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|
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).
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).
Domain of Applicability
Chromosomal aberrations indicating clastogenicity can occur in any eukaryotic or prokaryotic cell.
Regulatory Significance of the Adverse Outcome
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.
Bryce, S., Bemis, J., Mereness, J., Spellman, R., Moss, J., Dickinson, D., Schuler, M., Dertinger, S. (2014), Interpreting In VitroMicronucleus Positive Results: Simple Biomarker Matrix Discriminates Clastogens, Aneugens, and Misleading Positive Agents, Environ Mol Mutagen, 55:542-555.
Bryce, S., Bernacki, D., Bemis, J., Dertinger, S. (2016), Genotoxic mode of action predictions from a multiplexed flow cytometric assay and a machine learning approach, Environ Mol Mutagen, 57:171-189.
Dertinger, S.D., Camphausen, K., MacGregor, J.T., Torous, D.K., Alasevich, S., Cairns, S., Tometsko, C.R., Menard, C., Muanza, T., Chen, Y., Miller, R.K., Cederbrant, K., Sandelin, K., Ponten, I., Bolcsfoldi, G. (2004), Three-color labeling method for flow cytometric measurement of cytogenetic damage in rodent and human blood, Environ Mol Mutagen, 44:427-435.
Hendriks, G., Ataliah, M., Morolli, B., Calleja, F., Ras-Verloop, N., Huijskens, I., Raamsman, M., Van de Water, B., Vrieling, H. (2012), The ToxTracker assay: novel GFP reporter systems that provide mechanistic insight into the genotoxic properties of chemicals, Toxicol Sci, 125:285-298.
Hendriks, G., Derr, R., Misovic, B., Morolli, B., Calleja, F., Vrieling, H. (2016), The Extended ToxTracker Assay Discriminates Between Induction of DNA Damage, Oxidative Stress, and Protein Misfolding, Toxicol Sci, 150:190-203.\
Khoury, L., Zalko, D., Audebert, M. (2016), Evaluation of four human cell lines with distinct biotransformation properties for genotoxic screening, Mutagenesis, 31:83-96.
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.
Mitelman, F. (1982), Application of cytogenetic methods to analysis of etiologic factors in carcinogenesis, IARC Sci Publ, 39:481-496.
Savage, J.R. (1976), Classification and relationships of induced chromosomal structual changes, J Med Genet, 13:103-122.
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.
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.
Tucker, J.D., Preston, R.J. (1996), Chromosome aberrations, micronuclei, aneuploidy, sister chromatid exchanges, and cancer risk assessment, Mutat Res, 365:147-159.
Wink, S., Hiemstra, S., Huppelschoten, S., Danen, E., Niemeijer, M., Hendriks, G., Vrieling, H., Herpers, B., Van de Water, B. (2014), Quantitative high content imaging of cellular adaptive stress response pathways in toxicity for chemical safety assessment, Chem Res Toxicol, 27:338-355.