Large-sized polystyrene microplastics induce oxidative stress in AML12 cells

Abstract

Microplastics (MPs), particularly those exceeding 20 μm in diameter, are increasingly detected in environment and animal tissues, yet their cytotoxicity remains poorly understood. While existing studies focused on MPs with relatively small sizes (≤ 20 μm) or nanoplastics (NPs), the biological impacts of large-sized MPs and amino-modified MPs are underexplored. In this study, we investigated the oxidative stress (OS)-mediated responses of alpha mouse liver 12 (AML12) cells to 50 μm polystyrene MPs (PS-MPs) and polystyrene-amine modified MPs (PS-NH₂-MPs) at concentrations of 0, 0.05, 0.1, 0.2, and 0.5 mg/mL for a duration of 48 h. Our investigation particularly emphasised on responses of apoptosis and ferroptosis. Intriguingly, exposure to PS-MPs at concentrations below 0.5 mg/mL did not induce significant ferroptosis-related alterations. However, at 0.5 mg/mL, PS-MPs triggered a significant increase in intracellular reactive oxygen species and reduced cell viability, paralleled by upregulated Caspase-9 mRNA expression, suggesting OS-driven apoptotic priming. Notably, surface functionalization with PS-NH₂-MPs did not amplify these effects compared to pristine PS-MPs, indicating particle size dominate hepatocyte responses. These findings provide a toxicological paradigm for large-sized MPs, emphasising OS as a pivotal evaluation criterion for risk assessment. These results provide a novel perspective for environmental monitoring of MPs.

Keywords: Apoptosis; Ferroptosis; Large-sized microplastics; Oxidative stress; Polystyrene.

Fig. 1

Characterization of PS-MPs. (A) SEM image of PS-MPs. (B) Microscope image of PS-MPs with cells. Scale bar: 50 μm.

Fig. 2

Cell viability after exposure to PS-MPs. Statistical trend of cell viability after exposure with PS-MPs at 0, 0.05, 0.1, 0.2 and 0.5 mg/mL for (A) 24 h and (B) 48 h. n = 9. Compared to control: 0 mg/mL; Statistics are calculated as mean ± SD; ANOVA followed by Tukey test was used to assess differences groups; *P < 0.05, **P < 0.01.

Fig. 3

Effects of AML12 cells exposure to PS-MPs on oxidative stress. (A-D) The condition of cellular oxidative stress used GSH, MDA, SOD and CAT assay kits to detect after exposure with PS-MPs at 0, 0.05, 0.1, 0.2 and 0.5 mg/mL for 48 h. n = 3. Compared to control; Statistics are calculated as mean ± SD; ANOVA followed by Tukey test was used to assess differences groups; *P < 0.05, **P < 0.01.

Fig. 4

ROS levels of AML12 cells exposure to PS-MPs. (A) DCFH-DA staining was followed by fluorescence microscopy to detect the levels of ROS. The green fluorescent dots indicate ROS generation. (B) Detected ROS levels by flow cytometry after treatment. Scale bar: 50 μm. n = 3. Compared to control; Con: control group, 0 mg/mL PS-MPs; PS: 0.5 mg/mL PS-MPs; PS-NH₂: 0.5 mg/mL PS-NH₂-MP; Statistics are calculated as mean ± SD; *P < 0.05, **P < 0.01.

Fig. 5

Percentage of apoptotic cells analysis in AML12 cells. (A) Percentage of apoptotic cells was measured by flow cytometry after exposure to PS-MPs at 0, 0.05, 0.1, 0.2 and 0.5 mg/mL for 48 h. (B) Percentage of apoptotic cells was measured by flow cytometry after exposure to PS-MPs and PS-NH₂-MPs at 0.5 mg/mL. n = 3. Compared to control; Con: control group; 0 mg/mL PS-MPs; PS: 0.5 mg/mL PS-MPs; PS-NH₂: 0.5 mg/mL PS-NH₂-MPs; Statistics are calculated as mean ± SD; *P < 0.05, **P < 0.01.

Fig. 6

Apoptosis analysis in AML 12 cells. (A-B) Make Western blot analysis of P53, Bcl-2 and Bax after exposure to PS-MPs for 48 h. The blots have been cropped. Original blots are presented in Supplementary Fig. S2. (C) RT-qPCR analysis of P53, Bcl-2, Bax, CytC, Caspase-9, Caspase-3 and Cox-2 mRNA after exposure to PS-MPs for 48 h. n = 3. Compared to control; Con: control group, 0 mg/mL PS-MPs; PS: 0.5 mg/mL PS-MPs; PS-NH₂: 0.5 mg/mL PS-NH₂-MPs; Statistics are calculated as mean ± SD; *P < 0.05, **P < 0.01.

Fig. 7

Ferroptosis analysis in AML 12 cells. (A) Ferrous ion detection after exposure to PS-MPs at 0, 0.05, 0.1, 0.2 and 0.5 mg/mL for 48 h. (B) RT-qPCR analysis of GPX4, SLC7A11, ACSL4 and FTH1 mRNA after exposure to PS-MPs for 48 h. (C-D) Western blot analysis of GSS, GPX4 and SLC7A11. The blots have been cropped. Original blots are presented in Supplementary Fig. S2. n = 3. Compared to control; Con: control group, 0 mg/mL PS-MPs; PS: 0.5 mg/mL PS-MPs; PS-NH₂: 0.5 mg/mL PS-NH₂-MPs; Statistics are calculated as mean ± SD; *P < 0.05, **P < 0.01.