Real-time Monitoring of Intracellular ROS and Screening of Antioxidant Actives

Reactive oxygen species (ROS) are generated intracellularly and their accumulation in vascular endothelium has been proposed as a mechanism of tissue injury such as inflammation (1).

Objective: The purpose of this work is to develop a real-time approach to measure concentrations of intracellular ROS utilizing fluorescent dyes, and thus to screen free radical scavenging capacities of actives considered as potential inflammation-control reagents as well as dentifrices in vitro.

Methods: Two fluorescent dyes exhibiting a characteristic fluorescence intensity in oxidized or non-oxidized form, fluorescein (DCF-DA) and rhodamine (DHR123), were introduced to cultured cells, respectively, followed by addition of the ROS stimulants, hydrogen peroxide (H₂O₂) and lipopolysaccharide (LPS). The actives were added and cells were incubated at 37 ^oC. Fluorescence intensity was measured after various incubation times.

Results: Three leading actives, a-tocopherol, Substance 1 (S1) and Substance 2 (S2), exhibited strong antioxidant capacity. At 30 min 100 ppm of a-tocopherol reduced 44% of H₂O₂ induced oxidative stress. The reduction level of H₂O₂ free radical was 77% for S1 and 36% for S2. In terms of LPS induced oxidation, the reduction percentage was 30% for a-tocopherol, 70% for S1 and 8% for S2.

Conclusion: The study shows that ROS generation is scavenged by three antioxidant actives to various extents in the form of simple solutions in a time dependent fashion. This new approach can be extended to monitor in real time the ROS production and inhibition in individual cells by utilizing Optical LiveCell Array technology and microscopy imaging software.

(1). Brigham, K. L.; Meyrick, B.; Berry, L. C.; Repine, J. E. (1987) J. Appl. Physiol. 63, 840-850