Development of a high-throughput Gaussia luciferase reporter assay for the activation of the GADD45a gene by mutagens, promutagens, clastogens, and aneugens.

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.

Nongenotoxic apoptosis inducers do not produce misleading positive results in the TK6 cell-based GADD45a-GFP genotoxicity assay.

The in vitro mammalian genotoxicity tests identify some carcinogens not identified by the bacterial Ames test. However, historically they have produced rather more misleading predictions of carcinogenicity than the Ames test. This liability has been reduced in pharmaceutical testing by lowering the top-testing dose and rejecting data from excessively toxic doses. It also stimulated the development of new assays with inherently higher specificity. Among these, the GADD45a-GFP assay has been recognized as a maturing technology by the International Life Sciences Institute Health and Environmental Sciences Institute In Vitro Genetic Toxicity Emerging Technologies and New Strategies workgroup and has been concluded to be suitable for inclusion in a battery of high throughput screening by the U.K. Committee on Mutagenicity of Chemicals in Food, Consumer Products and the Environment. GADD45a is induced by compounds that cause damage to or missegregation of chromosomes, and is implicated in the stimulation of repair or apoptosis where damage is overwhelming. It is therefore important to understand whether this causes a liability in the assay to produce misleading positives for nongenotoxic inducers of apoptosis. Compounds hypothesized to stimulate apoptosis in the GADD45a-GFP assay or to induce GADD45a in the absence of genotoxic stress, such as p53 activators, NF-κB and Bcl-2 inhibitors were selected. Apoptosis induction was monitored using Annexin V binding and caspase 3/7 activation assays. The majority of compounds tested were negative in the GADD45a-GFP assay. The few that generated positive data were also found positive in concurrent comet assay and/or micronucleus tests. The data presented here demonstrate that the GADD45a-GFP assay is not vulnerable to the generation of misleading positive results by apoptosis inducers.

How accurate is in vitro prediction of carcinogenicity?

Positive genetic toxicity data suggest carcinogenic hazard, and this can stop a candidate pharmaceutical reaching the clinic. However, during the last decade, it has become clear that many non-carcinogens produce misleading positive results in one or other of the regulatory genotoxicity assays. These doubtful conclusions cost a lot of time and money, as they trigger additional testing of apparently genotoxic candidates, both in vitro and in animals, to discover whether the suggested hazard is genuine. This in turn means that clinical trials can be put on hold. This review describes the current approaches to the 'misleading positive' problem as well as efforts to reduce the use of animals in genotoxicity assessment. The following issues are then addressed: the application of genotoxicity testing screens earlier in development; the search for new or improved in vitro genotoxicity tests; proposed changes to the International Committee on Harmonisation guidance on genotoxicity testing [S2(R1)]. Together, developments in all these areas offer good prospects of a more rapid and cost-effective way to understand genetic toxicity concerns.

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.

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.

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.

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.

Clear and present danger? The use of a yeast biosensor to monitor changes in the toxicity of industrial effluents subjected to oxidative colour removal treatments.

Discharges of coloured effluents into surface waters provide conspicuous evidence of the impact of industry on the environment. The textile industry is an obvious candidate for sources of such discharges. Conventional treatment methods appear to alleviate this situation by removing colour, however the affect on toxicity is less obvious. The objective of this study was to examine the changes in effluent toxicity during the course of two alternative wastewater treatment methods, ozonation and electrochemical oxidation, using a novel toxicity biosensor, GreenScreen EM. The biosensor is capable of measuring both general acute toxicity (cytotoxicity), and more specifically genotoxicity, that is damage to a cell's DNA structure, replication or distribution, caused by substances that may be mutagenic and/or carcinogenic. The biosensor utilises a modified strain of the brewers yeast Saccharomyces cerevisiae, incorporating a gene encoding green fluorescent protein (GFP) linked to the inducible promoter of the DNA damage responsive RAD54 gene. Upon exposure to a genotoxin, the production of GFP is up-regulated in parallel with RAD54, and the resulting cellular fluorescence provides a measure of genotoxicity. Acute toxicity is simultaneously determined by monitoring relative total growth of the cell culture during incubation. The results presented in this paper show that a reduction in colouration does not necessarily correspond to a reduction in effluent toxicity.

DNA-damage induction of RAD54 can be regulated independently of the RAD9- and DDC1-dependent checkpoints that regulate RNR2.

DNA damage checkpoints regulate a number of physiological responses after DNA damage. The transcriptional level of many genes is specifically induced in response to genotoxic stress in a checkpoint-dependent manner. The regulation of DNA damage-induced transcription of RAD54 and RNR2 by RAD9, DDC1, DUN1, CRT1 and MBP1 was investigated in Saccharomyces cerevisiae, using green fluorescent protein reporter assays and Northern blots. RAD54 and RNR2 reporter activity in response to the DNA damaging agent, methyl methanesulphonate, was measured in ddc1-Delta, rad9-Delta, ddc1-Delta/rad9-Delta, dun1-Delta, crt1-Delta and mbp1-Delta mutants and was compared with that of the wild type. RAD9 and DDC1 were shown to be required for a full RNR2 transcriptional response, although with the double mutant, ddc1-Delta/rad9-Delta, no additive effect on RNR2 induction was observed. RAD54 promoter activity was not significantly reduced in either rad9-Delta or ddc1-Delta mutants and was only partially reduced in the rad9-Delta/ddc1-Delta strain, suggesting that DNA damage induction of RAD54 must depend on other genes, in addition to RAD9 and DDC1. In the dun1-Delta mutant, RNR2 promoter activity was lowered, whilst that of RAD54 was increased, confirming that DUN1 is required for transcriptional induction of RNR2, but is not required for damage-induced transcription of RAD54. Analysis of the crt1-Delta strain confirmed that RNR2 is regulated via the CRT1 repressor pathway, downstream of DUN1, but RAD54 is not. MBP1 was shown to be required for transcription of RNR2, but was not needed for transcription of RAD54. These results indicate that RNR2 and RAD54 are regulated in different ways.

Fluorescence polarization discriminates green fluorescent protein from interfering autofluorescence in a microplate assay for genotoxicity.

An unconventional use for the polarization optics, associated with a variety of commercially available fluorescence microplate readers, is reported. This novel application has allowed the discrimination of green fluorescent protein (GFP) fluorescence in genetically modified yeast cells from interfering autofluorescent species. The method exploits the unusually high fluorescence anisotropy of GFP compared to smaller fluorophores and autofluorescent species. The principle was successfully applied to resolve the induced GFP signal from that of autofluorescent test compounds, in an assay for genotoxic species. The use of fluorescence polarization enabled both proflavin and methapyrilene to be identified as genotoxic agents in the yeast assay. This would not have been possible using conventional fluorescence alone since these compounds were found to be intensely autofluorescent at the same wavelength as GFP and thus effectively mask the GFP signal.