Impact of Endocytosis and Lysosomal Acidification on the Toxicity of Copper Oxide Nano- and Microsized Particles: Uptake and Gene Expression Related to Oxidative Stress and the DNA Damage Response.

Wera Hubele,Bettina Maria Strauch,Andrea Hartwig

doi:10.3390/nano10040679

Wera Hubele, Bettina Maria Strauch + Show 1 more

Open Access

https://doi.org/10.3390/nano10040679

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Abstract

The toxicity of the copper oxide nanoparticles (CuO NP) has been attributed to the so-called “Trojan horse”-type mechanism, relying on the particle uptake and extensive intracellular release of copper ions, due to acidic pH in the lysosomes. Nevertheless, a clear distinction between extra- and intracellular-mediated effects is still missing. Therefore, the impact of the endocytosis inhibitor hydroxy-dynasore (OH-dyn), as well as bafilomycin A1 (bafA1), inhibiting the vacuolar type H+-ATPase (V-ATPase), on the cellular toxicity of nano- and microsized CuO particles, was investigated in BEAS 2 B cells. Selected endpoints were cytotoxicity, copper uptake, glutathione (GSH) levels, and the transcriptional DNA damage and (oxidative) stress response using the high-throughput reverse transcription quantitative polymerase chain reaction (RT-qPCR). OH-dyn markedly reduced intracellular copper accumulation in the cases of CuO NP and CuO MP; the modulation of gene expression, induced by both particle types affecting especially HMOX1, HSPA1A, MT1X, SCL30A1, IL8 and GADD45A, were completely abolished. BafA1 lowered the intracellular copper concentration in case of CuO NP and strongly reduced transcriptional changes, while any CuO MP-mediated effects were not affected by bafA1. In conclusion, the toxicity of CuO NP depended almost exclusively upon dynamin-dependent endocytosis and the intracellular release of redox-active copper ions due to lysosomal acidification, while particle interactions with cellular membranes appeared to be not relevant.

Highlights

Copper oxide nanoparticles (CuO NP) are increasingly applied as catalysts, additives in plastics, or as antimicrobial agents [1]
The modulation of zinc-binding structures which are present in several DNA repair and tumor suppressor proteins, and in redox-regulated transcription factors, may lead to an interference with the DNA damage response system, either directly or indirectly via Reactive oxygen species (ROS) as second messengers; this may further contribute to the toxicity of CuO NP [11,12]
CuO NP were approximately spherical, displayed a narrow size range (20–200 nm, with a calculated average diameter of 55 nm based on the specific surface area, and 146 nm in cell culture medium containing 10% Fetal calf serum (FCS) derived from the Dynamic light scattering (DLS) measurements), a surface area of 17.23 m2/g, and a zeta potential (ZP) of −13.1 mV in Dulbecco’s modified Eagle’s medium (DMEM)/FCS [7]

Summary

Introduction

Copper oxide nanoparticles (CuO NP) are increasingly applied as catalysts, additives in plastics, or as antimicrobial agents [1]. A CuO NP-induced intracellular copper overload was reported and proposed to be most decisive for adverse reactions [2,3,4,5,6,7,8], named as the “Trojan horse” mechanism [2,9]. A pronounced dose-dependent impact on copper uptake, an oxidative stress response, DNA damage response and apoptosis-associated genes, was observed in case of the CuO NP, concomitant with a distinct copper overload in the cytoplasm, and especially the nucleus. Along with the marked activation of the DNA damage response on the transcriptional level, concentration-dependent cytotoxicity, the induction of oxidative stress, cell cycle arrest, and apoptosis, were observed by CuO NP on the functional level in BEAS-2B cells [8]

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