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.

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.

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.

Boronic acids and their derivatives have been exploited for their pharmacological activity and their utility as intermediates in the synthesis of novel non-boron containing compounds. A recent study reported that boronic acids are bacterial mutagens. Here, results are reported from the testing of nine boronic acids using the pan-mechanistic eukaryotic GADD45a genotoxicity assays, BlueScreen HC and GreenScreen HC. Positive results were produced for one compound in GreenScreen and four compounds in BlueScreen. Only negative results were produced when tested with S9 metabolic activation. These data suggest that there is not a general genotoxic liability in eukaryotes, within this chemical domain. Furthermore, they are not potent eukaryotic genotoxins: positive results were produced only at concentrations between 1mM and 10mM. Their presence as low concentration contaminants or impurities would be unlikely to produce misleading positive results for a test material.

Ambient air particulate matter (PM)-associated reactive oxygen species (ROS) have been linked to a variety of altered cellular outcomes. In this study, three different PM samples from diesel exhaust particles (DEPs), urban dust standard reference material SRM1649a and air collected in Manchester have been tested for their ability to oxidise DNA in a cell-free assay, to increase intracellular ROS levels and to induce CYP1A1 gene expression in mammalian cells. In addition, the cytotoxicity and genotoxicity of PM were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and alkaline comet assay, respectively. All PM samples catalysed the Fenton reaction in a cell-free assay, but only DEP resulted in the generation of ROS as measured by dichlorodihydrofluorescein diacetate oxidation in mammalian cells. However, there was no evidence that increased ROS was a consequence of polycyclic aromatic hydrocarbon metabolism via CYP1A1 induction as urban dust, the Manchester dust samples but not DEP-induced CYP1A1 expression. Urban dust was more cytotoxic in murine embryonic fibroblasts (MEFs) than the other PM samples and also induced expression of GADD45a in the GreenScreen Human Cell assay without S9 activation suggesting the presence of a direct-acting genotoxicant. Urban dust and DEP produced comparable levels of DNA damage, as assessed by the alkaline comet assay, in MEFs at higher levels than those induced by Manchester PM. In conclusion, results from the cytotoxic and genotoxic assays are not consistent with ROS production being the sole determinant of PM-induced toxicity. This suggests that the organic component can contribute significantly to this toxicity and that further work is required to better characterise the extent to which ROS and organic components contribute to PM-induced toxicity.

Histone-deacetylase inhibitors (HDACi) are able to induce cell-cycle arrest, apoptosis and differentiation in a variety of tumour cell lines. The mechanisms leading to these cellular outcomes are not fully understood, however, it is has been proposed that induction of cell-cycle arrest might be a result of genotoxic stress. Despite the potential for genotoxic activity of this class of compounds, there are very few data available to provide evidence for this, either in vitro or in vivo. In this study, four HDACi, viz. trichostatin A, sodium butyrate, APHA compound 8 and apicidin, were tested in the human lymphoblastoid TK6 cell line-hosted GADD45a-GFP assay, which has high sensitivity and specificity in the detection of genotoxic carcinogens and in vivo genotoxicants. All four compounds produced positive genotoxicity results within the acceptable toxic dose range of the assay, with APHA compound 8 producing the weakest response. Taken alongside recent evidence demonstrating that GADD45a is not induced by non-genotoxic apoptogens, this study suggests that genotoxicity contributes to the anti-tumour activity of HDACi drugs.

Increased expression of the GADD45a gene is a very specific biomarker of genotoxin exposure in TK6 cells, and has been exploited in green fluorescent protein and luciferase reporter genotoxicity assays. A recent European Food Safety Authority suggested that the GADD45a-reporter genotoxicity assays might produce misleading positive results for non-steroidal anti-inflammatory drugs. This study was conceived to test the hypothesis that these drugs should be excluded from the applicability domain of the TK6 cell GADD45a GreenScreen HC and BlueScreen HC reporter assays. Data from published screening and validation studies have been reviewed, and new test data have been generated from 20 NSAIDS, from both reporter assays, both in the presence and absence of metabolic activation. The data fail to support this hypothesis: the high specificity of the GADD45a reporter assays is maintained amongst NSAIDs.

An automated approach for scoring in vitro micronuclei (MN) has been described in which flow cytometric analysis is combined with compound exposure, processing, and sampling in a single 96-well plate (Bryce SM et al. [2010]: Mutat Res 703:191-199). The current report describes protocol optimization and an interlaboratory assessment of the assay's transferability and reproducibility. In a training phase, the methodology was refined and collaborating laboratories were qualified by repeatedly testing three compounds. Second, a set of 32 chemicals comprised of reference genotoxicants and presumed non-genotoxicants was tested at each of four sites. TK6 cells were exposed to 10 closely spaced compound concentrations for 1.5- to 2-cell population doublings, and were then stained and lysed for flow cytometric analysis. MN frequencies were determined by evaluating ≥5,000 cells per replicate well, and several indices of cytotoxicity were acquired. The prevalence of positive results varied according to the MN-fold increase used to signify a genotoxic result, as well as the endpoint used to define a cytotoxicity limit. By varying these parameters, assay sensitivity and specificity values ranged from 82 to 98%, and 86 to 97%, respectively. In a third phase, one laboratory tested a further six genotoxicants and five non-genotoxic apoptosis inducers. In these experiments assay specificity was markedly improved when top concentration selection was based on two cytotoxicity endpoints—relative survival and quantification of ethidium monoazide-positive events. Collectively, the results indicate that the miniaturized assay is transferable across laboratories. The 96-well format consumes considerably less compound than conventional in vitro MN test methods, and the high information content provided by flow cytometry helps guard against irrelevant positive results arising from overt toxicity. Environ. Mol. Mutagen. 54:180–194, 2013. © 2013 Wiley Periodicals, Inc.

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.

An automated approach for scoring in vitro micronuclei (MN) has been described in which flow cytometric analysis is combined with compound exposure, processing, and sampling in a single 96‐well plate (Bryce SM et al. [2010]: Mutat Res 703:191‐199). The current report describes protocol optimization and an interlaboratory assessment of the assay's transferability and reproducibility. In a training phase, the methodology was refined and collaborating laboratories were qualified by repeatedly testing three compounds. Second, a set of 32 chemicals comprised of reference genotoxicants and presumed non‐genotoxicants was tested at each of four sites. TK6 cells were exposed to 10 closely spaced compound concentrations for 1.5‐ to 2‐cell population doublings, and were then stained and lysed for flow cytometric analysis. MN frequencies were determined by evaluating ≥5,000 cells per replicate well, and several indices of cytotoxicity were acquired. The prevalence of positive results varied according to the MN‐fold increase used to signify a genotoxic result, as well as the endpoint used to define a cytotoxicity limit. By varying these parameters, assay sensitivity and specificity values ranged from 82 to 98%, and 86 to 97%, respectively. In a third phase, one laboratory tested a further six genotoxicants and five non‐genotoxic apoptosis inducers. In these experiments assay specificity was markedly improved when top concentration selection was based on two cytotoxicity endpoints—relative survival and quantification of ethidium monoazide‐positive events. Collectively, the results indicate that the miniaturized assay is transferable across laboratories. The 96‐well format consumes considerably less compound than conventional in vitro MN test methods, and the high information content provided by flow cytometry helps guard against irrelevant positive results arising from overt toxicity. Environ. Mol. Mutagen. 54:180–194, 2013. © 2013 Wiley Periodicals, Inc.

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.

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.