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

• Published in: Particle and fibre toxicology 2017-08, Vol.14 (1), p.28-17, Article 28
• Main Authors: Strauch, Bettina Maria, Niemand, Rebecca Katharina, Winkelbeiner, Nicola Lisa, Hartwig, Andrea
• Format: Article
• Language: English
• Subjects: A549 Cells; Accumulation; Analysis; Apoptosis; Cancer; Cell cycle; Cell Nucleus - drug effects; Cell Nucleus - metabolism; Cell Survival - drug effects; Copper; Copper - analysis; Copper - chemistry; Copper - toxicity; Copper chloride; Copper oxide nanoparticles; Copper oxides; Copper uptake and intracellular distribution; Cytoplasm; Cytoplasm - drug effects; Cytoplasm - metabolism; Cytotoxicity; Deoxyribonucleic acid; DNA; DNA Damage; DNA damage response; DNA repair; Dose-Response Relationship, Drug; Gene expression; Gene expression profiling; Genes; Genetic aspects; GTP-binding protein; Health aspects; Health risks; High-throughput RT-qPCR; Homeostasis; Humans; Impact damage; In vitro methods and tests; Inflammation; Intracellular; Lungs; Metallothionein; Mode of action; Modulation; Nanoparticles; Nanoparticles - analysis; Nanoparticles - chemistry; Nanoparticles - toxicity; Nuclei; Oxidative stress; Oxidative Stress - drug effects; Particle Size; Physiological aspects; Political aspects; Properties; Proteins; Risk factors; Solubility; Toxicity; Transcription; Transcriptome - drug effects
• ISSN: 1743-8977; 1743-8977
• DOI: 10.1186/s12989-017-0209-1

Nano- 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 CuCl 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. Cytotoxicity of CuO NP was more pronounced when compared to CuO MP and CuCl 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 CuCl . Moreover, CuCl 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 CuCl 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. 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. 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 CuCl , relevant modulations of gene expression were restricted to cytotoxic concentrations provoking impaired copper homoeostasis.