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Updated: May 12, 2026
Evaluating In Vitro DNA Damage Using Comet Assay
Published on: October 11, 2017
Effects of ochratoxin A on DNA evaluatedin vitro with the single cell gel electrophoresis (Comet assay)
1Institut für Arbeitsphysiologie an der Universität Dortmund, Ardeystr. 67, 44139, Dortmund, Germany.
Abstract:
In cell cultures of Madin Darby canine kidney (MDCK) cells, the mycotoxin ochratoxin A (OTA) induced single strand breaks (ssb) in a concentration dependent manner detected with the single cell gel electrophoresis (Comet assay). When an external metabolizing enzyme system (S9-mix from rat liver) was added, this genotoxic effect was significantly stronger. By addition of methotrexate (MT), a substrate of the hepatic organic anion transporter, the effect of OTA can be completely blocked at concentrations >100 μM methotrexate.When DNA repair was inhibited by addition of cytosine arabinose (araC) and hydroxyurea (HU), the tail length in the Comet assay increased dramatically and all treated cells showed ssb. A further culture of the damaged cells in the absence of any supplement resulted in a complete repair of the damaged DNA within three hours.Compared with MDCK cells, primary cultured porcine urinary bladder epithelial cells (PUBECs) showed weaker effects in the Comet assay if treated with OTA. The presence of S9-mix did not significantly enhance the response. Methotrexate only partially reduced the OTA-induced effects, because in PUBECs methotrexate induced ssb at high concentrations. If DNA repair was inhibited, also in PUBECs clearly more ssb were induced by OTA, an effect which was reversible.These results demonstrate that OTA induces single strand breaks in vitro. The damaged DNA can be repaired more effectively in primary cultured epithelial cells (PUBECs) compared to cells of a cell line (MDCK cells). By competitive inhibition of OTA uptake, DNA damage can be prevented with suitable substrates.
Insights
The mycotoxin ochratoxin A (OTA) causes DNA damage in kidney cells, which is enhanced by liver enzymes but blocked by methotrexate. DNA repair is more effective in primary bladder cells than cell lines.
Area of Science:
- Toxicology
- Molecular Biology
- Cell Biology
Background:
- Mycotoxins, such as ochratoxin A (OTA), are common food contaminants.
- OTA is known to exhibit nephrotoxic and genotoxic effects.
- Understanding OTA's genotoxicity and cellular response is crucial for risk assessment.
Purpose of the Study:
- To investigate the genotoxic effects of OTA on kidney and bladder epithelial cells in vitro.
- To explore the role of metabolic activation and DNA repair mechanisms in OTA-induced DNA damage.
- To assess the potential of competitive inhibition in preventing OTA genotoxicity.
Main Methods:
- Single cell gel electrophoresis (Comet assay) to detect DNA single-strand breaks (ssb).
- Use of Madin Darby canine kidney (MDCK) cells and primary porcine urinary bladder epithelial cells (PUBECs).
- Inclusion of rat liver S9-mix for metabolic activation and inhibitors (methotrexate, cytosine arabinose, hydroxyurea) to study repair and uptake.
Main Results:
- OTA induced concentration-dependent DNA ssb in MDCK cells, enhanced by S9-mix.
- Methotrexate (MT) blocked OTA effects in MDCK cells, while it partially reduced effects in PUBECs due to MT's own genotoxicity.
- DNA repair was complete within three hours in MDCK cells and OTA-induced damage was reversible in PUBECs, with primary cells showing more effective repair.
Conclusions:
- OTA is confirmed to induce DNA single-strand breaks in vitro.
- Primary cultured epithelial cells (PUBECs) exhibit more effective DNA repair of OTA-induced damage compared to cell lines (MDCK).
- Competitive inhibition of OTA uptake using specific substrates can prevent DNA damage.
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