3Rs-friendly approach to exogenous metabolic activation that supports high-throughput genetic toxicology testing.
Activation, Metabolic
/ drug effects
Animals
Anisomycin
/ toxicity
Brefeldin A
/ toxicity
Cell Line
Cycloheximide
/ toxicity
DNA Damage
/ drug effects
High-Throughput Screening Assays
/ methods
Histones
/ genetics
Humans
Liver
/ drug effects
Mutagenicity Tests
/ methods
Mutagens
/ toxicity
Rats
Tumor Suppressor Protein p53
/ genetics
DNA damage
flow cytometry
metabolic activation
p53
γH2AX
Journal
Environmental and molecular mutagenesis
ISSN: 1098-2280
Titre abrégé: Environ Mol Mutagen
Pays: United States
ID NLM: 8800109
Informations de publication
Date de publication:
04 2020
04 2020
Historique:
received:
03
01
2020
revised:
29
01
2020
accepted:
05
02
2020
pubmed:
11
2
2020
medline:
30
10
2020
entrez:
11
2
2020
Statut:
ppublish
Résumé
MultiFlow® DNA Damage-p53, γH2AX, Phospho-Histone H3 is a miniaturized, flow cytometry-based assay that provides genotoxic mode of action information by distinguishing clastogens, aneugens, and nongenotoxicants. Work to date has focused on the p53-competent human cell line TK6. While mammalian cell genotoxicity assays typically supply exogenous metabolic activation in the form of concentrated rat liver S9, this is a less-than-ideal approach for several reasons, including 3Rs considerations. Here, we describe our experiences with low concentration S9 and saturating co-factors which were allowed to remain in contact with cells and test chemicals for 24 continuous hours. We exposed TK6 cells in 96-well plates to each of 15 reference chemicals over a range of concentrations, both in the presence and absence of 0.25% v/v phenobarbital/β-naphthoflavone-induced rat liver S9. After 4 and 24 hr of treatment cell aliquots were added to wells of a microtiter plate containing the working detergent/stain/antibody cocktail. After a brief incubation robotic sampling was employed for walk-away flow cytometric data acquisition. PROAST benchmark dose (BMD) modeling was used to characterize the resulting dose-response curves. For each of the 8 reference pro-genotoxicants studied, relative nuclei count, γH2AX, and/or p53 biomarker BMD values were order(s) of magnitude lower for 0.25% S9 conditions compared to 0% S9. Conversely, several of the direct-acting reference chemicals exhibited appreciably lower cytotoxicity and/or genotoxicity BMD values in the presence of S9 (eg, resorcinol). These results prove the efficacy of the low concentration S9 system, and indicate that an efficient and highly scalable multiplexed assay can effectively identify chemicals that require bioactivation to exert their genotoxic effects.
Substances chimiques
Histones
0
Mutagens
0
Tumor Suppressor Protein p53
0
Brefeldin A
20350-15-6
Anisomycin
6C74YM2NGI
Cycloheximide
98600C0908
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Langues
eng
Sous-ensembles de citation
IM
Pagination
408-432Subventions
Organisme : NIEHS NIH HHS
ID : R44 ES029014
Pays : United States
Informations de copyright
© 2020 Wiley Periodicals, Inc.
Références
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