Multiple-endpoint in vitro carcinogenicity test in human cell line TK6 distinguishes carcinogens from non-carcinogens and highlights mechanisms of action.

Current in vitro genotoxicity tests can produce misleading positive results, indicating an inability to effectively predict a compound's subsequent carcinogenic potential in vivo. Such oversensitivity can incur unnecessary in vivo tests to further investigate positive in vitro results, supporting the need to improve in vitro tests to better inform risk assessment. It is increasingly acknowledged that more informative in vitro tests using multiple endpoints may support the correct identification of carcinogenic potential. The present study, therefore, employed a holistic, multiple-endpoint approach using low doses of selected carcinogens and non-carcinogens (0.001-770 µM) to assess whether these chemicals caused perturbations in molecular and cellular endpoints relating to the Hallmarks of Cancer. Endpoints included micronucleus induction, alterations in gene expression, cell cycle dynamics, cell morphology and bioenergetics in the human lymphoblastoid cell line TK6. Carcinogens ochratoxin A and oestradiol produced greater Integrated Signature of Carcinogenicity scores for the combined endpoints than the "misleading" in vitro positive compounds, quercetin, 2,4-dichlorophenol and quinacrine dihydrochloride and toxic non-carcinogens, caffeine, cycloheximide and phenformin HCl. This study provides compelling evidence that carcinogens can successfully be distinguished from non-carcinogens using a holistic in vitro test system. Avoidance of misleading in vitro outcomes could lead to the reduction and replacement of animals in carcinogenicity testing.

High information content assays for genetic toxicology testing: A report of the International Workshops on Genotoxicity Testing (IWGT).

We live in an era of 'big data', where the volume, velocity, and variety of the data being generated is increasingly influencing the way toxicological sciences are practiced. With this in mind, a workgroup was formed for the 2017 International Workshops on Genotoxicity Testing (IWGT) to consider the use of high information content data in genetic toxicology assessments. Presentations were given on adductomics, global transcriptional profiling, error-reduced single-molecule sequencing, and cellular phenotype-based assays, which were identified as methodologies that are relevant to present-day genetic toxicology assessments. Presenters and workgroup members discussed the state of the science for these methodologies, their potential use in genetic toxicology, current limitations, and the future work necessary to advance their utility and application. The session culminated with audience-assisted SWOT (strength, weakness, opportunities, and threats) analyses. The summary report described herein is structured similarly. A major conclusion of the workgroup is that while conventional regulatory genetic toxicology testing has served the public well over the last several decades, it does not provide the throughput that has become necessary in modern times, and it does not generate the mechanistic information that risk assessments ideally take into consideration. The high information content assay platforms that were discussed in this session, as well as others under development, have the potential to address aspect(s) of these issues and to meet new expectations in the field of genetic toxicology.

A novel, integrated in vitro carcinogenicity test to identify genotoxic and non-genotoxic carcinogens using human lymphoblastoid cells.

Human exposure to carcinogens occurs via a plethora of environmental sources, with 70-90% of cancers caused by extrinsic factors. Aberrant phenotypes induced by such carcinogenic agents may provide universal biomarkers for cancer causation. Both current in vitro genotoxicity tests and the animal-testing paradigm in human cancer risk assessment fail to accurately represent and predict whether a chemical causes human carcinogenesis. The study aimed to establish whether the integrated analysis of multiple cellular endpoints related to the Hallmarks of Cancer could advance in vitro carcinogenicity assessment. Human lymphoblastoid cells (TK6, MCL-5) were treated for either 4 or 23 h with 8 known in vivo carcinogens, with doses up to 50% Relative Population Doubling (maximum 66.6 mM). The adverse effects of carcinogens on wide-ranging aspects of cellular health were quantified using several approaches; these included chromosome damage, cell signalling, cell morphology, cell-cycle dynamics and bioenergetic perturbations. Cell morphology and gene expression alterations proved particularly sensitive for environmental carcinogen identification. Composite scores for the carcinogens' adverse effects revealed that this approach could identify both DNA-reactive and non-DNA reactive carcinogens in vitro. The richer datasets generated proved that the holistic evaluation of integrated phenotypic alterations is valuable for effective in vitro risk assessment, while also supporting animal test replacement. Crucially, the study offers valuable insights into the mechanisms of human carcinogenesis resulting from exposure to chemicals that humans are likely to encounter in their environment. Such an understanding of cancer induction via environmental agents is essential for cancer prevention.

From the Cover: Does the Assessment of Nondisjunction Provide a More Sensitive Assay for the Detection of Aneugens?

The detection of aneugenic chemicals is important due to the implications of aneuploidy for human health. Aneuploidy can result from chromosome loss or nondisjunction due to chromosome mis-segregation at anaphase. Frequently, aneugens are detected using the in vitro micronucleus assay (IVM), with either centromere or kinetochore labeling. However, this method does not consider nondisjunction, the suggested predominant mechanism of spindle poison induced aneugenicity in primary human lymphocytes. Therefore, the IVM may be relatively insensitive in detecting aneuploidy. To investigate whether chromosome distribution analysis, specifically of nondisjunction, using chromosome-specific centromeric probes provides a more sensitive assay for aneugen detection, six reference aneugens with differing modes of action were tested on human lymphoblastoid TK6 cells. The results show that chromosome loss is a substantial part of the process leading to aneuploidy in TK6 cells. This differs from previous studies on human lymphocytes where nondisjunction has been described as the major mechanism of aneugenicity. However, in the current study more cells and types of aneugenic damage were analyzed. Although compound specific effects on nondisjunction were identified, chromosome distribution analysis did not provide increased sensitivity for the detection of aneugens: For the six reference aneugens examined, chromosome loss was shown at the same concentrations or lower than nondisjunction, even when nondisjunction levels were comparatively high. Therefore, in TK6 cells methods that detect chromosome loss, eg, the IVM, provide a more sensitive technique for the detection of aneugens than the measurement of nondisjunction.

Viral Vectors: The Road to Reducing Genotoxicity.

Viral vector use in gene therapy has highlighted several safety concerns, including genotoxic events. Generally, vector-mediated genotoxicity results from upregulation of cellular proto-oncogenes via promoter insertion, promoter activation, or gene transcript truncation, with enhancer-mediated activation of nearby genes the primary mechanism reported in gene therapy trials. Vector-mediated genotoxicity can be influenced by virus type, integration target site, and target cell type; different vectors have distinct integration profiles which are cell-specific. Non-viral factors, including patient age, disease, and dose can also influence genotoxic potential, thus the choice of test models and clinical trial populations is important to ensure they are indicative of efficacy and safety. Efforts have been made to develop viral vectors with less risk of insertional mutagenesis, including self-inactivating (SIN) vectors, enhancer-blocking insulators, and microRNA targeting of vectors, although insertional mutagenesis is not completely abrogated. Here we provide an overview of the current understanding of viral vector-mediated genotoxicity risk from factors contributing to viral vector-mediated genotoxicity to efforts made to reduce genotoxicity, and testing strategies required to adequately assess the risk of insertional mutagenesis. It is clear that there is not a 'one size fits all' approach to vector modification for reducing genotoxicity, and addressing these challenges will be a key step in the development of therapies such as CRISPR-Cas9 and delivery of future gene-editing technologies.

The clastogenicity of 4NQO is cell-type dependent and linked to cytotoxicity, length of exposure and p53 proficiency.

4-Nitroquinoline 1-oxide (4NQO) is used as a positive control in various genotoxicity assays because of its known mutagenic and carcinogenic properties. The chemical is converted into 4-hydroxyaminoquinoline 1-oxide and gives rise to three main DNA adducts, N-(deoxyguanosin-8-yl)-4AQO, 3-(desoxyguanosin-N (2)-yl)-4AQO and 3-(deoxyadenosin-N (6)-yl)-4AQO. This study was designed to assess the shape of the dose-response curve at low concentrations of 4NQO in three human lymphoblastoid cell lines, MCL-5, AHH-1 and TK6 as well as the mouse lymphoma L5178Y cell line in vitro. Chromosomal damage was investigated using the in vitro micronucleus assay, while further gene mutation and DNA damage studies were carried out using the hypoxanthine-guanine phosphoribosyltransferase forward mutation and comet assays. 4NQO showed little to no significant increases in micronucleus induction in the human lymphoblastoid cell lines, even up to 55±5% toxicity. A dose-response relationship could only be observed in the mouse lymphoma cell line L5178Y after 4NQO treatment, even at concentrations with no reduction in cell viability. Further significant increases in gene mutation and DNA damage induction were observed. Hence, 4NQO is a more effective point mutagen than clastogen, and its suitability as a positive control for genotoxicity testing has to be evaluated for every individual assay.

Evaluation of 4,4'-diaminodiphenyl ether in the rat comet assay: Part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative international validation study of in vivo rat alkaline comet assay.

As a part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative international validation study of the in vivo rat alkaline comet assay, 4,4'-diaminodiphenyl ether (DPE), a known rodent genotoxic carcinogen, was tested in this laboratory. Sprague Dawley rats (7-9 weeks of age) were given three oral doses of DPE, 24 and 21 h apart and liver or stomach sampled 3h after the final dose. Under the conditions of the test, no increases in DNA damage in liver and stomach were observed with DPE (up to 200 mg/kg/day). A dose-dependent decrease in DNA migration, compared to vehicle controls, was noted for DPE in rat stomach. Further analysis is required to elucidate fully whether this decrease is a consequence of the mode of action or due to the toxicity of DPE. What is perhaps surprising is the inability of the comet assay to detect a known rat genotoxic carcinogen in liver. Further investigation is needed to clarify whether this apparent lack of response results from limited tissue exposure or metabolic differences between species. This finding highlights a need for careful consideration of study design when evaluating assay performance as a measure of in vivo genotoxicity.

Genotoxins induce binucleation in L5178Y and TK6 cells.

Following the initial observation that methyl methanesulphonate induced binucleated cells in the AHH-1 line and a significant number of them contained micronuclei, human lymphoblastoid TK6 and mouse lymphoma L5178Y cells were treated with methyl methanesulphonate, methylnitrosourea, mitomycin C, cytosine arabinoside, colchicine and triton X. All except triton X induced binucleated cells in both lines but an increased micronucleus incidence in them was seen only in TK6. The two lines also differed in the numbers of binucleates in the control cultures with 2.0% and 0.5% in TK6 and L5178Y, respectively, and a much higher proportion of those in TK6 contained micronuclei. The differences in behaviour between the two cell lines could not clearly be ascribed to their P53 status. Colchicine induced binucleates in both cell lines but they did not contain increased numbers of micronuclei. The effect on binucleate incidence was not a non-specific cytotoxic response because no increase was seen with triton X even at highly cytotoxic concentrations. The initial concern that not scoring micronuclei in binucleated cells might lead to erroneous results in in vitro micronucleus tests not using a cytokinesis block, was not proven because all the genotoxins tested here induced significant increases in micronucleus frequency in mononuclear cells. When testing less potently active agents in in vitro micronucleus tests not employing a cytokinesis block, care should be taken to understand better this phenomenon and not to include these damaged cells until we do.

Recommendations, evaluation and validation of a semi-automated, fluorescent-based scoring protocol for micronucleus testing in human cells.

Micronucleus (MN) induction is an established cytogenetic end point for evaluating structural and numerical chromosomal alterations in genotoxicity testing. A semi-automated scoring protocol for the assessment of MN preparations from human cell lines and a 3D skin cell model has been developed and validated. Following exposure to a range of test agents, slides were stained with 4'-6-diamidino-2-phenylindole (DAPI) and scanned by use of the MicroNuc module of metafer 4, after the development of a modified classifier for selecting MN in binucleate cells. A common difficulty observed with automated systems is an artefactual output of high false positives, in the case of the metafer system this is mainly due to the loss of cytoplasmic boundaries during slide preparation. Slide quality is paramount to obtain accurate results. We show here that to avoid elevated artefactual-positive MN outputs, diffuse cell density and low-intensity nuclear staining are critical. Comparisons between visual (Giemsa stained) and automated (DAPI stained) MN frequencies and dose-response curves were highly correlated (R (2) = 0.70 for hydrogen peroxide, R (2) = 0.98 for menadione, R (2) = 0.99 for mitomycin C, R (2) = 0.89 for potassium bromate and R (2) = 0.68 for quantum dots), indicating the system is adequate to produce biologically relevant and reliable results. Metafer offers many advantages over conventional scoring including increased output and statistical power, and reduced scoring subjectivity, labour and costs. Further, the metafer system is easily adaptable for use with a range of different cells, both suspension and adherent human cell lines. Awareness of the points raised here reduces the automatic positive errors flagged and drastically reduces slide scoring time, making metafer an ideal candidate for genotoxic biomonitoring and population studies and regulatory genotoxic testing.

Micronucleus induction in the bone marrow of rats by pharmacological mechanisms. I: glucocorticoid receptor agonism.

A novel selective glucocorticoid receptor (GR) agonist, AZD2906, was found to increase the incidence of micronucleated immature erythrocytes (MIE) in the bone marrow of rats given two oral doses at the maximum tolerated level. Because GR agonists as a class are considered not to be genotoxic and AZD2906 showed no activity in the standard in vitro tests or in vivo in a rat liver comet assay, investigative studies were performed to compare AZD2906 with a reference traditional GR agonist, prednisolone. Emphasis was placed on blood and bone marrow parameters in these studies because GR activation has been reported to induce erythropoiesis which, in turn, is known to increase MIE in the bone marrow. Both compounds induced almost identical, small increases in micronucleus frequency at all doses tested. Directly comparable changes in haematological and bone marrow parameters were also seen with significant decreases in lymphoid cells in both compartments and significant increases in numbers of circulating neutrophils. Although no evidence of increased erythropoiesis was seen as increased immature erythrocyte numbers either in the blood or in the bone marrow, histopathological examination showed focal areas in the bone marrow where the erythroid population was enriched in association with an atrophic myeloid lineage. This could have been due to direct stimulation of the erythroid lineage or a secondary effect of myelosuppression inducing a rebound increase in erythropoiesis into the vacant haematopoietic cell compartment. It was concluded that the increased MIE frequencies induced by both AZD2906 and prednisolone are a consequence of their pharmacological effects on the bone marrow, either by directly inducing erythropoiesis or by some other unknown effect on cellular function, and do not indicate potential genotoxicity. This conclusion is supported by the lack of carcinogenic risk in man demonstrated by decades of clinical use of prednisolone and other GR agonists.

Chromosome aberration frequency in rat peripheral lymphocytes increases with repeated dosing with hexamethylphosphoramide or cyclophosphamide.

Although there are several in vivo tests for potential genotoxicity, with the possible exception of the transgenic rodent mutation models, none is specifically intended to assess increasing damage with chronic administration. In principle, peripheral blood lymphocytes would be expected to accumulate DNA damage with repeated dosing because the majority are not in active division and appear to have limited DNA repair capability, and they are exposed to plasma levels of test materials and metabolites. However, there appear to be no published reports confirming this principle. Therefore, in the current study, after optimising culture conditions for rat lymphocytes in this laboratory, rats were given oral doses of cyclophosphamide or hexamethylphosphoramide (HMPA) for up to 28 days and peripheral lymphocytes analysed for chromosome aberrations at various time points. The results clearly show that, for both compounds, doses that gave no significant increases in aberration frequency after 2 days induced clear increases after 15 days with further damage detectable after 28 doses. With HMPA, it was shown that DNA damage persisted for at least 10 days after cessation of treatment. These data show that repeat dose studies in the rat measuring chromosome aberration frequency in lymphocytes can give a genuine indication that genotoxicity may increase with chronic administration and, therefore, maybe useful in assessing the risk of potentially genotoxic substances.

The in vitro micronucleus assay.

The in vitro micronucleus test detects genotoxic damage in interphase cells. The in vitro micronucleus test provides an alterative to the chromosome aberration test, and because the in vitro micronucleus test examines cells at interphase, the assessment of micronuclei can be scored faster, as the analysis of damage is thought to be less subjective and is more amenable to automation.Micronuclei may be the result of aneugenic (whole chromosome) or clastogenic (chromosome breakage) damage. This chapter provides methods for mononucleate and binucleate micronucleus tests and the addition of centromeric labelling and a non-disjunction assay to investigate any potential aneugenic mode of action.

Cytogenetic in vivo assays in somatic cells.

Chromosome aberration assays are employed to detect the induction of chromosome breakage (clastogenesis) in somatic and germ cells by direct observation of the chromosomal damage during metaphase analysis, or by indirect observation of chromosomal fragments. Thus, various types of cytogenetic change can be detected such as structural chromosome aberrations (CA), sister chromatid exchanges (SCE), ploidy changes, and micronuclei. Following the induction of the chromosomal damage, most of the aberrations and abnormalities detected by these assays can be detrimental or even lethal to the cell. Their presence, however, indicates a potential to also induce more subtle and therefore transmissible chromosomal damage which survives cell division to produce heritable cytogenetic changes. Usually, induced cytogenetic damage is accompanied by other genotoxic damage such as gene mutations.

A rapid, semi-automated method for scoring micronuclei in mononucleated mouse lymphoma cells.

A semi-automated scoring system has been developed to provide rapid, accurate assessment of micronuclei in preparations of mononuclear mouse lymphoma L5178Y cells. Following exposure to a range of test agents, flat, single-cell preparations were produced from exponentially growing cultures by cytocentrifugation. Following staining with 4'-6-diamidino-2-phenylindole (DAPI), cells were scanned by use of the MicroNuc module of Metafer 4 v 3.4.102, after modifying the classifier developed for selecting micronuclei in binucleate cells to increase its sensitivity. The image gallery of all cells was then sorted to bring aberrant cells to the top of the gallery to assess visually the numbers of cells with micronuclei, as distinct from other debris. Slide quality was shown to be paramount in obtaining accurate results from an automated scan and the data obtained compared very well with the incidence of micronuclei scored conventionally by microscopy. Compared with manual scoring the time saving is considerable, as more than 2000 images are captured in approximately 2min, with subsequent visual assessment of aberrant cells in the image gallery taking about 1-2min/slide. By scanning all aberrant cells, the system also captures additional information on necrotic, apoptotic and fragmented cells. Although optimised for mouse lymphoma cells, it should be simple to adapt the method for any cell type growing in suspension.

The ability of the mouse lymphoma TK assay to detect aneugens.

There is some evidence that the mouse lymphoma TK assay (MLA) can detect aneugens, and this is accepted in the current International Conference on Harmonisation guidance for testing pharmaceuticals. However, whether or not it can be used as a reliable screen for aneugenicity has been the subject of debate. Consequently, aneugens with diverse mechanisms of action were tested in the MLA using 24-h exposure. No evidence of increased mutant frequency was seen with noscapine, diazepam or colchicine and increases were seen with taxol, carbendazim, econazole and chloral hydrate only at high levels of toxicity (for all but one taxol concentration survival reduced to ≤10% of control). None of these agents would be unequivocally classified as positive using currently accepted criteria. The largest increases in mutant number were seen with taxol and carbendazim; therefore, trifluorothymidine (TFT)-resistant clones resulting from treatment with them were cultured and analysed for chromosome 11 copy number using fluorescent in situ hybridisation (FISH) and loss of heterozygosity (LOH). High concentrations of these aneugens induced LOH at all loci examined indicating only one chromosome 11 was present but, perhaps surprisingly, all were found to have two copies of chromosome 11 using FISH. This would be consistent with loss of the tk(+) chromosome 11b with concomitant duplication of chromosome 11a, which has been proposed as a likely mechanism for induction of TFT-resistant clones. However, it was also surprising that analysis of centromere size showed that almost all the clones had both small and large centromeres, i.e. suggesting the presence of both chromosomes 11a and 11b. In conclusion, it appears that the TFT-resistant mutants resulting from treatment with toxic concentrations of some aneugens such as taxol and carbendazim have undergone complex genetic changes. However, these data show that the MLA cannot be used as a routine screen to detect aneugens.

Anomalous genotoxic responses induced in mouse lymphoma L5178Y cells by potassium bromate.

Potassium bromate (KBrO3) is a well-established rodent kidney carcinogen and its oxidising activity is considered to be a significant factor in its mechanism of action. Although it has also been shown to be clearly genotoxic in a range of in vivo and in vitro test systems, surprisingly, it is not readily detected in several cell lines using the standard alkaline Comet assay. However, previous results from this laboratory demonstrated huge increases in tail intensity by modifying the method to include incubation with either human 8-oxodeoxyguanosine DNA glycosylase-1 (hOGG1) or bacterial formamidopyrimidine DNA glycosylase (FPG) indicating that, as expected, significant amounts of 8-oxodeoxyguanosine (8-OHdG) were induced. The purpose of this work, therefore, was to investigate why KBrO3, in contrast to other oxidising agents, gives a relatively poor response in the standard Comet assay. Results confirmed that it is a potent genotoxin in mouse lymphoma L5178Y cells inducing micronuclei and mutation at the tk and hprt loci at relatively non-cytotoxic concentrations. Subsequent time-course studies demonstrated that substantial amounts of 8-OHdG appear to remain in cells 24h after treatment with KBrO3 but result in no increase in frank stand breaks (FSB) even though phosphorylated histone H2AX (gamma-H2AX) antibody labelling confirmed the presence of double-strand breaks. Using bromodeoxyuracil (BrdU) incorporation together with measured increases in cell numbers, L5178Y cells also appeared to go through the cell cycle with unrepaired hOGG1-recognisable damage. Since unrepaired 8-OHdG can give rise to point mutations through G:C-->T:A transversions, it was also surprising that mutation could not be detected at the Na+/K+ATPase locus as determined by ouabain resistance. Some increases in strand breakage could be seen in the Comet assay by increasing the unwinding time, but only at highly toxic concentrations and to a much smaller extent than would be expected from the magnitude of the other genotoxic responses. It was considered unlikely that these anomalous observations were due to the inability of L5178Y cells to recognise 8-OHdG because these cells were shown to express mOGG1 and have functional cleavage activity at the adducted site. It appears that the responses of L5178Y cells to KBrO3 are complex and differ from those induced by other oxidising agents.

The rat bone marrow micronucleus test--study design and statistical power.

Although the rodent bone marrow micronucleus test has been in routine use for over 20 years, little work has been published to support its experimental design and all this has used the mouse rather than the rat. When it was decided to change the strain of rat routinely used in this laboratory to the Han Wistar, a preliminary study was performed to investigate the possible factors influencing experimental variability and to use statistical tools to examine possible study designs. Subsequently, a historical database comprising of vehicle controls accumulated from 65 studies was used to establish test acceptance criteria and a strategy for analysing equivocal results. The following conclusions were made: (i) no statistically significant differences were observed in experimental variability within or between control animals; although not statistically significant, the majority of experimental variability seen was found to be between separate counts on the same slide, with minimal differences found between duplicate slides from the same rat or between individual rats; (ii) power analyses showed that, if an equivocal result is obtained after scoring 2000 immature erythrocytes (IE), it is appropriate to re-code the slides and score an additional 4000 IE, i.e. analysing a total of 6000 IE; no meaningful increase in statistical power is gained by scoring >6000 IE; this is consistent with the variability observed between separate counts on the same slide; (iii) there was no significant difference between the control micronucleated immature erythrocyte (MIE) values at 24 and 48 h after dosing or between males and females; therefore, if an unusually low control value at either time point results in apparent small increases in MIE in a treated group, it is valid to pool control values from both time points for clarification and (iv) similar statistical power can be achieved by scoring 2000 IE from seven rats or 4000 IE from five rats, respectively. However, this is based only on control animals and does not consider possible differences in responses between animals to treatment with a potential genotoxin. In order to minimize the possible influence of responders and non-responders, the preferred study design in this laboratory is to score 2000 IE from groups of seven rats. Study data obtained over time confirmed observations made in the control study. Also from an ethical viewpoint, clarifying equivocal responses by combining control data from the 24- and 48-h time points and/or increasing the number of IE scored per animal has minimized the numbers of repeat studies necessary to determine the genotoxic status of a novel compound. However, before any laboratory can use these procedures, experimental data must be generated to demonstrate their validity.