Exposure to genotoxic carcinogens leads to increased expression of the GADD45a gene in mammalian cells. This signature of genotoxic hazard has previously been exploited in the GreenScreen HC assay, in which GADD45a expression is linked to green fluorescent protein (GFP) expression in the human TK6 lymphoblastoid cell line. This article describes the development and validation of an alternative assay ("BlueScreen HC"), in which expression is linked to Gaussia luciferase (GLuc) expression, yielding a luminescent reporter, the preferred optical output in high-throughput screening. The coelentrazine substrate of GLuc is relatively unstable, and a new buffer is reported that improves its stability. A more sensitive method is demonstrated for the measurement of cell densities in the assay, using the fluorescent cyanine dye thiazole orange. A protocol amendment also allows the assessment of pro-genotoxicity using S9 liver extracts. Compounds from the European Centre for the Validation of Alternative Methods (ECVAM) recommended list for the assessment of new or improved genotoxicity assays were evaluated with and without S9 in the new assay. The new GLuc assay was as effective as the GFP assay in producing positive results for all classes of genotoxic carcinogen and negative results for all nongenotoxins tested.

The GADD45a-GFP (GreenScreen HC) reporter assay detects genotoxic damage in the human lymphoblastoid TK6 cell line and gives positive results for all classes of genotoxin, including mutagens, aneugens and clastogens. In this study, a collection of 75 marketed pharmaceuticals were tested in the assay. Compounds in the collection represent a broad range of chemical structures, pharmacologies and therapeutic indications, including neoplasia and viral infection where positive genotoxicity results are often associated with the pharmacological activity. Based on the results of this study, two main conclusions can be drawn: (i) the GreenScreen HC is more predictive of in vivo genotoxicity (88%) and genotoxic carcinogenicity (93%) data than the any of the other regulatory in vitro genotoxicity assay and (ii) no compounds were uniquely positive in the GADD45a-GFP assay. This analysis therefore provides additional evidence to support the use of the GADD45a-GFP assay as an effective tool either in early genotoxic liability identification or non-clinical safety assessment of candidate pharmaceuticals during development.

A recent ECVAM workshop considered how to reduce falsely predictive positive results when undertaking in vitro genotoxicity testing, and thus to avoid unnecessary follow-up with tests involving animals. As it was anticipated that modified versions of existing assays as well as new assays might contribute to a solution, an expert panel was asked to identify a list of chemicals that could be used in the evaluation of such assays. Three categories of test chemicals were chosen comprising a total of 62 compounds. This paper provides test results for these chemicals using the GreenScreen HC assay. All tests were carried out in triplicate, by multiple operators, with and without S9, using invariant protocols. Group 1 chemicals should be detected as positive in in vitro mammalian cell genotoxicity tests: 18/20 (90%) were reproducibly positive in GreenScreen HC. Group 2 chemicals should give negative results in in vitro genotoxicity tests: 22/23 (96%) were reproducibly negative in GreenScreen HC. Overall concordance for Groups 1 and 2 is 93%. Group 3 chemicals should give negative results in in vitro mammalian cell genotoxicity tests, but have been reported to induce chromosomal aberrations or Tk mutations in mouse lymphoma cells, often at high concentrations or at high levels of cytotoxicity: 13/17 (76%) were reproducibly negative in GreenScreen HC. Of the four positive compounds in Group 3, p-nitrophenol was only positive at the top dose (10mM), 2,4-DCP is an in vivo genotoxin, and two chemicals are antioxidant compounds that may be acting as pro-oxidants in the hyperoxic conditions of cell culture. Overall, these predictive figures are similar to those from other studies with the GreenScreen HC assay and confirm its high specificity, which in turn minimizes the generation of falsely predictive positive results.