R3ACT: An Oxygen-Controlled Biosensor-Based NAM to Support Animal-Free Chemical Safety Assessment

Area of Science:

• Toxicology and Chemical Safety
• Cellular and Molecular Biology
• Biomedical Engineering

Background:

• In vitro chemical safety testing often occurs at ambient oxygen, not physiological levels, potentially misinterpreting chemical effects.
• Redox balance and antioxidant responses are critical in human tissues but poorly modeled in standard assays.
• Existing methods lack the mechanistic detail to fully understand chemical interactions under physiologically relevant oxygen conditions.

Purpose of the Study:

• To introduce R3ACT (Redox & Antioxidant Capacity Tracker), an oxygen-controlled, animal-free live-cell assay for mechanistic profiling.
• To assess redox perturbation and antioxidant response activation in human cells under controlled oxygen.
• To evaluate the utility of R3ACT for prioritizing compounds in chemical and drug screening.

Main Methods:

• Developed R3ACT, integrating viability-gated concentration selection, oxygen-controlled plate-reader measurements, and high-content microscopy.
• Utilized cytosolic and mitochondrial HyPer7.2 biosensors for H₂O₂ dynamics and a POINTER Nrf2 transcriptional reporter.
• Employed deep-learning-assisted single-cell analysis for mechanistic profiling of chemical exposures.

Main Results:

• Zinc oxide and polystyrene nanoplastics showed reproducible Nrf2 activation, confirmed by microscopy.
• Perfluorooctanoic acid exhibited a borderline response, not confirmed by microscopy.
• Salicylic acid induced Nrf2 activation, interpreted as antioxidant response engagement, not skin sensitization.

Conclusions:

• R3ACT is an early-stage, oxygen-controlled assay supporting animal-free screening by providing mechanistic redox/Nrf2 profiles.
• The assay offers insights into cellular responses under physiologically relevant oxygen levels.
• Further validation is needed for regulatory applicability, including benchmarking and inter-laboratory studies.