Abstract
Several acellular assays are routinely used to measure oxidative stress elicited by engineered nanomaterials (ENMs), yet little comparative evaluations of such methods exist. This study compares for the first time the performance of the dichlorofluorescein (DCFH) assay which measures reactive oxygen species (ROS) generation, to that of the ferric-reducing ability of serum (FRAS) assay, which measures biological oxidant damage in serum. A diverse set of 28 commercially important and extensively characterized ENMs were tested on both the assays. Intracellular oxidative stress was also assessed on a representative subset of seven ENMs in THP-1 (phorbol 12-myristate 13-acetate matured human monocytes) cells. Associations between assay responses and ENM physicochemical properties were assessed via correlation and regression analysis. DCFH correlated strongly with FRAS after dose normalization for mass (R 2 = 0.78) and surface area (R 2 = 0.68). Only 10/28 ENMs were positive in DCFH versus 21/28 in FRAS. Both assays were strongly associated with specific surface area and transition metal content. Qualitatively, a similar response ranking was observed for acellular FRAS and intracellular reduced:oxidized glutathione ratio (GSH:GSSG) in cells. Quantitatively, weak correlation was found between intracellular GSSG and FRAS or DCFH (R 2 < 0.25) even after calculating effective dose to cells. The FRAS assay was more sensitive than DCFH, especially for ENMs with low to moderate oxidative damage potential, and may serve as a more biologically relevant substitute for acellular ROS measurements of ENMs. Further in vitro and in vivo validations of FRAS against other toxicological endpoints with larger datasets are recommended.
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Abbreviations
- DCFH/DCF:
-
Dichlorofluorescein
- FRAS:
-
Ferric reducing ability of serum
- ENM(s):
-
Engineered nanomaterial(s) with one or more dimensions <100 nm
- BOD:
-
Biological oxidative damage
- DDT:
-
Dithiothreitol
- ESR:
-
Electron spin resonance
- GSH:
-
Reduced glutathione
- GSSG:
-
Oxidized glutathione
- ROS:
-
Reactive oxygen species
- SSA:
-
Specific surface area
- TPTZ:
-
2,4,6-Tripyridyl-s-triazine
- CNTs:
-
Carbon nanotubes
- SWCNTs:
-
Single-wall carbon nanotubes
- MWCNTs:
-
Multi-wall carbon nanotubes
- SWCNHs-ox:
-
H2O2-oxidized single-wall carbon nanohorns
- ICP-MS:
-
Inductively coupled plasma-mass spectrometry
- INAA:
-
Instrumental neutron activation analysis
- OS:
-
Oxidative stress
- TEUs:
-
Trolox equivalent units, trolox is a water-soluble form of vitamin E
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Acknowledgments
This study was supported through the Nanoscale Science and Engineering Centers Program of the National Science Foundation # 0425826 and EEC-0425826 (Supplement). The authors would like to thank Joel Cohen from the Harvard School of Public Health, Boston for help with ISDD-based dosimetry,and Rohan Dagoankar for his assistance with the GSH/GSSG assay.
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The authors declare that they have no conflict of interest.
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Pal, A.K., Hsieh, SF., Khatri, M. et al. Screening for oxidative damage by engineered nanomaterials: a comparative evaluation of FRAS and DCFH. J Nanopart Res 16, 2167 (2014). https://doi.org/10.1007/s11051-013-2167-3
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DOI: https://doi.org/10.1007/s11051-013-2167-3