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Updated: Aug 30, 2026
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Published on: June 2, 2023
A new method for in vitro detection of microbially produced mitochondrial toxins
D Hoornstra1, M A Andersson, R Mikkola
1University of Helsinki, Department of Applied Chemistry and Microbiology, Division of Microbiology, PO Box 56, FIN-00014 University of Helsinki, Finland.
Abstract:
Sperm motility inhibition assay, earlier shown valuable for the detection of food poisoning non-protein toxins of Bacillus species was developed into an assay useful for specific detection of mitochondria damaging toxins. This was done by assessing the dissipation of the mitochondrial inner membrane transmembrane potential, Deltapsim under conditions where the plasma membrane permeability barrier remained intact. The Deltapsim was estimated as the intensity of orange JC-1 fluorescence in the mitochondrial sheath of the exposed spermatozoa. The plasma membrane integrity of the same cells was assessed by observing the exclusion of propidium iodide from the cytoplasm. Three types of mitochondrial toxic responses to microbially made bioactive substances were recognised. Mitochondrial toxicity by gramicidin (A, B, C, D), nigericin, salinomycin, narasin, monensin, calcimycin and antimycin A was characterised by gradual fading of the JC-1 fluorescence in the mitochondria. Dissipation of the Deltapsim by cereulide, valinomycin and enniatin (A, A1, B, B1) was visible as spotwise quenching of the mitochondrial JC-1 fluorescence. In addition these substances caused hyperpolarisation of the plasma membrane. Oligomycin (A, B, C), ionomycin and staurosporine inhibited the spermatozoan motility, but Deltapsim was fully preserved. Surfactin and lichenysin A caused mitochondrial damage at concentrations where the plasma membrane was also damaged.
Insights
This study refines a sperm motility assay to specifically detect mitochondrial toxins. The enhanced assay measures mitochondrial membrane potential, differentiating toxin effects for improved detection of cellular damage.
Area of Science:
- Biochemistry
- Toxicology
- Cell Biology
Background:
- Sperm motility inhibition assays can detect bacterial toxins.
- Mitochondria are key targets for various cellular toxins.
- Specific detection of mitochondrial toxins requires sensitive assays.
Purpose of the Study:
- To develop a sperm-based assay for specific detection of mitochondrial toxins.
- To characterize different types of mitochondrial toxic responses.
- To differentiate toxins based on their effects on mitochondrial membrane potential and plasma membrane integrity.
Main Methods:
- Assessing mitochondrial inner membrane potential (Deltapsim) using JC-1 fluorescence in spermatozoa.
- Evaluating plasma membrane integrity by propidium iodide exclusion.
- Classifying toxic responses based on fluorescence changes and membrane potential.
Main Results:
- Three distinct mitochondrial toxic responses were identified.
- Gramicidin, nigericin, and antimycin A caused gradual Deltapsim dissipation.
- Cereulide, valinomycin, and enniatin induced spotwise quenching of fluorescence and plasma membrane hyperpolarization.
- Oligomycin, ionomycin, and staurosporine inhibited motility without affecting Deltapsim.
- Surfactin and lichenysin A caused simultaneous mitochondrial and plasma membrane damage.
Conclusions:
- The modified sperm motility assay accurately detects and differentiates mitochondrial toxins.
- The assay distinguishes between toxins that dissipate mitochondrial membrane potential and those that inhibit motility.
- This method provides a sensitive tool for identifying specific mitochondrial toxicants.
