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. 2026 Feb 12;39(3):293–304. doi: 10.1021/acs.chemrestox.5c00308

The Mechanism of Combined Exposure of Polystyrene Microplastics and Cadmium Inducing Hepatic Injury through the Modulation of PI3K/AKT/mTOR-Mediated Autophagy

Shuting Li †,‡,§, Qian Zhang †,‡, Yue Hao †,‡, Xiaojing Liu ∥,⊥, Yansheng Li ∥,⊥, Lingjun Yan †,‡, Guowei Pan †,‡,*, Wei Sun †,‡,*
PMCID: PMC12997244  PMID: 41677865

Abstract

Recent studies indicate that microplastics and nanoplastics (MNPs) act as key vectors for contaminants including cadmium (Cd). However, the bioavailability induced by their interaction remains controversial. Since both MNPs and Cd primarily accumulate in the liver after ingestion by organisms, hepatotoxicity induced by coexposure to MNPs (100 mg/kg body weight (BW)), 100 nm and 1 μm polystyrene (PS), and Cd (5 mg/kg BW) was examined in this study. Single or combined exposure models were established, and gavage was performed 5 times a week for 5 weeks. We observed that polystyrene (PS) accumulated in the mice liver. In comparison to the control group, all exposure groups exhibited significantly increased serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, altered hepatic antioxidant enzyme activities, decreased P62 protein expression, and elevated Beclin-1 expression and LC3II/I ratios, indicating that PS alone or in combination with Cd disrupted liver structures and induced excessive autophagy and oxidative damage. Specifically, the 1 μm PS group induced significantly stronger hepatotoxic effects than the 100 nm PS group. In contrast, for 100 nm PS, although it was less toxic when administered alone, it significantly enhanced the Cd-induced liver injury. Notably, triple exposure to 100 nm PS, 1 μm PS, and Cd resulted in the most severe liver dysfunction, histopathological alterations, and activated cellular autophagy. Mechanistic investigations revealed that PS exposure alone or in combination with Cd triggered excessive autophagy and oxidative stress in hepatocytes by interfering with the PI3K/AKT/mTOR signaling pathway, thereby mediating liver injury. This study innovatively demonstrates that coexposure to different-sized PS particles and Cd can lead to complex liver injury patterns while particle size influences their combined hepatotoxicity with Cd.

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1. Introduction

In recent years, global plastic consumption has shown sustained growth, with annual plastic production reaching 300 million tons. Plastics degrade slowly due to physical, chemical, biological, and other factors, resulting in smaller-sized microplastics (MPs, ≤5 mm) and nanoplastics (NPs, ≤100 nm). Micro- and nanoplastics (MNPs) in the environment exhibit persistence and low buoyancy characteristics and are widely distributed across global ecosystems. They are ingested by organisms and accumulate via food chains, leading to different adverse effects.

Importantly, the biological effects of MNPs show size-dependent characteristics, with different MPs particle sizes causing different forms of cell damage. For instance, 1–10 μm MPs primarily induce programmed cell necrosis, while larger MPs (50–100 μm) mainly induce apoptosis. When compared to 200 and 500 nm polystyrene (PS) particles, 20 nm PS induced significant cytotoxicity in mice and human liver cell lines. In contrast, 500 nm PS demonstrated markedly enhanced pathological effects toward both hepatic and intestinal tissues when compared to 20 nm particles. Furthermore, reproductive toxicity assessment showed a distinct size-effect relationship, where 100 nm PS provoked more significant damage than 1 μm PS. However, 1 μm PS induced more severe anxiety-like behaviors and intestinal damage in mice when compared to 100 nm PS. These findings demonstrate that differently sized MNPs induce varied detrimental effects inside and outside of organisms. In nature, differently sized MNPs coexist, with interactions potentially enhancing cell uptake efficiency and inducing more severe biodegradation and biotoxicity effects. Earlier studies have demonstrated that coexposure to both 1 μm and 100 nm PS particles enhances reproductive toxicity in murine models when compared to single-size particle exposure. While exposure to either 50 or 500 nm PS alone impairs intestinal barrier function, the combined treatment was shown to induce more severe damage. Therefore, to better understand the potential impact and toxic mechanisms underpinning differently sized MNPs’ effects, both individually and in combination, more research is required.

MNPs exhibit a strong adsorption capacity for various hazardous substances, including heavy metals and persistent organic pollutants, owing to their hydrophobic properties and high surface area. This raises significant environmental concerns. Notably, among various heavy metals, cadmium (Cd) represents one of the most concerning soil contaminants; with low excretion and high toxicity rates, the metal poses serious threats to biological health. Additionally, toxic heavy metal contaminants, including Cd, are commonly found constituents in plastic stabilizers and lubricating agents. This promotes direct contact between MNPs and Cd, with complex interactions influencing environmental behaviors and bioavailability. , In previous research when PS and Cd were coincubated, Cd was adsorbed by PS, leading to physicochemical property changes in PS. Thus, such alterations could potentially facilitate MNPs and Cd ingestion by humans and other organisms. Additionally, differently sized MNPs induce different interactions in organisms; smaller MNPs enter cells more easily and interact more directly with Cd, thereby causing different combined effects. ,

The liver has crucial metabolic, synthesis, and detoxification roles and has also been reported to accumulate MNPs and Cd. PS and Cd cause liver toxicity by disrupting key liver enzymes, such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST). As a protective response following tissue damage, inflammation induced by PS and Cd is a common effect. During inflammation, imbalanced oxidative and antioxidative levels arise in cells with both states influencing each other. Additionally, reactive oxygen species generated during oxidative stress can promote autophagy activation, which reduces oxidative stress by engulfing oxidative products. However, in some cases, excessive autophagy exacerbates this stress, further damaging cell substances and structures and even inducing cell death. PI3K/AKT/mTOR signaling regulates many different biological processes, such as autophagy and apoptosis. Critically, both MNPs and Cd activate autophagy by modulating the PI3K/AKT/mTOR signaling. , However, there are no reports on whether combined exposure to MNPs and Cd can activate cellular autophagy via PI3K/AKT/mTOR signaling and alter the extent of oxidative damage, resulting in liver injury.

Currently, combined MNPs and Cd exposure effects remain an environmental concern. The potential hepatotoxicity induced by exposure to PS particles of varying sizes, either alone or in combination with Cd, remains a subject of active debate in real-world environmental conditions. Here, we examined these issues by investigating individual or combined exposure effects to differently sized PS particles and Cd in the livers of Kunming mice and normal human hepatocytes (MIHA cells). Additionally, we explored relationships among these exposure effects, autophagy, and oxidative stress, which were potentially mediated by PI3K/AKT/mTOR signaling.

2. Experimental Design and Procedures

2.1. PS and Chemicals

The study used polystyrene microspheres (100 nm and 1 μm; CAS No. 9003-53-6) purchased from Tianjin Goose Technology Co., Ltd. PS particles were dispersed in deionized water and treated by ultrasonication for 5 min to achieve a complete suspension. Analytical grade CdCl2 (CAS No. 10108-64-2, Macklin Biochemical Technology Co., Ltd., Shanghai, China; purity 99.99%) was dissolved in deionized water to achieve the desired concentration. Uniformly ultrasonically dispersed PS and Cd solutions were prepared to the desired concentrations and incubated at room temperature for 48 h for subsequent experiments.

To characterize PS microspheres, aliquots (10 μL) of a diluted PS suspension were deposited onto clean glass slides and air-dried at room temperature. Dried samples were then fixed to conductive carbon films and placed on the sample table of an ion sputtering instrument for approximately 30 s. Finally, the sample surface morphology was examined using scanning electron microscopy (SEM). Experimental details are described in our previous study.

2.2. Animals and Study Design

All animal experiments were performed in strict compliance with ethical standards established by the Institutional Animal Care and Use Committee of China Medical University (Approval No. CMU2021385), which reviewed and authorized both experimental protocols and animal handling procedures. We randomly divided 64 Kunming mice into the following groups: 1) control (deionized water), 2) 100 nm PS (100 mg/kg body weight [BW]), 3) 1 μm PS (100 mg/kg BW), 4) 100 nm PS + 1 μm PS (50 and 50 mg/kg BW, respectively), 5) Cd (5 mg/kg BW), 6) Cd + 100 nm PS (5 and 100 mg/kg BW, respectively), 7) Cd + 1 μm PS (5 and 100 mg/kg BW, respectively), and 8) Cd + 100 nm PS + 1 μm PS (5, 50, and 50 mg/kg BW, respectively). In terms of administered MNP doses, the human intake of approximately 5 g/week, as reported by Cox et al., was used and converted to a mouse equivalent dose using a body surface area-based dose conversion method. Based on the subacute toxicity study, a dose of 100 mg/kg/day was selected for oral gavage in mice. Mice underwent a 12 h fasting period, followed by daily gavage at 10 mL/kg BW, administered 5 days/week over a 5 week period. After exposure, mice were weighed, and blood was collected for serum analysis. Additionally, liver tissues were harvested, weighed, and preserved at −80 °C or underwent fixation in 4% paraformaldehyde. Following experimental interventions, liver coefficient values were derived by computing liver weight to final body weight ratios and converting the values to percentages.

2.3. Cell Culture

The MIHA human normal hepatocyte cell line was obtained from Fenghui (Hunan, China) and routinely cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin solution at 37 °C under a humidified atmosphere of 5% CO2.

2.4. Cd Content

Liver tissue samples were placed in a microwave digestion quartz tube with concentrated nitric acid and hydrogen peroxide and processed in a microwave digestion system. After acid digestion, samples were brought to 10 mL with distilled water and then analyzed for Cd using Inductively Coupled Plasma Mass Spectrometry (ICP-MS).

2.5. Accumulation of PS Microspheres in Mice Liver

Tissue specimens were positioned on the sample platform of an enhanced hyperspectral microscope with the light source intensity at 100%. Images were captured using a 60× objective lens with an exposure time of 0.25 μs. ENVI 4.8 was used to process the data. Regions of interest were selected to establish a spectral library for PS, and a filter spectral library using blank samples was used to eliminate false positives. Pixel-wise spectral matching was implemented using the Spectral Angle Mapper algorithm with the PS spectral library serving as reference data for the identification of spectrally identical regions in the acquired images. A 0.1 threshold was applied, and regions where pixels matched were marked in pseudo-red.

2.6. Cell Viability

After 24 h of culture, MIHA cells were exposed to PS and Cd by replacing the complete medium. 100 nm PS and 1 μm PS were prepared as follows: 0, 50, 100, 200, 300, and 400 μg/mL, and for Cd: 0, 1, 5, 10, 20, and 40 μM. A no-cell-blank control group was also included. After 24 h of incubation, cell viability was measured using CCK-8 assays in combination with absorbance detection.

2.7. Serum Biochemistry

Serum samples cryopreserved at −80 °C were gradually defrosted at 4 °C and subjected to centrifugation (10 min, 3000 rpm) for particulate removal. Clarified supernatant fractions were carefully aspirated for biochemical analysis. Hepatic transaminase activities, including alanine aminotransferase (ALT) and aspartate aminotransferase (AST), were quantified using commercial diagnostic kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

2.8. Histopathology

Following 24 h fixation in 4% paraformaldehyde solution buffered at pH 7.4, tissue specimens were progressively dehydrated through a graded ethanol series. Subsequent processing included paraffin infiltration and embedding using standard protocols. Serial sections of uniform thickness (5 μm) were prepared by microtomy for histological examination. Prior to staining, sections underwent deparaffinization through sequential immersion in xylene and rehydration in a descending ethanol gradient. Histological evaluation was performed using conventional hematoxylin and eosin (H&E) staining methodology. After neutral balsam mounting, sections were examined under bright-field microscopy.

2.9. Immunofluorescence

After antigen retrieval, specimens were treated with a LC3 antibody solution (Abconal, 1:200) under refrigerated conditions (4 °C) overnight. Next, a fluorescently labeled secondary antibody and TSA dye were sequentially applied, followed by high-temperature treatment (microwave). After p62 antibody incubation (Abconal, 1:200), sections were stained with fluorescent secondary antibodies and DAPI (nuclear labeling). Finally, samples were mounted in antifade mounting medium to quench fluorescence, observed under an inverted microscope, and fluorescence signals captured.

2.10. Oxidative Stress

For measurements, liver tissues were homogenized, and MIHA cells were lysed. Subsequently, superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and malondialdehyde (MDA) levels were measured according to the kit instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

2.11. Western Blotting

To process liver tissues and MIHA cells, RIPA lysis buffer was added to the samples along with protease and phosphatase inhibitors (Beyotime Institute of Biotechnology, Shanghai, China), after which homogenization was performed to extract the total proteins. Bicinchoninic acid protein assays (Beyotime Institute of Biotechnology, Shanghai, China) were used to measure protein concentrations. Aliquots containing 30–50 μg of total protein were resolved using discontinuous SDS-PAGE (8–12% gradient gels) under reducing conditions and subsequently electrotransferred onto polyvinylidene difluoride membranes (EMD Millipore Corporation, Billerica, MA, USA) using a standard wet transfer methodology. Membranes were blocked in 5% (w/v) nonfat dairy milk (Beyotime Institute of Biotechnology, Shanghai, China) in Tris-buffered saline containing 0.1% Tween-20 for 120 min at ambient temperature. Immunoblotting was conducted using the following primary antibodies (dilutions are indicated): LC3 (Abclonal, 1:1000), Beclin-1 (Abclonal, 1:1000), P62 (Abclonal, 1:1000), p-PI3K (Affinity, 1:1000), PI3K (Proteintech, 1:1000), p-AKT (Affinity, 1:1000), AKT (Proteintech, 1:1000), p-mTOR (Affinity, 1:1000), mTOR (Proteintech, 1:5000), GAPDH (Abclonal, 1:4000), and β-actin (Abclonal, 1:4000). Primary antibody binding was carried out at 4 °C for 16–18 h with gentle rocking, followed by a 1 h incubation with HRP-tagged secondary antibodies (Abclonal Technology) at ambient temperature. Chemiluminescent signals were developed by using commercial ECL reagents (Vazyme Biotech Co., Ltd., Nanjing, China) and digitally captured. Protein band intensities were quantified using ImageJ software with values normalized to endogenous reference proteins.

2.12. Statistical Analysis

Data were analyzed using IBM SPSS Statistics (v23.0), with quantitative data expressed as the arithmetic mean ± SD. Intergroup comparisons were conducted using single-factor ANOVA followed by Fisher’s LSD method for multiple comparisons, adopting 0.05 as the probability threshold for type I error determination. Visual data representations were created in Microsoft Excel (2021) and graphing tools (Prism 8.0; GraphPad Software).

3. Results

3.1. Characterization of PS Microspheres

Previously, SEM observations revealed that both 100 nm and 1 μm PS microspheres had uniform sizes and smooth surfaces. However, after mixing the 100 nm PS with Cd, cracks appeared on the microsphere surfaces and noticeable adhesion was observed between the microspheres, indicating that Cd adsorption had negatively affected the PS dispersion (Figure S1). This observation aligned with Hu et al., who observed that after mixing PS with Cd, solution dispersion decreased and the particle size distribution became uneven. As a result, Cd adsorption altered the PS properties.

3.2. Different PS Particle Sizes Induce Hepatic Toxicity in Mice

After the dosing period and to determine whether differently sized PS particles induced hepatic toxicity in mice, body and liver wet weights were recorded, and organ coefficients were calculated. When compared to the control group, liver organ coefficients in the 100 nm PS, 1 μm PS, and 100 nm PS + 1 μm PS groups all showed significant decreases (Figure S2A). PS particles were then visualized by using enhanced hyperspectral microscopy, which showed their accumulation in liver tissues (Figure D). Furthermore, serum ALT and AST levels were significantly increased in all PS groups when compared to the control group. Specifically, when compared to the 100 nm PS group, the 1 μm PS group exhibited significantly higher serum ALT and AST levels. Furthermore, when compared to the single PS particle size group, the 100 nm PS + 1 μm PS group showed significantly elevated serum ALT and AST levels (Figure A,B). Additionally, the control group showed an orderly liver cell arrangement without apparent lipid degeneration or vacuoles. Both the 100 nm PS and 1 μm PS groups exhibited lipid droplets in the tissues. However, in further comparison, the 1 μm PS group showed disordered liver cord arrangements, cell morphology changes, and more prominent lipid droplets. Furthermore, when compared to single-sized PS groups, the 100 nm PS + 1 μm PS group showed abnormal liver cell arrangements and more vacuoles of varying sizes (Figure C). Therefore, differently sized PS particles damaged liver tissue in mice and showed enhanced toxic effects from combined to differently sized PS particles.

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Differently sized PS particles induce hepatic toxicity in mice. (A) ALT and (B) AST levels. (C) H&E-stained sections. Scale bar = 50 and 20 μm. (D) PS particle accumulation (arrows) in the liver. Scale bar = 20 μm. Values are the mean ± SD (n = 3). *vs control group, #vs 100 nm PS group, and &vs 1 μm PS group (all P < 0.05).

3.3. Differently Sized PS Particles Induce Autophagy in Mice Liver

Liver tissue analysis revealed significant reductions in CAT and SOD activities across all PS-exposed groups relative to controls, concomitant with elevated GSH levels, demonstrating substantially induced oxidative stress. When compared to the 100 nm PS group, the 1 μm PS group showed significantly reduced SOD and CAT levels, but the levels were more pronounced in the 100 nm PS + 1 μm PS group when compared to the single-sized PS group (Figure A–C). We also evaluated differently sized PS particle effects on autophagy in liver samples. Immunofluorescence showed that when compared to the control group, LC3 (pro-autophagy gene) fluorescence intensity was increased in all PS groups, while P62 (antiautophagy factor) fluorescence intensity was decreased (Figure S2B-D). Western blotting showed that when compared to the control group, LC3II/I and Beclin-1 protein levels were elevated, whereas P62 levels were reduced in PS group samples. These changes were more significant in the 1 μm PS group when compared to the 100 nm PS group and even more pronounced in the 100 nm PS + 1 μm PS group when compared to the single-sized PS groups (Figure D,E).

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Differently sized PS particles induce autophagy in mice liver by affecting PI3K/AKT/mTOR signaling. (A–C) SOD, GSH, and CAT levels. (D, E) Representative Western blots and autophagy-related protein quantification. (F, G) Representative Western blots and PI3K/AKT/mTOR protein quantification. Values are the mean ± SD (n = 3). *vs control group, #vs 100 nm PS group, and &vs 1 μm PS group (all P < 0.05).

When compared to the control group, p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR protein levels were significantly decreased in the PS group liver samples. These changes were more pronounced in the 1 μm PS group when compared to the 100 nm PS group, and even more significant in the 100 nm PS + 1 μm PS group when compared to the single PS particle size groups (Figure F,G). Therefore, differently sized PS particles potentially induced hepatocyte autophagy by modulating PI3K/AKT/mTOR signaling, thereby affecting SOD, GSH, and CAT levels and exacerbating liver damage.

3.4. Combined Differently Sized PS Particles and Cd Exposure Induce Liver Toxicity in Mice

Based on the liver damage caused by exposure to differently sized PS particles, we further investigated the combined exposure effects to different PS particle sizes and Cd in the mice liver. Liver coefficients were significantly lower in the Cd and Cd + PS groups than in the control group (Figure S3A). When compared to the control group, Cd liver concentrations were significantly increased in the Cd and Cd + PS combined exposure groups. Furthermore, when compared to the Cd-only exposure group, the group exposed to both Cd and single-sized PS particles showed significant Cd reductions (Figure A). Additionally, after combined PS and Cd exposure, PS particles accumulated in the liver (the red area, as indicated by the arrow, Figure E). Furthermore, when compared to the control group, the Cd and Cd + PS combined exposure groups showed significantly increased serum ALT and AST levels. When compared to the Cd and Cd + 1 μm PS groups, the Cd + 100 nm PS and Cd + 100 nm PS + 1 μm PS groups showed significantly elevated serum ALT and AST levels. However, no significant differences in levels were recorded between the Cd + 1 μm PS and Cd groups (Figure B,C). In terms of cell morphology, hepatocytes in the control group were arranged in an orderly manner without significant fat degeneration. In the Cd and Cd + PS combined exposure groups, hepatocyte shapes were altered with varying lipid droplet sizes. In particular, when compared to groups exposed to combined single PS particle sizes and Cd, the Cd + 100 nm PS + 1 μm PS group showed increased variably sized lipid droplets in tissues (Figure D).

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Combined exposure to differently sized PS particles and Cd induces liver toxicity in mice. (A) Cd accumulation in liver. (B) ALT and (C) AST levels. (D) H&E-stained sections. Scale bar = 50 and 20 μm. (E) PS particle accumulation (arrows) in the liver. Scale bar = 20 μm. Values are the mean ± SD (n = 3). *vs control group, #vs Cd group, &vs Cd + 100 nm PS group, and $vs Cd + 1 μm PS group (all P < 0.05).

3.5. Combined Exposure to Differently Sized PS Particles and Cd Induces Autophagy in Mice Liver

Cd and Cd + PS exposure significantly decreased hepatic CAT and SOD activities but increased GSH levels relative to the controls. Additionally, when compared to the group exposed to a single PS particle size combined with Cd, the Cd + 100 nm PS + 1 μm PS group showed significantly decreased SOD and CAT levels (Figure A–C).

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Combined exposure to differently sized PS particles and Cd induces autophagy in mice liver. (A–C) SOD, GSH, and CAT levels. (D, E) Representative Western blots and autophagy-related protein quantification. (F, G) Representative Western blots and PI3K/AKT/mTOR protein quantification. Values are the mean ± SD (n = 3). *vs control group, #vs Cd group, &vs Cd + 100 nm PS group, and $vs Cd + 1 μm PS group (all P < 0.05).

Immunofluorescence assays showed significantly increased LC3 but decreased P62 fluorescence intensity in Cd-exposed and Cd + PS groups versus controls (Figure S3B-D). Western blotting showed that when compared to the control group, Cd and PS combined exposure groups exhibited increased LC3II/LC3I and Beclin-1 protein levels along with decreased P62 levels. These changes were more pronounced in the Cd + 100 nm PS group compared to the Cd + 1 μm PS and Cd groups. Moreover, the Cd + 100 nm PS + 1 μm PS group exhibited more pronounced alterations in protein expression levels (LC3II/LC3I, Beclin-1, and P62) compared to either the Cd-only group or groups exposed to Cd in combination with individual PS particle sizes (Figure D,E). Furthermore, p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR protein expression ratios were significantly lower in both the Cd-exposed group and the Cd + PS group compared to the control group. These decreases were more pronounced in the Cd + 100 nm PS + 1 μm PS group when compared to the Cd and single-sized PS particles combined with Cd groups. Additionally, significant decreases in p-PI3K/PI3K and p-mTOR/mTOR ratios were observed in the Cd + 100 nm PS group when compared to both the Cd + 1 μm PS and Cd-only groups (Figure F,G). Therefore, differently sized PS particles combined with Cd exposure potentially induced hepatocyte autophagy by modulating PI3K/AKT/mTOR signaling, thereby affecting SOD, GSH, and CAT levels and exacerbating liver damage.

3.6. The Impact of Combined Differently Sized PS Particles and Cd Exposure on MIHA Cell Viability

Using CCK-8 assays, exposure effects from 100 nm PS (Figure S4A), 1 μm PS (Figure S4B), and Cd (Figure S4C), or in combination (Figure S4D), on MIHA cell viability were assessed after 24 h. When the 100 nm PS group concentration was 100 μg/mL and the Cd concentration was 10 μM, the MIHA cell viability decreased to approximately 80%. Therefore, 100 μg/mL of 100 nm PS, 100 μg/mL of 1 μm PS, and 10 μM Cd were selected for subsequent assays. When compared to PS or Cd exposure, MIHA cell viability significantly decreased with Cd + 100 nm PS + 1 μm PS exposure.

3.7. Combined PS and Cd Exposure May Exacerbate Oxidative Damage via Autophagy

After supplementing the autophagy inhibitor 3-MA to cells, when compared to control cells, Cd + 100 nm PS + 1 μm PS treatment significantly upregulated the LC3II/I ratio while downregulating P62 protein levels. In the 3-MA-treated group, the LC3II/I ratio was markedly reduced, while the P62 protein expression showed a significant increase. When compared to the Cd + 100 nm PS + 1 μm PS group, LC3II/I and P62 levels were significantly decreased and increased, respectively, in the Cd + 100 nm PS + 1 μm PS + 3-MA group (Figure D,E). Next, when compared to control cells, Cd + 100 nm PS + 1 μm PS cells exhibited significantly decreased SOD and GSH levels and significantly increased MDA levels. Furthermore, when compared to Cd + 100 nm PS + 1 μm PS cells, Cd + 100 nm PS + 1 μm PS + 3-MA cells showed significantly increased SOD and GSH levels and significantly decreased MDA levels (Figure A–C). Thus, combined PS and Cd exposure appeared to induce excessive autophagy in mice liver, thereby exacerbating oxidative stress.

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Combined exposure to PS and Cd exacerbates oxidative damage via autophagy. (A–C) SOD, GSH, and MDA levels. (D, E) Representative Western blot and autophagy-related protein quantifications. Values are the mean ± SD (n = 3). *vs control group, #vs 3-MA group, and &vs Cd + 100 nm PS + 1 μm PS group (all P < 0.05).

3.8. The Role of PI3K/AKT/mTOR Signaling in Inducing Autophagy and Oxidative Stress in MIHA Cells Exposed to Combined PS and Cd

Compared to control cells, Cd + 100 nm PS + 1 μm PS treatment significantly reduced p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR levels. However, supplementation with the PI3K activator 740Y-P reversed this suppression. Conversely, in 740Y-P-treated cells, a significant increase in the p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR protein levels was recorded. The addition of 740Y-P to Cd + 100 nm PS + 1 μm PS-treated cells significantly elevated p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR protein ratios when compared to cells treated with Cd + 100 nm PS + 1 μm PS alone (Figure F,G). When compared to control cells, Cd + 100 nm PS + 1 μm PS cells showed significantly increased and decreased LC3II/I and P62 protein levels, respectively. In 740Y-P cells, a significant decrease and increase in LC3II/I and P62 protein levels, respectively, was recorded. When compared to Cd + 100 nm PS + 1 μm PS cells, Cd + 100 nm PS + 1 μm PS + 740Y-P cells exhibited a significant decrease and increase in LC3II/I and P62 protein levels, respectively (Figure D,E). Furthermore, exposure to Cd + 100 nm PS + 1 μm PS induced significant reductions in SOD and GSH levels, concomitant with elevated MDA levels relative to the controls. Compared to cells exposed to Cd + 100 nm PS + 1 μm PS alone, those cotreated with 740Y-P exhibited significantly upregulated SOD and GSH levels, accompanied by a marked decrease in MDA levels (Figure A–C). These observations further confirmed that combined PS and Cd exposure induced hepatocyte autophagy via PI3K/AKT/mTOR signaling, thereby regulating SOD, GSH, and MDA levels, increasing hepatic oxidative stress, and exacerbating liver damage.

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PI3K/AKT/mTOR actions in inducing autophagy and oxidative stress in MIHA cells exposed to combined PS and Cd. (A–C) SOD, GSH, and MDA levels. (D, E) Representative Western blots and autophagy-related protein quantification. (F, G) Representative Western blots and PI3K/AKT/mTOR protein quantification. Values are the mean ± SD (n = 3). *vs control group, #vs 740-YP group, and &vs Cd + 100 nm PS + 1 μm PS group (all P < 0.05).

4. Discussion

MNPs are widely distributed across the globe, where they are often ingested by organisms and accumulate via food chains. However, due to limited detection technologies, studies have only examined and detected larger-sized MPs in human tissues (e.g., intestines, lungs, liver, and placenta). , In animal models, both 50 nm PS and 500 nm PS particles were shown to accumulate in the spleen, kidneys, heart, liver, lungs, and blood. In our study, 100 nm PS and 1 μm PS particles accumulated in mice liver tissues as evidenced by hyperspectral imaging technology. From these findings, we examined liver function markers in serum and observed that exposure to 100 nm PS and 1 μm PS, either alone or in combination, significantly elevated ALT and AST levels, indicating impaired liver function. Moreover, H&E staining revealed that exposure to 100 nm PS and 1 μm PS, either alone or in combination, induced morphological changes accompanied by lipid droplets of varying sizes. These observations indicated that both 100 nm PS and 1 μm PS particles, individually or combined, exerted pathological damage in the liver. These findings were consistent with Mu et al., who showed that 5 μm PS particles affected liver function and induced pathological damage in mice. Additionally, by assessing oxidative stress-related markers, exposure to 100 nm PS and 1 μm PS, either alone or in combination, significantly altered SOD, GSH, and CAT levels, leading to oxidative damage in liver tissue, consistent with Meng et al. Our results further indicated that exposure to 100 nm PS and 1 μm PS, either individually or in combination, induced mice liver damage, while combined 100 nm PS and 1 μm PS exposure showed the most pronounced toxicity. Our results corroborate the findings of Liang et al., who demonstrated that combined exposure to 500 and 50 nm PS induced more severe intestinal toxicity compared to exposure to either size alone. The enhanced toxic effect observed when MNPs of two different sizes are combined may be attributed to the fact that larger particles may facilitate the cellular uptake of the smaller nanoparticles, leading to an increase in the total amount of particles internalized by cells. Also, by mixing, this can alter their physicochemical properties, thereby influencing their toxicity. Dramatically, liver toxicity induced by 1 μm PS was more severe in comparison to 100 nm PS. This finding was corroborated by Huang et al., who observed that in mice, 200 nm PS and 500 nm PS particles induced stronger liver and intestinal toxicity when compared to 20 nm PS particles. Although smaller particles are expected to exhibit stronger effects, the more serious effects of larger PS particles may be due to the level of internalization. − When the particle sizes are in a particular internalization range, smaller particles tend to be internalized more effectively than larger particles. The presence of larger particles, due to less internalization, will result in their accumulation in the organs, thereby exacerbating increased intestinal damage and intestinal permeability and inducing more serious toxicity. Additionally, after passing through intestinal monolayer cells, MNP particles may alter their physicochemical properties, potentially leading to other cell uptake mechanisms, such as endocytosis into hepatocytes, which could modify their toxic effects toward hepatocytes. Future in-depth validation studies are required to validate these hypotheses.

Considering the multifaceted nature of actual exposure scenarios, MNPs act as carriers, adsorbing Cd into organisms. Therefore, we examined Cd accumulation in liver tissues, which showed accumulation in the Cd and Cd and PS combined exposure groups. However, when compared to the Cd group, the liver Cd content in the Cd and PS combined exposure group decreased. This finding was consistent with Wen et al. and Zou et al., who suggested that PS and polyvinyl chloride microplastics reduced Cd accumulation in tissues. Lu et al. also reported that 5 μm PS particles increased Cd accumulation in zebrafish liver. Consequently, hepatotoxicity resulting from combined PS particle exposure does not invariably enhance the heavy metal bioavailability. This could be related to MPs’ chemical properties (size, nature, and concentration), metal type and concentration, as well as environmental factors (pH, ionic strength, and other dissolved substances). From our analyses of serum liver indicators, ALT and AST levels were significantly elevated in Cd and Cd and PS combined exposure groups, indicating abnormal liver function. From H&E staining, combined PS and Cd exposure altered liver cell shape and induced variably sized vacuoles containing fat droplets. Thus, this combination caused pathological damage to mice liver tissue. Additionally, significant SOD, GSH, and CAT alterations were identified in both the Cd and Cd and PS combined exposure groups, indicating oxidative damage. These results indicated that Cd exposure, or in combination with 100 nm PS or 1 μm PS, induced liver damage in mice. Furthermore, 100 nm PS particles significantly enhanced hepatotoxic effects due to Cd, consistent with Sun et al., which showed that 5 μm PS particles significantly enhanced Cd-induced liver damage. However, when compared to this result, our combined 1 μm PS and Cd exposure analysis did not show increased Cd hepatotoxic effects in mice, with no significant toxicity differences between groups, contrary to Wang et al. However, recently, Sheng et al. reported that hepatic toxicity induced by combined 1 μm PS and Cd exposure did not show any significant differences when compared to Cd exposure consistent with our findings. Furthermore, we showed that combined exposure to 100 nm PS, 1 μm PS, and Cd showed the most significant hepatotoxic effects. Study differences may not be only related to MNPs’ particle size but also possibly to MNPs and Cd exposure concentrations. Therefore, future investigations are required to explore the range of critical MNPs and Cd concentrations that promote or inhibit Cd toxicity.

Autophagy is a dynamic process which is rigorously regulated by cell nutrient availability and metabolic balance. Under stress conditions (e.g., cell starvation and hypoxia), autophagy is generally considered a protective mechanism that alleviates damage. Under starvation conditions, autophagy degrades cell components to provide additional energy and nutrients, ensuring cell survival. Under hypoxic conditions, autophagy eliminates damaged cell components, promotes cell survival, and mitigates damage by regulating oxidative stress. However, under certain circumstances, excessive autophagy exacerbates oxidative stress, further damaging cell substances and structures, and even inducing cell death. Our data indicated that PS and Cd exposure, alone or in combination, significantly increased LC3II/I and Beclin-1 protein levels in mice liver while decreasing P62 levels. Simultaneously, exposure disrupted antioxidant enzyme balance, increasing GSH levels and reducing SOD and CAT levels. Thus, PS and Cd exposure, alone or in combination, potentially induced excessive autophagy in mice liver, exacerbating oxidative stress. These results corroborate Zou et al.’s findings that MPs enhance Cd-induced nephrotoxicity via oxidative stress and autophagy. To further demonstrate combined PS and Cd exposure effects on autophagy, we examined LC3 and P62 protein levels after the addition of the autophagy inhibitor 3-MA to MIHA cells. We observed that 3-MA significantly reduced LC3II/I protein levels following combined PS and Cd exposure but increased P62 protein levels, enhanced SOD and GSH levels, and decreased MDA activity. This result is consistent with the findings of Wu et al. in a postoperative anxiety model, in which 3-MA supplementation was demonstrated to increase SOD activity while decreasing GSH activity. These observations further confirmed that combined PS and Cd exposure overactivated autophagy, exacerbating oxidative damage in the liver.

PI3K/AKT/mTOR signaling has key regulatory roles in autophagy across different cell types. PI3K primarily regulates inflammation and immune functions, activating the downstream signaling molecule AKT, which, in turn, activates mTOR to inhibit autophagy. Following individual or combined PS and Cd exposure, p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR protein levels were significantly decreased. This suggested that combined PS and Cd exposure may have induced autophagy by affecting the PI3K/AKT/mTOR pathway. We also validated the autophagy pathway by introducing the PI3K activator 740Y-P into MIHA cells and showed that it increased p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR protein levels following combined PS and Cd exposure. This exposure also reduced LC3II/I protein levels, increased P62 protein levels, and enhanced SOD and GSH levels but decreased MDA content. Therefore, Cd and PS combined exposure potentially induced excessive autophagy and oxidative stress in hepatocytes by affecting PI3K/AKT/mTOR signaling, thereby exacerbating liver damage.

There are some limitations of the present study. First, we used only commercially available PS microspheres at two particle sizes, which were uniform in shape with pristine or functionalized surfaces. In real-world environments, discarded plastics undergo weathering and other processes, leading to diverse MNP shapes, types, particle sizes, and physicochemical properties. However, due to a lack of standardized MNP references, toxicity studies are limited to commercial standards for this kind of research. Additionally, based on human exposure scenarios, we only investigated single MNPs and Cd concentrations individually with consideration for animal numbers (the 3R principle) as the first attempt. This inevitably limits the exploration of toxicity thresholds and concentration–response relationships. Future research involving different concentration combinations should be performed to systematically evaluate their interaction patterns (synergistic, additive, or antagonistic) and establish no-observed adverse-effect levels or benchmark concentrations, which are essential for accurate environmental risk assessments.

5. Conclusions

PS and Cd exposure, either individually or in combination, induced excessive autophagy and oxidative damage in mice liver. Specifically, 1 μm PS particles induced stronger liver damage when compared to 100 nm PS particles, but a combined 100 nm PS and Cd exposure induced stronger liver damage than a combined 1 μm PS and Cd exposure. Moreover, combined exposure to PS particles of different sizes caused liver damage that was stronger than exposure to a single PS size alone. Furthermore, the combined exposure of PS and Cd induced excessive autophagy and oxidative stress in hepatocytes by affecting PI3K/AKT/mTOR signaling, thereby affecting SOD, GSH, and CAT levels and exacerbating liver damage.

Supplementary Material

tx5c00308_si_001.pdf (527.6KB, pdf)

The research data sets generated in this study will be shared upon formal application to the corresponding author, provided the request aligns with our institution’s data access regulations and the ethical framework of this project.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.chemrestox.5c00308.

  • Detailed supplementation on animals and study design; surface characterization of 100 nm PS particles after mixing with Cd; effects of individual and combined exposure to PS and Cd on liver coefficient; immunofluorescence results of LC3 and P62 in liver tissue; impact of coexposure to PS particles of different sizes and Cd on the viability of MIHA cells (PDF)

S.L.: Data curation, Formal analysis, Validation, Writingoriginal draft. Q.Z.: Methodology. Y.H.: Validation. X.L.: Data curation. Y.L.: Data curation. L.Y.: Visualization, Supervision, Funding acquisition. G.P.: Visualization, Supervision. W.S.: Project administration, Validation, Writingreview and editing.

This research received funding from the following sources: National Natural Science Foundation of China (Award number: U21A20399), the Key R&D Program of Liaoning Province (Award number: 2019JH2/10300044), the Science and Technology Innovation Team Project of China Medical University 2022 (Award number: CXTD2022006), and the Key Laboratory Program of Shenyang City (Award number: 21-103-0-16).

The authors declare no competing financial interest.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

tx5c00308_si_001.pdf (527.6KB, pdf)

Data Availability Statement

The research data sets generated in this study will be shared upon formal application to the corresponding author, provided the request aligns with our institution’s data access regulations and the ethical framework of this project.

Articles from Chemical Research in Toxicology are provided here courtesy of American Chemical Society

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