Co-culture for comet assay: in vitro model for predictive genotoxicology

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• Created: Wednesday, 09 April 2014 14:34

Researchers from the Laboratoire de Fougères, ANSES, have recently reported on the genotoxicity of ingested compounds, such as those found in oral drugs and food contaminants.  The three compounds investigated in the study were: methylmethanesulfonate (MMS), benzo[a]-pyrene (BaP) and aflatoxin B1 (AFB1).

Considering the role of intestinal first-pass metabolism in the genotoxicity response of ingested compounds, the researchers investigated the feasibility of a co-culture system of intestinal Caco-2 cells and lymphoblastoid TK6 cells to test the predictive genotoxicity of ingested compounds.

Caco-2 cells were chosen because they display the morphological and biochemical characteristics of a human model for intestinal metabolism and transport studies. The Caco-2 cells can differentiate and form a monolayer. 

The P53-competent human TK6 cells are commonly used in genotoxicology testing as a target blood cell model for the micronucleus assay and in the thymidine kinase (TK) gene-mutation assay. Dividing TK6 cells were selected to mimic the blood compartment and target cells of toxic metabolites produced by enterocytes.

All three compounds were tested on the both cell lines (Caco-2 and TK6) within the co-culture system using the comet and the micronucleus assays.

After performing the comet assay, DNA was stained with propidium iodide (2.5 µg/ml in PBS) before examination of the slide with a fluorescence microscope equipped with a camera. At least two slides per dose and 100 cells per slide were analysed using Comet Assay IV (Perceptive Instruments, Suffolk, UK). The percentage of DNA in the comet tail (% tail DNA) was used to evaluate the extent of DNA damage.

MMS (10 µg/ml) showed positive results in the micronucleus assays in both cell lines, even though DNA damage was only detected in the Caco-2 cells with the comet assay. At concentrations of 0.5–50 µM, BaP induced dose-dependent comet and micronucleus formation at 24 hours in Caco-2 cells, but no DNA damage was observed in TK6 cells. Although AFB1 failed to induce comet formation, it resulted in a high level of micronuclei in both cell lines.

The researchers concluded that the results suggested that the Caco-2/TK6 co-culture model is suitable for modelling the role of intestinal biotransformation and transport processes in the genotoxic potential of oral drugs and food contaminants in target blood cells.  The researchers believe that the co-culture system contributes to the development of new in vitro models that help increase predictivity in genotoxicology.

Case study based on:
A co-culture system of human intestinal Caco-2 cells and lymphoblastoid TK6 cells for investigating the genotoxicity of oral compounds
Ludovic Le Hégarat, Sylvie Huet and Valérie Fessard, Mutagenesis, 2012 Nov;27(6):631-6.