In vivo genotoxicity testing strategies: Report from the 8th International Workshop on Genotoxicity Testing (IWGT).

The in vivo working group (WG) considered three topics: acceptable maximum doses for negative erythrocyte micronucleus (MN) tests, validation status of MN assays in non-hematopoietic tissues, and nuisance factors in the comet assay. The WG reached agreement on many issues, including: negative erythrocyte MN studies should be acceptable if dosing is conducted to Organisation for Economic Co-operation and Development (OECD) test guideline (TG) 474 recommendations and if sufficient bone marrow exposure is demonstrated; consensus on the evidence required to demonstrate "sufficient" exposure was not reached. The liver MN test using six-week-old rats is sufficiently validated to develop an OECD TG, but the impact of animal age warrants additional study. Ki-67 is a reliable marker for cellular proliferation in hepatocytes. The gastrointestinal tract MN test is useful for detecting poorly absorbed or rapidly degraded aneugens, and for genotoxic metabolites formed in the colon. Although current validation data are insufficient to support the development of an OECD TG, the methodologies are sufficient to consider as an appendix to OECD TG474. Comparison of comet assay results to laboratory historical control data (HCD) should not be used in data evaluation, unless the HCD distribution is demonstrated to be stable and the predominant source of HCD variation is due to animal, not study, factors. No universally acceptable negative control limit for any tissue was identified. Methodological differences in comet studies can result in variable data interpretations; more data are required before best practice recommendations can be made. Hedgehogs alone are unreliable indicators of cytotoxicity and additional investigations into cytotoxicity markers are required.

CDK8/19 inhibition plays an important role in pancreatic ß-cell induction from human iPSCs.

BACKGROUND: Transplantation of differentiated cells from human-induced pluripotent stem cells (hiPSCs) holds great promise for clinical treatments. Eliminating the risk factor of malignant cell transformation is essential for ensuring the safety of such cells. This study was aimed at assessing and mitigating mutagenicity that may arise during the cell culture process in the protocol of pancreatic islet cell (iPIC) differentiation from hiPSCs. METHODS: We evaluated the mutagenicity of differentiation factors used for hiPSC-derived pancreatic islet-like cells (iPICs). We employed Ames mutagenicity assay, flow cytometry analysis, immunostaining, time-resolved fluorescence resonance energy transfer-based (TR-FRET) cell-free dose-response assays, single-cell RNA-sequencing and in vivo efficacy study. RESULTS: We observed a mutagenic effect of activin receptor-like kinase 5 inhibitor II (ALK5iII). ALK5iII is a widely used ß-cell inducer but no other tested ALK5 inhibitors induced ß-cells. We obtained kinase inhibition profiles and found that only ALK5iII inhibited cyclin-dependent kinases 8 and 19 (CDK8/19) among all ALK5 inhibitors tested. Consistently, CDK8/19 inhibitors efficiently induced ß-cells in the absence of ALK5iII. A combination treatment with non-mutagenic ALK5 inhibitor SB431542 and CDK8/19 inhibitor senexin B afforded generation of iPICs with in vitro cellular composition and in vivo efficacy comparable to those observed with ALK5iII. CONCLUSION: Our findings suggest a new risk mitigation approach for cell therapy and advance our understanding of the ß-cell differentiation mechanism.

A comparison of the lowest effective concentration in culture media for detection of chromosomal damage in vitro and in blood or plasma for detection of micronuclei in vivo.

It is often assumed that genotoxic substances will be detected more easily by using in vitro rather than in vivo genotoxicity tests since higher concentrations, more cytotoxicity and static exposures can be achieved. However, there is a paucity of data demonstrating whether genotoxic substances are detected at lower concentrations in cell culture in vitro than can be reached in the blood of animals treated in vivo. To investigate this issue, we compared the lowest concentration required for induction of chromosomal damage in vitro (lowest observed effective concentration, or LOEC) with the concentration of the test substance in blood at the lowest dose required for biologically relevant induction of micronuclei in vivo (lowest observed effective dose, or LOED). In total, 83 substances were found for which the LOED could be identified or estimated, where concentrations in blood and micronucleus data were available via the same route of administration in the same species, and in vitro chromosomal damage data were available. 39.8 % of substances were positive in vivo at blood concentrations that were lower than the LOEC in vitro, 22.9 % were positive at similar concentrations, and 37.3 % of substances were positive in vivo at higher concentrations. Distribution analysis showed a very wide scatter of > 6 orders of magnitude across these 3 categories. When mode of action was evaluated, the distribution of clastogens and aneugens across the 3 categories was very similar. Thus, the ability to detect induction of micronuclei in bone marrow in vivo regardless of the mechanism for micronucleus induction, is clearly not solely determined by the concentration of test substance which induced chromosomal damage in vitro.

Analysis of historical negative control group data from the in vitro micronucleus assay using human lymphocytes.

A database of the micronuclei counts was built up for historical negative control data from human lymphocyte in vitro micronuclei tests (MnVit) carried out in 8 laboratories with experience of the method. The mean incidence of micronucleated cells (mnt)/1000 cells ranged from 2.2/1000 to 15.9/1000. There were no large differences in incidence between the presence or absence of S9 mix or between different treatment lengths. There was also little evidence that different solvents affected the numbers of micronuclei appreciably. A number of laboratories did show significant inter-experiment variability, indicating that there remained unidentified factors affecting frequencies. Donor variance may be one such factor. Inter-individual variability may explain some of these differences. The approximate 7.5-fold difference in mnt/1000 scores in a relatively small group of experienced laboratories illustrates the potential complications that can arise if a metric like a fold increase was considered the only biologically important finding. Although there is inherent variability between experiments, it was evident that within a laboratory the overall laboratory mean remains constant over time. It is believed that these findings will provide help to laboratories conducting studies using human lymphocytes in the MnVit and to those involved in the assessment of MnVit results.

Analysis of negative historical control group data from the in vitro micronucleus assay using TK6 cells.

The recent revisions of the Organisation for Economic Co-operation and Development (OECD) genetic toxicology test guidelines emphasize the importance of historical negative controls both for data quality and interpretation. The goal of a HESI Genetic Toxicology Technical Committee (GTTC) workgroup was to collect data from participating laboratories and to conduct a statistical analysis to understand and publish the range of values that are normally seen in experienced laboratories using TK6 cells to conduct the in vitro micronucleus assay. Data from negative control samples from in vitro micronucleus assays using TK6 cells from 13 laboratories were collected using a standard collection form. Although in some cases statistically significant differences can be seen within laboratories for different test conditions, they were very small. The mean incidence of micronucleated cells/1000 cells ranged from 3.2/1000 to 13.8/1000. These almost four-fold differences in micronucleus levels cannot be explained by differences in scoring method, presence or absence of exogenous metabolic activation (S9), length of treatment, presence or absence of cytochalasin B or different solvents used as vehicles. The range of means from the four laboratories using flow cytometry methods (3.7-fold: 3.5-12.9 micronucleated cells/1000 cells) was similar to that from the nine laboratories using other scoring methods (4.3-fold: 3.2-13.8 micronucleated cells/1000 cells). No laboratory could be identified as an outlier or as showing unacceptably high variability. Quality Control (QC) methods applied to analyse the intra-laboratory variability showed that there was evidence of inter-experimental variability greater than would be expected by chance (i.e. over-dispersion). However, in general, this was low. This study demonstrates the value of QC methods in helping to analyse the reproducibility of results, building up a 'normal' range of values, and as an aid to identify variability within a laboratory in order to implement processes to maintain and improve uniformity.

Poor recognition of O6-isopropyl dG by MGMT triggers double strand break-mediated cell death and micronucleus induction in FANC-deficient cells.

Isopropyl methanesulfonate (IPMS) is the most potent genotoxic compound among methanesulfonic acid esters. The genotoxic potential of alkyl sulfonate esters is believed to be due to their alkylating ability of the O6 position of guanine. Understanding the primary repair pathway activated in response to IPMS-induced DNA damage is important to profile the genotoxic potential of IPMS. In the present study, both chicken DT40 and human TK6 cell-based DNA damage response (DDR) assays revealed that dysfunction of the FANC pathway resulted in higher sensitivity to IPMS compared to EMS or MMS. O6-alkyl dG is primarily repaired by methyl guanine methyltransferase (MGMT), while isopropyl dG is less likely to be a substrate for MGMT. Comparison of the cytotoxic potential of IPMS and its isomer n-propyl methanesulfonate (nPMS) revealed that the isopropyl moiety avoids recognition by MGMT and leads to higher cytotoxicity. Next, the micronucleus (MN) assay showed that FANC deficiency increases the sensitivity of DT40 cells to MN induction by IPMS. Pretreatment with O6-benzyl guanine (OBG), an inhibitor of MGMT, increased the MN frequency in DT40 cells treated with nPMS, but not IPMS. Lastly, IPMS induced more double strand breaks in FANC-deficient cells compared to wild-type cells in a time-dependent manner. All together, these results suggest that IPMS-derived O6-isopropyl dG escapes recognition by MGMT, and the unrepaired DNA damage leads to double strand breaks, resulting in MN induction. FANC, therefore, plays a pivotal role in preventing MN induction and cell death caused by IPMS.

Incorporation of metabolic activation potentiates cyclophosphamide-induced DNA damage response in isogenic DT40 mutant cells.

Elucidating the DNA repair pathways that are activated in the presence of genotoxic agents is critical to understand their modes of action. Although the DT40 cell-based DNA damage response (DDR) assay provides rapid and sensitive results, the assay cannot be used on genotoxic compounds that require metabolic activation to be reactive. Here, we applied the metabolic activation system to a DDR and micronucleus (MN) assays in DT40 cells. Cyclophosphamide (CP), a well-known cross-linking agent requiring metabolic activation, was preincubated with liver S9 fractions. When DT40 cells and mutant cells were exposed to the preactivated CP, CP caused increased cytotoxicity in FANC-, RAD9-, REV3- and RAD18-mutant cells compared to isogenic wild-type cells. We then performed a MN assay on DT40 cells treated with preactivated CP. An increase in the MN was observed in REV3- and FANC-mutant cells at lower concentrations of activated CP than in the parental DT40 cells. These results demonstrated that the incorporation of metabolic preactivation system using S9 fractions significantly potentiates DDR caused by CP in DT40 cells and their mutants. In addition, our data suggest that the metabolic preactivation system for DDR and MN assays has a potential to increase the relevance of this assay to screening various compounds for potential genotoxicity.

Mitotic slippage underlies the relationship between p53 dysfunction and the induction of large micronuclei by colcemid.

Micronuclei induced by aneugens are larger than those induced by clastogens in both in vitro and in vivo micronucleus (MN) assays. p53 dysfunction increases the formation of large micronuclei following treatment with aneugens; this study sought to identify the mechanisms responsible for this. Treatment with colcemid, both a mitotic inhibitor and an aneugen, induced MN containing two or more chromosomes more frequently in NH32 cells, in which p53 function is compromised, than in TK6 cells, in which p53 is functional. This indicates that p53 dysfunction enhances aneugen-induced chromosome loss or perturbs apoptosis, resulting in the formation of large MN. To examine the former hypothesis, the incidence of chromosome malsegregation in colcemid-treated TK6 and NH32 cells was compared using the cytokinesis-block MN assay. The incidence of chromosome non-disjunction was higher in NH32 cells than in TK6 cells, whereas the incidence of MN containing two or more chromosomes was similar between the two cell lines. To address the involvement of apoptosis in cell cycle progression, examination of chromosome 8 distribution revealed that more mononuclear NH32 than TK6 cells were tetraploid after prolonged mitotic inhibition, which indicated that the more number of NH32 cells may have bypassed the spindle assembly checkpoint via mitotic slippage and progression into the next interphase. Cells that underwent mitotic slippage were likely to contain lagging chromosomes formed via chromosome malsegregation, resulting in MN separated from the main nucleus. The number of TK6 cells containing large MN following colcemid treatment was increased by treatment with a caspase inhibitor in a dose-dependent manner, indicating that TK6 cells with MN normally undergo apoptosis. In conclusion, these findings indicate that mitotic slippage and perturbed apoptosis contribute to the induction of large MN in p53-compromised cells following treatment with colcemid.

Difference in susceptibility to morphological changes in the nucleus to aneugens between p53-competent and p53-abrogated lymphoblastoid cell lines (TK6 and NH32 cells) in the in vitro micronucleus assay.

We previously reported that the proportion of large-size micronuclei (MN) can be a reliable parameter to discriminate aneugens from clastogens in the in vitro MN assay using Chinese hamster lung cells. The frequencies of polynuclear (PN) and mitotic (M) cells are also supposed to be useful parameters for the same purpose since they are known to be increased by aneugens. In the present study, we investigated whether morphological observations of the cell nucleus can be applied for the in vitro MN assay using the p53-competent human lymphoblastoid cell line, TK6 cells. Our present MN assay with six clastogens and six aneugens revealed that the frequencies of large-size MN or PN cells cannot distinguish aneugens from clastogens, while the frequencies of M cells can distinguish them, suggesting that the M-cell frequency is a recommended parameter to determine a mode of action for MN induction in the in vitro MN assay using TK6 cells. Our further investigation using p53-null mutant NH32 cells showed that the frequencies of large-size MN or PN cells induced by aneugen treatments were higher than those in TK6 cells but not by clastogen treatments. These findings suggest that p53 abrogation promotes the susceptibility for morphological changes in the nucleus to aneugens and that morphological observation of the cell nucleus including size-classifying MN counting could distinguish aneugens from clastogens in the MN assay using NH32 cells.

Involvement of p53 function in different magnitude of genotoxic and cytotoxic responses in in vitro micronucleus assays.

In in vitro micronucleus (MN) assays the sensitivity to MN induction or cytotoxicity can vary depending on the kind of cells employed. This study was conducted to examine the involvement of the p53 function in the different sensitivities between Chinese hamster lung (CHL) cells and human lymphoblastoid TK6 cells in MN assays. MN induction and cytotoxicity were compared using MN-inducing chemicals reported as DNA reactive clastogens, non-DNA reactive clastogens or aneugens. The study revealed that the maximum levels of MN induction in p53-compromised CHL cells were higher than those in p53-competent TK6 cells, but MN were significantly induced in TK6 cells at lower concentrations than in CHL cells. Most of the test chemicals produced a more severe cytotoxicity in TK6 cells, suggesting TK6 cells are more sensitive for cytotoxicity than CHL cells. An additional experiment with 9 MN inducers revealed that the magnitude of MN induction and cytotoxicity were comparable between p53-competent TK6 cells and its p53-null mutant NH32 cells at the same concentrations. Furthermore, the MN frequencies induced by methylmethane sulfonate, aphidicolin and hydroxyurea in NH32 cells were identical to those in TK6 cells at different recovery times. From these results, it is suggested that the p53 abrogation does not explain the difference in sensitivity to MN induction or cytotoxicity between CHL and TK6 cells. In this regard, p53 abrogated NH32 cells can be an option for the in vitro MN assay.

Comparison of four different treatment conditions of extended exposure in the in vitro micronucleus assay using TK6 lymphoblastoid cells.

In the OECD Guideline 487, a total of four extended exposure treatment conditions are proposed for the in vitro micronucleus (MNvit) assay in the presence and absence of a cytokinesis block and with or without a recovery period. This guideline also states that rodent cell lines and human lymphocytes can be used as shown by many validated studies but that human cell lines such as TK6 and HepG2 are not yet validated. In this present study each extended exposure condition was characterized by investigation using TK6 cells and nine chemicals known to be able to induce micronucleus (MN) in rodent cell lines. The results revealed two concerns: six chemicals did not show significant MN induction in the 'cytokinesis block without recovery period'; two aneugens showed no dose-dependent cytotoxicity in the 'cytokinesis block with recovery period'. Further investigation revealed that 3-4 times higher spontaneous MN frequency than that in the other conditions is a possible reason for the low sensitivity, and this high spontaneous MN frequency was not observed in Chinese hamster lung cells under the identical treatment condition. With regard to the two conditions without cytokinesis block, two negative substances were evaluated and found to be negative, suggesting the validity of the TK6 test system for these conditions. Although our findings showed a few concerns for the treatment with cytokinesis block, the TK6 cells were considered to be a reliable cell line to be used for detecting potential inducers of MN in the in vitro micronucleus assay based on the overall results.

An in vitro micronucleus assay with size-classified micronucleus counting to discriminate aneugens from clastogens.

In the in vitro micronucleus (MN) assay, genotoxic chemicals can be characterized as aneugens and clastogens by the presence and absence of kinetochore protein or centromere regions in the micronuclei, respectively. Aneugens preferentially induce kinetochore- or centromere-positive micronuclei which can be detected by the immunofluorescence staining method or the fluorescence in situ hybridization (FISH) method. Both methods are robust and reliable; however, these assays require a definite period of time and cost to obtain a result that suggests that the genotoxic chemicals cause aneuploidy. This is why these methods are not adequate to evaluate dozens of chemicals which are mixtures of aneugens and clastogens. To evaluate a batch of chemicals, a quicker and more convenient assay is desirable. In the present study, we examined whether the size-classified counting of MN is as effective as the FISH method to characterize aneuploidy in the in vitro MN assay using Chinese hamster lung (CHL) cell lines. As aneugens, 9 substances (colcemid, vincristine sulfate, paclitaxel, thiabendazole, diethylstilbestrol, griseofulvin, bisphenol A, fisetin and okadaic acid) were used; as clastogens 6 substances (methylmethane sulfonate, N-methyl-N'-nitro-N-nitroso-guanidine, etoposide, mitomycin C, hydroxyurea and actinomycin D) were used. The size-classified counting revealed that all the 9 aneugens increased both the frequency and proportion of large-size MN as compared with the vehicle control. Although N-methyl-N'-nitro-N-nitroso-guanidine, etoposide and mitomycin C increased the frequency, no increase was observed in the proportion. Meanwhile, with the FISH method, all the aneugens induced centromere-positive micronuclei but the clastogens did not. Based on these results, it is considered that the frequency of large-size MN in the in vitro MN assay is an alerting index for aneugenic effects and that its proportion is a simple and reliable index which is as effective as the FISH analysis for discrimination of aneugens from clastogens.

Mutations induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in the lacZ and cII genes of Muta Mouse.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) found in chewing tobacco, snuff, cigarettes, and cigars is a tobacco-specific nitrosamine and classified as a possible human carcinogen (Class 2B) by the International Agency for Research on Cancer (IARC). NNK given intraperitoneally was seen to induce lung and liver adenomas. To evaluate the genotoxicity of NNK in vivo, NNK was intraperitoneally administered to Muta Mouse at two concentrations (125 and 250 mg/kg, once a week for 4 weeks) followed by the measurement of mutant frequencies in the lacZ and cII genes from lung and liver in the same mice. Characterization of the types of the mutation was determined by sequencing the cII genes from mutant plaques. The mutant frequencies in both target genes from both organs dose-dependently increased up to 10 times compared to those of the control group. For the types of mutations, the ratio of the G:C to A:T mutation in the total number of mutants was less than the ratio of A:T to T:A and A:T to C:G transversion, contrary to a previous report. The A:T to T:A transversion was the most highly induced mutation both in the lung and liver cII genes. The increasing rate of mutant frequencies in lung and liver over the vehicle control was 55 and 56 times, respectively, while the increasing rate of G:C to A:T transition was only 1.9 and 2.8 times, respectively. These observations show that NNK predominantly induces DNA adducts leading to A:T to T:A and/or A:T to C:G mutations in the transgene.