No abstract

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.

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.

Complement receptor type 2 (CR2/CD21), in association with CD19, plays an important role in enhancing mature B cell responses to opsonized Ags. We have shown that mice expressing a human CR2/CD21 (hCR2/CD21) transgene during the CD43+/CD25− late pro-B cell stage of B cell development demonstrate marked changes in subsequent B cell ontogeny. In the present study, we show that the humoral immune response to the T cell-dependent Ag, sheep RBC, is muted severely in a manner inversely proportional to B cell expression level of hCR2. Individual Ag-specific IgG isotypes vary in the degree to which they are affected but all are reduced while IgM titers are normal. A substantial reduction in germinal centers, both in size and frequency, in the spleens of immunized hCR2 transgenic mice demonstrates a failure to maintain germinal center reaction. However, both IgM expression levels and LPS-proliferative responses appear fully intact in B cells from hCR2-positive mice, suggesting that this alteration in B cell phenotype is different qualitatively from that of specific Ag-defined anergy models. These data suggest that the unresponsiveness to T-dependent Ags displayed by hCR2-positive B cells is linked to an increase in the level of stimulus required to propel the B cell into a fully activated state and thus a normal humoral immune response to Ags. We conclude that this phenotype and these mice may offer an additional means to dissect mechanisms underlying B cell tolerance and Ag responsiveness both in bone marrow and periphery.

No abstract

There is a pressing need to develop rapid yet accurate screening assays for the identification of genotoxic liability and for early hazard assessment in drug discovery. The GADD45a-GFP human cell-based genotoxicity assay (GreenScreen HC) has been reformatted to test 12 compounds per 96-well microplate in a higher throughput, automated screening mode and the protocol applied to the analysis of 1266 diverse, pharmacologically active compounds. Testing from a fixed starting concentration of 100 μM and over 3 serial dilutions, the hit rates for genotoxicity (7.3%) and cytotoxicity (33%) endpoints of the assay have been determined in a much wider chemical space than previously reported. The degree of interference from color, autofluorescence, and low solubility has also been assessed. The assay results have been compared to an in silico approach to genotoxicity assessment using Derek for Windows software. Where carcinogenicity data were available, GreenScreen HC demonstrated a higher specificity than in silico methods while identifying genotoxic species that were not highlighted for genotoxic liability in structure-activity relationship software. Higher throughput screening from a fixed, low concentration reduces sensitivity to less potent genotoxins, but the maintenance of the previously reported high specificity is essential in early hazard assessment where misclassification can lead to the needless rejection of potentially useful compounds in drug development. (Journal of Biomolecular Screening 2009:16-30)

The genotoxicity of a library of 70 flavour and fragrance substances having a high proportion of in vivo and/or carcinogenicity test data has been assessed using the GADD45a-GLuc 'BlueScreen HC' genotoxicity assay, with and without exogenous metabolic activation. There are only limited genotoxicity and carcinogenicity study data for compounds in this applicability domain, but this study allowed the following conclusions: (i) The BlueScreen HC results are highly predictive of positive results from regulator-required in vitro genotoxicity assays for the test set of materials; the moderate negative predictivity of BlueScreen HC from the in vitro test set of material is mainly due to the high rate of false positive in regulatory in vitro mammalian tests. (ii) BlueScreen HC negative results are predictive of negative in vivo results and provide a specific prediction of in vivo genotoxicity assay results. (iii) In this applicability domain, which comprises a large proportion of relatively low molecular weight molecules, a 1mM testing limit maintains the sensitivity of the assay, and increases specificity. (iv) The predictive capacity and specificity to in vivo genotoxins and carcinogens, coupled to a microplate format with low compound requirement supports further investigation of the BlueScreen HC assay as a useful tool in prioritizing the assessment of new F&F materials and in filling data gaps on materials with no or limited regulatory test data for genotoxicity.