, participated in the the conception of the thesis and drafted the manuscript. Y.Z. carried out Data visualization. X.L. designed experiments. K.L., H.L., W.L., Y.S. and Z.X. provided laboratory support. L.Y., L.T., and B.L. conceived of the study, coordination and helped to draft the manuscript. All authors read and approved the final manuscript.

Acknowledgments

The authors would like to thank Dr. Bencheng Lin, Dr. Lei Tian and Dr. Licheng Yan for their Ideation and writing assistance.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The data underlying this article are available in the article and in its online supplementary material. If you can’t find it, contact the corresponding author.