The influence of exogenous metabolism on the specificity of in vitro mammalian genotoxicity tests.

A two-part study was designed to determine whether the inclusion of the rodent liver 'S9' exogenous metabolic activating system contributes to the generation of misleading positive results by the regulator-required in vitro mammalian genotoxicity tests. The mono-oxygenase enzymes in S9 produce direct-acting DNA-reactive electrophiles, and are included in in vitro genotoxicity tests to enhance the detection of substances which only become genotoxic following metabolism. However, as the S9 system lacks 'detoxifying' phase 2 factors it was hypothesised that increased chemical metabolism per se may lead to an increase in irrelevant S9 test outcomes in safety assessment. To test this, 89 compounds with positive or negative carcinogenicity data were identified, which produced negative Ames test data (+/- S9), and only produced positive in vitro mammalian test data in the presence of S9. This allowed a determination of whether or not misleading predictions of carcinogenicity by the in vitro mammalian tests were more or less prevalent in the presence of S9. A subset of these compounds was then tested with and without S9 in the GADD45a-GFP genotoxicity test, in order to determine whether misleading in vitro mammalian positive results were generally more prevalent with S9, or reflected particular tests' liabilities. This study suggests that the use of S9 metabolic activation in in vitro genotoxicity tests does not increase the prevalence of misleading positive results in in vitro mammalian genotoxicity assays, at least amongst Ames negative compounds.

Development of an in vitro PIG-A gene mutation assay in human cells.

Mutagens can be carcinogens, and traditionally, they have been identified in vitro using the Salmonella 'Ames' reverse mutation assay. However, prokaryotic DNA packaging, replication and repair systems are mechanistically very different to those in the humans we inevitably seek to protect. Therefore, for many years, mammalian cell line genotoxicity assays that can detect eukaryotic mutagens as well as clastogens and aneugens have been used. The apparent lack of specificity in these largely rodent systems, due partly to their mutant p53 status, has contributed to the use of animal studies to resolve data conflicts. Recently, silencing mutations at the PIG-A locus have been demonstrated to prevent glycophosphatidylinositol (GPI) anchor synthesis and consequentially result in loss of GPI-anchored proteins from the cell's extracellular surface. The successful exploitation of this mutant phenotype in animal studies has triggered interest in the development of an analogous in vitro PIG-A mutation screening assay. This article describes the development of a robust assay design using metabolically active human cells. The assay includes viability and cell membrane integrity assessment and conforms to the future ideas of the 21st-century toxicology testing.

The involvement of WT1 in the regulation of GADD45a in response to genotoxic stress.

Expression of the human GADD45a gene is increased in TK6 cells exposed to mutagens, clastogens and aneugens. It is known to be regulated through both p53-dependent and p53-independent pathways and WT1 has been implicated in both cases. This article reports an investigation into the effect that mutations in the WT1 and p53 response elements of the gene have on GADD45a expression. This was conducted in both p53 wild-type (TK6) and mutant (WI-L2-NS) human B lymphoblastoid cell lines. Gene expression was monitored using a GADD45a-green fluorescent protein reporter assay. Mutant cell lines were exposed to the mechanistically diverse genotoxins methyl methanesulphonate, cisplatin and mitomycin C (direct acting), hydroxyurea, aphidicolin and 5'fluorouracil (inhibitors of nucleotide/DNA synthesis) and benomyl (aneugen). In all cases, the induction of the reporter was reduced in the mutants compared with wild-type. These results provide experimental evidence for the implied role of WT1 in both p53-dependent and p53-independent pathways of GADD45a regulation and further insight into the mechanism of GADD45a induction by genotoxins.

GADD45a-GFP GreenScreen HC assay results for the ECVAM recommended lists of genotoxic and non-genotoxic chemicals for assessment of new genotoxicity tests.

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.

A pre-validation transferability study of the GreenScreen HC GADD45a-GFP assay with a metabolic activation system (S9).

A new protocol has recently been developed and validated for the GreenScreen HC GADD45a-GFP genotoxicity reporter assay, enabling the incorporation of an S9 metabolic activation system into the assay. The S9 protocol employs flow-cytometric methodology for the detection of both reporter GFP fluorescence and propidium iodide fluorescence for the estimation of cellular viability. In the spirit of assay validation by bodies such as the European Centre for the Validation of Alternative Methods (ECVAM), the adapted metabolic activation protocol for the GADD45a-GFP assay has been undergoing 'pre-validation'. Results of phases I and II of this pre-validation, namely protocol refinement and protocol transfer, respectively, are presented here. In phase I the protocol was transferred to a second laboratory for initial assessment of method portability and subsequent refinement of the protocol. In phase II, the protocol was then transferred to two further laboratories along with the elaborated standard operating-procedure (SOP) for further assessment of transferability. The three transfer sites then undertook an assessment of the method's reproducibility by testing eight compounds. The outcome of the study was a refined protocol that was found to be highly transferable. It yielded 100% agreement in results between all four laboratories.

Development and validation of a higher throughput screening approach to genotoxicity testing using the GADD45a-GFP GreenScreen HC assay.

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 AmicroM 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.

Assessment of the genotoxicity of S9-generated metabolites using the GreenScreen HC GADD45a-GFP assay.

Genotoxicity can be assessed by monitoring expression of a GADD45a-GFP reporter in the human lymphoblastoid cell line TK6. A flow cytometric method has been developed to effectively distinguish GFP fluorescence from coloured and fluorescent test samples as well from the S9 liver extracts used to generate metabolites from pro-genotoxins. The method includes the use of propidium iodide exclusion for the determination of cellular viability. This paper describes the method development, the derivation of decision thresholds for the identification of genotoxins using the method, and presents data from a 56-compound validation study of the method. The results illustrate that the method permitted the detection of the majority of pro-genotoxins tested and, importantly, the high specificity of the GADD45a-GFP assay was maintained.

Analysis of 75 marketed pharmaceuticals using the GADD45a-GFP 'GreenScreen HC' genotoxicity assay.

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.

Evaluation of high-throughput genotoxicity assays used in profiling the US EPA ToxCast chemicals.

Three high-throughput screening (HTS) genotoxicity assays-GreenScreen HC GADD45a-GFP (Gentronix Ltd.), CellCiphr p53 (Cellumen Inc.) and CellSensor p53RE-bla (Invitrogen Corp.)-were used to analyze the collection of 320 predominantly pesticide active compounds being tested in Phase I of US. Environmental Protection Agency's ToxCast research project. Between 9% and 12% of compounds were positive for genotoxicity in the assays. However, results of the varied tests only partially overlapped, suggesting a strategy of combining data from a battery of assays. The HTS results were compared to mutagenicity (Ames) and animal tumorigenicity data. Overall, the HTS assays demonstrated low sensitivity for rodent tumorigens, likely due to: screening at a low concentration, coverage of selected genotoxic mechanisms, lack of metabolic activation and difficulty detecting non-genotoxic carcinogens. Conversely, HTS results demonstrated high specificity, >88%. Overall concordance of the HTS assays with tumorigenicity data was low, around 50% for all tumorigens, but increased to 74-78% (vs. 60% for Ames) for those compounds producing tumors in rodents at multiple sites and, thus, more likely genotoxic carcinogens. The aim of the present study was to evaluate the utility of HTS assays to identify potential genotoxicity hazard in the larger context of the ToxCast project, to aid prioritization of environmentally relevant chemicals for further testing and assessment of carcinogenicity risk to humans.

Interlaboratory assessment of the GreenScreen HC GADD45a-GFP genotoxicity screening assay: an enabling study for independent validation as an alternative method.

Sixteen coded compounds were blind-tested at 4 laboratories using the recently described GADD45a-GFP genotoxicity assay. The compounds were chosen to include non-genotoxic compounds as well as weak and strong genotoxins. None of the compounds required metabolic activation in order to exhibit genotoxic effects. The participating laboratories included 2 global pharmaceutical companies, a global consumer goods company and the Gentronix laboratory in Manchester. Each compound was tested 4 times on different days following a protocol previously described. The tests were carried out after a 3-day training period from the parent lab (Manchester). Following the exclusion of data from tests with positive control failures and data series with 'spikes', 92% of assays gave the correct result: non-genotoxins giving negative results and genotoxins giving positive results. There were no randomly distributed problems suggesting that differences between the results from different sites reflected the use of different instruments, procedural differences and operator experience. In naïve operator laboratories the quality of data improved with operator practice. It was concluded that simple clarification of the protocol would provide the level of reliability required for widespread use of the assay in hazard assessment.