Integrating aggregate exposure pathway and adverse outcome pathway for micro/nanoplastics: A review on exposure, toxicokinetics, and toxicity studies.

Micro/nanoplastics (MNPs) have emerged as a significant environmental concern due to their widespread distribution and potential adverse effects on human health and the environment. In this study, to integrate exposure and toxicity pathways of MNPs, a comprehensive review of the occurrence, toxicokinetics (absorption, distribution, and excretion [ADE]), and toxicity of MNPs were investigated using the aggregate exposure pathway (AEP) and adverse outcome pathway (AOP) frameworks. Eighty-five papers were selected: 34 papers were on detecting MNPs in environmental samples, 38 papers were on the ADE of MNPs in humans and fish, and 36 papers were related to MNPs toxicity using experimental models. This review not only summarizes individual studies but also presents a preliminary AEP-AOP framework. This framework offers a comprehensive overview of pathways, enabling a clearer visualization of intricate processes spanning from environmental media, absorption, distribution, and molecular effects to adverse outcomes. Overall, this review emphasizes the importance of integrating exposure and toxicity pathways of MNPs by utilizing AEP-AOP to comprehensively understand their impacts on human and ecological organisms. The findings contribute to highlighting the need for further research to fill the existing knowledge gaps in this field and the development of more effective strategies for the safe management of MNPs.

Alleviation of neurotoxicity induced by polystyrene nanoplastics by increased exocytosis from neurons.

Nanoplastics (NPs) are potentially toxic and pose a health risk as they can induce an inflammatory response and oxidative stress at cellular and organismal levels. Humans can be exposed to NPs through various routes, including ingestion, inhalation, and skin contact. Notably, uptake into the body via inhalation could result in brain accumulation, which may occur directly across the blood-brain barrier or via other routes. NPs that accumulate in the brain may be endocytosed into neurons, inducing neurotoxicity. Recently, we demonstrated that exposure to polystyrene (PS)-NPs reduces the viability of neurons. We have also reported that inhibiting the retrograde transport of PS-NPs by histone deacetylase 6 (HDAC6) prevents their intracellular accumulation and promotes their export in mouse embryonic fibroblasts. However, whether HDAC6 inhibition can improve neuronal viability by increasing exocytosis of PS-NPs from neurons remains unknown. In this study, mice were intranasally administered fluorescent PS-NPs (PS-YG), which accumulated in the brain and showed potential neurotoxic effects. In cultured neurons, the HDAC6 inhibitor ACY-1215 reduced the fluorescence signal detected from PS-YG, suggesting that the removal of PS-YG from neurons was promoted. Therefore, these results suggest that blocking the retrograde transport of PS-NPs using an HDAC6 inhibitor can alleviate the neurotoxic effects of PS-NPs that enter the brain.

Effect of Early-Life Exposure of Polystyrene Microplastics on Behavior and DNA Methylation in Later Life Stage of Zebrafish.

Microplastic contamination has received increasing attention in recent years, and concern regarding the toxicity of microplastics to the environment and humans has increased. In this study, we investigated the neurodevelopmental toxicity of polystyrene microplastics (PSMPs) in the zebrafish Danio rerio under different exposure scenarios. Zebrafish were exposed to PSMPs during embryonic stage and then allowed the fish to recover. The neurodevelopmental toxic responses were investigated using fish behavior and behavior-related gene expression. Early-life exposure to PSMPs did not alter fish behavior at the early stage; however, it led to hyperactivity later life stage. Generally, oxidative stress (i.e., sod2 and nrf2a)- and nervous system (i.e., slc6a4b, npy, and nrbf2)-related gene expression increased in all PSMPs-exposed fish. DNA hypomethylation was observed in fish challenged for a second time using the same PSMPs. Taken together, the current results imply that PSMPs have neurodevelopmental toxic potential when introduced early in life.

Stress Response of Mouse Embryonic Fibroblasts Exposed to Polystyrene Nanoplastics.

Polystyrene (PS) nanoplastic exposure has been shown to affect the viability of neuronal cells isolated from mouse embryonic brains. However, the viability of mouse embryonic fibroblasts (MEFs) was not affected although PS nanoplastics accumulated in the cytoplasm. It is currently unknown whether MEFs do not respond to PS nanoplastics or their cellular functions are altered without compromising viability. Here, we found that PS nanoplastics entered the cells via endocytosis and were then released into the cytoplasm, probably by endosomal escape, or otherwise remained in the endosome. Oxidative and inflammatory stress caused by intracellular PS nanoplastics induced the antioxidant response pathway and activated the autophagic pathway. However, colocalization of the autophagic marker LC3B and PS nanoplastics suggested that PS nanoplastics in the cytoplasm might interfere with normal autophagic function. Furthermore, autophagic flux could be impaired, probably due to accumulation of PS nanoplastic-containing lysosomes or autolysosomes. Intriguingly, the level of accumulated PS nanoplastics decreased during prolonged culture when MEFs were no longer exposed to PS nanoplastics. These results indicate that accumulated PS nanoplastics are removed or exported out of the cells. Therefore, PS nanoplastics in the cytoplasm affect cellular functions, but it is temporal and MEFs can overcome the stress caused by PS nanoplastic exposure.

Inhalation toxicity of polystyrene micro(nano)plastics using modified OECD TG 412.

Recently, there have been reports that many microplastics are found in the air, which has raised concerns about their toxicity. To date, however, only limited research has investigated the effects of micro(nano)plastics on human health, and even less the potential for inhalation toxicity. To fill this research gap, we investigated the potential inhalation toxicity of micro(nano)plastics using a modified OECD Guideline for Testing of Chemicals No. 412 '28-Day (subacute) inhalation toxicity study' using a whole-body inhalation system. Sprague-Dawley rats were exposed to three different exposure concentrations of polystyrene micro(nano)plastics (PSMPs), as well as control, for 14 days of inhalation exposure. After 14 days, alterations were observed on sevral endpoints in physiological, serum biochemical, hematological, and respiratory function markers measured on the samples exposed to PSMPs. However, no concentration-response relationships were observed, suggesting that these effects may not be definitively linked to exposure of PSMPs. On the other hand, the expression of inflammatory proteins (TGF-ß and TNF-α) increased in the lung tissue in an exposure concentration-dependent manner. The overall results indicate that 14-day inhalation exposure of PSMPs to rats has a more pronounced effect at the molecular level than at the organismal one. These results suggest that if the exposure sustained, alterations at the molecular level may lead to subsequent alterations at the higher levels, and consequently, the health risks of inhalation exposed micro(nano)plastics should not be neglected.

Development of AOP relevant to microplastics based on toxicity mechanisms of chemical additives using ToxCast™ and deep learning models combined approach.

Various additives are used in plastic products to improve the properties and the durability of the plastics. Their possible elution from the plastics when plastics are fragmented into micro- and nano-size in the environment is suspected to one of the major contributors to environmental and human toxicity of microplastics. In this context, to better understand the hazardous effect of microplastics, the toxicity of chemical additives was investigated. Fifty most common chemicals presented in plastics were selected as target additives. Their toxicity was systematically identified using apical and molecular toxicity databases, such as ChemIDplus and ToxCast™. Among the vast ToxCast assays, those having intended gene targets were selected for identification of the mechanism of toxicity of plastic additives. Deep learning artificial neural network models were further developed based on the ToxCast assays for the chemicals not tested in the ToxCast program. Using both the ToxCast database and deep learning models, active chemicals on each ToxCast assays were identified. Through correlation analysis between molecular targets from ToxCast and mammalian toxicity results from ChemIDplus, we identified the fifteen most relevant mechanisms of toxicity for the understanding mechanism of toxicity of plastic additives. They are neurotoxicity, inflammation, lipid metabolism, and cancer pathways. Based on these, along with, previously conducted systemic review on the mechanism of toxicity of microplastics, here we have proposed potential adverse outcome pathways (AOPs) relevant to microplastics pollution. This study also suggests in vivo and in vitro toxicity database and deep learning model combined approach is appropriate to provide insight into the toxicity mechanism of the broad range of environmental chemicals, such as plastic additives.

Identification of adverse outcome pathway related to high-density polyethylene microplastics exposure: Caenorhabditis elegans transcription factor RNAi screening and zebrafish study.

To gain insight into the human health implications of microplastics, in this study, we investigated the possible mechanisms affecting the toxicity of high-density polyethylene (HDPE) in the nematode Caenorhabditis elegans using RNAi screening and a bioinformatics-based unbiased approach. The candidate pathways identified from C. elegans study were also confirmed using vertebrate model, zebrafish, Danio rerio and human relevance was then inferred using Comparative Toxicogenomics Database (CTD) analysis. Prior to evaluating the toxicity, label-free Raman mapping was conducted to investigate whether or not the organisms could uptake HDPE. C. elegans transcription factor RNAi screening results showed that the nucleotide excision repair (NER) and transforming growth factor-beta (TGF-ß) signaling pathways were significantly associated with HDPE exposure, which was also confirmed in zebrafish model. Gene-disease interaction analysis using the CTD revealed the possible human health implications of microplastics. Finally, based on this finding, related AOPs were identified from AOP Wiki (http://aopwiki.org), which are "Peroxisome proliferator-activated receptors γ inactivation leading to lung fibrosis" and "AFB1: Mutagenic Mode-of-Action leading to Hepatocellular Carcinoma". Further studies are needed for the validation of these AOPs with various microplastics.

Neurotoxic potential of polystyrene nanoplastics in primary cells originating from mouse brain.

Polystyrene (PS) and chemically modified compounds in the PS family have long been used in commercial and industrial fields. However, it is poorly understood whether nanoscale-PS microplastic or PS nanoplastic exposure leads to perturbations in fundamental cellular functions, such as proliferation, differentiation, and apoptosis. Herein, we cultured three types of primary cells, including mouse embryonic fibroblasts (MEFs), mixed neuronal cells isolated from embryonic cortex, and cortical astrocytes, and investigated the effects of their exposure to PS nanoplastics with a 100 nm diameter. Although PS nanoplastic exposure did not affect the viability of MEFs or astrocytes, it significantly reduced the viability of mixed neuronal cells. Consistent with the observed effect on cellular viability, levels of the apoptosis marker, cleaved caspase-3, were elevated exclusively in mixed neuronal cells. To investigate whether cells uptake PS nanoplastics into the cytoplasm, we exposed MEFs and neurons to fluorescent PS latex beads and monitored fluorescence over time. We found that PS nanoplastics were deposited and accumulated in the cytoplasm in a concentration-dependent manner. Although astrocytes were not apoptotic upon exposure to PS nanoplastics, they underwent reactive astrocytosis, with increased levels of lipocalin-2 and proinflammatory cytokines. Therefore, our findings suggested that the vulnerability of cells to the deposition and accumulation of PS nanoplastics in the cytoplasm was dependent on cell type. Furthermore, based on our data from primary cells originating from mouse brains, we suggest that reactive astrocytosis may contribute to the neuronal apoptosis seen in defective neurons with PS nanoplastics accumulated in the cell body.

Adverse outcome pathways potentially related to hazard identification of microplastics based on toxicity mechanisms.

Increasing concern over microplastics has recently brought increased attention to studies on microplastic toxicity. Here, we conduct a systematic review on toxicity of microplastics that focuses on identifying data gaps in the mechanisms of microplastic toxicity. We observe that microplastic toxicology research thus far has focused on ecotoxicity using apical endpoints and only a few studies deal with toxicity mechanisms. Based on this review, we propose putative Adverse Outcome Pathways (AOPs) applicable to microplastic management to understand microplastic toxicity. We matched toxicity mechanisms and apical endpoints to a key event (KE) and adverse outcome (AO) information from the AOP Wiki. Overall, our results suggest that the molecular initiating event (MIE) was reactive oxygen species (ROS) formation and the AO was increased mortality, decreased growth and feeding, and reproduction failure. However, there are a limited number of studies on toxicity mechanisms of microplastics and, therefore, evidence concerning the relationship between KEs is not sufficient. Clearly, more studies on toxicity mechanisms are required to fill these gaps in data. This study also suggests that the AOP framework is a suitable tool to integrate existing data from various literature sources and can identify data gaps in microplastic toxicity mechanisms.

Ecotoxicity of bare and coated silver nanoparticles in the aquatic midge, Chironomus riparius.

Although sediment is generally considered to be the major sink for nanomaterials in aquatic environments, few studies have addressed the ecotoxicity of nanomaterials in the presence of sediment. In the present study, the ecotoxicity of silver nanoparticles (AgNPs) with a range of organic coatings was examined in a freshwater sediment-dwelling organism, Chironomus riparius, using acute and chronic ecotoxicity endpoints, including molecular indicators. The toxicity of AgNPs coated with different organic materials, such as polyvinylpyrrolidone, gum arabic, and citrate, to C. riparius was compared with that of bare-AgNPs and AgNO3 (ionic silver). Total silver concentration was also measured to monitor the behavior of the AgNPs in water and sediment and to determine how ion dissolution affects the toxicity of all AgNPs. The coated- and bare-AgNPs caused DNA damage and oxidative stress-related gene expression. In addition, the bare-AgNPs and AgNO3 had a significant effect on development and reproduction. The surface coatings generally mitigated the toxicity of AgNPs to C. riparius, which can be explained by the reduced number of ions released from coated-AgNPs. Citrate-AgNPs caused the most significant alteration at the molecular level, but this did not translate to higher-level effects. Finally, comparing previously conducted studies on AgNP-induced gene expression without sediments, the authors show that the presence of sediment appears to mitigate the toxicity of AgNPs.

A systems toxicology approach to the surface functionality control of graphene-cell interactions.

The raised considerable concerns about the possible environmental health and safety impacts of graphene nanomaterials and their derivatives originated from their potential widespread applications. We performed a comprehensive study about biological interaction of grapheme nanomaterials, specifically in regard to its differential surface functionalization (oxidation status), by using OMICS in graphene oxide (GO) and reduced graphene oxide (rGO) treated HepG2 cells. Differential surface chemistry (particularly, oxidation - O/C ratio) modulates hydrophobicity/philicity of GO/rGO which in turn governs their biological interaction potentiality. Similar toxic responses (cytotoxicity, DNA damage, oxidative stress) with differential dose dependency were observed for both GO and rGO but they exhibited distinct mechanism, such as, hydrophilic GO showed cellular uptake, NADPH oxidase dependent ROS formation, high deregulation of antioxidant/DNA repair/apoptosis related genes, conversely, hydrophobic rGO was found to mostly adsorbed at cell surface without internalization, ROS generation by physical interaction, poor gene regulation etc. Global gene expression and pathway analysis displayed that TGFß1 mediated signaling played the central role in GO induced biological/toxicological effect whereas rGO might elicited host-pathogen (viral) interaction and innate immune response through TLR4-NFkB pathway. In brief, the distinct biological and molecular mechanisms of GO/rGO were attributed to their differential surface oxidation status.

Comparative toxicity of silver nanoparticles on oxidative stress and DNA damage in the nematode, Caenorhabditis elegans.

This study examined the effects of polyvinylpyrrolidone (PVP) surface coating and size on the organismal and molecular toxicity of silver nanoparticles (AgNPs) on the nematode, Caenorhabditis elegans. The toxicity of bare AgNPs and 8 and 38 nm PVP-coated AgNPs (PVP8-AgNPs, PVP38-AgNPs) were compared. The toxicity of AgNO3 was also tested because ion dissolution and particle-specific effects are often important characteristics determining Ag nanotoxicity. Comparative toxicity across AgNO3 and the three different types of AgNPs was first evaluated using a C. elegans mortality test by a direct comparison of the LC50 values. Subsequently, mutant screening followed by oxidative stress, mitochondrial toxicity and DNA damage assays were carried out at equitoxic (LC10 and LC50) concentrations to further assess the toxicity mechanism of AgNO3 and AgNPs. AgNO3 and bare AgNPs had similar toxicities, whereas PVP coating reduced the toxicity of the AgNPs significantly. Of the PVP-AgNPs, the smaller NPs were more toxic. Different groups of mutants responded differently to AgNO3 and AgNPs, which indicates that their toxicity mechanism might be different. AgNO3 and bare AgNPs induced mitochondrial membrane damage. None of the silver materials tested caused detectable polymerase-inhibiting DNA lesions in either the nucleus or mitochondria as measured by a quantitative PCR assay, but AgNO3, bare AgNPs and PVP8-AgNPs induced oxidative DNA damage. These results show that coatings on the AgNPs surface and the particle size make a clear contribution to the toxicity of the AgNPs, and oxidative stress-related mitochondrial and DNA damage appear to be potential mechanisms of toxicity.