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

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12989-017-0209-1) contains supplementary material, which is available to authorized users.

Cytotoxicity of vanadium oxide nanoparticles and titanium dioxide‐coated vanadium oxide nanoparticles to human lung cells

“…Bronchial epithelial cell line (BEAS‐2B) was kindly provided by Dr. Baoxin Bai of Shanghai East Hospital, China. They are the two mostly used lung cell models in literature (Louro et al, ; Strauch, Niemand, Winkelbeiner, & Hartwig, ) and similar in many aspects, but they have differences in phenotype and proliferation. A549 is a cancer cell belongs to type II lung epithelial cells and can differentiate to other lung cells, while BEAS‐2B is a non‐cancer cell.…”

“…When conducting toxicological assessments on lungs, many cell types could be used, though they have differences in phenotypes, morphologies, proliferation rates, mRNA and protein expressions, antioxidant capacities, pro‐inflammatory states, and cell cycle distributions (Lujan, Criscitiello, Hering, & Sayes, ). Here, both human alveolar epithelial cell line A549 and human bronchial epithelial cell line BEAS‐2B were used, for they are the two mostly used lung cell models in literature (Louro et al, ; Strauch, Niemand, Winkelbeiner, & Hartwig, ). A549 cells maintain many morphological and biochemical characteristics of pneumocytes type II (Lieber, Smith, Szakal, Nelson‐Rees, & Todaro, ) and BEAS‐2B cells are targets of inhalation and play an important role in the maintenance of mucosal integrity as mechanical barriers against various particulate (Takizawa, ).…”

Essentielle Spurenelemente und toxische Metallverbindungen

“…Während bei oraler Aufnahme die Resorption über den Magen‐Darm‐Trakt reguliert wird und nicht internalisierte Anteile über die Fäzes ausgeschieden werden, sind ähnlich effektive Regulationsmechanismen nach Inhalation nicht zu erwarten. Dies trifft beispielsweise für metallhaltige Nanopartikel zu, die sich in der Lunge ablagern und nach Endozytose aufgrund des sauren pH‐Wertes in den Lysosomen zu einer ausgeprägten intrazellulären Freisetzung von Metallionen führen können, die in sehr hohen, unter physiologischen Bedingungen nicht erreichbaren Metallionenkonzentrationen resultieren .…”

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

“…Far weaker effects were observed after treatment with micro-sized CuO particles, or with water-soluble copper chloride. Furthermore, 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].Following up on these observations, different aspects still need to be clarified. Even though the observations may well be explained by a Trojan horse-type mechanism described above, a clear link, as well as the exclusion of other direct cellular interactions of the NP, are still missing.…”

“…Accordingly, the modulation of zinc-binding structures which are present in several DNA repair and tumor suppressor proteins, but also in redox-regulated transcription factors, may lead to an interference with the DNA damage response system, either directly or indirectly via ROS as second messengers; this may further contribute to the toxicity of CuO NP [11,12]. The modulation of gene expression by CuO NP was investigated in detail in a previous study of our working group in adenocarcinoma A549 and BEAS-2B lung cells [8]. Gene expression profiling was performed by a high-throughput reverse transcription quantitative polymerase chain reaction (RT-qPCR) technique, quantifying the impact of 96 samples in parallel on the expression of 95 genes crucial for maintaining genomic stability.…”