Unusual structure-genotoxicity relationship in mouse lymphoma cells observed with a series of kinase inhibitors.

During development of a novel kinase inhibitor for an anti-inflammatory therapy at AstraZeneca UK, the lead compound was found to be potently active in the mouse lymphoma assay (MLA). This was not believed to be due to primary pharmacology because structural alert relationships and a negative Ames test indicated that the compound was unlikely to form DNA adducts. A number of investigations were performed to assess whether mammalian cell genotoxicity was inherent to the chemical series. The in vitro micronucleus assay (MN(vit)) combined with a semi-automated analysis system, was used as a high-throughput screen. A number of additional compounds were selected for testing, all with different substituents around a core isoquinolinone. These modifications did not affect the kinase and non-kinase selectivity of the compounds. Several of these compounds were positive in the MN(vit), however, two compounds were found to be negative and these were also confirmed to be negative in the MLA. It was considered possible that topoisomerase II or off-target kinase inhibition may have been responsible for the observed mammalian cell genotoxicity. The present investigations show how an iterative chemical design, along with genotoxicity screening by use of a semi-automated MN(vit), can identify and remove the genotoxic hazard from pharmaceutical projects at an early stage of development, and produce high-quality molecules suitable for further progression.

The incidence of positive results in the mouse lymphoma TK assay (MLA) in pharmaceutical screening and their prediction by MultiCase MC4PC.

There are published data indicating that the mouse lymphoma TK assay (MLA) has an unacceptably high incidence of positive results, hence it was decided to review the MLA data generated in this laboratory for potential drug candidates. Of the 355 compounds tested, only 52 (15%) gave positive results so, even if it is assumed that all of these are non-carcinogens, the incidence of 'false positive' predictions of carcinogenicity is much lower than the 61% apparent from analysis of the literature. Furthermore, only 19 compounds (5%) were positive by a mechanism that could not be associated with the compounds primary pharmacological activity or positive responses in other genotoxicity assays. It should be noted that the majority of these compounds were not bacterial mutagens so, in most cases, the positive results were an additional indicator of genotoxicity. However, data are not available to assess any risk they might present. At least for pharmaceuticals, it appears that the MLA does not generate as many positive results as is commonly believed, and it is against this incidence that the performance of other in vitro genotoxicity tests should be compared. The predictive accuracy of the program MultiCase MC4PC was also examined using these results. The sensitivity and specificity were found to be 62 and 38%, respectively; in fact, 62% of all compounds were predicted to be positive irrespective of whether they were actually positive or negative. It was concluded that, in its current state of development, M4PC cannot be considered sufficiently accurate to be used to predict the activity of pharmaceuticals in the MLA.

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.

Etoposide, cadmium chloride, benzo[a]pyrene, cyclophosphamide and colchicine tested in the in vitro mammalian cell micronucleus test (MNvit) in the presence and absence of cytokinesis block using L5178Y mouse lymphoma cells and 2-aminoanthracene tested in MNvit in the absence of cytokinesis block using TK6 cells at AstraZeneca UK, in support of OECD draft Test Guideline 487.

At the laboratories of AstraZeneca, Alderley Park, UK the reference genotoxic agents etoposide (a topoisomerase II inhibitor), cadmium chloride (an inorganic carcinogen), colchicine (an aneugen that inhibits tubulin polymerisation), benzo[a]pyrene (a metabolism dependent reference genotoxin) and cyclophosphamide (a metabolism dependent reference genotoxin) were tested in the in vitro micronucleus assay (MNvit), using mouse lymphoma L5178Y cells, with and without cytokinesis block. Further, 2-aminoanthracene (a metabolism dependent weak clastogen) was tested in the MNvit, using TK6 cells, without cytokinesis block. This was done in support of the toxicity (cell death and cytostasis) measures recommended in the late 2007 version of the draft OECD Test Guideline 487 for the testing of chemicals. All six reference agents were positive in the MNvit without cytokinesis block at concentrations giving approximately 50% toxicity or less as defined by draft Test Guideline 487 recommended measures, relative population doublings and relative increase in cell counts. Furthermore, the five agents tested with cytokinesis block were positive in the MNvit at concentrations giving approximately 50% toxicity or less as assessed by replicative index. Accordingly, this work supports the premise that relative population doublings and relative increase in cell counts are appropriate measures of toxicity for the non-cytokinesis-blocked in vitro micronucleus assay.

Comparison of different methods for an accurate assessment of cytotoxicity in the in vitro micronucleus test. II: Practical aspects with toxic agents.

Appropriate measures of cytotoxicity need to be used when selecting test concentrations in in vitro genotoxicity assays. Underestimation of toxicity may lead to inappropriately toxic concentrations being selected for analysis, with the potential for generation of irrelevant positive results. As guidance for the in vitro micronucleus test is being developed, it is clearly important to compare the different measures of cytotoxicity that can be used both with and without cytokinesis blocking. Therefore, relative cell counts (RCC), relative increase in cell counts (RICC) and relative population doubling (RPD) for treatments without cytokinesis block were compared with replication index (RI) for treatments with cytokinesis block, and the corresponding induction of micronucleated cells was evaluated. A wide range of chemicals and gamma irradiation were used, and in almost all cases, RCC underestimated cytotoxicity when compared with all other measures such that RCC would have resulted in the selection of inappropriately high concentrations for micronuclei analysis. In the absence of cytokinesis block, RICC or RPD is more comparable with RI with cytokinesis block, and therefore considered more appropriate measure of survival. Furthermore, using these estimations of cytotoxicity and the limit of 50% survival, all the mutagens and aneugens tested were appropriately identified as positive in the in vitro micronucleus assay. Accordingly, it was clear that testing beyond 50% survival was not necessary to identify the potential of these agents to induce micronuclei.

Mouse lymphoma thymidine kinase gene mutation assay: meeting of the International Workshop on Genotoxicity Testing, San Francisco, 2005, recommendations for 24-h treatment.

The Mouse Lymphoma Assay (MLA) Workgroup of the International Workshop on Genotoxicity Testing (IWGT), comprised of experts from Japan, Europe and the United States, met on September 9, 2005, in San Francisco, CA, USA. This meeting of the MLA Workgroup was devoted to reaching a consensus on issues involved with 24-h treatment. Recommendations were made concerning the acceptable values for the negative/solvent control (mutant frequency, cloning efficiency and suspension growth) and the criteria to define an acceptable positive control response. Consensus was also reached concerning the use of the global evaluation factor (GEF) and appropriate statistical trend analysis to define positive and negative responses for the 24-h treatment. The Workgroup agreed to continue their support of the International Committee on Harmonization (ICH) recommendation that the MLA assay should include a 24-h treatment (without S-9) in those situations where the short treatment (3-4 h) gives negative results.

Mouse lymphoma thymidine kinase gene mutation assay: follow-up meeting of the International Workshop on Genotoxicity Testing--Aberdeen, Scotland, 2003--Assay acceptance criteria, positive controls, and data evaluation.

The Mouse Lymphoma Assay (MLA) Workgroup of the International Workshop on Genotoxicity Testing (IWGT), comprised of experts from Japan, Europe, and the United States, met on August 29, 2003, in Aberdeen, Scotland, United Kingdom. This meeting of the MLA Workgroup was devoted to reaching a consensus on the appropriate approach to data evaluation and on acceptance criteria for both the positive and negative/vehicle controls. The Workgroup reached consensus on the acceptance criteria for both the agar and microwell versions of the MLA. Recommendations include acceptable ranges for mutant frequency, cloning efficiency, and suspension growth of the negative/vehicle controls and on criteria to define an acceptable positive control response. The recommendation for the determination of a positive/negative test chemical response includes both the requirement that the response exceeds a defined value [the global evaluation factor (GEF)] and that there also be a positive dose-response (evaluated by an appropriate statistical method).

Mouse lymphoma thymidine kinase gene mutation assay: follow-up International Workshop on Genotoxicity Test Procedures, New Orleans, Louisiana, April 2000.

The Mouse Lymphoma Assay (MLA) Workgroup of the International Workshop on Genotoxicity Test Procedures held a second harmonization meeting just prior to the U.S. Environmental Mutagen Society Meeting in New Orleans, LA, in April 2000. The discussion focused on several important aspects of the MLA, including: 1) cytotoxicity measures and their determination, 2) use of a 24-hr treatment, 3) the ability of the assay to detect aneugens, and 4) concentration selection. Prior to the meeting the group developed Microsoft Excel Workbooks for data entry. Ten laboratories entered their data into the workbooks (primarily as coded chemicals). The Excel Workbooks were used to facilitate data analysis by generating an extensive set of graphs that were evaluated by the meeting participants. Based on the Workgroup's previous agreement that a single cytotoxicity measure should be established for both the microwell and soft agar versions of the assay, the Workgroup analyzed the submitted data and unanimously agreed that the relative total growth (RTG) should be used as the cytotoxicity measure for concentration selection and data evaluation. The Workgroup also agreed that the various cytotoxicity measures should be calculated using the same methods regardless of whether the soft agar or microwell version of the assay was used. In the absence of sufficient data to make a definitive determination, the Workgroup continued to endorse the International Committee on Harmonization recommendation for the use of 24-hr treatment and made some specific 24-hr treatment protocol recommendations. The Workgroup recognized the ability of the MLA to detect at least some aneugens and also developed general guidance and requirements for appropriate concentration selection.

Mouse lymphoma thymidine kinase gene mutation assay: International Workshop on Genotoxicity Tests Workgroup report--Plymouth, UK 2002.

The Mouse Lymphoma Assay (MLA) Workgroup of the International Workshop on Genotoxicity Tests (IWGT) met on June 28th and 29th, 2002, in Plymouth, England. This meeting of the MLA group was devoted to discussing the criteria for assay acceptance and appropriate approaches to data evaluation. Prior to the meeting, the group conducted an extensive analysis of data from both the microwell and soft agar versions of the assay. For the establishment of criteria for assay acceptance, 10 laboratories (6 using the microwell method and 4 using soft agar) provided data on their background mutant frequencies, plating efficiencies of the negative/vehicle control, cell suspension growth, and positive control mutant frequencies. Using the distribution curves generated from this data, the Workgroup reached consensus on the range of values that should be used to determine whether an individual experiment is acceptable. In order to establish appropriate approaches for data evaluation, the group used a number of statistical methods to evaluate approximately 400 experimental data sets from 10 laboratories entered into a database created for the earlier MLA Workshop held in New Orleans [Environ. Mol. Mutagen. 40 (2002) 292]. While the Workgroup could not, during this meeting, make a final recommendation for the evaluation of data, a general strategy was developed and the Workgroup members agreed to evaluate this new proposed approach using their own laboratory data. This evaluation should lead to a consensus global approach for data evaluation in the near future.

Parameterized algorithmics for finding connected motifs in biological networks.

We study the NP-hard LIST-COLORED GRAPH MOTIF problem which, given an undirected list-colored graph G = (V, E) and a multiset M of colors, asks for maximum-cardinality sets S ⊆ V and M' ⊆ M such that G[S] is connected and contains exactly (with respect to multiplicity) the colors in M'. LIST-COLORED GRAPH MOTIF has applications in the analysis of biological networks. We study LIST-COLORED GRAPH MOTIF with respect to three different parameterizations. For the parameters motif size |M| and solution size |S|, we present fixed-parameter algorithms, whereas for the parameter |V| - |M|, we show W[1]-hardness for general instances and achieve fixed-parameter tractability for a special case of LIST-COLORED GRAPH MOTIF. We implemented the fixed-parameter algorithms for parameters |M| and |S|, developed further speed-up heuristics for these algorithms, and applied them in the context of querying protein-interaction networks, demonstrating their usefulness for realistic instances. Furthermore, we show that extending the request for motif connectedness to stronger demands, such as biconnectedness or bridge-connectedness leads to W[1]-hard problems when the parameter is the motif size |M|.

Haplotype inference constrained by plausible haplotype data.

The haplotype inference problem (HIP) asks to find a set of haplotypes which resolve a given set of genotypes. This problem is important in practical fields such as the investigation of diseases or other types of genetic mutations. In order to find the haplotypes which are as close as possible to the real set of haplotypes that comprise the genotypes, two models have been suggested which are by now well-studied: The perfect phylogeny model and the pure parsimony model. All known algorithms up till now for haplotype inference may find haplotypes that are not necessarily plausible, i.e., very rare haplotypes or haplotypes that were never observed in the population. In order to overcome this disadvantage, we study in this paper, a new constrained version of HIP under the above-mentioned models. In this new version, a pool of plausible haplotypes H is given together with the set of genotypes G, and the goal is to find a subset H ⊆ H that resolves G. For constrained perfect phlogeny haplotyping (CPPH), we provide initial insights and polynomial-time algorithms for some restricted cases of the problem. For constrained parsimony haplotyping (CPH), we show that the problem is fixed parameter tractable when parameterized by the size of the solution set of haplotypes.

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.

Cytotoxicity in cultured mammalian cells is a function of the method used to estimate it.

Up to prescribed limits, the maximum test compound concentrations used in mammalian cell genotoxicity assays in vitro are determined by cytotoxicity, unless limited by solubility in solvents or culture medium. However, 'cytotoxicity' is different in the various test systems, both in the methods used to estimate it and the levels of toxicity that must be achieved. For example, in cytogenetic assays, the acceptable level of toxicity is defined as a 'significant reduction (>50%)' in cell number, culture confluency or mitotic index (MI) (OECD 473, ICH S2A), whereas mutation tests require relative total growth or cloning efficiency (CE) to be reduced by 80-90% (OECD 476, ICH S2A). In this study using mouse lymphoma cells, it was shown that, for a variety of agents with differing modes of action, cytotoxicity varies considerably depending on the method used to estimate it. Specifically, trypan blue exclusion, MI and binucleate incidence all grossly underestimate cytotoxicity in comparison with cell growth or CE. If the performance of different test systems is to be compared, or if data from different assays are to be used for the meaningful assessment of a novel chemical entity, it is essential that similar methods to determine cytotoxicity are used for them all. The purpose of this paper is not to recommend a specific method to determine cytotoxicity, although it can be argued that any such method must quantify the proportion of cells capable of division following treatment, but rather to draw attention to the fact that apparent toxicity depends upon the method used to estimate it.

In vivo genotoxicity studies with 3-monochloropropan-1,2-diol.

3-Monochloropropan-1,2-diol (3-MCPD) is a contaminant of polyamine flocculants used in the production of drinking water, but more significantly for human exposure it can arise also in certain foodstuffs containing acid-hydrolysed vegetable protein. It is carcinogenic in the rat, producing tumours in males in the testes, mammary gland and the preputial gland and also kidney tumours in both sexes. It has given positive results in in vitro mutagenicity studies, but there have been no satisfactorily conducted in vivo studies in somatic cells published in the peer reviewed literature. As a result, and because of the absence of appropriate in vivo evidence, several international regulatory agencies had previously judged it prudent to assume that 3-MCPD possessed mutagenic activity in vivo and considered 3-MCPD to be a genotoxic carcinogen. We present in this paper results from two in vivo mutagenicity studies with 3-MCPD, namely a bone marrow micronucleus test in the rat and unscheduled DNA synthesis in the rat liver, both conducted in accordance with relevant OECD protocols. These studies show that 3-MCPD does not possess genotoxic activity in vivo in the tissues examined. On the basis of these findings, and along with evidence that tumours may be induced by mechanisms involving either hormonal disturbances or sustained cytotoxicity, we believe that 3-MCPD may now be considered to be carcinogenic to rodents via a non-genotoxic mechanism.