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

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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.

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

  1. Biomedical Engineering and Biotechnology Program, University of Massachusetts Lowell, Lowell, MA, 01854, USA

    Anoop K. Pal, Madhu Khatri & Dhimiter Bello

  2. Department of Clinical Laboratory and Nutritional Sciences, University of Massachusetts Lowell, 3 Solomont Way, Lowell, MA, 01854, USA

    Shu-Feng Hsieh & Eugene J. Rogers

  3. Department of Biological Sciences, University of Massachusetts Lowell, 198 Riverside Street, Lowell, MA, 01854, USA

    Peter Gaines

  4. Department of Plastics Engineering, University of Massachusetts Lowell, 185 Riverside Street, Lowell, MA, 01854, USA

    Daniel F. Schmidt

  5. Department of Work Environment, University of Massachusetts Lowell, One University Avenue, Lowell, MA, 01854, USA

    Dhimiter Bello

  6. Center for High-rate Nanomanufacturing, University of Massachusetts Lowell, Lowell, MA, 01854, USA

    Peter Gaines, Daniel F. Schmidt, Eugene J. Rogers & Dhimiter Bello

  7. Department of Mechanical, Industrial, and Manufacturing Engineering, Northeastern University, 360 Huntington Avenue, Boston, MA, 02115, USA

    Jacqueline A. Isaacs

  8. Department of Environmental Health, Harvard School of Public Health, 677 Huntington Avenue, Boston, MA, 02115, USA

    Philip Demokritou

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  1. Anoop K. Pal
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  2. Shu-Feng Hsieh
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Correspondence to Anoop K. Pal or Dhimiter Bello.

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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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