Key Event Title
|Level of Biological Organization|
Key Event Components
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP|
|Oxidative DNA damage, chromosomal aberrations and mutations||AdverseOutcome|
|Ionizing energy leading to lung cancer||KeyEvent|
|All life stages||High|
Key Event Description
Chromosomal aberrations describe the structural damage to chromosomes that result from breaks along the DNA and may lead to deletion, addition, or rearrangement of sections in the chromosome. Chromosomal aberrations can be divided in two major categories: chromatid-type or chromosome-type depending on whether one or both chromatids are involved, respectively. They can be further classified as rejoined or non-rejoined aberrations. Rejoined aberrations include translocations, insertions, dicentrics and rings, while unrejoined aberrations include acentric fragments and breaks (Savage, 1976). Some of these aberrations are stable (i.e., reciprocal translocations) and can persist for many years (Tucker and Preston, 1996). Others are unstable (i.e., dicentrics, acentric fragments) and decline at each cell division because of cell death (Boei et al., 1996). These events may be detectable after cell division and such damage to DNA is irreversible. Chromosomal aberrations are associated with cell death and carcinogenicity (Mitelman, 1982).
Chromosomal aberrations (CA) refer to a missing, extra or irregular portion of chromosomal DNA. These DNA changes in the chromosome structure may be produced by different double strand break (DSB) repair mechanisms (Obe et al., 2002).
There are 4 main types of CAs: deletions, duplications, translocations, and inversions. Deletions happen when a portion of the genetic material from a chromosome is lost. Terminal deletions occur when an end piece of the chromosome is cleaved. Interstitial deletions arise when a chromosome breaks in two separate locations and rejoins incorrectly, with the center piece being omitted. Duplications transpire when there is any addition or rearrangement of excess genetic material; types of duplications include transpositions, tandem duplications, reverse duplications, and displaced duplications (Griffiths et al., 2000). Translocations result from a section of one chromosome being transferred to a non-homologous chromosome (Bunting and Nussenzweig, 2013). When there is an exchange of segments on two non-homologous chromosomes, it is called a reciprocal translocation. Inversions occur in a single chromosome and involve both of the ends breaking and being ligated on the opposite ends, effectively inverting the DNA sequence.
A fifth type of CA that can occur in the genome is the copy number variant (CNV). CNVs, which may comprise greater than 10% of the human genome (Shlien et al., 2009; Zhang et al., 2016; Hastings et al., 2009), are deletions or duplications that can vary in size from 50 base pairs (Arlt et al., 2012; Arlt et al., 2014; Liu et al., 2013) up into the megabase pair range (Arlt et al., 2012; Wilson et al., 2015; Arlt et al., 2014; Zhang et al., 2016). CNV regions are especially enriched in large genes and large active transcription units (Wilson et al., 2015), and are of particular concern when they cause deletions in tumour suppressor genes or duplications in oncogenes (Liu et al., 2013; Curtis et al., 2012). There are two types of CNVs: recurrent and non-recurrent. Recurrent CNVs are thought to be produced through a recombination process during meiosis known as non-allelic homologous recombination (NAHR) (Arlt et al., 2012; Hastings et al., 2009). These recurrent CNVs, also called germline CNVs, could be inherited and are thus common across different individuals (Shlien et al., 2009; Liu et al., 2013). Non-recurrent CNVs are believed to be produced in mitotic cells during the process of replication. Although the mechanism is not well studied, it has been suggested that stress during replication, in particular stalling replication forks, prompt microhomology-mediated mechanisms to overcome the replication stall, which often results in duplications or deletions. Two models that have been proposed to explain this mechanism include the Fork Stalling and Template Switching (FoSTeS) model, and the Microhomology-Mediated Break-Induced Replication (MMBIR) model (Arlt et al., 2012; Wilson et al., 2015; Lee et al., 2007; Hastings et al., 2009).
CAs can be classified according to whether the chromosome or chromatid is affected by the aberration. Chromosome-type aberrations (CSAs) include chromosome-type breaks, ring chromosomes, marker chromosomes, and dicentric chromosomes; chromatid-type aberrations (CTAs) refer to chromatid breaks and chromatid exchanges (Bonassi et al., 2008; Hagmar et al., 2004). When cells are blocked at the cytokinesis step, CAs are evident in binucleated cells as micronuclei (MN; small nucleus-like structures that contain a chromosome or a piece of a chromosome that was lost during mitosis) and nucleoplasmic bridges (NPBs; physical connections that exist between the two nuclei) (El-Zein et al., 2014). Other CAs can be assessed by examining the DNA sequence, as is the case when detecting copy number variants (CNVs) (Liu et al., 2013).
OECD defines clastogens as ‘any substance that causes structural chromosomal aberrations in populations of cells or organisms’.
How It Is Measured or Detected
Chromosome aberrations are typically measured after cell division.
- Micronucleus detection:
- Micronuclei are DNA fragments that are not incorporated in the nucleus during cell division. Micronucleus induction indicates chromosomal breakage and irreversible damage.
- Traditional (microscopy-based) micronucleus assay; OECD guidelines for both in vivo (#474) and in vitro (#487) testing are available (OECD, 2014; OECD, 2016b)
- In vivo and in vitro flow cytometry-based, automated micronuclei measurements (Dertinger et al., 2004; Bryce et al., 2014)
- High content imaging (Shahane et al., 2016)
- DNA can be stained using fluorescent dyes and micronuclei can be scored in microscope images.
- Chromosomal aberration test
- OECD guidelines exist for both in vitro (#473) and in vivo (#475 and #483) testing (OECD, 2015; OECD, 2016a; OECD, 2016c)
- In vitro, the cell cycle is arrested at metaphase after 1.5 cell cycle following 3-6 hour exposure
- In vivo, the test chemically is administered as a single treatment and bone marrow is collected 18-24 hrs later (#475) while testis is collected 24-48 hrs later (#483). The cell cycle is arrested with a metaphase-arresting chemical (e.g., colchicine) 2-5 hours before cell collection.
- Once cells are fixed and stained on microscope slides, chromosomal aberrations are scored
- Indirect measurement of clastogenicity via protein expression:
- Flow cytometry-based quatification of γH2AX foci and p53 protein expression (Bryce et al., 2016).
- Prediscreen Assay– In-Cell Western -based quantification of γH2AX (Khoury et al., 2013, Khoury et al., 2016)
- Green fluorescent protein reporter assay to detect the activation of stress signaling pathways, including DNA damage signaling including a reporter porter that is associated with DNA double strand breaks (Hendriks et al., 2012; Hendriks et al., 2016; Wink et al., 2014).
|Assay Name||References||Description||OECD Approved Assay|
|Fluorescent In Situ Hybridization (FISH)||
Beaton et al., 2013; Pathak et al., 2017
|Fluorescent assay of condensed chromosomes that can detect CAs through chromosome painting and microscopic analysis||N/A|
|Cytokinesis Block Micronucleus (CBMN) Assay with Microscopy||Fenech, 2000||Cells are cultured with cytokinesis blocked, fixed to slides, and undergo MN quantification using microscopy||Yes (No.487)|
|CBMN with Imaging Flow Cytometry||Rodrigues et al., 2015||Cells are cultured with cytokinesis blocked, fixed in solution, and imaged with flow cytometry to quantify MN||N/A|
|Dicentric Chromosome Assay (DCA)||Abe et al., 2018||Cells are fixed on microscope slides, chromosomes are stained, and the number of dicentric chromosomes are quantified||N/A|
|Array Comparative Genomic Hybridization (aCGH) or SNP Microarray||
Adewoye et al., 2015; Wilson et al., 2015; Arlt et al., 2014; Redon et al., 2006; Keren, 2014; Mukherjee, 2017
|CNVs are detected in single-stranded and fluorescently-tagged DNA using a microarray plate with fixed, known DNA (or SNP) probes; This method, however, is unable to detect balanced CAs, such as inversions||N/A|
|Next Generation Sequencing (NGS): Whole Genome Sequencing (WGS) or Whole Exome Sequencing (WES)||
Liu, 2013; Shen, 2016; Mukherjee, 2017
|CNVs are detected by fragmenting the genome and using NGS to sequence either the entire genome (WGS), or only the exome (WES); Challenges with this methodology include only being able to detect CNVs in exon-rich areas if using WES, the computational investment required for the storage and analysis of these large datasets, and the lack of computational algorithms available for effectively detecting somatic CNVs||N/A|
Domain of Applicability
Chromosomal aberrations indicating clastogenicity can occur in any eukaryotic or prokaryotic cell. However, dose-response curves can differ depending on the cell cycle stage when the DSB agent was introduced (Obe et al., 2002).
Evidence for Perturbation by Stressor
Regulatory Significance of the Adverse Outcome
Abe, Y et al. (2018), “Dose-response curves for analyzing of dicentric chromosomes and chromosome translocations following doses of 1000 mGy or less, based on irradiated peripheral blood samples from five healthy individuals”, J Radiat Res. 59(1), 35-42. doi:10.1093/jrr/rrx052
Adewoye, A.B.et al. (2015), “The genome-wide effects of ionizing radiation on mutation induction in the mammalian germline”, Nat. Commun. 6:66-84. doi: 10.1038/ncomms7684.
Arlt MF, Wilson TE, Glover TW. (2012), “Replication stress and mechanisms of CNV formation”, Curr Opin Genet Dev. 22(3):204-10. doi: 10.1016/j.gde.2012.01.009.
Arlt, MF. Et al. (2014), “Copy number variants are produced in response to low-dose ionizing radiation in cultured cells”, Environ Mol Mutagen. 55(2):103-13. doi: 10.1002/em.21840.
Beaton, L. A. et al. (2013), “Investigating chromosome damage using fluorescent in situ hybridization to identify biomarkers of radiosensitivity in prostate cancer patients”, Int J Radiat Biol. 89(12): 1087-1093. doi:10.3109/09553002.2013.825060
Boei, J.J., Vermeulen, S., Natarajan, A.T. (1996), “Detection of chromosomal aberrations by fluorescence in situ hybridization in the first three postirradiation divisions of human lymphocytes”, Mutat Res, 349:127-135. Doi: 10.1016/0027-5107(95)00171-9.
Bonassi, S. (2008),”Chromosomal aberration frequency in lymphocytes predicts the risk of cancer: results from a pooled cohort study of 22 358 subjects in 11 countries”, Carcinogenesis. 29(6):1178-83. doi: 10.1093/carcin/bgn075.
Bryce, S. et al. (2014), “Interpreting In VitroMicronucleus Positive Results: Simple Biomarker Matrix Discriminates Clastogens, Aneugens, and Misleading Positive Agents”, Environ Mol Mutagen, 55:542-555. Doi:10.1002/em.21868.
Bryce, S. et al.(2016), “Genotoxic mode of action predictions from a multiplexed flow cytometric assay and a machine learning approach”, Environ Mol Mutagen, 57:171-189. Doi: 10.1002/em.21996.
Bunting, S. F., & Nussenzweig, A. (2013), “End-joining, translocations and cancer”, Nature Reviews Cancer.13 (7): 443-454. doi:10.1038/nrc3537
Curtis, C. et al. (2012), “The genomic and transcriptomic architecture of 2,000 breast tumours reveals novel subgroups”, Nature. 486(7403):346-52. doi: 10.1038/nature10983.
Dertinger, S.D. et al.(2004), “Three-color labeling method for flow cytometric measurement of cytogenetic damage in rodent and human blood”, Environ Mol Mutagen, 44:427-435. Doi: 10.1002/em.20075.
El-Zein, RA. Et al. (2014), “The cytokinesis-blocked micronucleus assay as a strong predictor of lung cancer: extension of a lung cancer risk prediction model”, Cancer Epidemiol Biomarkers Prev. 23(11):2462-70. doi: 10.1158/1055-9965.EPI-14-0462.
Fenech, M. (2000), “The in vitro micronucleus technique”, Mutation Research. 455(1-2), 81-95. Doi: 10.1016/s0027-5107(00)00065-8
Griffiths, A. J. F., Miller, J. H., & Suzuki, D. T. (2000), “An Introduction to Genetic Analysis”, 7th edition. New York: W. H. Freeman. Available from: https://www.ncbi.nlm.nih.gov/books/NBK21766/
Hagmar, L. et al. (2004), “Impact of types of lymphocyte chromosomal aberrations on human cancer risk: results from Nordic and Italian cohorts”, Cancer Res. 64(6):2258-63.
Hastings PJ, Ira G & Lupski JR. (2009), “A microhomology-mediated break-induced replication model for the origin of human copy number variation”. PLoS Genet. 2009 Jan;5(1): e1000327. doi: 10.1371/journal.pgen.1000327.
Hendriks, G. et al. (2012), “The ToxTracker assay: novel GFP reporter systems that provide mechanistic insight into the genotoxic properties of chemicals”, Toxicol Sci, 125:285-298. Doi: 10.1093/toxsci/kfr281.
Hendriks, G. et al. (2016), “The Extended ToxTracker Assay Discriminates Between Induction of DNA Damage, Oxidative Stress, and Protein Misfolding”, Toxicol Sci, 150:190-203. Doi: 10.1093/toxsci/kfv323.
Keren, B. (2014),”The advantages of SNP arrays over CGH arrays”, Molecular Cytogenetics.7( 1):I31. Doi: 10.1186/1755-8166-7-S1-I31.
Khoury, L., Zalko, D., Audebert, M. (2016), “Evaluation of four human cell lines with distinct biotransformation properties for genotoxic screening”, Mutagenesis. 31:83-96. Doi: 10.1093/mutage/gev058.
Khoury, L., Zalko, D., Audebert, M. (2013), “Validation of high-throughput genotoxicity assay screening using cH2AX in-cell Western assay on HepG2 cells”, Environ Mol Mutagen, 54:737-746. Doi: 10.1002/em.21817.
Lee JA, Carvalho CM, Lupski JR. (2007). “Replication mechanism for generating nonrecurrent rearrangements associated with genomic disorders”, Cell. 131(7):1235-47. Doi: 10.1016/j.cell.2007.11.037.
Liu B. et al. (2013). “Computational methods for detecting copy number variations in cancer genome using next generation sequencing: principles and challenges”, Oncotarget. 4(11):1868-81. Doi: 10.18632/oncotarget.1537.
Mitelman, F. (1982), “Application of cytogenetic methods to analysis of etiologic factors in carcinogenesis”, IARC Sci Publ, 39:481-496.
Mukherjee. S. et al. (2017), “Addition of chromosomal microarray and next generation sequencing to FISH and classical cytogenetics enhances genomic profiling of myeloid malignancies, Cancer Genet. 216-217:128-141. doi: 10.1016/j.cancergen.2017.07.010.
Obe, G. et al. (2002), “Chromosomal Aberrations: formation, Identification, and Distribution”, Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis. 504(1-2), 17-36. Doi: 10.1016/s0027-5107(02)00076-3.
Savage, J.R. (1976), “Classification and relationships of induced chromosomal structual changes”, J Med Genet, 13:103-122. Doi: 10.1136/jmg.13.2.103.
OECD. (2016a), “In Vitro Mammalian Chromosomal Aberration Test 473.”
OECD. (2016b), “Test No. 474: Mammalian Erythrocyte Micronucleus Test. OECD Guideline for the Testing of Chemicals, Section 4.”Paris: OECD Publishing.
OECD. (2016c). Test No. 475: Mammalian Bone Marrow Chromosomal Aberration Test. OECD Guideline for the Testing of Chemicals, Section 4. Paris: OECD Publishing.
OECD. (2015). Test No. 483: Mammalian Spermatogonial Chromosomal Aberration Test. Paris: OECD Publishing.
OECD. (2014). Test No. 487: In Vitro Mammalian Cell Micronucleus Test. Paris: OECD Publishing.
Pathak, R., Koturbash, I., & Hauer-Jensen, M. (2017), “Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization (mFISH) and Spectral Karyotyping (SKY) in Irradiated Mice”, J Vis Exp(119). doi:10.3791/55162.
Redon, R. et al. (2006), “Global variation in copy number in the human genome”, Nature. 444(7118):444-54. 10.1038/nature05329.
Rodrigues, M. A., Beaton-Green, L. A., & Wilkins, R. C. (2016), “Validation of the Cytokinesis-block Micronucleus Assay Using Imaging Flow Cytometry for High Throughput Radiation Biodosimetry”, Health Phys. 110(1): 29-36. doi:10.1097/HP.0000000000000371
Shahane S, Nishihara K, Xia M. (2016), “High-Throughput and High-Content Micronucleus Assay in CHO-K1 Cells”, In: Zhu H, Xia M, editors. High-Throughput Screening Assays in Toxicology. New York, NY: Humana Press. p 77-85.
Shen.TW, (2016),”Concurrent detection of targeted copy number variants and mutations using a myeloid malignancy next generation sequencing panel allows comprehensive genetic analysis using a single testing strategy”, Br J Haematol. 173(1):49-58. doi: 10.1111/bjh.13921.
Shlien A, Malkin D. (2009), “Copy number variations and cancer”, Genome Med. 1(6):62. doi: 10.1186/gm62.
Tucker, J.D., Preston, R.J. (1996), “Chromosome aberrations, micronuclei, aneuploidy, sister chromatid exchanges, and cancer risk assessment”, Mutat Res, 365:147-159.
Wilson, TE. et al. (2015), “Large transcription units unify copy number variants and common fragile sites arising under replication stress”, Genome Res. 25(2):189-200. doi: 10.1101/gr.177121.114.
Wink, S. et al. (2014), “Quantitative high content imaging of cellular adaptive stress response pathways in toxicity for chemical safety assessment”, Chem Res Toxicol, 27:338-355.
Zhang N, Wang M, Zhang P, Huang T. 2016. Classification of cancers based on copy number variation landscapes. Biochim Biophys Acta. 1860(11 Pt B):2750-5. doi: 10.1016/j.bbagen.2016.06.003.