Comparison between micro- and nanosized copper oxide and water soluble copper chloride: interrelationship between intracellular copper concentrations, oxidative stress and DNA damage response in human lung cells

Bettina Maria Strauch,Nicola Lisa Winkelbeiner,Rebecca Katharina Niemand,Andrea Hartwig

doi:10.1186/s12989-017-0209-1

Bettina Maria Strauch, Nicola Lisa Winkelbeiner + Show 2 more

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https://doi.org/10.1186/s12989-017-0209-1

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Abstract

BackgroundNano- and microscale copper oxide particles (CuO NP, CuO MP) are applied for manifold purposes, enhancing exposure and thus the potential risk of adverse health effects. Based on the pronounced in vitro cytotoxicity of CuO NP, systematic investigations on the mode of action are required. Therefore, the impact of CuO NP, CuO MP and CuCl2 on the DNA damage response on transcriptional level was investigated by quantitative gene expression profiling via high-throughput RT-qPCR. Cytotoxicity, copper uptake and the impact on the oxidative stress response, cell cycle regulation and apoptosis were further analysed on the functional level.ResultsCytotoxicity of CuO NP was more pronounced when compared to CuO MP and CuCl2 in human bronchial epithelial BEAS-2B cells. Uptake studies revealed an intracellular copper overload in the soluble fractions of both cytoplasm and nucleus, reaching up to millimolar concentrations in case of CuO NP and considerably lower levels in case of CuO MP and CuCl2. Moreover, CuCl2 caused copper accumulation in the nucleus only at cytotoxic concentrations. Gene expression analysis in BEAS-2B and A549 cells revealed a strong induction of uptake-related metallothionein genes, oxidative stress-sensitive and pro-inflammatory genes, anti-oxidative defense-associated genes as well as those coding for the cell cycle inhibitor p21 and the pro-apoptotic Noxa and DR5. While DNA damage inducible genes were activated, genes coding for distinct DNA repair factors were down-regulated. Modulation of gene expression was most pronounced in case of CuO NP as compared to CuO MP and CuCl2 and more distinct in BEAS-2B cells. GSH depletion and activation of Nrf2 in HeLa S3 cells confirmed oxidative stress induction, mainly restricted to CuO NP. Also, cell cycle arrest and apoptosis induction were most distinct for CuO NP.ConclusionsThe high cytotoxicity and marked impact on gene expression by CuO NP can be ascribed to the strong intracellular copper ion release, with subsequent copper accumulation in the cytoplasm and the nucleus. Modulation of gene expression by CuO NP appeared to be primarily oxidative stress-related and was more pronounced in redox-sensitive BEAS-2B cells. Regarding CuCl2, relevant modulations of gene expression were restricted to cytotoxic concentrations provoking impaired copper homoeostasis.

Highlights

Nano- and microscale copper oxide particles (CuO NP, CuO MP) are applied for manifold purposes, enhancing exposure and the potential risk of adverse health effects
The high cytotoxicity and marked impact on gene expression by CuO NP can be ascribed to the strong intracellular copper ion release, with subsequent copper accumulation in the cytoplasm and the nucleus
Particle characterization The particulate copper compounds CuO NP and CuO MP were characterized in detail with respect to size, surface area and charge, chemical composition, crystallinity and morphology via dynamic light scattering (DLS), scanning electron microscopy (SEM), Brunauer-EmmetTeller (BET) analysis, zeta potential (ZP), inductively coupled plasma mass spectrometry (ICP-MS), energydispersive X-ray spectroscopy (EDX) as well as X-ray diffraction (XRD) as previously described

Summary

Introduction

Nano- and microscale copper oxide particles (CuO NP, CuO MP) are applied for manifold purposes, enhancing exposure and the potential risk of adverse health effects. Cytotoxicity, copper uptake and the impact on the oxidative stress response, cell cycle regulation and apoptosis were further analysed on the functional level. While the redox-activity is required for essential functions, copper ions may be toxic due to the catalysis of Fenton-type reactions, which may lead to the formation of highly reactive hydroxyl radicals, provoking oxidative damage to lipids, proteins and DNA [7]. While copper uptake occurs usually via food and drinking water by the oral route, which is tightly regulated, the situation may be different in case of inhalation of copper fumes or aerosols in exposed workers, leading potentially to cellular and/or systemic copper overload

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