In vitro evaluation of copper oxide nanoparticle-induced cytotoxicity and oxidative stress using human embryonic kidney cells

Abstract

This study aimed to evaluate the in vitro cytotoxicity and oxidative stress induced by the copper oxide nanoparticles (CuO NPs) in human embryonic kidney cell line (HEK-293) cells following exposure. CuO NPs size <50 nm were used in this study. HEK-293 cell cultures were exposed to different concentrations of CuO NPs between 3 µg/ml and 300 µg/ml and quartz (known as cytotoxic agent) and assessed for cell viability-mitochondrial function (MTT assay), cell membrane damage (lactate dehydrogenase (LDH) assay), reduced glutathione (GSH), interleukin-8 (IL-8), and lipid peroxidation levels. The IC₅₀ value of NPs was found to be 65.5 µg/ml. Exposure of HEK cells to CuO NPs (10–300 µg/ml) resulted in concentration-dependent cell membrane damage, increased production of IL-8, increased thiobarbituric acid reactive substance (TBARS), and decreased intracellular GSH levels. The significant increases in IL-8, TBARS, and LDH levels along with decreased GSH levels indicated induction of oxidative stress in cells. Our preliminary data suggest that oxidative stress might contribute to CuO NPs-induced cytotoxicity in HEK-293 cells.

Keywords

Nanoparticles copper oxide copper oxide nanoparticles human embryonic kidney-293 cells oxidative stress cytotoxicity

Introduction

Nanoparticles (NPs; <100 nm size) are widely used in almost all fields because of their unique physicochemical characteristics (Duan and Li, 2013; Linic et al., 2015; Uclésa, et al., 2015). However, usage of these NPs brings challenges to the environment and to humans. With sizes smaller than cellular organelles, NPs can easily penetrate through basic biological structures (Lai et al., 2018).

Copper oxide NPs (CuO NPs) have attracted attention and have been commonly used in industrial and commercial fields like medicine and engineering for their photovoltaic and photoconductive properties (Jo et al., 2015). Further use for CuO NPs has been employed in the pharmaceutical industry especially in the production of antimicrobial fabric treatments for the prevention of infections caused by Escherichia coli and methicillin-resistant Staphylococcus aureus (Assadian et al., 2018).

During production and use, CuO NPs are prone to diffuse through ambient air as aerosols and be retained in the lungs for a long time after inhalation (Larsen et al., 2016). Previously, we studied and reported on the pulmonary toxicity of CuO NPs in rats following exposure through intratracheal instillation (Rani et al., 2013). In vitro studies have demonstrated that CuO NPs induce cytotoxic, genotoxic, and oxidative stress against lung epithelial cells (Ahamed et al., 2015; Hanagata et al., 2011; Ivask et al., 2015). To futher explosre these findings, this study investigated the in vitro cytotoxicity and oxidative stress induced by the CuO NPs using human embryonic kidney cell line (HEK-293) cells.

Materials and methods

Chemicals

Fetal bovine serum (FBS), penicillin, amphotericin B, Dulbecco’s modified eagle’s medium (DMEM), and streptomycin were purchased from Himedia (Mumbai, India). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2-thiobarbituric acid, bovine serum albumin, and tetraethoxypropane were purchased from Sigma-Aldrich Company (St Louis, Missouri, USA). Quartz fine powder (>230 mesh; 58–68 µm; 99.94% purity) was obtained from SD fine chemicals (Mumbai, India). The glutathione (GSH), interleukin-8 (IL-8), and lactate dehydrogenase (LDH) assay kits were purchased from Ray Biotech, Inc. (New Delhi, India).

Copper oxide NPs

The test CuO nanomaterials (size < 50 nm, surface area of 29 m²/g, crystalline in shape, diameter < 50 nm, length < 50 nm) were obtained from Sigma-Aldrich. Both CuO and quartz NPs were suspended in phosphate buffered saline (PBS), and final concentrations were made in the cultured media (DMEM) without serum for the uniform dispersion of NPs followed by a brief sonication prior to exposure to the cells.

Exposure of CuO NPs to cell cultures

HEK-293 cells were purchased from the cell bank of the National Centre for Cell Science (Pune, Maharashtra, India). Cells were cultured in a full DMEM medium containing 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin, and incubated at 37°C with 5% carbon dioxide (CO₂). Different concentrations of NP suspensions in the cell culture medium without serum were prepared, and cells free of CuO NPs were used as controls throughout each assay. Tests for cytotoxicity and cell viability (MTT assay), LDH release, cytokine production (IL-8), lipid peroxidation products (thiobarbituric acid reactive substances (TBARSs)), and quantification of intracellular GSH levels were performed on the HEK-239 cell culture system.

Assessment of cytotoxicity

The MTT assay was used to measure both mitochondrial function and cell viability (Denizot and Lang, 1986). HEK-293 cells were plated onto a 96-well plate at a density of 1.0 × 10⁴ cells/each well. After 48 h of treatment with different concentrations (3–300 µg/ml) of NPs, the cells were incubated with MTT (2.5 mg/ml) for 2 h. Following the reported method (Anreddy et al., 2010), the plate was read at 570 nm for optical density that is directly correlated with cell quantity.

Evaluation of oxidative stress markers

Assessment of NP-induced oxidative stress was done by estimating cell membrane damage (LDH assay), reduced GSH, and lipid peroxidation levels in HEK-293 cells following exposure to NPs (10–300 µg/ml) using methodology published previously (Anreddy et al., 2010). The test doses of NPs used in all experimental procedures were selected based on a literature review (Anreddy et al., 2010) and may simulate or mimic doses of airborn exposure of NPs to human volunteers.

LDH release

Cells were seeded onto 24-well plates, exposed to increasing concentrations of CuO NPs suspensions (10–300 µg/ml). After 48 h of incubation, the plates were centrifuged at 968 xg. The media were transferred into fresh 24-well plates and analyzed for LDH release as described in the study by Hussain and Frazier (2002). Each experiment was done in triplicate. Cytotoxicity was expressed relative to the basal LDH release by untreated control cells.

Production of IL-8

Cells were seeded onto 24-well plates and exposed to increasing concentrations of CuO NPs suspensions (10–300 g/ml). After 48 h of incubation, plates were centrifuged at 968 xg. The media were transferred into fresh 24-well plates and analyzed for LDH release as described by Hussain and Frazier (2002). Each experiment was done in triplicate.

Quantification of intracellular GSH levels

Cellular levels of reduced GSH were determined using a GSH-400 colorimetric assay kit (Ray Biotech Inc., New Delhi, India). The method is based on a chemical reaction between GSH and 5,5-dithiobis (2-nitrobenzoic acid) to generate glutathione disulfide and 2-nitro-5-thiobenzoic acid, a yellow colored product. Thus, GSH concentration in a sample solution was determined by measurement at 412-nm absorbance (Akerboom and Sies, 1981). HEK-293 cells were plated onto a 24-well plate at a density of 2 × 10⁴ cells/ml. After 48 h of exposure to CuO NPs, the cells were washed twice in ice-cold PBS and then homogenized in 400 µl of 0.5% Triton X-100. The cell homogenate was centrifuged at 3000 × g at 4°C for 10 min. The assay was performed on 200-µl centrifugation supernatants according to the manufacturer’s protocol, and the absorbance of the supernatant was measured at 400 nm using an ultraviolet–visible spectrophotometer (Elico, Hyderabad, Telangana, India). GSH level was calculated and expressed as the percentage of control.

Estimation of lipid peroxidation

The Malondialdehyde (MDA) content, a measure of lipid peroxidation, was assayed based on the formation of TBARS (Ohkawa et al., 1979). HEK-293 cells were plated onto a 24-well plate at a density of 1 × 10⁵ cells/well. After 48 h of exposure to CuO NPs (10–300 µg/ml), the cells were washed with ice-cold PBS and homogenized in 400 µl of 0.5% Triton X-100. The cell homogenates were used in the TBARS assay.

Statistical analysis

Experimental data were expressed as mean ± standard deviation. Statistical analysis was performed using one-way analysis of variance and Dunnett’s test. Results with p < 0.05 were considered to be statistically significant.

Results

Cytotoxicity of CuO NPs

Exposure of CuO NPs and quartz (3–300 µg/ml) to HEK-293 cells for 48 h produced a dose-dependent decrease in cell viability, and the results are shown in Figure 1. Significant cell death (cytotoxicity) was observed with CuO NPs, and the IC₅₀ value (concentration of NPs to induce 50% cell mortality) was found as 65.5 µg/ml, which was comparable to that of quartz (37.8 µg/ml).

Oxidative stress markers

Cell membrane damage induced by CuO NPs was monitored by the LDH leakage assay. Exposure of HEK 293 cells to CuO NPs (10–300 µg/ml) for 48 h resulted in a significant (p < 0.01) increase in LDH release into the media (Figure 2). CuO NP caused cell death, and membrane damage resulted in LDH leakage (Figure 2). Similar to quartz, exposure of cells to CuO NPs (10–300 µg/ml) resulted in a dose-dependent increase in IL-8 release (Figure 3), indicating an inflammation response of NP to kidney cells. As shown in Figure 4, CuO NPs decreased GSH levels in the cells in a concentration-dependent manner. Results demonstrated a significant decrease of GSH levels in CuO-NPs exposed cells. The MDA content, a measure of lipid peroxidation, was assayed in the form of TBARS, a major indicator of oxidative stress. As shown in Figure 5, exposure of NP to HEK-293 cells resulted in significant (p < 0.05) increases in TBARS levels in a concentration-dependent manner.

Discussion

In this study, investigations were carried out to evaluate the in vitro cytotoxicity and oxidative stress induced by CuO NPs in HEK-293 cells following exposure. In this study, it was found that exposure of HEK-293 cells to CuO NPs (3–300 µg/ml) caused a dose-dependent cytotoxicity with an IC₅₀ value as 65.5µg/ml. Results showed that exposure of CuO NPs to HEK cells produced dose-dependent cell membrane damage, reduced intracellular GSH levels, increased IL-8 production, and elevated lipid peroxidation products, indicating that oxidative stress contributed to the cytotoxicity induced by the NPs. These results were supported by other published reports (Anreddy et al., 2010; Reddy and Srividya, 2018). Moreover, results from the present study are consistent with previous results suggesting that toxicity of CuO NPs are mediated through reactive oxygen species (ROS) generation and oxidative stress (Alarifi et al., 2013; Fahmy and Stephania, 2009). Based on the previous results of similar studies (Alarifi et al., 2013; Fahmy and Stephania, 2009), it was extrapolated that CuO NP–induced cytotoxicity may be mediated through the ROS generation and oxidative stress.

Previously, the in vivo pulmonary toxicity of CuO NPs in rats was reported in the study by Rani et al., 2013. It was reported that CuO NPs induced dose-dependent toxicity and oxidative stress in rats by generation of ROS by decreasing the levels of antioxidants superoxide dismutase and catalase. Thus, it was confirmed that CuO NPs induced toxicity and oxidative stress both in vivo as well as in vitro. In support of this, Anreddy (2018) reported oxidative stress and liver toxicity by the CuO NPs following oral exposure in rats (Anreddy, 2018). Similarly, Reddy and Srividya (2018) reported that metal oxide NPs (zinc oxide) produces dose-dependent cytotoxicity against various human cell lines and found the significant decreases in cell viability following exposure of these NPs (Reddy and Srividya, 2018).

Recently, Assadian et al. (2018) also reported on the cytotoxicity of CuO NPs in a concentration-dependent manner (with an IC₅₀ of 382 μM) against blood lymphocytes, and this cytotoxicity was associated with significant increases of intracellular ROS levels with effective induction of oxidative stress. Jing et al. (2015) also reported dose-dependent toxicity of CuO NPs in lung epithelial cells, and this cytotoxicity was related to the NPs-induced significant oxidative stress (Ng et al., 2017). The capability of NPs to produce free radicals is one of the primary mechanisms of NPs toxicity (Deng et al., 2009; Lin et al., 2009; Yang et al., 2009). NP exposure may result in oxidative stress, inflammation, and consequent damage to proteins, membranes, and DNA (Akhtar et al., 2016; Hu et al., 2009).

In conclusion, exposure of HEK-293 cell cultures to CuO NPs causes significant dose-dependent cytotoxicity, cell membrane damage, reduced intracellular GSH levels, increased IL-8 production, and elevated lipid peroxidation products. Our preliminary data suggest that oxidative stress might contribute to CuO NPs cytotoxicity. To determine whether induction of apoptosis is involved in CuO NPs toxicity, further studies are underway. More extensive studies would be needed to verify the safety issues related to increased usage of CuO NPs by consumers.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Anreddy Rama Narsimha Reddy

References

Ahamed

Akhtar

Alhadlaq

. (2015) Assessment of the lung toxicity of copper oxide nanoparticles: current status. Nanomedicine 10: 2365–2377.

Akerboom

TPM

Sies

(1981) Assay of glutathione, glutathione disulfide, and glutathione mixed disulfides in biological samples. Methods in Enzymology 77: 373–382.

Akhtar

Kumar

Alhadlaq

. (2016) Dose-dependent genotoxicity of copper oxide nanoparticles stimulated by reactive oxygen species in human lung epithelial cells. Toxicology Industrial Health 32(5): 809–821.

Alarifi

Ali

Verma

. (2013) Cytotoxicity and genotoxicity of copper oxide nanoparticles in human skin keratinocytes cells. International Journal of Toxicology 32(4): 296–307.

Anreddy

RNR

(2018) Copper oxide nanoparticles induces oxidative stress and liver toxicity in rats following oral exposure. Toxicological Reports 5: 903–904.

Anreddy

RNR

Reddy

Krishna

. (2010) Multi wall carbon nanoparticles induce oxidative stress and cytotoxicity in human embryonic kidney (HEK293) cells. Toxicology 272: 11–16.

Assadian

Zarei

Gilani

. (2018) Toxicity of copper oxide (CuO) nanoparticles on human blood lymphocytes. Biological Trace Elements Research 184(2): 350–357.

Deng

Luan

Chen

. (2009) Nanosized zinc oxide particles induce neural stem cell apoptosis. Nanotechnology 20: 115101.

Denizot

Lang

(1986) Rapid colorimetric assay for cell growth and survival: modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. Journal of Immunological Methods 89: 271–277.

10.

Duan

(2013) Physicochemical characteristics of nanoparticles affect circulation, biodistribution, cellular internalization, and trafficking. Small 9: 1521–1532.

11.

Fahmy

Stephania

. (2009) Copper nanoparticles induce oxidative stress and cytotoxicity in airway epithelial cells. Toxicol In Vitro 23: 1365–1371.

12.

Hanagata

Zhuang

Connolly

. (2011) Molecular responses of human lung epithelial cells to the toxicity of copper oxide nanoparticles inferred from whole genome expression analysis. ACS Nano 5: 9326–9338.

13.

Cook

Wang

. (2009) In vitro evaluation of cytotoxicity of engineered metal oxide nanoparticles. Total Environment 407: 3070–3072.

14.

Hussain

Frazier

(2002) Cellular toxicity of hydrazine in primary rat hepatocytes. Toxicological Sciences 2: 424–432.

15.

Ivask

Titma

Visnapuu

. (2015) Toxicity of 11 metal oxide nanoparticles to three mammalian cell types in vitro. Current Topics in Medicinal Chemistry 15: 1914–1929.

16.

Jing

Jae

Thomas

. (2015) Toxicity of copper oxide nanoparticles in lung epithelial cells exposed at the air–liquid interface compared with in vivo assessment. Toxicology in Vitro 29(3): 502–511.

17.

Kim

Lee

. (2015) Size, surface charge, and shape determine therapeutic effects of nanoparticles on brain and retinal diseases. Nanomedicine 11: 1603–1611.

18.

Lai

Yong

. (2018) Intranasal delivery of copper oxide nanoparticles induces pulmonary toxicity and fibrosis in C57BL/6 mice. Scientific Reports 8: 4499.

19.

Larsen

Jackson

Poulsen

. (2016) Airway irritation, inflammation, and toxicity in mice following inhalation of metal oxide nanoparticles. Nanotoxicology 10: 1254–1262.

20.

Lin

Huang

. (2009) Toxicity of nano- and micro-sized ZnO particles in human lung epithelial cells. Journal of Nanoparticle Research 11: 25–39.

21.

Linic

Aslam

Boerigter

. (2015) Photochemical transformations on plasmonic metal nanoparticles. Nature materials 14: 567–576.

22.

Yong

Hande

. (2017) Zinc oxide nanoparticles exhibit cytotoxicity and genotoxicity through oxidative stress responses in human lung fibroblasts and Drosophila melanogaster. International Journal of Nanomedicine 12: 1621–1637.

23.

Ohkawa

Ohishi

Yagi

(1979) Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical Biochemistry 95: 351–358.

24.

Rani

Kumar

. (2013) Pulmonary toxicity of copper oxide (CuO) nanoparticles in rats. Journal of Medical Sciences 13: 571–577.

25.

Reddy

ARN

Srividya

(2018) Evaluation of in vitro cytotoxicity of zinc oxide (ZnO) nanoparticles using human cell lines. Journal of Toxicology and Risk Assessment 4: 009.

26.

Uclésa

López

Hernando

. (2015) Application of zirconium dioxide nanoparticle sorbent for the clean-up step in post-harvest pesticide residue analysis. Talanta 144: 51–61.

27.

Yang

Liu

Yang

. (2009) Comparative study of cytotoxicity, oxidative stress and genotoxicity induced by four typical nanomaterials: the role of particle size, shape and composition. Journal of Applied Toxicology 29: 69–78.