Exposure to polystyrene nanoparticles leads to dysfunction in DNA repair mechanisms in Caco-2 cells

Abstract

Background: Recent studies have highlighted the critical health implications of environmental exposure to nanoplastics, particularly concerning their effects on human gastrointestinal cells. In this study, we used human colorectal adenocarcinoma (Caco-2) cells to investigate the exposure of polystyrene nanoparticles (PNPs) to cellular processes and DNA repair.

Methods: We exposed Caco-2 cells to various concentrations of PNPs and monitored cytotoxicity, ROS levels, PARP-1-dependent apoptosis, DNA damage, and changes in DNA damage response (DDR) gene expression.

Results: The results indicated that although PNPs did not directly cause SSBs or DSBs, as evidenced by comet assays and γH2AX staining, they induced oxidative stress and significantly altered the expression of genes required for DDR. In particular, critical genes involved in the base excision repair (BER) pathway and DSBs repair were downregulated, suggesting a potential impairment of the cell's ability to repair oxidative DNA damage.

Conclusions: This study highlights the sublethal effects of nanoplastics on intestinal barrier cells. It underscores the possible risks of exposure to these environmental contaminants, which can lead to genome instability and other long-term health consequences.

Keywords: Apoptosis; DNA damage response; DNA-DSBs; DNA-SSBs; PARP; Polystyrene nanoparticles.

Fig. 1

Cell viability and survival fraction after exposure of Caco-2 cells to PNPs (0, 50, 100, 400, 800, and 1200 µg/mL). A: Trypan blue exclusion test after 3 h, 24 h, and 48 h incubation with PNPs. Data were expressed as a percentage; the bars correspond to the mean ± SEM (n = 6). B: The colony formation assay: representative images of colonies stained with Coomassie blue for nontreated and after 24-hour treatment of cells with PNPs. C: The panel represents decreased colony survival fractions of Caco-2 cells exposed to PNPs compared to the unexposed cells. The colonies were analyzed after 9 days of incubation in 5% CO₂ and 37 °C and counted using countPHICS software. Survival Fraction (SF) was considered using the formula SF = (plating efficiency of tested cells/plating efficiency of control cells x 100%). Data were expressed as a percentage; the bars correspond to the mean ± SEM (n = 3). P-values were considered significant: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001

Fig. 2

Caco-2 PNPs exposure (100 µg/mL, 24 h) on cleaved PARP-1 apoptosis. A: Multicolor flow cytometric representative images of Cleaved PARP (Asp214) PE versus BrdU PerCP-Cy™5.5 profile. B: Data were expressed as a percentage; the point bars represent the mean ± SEM (n = 4) for apoptosis. 2-hour incubation with etoposide (ETOP), was used as a positive control with a final concentration of 50 µM. The fragment of cleaved-PARP1 (89 kDa) was used as a marker of apoptosis (PE mouse anti-cleaved PARP (Asp214) antibody). P-values were considered significant: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001

Fig. 3

Flow cytometry identification of cell cycle changes with anti-BrdU antibody in Caco-2 cells after PNPs exposure (100 µg/mL, 24 h). 2-hour incubation with etoposide (ETOP), was used as a positive control with a final concentration of 50 µM. The cells were labeled with 10 µM BrdU for 1 h and stained with BrdU PerCP-Cy™5.5 antibody. Flow cytometry representative images (A panel) of DAPI versus BrdU PerCP-Cy™5.5 staining profile (% of Caco-2 cells in each phase) and relative quantification (B panel). Cells were gated correctly: G0/G1 phase, S phase, and G2/M phase. Data were demonstrated as percentages of cells and the bars correspond to the mean ± SEM (n = 4). P-values were considered significant: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001

Fig. 4

Caco-2 PNPs exposure (0, 50, 100, 400, 800, and 1200 µg/mL) on intracellular ROS levels. 1.5 mM H₂O₂ was used as a positive control, [a.u.] = arbitrary units. The data was normalized to the control and presented as mean ± SEM (n = 3) for ROS level analysis (H2DCFDA probe). P-values were considered significant: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001

Fig. 5

Identification of SSBs and DSBs in Caco-2 cells after PNPs exposure (100 µg/mL, 24 h) with alkaline comet assay. 50 µM etoposide (2 h incubation) was used as a positive control. A: Representative comets with a comet tail (fragmented DNA) and a head (intact DNA), visualized under 40x immersion oil, confocal microscope Leica SP5 with SYBR SAFE™ staining using fluorescein filter with maximum excitation/emission 496 nm/522 nm). B: Quantification of comet assay images was performed using OpenComet v1.3.1 software for % of DNA in the head and % of DNA in the tail (box plots). Results are based on the analysis of at least 50 comets per replicate (n = 3). P-values were considered significant: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001

Fig. 6

Flow cytometry identification of DNA double-strand breaks in Caco-2 cells after PNPs exposure (100 µg/mL, 24 h). 2-hour incubation with etoposide (ETOP), was used as a positive control with a final concentration of 50 µM. A: Flow cytometry representative images of BrdU PerCP-Cy™5.5 and H2AX (pS139) Alexa Fluor^® 647 (% indicating the proportion of DSBs in the presented experiment). B: The distribution of γH2AX in Caco-2 cells (in control and PNPs-treated Caco-2 cells); the bars correspond to the mean ± SEM (n = 4). P-values were considered significant: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001

Fig. 7

Real-time PCR analysis of the normalized expression of DDR signaling genes after PNPs exposure (100 µg/mL, 24 h). A: Cluster heatmap, red signifies a relatively high gene expression level, while green signifies a low level. The data were analyzed (n = 3) and clustered by targets using the Reference Gene Selection Tool from Bio-Rad CFX Maestro software. B: Bar plot of the relative expression of all genes (belonging to SSBR and DSBR) calculated via the ΔΔCt method. The bars correspond to the mean ± SEM (n = 3). P-values were considered significant: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001