Metabolomics of silver nanoparticles toxicity in HaCaT cells: structure–activity relationships and role of ionic silver and oxidative stress

Abstract

The widespread use of silver nanoparticles (AgNPs) is accompanied by a growing concern regarding their potential risks to human health, thus calling for an increased understanding of their biological effects. The aim of this work was to systematically study the extent to which changes in cellular metabolism were dependent on the properties of AgNPs, using NMR metabolomics. Human skin keratinocytes (HaCaT cells) were exposed to citrate-coated AgNPs of 10, 30 or 60 nm diameter and to 30 nm AgNPs coated either with citrate (CIT), polyethylene glycol (PEG) or bovine serum albumin (BSA), to assess the influence of NP size and surface chemistry. Overall, CIT-coated 60 nm and PEG-coated 30 nm AgNPs had the least impact on cell viability and metabolism. The role of ionic silver and reactive oxygen species (ROS)-mediated effects was also studied, in comparison to CIT-coated 30 nm particles. At concentrations causing an equivalent decrease in cell viability, Ag⁺ions produced a change in the metabolic profile that was remarkably similar to that seen for AgNPs, the main difference being the lesser impact on the Krebs cycle and energy metabolism. Finally, this study newly reported that while down-regulated glycolysis and disruption of energy production were common to AgNPs and H₂O₂, the impact on some metabolic pathways (GSH synthesis, glutaminolysis and the Krebs cycle) was independent of ROS-mediated mechanisms. In conclusion, this study shows the ability of NMR metabolomics to define subtle biochemical changes induced by AgNPs and demonstrates the potential of this approach for rapid, untargeted screening of pre-clinical toxicity of nanomaterials in general.

• AgNPs
• keratinocytes
• metabolism
• NMR spectroscopy
• reactive oxygen species

Acknowledgements

Tiago Pedrosa is thanked for laboratory assistance, Dr António Barros for helpful advice regarding statistical analysis, and Prof. Ruth Duncan for insightful discussions. Dr Manfred Spraul, Bruker BioSpin (Germany) is thanked for access to software and spectral database. The Portuguese National NMR (PTNMR) Network, supported with FCT funds, is also acknowledged.

Declaration of interest

No conflict of interest to declare. Funding institutions had no role in study design, data collection or analysis, decision to publish, or manuscript preparation. This study was developed in the scope of the projects CICECO-Aveiro Institute of Materials (Ref. FCT UID/CTM/50011/2013) and CESAM (Ref. FCT UID/AMB/50017/2013), financed by national funds through the FCT/MEC and when applicable co-financed by the European Regional Development Fund (FEDER) under the PT2020 Partnership Agreement. Funding to the project FCOMP-01-0124-FEDER-021456 (Ref. FCT PTDC/SAU-TOX/120953/2010) by FEDER through COMPETE and by national funds through FCT, and the FCT-awarded grants (SFRH/BD/79494/2011; SFRH/BD/81792/2011; SFRH/BPD/48853/2008) are acknowledged. I.F.D acknowledges FCT/MCTES for a research contract under the Program “Investigador FCT” 2014.

Supplementary material available online

Supplementary Figures S1–S5