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Interference of engineered nanoparticles with in vitro toxicity assays

  • Inorganic compounds
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Abstract

Accurate in vitro assessment of nanoparticle cytotoxicity requires a careful selection of the test systems. Due to high adsorption capacity and optical activity, engineered nanoparticles are highly potential in influencing classical cytotoxicity assays. Here, four common in vitro assays for oxidative stress, cell viability, cell death and inflammatory cytokine production (DCF, MTT, LDH and IL-8 ELISA) were assessed for validity using 24 well-characterized engineered nanoparticles. For all nanoparticles, the possible interference with the optical detection methods, the ability to convert the substrates, the influence on enzymatic activity and the potential to bind proinflammatory cytokines were analyzed in detail. Results varied considerably depending on the assay system used. All nanoparticles tested were found to interfere with the optical measurement at concentrations of 50 μg cm−2 and above when DCF, MTT and LDH assays were performed. Except for Carbon Black, particle interference could be prevented by altering assay protocols and lowering particle concentrations to 10 μg cm−2. Carbon Black was also found to oxidize H2DCF-DA in a cell-free system, whereas only ZnO nanoparticles significantly decreased LDH activity. A dramatic loss of immunoreactive IL-8 was observed for only one of the three TiO2 particle types tested. Our results demonstrate that engineered nanoparticles interfere with classic cytotoxicity assays in a highly concentration-, particle- and assay-specific manner. These findings strongly suggest that each in vitro test system has to be evaluated for each single nanoparticle type to accurately assess the nanoparticle toxicity.

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Abbreviations

H2DCF-DA:

2′,7′-Dichlorodihydrofluorescein diacetate

DCF:

2′,7′-Dichlorofluorescein

MTT:

(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide)

INT:

(2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride)

MTS:

3-(4,5-Dimethylthiazole-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoliumin

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Acknowledgments

This work was supported by grants of the German Federal Ministry of Education and Research (BMBF projects NanoCare and Cell@Nano) and the state NRW (NanoPaCT). We thank Birgit Phillip for excellent technical assistance.

Conflict of interest

The authors declare that they have no conflict of interest.

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Author notes

  1. Alexandra Kroll

    Present address: Eawag, Swiss Federal Institute of Aquatic Science and Technology, Dübendorf, Switzerland

Authors and Affiliations

  1. Biomedical Technology Center, Westfälische Wilhelms-Universität, Albert-Schweitzer-Campus 1 A14, 48149, Münster, Germany

    Alexandra Kroll, Mike Hendrik Pillukat, Daniela Hahn & Jürgen Schnekenburger

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  1. Alexandra Kroll
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Correspondence to Jürgen Schnekenburger.

Additional information

This article is published as a part of the Special Issue “Nanotoxicology II” on the ECETOC Satellite workshop, Dresden 2010 (Innovation through Nanotechnology and Nanomaterials + Current Aspects of Safety Assessment and Regulation).

Alexandra Kroll and Mike Hendrik Pillukat contributed equally to this article.

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Kroll, A., Pillukat, M.H., Hahn, D. et al. Interference of engineered nanoparticles with in vitro toxicity assays. Arch Toxicol 86, 1123–1136 (2012). https://doi.org/10.1007/s00204-012-0837-z

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  • DOI: https://doi.org/10.1007/s00204-012-0837-z

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