A Sensitive, Cell-Based Assay for Measuring Low-Level Biological Activity of α-Amanitin.

Area of Science:

• Toxicology
• Molecular Biology
• Biochemistry

Background:

• Alpha-amanitin from *Amanita* mushrooms is a potent toxin responsible for fatal poisonings due to its odorless and tasteless nature.
• Understanding the toxin's stability is crucial for risk assessment and potential mitigation strategies.

Purpose of the Study:

• To investigate the thermal stability and biological activity of alpha-amanitin using novel cell-based assays.
• To develop sensitive methods for detecting alpha-amanitin activity and its inhibition of RNA polymerase II.

Main Methods:

• Development of three cell-based assays: MTT-formazan for viability, RT-qPCR for viral replication inhibition, and GFP expression for toxin activity.
• Testing alpha-amanitin's activity after thermal stress (90°C for 16h, microwave for 3 min) in HEK293 and Vero cells.

Main Results:

• MTT assay detected 10 μg/mL; RT-qPCR detected 100 ng/mL, showing significant adenovirus transcription inhibition.
• GFP assay demonstrated alpha-amanitin retained biological activity after heat and microwave treatment in HEK293 cells, with slight reduction in Vero cells.

Conclusions:

• Alpha-amanitin exhibits significant thermal stability, retaining biological activity after substantial heat exposure.
• The developed cell-based assays are sensitive tools for detecting alpha-amanitin activity and can be adapted for other RNA polymerase inhibitor toxins.