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

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

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 (R2 = 0.78) and surface area (R2 = 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 (R2 < 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.

Keywords

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

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

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)

References

  1. Ayres JG, Borm P, Cassee FR, Castranova V, Donaldson K, Ghio A, Harrison RM, Hider R, Kelly F, Kooter IM, Marano F, Maynard RL, Mudway I, Nel A, Sioutas C, Smith S, Baeza-Squiban A, Cho A, Duggan S, Froines J (2008) Evaluating the toxicity of airborne particulate matter and nanoparticles by measuring oxidative stress potential: a workshop report and consensus statement. Inhal Toxicol 20:75–99CrossRefGoogle Scholar
  2. Bello D, Hsieh S-F, Schmidt D, Rogers E (2009) Nanomaterials properties versus biological oxidative damage: implications for toxicity screening and exposure assessment. Nanotoxicology 3:249–261CrossRefGoogle Scholar
  3. Bihari P, Vippola M, Schultes S, Praetner M, Khandoga A, Reichel C, Coester C, Tuomi T, Rehberg M, Krombach F (2008) Optimized dispersion of nanoparticles for biological in vitro and in vivo studies. Part Fibre Toxicol 5:14CrossRefGoogle Scholar
  4. Bonini MG, Rota C, Tomasi A, Mason RP (2006) The oxidation of 2′,7′-dichlorofluorescin to reactive oxygen species: a self-fulfilling prophesy? Free Radic Biol Med 40:968–975CrossRefGoogle Scholar
  5. Borm PJ, Kelly F, Kunzli N, Schins RP, Donaldson K (2007) Oxidant generation by particulate matter: from biologically effective dose to a promising, novel metric. Occup Environ Med 64:73–74CrossRefGoogle Scholar
  6. Brunauer S, Emmett PH, Teller E (1938) Adsorption of gases in multimolecular layers. J Am Chem Soc 60:309–319CrossRefGoogle Scholar
  7. Chen X, Zhong Z, Xu Z, Chen L, Wang Y (2010) 2′,7′-Dichlorodihydrofluorescein as a fluorescent probe for reactive oxygen species measurement: forty years of application and controversy. Free Radic Res 44:587–604CrossRefGoogle Scholar
  8. Cohen J, Deloid G, Pyrgiotakis G, Demokritou P (2012) Interactions of engineered nanomaterials in physiological media and implications for in vitro dosimetry. Nanotoxicology 7(4):417–431CrossRefGoogle Scholar
  9. Doak SH, Griffiths SM, Manshian B, Singh N, Williams PM, Brown AP, Jenkins GJ (2009) Confounding experimental considerations in nanogenotoxicology. Mutagenesis 24:285–293CrossRefGoogle Scholar
  10. Donaldson K, Tran L, Jimenez L, Duffin R, Newby D, Mills N, MacNee W, Stone V (2005) Combustion-derived nanoparticles: a review of their toxicology following inhalation exposure. Part Fibre Toxicol 2:10CrossRefGoogle Scholar
  11. Foucaud L, Wilson MR, Brown DM, Stone V (2007) Measurement of reactive species production by nanoparticles prepared in biologically relevant media. Toxicol Lett 174:1–9CrossRefGoogle Scholar
  12. Gou N, Gu AZ (2011) A new transcriptional effect level index (TELI) for toxicogenomics-based toxicity assessment. Environ Sci Technol 45:5410–5417CrossRefGoogle Scholar
  13. Guo L, Morris DG, Liu X, Vaslet C, Hurt RH, Kane AB (2007) Iron bioavailability and redox activity in diverse carbon nanotube samples. Chem Mater 19:3472–3478CrossRefGoogle Scholar
  14. Hilding J, Grulke EA, George Zhang Z, Lockwood F (2003) Dispersion of carbon nanotubes in liquids. J Dispers Sci Technol 24:1–41CrossRefGoogle Scholar
  15. Hinderliter P et al (2010) ISDD: a computational model of particle sedimentation, diffusion and target cell dosimetry for in vitro toxicity studies. Part Fibre Toxicol 7(1):36CrossRefGoogle Scholar
  16. Hoet P, Nemery HB, Napierska D (2013) Intracellular oxidative stress caused by nanoparticls: what do we measure with the dichlorofluorescein assay? Nanotoday 8(3):223–227CrossRefGoogle Scholar
  17. Holder AL, Goth-Goldstein R, Lucas D, Koshland CP (2012) Particle-induced artifacts in the MTT and LDH viability assays. Chem Res Toxicol 25(9):1885–1892CrossRefGoogle Scholar
  18. Hsieh SF, Bello D, Schmidt DF, Pal AK, Rogers EJ (2012) Biological oxidative damage by carbon nanotubes: fingerprint or footprint? Nanotoxicology 6:61–76CrossRefGoogle Scholar
  19. Hsieh SF, Bello D, Schmidt D, Pal A, Stella A, Isaacs J, Rogers E (2013) Mapping the biological oxidative damage of engineered nanomaterials. Small 9(9–10):1853–1865CrossRefGoogle Scholar
  20. Hussain S, Boland S, Baeza-Squiban A, Hamel R, Thomassen LC, Martens JA, Billon-Galland MA, Fleury-Feith J, Moisan F, Pairon JC, Marano F (2009) Oxidative stress and proinflammatory effects of carbon black and titanium dioxide nanoparticles: role of particle surface area and internalized amount. Toxicology 260:142–149CrossRefGoogle Scholar
  21. Karajanagi SS, Vertegel AA, Kane RS, Dordick JS (2004) Structure and function of enzymes adsorbed onto single-walled carbon nanotubes. Langmuir 20:11594–11599CrossRefGoogle Scholar
  22. Koike E, Kobayashi T (2006) Chemical and biological oxidative effects of carbon black nanoparticles. Chemosphere 65:946–951CrossRefGoogle Scholar
  23. Lewinski N, Colvin V, Drezek R (2008) Cytotoxicity of nanoparticles. Small 4:26–49CrossRefGoogle Scholar
  24. Lu S, Duffin R, Poland C, Daly P, Murphy F, Drost E, Macnee W, Stone V, Donaldson K (2009) Efficacy of simple short-term in vitro assays for predicting the potential of metal oxide nanoparticles to cause pulmonary inflammation. Environ Health Perspect 117:241–247Google Scholar
  25. Mahmoudi A, Nazari K, Mohammadian N, Moosavi-Movahedi AA (2003) Effect of Mn2+, Co2+, Ni2+, and Cu2+ on horseradish peroxidase: activation, inhibition, and denaturation studies. Appl Biochem Biotechnol 104:81–94CrossRefGoogle Scholar
  26. Maynard A (2006) Nanotechnology: a research strategy for addressing risk. Woodrow Wilson International Center for Scholars, WashingtonGoogle Scholar
  27. Meng H, Xia T, George S, Nel AE (2009) A predictive toxicological paradigm for the safety assessment of nanomaterials. ACS Nano 3:1620–1627CrossRefGoogle Scholar
  28. Mills NL, Donaldson K, Hadoke PW, Boon NA, MacNee W, Cassee FR, Sandstrom T, Blomberg A, Newby DE (2009) Adverse cardiovascular effects of air pollution. Nat Clin Pract Cardiovasc Med 6:36–44CrossRefGoogle Scholar
  29. Montes-Burgos I, Walczyk D, Hole P, Smith J, Lynch I, Dawson K (2010) Characterisation of nanoparticle size and state prior to nanotoxicological studies. J Nanopart Res 12:47–53CrossRefGoogle Scholar
  30. Moore VC, Strano MS, Haroz EH, Hauge RH, Smalley RE, Schmidt J, Talmon Y (2003) Individually suspended single-walled carbon nanotubes in various surfactants. Nano Lett 3:1379–1382CrossRefGoogle Scholar
  31. Nel A, Xia T, Mädler L, Li N (2006) Toxic potential of materials at the nanolevel. Science 311:622–627CrossRefGoogle Scholar
  32. Nel A, Xia T, Meng H, Wang X, Lin S, Ji Z, Zhang H (2012) Nanomaterial toxicity testing in the 21st century: use of a predictive toxicological approach and high-throughput screening. Acc Chem Res 46(3):607–621CrossRefGoogle Scholar
  33. Pal AK, Bello D, Budhlall B, Milton DK (2012) The limited utility of the dichlorofluorescin (DCFH) assay for measuring oxidative stress elicited by engineered nanomaterials. Dose Response 10:308–330CrossRefGoogle Scholar
  34. Pierzchała K, Lekka M, Magrez A, Kulik AJ, Forró L, Sienkiewicz A (2012) Photocatalytic and phototoxic properties of TiO2-based nanofilaments: ESR and AFM assays. Nanotoxicology 6:813–824CrossRefGoogle Scholar
  35. Rahman I, Kode A, Biswas SK (2007) Assay for quantitative determination of glutathione and glutathione disulfide levels using enzymatic recycling method. Nat Protoc 1:3159–3165CrossRefGoogle Scholar
  36. Rogers EJ, Bello D, Hsieh S (2008) Oxidative stress as a screening metric of potential toxicity by nanoparticles and ariborne particulate matter. Inhal Toxicol 20:895CrossRefGoogle Scholar
  37. Rota C, Chignell CF, Mason RP (1999a) Evidence for free radical formation during the oxidation of 2′-7′-dichlorofluorescin to the fluorescent dye 2′-7′-dichlorofluorescein by horseradish peroxidase: possible implications for oxidative stress measurements. Free Radic Biol Med 27:873–881CrossRefGoogle Scholar
  38. Rota C, Fann YC, Mason RP (1999b) Phenoxyl free radical formation during the oxidation of the fluorescent dye 2′,7′-dichlorofluorescein by horseradish peroxidase. Possible consequences for oxidative stress measurements. J Biol Chem 274:28161–28168CrossRefGoogle Scholar
  39. Rothen-Rutishauser B, Brown DM, Piallier-Boyles M, Kinloch IA, Windle AH, Gehr P, Stone V (2010) Relating the physicochemical characteristics and dispersion of multiwalled carbon nanotubes in different suspension media to their oxidative reactivity in vitro and inflammation in vivo. Nanotoxicology 4:331–342CrossRefGoogle Scholar
  40. Rushton EK, Jiang J, Leonard SS, Eberly S, Castranova V, Biswas P, Elder A, Han X, Gelein R, Finkelstein J, Oberdörster G (2010) Concept of assessing nanoparticle hazards considering nanoparticle dosemetric and chemical/biological response metrics. J Toxicol Environ Health A 73:445–461CrossRefGoogle Scholar
  41. Saran N, Parikh K, Suh DS, Munoz E, Kolla H, Manohar SK (2004) Fabrication and characterization of thin films of single-walled carbon nanotube bundles on flexible plastic substrates. J Am Chem Soc 126:4462–4463CrossRefGoogle Scholar
  42. Sauvain J-J, Deslarzes S, Riediker M (2008) Nanoparticle reactivity toward dithiothreitol. Nanotoxicology 2:121–129CrossRefGoogle Scholar
  43. Schulte PA, Schubauer-Berigan MK, Mayweather C, Geraci CL, Zumwalde R, McKernan JL (2009) Issues in the development of epidemiologic studies of workers exposed to engineered nanoparticles. J Occup Environ Med 51:323–335CrossRefGoogle Scholar
  44. Shvedova AA, Kagan VE, Fadeel B (2010) Close encounters of the small kind: adverse effects of man-made materials interfacing with the nano-cosmos of biological systems. Annu Rev Pharmacol Toxicol 50:63–88CrossRefGoogle Scholar
  45. Valko M, Morris H, Cronin MT (2005) Metals, toxicity and oxidative stress. Curr Med Chem 12:1161–1208CrossRefGoogle Scholar
  46. Vertegel AA, Siegel RW, Dordick JS (2004) Silica nanoparticle size influences the structure and enzymatic activity of adsorbed lysozyme. Langmuir 20:6800–6807CrossRefGoogle Scholar
  47. Wang H, Joseph JA (1999) Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader. Free Radic Biol Med 27:612–616CrossRefGoogle Scholar
  48. Wang G, Zhang J, Dewilde AH, Pal AK, Bello D, Therrien JM, Braunhut SJ, Marx KA (2012) Understanding and correcting for carbon nanotube interferences with a commercial LDH cytotoxicity assay. Toxicology 299:99–111CrossRefGoogle Scholar
  49. WWICS (2009) Woodrow Wilson International Centre for Scholars, vol 2012Google Scholar
  50. Xia T, Kovochich M, Brant J, Hotze M, Sempf J, Oberley T, Sioutas C, Yeh JI, Wiesner MR, Nel AE (2006) Comparison of the abilities of ambient and manufactured nanoparticles to induce cellular toxicity according to an oxidative stress paradigm. Nano Lett 6:1794–1807CrossRefGoogle Scholar
  51. Xia T, Li N, Nel AE (2009) Potential health impact of nanoparticles. Annu Rev Public Health 30:137–150CrossRefGoogle Scholar
  52. Yang H, Liu C, Yang D, Zhang H, Xi Z (2009) Comparative study of cytotoxicity, oxidative stress and genotoxicity induced by four typical nanomaterials: the role of particle size, shape and composition. J Appl Toxicol 29:69–78CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Anoop K. Pal
    • 1
  • 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
  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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