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Copper oxide nanoparticle toxicity profiling using untargeted metabolomics

View Article: PubMed Central - PubMed

ABSTRACT

Background: The rapidly increasing number of engineered nanoparticles (NPs), and products containing NPs, raises concerns for human exposure and safety. With this increasing, and ever changing, catalogue of NPs it is becoming more difficult to adequately assess the toxic potential of new materials in a timely fashion. It is therefore important to develop methods which can provide high-throughput screening of biological responses. The use of omics technologies, including metabolomics, can play a vital role in this process by providing relatively fast, comprehensive, and cost-effective assessment of cellular responses. These techniques thus provide the opportunity to identify specific toxicity pathways and to generate hypotheses on how to reduce or abolish toxicity.

Results: We have used untargeted metabolome analysis to determine differentially expressed metabolites in human lung epithelial cells (A549) exposed to copper oxide nanoparticles (CuO NPs). Toxicity hypotheses were then generated based on the affected pathways, and critically tested using more conventional biochemical and cellular assays. CuO NPs induced regulation of metabolites involved in oxidative stress, hypertonic stress, and apoptosis. The involvement of oxidative stress was clarified more easily than apoptosis, which involved control experiments to confirm specific metabolites that could be used as standard markers for apoptosis; based on this we tentatively propose methylnicotinamide as a generic metabolic marker for apoptosis.

Conclusions: Our findings are well aligned with the current literature on CuO NP toxicity. We thus believe that untargeted metabolomics profiling is a suitable tool for NP toxicity screening and hypothesis generation.

Electronic supplementary material: The online version of this article (doi:10.1186/s12989-016-0160-6) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus

Characterization of CuO NPs. Including (a) dissolution of 10 μg/ml CuO NPs (maximum potential Cu ion release = 8 μg/ml, and would be represented as 100 % dissolution), black entries represent Cu ions detected after incubation of CuO NPs in CCM for increasing lengths of time, green represents Cu ions detected in CCM alone, red represents CuCl2 control; b transmission electron microscopy (TEM) micrographs of CuO NPs, scale bars are shown at bottom right of each image; c size distribution histogram of CuO NP hydrodynamic diameter when suspended in water, determined by DLS; d size distribution histogram of CuO NP hydrodynamic diameter when suspended in CCM, determined by DLS; e size distribution histogram of CuO NP diameter, determined by TEM
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Fig1: Characterization of CuO NPs. Including (a) dissolution of 10 μg/ml CuO NPs (maximum potential Cu ion release = 8 μg/ml, and would be represented as 100 % dissolution), black entries represent Cu ions detected after incubation of CuO NPs in CCM for increasing lengths of time, green represents Cu ions detected in CCM alone, red represents CuCl2 control; b transmission electron microscopy (TEM) micrographs of CuO NPs, scale bars are shown at bottom right of each image; c size distribution histogram of CuO NP hydrodynamic diameter when suspended in water, determined by DLS; d size distribution histogram of CuO NP hydrodynamic diameter when suspended in CCM, determined by DLS; e size distribution histogram of CuO NP diameter, determined by TEM

Mentions: CuO NP characteristics are given in Table 1 and Fig. 1. The primary particle size was determined to be 28.2 nm (±13.7) by TEM. However, agglomerates of 172 nm (±7.6) or 214.2 nm (±14.8) were shown to form when CuO NPs were suspended in water or CCM, respectively. In CCM the particle suspension was shown to contain three distinct peaks of agglomerate sizes (Fig. 1d), also highlighted by a high polydispersity index (PDI) of 0.507; in water the PDI was far lower, at 0.188, and only one size distribution peak was observed (Fig. 1c). When in water, the CuO NPs presented a positive surface charge of +35.9 mV (±1.3), which became negative when suspended in CCM (−10.5 mV ±0.1); which is likely due to the coating of NPs with protein components of the CCM, as has previously been shown [34]. As CuO NPs are metal oxide NPs which possess the capacity to release ions, the concentration of Cu2+ ions in solution was also assessed (Fig. 1a). The dissolution was studied under the same conditions as those of the cellular experiments, therefore within our CCM and during a time course of 0, 1, 3, 6, 12, 24 and 48 h. We observed a time dependent dissolution. A rapid dissolution was observed between 3 and 6 h. To correlate with the metabolome experiments, a CuO NP concentration of 10 μg/ml was used, therefore a concentration of Cu2+ ions equivalent to 8 μg/ml was expected if the NPs dissolved completely; which was observed within 24 h of incubation. CCM served as negative control, and was found to be < 0.05 μg/ml, and as positive control a copper (II) chloride (CuCl2 · 2H2O) solution was used, this was suspended at 21.45 μg/ml to give a final copper concentration of 8 μg/ml. Although optimal CuO dissolution has been shown under highly acidic or highly basic conditions [43], we have shown, as have others [28], that CuO NPs release significant levels of Cu2+ ions in buffered CCM.Table 1


Copper oxide nanoparticle toxicity profiling using untargeted metabolomics
Characterization of CuO NPs. Including (a) dissolution of 10 μg/ml CuO NPs (maximum potential Cu ion release = 8 μg/ml, and would be represented as 100 % dissolution), black entries represent Cu ions detected after incubation of CuO NPs in CCM for increasing lengths of time, green represents Cu ions detected in CCM alone, red represents CuCl2 control; b transmission electron microscopy (TEM) micrographs of CuO NPs, scale bars are shown at bottom right of each image; c size distribution histogram of CuO NP hydrodynamic diameter when suspended in water, determined by DLS; d size distribution histogram of CuO NP hydrodynamic diameter when suspended in CCM, determined by DLS; e size distribution histogram of CuO NP diameter, determined by TEM
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Fig1: Characterization of CuO NPs. Including (a) dissolution of 10 μg/ml CuO NPs (maximum potential Cu ion release = 8 μg/ml, and would be represented as 100 % dissolution), black entries represent Cu ions detected after incubation of CuO NPs in CCM for increasing lengths of time, green represents Cu ions detected in CCM alone, red represents CuCl2 control; b transmission electron microscopy (TEM) micrographs of CuO NPs, scale bars are shown at bottom right of each image; c size distribution histogram of CuO NP hydrodynamic diameter when suspended in water, determined by DLS; d size distribution histogram of CuO NP hydrodynamic diameter when suspended in CCM, determined by DLS; e size distribution histogram of CuO NP diameter, determined by TEM
Mentions: CuO NP characteristics are given in Table 1 and Fig. 1. The primary particle size was determined to be 28.2 nm (±13.7) by TEM. However, agglomerates of 172 nm (±7.6) or 214.2 nm (±14.8) were shown to form when CuO NPs were suspended in water or CCM, respectively. In CCM the particle suspension was shown to contain three distinct peaks of agglomerate sizes (Fig. 1d), also highlighted by a high polydispersity index (PDI) of 0.507; in water the PDI was far lower, at 0.188, and only one size distribution peak was observed (Fig. 1c). When in water, the CuO NPs presented a positive surface charge of +35.9 mV (±1.3), which became negative when suspended in CCM (−10.5 mV ±0.1); which is likely due to the coating of NPs with protein components of the CCM, as has previously been shown [34]. As CuO NPs are metal oxide NPs which possess the capacity to release ions, the concentration of Cu2+ ions in solution was also assessed (Fig. 1a). The dissolution was studied under the same conditions as those of the cellular experiments, therefore within our CCM and during a time course of 0, 1, 3, 6, 12, 24 and 48 h. We observed a time dependent dissolution. A rapid dissolution was observed between 3 and 6 h. To correlate with the metabolome experiments, a CuO NP concentration of 10 μg/ml was used, therefore a concentration of Cu2+ ions equivalent to 8 μg/ml was expected if the NPs dissolved completely; which was observed within 24 h of incubation. CCM served as negative control, and was found to be < 0.05 μg/ml, and as positive control a copper (II) chloride (CuCl2 · 2H2O) solution was used, this was suspended at 21.45 μg/ml to give a final copper concentration of 8 μg/ml. Although optimal CuO dissolution has been shown under highly acidic or highly basic conditions [43], we have shown, as have others [28], that CuO NPs release significant levels of Cu2+ ions in buffered CCM.Table 1

View Article: PubMed Central - PubMed

ABSTRACT

Background: The rapidly increasing number of engineered nanoparticles (NPs), and products containing NPs, raises concerns for human exposure and safety. With this increasing, and ever changing, catalogue of NPs it is becoming more difficult to adequately assess the toxic potential of new materials in a timely fashion. It is therefore important to develop methods which can provide high-throughput screening of biological responses. The use of omics technologies, including metabolomics, can play a vital role in this process by providing relatively fast, comprehensive, and cost-effective assessment of cellular responses. These techniques thus provide the opportunity to identify specific toxicity pathways and to generate hypotheses on how to reduce or abolish toxicity.

Results: We have used untargeted metabolome analysis to determine differentially expressed metabolites in human lung epithelial cells (A549) exposed to copper oxide nanoparticles (CuO NPs). Toxicity hypotheses were then generated based on the affected pathways, and critically tested using more conventional biochemical and cellular assays. CuO NPs induced regulation of metabolites involved in oxidative stress, hypertonic stress, and apoptosis. The involvement of oxidative stress was clarified more easily than apoptosis, which involved control experiments to confirm specific metabolites that could be used as standard markers for apoptosis; based on this we tentatively propose methylnicotinamide as a generic metabolic marker for apoptosis.

Conclusions: Our findings are well aligned with the current literature on CuO NP toxicity. We thus believe that untargeted metabolomics profiling is a suitable tool for NP toxicity screening and hypothesis generation.

Electronic supplementary material: The online version of this article (doi:10.1186/s12989-016-0160-6) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus