Mick D. Fellows

New approaches to advance the use of genetic toxicology analyses for human health risk assessment

Genetic toxicology testing has a crucial role in the safety assessment of new and existing substances of societal value by reducing/eliminating human exposure to potential somatic and germ cell mutagens. Genetic toxicology assays have historically been used in a qualitative manner to arrive at the binary decision of ‘yes’ or ‘no’ with regards to the mutagenic potential. However, the field is currently at a crossroads, with new methods being developed and new proposals being made to use genetic toxicity data in a more quantitative manner. Technological advances have made it possible to perform high-content, high-throughput and high-precision analysis to increase the number of “scored” events leading to increased statistical precision of the endpoint under evaluation. Automated flow cytometry and image analysis are providing significant advantages for the evaluation of gene mutations as well as cytogenetic damage both in vitro and in vivo. In addition, statistical methods such as the benchmark dose (BMD) approach can be used to identify point of departure (PoD) metrics for use in human health risk assessments, including estimation of reference dose (RfD) and margins of exposure (MOE) from in vivo data. Here we provide new data to compare different in vitro micronucleus approaches, observing that the flow based assay performs very well in defining a PoD for methyl methanesulfonate. We also present reanalysis of published in vivo Pig-a gene mutation data, to show how covariate analysis increases precision and reduces the effects of outliers when defining BMD values. Furthermore, we show how in vivo BMD metrics can be used to define RfD values, and then provide comparisons to other human exposure limit values such as permitted daily exposure (PDE). Finally, the principles of empirical correlation using BMD metrics are presented, with methods for derivation of BMD values for endpoint B, when using data from only endpoint A. These developments are opening the possibility of genetic toxicity data being used as an apical endpoint to define negligible risk in human health risk assessments. Expert groups consisting of stakeholders representing academia, industry and the government are now developing guidance on transforming genetic toxicology testing from a qualitative to a quantitative science, keeping in mind the 3R principles of animal welfare.

The spectral karyotype of L5178Y TK⁺/⁻ mouse lymphoma cells clone 3.7.2C and factors affecting mutant frequency at the thymidine kinase (tk) locus in the microtitre mouse lymphoma assay.

There has been much discussion on acceptable spontaneous mutant frequencies in the mouse lymphoma assay (MLA). This culminated in the International Workshop on Genotoxicity Testing (IWGT) recommended control limits for the microtitre version of 50–170 mutants/106 viable cells, which has now been included in the draft Organization for Economic Co‐Operation and Development guideline for assays investigating mammalian cell gene mutation at the tk locus. Some of the factors affecting mutant frequency have been investigated. It was shown that when culturing methotrexate cleansed TK+/− cells, a spontaneous mutant frequency of ∼100 mutants/106 viable cells was achieved after only 26 doublings. However, after further culturing for ∼6 months the spontaneous mutant frequency only gradually increased. Culturing for this time did not affect the karyotype of the cell in so much as the modal chromosome number remained stable. The spontaneous mutant frequency could effectively be manipulated by cleansing with various concentrations of methotrexate. The necessity for using appropriately heat‐inactivated horse serum was confirmed. Finally, following treatment with 4‐nitroquinoline‐N‐oxide, cells did not preferentially survive when plated at high cell densities (1.6 cells plus 2,000 feeder cells/well) versus cells at low density (1.6 cells/well). It was considered that these findings confirm that the dynamics of spontaneous mutant formation in the MLA are complex. However, the karyotype of L5178Y cells is remarkably stable and assuming investigators are using cells with appropriate provenance and good culturing technique, it is clear that the IWGT recommendations are achievable. Environ. Mol. Mutagen. 55:35–42, 2014.

The utility of metabolic activation mixtures containing human hepatic post-mitochondrial supernatant (S9) for in vitro genetic toxicity assessment

In vitro genotoxicity assessment routinely employs an exogenous metabolic activation mixture to simulate mammalian metabolism. Activation mixtures commonly contain post-mitochondrial liver supernatant (i.e. S9) from chemically induced Sprague Dawley rats. Although Organization for Economic Cooperation and Development (OECD) test guidelines permit the use of other S9 preparations, assessments rarely employ human-derived S9. The objective of this study is to review and evaluate the use of human-derived S9 for in vitro genetic toxicity assessment. All available published genotoxicity assessments employing human S9 were compiled for analysis. To facilitate comparative analyses, additional matched Ames data using induced rat liver S9 were obtained for certain highly cited chemicals. Historical human and induced rat S9 quality control reports from Moltox were obtained and mined for enzyme activity and mutagenic potency data. Additional in vitro micronucleus data were experimentally generated using human and induced rat S9. The metabolic activity of induced rat S9 was found to be higher than human S9, and linked to high mutagenic potency results. This study revealed that human S9 often yields significantly lower Salmonella mutagenic potency values, especially for polycyclic aromatic hydrocarbons, aflatoxin B1 and heterocyclic amines (~3- to 350-fold). Conversely, assessment with human S9 activation yields higher potency for aromatic amines (~2- to 50-fold). Outliers with extremely high mutagenic potency results were observed in the human S9 data. Similar trends were observed in experimentally generated mammalian micronucleus cell assays, however human S9 elicited potent cytotoxicity L5178Y, CHO and TK6 cell lines. Due to the potential for reduced sensitivity and the absence of a link between enzyme activity levels and mutagenic potency, human liver S9 is not recommended for use alone in in vitro genotoxicity screening assays; however, human S9 may be extremely useful in follow-up tests, especially in the case of chemicals with species-specific metabolic differences, such as aromatic amines.

Correlation of In Vivo Versus In Vitro Benchmark Doses (BMDs) Derived From Micronucleus Test Data: A Proof of Concept Study

In this study, we explored the applicability of using in vitro micronucleus (MN) data from human lymphoblastoid TK6 cells to derive in vivo genotoxicity potency information. Nineteen chemicals covering a broad spectrum of genotoxic modes of action were tested in an in vitro MN test using TK6 cells using the same study protocol. Several of these chemicals were considered to need metabolic activation, and these were administered in the presence of S9. The Benchmark dose (BMD) approach was applied using the dose-response modeling program PROAST to estimate the genotoxic potency from the in vitro data. The resulting in vitro BMDs were compared with previously derived BMDs from in vivo MN and carcinogenicity studies. A proportional correlation was observed between the BMDs from the in vitro MN and the BMDs from the in vivo MN assays. Further, a clear correlation was found between the BMDs from in vitro MN and the associated BMDs for malignant tumors. Although these results are based on only 19 compounds, they show that genotoxicity potencies estimated from in vitro tests may result in useful information regarding in vivo genotoxic potency, as well as expected cancer potency. Extension of the number of compounds and further investigation of metabolic activation (S9) and of other toxicokinetic factors would be needed to validate our initial conclusions. However, this initial work suggests that this approach could be used for in vitro to in vivo extrapolations which would support the reduction of animals used in research (3Rs: replacement, reduction, and refinement).

Nitroarenes as Antitubercular Agents: Stereoelectronic Modulation to Mitigate Mutagenicity

Nitroarenes are less preferred in drug discovery due to their potential to be mutagenic. However, several nitroarenes were shown to be promising antitubercular agents with specific modes of action, namely, nitroimidazoles and benzothiazinones. The nitro group in these compounds is activated through different mechanisms, both enzymatic and non‐enzymatic, in mycobacteria prior to binding to the target of interest. From a whole‐cell screening program, we identified a novel lead nitrobenzothiazole (BT) series that acts by inhibition of decaprenylphosphoryl‐β‐d‐ribose 2′‐epimerase (DprE1) of Mycobacterium tuberculosis (Mtb). The lead was found to be mutagenic to start with. Our efforts to mitigate mutagenicity resulted in the identification of 6‐methyl‐7‐nitro‐5‐(trifluoromethyl)‐1,3‐benzothiazoles (cBTs), a novel class of antitubercular agents that are non‐mutagenic and exhibit an improved safety profile. The methyl group ortho to the nitro group decreases the electron affinity of the series, and is hence responsible for the non‐mutagenic nature of these compounds. Additionally, the co‐crystal structure of cBT in complex with Mtb DprE1 established the mode of binding. This investigation led to a new non‐mutagenic antitubercular agent and demonstrates that the mutagenic nature of nitroarenes can be solved by modulation of stereoelectronic properties.

In vivo CRISPR-Cas gene editing with no detectable genome-wide off-target mutations

CRISPR-Cas genome-editing nucleases hold substantial promise for human therapeutics1–5 but identifying unwanted off-target mutations remains an important requirement for clinical translation6, 7. For ex vivo therapeutic applications, previously published cell-based genome-wide methods provide potentially useful strategies to identify and quantify these off-target mutation sites8–12. However, a well-validated method that can reliably identify off-targets in vivo has not been described to date, leaving the question of whether and how frequently these types of mutations occur. Here we describe Verification of In Vivo Off-targets (VIVO), a highly sensitive, unbiased, and generalizable strategy that we show can robustly identify genome-wide CRISPR-Cas nuclease off-target effects in vivo. To our knowledge, these studies provide the first demonstration that CRISPR-Cas nucleases can induce substantial off-target mutations in vivo, a result we obtained using a deliberately promiscuous guide RNA (gRNA). More importantly, we used VIVO to show that appropriately designed gRNAs can direct efficient in vivo editing without inducing detectable off-target mutations. Our findings provide strong support for and should encourage further development of in vivo genome editing therapeutic strategies.

In vivo CRISPR editing with no detectable genome-wide off-target mutations

CRISPR–Cas genome-editing nucleases hold substantial promise for developing human therapeutic applications1–6 but identifying unwanted off-target mutations is important for clinical translation7. A well-validated method that can reliably identify off-targets in vivo has not been described to date, which means it is currently unclear whether and how frequently these mutations occur. Here we describe ‘verification of in vivo off-targets’ (VIVO), a highly sensitive strategy that can robustly identify the genome-wide off-target effects of CRISPR–Cas nucleases in vivo. We use VIVO and a guide RNA deliberately designed to be promiscuous to show that CRISPR–Cas nucleases can induce substantial off-target mutations in mouse livers in vivo. More importantly, we also use VIVO to show that appropriately designed guide RNAs can direct efficient in vivo editing in mouse livers with no detectable off-target mutations. VIVO provides a general strategy for defining and quantifying the off-target effects of gene-editing nucleases in whole organisms, thereby providing a blueprint to foster the development of therapeutic strategies that use in vivo gene editing.A strategy developed to define off-target effects of gene-editing nucleases in whole organisms is validated and leveraged to show that CRISPR–Cas9 nucleases can be used effectively in vivo without inducing detectable off-target mutations.

The Lack of Mutagenic Potential of a Guanine-Rich Triplex Forming Oligonucleotide in Physiological Conditions

Triplex forming oligonucleotides (TFOs) bind in the major groove of DNA duplex in a sequence-specific manner imparted by Hoogsteen hydrogen bonds. There have been several reports demonstrating the ability of guanine-rich TFOs to induce targeted mutagenesis on an exogenous plasmid or an endogenous chromosomal locus. In particular, a 30mer guanine-rich triplex forming oligonucleotide, AG30, optimally designed to target the supFG1 reporter gene was reported to be mutagenic in the absence of DNA reactive agents in cultured cells and in vivo. Here, we investigated the mutagenic potential of AG30 using the supFG1 shuttle vector forward mutation assay under physiological conditions. We also assessed the triplex binding potential of AG30 alongside cytotoxic and mutagenic assessment. In a cell free condition, AG30 was able to bind its polypurine target site in the supFG1 gene in the absence of potassium chloride and also aligned with a 5-fold increase in the mutant frequency when AG30 was pre-incubated with the supFG1 plasmid in the absence of potassium prior to transfection into COS-7 cells. However, when we analyzed triplex formation of AG30 and the supFG1 target duplex at physiological potassium levels, triplex formation was inhibited due to the formation of competing secondary structures. Subsequent assessment of mutant frequency under physiological conditions, by pre-transfecting COS-7 cells with the supFG1 plasmid prior to AG30 treatment led to a very small increase (1.4-fold) in the mutant frequency. Transfection of cells with even higher concentrations of AG30 did result in an elevated mutagenic response but this was also seen with a scrambled sequence, and was therefore considered unlikely to be biologically relevant as an associated increase in cytotoxicity was also apparent. Our findings also provide further assurance on the low potential of triplex-mediated mutation as a consequence of unintentional genomic DNA binding by therapeutic antisense oligonucleotides.

The Mouse Lymphoma TK Assay

Abstract The mouse lymphoma L5178Y tk+/− assay (MLA) is capable of identifying both gene mutation and clastogenicity and is included in many regulatory in vitro genotoxicity testing paradigms. L5178Y clone 3.7.2C had been isolated by the 1970s. Being heterozygous at the thymidine kinase locus (tk+/−), forward mutation to tk−/− is identified by growth in the presence of the toxic base analogue trifluorothymidine. The karyotype has been established and cells of known provenance are available from The European Collection of Animal Cell Cultures (ECACC) and The Japanese Collection of Research Bioresources Cell Bank (JCRB). Details of testing methodologies in agar and microwell are discussed in this chapter, along with acceptance and evaluation criteria. When conducted using modern protocols, avoiding nonphysiological conditions, and using cells of good provenance, the MLA is a robust in vitro genotoxicity test that does not seem to be prone to the high number of unexpected positives that have been previously suggested by retrospective reviews.

Abstract 5413: Is a non-conjugated triple-helix forming oligonucleotide targeting the genomic HPRT locus capable of sequence specific mutagenesis

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Oligonucleotide strands have been shown to be capable of binding to duplex DNA in a sequence specific manner to form a triple-helix or triplex structure. Such phenomenons have been shown to induce mutation, as demonstrated using plasmid-based reporter constructs (Wang et al 1996, Science, 271, p802). These studies have engendered concern from the European Medicines Agency (EMEA) in regard to biotechnology derived pharmaceuticals such that antisense oligonucleotides may also form triplex structures in a sequence specific manner at genomic DNA with potentially mutagenic effects. In this study, we have examined the mutagenic potential of a non-conjugated triplex forming oligonucleotide (TFO27) targeting the genomic hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus. The mutagenicity of TFO27 was assessed using the human lymphoblastoid TK6 cell line through 6-thioguanine resistance (6TGr). Electrophoretic mobility shift assays were used to demonstrate triplex formation by TFO27 at the target motif at sub-nanomolar concentrations. As expected, a control oligonucleotide, SCR27, failed to form a triplex at the target motif. Subsequently, a range of transfection facilitators were evaluated for optimal delivery of a fluorescently labelled oligonucleotide probe. In preliminary experiments treating 3×106 cells, TFO27 appeared to produce a dose dependent increase in 6TGr mutants. Furthermore, TFO27 failed to induce mutation at the non-targeted thymidine kinase (TK) locus suggesting locus specificity for its mode of action. Moreover, SCR27 failed to induce mutation at both loci. However, TFO27 failed to induce mutation at the HPRT locus in experiments treating 10×106 cells. Increasing the oligonucleotide concentration had no effect on the number of 6TGr recovered. These contradictory findings are difficult to explain, but emphasise the importance of using appropriate target cell numbers in mutation assays. Overall, our data would suggest that triplex formation could occur in cells but chromatin structure, intranuclear pH, ion concentrations and nucleases are all potential barriers that probably influence the dynamics of triplex formation and stability. In view of these factors, we suggest that studies of the mutagenic potential of triplex formation are better performed at genomic loci rather than extra-chromosomal test systems. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5413. doi:10.1158/1538-7445.AM2011-5413