In vitro human cell‐based aneugen molecular mechanism assay

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., Toxi...

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Bibliographic Details
Published in:Environmental and molecular mutagenesis 2022-03, Vol.63 (3), p.151-161
Main Authors: Hall, Nikki E., Tichenor, Kyle, Bryce, Steven M., Bemis, Jeffrey C., Dertinger, Stephen D.
Format: Article
Language:English
Subjects:
Acid dyes
aneugen
Aneugens - toxicity
Antibodies
Aurora kinase
aurora kinase inhibitors
Data analysis
Destabilization
Flow cytometry
Fluorescence
Histone H3
Histones
Humans
Kinases
Micronucleus Tests - methods
Microtubules
Molecular modelling
Mutagens - pharmacology
Nucleic acids
Paclitaxel
Paclitaxel - pharmacology
Taxol
TK6 cells
Tubulin
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Summary: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 Taxol‐associated 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.
ISSN:0893-6692
1098-2280
DOI:10.1002/em.22480