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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.


Apoptosis (caspase-3 and −7 activity) in response to CuO NPs, staurosporine (STS) and camptothecin (CPT). A549 cells treated with (a) CuO NPs at 0, 2.5, 5.0, 10 and 20 μg/ml, (b) STS at 0.5, 1, and 2 μM, or (c) CPT at 8, 16, and 32 μg/ml; for 3, 6, 12 and 24 h and assessed for caspase-3 and −7 activity. Data is expressed as % enzyme activity compared to medium only treated cells, and each data point represents the mean ± SEM (n = 3). Statistical significance was determined by ANOVA with Tukey posthoc, and + for 6 h, ^ for 12 h and * for 24 h, when p < 0.05 compared to medium only treated cells
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Fig6: Apoptosis (caspase-3 and −7 activity) in response to CuO NPs, staurosporine (STS) and camptothecin (CPT). A549 cells treated with (a) CuO NPs at 0, 2.5, 5.0, 10 and 20 μg/ml, (b) STS at 0.5, 1, and 2 μM, or (c) CPT at 8, 16, and 32 μg/ml; for 3, 6, 12 and 24 h and assessed for caspase-3 and −7 activity. Data is expressed as % enzyme activity compared to medium only treated cells, and each data point represents the mean ± SEM (n = 3). Statistical significance was determined by ANOVA with Tukey posthoc, and + for 6 h, ^ for 12 h and * for 24 h, when p < 0.05 compared to medium only treated cells

Mentions: Caspase-3 and −7 activity was assessed in response to CuO NPs at 0–20 μg/ml (Fig. 6a), and to two further substances, namely staurosporine (STS) (Fig. 6b) and camptothecin (CPT) (Fig. 6c). The addition of the second two substances (STS and CPT) would allow us to gain confidence in metabolic markers which we may propose as generic markers for apoptosis. Cells were exposed to 0.5, 1, and 2 μM STS, or 8, 16, and 32 μg/ml CPT, for 3, 6, 12, and 24 h. A dose- and time-dependent increase in caspase activity was observed with exposure of CuO NPs, with significant increases found in response to 10 μg/ml (12 and 24 h) and 20 μg/ml (6, 12 and 24 h) CuO NPs. STS induced a clear dose- and time-dependent increase in caspase activity when compared to untreated cells, with significantly increased activity in response to 1 μM (12 and 24 h) and 2 μM (6, 12, and 24 h). The response to CPT was similar, although increased activity was observed at all concentrations tested, 8 μg/ml (24 h), 16 μg/ml (24 h), 32 μg/ml (6, 12, and 24 h). However, although both substances induced apoptosis, indicated by a strong caspase activity, STS was shown to induce an early activation of caspases at low concentrations (in respect to the dose range used here), while CPT needed higher concentrations (in respect to the dose range used here) and a clear dose response was only observed during the latter time points. Therefore, these response characteristics were taken into account for the study design of the subsequent metabolomics experiment.Fig. 6


Copper oxide nanoparticle toxicity profiling using untargeted metabolomics
Apoptosis (caspase-3 and −7 activity) in response to CuO NPs, staurosporine (STS) and camptothecin (CPT). A549 cells treated with (a) CuO NPs at 0, 2.5, 5.0, 10 and 20 μg/ml, (b) STS at 0.5, 1, and 2 μM, or (c) CPT at 8, 16, and 32 μg/ml; for 3, 6, 12 and 24 h and assessed for caspase-3 and −7 activity. Data is expressed as % enzyme activity compared to medium only treated cells, and each data point represents the mean ± SEM (n = 3). Statistical significance was determined by ANOVA with Tukey posthoc, and + for 6 h, ^ for 12 h and * for 24 h, when p < 0.05 compared to medium only treated cells
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5017021&req=5

Fig6: Apoptosis (caspase-3 and −7 activity) in response to CuO NPs, staurosporine (STS) and camptothecin (CPT). A549 cells treated with (a) CuO NPs at 0, 2.5, 5.0, 10 and 20 μg/ml, (b) STS at 0.5, 1, and 2 μM, or (c) CPT at 8, 16, and 32 μg/ml; for 3, 6, 12 and 24 h and assessed for caspase-3 and −7 activity. Data is expressed as % enzyme activity compared to medium only treated cells, and each data point represents the mean ± SEM (n = 3). Statistical significance was determined by ANOVA with Tukey posthoc, and + for 6 h, ^ for 12 h and * for 24 h, when p < 0.05 compared to medium only treated cells
Mentions: Caspase-3 and −7 activity was assessed in response to CuO NPs at 0–20 μg/ml (Fig. 6a), and to two further substances, namely staurosporine (STS) (Fig. 6b) and camptothecin (CPT) (Fig. 6c). The addition of the second two substances (STS and CPT) would allow us to gain confidence in metabolic markers which we may propose as generic markers for apoptosis. Cells were exposed to 0.5, 1, and 2 μM STS, or 8, 16, and 32 μg/ml CPT, for 3, 6, 12, and 24 h. A dose- and time-dependent increase in caspase activity was observed with exposure of CuO NPs, with significant increases found in response to 10 μg/ml (12 and 24 h) and 20 μg/ml (6, 12 and 24 h) CuO NPs. STS induced a clear dose- and time-dependent increase in caspase activity when compared to untreated cells, with significantly increased activity in response to 1 μM (12 and 24 h) and 2 μM (6, 12, and 24 h). The response to CPT was similar, although increased activity was observed at all concentrations tested, 8 μg/ml (24 h), 16 μg/ml (24 h), 32 μg/ml (6, 12, and 24 h). However, although both substances induced apoptosis, indicated by a strong caspase activity, STS was shown to induce an early activation of caspases at low concentrations (in respect to the dose range used here), while CPT needed higher concentrations (in respect to the dose range used here) and a clear dose response was only observed during the latter time points. Therefore, these response characteristics were taken into account for the study design of the subsequent metabolomics experiment.Fig. 6

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.