Screening for oxidative damage by engineered nanomaterials: a comparative evaluation of FRAS and DCFH

  • Anoop K. PalEmail author
  • Shu-Feng Hsieh
  • Madhu Khatri
  • Jacqueline A. Isaacs
  • Philip Demokritou
  • Peter Gaines
  • Daniel F. Schmidt
  • Eugene J. Rogers
  • Dhimiter BelloEmail author
Research Paper


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.


Oxidative stress Engineered nanomaterials ROS Glutathione DCFH FRAS ESR Nanotechnology Environmental and health effects 





Ferric reducing ability of serum


Engineered nanomaterial(s) with one or more dimensions <100 nm


Biological oxidative damage




Electron spin resonance


Reduced glutathione


Oxidized glutathione


Reactive oxygen species


Specific surface area




Carbon nanotubes


Single-wall carbon nanotubes


Multi-wall carbon nanotubes


H2O2-oxidized single-wall carbon nanohorns


Inductively coupled plasma-mass spectrometry


Instrumental neutron activation analysis


Oxidative stress


Trolox equivalent units, trolox is a water-soluble form of vitamin E



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.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11051_2013_2167_MOESM1_ESM.docx (200 kb)
Supplementary material 1 (DOCX 200 kb)
11051_2013_2167_MOESM2_ESM.docx (22 kb)
Supplementary material 2 (DOCX 21 kb)
11051_2013_2167_MOESM3_ESM.docx (74 kb)
Supplementary material 3 (DOCX 73 kb)


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Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Anoop K. Pal
    • 1
    Email author
  • Shu-Feng Hsieh
    • 2
  • Madhu Khatri
    • 1
  • Jacqueline A. Isaacs
    • 7
  • Philip Demokritou
    • 8
  • Peter Gaines
    • 3
    • 6
  • Daniel F. Schmidt
    • 4
    • 6
  • Eugene J. Rogers
    • 2
    • 6
  • Dhimiter Bello
    • 1
    • 5
    • 6
    Email author
  1. 1.Biomedical Engineering and Biotechnology ProgramUniversity of Massachusetts LowellLowellUSA
  2. 2.Department of Clinical Laboratory and Nutritional SciencesUniversity of Massachusetts LowellLowellUSA
  3. 3.Department of Biological SciencesUniversity of Massachusetts LowellLowellUSA
  4. 4.Department of Plastics EngineeringUniversity of Massachusetts LowellLowellUSA
  5. 5.Department of Work EnvironmentUniversity of Massachusetts LowellLowellUSA
  6. 6.Center for High-rate NanomanufacturingUniversity of Massachusetts LowellLowellUSA
  7. 7.Department of Mechanical, Industrial, and Manufacturing EngineeringNortheastern UniversityBostonUSA
  8. 8.Department of Environmental HealthHarvard School of Public HealthBostonUSA

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