In vitro human cell‐based aneugen molecular mechanism assay

Abstract

This laboratory previously described an in vitro human cell-based assay and data analysis scheme that discriminates common molecular targets responsible for chemical-induced in vitro aneugenicity: tubulin destabilization, tubulin stabilization, and inhibition of Aurora kinases (Bernacki et al., Toxicol. Sci. 170 [2019] 382-393).The current report describes updated procedures that simplify benchtop processing and data analysis methods. For these experiments, human lymphoblastoid TK6 cells were exposed to each of 25 aneugens over a range of concentrations in the presence of fluorescent paclitaxel (488 Taxol). After a 4 h treatment period, cells were lysed and nuclei were stained with a nucleic acid dye and labeled with fluorescent antibodies against phospho-histone H3 (p-H3). Flow cytometric analyses revealed several unique signatures: tubulin stabilizers caused increased frequencies of p-H3-positive events with concentration-dependent increases in 488 Taxolassociated fluorescence; tubulin destabilizers caused increased frequencies of p-H3-positive events with concomitant decreases in 488 Taxol-associated fluorescence; and Aurora kinase B inhibitors caused reduced frequencies of p-H3-positive events and lower median fluorescent intensities of p-H3-positive events. These results demonstrate a simple rubric based on 488 Taxol-and p-H3-associated metrics can reliably discriminate between several commonly encountered aneugenic molecular mechanisms.

Adverse (geno)toxic effects of bisphenol A and its analogues in hepatic 3D cell model

“…In addition to BPAF, BPA also increased the frequency of p-H3-positive cells compared with control, but only at a low concentration (0.1 μM). The combination of γH2AX/phospho-histone p-H3 assays, allows us to distinguish whether a compound is clastogenic or aneugenic, which is not possible using the in vitro micronucleus assay ( Bryce, 2014 ) unless CREST antibodies or FISH probes are used Hall et al, 2022 . The results on HepG2 spheroids suggest that BPAF, which induced γH2AX and histone p-H3 positive events, acted as an aneugenic agent, whereas for BPC and BPA there is an indication that they may have an aneugenic effect, but this should be further confirmed.…”

Visualization strategies to aid interpretation of high‐dimensional genotoxicity data

“…Interested readers are directed to recent reviews by Krewski et al (2022) and Pognan et al (2023). With the discipline of genetic toxicology working toward providing more detailed information, for example about test substances’ predominant genotoxic mode of action or mechanism, high‐information content genotoxicity assays have proliferated (Bryce et al, 2016, 2017, 2018; Chapman et al, 2021; Corton et al, 2018; Hall et al, 2022; Hendriks et al, 2012; Salk & Kennedy, 2020; Sun et al, 2022; Wilson et al, 2021).…”