Impact of Short-Term Exposure to Non-Functionalized Polystyrene Nanoparticles on DNA Methylation and Gene Expression in Human Peripheral Blood Mononuclear Cells

Abstract

The aim of the present study was to investigate the concentration- and size-dependent effects of non-functionalized polystyrene nanoparticles (PS-NPs) of varying diameters (29 nm, 44 nm, and 72 nm) on specific epigenetic modifications and gene expression profiles related to carcinogenesis in human peripheral blood mononuclear cells (PBMCs) in vitro. This in vitro human-cell-based model is used to investigate the epigenetic effect of various environmental xenobiotics. PBMCs were exposed to PS-NPs at concentrations ranging from 0.001 to 100 µg/mL for 24 h period. The analysis encompassed epigenetic DNA modifications, including levels of 5-methyl-2'-deoxycytidine (5-mdC) and 5-(hydroxymethyl)-2'-deoxycytidine (5-hmdC), as well as the levels of 2'-deoxyuridine (dU) and 5-(hydroxymethyl)-2'-deoxyuridine (5-hmdU) by mass spectrometry methods, methylation in the promoter regions of selected tumor suppressor genes TP53 (P53), CDKN2A (P16), and CDKN1A (P21) and proto-oncogenes (CCND1, BCL2, BCL6), along with the expression profile of the indicated genes by real-time PCR assays. The results obtained revealed no significant changes in global DNA methylation/demethylation levels in PBMCs after short-term exposure to non-functionalized PS-NPs. Furthermore, there were no changes observed in the level of dU, a product of cytosine deamination. However, the level of 5-hmdU, a product of both 5-hmdC deamination and thymine oxidation, was increased at the highest concentrations of larger PS-NPs (72 nm). None of the PS-NPs caused a change in the methylation pattern of the promoter regions of the TP53, CDKN2A, CDKN1A, CCND1, BCL2 and BCL6 genes. However, gene profiling indicated that PS-NPs with a diameter of 29 nm and 44 nm altered the expression of the TP53 gene. The smallest PS-NPs with a diameter of 29 nm increased the expression of the TP53 gene at a concentration of 10 µg/mL, while PS-NPs with a diameter of 44 nm did so at a concentration of 100 µg/mL. An increase in the expression of the CDKN2A gene was also observed when PBMCs were exposed to PS-NPs with 29 nm in diameter at the highest concentration. The observed effect depended on both the concentration and the size of the PS-NPs.

Keywords: epigenetic DNA modifications; peripheral blood mononuclear cells; polystyrene nanoparticles; proto-oncogenes; suppressor genes.

Figure 1

The level of (A) 5-mdC and (B) 5-hmdC per 10³ dN (10³ deoxyribonucleotides) in human PBMCs incubated with PS-NPs of 29, 44, and 72 nm in diameter (0.001–100 μg/mL) for 24 h period. The data are presented as mean ± SD, n = 4 independent experiments. Statistical analysis was conducted using one-way ANOVA or Kruskal–Wallis test.

Figure 2

The level of (A) dU and (B) 5-hmdU per 10⁶ dN (10⁶ deoxyribonucleotides) in human PBMCs incubated with PS-NPs of 29, 44, and 72 nm in diameter (0.001–100 μg/mL) for 24 h period. The data are presented as mean ± SD, n = 4 independent experiments. Statistical analysis was conducted using one-way ANOVA or Kruskal–Wallis test, followed by relevant post hoc; * p < 0.05.

Figure 3

Methylation of suppressor genes (TP53, CDKN2A, CDKN1A) in human PBMCs incubated with PS-NPs of 29, 44, and 72 nm in diameter (0.001–100 μg/mL) for 24 h period. The data are presented as mean ± SD, n = 3 to 4 independent experiments. The promoter methylation experiments for 29, 44, and 72 nm were performed on separate plates, each including a respective control group (cells incubated in medium only). Consequently, baseline MI levels varied for some genes across different PS-NP sizes. Statistical analysis was conducted using one-way ANOVA or Kruskal–Wallis test.

Figure 4

Methylation of proto-oncogenes (CCND1, BCL2, BCL6) in human PBMCs incubated with PS-NPs of 29, 44, and 72 nm in diameter (0.001–100 μg/mL) for 24 h period. The data are presented as mean ± SD, n = 3 to 4 independent experiments. The promoter methylation experiments for 29, 44, and 72 nm were performed on separate plates, each including a respective control group (cells incubated in medium only). Consequently, baseline MI levels varied for some genes across different PS-NP sizes. Statistical analysis was conducted using one-way ANOVA or Kruskal–Wallis test.

Figure 5

Expression of suppressor genes (TP53, CDKN2A and CDKN1A) in human PBMCs incubated with PS-NPs of 29, 44, and 72 nm in diameter (0.001–100 μg/mL) for 24 h period. The data are presented as mean ± SD, n = 3 to 4 independent experiments. Statistical analysis was conducted using one-way ANOVA or Kruskal–Wallis test, followed by relevant post hoc test, * p < 0.05, ** p < 0.001.

Figure 6

Expression of proto-oncogenes (CCND1, BCL2, BCL6) in human PBMCs incubated with PS-NPs of 29, 44, and 72 nm in diameter (0.001–100 μg/mL) for 24 h period. The data are presented as mean ± SD, n = 3 to 4 independent experiments. Statistical analysis was conducted using one-way ANOVA or Kruskal–Wallis test.

Figure 7

Flow chart of experimental design.