A sensitive cell-based assay for measuring low-level biological activity of a-amanitin

Authors: Rasooly, Reuven; Do, Paula; He, Xiaohua; Hernlem, Bradley - Brad

Submitted to: International Journal of Molecular Sciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/14/2023
Publication Date: 11/16/2023
Citation: Rasooly, R., Do, P.M., He, X., Hernlem, B.J. 2023. A sensitive cell-based assay for measuring low-level biological activity of a-amanitin. International Journal of Molecular Sciences. 24(22). Article e16402. https://doi.org/10.3390/ijms242216402.
DOI: https://doi.org/10.3390/ijms242216402

Interpretive Summary: Alpha-Amanitin is one of the main toxins responsible for poisoning and death of persons eating misidentified mushrooms. It has no smell or taste and works by stopping the victim’s body from making new proteins needed for life. Common tests for this toxin measure the amount of the chemical but do not measure whether the toxin is in an active, deadly form. We developed new methods to test for alpha-amanitin activity using living cells to measure its effect on new protein formation. We improved the method and made it simpler, finally using cells that make a glowing green protein. Cooking is not known to make poisonous mushrooms safe to eat but some people using chemical tests have found that heat can reduce the amount of alpha-amanitin. Using our activity method, we showed that the toxin was still active after heating for 16 h at 90°C or microwaving for 3 minutes. The importance of this work is in food safety and in food processing.

Technical Abstract: Alpha-Amanitin is one of the primary toxins in poisonous mushrooms. Being odorless and tasteless, it is an important cause of death from consumption of misidentified mushrooms. The toxin functions by inhibiting RNA polymerase II, preventing protein synthesis. Amanitins are traditionally characterized by their thermal stability but recent works by researchers using chemical detection methods such as HPLC have reported degradation of alpha-amanitin during cooking and boiling. However, this chromatography detection method provides no information on the biological activity of the toxin. We harnessed alpha-amanitin’s inhibition effect on RNA polymerase II and developed new cell-based assays to measure alpha-amanitin activity; initially using MTT-formazan cell viability assay. To increase the sensitivity of detection by 100 times we turned to quantitative PCR and examined inhibition of viral replication. We made this activity assay easier to perform by generating an adenovirus that expressed GFP in transduced cells. After treatments with alpha-amanitin, GFP fluorescence emission inhibition was quantified. Using this activity assay, we found that heating alpha-amanitin in aqueous solution at 90°C for 16 h or microwave treatment for 3 minutes did not reduce alpha-amanitin biological activity when tested in HEK cells, but slight reduction was observed when tested in Vero cells.