HTS compatible assay for antioxidative agents using primary cultured hepatocytes

Area of Science:

• Biochemistry
• Cell Biology
• Pharmacology

Background:

• Reactive oxygen species (ROS) play a critical role in oxidative stress and associated pathological alterations.
• There is a significant need for novel compounds with antioxidant properties to combat oxidative stress.
• High-throughput screening (HTS) requires accurate and reliable assays for identifying potential drug candidates.

Purpose of the Study:

• To establish a cell-based assay system using primary cultured rat hepatocytes compatible with HTS.
• To develop a reliable method for screening substance libraries for compounds with antioxidant potential.
• To validate the assay's ability to accurately detect biologically active compounds, such as fisetin.

Main Methods:

• Primary cultured rat hepatocytes were treated with tert-butyl hydroperoxide (t-BHP) to induce oxidative stress and ROS production.
• A novel cell-based assay system was developed and optimized for HTS.
• Measurements were conducted using the Mithras LB 940 Multilabel Reader, with Z' factors assessed for reliability.

Main Results:

• The developed assay system demonstrated high reliability and accuracy in detecting compounds with antioxidant potential.
• The system successfully identified fisetin, a known antioxidative compound, from a substance library.
• Consistently high Z' factors were obtained, indicating the assay's robustness.

Conclusions:

• A versatile and reliable HTS system for screening antioxidant compounds using rat hepatocytes was successfully established.
• The assay system is suitable for identifying biologically active compounds with high confidence.
• The protocol can be readily adapted for screening other types of biologically active substances beyond antioxidants.