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Assessment of the lung toxicity of copper oxide nanoparticles: current status
Copper oxide nanoparticles (CuO NPs) are being used in several industrial and commercial products. Inhalation is one of the most significant routes of metal oxide NP exposure. Hence, the toxicity of CuO NPs in lung tissues is of great concern. In vitro studies have indicated that CuO NPs induce cytotoxicity, oxidative stress and genetic toxicity in cultivated human lung cells. Leaching of Cu ions, reactive oxygen species generation and autophagy appear to be the underlying mechanisms of Cu NP toxicity in lung cells. In vivo studies on the lung toxicity of CuO NPs are largely lacking. Some studies have shown that intratracheal instillation of CuO NPs induced oxidative stress, inflammation and neoplastic lesions in rats. This review critically assessed the current findings of the toxicity of CuO NPs in the lung. Keywords: apoptosis • autophagy • copper oxide nanoparticles • Cu ions • lung toxicity • oxidative stress • ROS
Nanotechnology provides the opportunity for the development of new materials and/or devices in the nanometer size range (1–100 nm) that can be utilized in various applications. This new technology has promised to improve almost all areas of human life, including the aviation sector, electronics, environmental remediation and the medical healthcare sector [1,2] . Nanoparticles (NPs) exhibit unique physicochemical characteristics compared with what they exhibit in their bulk materials. These properties of NPs come from their high surface area-to-volume ratio and different surface structures [3,4] . New characteristics of NPs, including quantum effects, make NPs suitable for various applications in modern industries. However, these unique characteristics of NPs may also pose adverse human and environmental health effects [5,6] . Being smaller than cellular organelles and cells, NPs can penetrate basic biological structures that may, in turn, disrupt their normal structure and function [7,8] . Metal oxide NPs belong to a family of nanoscale materials that have been produced on a large scale for many products, and
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they hold great promise for future applications [3,5,9–11] . The NPs could easily enter the human body via different routes such as inhalation, ingestion and dermal contact. Additionally, inhalation, at least from an occupational point of view, or from a commonly environmental one, is likely to be one of the most significant routes of exposure. Studies have shown that NPs are present in ambient air [3,12] . These NPs have a potentially high efficiency for deposition in the respiratory system [13] , are retained in the lungs for a long period [14] , may reach the central nervous system as inhaled NPs [13,15] and induce a stronger oxidative stress and inflammatory response [16,17] . In this review, we critically assessed the current findings concerning CuO NP exposure and associated toxicity in the lungs.
Maqusood Ahamed*,1, Mohd Javed Akhtar1, Hisham A Alhadlaq1,2 & Salman A Alrokayan3 1 King Abdullah Institute for Nanotechnology, King Saud University, P.O. Box 2454, Riyadh 11451, Saudi Arabia 2 Department of Physics & Astronomy, College of Science, King Saud University, Riyadh, Saudi Arabia 3 Department of Biochemistry, College of Science, King Saud University, Riyadh, Saudi Arabia *Author for correspondence: Tel.: +966 469 8781 Fax: +966 467 0664 maqusood@ gmail.com
Application of CuO nanoparticles CuO has been a hot topic among the investigations on transition metal oxides because of its interesting properties [18,19] . As a semiconducting material with a monoclinic structure, CuO has attracted particular attention
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Special Report Ahamed, Akhtar, Alhadlaq & Alrokayan in the field of nanotechnology. It possesses useful photovoltaic and photoconductive properties because CuO crystal structures have a narrow band gap [20] . CuO NPs have several industrial and commercial applications. CuO NPs are being utilized in semiconductors, electronic chips, heat transfer nanofluids, gas sensors, catalysts, solar cells and lithium batteries [21,22] . CuO NPs can aid in the synthesis of fullerenes and can be used in energetic materials such as explosives and propellants [23–25] . CuO NPs are employed as additives in inks, plastics, lubricants and metallic coatings as well as in different skin products [26,27] . CuO NPs have been shown to inhibit the growth of microorganisms and exert antiviral properties [28,29] . Therefore, CuO NPs are being used in textiles such as face masks, wound dressings and socks to give them biocidal activity [30] . Due to their high industrial and commercial demand, CuO NPs are being produced on a large scale and are expected to expand in the coming years. There is an undisputed consensus that the risk for the environment and humans to come into contact with CuO NPs will increase significantly. Therefore, the health safety of CuO NP exposure has become a major issue
for scientific and regulatory institutions. Figure 1 summarizes the wide-spread application of CuO NPs and their possible human and environmental health effects. Physicochemical characterization of CuO nanoparticles before toxicity studies A key challenge in predicting the potential toxicity of NPs is their complex nature. It has been suggested by scientific communities that the physicochemical behavior of NPs should be properly characterized before their toxicity studies. An explanation of the different physical and chemical characteristics, including size, morphology, crystal structure, purity, surface chemistry and coatings, which result from the various synthesis techniques, needs to be understood in a biological context to improve the existing methods and develop new assays with appropriate quality assurance controls at both the academic research and manufactured engineering levels [31] . Characterization of these properties also applies in the toxicity studies of CuO NPs (Figure 2) . Agglomeration and stability of NPs in aqueous suspension are major challenges in nanotoxicity
Human health effects Lung toxicity Skin toxicity Liver toxicity Neurotoxicity Kidney toxicity Reproductive toxicity
CuO NP applications Catalyst Gas Sensor Microelectronic Heat transfer fluid Solar energy convertor Intrauterine conceptive device Antimicrobials preparation Semiconductor device Cosmetic Battery Doping
CuO NPs
Environmental health effects Plant toxicity Green algae toxicity Mussel toxicity Juvenile carp toxicity Protozoa toxicity Microbial toxicity Yeast toxicity
Figure 1. CuO nanoparticle applications and potential health effects. CuO NPs are being utilized as industrial catalysts in manufacturing processes and are heavily utilized in semiconductor devices, gas sensors, batteries, solar energy converters, microelectronics and heat transfer fluids, among others. Due to their antimicrobial properties CuO NPs are also employed in textiles, paints, plastics and food containers. The increasing production of CuO NPs has led to major concerns regarding the potential toxicity to the environment and human health. NP: Nanoparticle.
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Assessment of the lung toxicity of copper oxide nanoparticles: current status
Physico-chemical characterization of CuO nanoparticles
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Factors for consideration
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Shape
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Size 300
Crystal structure
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Surface charge
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Surface morphology
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Functionalization
6000 (111) 5000
Chemical composition (purity)
Intensity (a.u.)
Intensity (a.u.)
(002) 4000
Agglomeration
3000 (202) 2000 (110)
1000
Hydrodynamic size Zeta potential
0
30
40 2 theta degree
50
Ionic dissolution
Figure 2. Physicochemical characteristics of CuO nanoparticles are necessary before toxicity studies.
research [32] . Once the NPs were introduced to aqueous media, the sizes changed to often times higher than those of the primary size [33] . For example, our previous studies have shown that, once CuO NPs are introduced into cell culture media, their hydrodynamic sizes changed to around ten-times higher than those observed from CuO nanopowder [34,35] . The higher size of CuO NPs in aqueous suspension compared with the primary size of dry powder might be due to the tendency of particles to agglomerate in an aqueous state. The same finding for ZnO NPs is reported by other investigators [36] and has been discussed in our previous publication [37] . For example, during dynamic light scattering measurement for the hydrodynamic size of NPs, there is a tendency of NPs to agglomerate in the aqueous state, thereby giving the size of clustered particles rather than individual particles. Agglomeration of NPs in aqueous suspension is also influenced by various factors such as the presence of protein in the culture media. Studies have shown that the presence of protein in aqueous suspension leads to the formation of a ‘protein corona’ on the surface of NPs that reduces the agglomeration of NPs [38] . In earlier work, we found that the hydrodynamic size of CuO NPs was lower in complete cell culture medium (DMEM with 10% fetal bovine serum) than in distilled water [39] . Therefore, not only the primary size of NPs but also the behavior of NPs in culture media could be used as characteristic parameters to study the biological response of CuO NPs. Dissolution of CuO NPs into Cu ions in aqueous suspension is also an important factor that can
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affect the toxicological response of this material. This point is briefly discussed in a later part of this review. Lung toxicity of CuO nanoparticles: in vitro studies Cell lines provide sensitive tools for high-throughput toxicity screening and have the potential to reduce the use of animals in toxicological testing [40] . They can be used for the collection of mechanistic data that could potentially be used for risk assessment. We are aware of the potential limitations of the in vitro systems for the evaluation of the toxic response of chemicals/ or NPs [41] . Because of the limited metabolic capacity of the in vitro models, the biotransformation of a chemical in vitro may be minimal compared with that in in vivo systems. However, despite their known limited metabolic capacity, in vitro cell lines still represent promising tools for the development of high-throughput, predictive and mechanism-based assays to evaluate the potential toxicity of agents such as NPs [42] . In this section, we evaluated the toxicity of CuO NPs in cultured human lung cell lines. Previous studies on the in vitro lung toxicity of CuO NPs are summarized in Table 1. Karlsson et al. [43] focused on different metal oxide (CuO, TiO2, ZnO, CuZnFe2O4, Fe3O4 and Fe2O3) NPs, and the toxicity was compared with that of carbon NPs and multiwalled carbon nanotubes in human lung epithelial (A549) cells. They observed that there was a high variation among different NPs concerning their ability to cause toxic effects. Importantly, CuO NPs were most potent among metal oxide NPs regarding cytotoxicity and DNA damage.
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185–276 nm No
300 nm
NR
300 nm
200 nm
20–40 nm
34 nm
30 nm
47 nm
50 nm
20–40 nm
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No
No
No
No
Stabilized with BSA
No
No
No
No
2–24 h
8, 12 and 24 h
18 h
18 h
24 h
CuO NPs showed much higher toxicity than CuO bulk particles. CuO NPs first generate ROS, which subsequently induces the expression of p38 and p53 and ultimately causes DNA damage
Induced cytotoxicity through autophagic pathway
NPs of CuO were much more cytotoxic, genotoxic and showed much higher ability to cause mitochondrial depolarization as compared with microparticles of CuO
ROS generation and DNA damage
DNA damage, micronuclei induction and ROS generation
Induction of cytotoxic, genotoxic and oxidative stress response
Major outcomes
5h
40 and 80 μg/ml
25 μg/ml
18 h
24 h
0–100 μg/ml 24 h
4–400 μg/ cm2
A549
Released Cu2+ involved in regulation of genes
Cu2+ contributes to toxicity
Dissolved Cu2+ ions did not induce toxicity
Cu2+ partially contributed to the toxicity
Dissolved Cu2+ ions contributed less than half of the total toxicity caused by CuO NPs
NR
NR
NR
NR
NR
Role of dissolved Cu2+
4 and 18 h CuO NPs caused much higher cytotoxicity and DNA damage than microparticles
Cu ions caused much lower toxicity than particles
Cu2+ induced toxic response
CuO NP toxicity (DNA damage and cell death) Cu2+ are responsible for is predominantly mediated by intracellular toxicity uptake and subsequent release of copper ions
DNA microarray analysis showed that more 1200 genes were regulated due to CuO NP exposure
Cytotoxicity is a function of particle surface charge and metal ion dissolution from CuO NPs
Induction of oxidative stress and cytotoxicity
1–100 μg/ml 3 and 24 h Cytotoxicity, oxidative stress and severe ultrastructural damages especially cell membrane and mitochondria
10–100 mg/ ml
30 μg/ml
40 and 80 μg/ml
40 and 80 μg/ml
5–15 μg/ml
10–50 μg/ml 24 h
Exposure time
BEAS-2B 0–200 μg/ml Overnight Cytotoxicity and oxidative stress response of CuO NPs were due to solubility of particles
BEAS2B and A549
A549
BEAS2B and A549
HEp-2
A549
A549
A549, H1650, CNE-2Z
A549
A549
A549
A549
Doses
[9]
[49]
[48]
[47]
[46]
[16]
[17]
[45]
[44]
[4]
[43]
[35]
[34]
Ref.
A549: Human lung epithelial cells; BEAS-2B: Human bronchial epithelial; BSA: Bovine serum albumin; CNE-2Z: Human nasopharyngeal carcinoma; DLS: Dynamic light scattering; H1650: Human nonsmall-cell lung cancer; HEp-2: Human larynx epithelial; NP: Nanoparticle; NR: Not reported; ROS: Reactive oxygen species; TEM: Transmission electron microscopy.
500 nm
NR
