Anthony M. Lynch

Quantification of the carcinogens 2-amino-3,8-dimethyl- and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in food using a combined assay based on gas chromatography—negative ion mass spectrometry

A gas chromatographic-mass spectrometric assay has been developed for the measurement of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in food. Stable isotope-labelled analogues of MeIQx and PhIP are used as internal standards and the synthesis of deuterated PhIP is described. The mass spectrometer is operated in the electron-capture negative ion chemical ionisation mode and the amines are chromatographed as their di-3,5-bistrifluoromethylbenzyl derivatives. All three compounds can be measured in a single chromatographic run and detection limits of 0.05, 0.1 and 0.2 ng/g for MeIQx, DiMeIQx and PhIP, respectively, in food are obtained. Various home-cooked and commercially prepared foodstuffs were analysed with this assay and several were found to contain measurable amounts of one or more of the three amines. These results are presented and discussed.

Early events in the mammalian response to DNA double-strand breaks

Physical and chemical agents that induce DNA double-strand breaks (DSBs) are among the most potent mutagens. The mammalian cell response to DSB comprises a highly co-ordinated, yet complex network of proteins that have been categorized as sensors, signal transducers, mediators and effectors of damage and repair. While this provides an accessible classification system, review of the literature indicates that many proteins satisfy the criteria of more than one category, pointing towards a series of highly co-operative pathways with overlapping function. In summary, the MRE11-NBS1-RAD50 complex is necessary for achieving optimal activation of ataxia-telangiectasia-mutated (ATM) kinase, which catalyses a phosphorylation-mediated signal transduction cascade. Among the subset of proteins phosphorylated by ATM are histone H2AX (H2AX), mediator of damage checkpoint protein 1, nibrin (NBS1), P53-binding protein 1 and breast cancer protein 1, all of which subsequently redistribute into DSB-containing sub-nuclear compartments. Post-translational modification of DSB responding proteins achieves a rapid and reversible change in protein behaviour and mediates damage-specific interactions, hence imparting a high degree of vigilance to the cell. This review highlights events fundamental in maintaining genetic integrity with emphasis on early stages of the DSB response.

The in vivo Pig-a assay: A report of the International Workshop On Genotoxicity Testing (IWGT) Workgroup.

The in vivo Pig-a assay uses flow cytometry to measure phenotypic variants for antibody binding to cell surface glycosylphosphatidylinositol (GPI)-anchored proteins. There is good evidence suggesting that the absence of antibody binding is the result of a mutation in the endogenous X-linked Pig-a gene, which forms the rationale for the assay. Although the assay has been performed with several types of hematopoietic cells and in a variety of mammalian species, including humans, currently it is optimized only for measuring CD59-deficient (presumed Pig-a mutant) erythrocytes in the peripheral blood of rats. An expert workgroup formed by the International Workshop on Genotoxicity Testing considered the state of assay development and the potential of the assay for regulatory use. Consensus was reached on what is known about the Pig-a assay and how it should be conducted, and recommendations were made on additional data and refinements that would help to further enhance the assay for use in hazard identification and risk assessment.

New and Emerging Technologies for Genetic Toxicity Testing

The International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute (HESI) Project Committee on the Relevance and Follow‐up of Positive Results in In Vitro Genetic Toxicity (IVGT) Testing established an Emerging Technologies and New Strategies Workgroup to review the current State of the Art in genetic toxicology testing. The aim of the workgroup was to identify promising technologies that will improve genotoxicity testing and assessment of in vivo hazard and risk, and that have the potential to help meet the objectives of the IVGT. As part of this initiative, HESI convened a workshop in Washington, DC in May 2008 to discuss mature, maturing, and emerging technologies in genetic toxicology. This article collates the abstracts of the New and Emerging Technologies Workshop together with some additional technologies subsequently considered by the workgroup. Each abstract (available in the online version of the article) includes a section addressed specifically to the strengths, weaknesses, opportunities, and threats associated with the respective technology. Importantly, an overview of the technologies and an indication of how their use might be aligned with the objectives of IVGT are presented. In particular, consideration was given with regard to follow‐up testing of positive results in the standard IVGT tests (i.e., Salmonella Ames test, chromosome aberration assay, and mouse lymphoma assay) to add weight of evidence and/or provide mechanism of action for improved genetic toxicity risk assessments in humans. Environ. Mol. Mutagen., 2011.

International Pig-a gene mutation assay trial: Evaluation of transferability across 14 laboratories†‡

A collaborative international trial was conducted to evaluate the reproducibility and transferability of an in vivo mutation assay based on the enumeration of CD59‐negative rat erythrocytes, a phenotype that is indicative of Pig‐a gene mutation. Fourteen laboratories participated in this study, where anti‐CD59‐PE, SYTO 13 dye, and flow cytometry were used to determine the frequency of CD59‐negative erythrocytes (RBCCD59−) and CD59‐negative reticulocytes (RETCD59−). To provide samples with a range of mutant phenotype cell frequencies, male rats were exposed to N‐ethyl‐N‐nitrosourea (ENU) via oral gavage for three consecutive days (Days 1–3). Each laboratory studied 0, 20, and 40 mg ENU/kg/day (n = 5 per group). Three sites also evaluated 4 mg/kg/day. At a minimum, blood samples were collected three times: predosing and on Days 15 and 30. Blood samples were processed according to a standardized sample processing and data acquisition protocol, and three endpoints were measured: %reticulocytes, frequency of RETCD59−, and frequency of RBCCD59−. The methodology was found to be reproducible, as the analysis of technical replicates resulted in experimental coefficients of variation that approached theoretical values. Good transferability was evident from the similar kinetics and magnitude of the dose‐related responses that were observed among different laboratories. Concordance correlation coefficients showed a high level of agreement between the reference site and the test sites (range: 0.87–0.99). Collectively, these data demonstrate that with adequate training of personnel, flow cytometric analysis is capable of reliably enumerating mutant phenotype erythrocytes, thereby providing a robust in vivo mutation assay that is readily transferable across laboratories. Environ. Mol. Mutagen. 2011.

Interlaboratory evaluation of a flow cytometric, high content in vitro micronucleus assay.

An international, multi-lab trial was conducted to evaluate a flow cytometry-based method for scoring micronuclei in mouse lymphoma L5178Y cells [S.L. Avlasevich, S.M. Bryce, S.E. Cairns, S.D. Dertinger, In vitro micronucleus scoring by flow cytometry: differential staining of micronuclei versus apoptotic and necrotic chromatin enhances assay reliability, Environ. Mol. Mutagen. 47 (2006) 56-66]. A reference laboratory investigated the potential of six chemicals to induce micronuclei -- the genotoxicants mitomycin C (MMC), etoposide (ETOPO), and vinblastine (VB), and the non-genotoxicants sucrose (SUC), staurosporine (STS), and dexamethasone (DEX). The latter two non-genotoxicants were selected as extreme challenges to the assay because of their potent apoptogenic activity. Three collaborating laboratories were supplied with prototype In Vitro MicroFlow kits, and each was assigned one genotoxicant and one non-genotoxicant. Cells were treated continuously for 24h over a range of concentrations up to 5 mg/ml, or overtly cytotoxic concentrations. Micronuclei were scored via standard microscopy and flow cytometry. In addition to enumerating micronucleus frequencies, a cytotoxicity measurement that is simultaneously acquired with the flow cytometric micronucleus scoring procedure was evaluated (Flow-NBR). With this method, latex particles served as counting beads, and facilitated relative survival measurements that exclude the presence of dead/dying cells. For comparison purposes, additional cytotoxicity endpoints were measured, including several that are based on cell number, and others that reflect compromised membrane integrity, including dye permeability and/or phospholipid distribution. Key findings for this set of compounds include the following: (1) significant discrepancies in top concentration selection were found when cytotoxicity measurements were based on different methods, with the Flow-NBR approach tending to be the most sensitive, (2) both microscopy- and flow cytometry-based scoring methods detected concentration-dependent micronucleus formation for the three genotoxic agents studied, with good agreement between the reference laboratory and the collaborating laboratories, and (3) whereas flow cytometric analyses showed no significant increases for the non-genotoxicants when top concentration selection was based on Flow-NBR, significantly elevated micronucleus frequencies were observed for concentrations that were chosen based on less-sensitive cytotoxicity assays. Collectively, these results indicate that rapid assessment of genotoxicity can be accomplished with a relatively simple flow cytometric technique, and that the scoring system is transferable across laboratories. Furthermore, a concurrent assessment of cytotoxicity, Flow-NBR, may help reduce the occurrence of irrelevant positive results, as it may represent a more appropriate means for choosing top concentration levels. Finally, the data presented herein reinforce concerns about the manner in which cytotoxicity limits are described in guidance documents, since these recommendations tend to cite fixed cut-off values without reference to methodology.

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.

International Pig‐a gene mutation assay trial (Stage III): Results with N‐methyl‐N‐nitrosourea

N‐methyl‐N‐nitrosourea (MNU) was evaluated in the in vivo Pig‐a mutation assay as part of an International Collaborative Trial to investigate laboratory reproducibility, 28‐day study integration, and comparative analysis with micronucleus (MN), comet, and clinical pathology endpoints. Male Sprague Dawley rats were treated for 28 days with doses of 0, 2.5, 5, and 10 mg MNU/kg/day in two independent laboratories, GlaxoSmithKline (GSK) and Bristol Myers Squibb (BMS). Additional studies investigated the low‐dose region (<2.5 mg/kg/day). Reticulocytes were evaluated for Pig‐a phenotypic mutation, CD59‐negative reticulocytes/erythrocytes (RETsCD59−/ RBCsCD59−) on Days 1, 4, 15, 29, 43, and 57, and for micronucleated reticulocytes (MN‐RETs) on Days 4 and 29. Comet analysis was conducted for liver and whole blood, and hematology and clinical chemistry was investigated. Dose‐dependent increases in the frequency of RETsCD59− and RBCsCD59− were observed by Day 15 or 29, respectively. Dose‐dependent increases were observed in %MN‐RET on Days 4 and 29, and in mean %tail intensity in liver and in blood. Hematology/clinical chemistry data demonstrated bone marrow toxicity. Data comparison between GSK and BMS indicated a high degree of concordance with the Pig‐a mutation assay results, consistent with previous observations with MNU and N‐ethyl‐N‐nitrosourea. These data confirm that complementary genotoxicity endpoints can be effectively incorporated into routine toxicology studies, a strategy that can provide information on gene mutation, chromosome damage, and DNA strand breaks in a single repeat dose rodent study. Collectively, this would reduce animal usage while providing valuable genetic toxicity information within the context of other toxicological endpoints. Environ. Mol. Mutagen., 2011.

Flow cytometric analysis of micronuclei in mammalian cell cultures: past, present and future

The relative simplicity of the in vitro micronucleus (MNvit) endpoint has made it amenable to several automated scoring approaches. Flow cytometry is one such scoring platform that has been successfully employed. This review describes the origins of the MNvit assay, as well as the evolution and properties of flow cytometry-based scoring systems. While the current state-of-the-art methods acquire micronucleus (MN) frequency data very efficiently, it is becoming clear that they also endow the assay with high information content. For instance, simultaneous with MN frequency determinations, several additional endpoints are acquired that provide insights into cytotoxicity, cell cycle perturbations and, in the event of MN induction, information about genotoxic mode of action. This review concludes with a discussion regarding data gaps and also recommendations for additional work that is needed to more fully realise the potential of flow cytometric MNvit scoring.

Can in vitro mammalian cell genotoxicity test results be used to complement positive results in the Ames test and help predict carcinogenic or in vivo genotoxic activity? I. Reports of individual databases presented at an EURL ECVAM Workshop

Positive results in the Ames test correlate well with carcinogenic potential in rodents. This correlation is not perfect because mutations are only one of many stages in tumour development. Also, situations can be envisaged where the mutagenic response may be specific to the bacteria or the test protocol, e.g., bacterial-specific metabolism, exceeding a detoxification threshold, or the induction of oxidative damage to which bacteria may be more sensitive than mammalian cells in vitro or tissues in vivo. Since most chemicals are also tested for genotoxicity in mammalian cells, the pattern of mammalian cell results may help identify whether Ames-positive results predict carcinogenic or in vivo mutagenic activity. A workshop was therefore organised and sponsored by the EU Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) to investigate this further. Participants presented results from other genotoxicity tests with Ames-positive compounds. Data came from published, regulatory agency, and industry sources. The question was posed whether negative results in mammalian cell tests were associated with absence of carcinogenic or in vivo genotoxic activity despite a positive Ames test. In the limited time available, the presented data were combined and an initial analysis suggested that the association of negative in vitro mammalian cell test results with lack of in vivo genotoxic or carcinogenic activity could have some significance. Possible reasons why a positive Ames test may not be associated with in vivo activity and what additional investigations/tests might contribute to a more robust evaluation were discussed. Because a considerable overlap was identified among the different databases presented, it was recommended that a consolidated database be built, with overlapping chemicals removed, so that a more robust analysis of the predictive capacity for potential carcinogenic and in vivo genotoxic activity could be derived from the patterns of mammalian cell test results obtained for Ames-positive compounds.