3Rs‐friendly approach to exogenous metabolic activation that supports high‐throughput genetic toxicology testing

Abstract

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. K E Y W O R D SDNA damage, flow cytometry, metabolic activation, γH2AX, p53

The use of benchmark dose uncertainty measurements for robust comparative potency analyses

“…The data were derived from a previously published article in which 15 compounds were studied using the in vitro MultiFlow® DNA Damage Assay in the presence and absence of low dose (0.25% vol/vol) S9 (Tian et al 2020). The in vitro MultiFlow DNA Damage Assay multiplexes several biomarkers that are responsive to diverse forms of DNA damage into a single flow cytometric analysis.…”

“…Herein we consider a previously published in vitro genotoxicity dataset where the authors performed BMD analyses and drew conclusions without considering the uncertainty associated with the BMD measurements. Tian et al (2020) investigated the use of phenobarbital/β‐napthoflavone‐induced rat liver S9 at maximal non‐cytotoxic concentration (0.25% vol/vol final) in a flow cytometry based multiplexed DNA damage response assay. The laboratory was able to maintain the S9 enzyme/co‐factor mix with cells and test compound for the entire exposure period of 24 hr in the TK6‐based assay.…”

“…To our knowledge, there are no published reports that critique the use of a BMD point estimate, and how this differs from use of BMD CIs in comparative analysis between experimental conditions. Herein, the Tian et al (2020) data is re‐analyzed to further inform use of the same in vitro genotoxicity/low concentration S9 dataset. Primarily, we stress that the use of a BMD point estimate value does not provide an accurate representation of the likely potency range of the test compound.…”

“…This current report focuses on reanalysis and augmentation of the previously published Tian et al (2020) dataset. Specifically, we convey the BMD uncertainty measurements that relate to the relative genotoxic potency of the 10 clastogenic compounds.…”

“…Specifically, we convey the BMD uncertainty measurements that relate to the relative genotoxic potency of the 10 clastogenic compounds. To this end, we calculate “S9 potency ratio CIs” using the BMD uncertainty measurements (BMDL and BMDU) between S9 exposure conditions (the presence and absence of S9) and utilize said ratios to derive robust potency conclusions as a follow up to the Tian et al (2020) work. Readers are encouraged to refer to the original Tian and colleagues' article for further context (Tian et al, 2020).…”