EN

María Moreno-Villanueva

doi:10.14573/altex.2011.4.295

Abstract

Genotoxicity tests are essential to identify compounds that have a potential to compromise not only the environment but also human and animal health, including compounds that increase the risk of cancer. At present, no single test is capable of detecting all types of genotoxic effects; therefore a battery of in vitro and, if positive, in vivo tests is necessary to determine the genotoxicity of a substance. However, the respective specificities of current assays have been criticized for their high percentage of false positive results. We recently published an automated version of the "Fluorimetric detection of Alkaline DNA Unwinding" (FADU) assay for measuring DNA strand breaks in human peripheral blood mononuclear cells or in cell lines. Using this new technology we show detection of DNA strand breaks in cells treated with several compounds known to induce DNA strand breaks by various mechanisms. We also tested toxic compounds that were not expected to induce DNA strand breaks; these were negative in the assay as expected. Finally, we included zinc oxide nanoparticles of high production volume to explore further fields of potential FADU applications. The main advantages of this assay are high reproducibility, easy handling, lack of operator bias, high-throughput, speed, and low cost.

Highlights

Environmental agents, or medicinal drugs may induce different types of DNA damage in organisms, which may lead to mutations, genetic instability, and epigenetic alterations and may cause a variety of diseases, including cancer or even death
The European Centre for the Validation of Alternative Methods (ECVAM) is working on the validation of the comet assay, and the European Comet Assay Validation Group (ECVAG) recently reported an inter-laboratory coefficient of variation (CV) of 47%, which could be reduced to 28% using reference standards (Forchhammer et al, 2010)
The results indicate an increase in both DNA strand breaks and number of apoptotic/necrotic cells in a timedependent manner (Fig. 4A,B)

Summary

Introduction

Environmental agents, or medicinal drugs may induce different types of DNA damage in organisms, which may lead to mutations, genetic instability, and epigenetic alterations and may cause a variety of diseases, including cancer or even death. Current regulatory measures to identify genotoxic compounds include the following: bacterial reverse mutation (Ames) test, in vitro cell gene mutation test, in vitro chromosomal aberration test, in vitro mammalian cell micronucleus test, in vitro sister chromatid exchange test, in vitro unscheduled DNA synthesis test, Saccharomyces cerevisiae gene mutation assay, and Saccharomyces cerevisiae mitotic recombination assay. Multiple DNA repair pathways have evolved to minimize the negative effects of DNA damage. Several methods have been established to investigate DNA lesions and different DNA repair pathways in the cell. The most commonly used method to measure DNA damage is the comet assay, which detects DNA strand breaks and alkaline labile sites in individual cells (Olive and Banáth, 2006)

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Conclusion