Validation of a high-throughput in vitro alkaline elution/rat hepatocyte assay for DNA damage

In vitro alkaline elution is a sensitive and specific short term assay which measures DNA strand breakage in a mammalian test system (primary rat hepatocytes). This lab has previously demonstrated the performance of the assay with known genotoxic and nongenotoxic compounds. The methodology employed has relatively low sample throughput and is labor-intensive, requiring a great deal of manual processing of samples in a format that is not amenable to automation. Here, we present an automated version of the assay. This high-throughput alkaline elution assay (HT-AE) was made possible through 3 key developments: (1) DNA quantitation using PicoGreen (R) and OliGreen (R) fluorescent DNA binding dyes; (2) design and implementation of a custom automation system; and (3) reducing the assay to a 96-well plate format. The assay can now be run with 5-50 mg of test compound. HT-AE was validated in a similar manner as the original assay, including assessment of non-genotoxic and non-carcinogenic compounds and evaluation. of cytotoxicity to avoid confounding effects of toxicity-associated DNA degradation. The validation test results from compounds of known genotoxic potential were used to set appropriate criteria to classify alkaline elution results for genotoxicity. (c) 2007 Elsevier B.V. All rights reserved.