| Molecular Initiating Event (MIE) |
Increases in Oxidative DNA damage |
This KE includes:
A broad range of lesions including direct breakage of the phosphate backbone of DNA causing strand breaks Oxidation of nitrogenous bases of the DNA. Notably, oxidative guanine lesions (e.g.,8‐Oxo‐2′‐deoxyguanosine (8‐oxo‐dG), 2,6‐diamino‐4‐hydroxy‐5‐formamidopyrimidine (FapyG)) are the most extensively studied and known to be the most abundant compared to other oxidative base lesions.
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Relative quantification methods
Modified comet assay (Fpg or hOGG1 enzyme digestion of DNA prior to electrophoresis) (Moller et al., 2017) Enzyme‐linked immunosorbant assays (ELISA) (Breton et al., 2003)
32P‐labelling (Collins, 2000) (has generally been replaced with newer assays)
Absolute quantification methods
High performance liquid chromatography coupled to electrochemical detection (HPLC‐EC) (Chepelev et al., 2015) Liquid chromatography (LC) coupled with mass spectrometry (MS) (Collins, 2000)
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| Key Event (KE) |
Inadequate DNA repair |
This KE includes:
Lack of DNA repair (accumulation of unrepaired damage) Incorrect or error‐prone DNA repair (insertion of incorrect base, joining two incorrect ends by NHEJ) Incomplete DNA repair (accumulation of repair intermediates)
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The KE can be inferred from retention of DNA lesions or increase in mutations and chromosomal aberrations (AO1 & AO2) which would indicate lack of repair, incomplete repair, or incorrect repair. Thus, methods of measuring this KE are mostly indirect (e.g., time‐course measurement of DNA lesions following exposure, dose–response experiments).
Direct methods would include assays that measure a cell's capability to repair DNA damage (e.g., introducing fluorescent reporter construct containing a specific lesion [Mao et al., 2011; Chaim et al., 2017; Nagel et al., 2019]).
Models of DNA repair deficiency such as knock‐out cell lines and rodent models (e.g., Agg
−/− mice [Kay et al., 2021]) can be used to determine which pathway is critical for repairing the genotoxic lesion (Olivieri et al., 2020).
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| Key Event (KE) |
Increases in DNA strand breaks |
This KE includes:
Increase in both single strand breaks (SSB) and double strand breaks (DSB) Strand breaks can arise during excision repair (e.g., BER, NER), during replication and transcription (e.g., topoisomerase), or directly due to chemical insult (e.g., collapsed replication fork) Two SSBs in close proximity to each other on opposite strands can lead to a DSB
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Comet assay (neutral and alkaline) (Collins, 2004; Ge et al., 2021; Ngo et al., 2021; OECD, 2014b) γ‐H2AX foci detection methods (e.g., fluorescent immunostaining and detection by flow cytometry, microscopy, in‐cell Western, and ELISA) (Bryce et al., 2016; Garcia‐Canton et al., 2013; Khoury et al., 2013) Pulsed field gel electrophoresis (Kawashima et al., 2017) Transcriptomic biomarkers of DNA damage (e.g., TGx‐DDI biomarker [Li et al., 2017])
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| Adverse Outcome (AO) |
Increases in Mutations |
This KE includes:
Permanent alterations in DNA sequence The alteration may involve a few bases (e.g., single nucleotide variants or small insertions/deletions) or a larger segment of DNA (e.g., incorrect rejoining of DSB, loss or insertion of large fragments)
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Various gene mutation assays (e.g., HPRT, TK, PIG‐A/O assays) (Dobrovolsky et al., 2014; Gollapudi et al., 2015) Transgenic rodent mutation assays (e.g., bacterial transfection with reporter gene, fluorescence tag expression in transgenic mice) (Lambert et al., 2005; OECD, 2020b) PCR methods (Allele‐specific competitive blocker‐polymerase chain reaction [ACB‐PCR] for using mutant‐specific primers to amplify point mutations in the target allele; single molecule PCR) (Banda et al., 2013) Error‐corrected next generation sequencing (Salk & Kennedy, 2020) and similar technologies
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Adverse Outcome (AO)
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Increases in Chromosomal aberrations |
This KE includes:Chromatid‐type and chromosome‐type structural aberrations such as translocations, inversions, rings, and fragmentation.
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Micronucleus detection (scoring by light or fluorescent microscopy and flow cytometry) (Bryce et al., 2014; OECD, 2014a; OECD, 2016b) Chromosomal aberration test (metaphase spread examined by microscopy and aberrations are scored) (OECD, 2016a; OECD, 2016c; OECD et al., 2015) Indirect detection methods such as γH2AX foci and p53 protein expression assays (flow cytometric and in‐cell Western blotting), and fluorescent protein reporter assays for stress signaling pathway activation (Bryce et al., 2016; Hendriks et al., 2012; Khoury et al., 2013)
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