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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.


Heatmap showing log2 fold changes for treatments of staurosporine (STS), camptothecin (CPT) and copper oxide nanoparticles (CuO NPs) for the various time points (n = 3), the levels of identification are indicated by 1identified by reference substance (MS2 and RT), 1aidentified by reference substance (only RT), 2identified by comparison of MS2 spectra with reference databases such as Metlin. The dendrogram on the left hand side shows a clustering of similarly regulated metabolites upon treatment with STS/CPT/CuO NPs
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Fig8: Heatmap showing log2 fold changes for treatments of staurosporine (STS), camptothecin (CPT) and copper oxide nanoparticles (CuO NPs) for the various time points (n = 3), the levels of identification are indicated by 1identified by reference substance (MS2 and RT), 1aidentified by reference substance (only RT), 2identified by comparison of MS2 spectra with reference databases such as Metlin. The dendrogram on the left hand side shows a clustering of similarly regulated metabolites upon treatment with STS/CPT/CuO NPs

Mentions: All three apoptotic agents, CuO NPs, STS, and CPT, induced a differential expression of metabolites, presented as a heat map in Fig. 8. However, although frequently similar, the dendrograms indicated that the induction patterns generated by each agent were also often markedly different. The colour coding in Fig. 8 indicates the extent of upregulation (red) or downregulation (blue) of a certain metabolite and further elucidates trends, such as successive downregulation of sphingosine over time upon treatment with STS. An accumulation of the various amino acids was observed for all three treatments. However, although a strong time-dependent upregulation was observed in response to CuO NPs, MTA was unaltered in response to STS or CPT. Furthermore, GPC regulation in response to STS was shown to have no association with exposure time, while for both CuO NPs and CPT a strong time-dependency was evident. Other metabolites differentially regulated in all three treatments included glutathione, sphingosine, phosphocreatine, and methylnicotinamide (MNA).Fig. 8


Copper oxide nanoparticle toxicity profiling using untargeted metabolomics
Heatmap showing log2 fold changes for treatments of staurosporine (STS), camptothecin (CPT) and copper oxide nanoparticles (CuO NPs) for the various time points (n = 3), the levels of identification are indicated by 1identified by reference substance (MS2 and RT), 1aidentified by reference substance (only RT), 2identified by comparison of MS2 spectra with reference databases such as Metlin. The dendrogram on the left hand side shows a clustering of similarly regulated metabolites upon treatment with STS/CPT/CuO NPs
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig8: Heatmap showing log2 fold changes for treatments of staurosporine (STS), camptothecin (CPT) and copper oxide nanoparticles (CuO NPs) for the various time points (n = 3), the levels of identification are indicated by 1identified by reference substance (MS2 and RT), 1aidentified by reference substance (only RT), 2identified by comparison of MS2 spectra with reference databases such as Metlin. The dendrogram on the left hand side shows a clustering of similarly regulated metabolites upon treatment with STS/CPT/CuO NPs
Mentions: All three apoptotic agents, CuO NPs, STS, and CPT, induced a differential expression of metabolites, presented as a heat map in Fig. 8. However, although frequently similar, the dendrograms indicated that the induction patterns generated by each agent were also often markedly different. The colour coding in Fig. 8 indicates the extent of upregulation (red) or downregulation (blue) of a certain metabolite and further elucidates trends, such as successive downregulation of sphingosine over time upon treatment with STS. An accumulation of the various amino acids was observed for all three treatments. However, although a strong time-dependent upregulation was observed in response to CuO NPs, MTA was unaltered in response to STS or CPT. Furthermore, GPC regulation in response to STS was shown to have no association with exposure time, while for both CuO NPs and CPT a strong time-dependency was evident. Other metabolites differentially regulated in all three treatments included glutathione, sphingosine, phosphocreatine, and methylnicotinamide (MNA).Fig. 8

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.