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Acellular Comet Detects DNA Reactivity

In response to regulatory requests for DNA reactivity data, Helix3 developed the acellular comet assay with positive controls for detecting specific forms of DNA damage such as strand breaks, alkali labile sites, and crosslinks without the potentially confounding effects of cytotoxicity, cell division, or excision repair. This assay can be used as a fast and reproducible method to screen early development stage compounds for DNA reactivity that could induce a positive response in the standard battery of genotoxicity tests. Alternatively, it may be used as additional weight of evidence to demonstrate the absence of DNA reactivity for highly cytotoxic exposures. The alkylating agent EMS and the highly cytotoxic Chlorambucil induce dramatic and dose-related increases in DNA migration. Meanwhile, the DNA-DNA crosslinker cisplatin and the protein-DNA crosslinker formaldehyde both induce dramatic and dose-related decreases. The repair inhibitor Aphidicolin did not have any direct effect on DNA migration as it indirectly induces strand breaks by inhibiting the completion of excision repair. While the topoisomerase II inhibitor Etoposide appeared to directly interact with DNA despite the absence of cell division or metabolism. But high inter-and intra-variability appears to indicate the presence of opposing mechanistic influences on DNA migration. And in the vehicle,s dH2O induced consistently higher migration than DMSO which was considered most likey due to hydrolysis induced by the dH2O.


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