Can Standard Genotoxicity Tests be Applied to Nanoparticles?

Abstract

Experiments were conducted to determine the validity of two common genotoxicity testing procedures, the comet assay and the micronucleus (MN) test, when applied to nanoparticles (NP). The comet assay is used to detect strand breaks (SB) induced in cellular DNA. There is a possibility of obtaining false positive results, if residual NP remain in proximity to the virtually naked DNA that results from lysis of agarose-embedded cells, and react with this DNA in ways that do not occur with chromatin in intact cells. However, data showed that if NP are deliberately present at high concentration with lysed cells, there is no change in SB with a range of NP. Only oleic acid-coated Fe₃O₄ NP induced damage, as these particles also produced equivalent alterations in whole cells. A modification of the comet assay incorporates digestion of DNA with lesion-specific endonucleases, notably formamidopyrimidine DNA glycosylase (FPG), which detects oxidized purines. Again there is a concern regarding the presence of residual NP with DNA of lysed cells, but this time because of the risk of false negative results if NP interfere with the FPG reaction. However, it was found that incubation of cells with NP before treatment with a known 8-oxoguanine-inducing agent does not lead to any decrease in the yield of FPG-sensitive sites. Chromosomal damage is detected with the MN assay, which depends on the use of cytochalasin B (CB) to prevent cell division and accumulates binucleate cells. It is known that CB also inhibits endocytosis, and thus might prevent NP uptake. Data demonstrated that if NP are added to cells together with CB, fewer MN are induced. It is therefore necessary to treat cells with NP prior to CB in order to avoid interference and possible false negative results.

Acknowledgments

Zuzana Magdolenova and Yolanda Lorenzo made equal contributions to this work. The work was supported by EC FP7 [Health-2007-1.3-4, contract 201335] and by the Polish-Norwegian Research Fund [PRNF/122-A I-1/07]. We thank Prof. Francelyne Marano and Dr. Sonja Boland for providing the dispersion protocol, and Dr. Giulio Pojana, Dr. Dagmar Bilanicova, and Prof. Antonio Marcomini for help with characterization of NP.