Aneugen Versus Clastogen Evaluation and Oxidative Stress-Related Mode-of-Action Assessment of Genotoxic Compounds Using the ToxTracker Reporter Assay

Abstract

<jats:title>Abstract</jats:title> <jats:p>Understanding the mode-of-action (MOA) of genotoxic compounds and differentiating between direct DNA interaction and indirect genotoxicity is crucial for their reliable safety assessment. ToxTracker is a stem cell-based reporter assay that detects activation of various cellular responses that are associated with genotoxicity and cancer. ToxTracker consists of six different GFP reporter cell lines that can detect the induction of DNA damage, oxidative stress and protein damage in a single test. The assay can thereby provide insight into the MOA of compounds. Genotoxicity is detected in ToxTracker by activation of two independent GFP reporters. Activation of the Bscl2-GFP reporter is associated with induction of DNA adducts and subsequent inhibition of DNA replication and the Rtkn-GFP reporter is activated following the formation of DNA double-strand breaks. Here we show that the differential activation of these two genotoxicity reporters could be used to further differentiate between a DNA reactive and clastogenic or a non-DNA-reactive aneugenic mode-of-action of genotoxic compounds. For further classification of aneugenic and clastogenic compounds, the ToxTracker assay was extended with cell cycle analysis and aneuploidy assessment. The extension was validated using a selection of 16 (genotoxic) compounds with a well-established mode-of-action. Furthermore, indirect genotoxicity related to the production of reactive oxygen species (ROS) was investigated using the DNA damage and oxidative stress ToxTracker reporters in combination with different ROS scavengers. With these new extensions, ToxTracker was able to accurately classify compounds as genotoxic or non-genotoxic, and could discriminate between DNA reactive compounds, aneugens and indirect genotoxicity caused by oxidative stress.</jats:p>

In vitro human cell‐based aneugen molecular mechanism assay

“…In general, in vitro assays currently used to study aneugenicity have concentrated on hazard identification, or to distinguish between micronucleus induction occurring via clastogenicity versus aneugenicity (Brandsma et al, 2020;Bryce et al, 2016Bryce et al, , 2017Bryce et al, , 2018Eastmond & Tucker, 1989;Khoury et al, 2016;Kirsch-Volders et al, 2003). The simplified AMM assay and data analysis rubric described herein is capable of elucidating common genotoxic mechanisms responsible for in vitro aneugenicity.…”

“…It is not surprising then that many genotoxicity assays have been developed to deliver greater insights into MoA, especially in regard to clatogenicity versus aneugenicity. Examples include tubulin polymerization assays (Mirigian et al, 2013), a combination γH2AX/phospho‐histone H3 assay (Khoury et al, 2016), the MultiFlow® DNA Damage Assay (Bryce et al, 2016, 2017, 2018), ToxTracker‐ACE (Brandsma et al, 2020), MEGA‐Screen (Wilson et al, 2021), iScreen (Sun et al, 2021), and TubulinTracker (Geijer et al, 2022).…”

Deducing the cellular mechanisms associated with the potential genotoxic impact of gold and silver engineered nanoparticles upon different lung epithelial cell lines in vitro

“…chromosome fragmentation). Substances deemed to be DNA reactive clastogens are generally considered not to have any safe level of human exposure, as they often pose a carcinogenic threat even at very low-doses (Kirkland et al 2016;Brandsma et al 2020). In line with elevated cytostasis and a 6-fold increase in the number of mononucleated cells present, it is hypothesized that the significant genotoxicity caused by Au ENP exposure is inducing a cell cycle stall to allow for DNA repair to occur before cell proliferation can resume.…”

Commentary: Novel strategies and new tools to curtail the health effects of pesticides

“…Yet the toxicological studies currently supporting pesticide registrations are not always capable of identifying the full range of metabolic and genetic mechanisms leading to, causing, or exacerbating DNA damage [ 154 ]. New methods that can identify genotoxic compounds with high accuracy and characterize their oxidative stress related mode, or modes of action are already available [ 155 ]. While bacterial mutation assays (Ames tests) mentioned above were able to detect genotoxic potential in 46% of a set of test compounds, new assays did so with 95% accuracy [ 156 ].…”