2022 Volume 97 Issue 6 Pages 261-269
Chromosomal damage occurs both endogenously and exogenously and is a crucial factor in the induction of carcinogenesis. Chemically induced chromosomal damage is mainly exogenous. The OECD has developed methods to detect chemicals that induce chromosomal damage so as to identify hazardous substances and limit their exposure to humans. The development and improvement of in vitro mammalian cell methods have been the focus of recent research, as these techniques have higher throughput than in vivo animal methods and are cruelty-free. In vitro mammalian cell methods are highly sensitive and widely used. Nevertheless, they have a high frequency of misleading positive test results, causing the wastage of vital raw materials and pharmaceutical agents, and necessitating additional in vivo animal tests. Therefore, the improvement of in vitro mammalian cell methods is required. Novel methodologies have been proposed and developed for robust animal-free evaluation. As they include omics and AI approaches that use big data, they may enable objective, multidirectional interpretation when applied in combination with current in vitro experimental techniques. We review the existing approaches toward improving chromosome damage detection and introduce innovative techniques that facilitate animal-free testing. The current and latest evaluation methods can support the protection of public health as well as the development of promising chemicals that enrich our lives.
Gene mutations and chromosomal breaks cause genome rearrangements in cancer cells (Kasparek and Humphrey, 2011). Chromothripsis is induced by micronuclei and involves genome rearrangements (Zhang et al., 2013, 2015). Endogenous and exogenous chromosomal damage, breaks and aberrations trigger micronuclei and may initiate carcinogenesis (Bonassi et al., 2004).
According to an epidemiological survey, dietary habits, tobacco and alcohol consumption, occupational exposure to various chemicals, and lifestyle are potential carcinogenic risk factors (Wynder and Gori, 1977). Exposure to various substances such as carcinogenic compounds may induce exogenous chromosomal breaks. Chemical agents that damage genetic information by causing mutations (e.g., gene mutation and chromosomal aberrations or damage) are defined as “genotoxic” or “mutagenic.” Strictly speaking, substances that damage chromosomes are “clastogenic” (Phillips and Arlt, 2009). In general, carcinogenic compounds may have a genotoxic or epigenetic (non-genotoxic) mode of action. The former may induce carcinogenicity even at low exposure levels (no threshold). By contrast, non-genotoxic carcinogens have controllable exposure thresholds (Nohmi, 2018). Accurate detection and identification of genotoxic chemicals is vital to avoid exposures that could result in genotoxicity-induced carcinogenicity and to reduce cancer risk.
The Organisation for Economic Co-operation and Development (OECD) has published official test guidelines (TGs) for detecting genotoxic chemicals (Table 1). They consist of in vitro methods (cell-based tests) and in vivo methods (animal-based tests). In vitro methods such as chromosomal aberration tests (TG473) (OECD, 2016b) and micronucleus tests (TG487) (OECD, 2016d) using mammalian and human cell lines have been used to detect clastogens that induce chromosomal damage. These methods are well established, have high throughput, and are animal cruelty-free compared to in vivo methods such as in vivo micronucleus tests using mice (TG474) (OECD, 2016c). Hence, they have been widely used in combinations (test batteries) to elucidate the mechanisms of different genotoxic compounds (Eastmond et al., 2009). A combination of a bacterial gene mutation assay (Ames test, TG471) and an in vitro micronucleus test (TG487) has been used for the highly sensitive detection of rodent carcinogens and in vivo genotoxins (Kirkland et al., 2011; Morita et al., 2016). However, this methodology sometimes generates misleading positives, possibly by provoking physiological irritation. Accurately detecting chemically induced chromosomal damage is a research priority in genetic toxicology.
| Endpoint | In vitro | In vivo |
|---|---|---|
| Gene mutation | TG471: Ames test (bacterial reverse mutation assay) | TG488: Transgenic rodent gene mutation assay |
| TG476: HPRT assay | ||
| TG490: Mouse lymphoma assay and TK6 assay | ||
| Chromosomal aberration | TG473: Chromosomal aberration test | TG474: Mammalian erythrocyte micronucleus test |
| TG487: Micronucleus test | ||
| TG490: Mouse lymphoma assay | TG475: Mammalian bone marrow chromosomal aberration test |
Recently, in silico evaluation methods have attracted research attention as they are cost-effective, rapid and cruelty-free. The regulatory aspects of using non-animal approaches and new approach methodologies (NAMs) integrated with in silico technologies were reported recently (European Chemicals Agency, 2017; Interagency Coordinating Committee on the Validation of Alternative Methods, 2018). The definition of the relevant terms slightly differs between regulatory agencies. Hence, NAMs refer to any non-animal-based risk assessment approach. Regulatory acceptance of these methods is low thus far. However, certain technologies have gradually been accepted and adopted for genotoxin detection. Quantitative structure–activity relationships (QSARs) are based on the associations between molecular properties and toxicity (structure–activity relationships [SARs]). QSARs have been implemented for decades. More recently, they have been used to evaluate impurities in pharmaceutical agents (International Council on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, 2013) and pesticide residues (European Food Safety Authority, 2016). Thus, in silico methods could effectively screen and predict causal factors using relationships between compound structures and genotoxicity mechanisms. Currently, in silico evaluation is applied mainly to detect bacterial mutagenicity (ICH, 2013). Probably, in silico models could also effectively evaluate chemically induced chromosomal damage.
In the present review, we focus on the methodologies and issues of the current genotoxicity detection methods and on strategies to improve the current methods, their biological relevance, and also the application of new methodologies to evaluate chemically induced chromosomal damage and DNA breaks that can lead to mutations and chromosome rearrangements. The current and new methods clarify genotoxic mechanisms and reflect in vivo conditions without actual animal tests. They enable the detection of chemicals that induce chromosomal instability and the screening of other relatively safe substances that can improve human quality of life and protect public health. This review also evaluates the various available techniques for detecting chemically induced chromosomal damage.
Misleading (irrelevant) positives have often been reported for in vitro mammalian cell tests (Kirkland et al., 2007). Additional in vivo tests are usually recommended to determine whether a positive in vitro test result truly indicates that the target compound is genotoxic (Eastmond et al., 2009). The inaccuracy of certain in vitro tests may necessitate undesirable in vivo tests. Animal tests are not permitted for cosmetic products or their raw materials according to EU regulations (The European Parliament and the Council of the European Union, 2003, 2009). Therefore, the difficulty in distinguishing between true and misleading positives impedes any further development of potentially useful chemicals for cosmetic applications. Certain countermeasures have been implemented, including the improvement of in vitro tests, pre-screening of misleading positives among all positive results, and follow-up evaluation of positive results, as shown in Fig. 1.
Countermeasures for avoiding misleading positives. (i) Improvement of in vitro tests, (ii) pre-screening of misleading positives among all positive results, and (iii) follow-up evaluation of positive results. TG: test guidelines; RS-MN: 3D reconstructed human skin micronucleus; ITS: Integrated Testing Strategy; MoA: mode of action; IATA: Integrated Approaches to Testing and Assessment.
Misleading positives may be caused by in vitro-specific conditions, such as very toxic conditions, inadequate exposure level and lack of a DNA repair system in the cell line. Therefore, the application of new cytotoxicity measures that take into account inhibition of the cell cycle, optimization of the top concentration for exposure, and the use of DNA repair system-competent cell lines are considered to improve the testing methods. Thus, several studies endeavored to identify appropriate cytotoxicity measures, cell types and top concentrations to reduce misleading positives of in vitro mammalian tests (Lorge et al., 2008; Fowler et al., 2012a). Based on the results from such studies, the OECD has updated its TGs. The revised guidelines may be more effective at reducing misleading positives (Fowler et al., 2012a; Morita et al., 2014; Fujita et al., 2016b, 2016c; Chapman et al., 2021). This section introduces the three measures (cytotoxicity, top concentration and p53 competency) in the new OECD guidelines.
Cytotoxicity indicesRetrospective evaluation showed that experimental conditions that potentially induce severe cytotoxicity could cause misleading positives (Kirkland et al., 2005; Morita et al., 2012). The OECD has developed cytotoxicity indices, including cell cycle delay, relative increase in cell count (RICC) and relative population doubling (RPD), instead of relative cell counts (RCC) (OECD, 2016d). These new indices may estimate cytotoxicity more reliably than RCC as they consider cell division and cell cycle arrest. The cytotoxicity indices are represented by Eqs. (1), (2), (3) below:
Relative cell count (RCC)
| (1) |
Relative increase in cell count (RICC)
| (2) |
Relative population doubling (RPD)
| (3) |
Fowler et al. (2012a) evaluated the new indices on known chemicals and reported that they effectively lowered the incidence of misleading positives compared to the previously adopted cytotoxicity indices.
Top concentrationTo reduce misleading positives resulting from unphysiological cell culture conditions (low pH, high toxicity) caused by high chemical exposure (Morita et al., 2012), the OECD changed the optimal top concentrations from ‘10 mM or 5 mg/ml’ to ‘10 mM or 2 mg/ml’ (whichever is lower) in the current OECD test guidelines. Morita et al. (2012, 2014) retrospectively analyzed previously reported data and assessed the effects of the new guidelines on the sensitivity (no. of true genotoxins/no. of tested positive chemicals) and specificity (no. of non-genotoxins/no. of tested negative chemicals) of rodent carcinogen detection. The authors found that the new guidelines influenced neither parameter but slightly decreased the number of misleading positives. Hence, the new guidelines are considered more appropriate (Morita et al., 2014).
p53 competencyThe p53 protein is a tumor suppressor that participates in cell division, DNA repair and apoptosis. Since most positive results have been obtained using p53-defective rodent cell lines in genotoxicity tests, p53 deficiency may have led to misleading positives. Fowler et al. (2012b) compared the results for rodent cell lines (V79, CHL, CHO) against those for p53-competent human cells (human peripheral blood lymphocytes, TK6, HepG2). The p53-defective cells were more susceptible to cytotoxicity than the p53-competent cells. Although both cell types were equally sensitive in terms of genotoxin detection, the p53 deficiency is probably an important factor underlying misleading positive results because it can be a trigger of cytotoxicity. Consequently, the OECD addressed p53 status in their current TGs (OECD, 2016d).
Pre-screening of misleading positives among all positive test resultsThe updated OECD test guidelines (OECD, 2014) with revised cytotoxicity indices, top concentration and p53 competency (cell types) are intended to reduce misleading positives. Therefore, chemicals that were deemed genotoxic according to previous guidelines should be re-evaluated. However, re-testing problematic chemicals with potentially misleading positive results is time-consuming. Hence, a retrospective evaluation of previous test results is required.
Retrospective evaluation of positive results in older in vitro evaluation methodsMorita et al. (2014) used a comprehensive retrospective evaluation to assess the current top concentration and reported a slight reduction in misleading positives but stable sensitivity. However, it was thought that the impact of changes in cytotoxicity and p53 status could be assessed using data from previous tests. To estimate the cytotoxicity of previously evaluated compounds using the new TGs, Fujita et al. (2016b) developed transformation formulas from RCC (previous index) to RICC (present index) using D = doubling time and E = experimental time:
| (4) |
| (5) |
When Chinese hamster lung (CHL/IU) cells, which are typically used for chromosomal aberration tests and micronucleus tests (D = 15 h), are used and the experimental time is 24 h, Eqs. (4) and (5) become Eqs. (6) and (7), respectively:
| (6) |
| (7) |
Greenwood et al. (2004) calculated population doubling (PD), which can transform RCC into RPD. They could estimate the initial number of cells from the final number of cells using control cultures or published data. In practice, however, it is difficult to obtain raw cell number data as they are not usually provided in the reports. Fujita et al. (2016b) reported that only D and E were required to transform RCC to RICC/RPD. Thence, they developed formulas to estimate RICC/RPD from RCC in the absence of cell count data. If positive results occurred only when severe cytotoxicity was detected (RCC < 60), then the results could change from positive to negative under the current test condition (Table 2; modified from Fujita et al. (2016b)). Of the 14 chemicals expected to return negative test results according to the transformation formulas, 11 were confirmed negative after they were re-tested under the current guidelines (Fujita et al., 2016a, 2016c). Thus, cytotoxicity is an important parameter for accurate in vitro mammalian cell genotoxicity testing.
| Cytotoxicity measurement*1 | RICC (%) | RPD (%) | eRCC (%)*3 from RICC (%) | eRCC (%)*3 from RPD (%) |
|---|---|---|---|---|
| Suitable cell viability*2 | 40–50 | 40–50 | 60–66 | 51–57 |
| Excessive cell viability*2 | < 40 | < 40 | < 60 | < 51 |
*1CHL/IU cells; doubling time = 15 h; experimental time = 24 h; *2cell viability: RICC, RPD or eRCC; *3eRCC: estimated RCC.
Cytotoxicity transformation formulas objectively assess the actual cytotoxicity of previous positives and estimate outcomes under new TGs, and they are useful in safety evaluation (Honda et al., 2018). The International Agency for Research on Cancer (IARC) (IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2019) and the Flavor and Extract Manufacturers Association (FEMA) Expert Panel (https://www.femaflavor.org/gras) have cited our chemical genotoxicity evaluation approach and conclusions. They concur that cytotoxicity is an essential factor in reviewing previous test data (Gooderham et al., 2020). Yamamura et al. (2018) used the foregoing transformation formulas for the accurate estimation of in vitro exposure levels in past data under current test guidelines. They successfully compared in vivo and in vitro exposure levels that were heretofore unreported. It is hoped that the regulatory acceptance of these retrospective evaluation methods as NAMs will increase over time.
In vivo animal tests have traditionally been the standard for verifying misleading positives from in vitro mammalian cell tests. However, new in vitro methods attempt to account for species differences, modes of action and in vivo exposure conditions. Ellinger-Ziegelbauer et al. (2009) evaluated the utility of toxicogenomic analyses by examining gene expression profiles in TK6 cells exposed to genotoxins. The authors distinguished compounds that induce DNA adducts and double-strand breaks from those that interfere with mitotic spindle function or induce cytotoxicity by analyzing the expression of 47 genes, including p53 target genes (e.g., CDKN1A). Buick et al. (2020) applied human hepatoma-derived (HepaRG) cells to an in vitro micronucleus test. HepaRG cells synthesize phase I and II metabolic enzymes as key metabolic enzymes in vivo. The authors devised the transcriptome-based assay TGx-DDI, which distinguishes chemicals that induce chromosomal damage from those that do not, using the expression of 64 biomarker genes (including p53 target genes) which were previously developed using TK6 cells (Li et al., 2015). Doktorova et al. (2014) conducted a toxicogenomic assay on triclosan using HepaRG cells. Triclosan was tested positive in an in vitro chromosomal aberration test using V79 cells but negative in a micronucleus test using mice (bone marrow) (Scientific Committee on Consumer Safety, 2011). The authors demonstrated that triclosan does not react with DNA, and in vitro methods such as toxicogenomics can effectively follow up in vitro positives. As a meta-analysis identified 32 genomic signatures (gene expression) of in vivo genotoxicity as general signatures to discriminate between genotoxic and non-genotoxic chemicals and compare in vivo outcomes (Auerbach, 2016), these in vivo signatures can be useful to compare the results from in vitro methods and interpret misleading positives. Other omics technologies such as toxicoproteomics have been employed to interpret positive results in genotoxicity evaluations on cell lines, and protein markers of oxidative stress were identified to discriminate misleading positives in continuous treatment conditions (Yasui et al., 2021). Thus, these new technologies could discriminate partly genotoxic substances from misleading positives based on the underlying molecular mechanisms.
For the cosmetics field, to consider the exposure route, 3D skin-based mammalian cell assays have been developed and validated. They presented 80% accuracy in their in vivo genotoxicity results (Pfuhler et al., 2021a) and were used to follow up on in vitro mammalian cell test positives (Pfuhler et al., 2021a, 2021b). In vitro tests, using human cell lines, that consider the in vivo exposure conditions in tandem with other in vitro biological assays, can follow up and help interpret in vitro positives without in vivo animal testing. Therefore, omics technologies and 3D models that consider modes of action and in vivo exposure conditions (e.g., considering exposure routes and using cells that synthesize in vivo-like metabolic enzymes) may suffice to clarify and interpret in vitro testing data and perform reliable genotoxicity evaluations. ToxTracker is an accurate genotoxicity assay that works by detecting green fluorescent protein (GFP) reporters for key proteins (e.g., Rtkn-GFP can detect DNA double-strand breaks) (Hendriks et al., 2012, 2016). It detects DNA damage, oxidative stress and protein damage, and can help to interpret misleading positives.
Although these methodologies should be useful for follow-up of misleading positives, there are still several limitations; for example, the in vivo ADME (adsorption, distribution, metabolism and excretion) conditions are hard to replicate in in vitro systems. Thus, further investigations for in vivo–in vitro extrapolation are required to achieve a robust assessment for misleading positives.
Micronucleus tests detect aneugenicity and clastogenicity as micronuclei (OECD, 2014). Aneugenicity, which is mostly induced by tubulin-binding chemicals (Lynch et al., 2019), is considered a cause of genetic disorders, cancer and infertility (Ben-David and Amon, 2019; Pacchierotti et al., 2019). ICH guidelines state that aneugenicity exhibits a nonlinear dose–response and may be used to establish the exposure threshold (ICH, 2011). An OECD test guideline (TG487) recommends the detection of kinetochores by anti-kinetochore antibody immunostaining (CREST staining) or by fluorescence in situ hybridization (FISH) with centromeric/telomeric probes to confirm whether the micronuclei originate from clastogenic and/or aneugenic events (OECD, 2016d). This information helps to confirm the mechanisms and define the exposure thresholds of promising pharmaceutical agents. In the future, new in silico prediction methods with structure alerts for aneugenicity are expected. In addition, application of the newly introduced Integrated Approaches to Testing and Assessment (IATA) is also desirable to re-consider the risk for human health of aneugens as well as non-DNA-reactive clastogens.
Several in silico methods have been developed to predict gene mutation endpoints, such as the Ames test (Benigni et al., 2020; Honma, 2020). As these tools are considered reliable and acceptable, they have been adopted to assess the genotoxicity of impurities in pharmaceutical agents and to reduce potential carcinogenic risk (ICH, 2013). In silico methods have also been developed to detect chromosomal aberrations, but they have not yet been used by regulatory agencies because the mechanisms that induce chromosomal aberrations are more complex than those that induce mutations. In practice, however, the prediction performance of certain in silico assays is adequate (Morita et al., 2019) but nonetheless inferior to that of in silico models for the Ames test (Tcheremenskaia and Benigni, 2021). In vitro and in vivo test data have been used as training datasets in model development. Therefore, the models might learn and incorporate previous misleading positive results and incorrectly predict toxicity (Hasselgren et al., 2019; Morita et al., 2019; Benigni et al., 2020). Canipa et al. (2016) evaluated the prediction performance of existing in vitro structural alerts by applying previous in vivo test results. The authors updated 32 in vitro structural alerts that cover in vivo mechanisms. Fujita et al. (2020) attempted to update datasets by separating true from misleading positives in vitro. They focused on cytotoxicity effects and adopted regularized regression models to improve the quality of the datasets. They developed a novel in silico method that identifies target compounds that could generate misleading positives under test conditions.
IATA is a strategic framework that was recently introduced for chemical toxicity evaluation (OECD, 2016a). IATA incorporates and integrates toxicity, physicochemical, pharmacokinetic and bioactivity data for target chemicals (Tcheremenskaia and Benigni, 2021). IATA for carcinogenicity detection was developed using genotoxicity testing results and chemical properties (Petkov et al., 2016; Fujita et al., 2019). Fujita et al. (2019) developed an Integrated Testing Strategy (model) for carcinogenicity evaluation using mutation and chromosomal damage assay data as well as chemical molecular weights and partial chemical structure information. Chromosomal damage was selected as the second most important factor after gene mutation to distinguish carcinogens in the final testing models developed by the random forest method. Hence, the integrated testing model using chromosomal damage information effectively detected carcinogenicity. Petkov et al. (2016) developed an IATA scheme to predict genotoxic carcinogens using both in vivo and in vitro genotoxicity test results that include chromosomal damage information. They showed that chromosomal aberration is an important factor covering all carcinogen mechanisms. Thus, chromosomal aberration is expected to be used to identify genotoxic carcinogens correctly in the IATA approach. Overall, the IATA approach can effectively predict carcinogenicity by using genotoxicity testing results.
A retrospective evaluation of the rodent carcinogen database revealed that the chromosomal aberration test contributed to the high sensitivity of the carcinogenicity evaluation (Morita et al., 2016). Moreover, the chromosomal aberration endpoint may be used to validate a positive bacterial gene mutation assay result and distinguish non-carcinogenic compounds (Fujita et al., 2019). Thus, chromosomal damage could be an important endpoint in detecting genotoxic carcinogens and help to differentiate genotoxicity and carcinogenicity mechanisms.
There is evidence that chromosome abnormalities are related to cancer prognosis and chemotherapy responses (Kou et al., 2020). Mutation signatures associated with cancers have been intensively investigated. Error-corrected sequencing technologies have evolved to a level that can now be used in genotoxicity evaluations (Matsumura et al., 2019; Valentine et al., 2020; You et al., 2020; Abascal et al., 2021). However, these modalities cannot yet detect structural aberrations, including large deletions. Thus, new sequencing technologies that can decipher chromosomal aberrations and genomic instability in carcinogenesis would be required to assess genome safety.
Examining chromosomal damage is becoming important in other areas besides chemical genotoxicity assessment. Genome editing techniques such as CRISPR/Cas9 have been adopted for drug development (Scott, 2018). These genome editing techniques may contribute to the development of novel treatments for hitherto incurable diseases. Nevertheless, Leibowitz et al. (2021) reported that CRISPR/Cas9 treatment may itself induce micronuclei and chromosomal damage that can trigger chromothripsis. To evaluate the safety of a new therapy with genome editing, detecting chromosomal damage and aberrations will be necessary as a quality control to ensure genome safety before chemical treatment. In the future, innovative methodologies, such as IATA, could be applied to assess and contribute to developing novel anticancer/antitumor agents.
The detection of chromosomal damage is vital for the assessment of both genotoxicity and carcinogenicity. Various detection methods have been developed and have contributed to the protection of public health. However, genotoxicity assessment is complex. It must consider environmental, social and corporate governance as well as animal welfare factors. To these ends, extensive research has been conducted, and NAMs have been developed. Much attention has been allocated to omics and AI methods that use big data. We believe that these innovative methodologies combined with existing methods will provide a robust and reliable assessment of genotoxicity, including chromosomal aberrations and carcinogenicity.
Y. F. is a former employee of the Kao Corporation, Tokyo, Japan.
The authors thank Dr. Asako Furukohri for helpful comments on this review.
