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Alternatives to Laboratory Animals - ATLA

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The Comet Assay with MCL-5 Cells as an Indicator of Genotoxic Treatment with Chemicals and Cigarette Smoke Condensate

Lucie Wolz, Günter Krause and Gerhard Scherer

The metabolically competent human lymphoblastoid cell line MCL-5 was treated with a panel of mutagens to assess the induction of DNA damage. Treatment effects were observed by monitoring cell proliferation and by single-cell gel electrophoresis (SCGE). The direct-acting mutagens benzo[a]pyrene-7,8-diol 9,10-epoxide (BPDE) and 1-methyl-3-nitro-1-nitrosoguanidine (MNNG), as well as pro-mutagens requiring metabolic activation, i.e. benzo[a]pyrene (BaP), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 4-N-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and cigarette-smoke condensate (CSC), were assayed by SCGE. Assay schemes were adapted for the MCL-5 cell line and for low levels of strand break induction, by inclusion of the DNA synthesis inhibitors cytosine arabinoside and hydroxyurea, and by extending the electrophoresis time. For all mutagens tested, dose-dependent increases of median and average tail moment values among 50 nucleoids per slide were observed. The determining factors for selecting the treatment doses for mutation-induction experiments were the solubility of BaP and PhIP in the exposure medium, and the cytotoxicity exhibited by BPDE, MNNG and CSC. Induction of DNA strand breaks was obtained at mutagen concentrations permitting suf ficient cell proliferation, except in the case of MNNG.