Comparison of in vitro high content screening methods for drug genotoxicity
Kuokkanen, Emmi (2021)
Kuokkanen, Emmi
2021
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Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi-fe2021042511768
https://urn.fi/URN:NBN:fi-fe2021042511768
Tiivistelmä
Detection of chemicals that induce damage to the DNA is an important aspect in drug development. The recognition of these chemicals is based on in vitro studies followed by in vivo studies, the latter being both expensive and ethically questionable. An early, accurate prediction of the genotoxic properties of these chemicals is therefore highly desired.
The phosphorylated histones yH2AX and pH3 are well established genotoxicity markers, yH2AX expressed upon double-stranded DNA damage caused by clastogens, and pH3 accumulating as a cause of aneuploidy in mitosis caused by aneugens. The detection of these, in combination with a translocation of p53 to the nucleus in response to DNA damage, is one example of markers used for high content genotoxicity analyses. In this study, these three markers were used to evaluate the genotoxic predictivity of 16 reference compounds with known properties in TK6 and HepG2 cells by two different high content methods for genotoxicity screening. The cells were exposed to the compounds over a range of concentrations for 4 and 24 hours. The first method used was a validated flow cytometry-based DNA damage assay MultiFlow® which was used with both TK6 and HepG2 cells. The other method was an imaging-based high content analysis method that was set up in this study and tested on HepG2 cells only. The aim was to compare these two methods and see which method could predict the genotoxic potential more accurately.
Using the MultiFlow® method with TK6 cells and previously determined cut-off values for genotoxicity, 14 out of 16 compounds were predicted correctly. With the imaging-based method with HepG2 cells the corresponding predictivity was 15 out of 16. By adapting the imaging-based method to include a 48-hour incubation with the reference compounds all compounds could be predicted correctly. This prediction did, however, not include p53 as a criterion and it wastherefore not considered as a significant marker in HepG2 cells in this study. Based on these results the imaging-based genotoxicity assay with HepG2 cells was considered a promising alternative for genotoxicity testing.
The phosphorylated histones yH2AX and pH3 are well established genotoxicity markers, yH2AX expressed upon double-stranded DNA damage caused by clastogens, and pH3 accumulating as a cause of aneuploidy in mitosis caused by aneugens. The detection of these, in combination with a translocation of p53 to the nucleus in response to DNA damage, is one example of markers used for high content genotoxicity analyses. In this study, these three markers were used to evaluate the genotoxic predictivity of 16 reference compounds with known properties in TK6 and HepG2 cells by two different high content methods for genotoxicity screening. The cells were exposed to the compounds over a range of concentrations for 4 and 24 hours. The first method used was a validated flow cytometry-based DNA damage assay MultiFlow® which was used with both TK6 and HepG2 cells. The other method was an imaging-based high content analysis method that was set up in this study and tested on HepG2 cells only. The aim was to compare these two methods and see which method could predict the genotoxic potential more accurately.
Using the MultiFlow® method with TK6 cells and previously determined cut-off values for genotoxicity, 14 out of 16 compounds were predicted correctly. With the imaging-based method with HepG2 cells the corresponding predictivity was 15 out of 16. By adapting the imaging-based method to include a 48-hour incubation with the reference compounds all compounds could be predicted correctly. This prediction did, however, not include p53 as a criterion and it wastherefore not considered as a significant marker in HepG2 cells in this study. Based on these results the imaging-based genotoxicity assay with HepG2 cells was considered a promising alternative for genotoxicity testing.