Copper oxide nanoparticles (CuO NP) are increasingly applied as catalysts, additives in plastics, or as antimicrobial agents [1
]. Thus, a detailed toxicological knowledge is important as a prerequisite to ensure an appropriate risk assessment due to enhanced exposure and the potential risk of adverse health effects, especially with respect to occupational exposure. Pronounced cytotoxic and genotoxic effects of CuO NP, compared to other metal-based nanoparticles, bigger counterparts of the same chemical composition, as well as water soluble copper compounds, was repeatedly demonstrated in various cell culture models. As an underlying reason, a CuO NP-induced intracellular copper overload was reported and proposed to be most decisive for adverse reactions [2
], named as the “Trojan horse” mechanism [2
According to this theory, the particles are taken up via endocytic pathways, delivering large amounts of CuO within vesicles, which are further processed into acidic lysosomes, leading to an enhanced dissolution of the particles, and resulting in an extensive intracellular overload with copper ions. Thus, strict homeostatic control of copper uptake via transporters like high-affinity copper transporter 1 (hCTR1) is bypassed, and redox-active copper ions provoke the oxidative stress induced by hydroxyl radicals via Fenton-type reactions, causing oxidative damage to DNA, lipids and proteins [10
]. Furthermore, the high thiol affinity of copper ions turns redox-sensitive cysteines, present for example in zinc-binding protein structures, but also in the active sites of several enzymes, into important targets of copper toxicity. 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
]. 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. Selected genes were related to metal homeostasis, an (oxidative) stress response as well as DNA repair, cell cycle regulation and apoptosis [13
]. 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. 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. Supporting evidence is provided by the demonstration of CuO NP uptake via different endocytic pathways [14
]. Nevertheless, the distinction between the potential effects induced by the particles extracellularly, e.g., via interactions with membrane receptors, and effects arising due to intracellular copper ion release in acidic lysosomes, remains to be established.
Therefore, within the present study, the impact of the dynamin-dependent endocytosis inhibitor hydroxy-dynasore (OH-dyn) [19
], and the specific inhibitor of the vacuolar type H+
-ATPase (V-ATPase) bafilomycin A1 (bafA1) (which prevents lysosomal acidification [21
]), on the cellular copper uptake, cytotoxicity, and especially the modulation of gene expression by CuO NP, was investigated. Since the lung is the major target of CuO NP toxicity, experiments were performed in epithelial bronchial non-tumorigenic BEAS-2B cells, widely used for mechanistic in-vitro studies associated with lung toxicity, including lung carcinogenesis, and well suited for NP-induced gene expression analyses [8
]. The effects of CuO NP were compared to CuO MP and water-soluble CuCl2
to distinguish between the particle, nanoparticle or copper ion-mediated effects, and to identify potential unspecific or side-effects of the inhibitor substances.
Our results confirmed a relevant participation of dynamin-dependent endocytosis in the uptake of CuO NP and CuO MP by the use of OH-dyn. Furthermore, bafA1 was effective in preventing CuO NP-induced cytotoxicity and glutathione (GSH) depletion, coincident with decreased intracellular copper accumulation assumed to result from the stalled uptake of CuO NP, and thus diminishing the release of redox-reactive copper ions in less acidic lysosomes. The gene expression analyses confirmed that the CuO NP-mediated modulation of gene expression greatly depends on both endocytosis and lysosomal acidification, and consequently on intracellular copper ion release.
2. Materials and Methods
Chemicals, including agarose, salts, glycerol, leupeptine, phenylmethanesulfonyl-fluoride (PMSF), bovine serum albumin, acids, snap-on lid glasses and stirring bars, were obtained from Carl Roth GmbH (Karlsruhe, Germany). CuO NP (#544868, Lot #MKAA0633), CuO MP (#208841, Lot #MKAA1788) and copper chloride were purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). All PCR consumables, including PCR tubes, strips and reaction tubes and tubules, as well as cell culture dishes and flasks, were obtained from Sarstedt (Nuembrecht, Germany). The primer pairs were synthesized by Eurofins (Ebersberg, Germany) or Fluidigm (San Francisco, CA, USA). OH-dyn, Dulbecco’s modified Eagle’s medium (DMEM), dimethyl sulfoxide (DMSO), trypsin, amphotericin B, trypsin inhibitor from glycine max (soybean) (SBTI) and penicillin-streptomycin solutions are products of Sigma-Aldrich. Fetal calf serum (FCS) and LHC-9 media are products of Invitrogen GmbH (Darmstadt, Germany). BafA1 was purchased from Santa Cruz Biotechnology (Heidelberg, Germany). Human fibronectin was obtained from Biopur (Reinach, Switzerland) and collagen from Roche (Mannheim, Germany). DNA suspension buffer, PCR-certified water and TE buffer were obtained from Teknova (Hollister, CA, USA). 2X Assay Loading Reagent and 20X DNA Binding Dye Sample Loading Reagent were purchased from Fluidigm (San Francisco, CA, USA). Bio-Rad (Munich, Germany) provided the 2X SsoFastTM EvaGreen® Supermix with Low ROX and the 2X SYBR Green Supermix. The 2X TaqMan® PreAmp Master Mix was obtained from Applied Biosystems (Darmstadt, Germany) and the exonuclease I from New England Biolabs (Frankfurt am Main, Germany).
2.2. Particle and CuCl2 Incubation Suspensions and Dilutions
Fresh CuO NP and CuO MP suspensions, as well as CuCl2 dilutions, were prepared for each experiment. Particles, received as dry powder, were aliquoted by weighing into 1.5 mL polystyrene reaction tubes. Stock solutions of 1 mg/mL CuO were prepared in an endotoxin-free snap-on glass, containing a stirring bar in DMEM containing 10% FCS. Dilutions in the range of 5, 10, 20 and 50 µg/mL were prepared by adding aliquots of the stirring stock solution into snap-on lid glasses filled with adequate volumes of fresh medium. Stirring took place at 900 rpm and room temperature on a multiphase stirrer (Variomag Poly, Carl Roth GmbH, Karlsruhe, Germany). CuCl2 was dissolved in bi-distilled H2O (100 mM) and sterile-filtered. The dilution to 630 µM corresponding to 50 µg/mL CuO was prepared from the stock solution by dilution in an adequate amount of DMEM containing 10% FCS directly before incubation. Based on the copper content, 1 µg/mL CuO is equal to 0.2 µg/cm2 CuO and 12.6 µM Cu2+ in case of complete dissolution.
2.3. Cell Culture and Incubation
Human lung bronchial epithelial BEAS-2B cells (ATCC CRL-9609), immortalized with SV40 large T-antigen, were kindly provided by Dr. Carsten Weiss (Karlsruhe Institute of Technology, Karlsruhe, Germany). They were grown as monolayers in coated cell culture dishes (100 mm, 58 cm2, coated with 10 µg/mL human fibronectin, 30 µg/mL collagen and 10 µg/mL bovine serum albumin in phosphate-buffered saline (PBS)) in 12 mL LHC-9 medium containing 2.5 µg/mL amphotericin B. Cells were incubated at 37 °C in a humidified atmosphere of 5% CO2 in air. For all of these experiments, the cells were seeded at a density of 16,600 cells/cm2. After 48 h the supernatant was removed from the logarithmically growing cells, and replaced by the particle or CuCl2 incubation suspensions (0.2 mL/cm2) in DMEM containing 10% FCS, as described for the respective experiments. For co-incubation with the endocytosis inhibitor, BEAS-2B cells were pre-incubated with 100 µM OH-dyn for 30 min, and afterwards co-treated with CuO NP, CuO MP or CuCl2 and OH-dyn for 8 h. Co-incubation of the copper compounds with 100 nM bafA1 occurred for 24 h without pre-treatment.
Since stock solutions of OH-dyn (20 mM) and bafA1 (50 µM) were prepared in DMSO, the same amount of DMSO was added to the respective controls without the inhibitor substances.
2.4. Cell Number
Logarithmically growing BEAS-2B cells were incubated for 24 h with 10 µg/mL CuO NP, 50 µg/mL CuO MP or 630 µM CuCl2, with or without 100 nM bafA1 or 100 µM OH-dyn, respectively, trypsinized and collected in DMEM containing 10% FCS. Cell number was determined via Casy® cell counter (OLS OMNI Life Science GmbH and Co. KG, Bremen, Germany).
2.5. Gene Expression Analyses
0.5–1 × 106
logarithmically growing BEAS-2B cells were treated with different concentrations of CuO NP, CuO MP or CuCl2
, with or without 100 µM OH-dyn or 100 nM bafA1 for 8 h or 24 h, respectively, in DMEM containing 10% FCS. Subsequently, gene expression analyses via high-throughput RT-qPCR with Fluidigm dynamic arrays on the BioMarkTM System were performed, as described previously [13
]. For normalization, five potential reference genes were available (ACTB
). Finally, potential alterations of the transcript levels of the target genes under investigation were displayed as fold change compared to the respective control group, by calculating relative quantities corresponding to the ΔΔCq method [23
2.6. Cellular Copper Uptake
Logarithmically growing BEAS-2B cells were treated with 10 µg/mL CuO NP, 50 µg/mL CuO MP or 630 µM CuCl2, with or without 100 µM OH-dyn or 100 nM bafA1, respectively, for 8 h or 24 h, respectively, in DMEM containing 10% FCS. The cells were trypsinized, collected in ice-cold PBS containing 10% FCS, and washed twice with PBS. Cell number and cell volume were determined via Casy® cell counter. To quantify the intracellular copper levels in the soluble cell fractions, and thus to eliminate particles attached at the outer cell membrane, the cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (0.01 M Tris pH 7.6, 0.15 M NaCl, 0.001 M ethylenediaminetetraacetic acid (EDTA), 1% (v/v) Trition-X 100, 1% (v/v) desoxycholic acid, sodium salt, 0.01% sodium dodecyl sulfate (SDS) 0.001 M phenylmethylsulfonyl fluoride (PMSF), 1 × protease-inhibitor mixture) for 30 min before centrifugation at 14,000 × g (1 h, 4 °C), thereby precipitating the cell membranes. The supernatant contained the soluble cell fraction of both the cytoplasm and nucleus, and its copper content was determined using GF-AAS (Perkin Elmer Atomic Absorption Spectrometer PinAAcle 900T, Rodgau, Germany). The solution was evaporated at 95 °C, incubated with ashing mixture, 65% HNO3/30% H2O2 (1/1), evaporated at 95 °C again, and resolved in water. Copper content was normalized to cell number and cell volume.
2.7. Quantification of Intracellular Glutathione
The quantification of intracellular glutathione levels was performed according to the method established by Tietze et al. [24
]. Briefly, logarithmically growing BEAS-2B cells were incubated for 2 h with 10 µg/mL CuO NP, with or without 100 nM BafA1, respectively, in DMEM containing 10% FCS. The cells were trypsinized, collected in ice-cold PBS containing 10% FCS, washed with PBS, analyzed via Casy®
cell counter for cell number and cell volume and collected by centrifugation. 1 × 106
cells were collected in KP buffer (0.1 M KH2
, 0.1 M K2
, 1 mM EDTA, pH 7.4). Cells were lyzed by 2 freeze-and-thawing cycles followed by sonification, acidification with sulphosalicyclic acid and vortexing. Cells were centrifuged at 16,000 rpm for 20 min at 4 °C prior to measuring total GSH in the supernatant by reducing oxidized GSH content using GR enzyme (4 U/mL) and the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) (0.3 mM). GSH reacts with 5,5’-dithiobis-2-nitrobenzoic acid (DTNB) to form 2-nitro-5-thiobenzoate (TNB). The change in TNB absorbance was measured at 412 nm using a plate reader (Tecan). Data were compared to GSH standard calibration curves and normalized to cell volume.
Differences between control and treated samples were analyzed by one-way analysis of variance (ANOVA), followed by Dunnett’s T post hoc test, and the differences between the different incubation conditions with and without OH-dyn or bafA1 were analysed by T-test, respectively.
Within the present study, the impact of the dynamin-dependent endocytosis inhibitor OH-dyn [19
], and the vacuolar-type H+
-ATPase inhibitor bafA1 attenuating lysosomal acidification [21
] on cytotoxicity, cellular copper uptake, and especially the modulation of cellular signaling pathways involved in the maintenance of genomic stability by CuO NP, as compared to CuO MP, was investigated.
As a prerequisite to estimate the impact of endocytosis, to quantify copper uptake and to discriminate between the cellular effects provoked by copper particle interactions at the outer cell membrane, and those derived from intracellular copper, a post-treatment protocol was applied. Within this procedure, the cells were lysed, and the membranes precipitated by centrifugation. Subsequently, the copper levels were quantified in the soluble cell fraction, excluding copper particles attached to the cell surface, which are not easily detached by washing procedures, thereby avoiding any overestimation of copper uptake. Intracellular copper concentrations were determined by AAS, and calculated based on the cell volume of BEAS 2B cells [8
]; no discrimination between copper particles and copper ions is possible by this approach.
In the absence of OH-dyn or bafA1, a pronounced copper accumulation up to 1 mM after 8 h or 24 h treatment with CuO NP was observed, while a lower increase in cellular copper was evident after incubation with CuO MP or CuCl2
, confirming previous observations in this cell line [8
]. In case of CuCl2
, neither OH-dyn nor bafA1 affected the intracellular copper content, thus excluding any unspecific interactions between the inhibitors and copper uptake or homeostasis.
On the contrary, OH-dyn caused a pronounced reduction of intracellular copper accumulation in the case of CuO NP and—even more pronounced—in the case of CuO MP, confirming their uptake via endocytosis. Indeed, dynamin-dependent endocytosis was identified as the main route for the internalization of the CuO NP, since a reduction of intracellular copper levels of about 50% was observed; considering the solubility of about 30% of the CuO NP in the cell culture medium (DMEM/FKS) within the 8 h time frame [7
], at least part of the residual increase in intracellular copper is likely to result from extracellularly-released copper ions taken up via respective ion transporters. This assumption is also in agreement with the stronger inhibition of OH-dyn towards CuO MP uptake, since almost no solubility in the same cell culture medium was observed [7
]. Nevertheless, a small impact of dynamin-independent endocytosis not inhibited by OH-dyn cannot be excluded [19
In contrast to OH-dyn, an impact of the “lysosomal neutralizer” bafA1 was restricted to CuO NP, diminishing the amount of intracellular copper accumulation to about 60%. On the first sight, this observation is surprising, since this inhibitor would only be expected to inhibit solubilization and copper ion release in the lysosomal environment, and by applying AAS to quantify intracellular copper content, no discrimination between particulate and ionic copper is possible. However, if the lysosomes do not get acidified, the complete endocytic process is disturbed, and the internalized particles remain within the lysosomes, thereby preventing the recycling of protein or lipids essential for this process [28
]. Consequently, endocytosis of the particles is slowed down and reduced during the incubation period of 24 h. The impact of the diminished release of redox-reactive copper ions by CuO NP in less acidic lysosomes became obvious when considering the other endpoints investigated within this study. Thus, co-treatment with bafA1 strongly reduced the cytotoxicity of CuO NP and prevented the CuO NP-induced GSH depletion after 2 h, indicating that oxidative stress occurs due to increased intracellular levels of copper ions. In support of this theory, an enhanced toxicity of CuO NP due to the increased intracellular solubility in an acidic environment resulting in elevated levels of copper ions, was also reported by other working groups [30
]. In contrast to CuO NP, no impact of bafA1 was observed in case of CuO MP, in agreement with the low solubility of CuO MP in artificial lysosomal fluid (ALF), as compared to CuO NP published previously [7
In the absence of either inhibitor, gene expression profiling after 8 h treatment with CuO NP revealed a strictly concentration-dependent induction of mainly so-called early response genes, namely MT1X
, and TXNRD1
, revealing elevated intracellular copper levels, the induction of oxidative stress, inflammation, DNA damage, inflammatory response, as well as an activation of the transcription factors AP-1 and MTF-1 [32
]. A similar pattern was observed in case of CuO MP, however, far less pronounced. These observations highly corresponded to the previously published, detailed gene expression analyses with the same copper particles [8
]. However, co-treatment with OH-dyn completely diminished the CuO NP- as well as CuO MP-induced gene inductions. This indicates that the observed changes in gene expression can be exclusively assigned to intracellular copper, with no impact of particle interactions with the outer membrane.
Gene expression analyses after 24 h exposure to CuO particles revealed almost the same pattern as compared to the 8 h treatment; however, respective modulations were more pronounced. In the presence of bafA1, CuO NP-mediated gene inductions were reduced to a high degree by bafA1 in the case of the ROS-inducible HMOX1
, genotoxic stress-inducible GADD45A
, pro- inflammatory IL8
, as well as the copper uptake related MT1X
. Thus, with respect to the modulation of those stress responsive genes, the acidification of lysosomes, and in consequence the release of copper ions, were of major importance, signaling elevated levels of ROS, with the consequence of oxidative and genotoxic stress, as well as inflammatory effects [34
]. These protective interactions in the presence of bafA1 correspond to the reduced intracellular copper accumulation, the prevented GSH-depletion and reduced cytotoxicity of CuO NP in the presence of bafA1 on the functional level.
On the other hand, no impact of bafA1 was observed concerning the transcriptional modulation of the pro-apoptotic gene PMAIP1
, the proto-oncogene coding JUN
assigned to AP-1 activation [32
] and the anti-oxidative genes GCLC
mainly induced by activated Nrf2 [40
]. Thus, the amount of intracellular copper and ROS generation in the presence of bafA1 seemed still sufficient for the activation of these genes, which may again be explained by uptake of copper ions released extracellularly from the CuO NP, but also from low level intracellular copper ion release in the presence of bafA1. In case of CuO MP, except for HSPA1A
, no impact of bafA1 on gene expression alterations was observed, in agreement with its low solubility, even in the acidic lysosomal environment.