Effect of silver nanoparticles (AgNPs) exposure on microRNA expression and global DNA methylation in endothelial cells EA.hy926

https://doi.org/10.1016/j.etap.2020.103543Get rights and content

Highlights

  • AgNPs decrease the viability of EA.hy926 cells.

  • AgNPs modify the expression of miR-126, 146 and 155 and endothelial markers as VCAM-1.

  • AgNPs induce a hypermethylation state of genomic DNA.

Abstract

The aim of this study was to determine the effect of AgNPs on the epigenome of endothelial cells EA.hy926, including the levels of expression of microRNAs (miRNAs) and global DNA methylation patterns. In addition, evaluation of the expression of inflammatory genes and the levels of VCAM-1 protein (miRNA-126 target) was performed. The expression levels of analyzed miRNAs (microRNAs-126, 155 and 146) were reduced significantly and there were not observed changes in inflammatory gene expression. Regarding the levels of protein vascular cell adhesion molecule 1 (VCAM-1), they increase significantly to 0.5 μM AgNPs at 24 h of exposure. As far as DNA methylation is concerned, we found that AgNPs induce a state of global hyper-methylation. In conclusion, it was demonstrated that direct contact between AgNPs and endothelial cells resulted in the dysregulation of highly enriched and vastly functional miRNAs and DNA hypermethylation, that may have multiple effects on endothelium function and integrity.

Introduction

The nanotechnology has emerged as one of the major technological progress due to its impact on electronics, medical health care, engineering, environmental remediation, consumer products and other, through the use of atomic-scale tailoring of materials (Fu et al., 2014; Hubbs et al., 2013; Medina et al., 2007; Radomska et al., 2016; Science and Council, 2000). In this regard, metallic nanoparticles have attracted significantly the scientific attention for their diversity and their wide-spread applications (Ahn et al., 2013; Luo et al., 2015; Mu and Sprando, 2010; Tourinho et al., 2012; Zhang et al., 2009). In this context, silver nanoparticles (AgNPs) are one of the most regularly used nanomaterials in consumer goods because of their potential antibacterial effect (Dizaj et al., 2014). AgNPs can be found mainly in biomedical applications, food preservation, and the textile industry (Schluesener and Schluesener, 2013; You et al., 2012). The use of AgNPs in a large number of products are significantly increasing the potential exposure to these compounds in humans. Despite the above, it is still limited the biological and toxicological information available about these nanoparticles and their effects on human health (Bondarenko et al., 2013; Fard et al., 2015).

Current studies have shown that AgNPs can easily enter the bloodstream and, therefore, be in contact with the endothelial cells of the blood vessels. Endothelial cells are a biological barrier that plays an essential role in the regulation, maintenance and control of vascular function (Sun et al., 2016). The compilation of in vitro and in vivo studies have shown that AgNPs are cytotoxic in endothelial cells, causing several effects, including reactive oxygen species (ROS) production, apoptosis, disruption of signal pathways, DNA damage, inflammation, endothelial activation, and injury (Chen et al., 2016; Kang et al., 2011; Shi et al., 2014; Sun et al., 2017). Consequently, it is crucial to develop new methods to evaluate the toxicity of these nanoparticles in order to understand better the mechanisms by which these effects occur; this is of great importance in endothelial cells because the dysfunction of these may be the leading cause for the development of cardiovascular diseases.

In this regard, epigenetics can help us to understand how AgNPs alter protein synthesis and inhibition molecules, and how they carry out their adverse effects. The DNA methylation and microRNAs (miRNAs) are epigenetic mechanisms that can help at the understanding of biology and medicine of AgNPs, and its implications, for future research. In the last decade, miRNAs have been described as important regulatory molecules in genetic expression at the post-translational level. These miRNAs are small non-coding RNA molecules (containing about 20 nucleotides) involved in many cellular processes, and its regulation is an emerging epigenetic mechanism (Bartel, 2009). Changes in the expression profile of miRNAs can globally modify the production of certain proteins, such as inflammatory cytokines; as well as the course of vital biological processes like growth, death, development and cell differentiation (Asirvatham, 2008; Saliminejad et al., 2019; Tsuchiya et al., 2006). Different studies have identified the participation of miRNA-126, miRNA-146 and miRNA-155 in the regulation of different cardiovascular processes, for example, angiogenesis, endothelial cell function, vascular repair, leukocyte recruitment and protective/anti-inflammatory role (Chistiakov et al., 2016; Faraoni et al., 2009; Wei et al., 2013). On the other hand, DNA methylation patterns, in CpG islands, modulate the transcription of DNA and play an essential role in the regulation of gene expression (Curradi et al., 2002). Some effects on global DNA methylation in HepG2 cells by AgNPs have been reported (Brzóska et al., 2019). However, their effect on endothelial cells is unknown.

Thus, the increase in the production and use of metallic NPs, creates an imminent need to evaluate potential health risks and, consequently, to develop prevention strategies for the proper handling of these materials. Therefore, the present study aims to determine the in vitro effect of AgNPs on the epigenome, including the expression of miRNAs (miRNA-126, miRNA-146, and miRNA-155) and global DNA methylation patterns.

Section snippets

Silver nanoparticles

AgNPs tested in this study were synthesized and characterized previously by our working group as described by Orta et al. (2015a). These AgNPs have a mean size below 2 nm (1.8 ± 0.3 nm in diameter), with a narrow size distribution. The zeta-potential of AgNPs in aqueous solution was -23.8 ± 1.54 mV (Orta et al., 2015a, b).

Cell culture

The human endothelial cell line (EA.hy926 cells) expresses highly differentiated functions, that are characteristic of human vascular endothelium (Edgell et al., 1983, 1990).

Cell viability assay

Cytotoxic effects of AgNPs on EA.hy926 cells, dosed at different concentrations, were evaluated by measuring cell viability (using Resazurin assay) at different times after treatments. Fig. 1 shows the viability of the EA.hy926 cells after AgNPs treatments. According to ISO 10993-5, 2009, the cell viability higher than 80 % is considered non-cytotoxic for "in vitro" assays. In this regard, non-cytotoxic properties of AgNPs were observed after EA.hy926 cells were dosed with 0.1, 0.5, 1.0, 5.0,

Discussion

The use of nanomaterials in diverse applications (medical, industrial, textile, among others), has been increased in recent years. However, the adverse effects that may be caused by exposure to these nanomaterials are not fully understood. Therefore, a complete understanding of their adverse effects is necessary. Recently, AgNPs have gained an augmented interest because they have been used for a wide variety of products for human consumption (Vance et al., 2015). Although, cytotoxicity of these

Conclusion

In this study, it was demonstrated that direct contact between AgNPs and endothelial cells resulted in dysregulation of highly enriched and vastly functional miRNAs (-126, -155, and -146), and pattern DNA methylation that may have multiple effects on endothelium function and integrity. However, further studies should be carried out to elucidate the mechanism involved in miRNA change expression and to understand the real meaning of this miRNA dysregulation on endothelium homeostasis, as well as

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

CRediT authorship contribution statement

A.K. González-Palomo: Methodology, Writing - original draft, Supervision. K. Saldaña-Villanueva: Methodology, Conceptualization. J.D. Cortés-García: Methodology, Formal analysis. J.C. Fernández-Macias: Writing - review & editing. K.B. Méndez-Rodríguez: Methodology. I.N. Pérez Maldonado: Resources, Supervision.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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