API
Event: 1194
Key Event Title
Short name
Increase, DNA DamageBiological Context
| Level of Biological Organization |
|---|
| Molecular |
Cell term
| Cell term |
|---|
| eukaryotic cell |
Organ term
Key Event Components
| Process | Object | Action |
|---|---|---|
| deoxyribonucleic acid | functional change |
Key Event Overview
AOPs Including This Key Event
| AOP Name | Role of event in AOP |
|---|---|
| ER activation to breast cancer | KeyEvent |
| ROS production leading to reproduction decline | KeyEvent |
| Uncoupling of OXPHOS leading to growth inhibition (4) | KeyEvent |
| Increased DNA damage leading to breast cancer | MolecularInitiatingEvent |
| RONS leading to breast cancer | AdverseOutcome |
Stressors
Taxonomic Applicability
Life Stages
Sex Applicability
Key Event Description
DNA nucleotide damage, single, and double strand breaks occur in the course of cellular operations such as DNA repair and replication and can be induced directly and in neighboring “bystander” cells by internal or external stressors like reactive oxygen species, chemicals, and radiation. Ionizing radiation and RONS such as hydroxyl radicals or peroxide can create a range of lesions (a change in molecular structure) in the base of the nucleotide, with guanine particularly vulnerable because of its low redox potential (David, O'Shea et al. 2007). The same stressors can also break the sugar (deoxyribose)-phosphate backbone creating a single strand break. Simultaneous proximal breaks in both strands of DNA form double strand breaks, which are considered to be more destructive and mutagenic than lesions or single strand breaks. Double strand breaks can generate chromosomal abnormalities including changes in chromosomal number, breaks and gaps, translocations, inversions, and deletions (Yang, Craise et al. 1992; Haag, Hsu et al. 1996; Ponnaiya, Cornforth et al. 1997; Yang, Georgy et al. 1997; Unger, Wienberg et al. 2010; Behjati, Gundem et al. 2016; Morishita, Muramatsu et al. 2016).
However, DNA lesions and single strand breaks can also be destructive and mutagenic. Lesions can lead to point mutations (David, O'Shea et al. 2007) or single strand breaks (Regulus, Duroux et al. 2007). Lesions and single strand breaks can also promote the formation of double strand breaks: replication fork collapse and double strand breaks sometimes occur during mitosis when the replisome encounters an unrepaired single strand break (Kuzminov 2001), and clustered lesions and closely opposed single strand breaks can also form double strand breaks (Chaudhry and Weinfeld 1997; Vispe and Satoh 2000; Shiraishi, Shikazono et al. 2017). Complex damage consists of any combination of closely opposed DNA lesions, abasic sites, crosslinks, single, or double strand breaks in proximity. While classically induced by ionizing radiation, there is also evidence that it can be induced by oxidative activity (Sharma, Collins et al. 2016) or even by a single oxidizing particle (Ravanat, Breton et al. 2014). Complex damage is more difficult to repair (Kuhne, Rothkamm et al. 2000; Stenerlow, Hoglund et al. 2000; Pinto, Prise et al. 2005; Rydberg, Cooper et al. 2005).
DNA damage and resulting repair activity can trigger a halt in the cell cycle, cell death (apoptosis), and cause permanent changes to DNA including deletions, translocations, and sequence changes. DNA damage is also associated with an increase in genomic instability - the new appearance of DNA damage including double strand breaks, mutations, and chromosomal damage following repair of initial damage in affected cells or in clonal descendants or neighbors of DNA damaged cells. The mechanism behind this long term DNA damage is not clear, but telomere erosion appears to play a major role (Murnane 2012; Sishc, Nelson et al. 2015). Genomic instability is more common and longer lasting following complex damage (Ponnaiya, Cornforth et al. 1997), and is influenced by multiple factors including variants in DNA repair genes (Ponnaiya, Cornforth et al. 1997; Yu, Okayasu et al. 2001; Yin, Menendez et al. 2012), RONS (Dayal, Martin et al. 2008), estrogen (Kutanzi and Kovalchuk 2013), caspases (Liu, He et al. 2015), and telomeres (Sishc, Nelson et al. 2015).
How It Is Measured or Detected
DNA damage can be studied in isolated DNA, fixed cells, or living cells. Types of damage that can be detected include single and double strand breaks, nucleotide damage, complex damage, and chromosomal or telomere damage. The OECD test guideline for DNA synthesis Test No. 486 (OECD 1997) detects nucleotide excision repair, so it will reflect the formation of bulky DNA adducts but not the majority of oxidative damage to nucleotides, which is typically repaired via the Base Excision Repair pathway. The OECD test guideline alkaline comet assay Test No. 489 (OECD 2016) detects single and double strand breaks, including those arising from repair as well as some (alkali sensitive) nucleotide lesions including some lesions from oxidative damage. OECD tests for chromosomal damage and micronuclei Test No. 473, 475, 483, and 487 measure longer term effects of DNA damage but these tests require the damaged cell to subsequently undergo replication (OECD 2016; OECD 2016; OECD 2016; OECD 2016). They can therefore reflect a wider range of sources of DNA damage including changes in mitosis. Finally, tests for mutations reveal past DNA damage that resulted in a heritable change, and these are described in the key event ‘Increase in Mutation’.
Many other (non-test guideline) techniques have been used to examine specific forms of DNA damage (Table 1). Double strand breaks are commonly reported because of the significant risk attributed to breaks and the relative ease of detecting and quantifying them. Historically, single and double strand breaks were measured using gel electrophoresis, but are now commonly visualized microscopically using fluorescent or other labeled probes for double and single strand break repair such as H2AX and XRCC2. Base lesions can also be detected using labeled probes for base excision repair enzymes, or by chemical methods such as mass spectroscopy. Refinements on these methods can be used to characterize complex or clustered damage, in which various forms of damage occur in close proximity on a DNA molecule (Lorat, Timm et al. 2016; Nikitaki, Nikolov et al. 2016).
Certain challenges are common to all methods of detecting DNA damage. In the time required to initiate the detection method, some DNA may already be repaired, leading to undercounting of damage. On the other hand, apoptotic DSBs may be incorrectly included in a measurement of direct (non-apoptotic) induction of DSB damage unless controlled. All methods have difficulty distinguishing individual components of clustered lesions, and microscopic methods may undercount disparate breaks that are processed together in repair centers (Barnard, Bouffler et al. 2013). Methods that use isolated DNA (gel electrophoresis, analytical chemistry) are vulnerable to artifacts and must ensure that the DNA sample is protected from oxidative damage during extraction (Pernot, Hall et al. 2012; Barnard, Bouffler et al. 2013; Ravanat, Breton et al. 2014).
Table 1. Common methods of detecting DNA damage
|
Target |
Name |
Method |
Strengths/Weaknesses |
|
Nucleotide damage |
Single cell gel electrophoresis (comet assay) with restriction enzymes (Collins 2004) |
Gel electrophoresis
|
A variant of the comet assay in which restriction enzymes allow the identification of different types of nucleotide damage. The comet assay is more sensitive than PFGE, detecting damage from 0.1 Gy ionizing radiation (Pernot, Hall et al. 2012). A reproducible high-throughput application of the assay is available (Ge, Prasongtanakij et al. 2014; Sykora, Witt et al. 2018), and the test requires only a small (single cell) sample. Requires destruction of the cell. |
|
Nucleotide damage |
Labeled probes including Biotrin OxyDNA and anti- 8-oxoguanine-DNA glycosylase (OGG1) for oxidative damage and AP endonuclease (APE1) for Base Excision Repair of less bulky lesions such as oxidative damage. |
Microscopy, FACS |
Most useful with FACS or other measures of average or relative intensity, as locations and numbers of damaged nucleotides can be difficult to distinguish using fluorescence microscopy. (Ogawa, Kobayashi et al. 2003; Nikitaki, Nikolov et al. 2016). |
|
Nucleotide damage |
High performance liquid chromatography (HPLC), tandem mass spectrometry (MS/MS) |
Analytical chemistry |
Capable of quantifying low levels of specific nucleotide lesions (Madugundu, Cadet et al. 2014; Ravanat, Breton et al. 2014). Requires destruction of the cell. |
|
Nucleotide damage |
Unscheduled DNA synthesis test OECD Test Guideline 486 (OECD 1997) |
Autoradiography |
Measures DNA damage that is repaired using Nucleotide Excision Repair - mostly bulky adducts (OECD (Organisation for Economic Co-operation and Development) 2016). |
|
Non-specific DNA strand breaks |
Single cell gel electrophoresis (comet assay), alkali conditions OECD Test Guideline 489 (OECD 2016) |
Gel electrophoresis |
When used in alkali conditions, the comet assay reveals single and double strand breaks and alkali-sensitive nucleotide lesions. See single cell gel electrophoresis (comet assay) with restriction enzymes above for further comments.
|
|
Single strand breaks |
Labeled probe pXRCC1 (Lorat, Brunner et al. 2015) |
Microscopy |
Fluorescent probes can label single strand breaks in cells, while immunogold labeling is able to distinguish multiple single strand breaks in clusters (Lorat, Timm et al. 2016; Nikitaki, Nikolov et al. 2016). |
|
Double strand breaks |
Single cell gel electrophoresis (comet assay), neutral conditions |
Gel electrophoresis |
Neutral conditions help minimize the release of single strand breaks coiled DNA and alkali lesions, allowing the measurement of double strand breaks. Since single strand breaks can still appear, assay is not very sensitive or specific to double strand breaks (Pernot, Hall et al. 2012). See single cell gel electrophoresis (comet assay) with restriction enzymes above for further comments. |
|
Double strand breaks |
Pulsed field gel electrophoresis (PFGE) |
Gel electrophoresis |
Permits the quantitative measurement of double strand breaks, and can be combined with immunoblotting to detect DNA-associated proteins (Lobrich, Rydberg et al. 1995; Kawashima, Yamaguchi et al. 2017). Considered less sensitive than comet assay, but detected damage from 0.25 Gy ionizing radiation (Gradzka and Iwanenko 2005). Requires destruction of the cell. |
|
Double strand breaks |
Labeled probes including phosphorylated H2AX, 53BP1, Ku70, ATM (Lorat, Brunner et al. 2015) |
Microscopy |
Fluorescent probes can label individual double breaks in cells allowing for quantification, with immunogold labeling resolving breaks in clusters (Lorat, Timm et al. 2016; Nikitaki, Nikolov et al. 2016). Sensitive: detects damage from 0.001 Gy ionizing radiation (Rothkamm and Lobrich 2003; Ojima, Ban et al. 2008). |
|
Chromosomal damage |
Chromosomal aberrations and micronuclei OECD Test Guidelines 473, 475, 483, and 487 (OECD 2016; OECD 2016; OECD 2016; OECD 2016) |
Microscopy |
Detects major DNA damage resulting from large breaks and rearrangements, or mitotic failures. Damage does not appear until DNA undergoes mitosis, so slower and limited to damage in replicating cells. Insensitive tosmall deletions and substitutions. |
Domain of Applicability
Evidence for Perturbation by Stressor
Overview for Molecular Initiating Event
?
Stressors include:
Ionizing radiation
Estrogen
Ionizing Radiation
When ionizing radiation enters a cell and interacts with cellular components including double stranded DNA, it releases energy that leads to DNA damage. This energy ejects electrons from atoms and molecules, and these electrons can produce more electrons, directly ionize DNA, or radiolyze water to form hydroxyl molecules which damage DNA (Hutchinson 1985; Ward 1988; Ravanat, Breton et al. 2014). DNA damage observed after IR includes oxidized base, sugar (deoxyribose), and phosphate lesions, single and double strand breaks, and cross-linking (Ward 1988; Roots, Holley et al. 1990; Haegele, Wolfe et al. 1998; Pouget, Frelon et al. 2002; Rothkamm and Lobrich 2003). DNA damage from IR can occur in a clustered pattern, even from a single particle or photon (Sutherland, Bennett et al. 2002). The type and amount of DNA damage depends on both the quality and dose of radiation. Higher LET radiation such as alpha particles generates more complex clusters of damage including more frequent double strand breaks (Ottolenghi, Merzagora et al. 1997; Rydberg, Heilbronn et al. 2002; Watanabe, Rahmanian et al. 2015; Nikitaki, Nikolov et al. 2016) and other chromosomal abnormalities (Yang, Georgy et al. 1997; Anderson, Stevens et al. 2002), while lower LET radiation (gamma rays, X-rays) generates more oxidized base damage and single strand breaks (Douki, Ravanat et al. 2006).
Damage is also observed in DNA in cells not directly in the path of ionizing radiation, or at a delay following exposure. Indirect or bystander effects are mediated by multiple factors including RONS (Yang, Asaad et al. 2005), TGF-β (Dickey, Baird et al. 2009), and other cytokines (Havaki, Kotsinas et al. 2015). DNA damage following ionizing radiation in directly and indirectly damaged cells is repaired over the first few hours or days (Nikitaki, Nikolov et al. 2016), but long term DNA damage can reoccur as genomic instability weeks, months, or even years after the initial exposure and persist in subsequent generations of cells in vivo (Pazhanisamy, Li et al. 2011; Datta, Suman et al. 2012; Mukherjee, Coates et al. 2012; Snijders, Marchetti et al. 2012) and in vitro (Moore, Marsden et al. 2005; Natarajan, Gibbons et al. 2007; Buonanno, de Toledo et al. 2011; Bensimon, Biard et al. 2016).
Double strand breaks occur linearly with dose between 0.001 Gy (the lowest dose at which an effect has been reliably observed) to over 80 Gy in irradiated cells (Rydberg, Heilbronn et al. 2002; Rothkamm and Lobrich 2003; Yang, Asaad et al. 2005; Asaithamby and Chen 2009). Some low dose studies find a steeper slope between 0.001 and 0.01 Gy for X-rays (although not gamma rays), possibly due to underassessment at higher doses or to a bystander effect superimposed on a linear response (Ojima, Ban et al. 2008; Beels, Werbrouck et al. 2010). Clustered DNA damage also occurs linearly from at least 0.05 Gy (the lowest dose tested) (Sutherland, Bennett et al. 2002), and single strand breaks and alkali sensitive lesions are linear with dose in isolated DNA (Roots, Holley et al. 1990). Chromosomal aberrations appear to be linear or supralinear with dose for low LET radiation (Yang, Georgy et al. 1997; Ryu, Kim et al. 2016) and linear with dose for high LET radiation (Yang, Georgy et al. 1997; Jones, Riggs et al. 2007) at doses examined as low as 0.01 Gy (Schiestl, Khogali et al. 1994; Iwasaki, Takashima et al. 2011). DNA damage measured in bystander cells 1 hour to 3 days after exposure is dose-dependent at low doses (0.001-0.005 Gy), but may approach a maximum between 0.005 and 0.1 Gy (Yang, Anzenberg et al. 2007; Ojima, Ban et al. 2008).
Estrogen
Metabolites created through the oxidative metabolism of estrogens form pro-mutagenic adducts with guanine and adenine. These adducts rapidly undergo depurination leaving abasic sites that can contribute to point mutations or to double strand breaks and further errors if not correctly repaired prior to replication (Cavalieri, Chakravarti et al. 2006; Savage, Matchett et al. 2014; Yager 2015; Yasuda, Sakakibara et al. 2017). The metabolic cycling of the same estrogen metabolites contributes to the formation of ROS, which can oxidatively damage DNA. However, this does not appear to be the major mechanism of DNA damage by estrogen (Cavalieri, Chakravarti et al. 2006). The creation of estrogen metabolites depend on an imbalance in estrogen synthesis and metabolism. DNA damage and mutation is enhanced under conditions that promote estrogen synthesis or inhibit further metabolism including the inactivation of the DNA-damaging metabolites (Cavalieri, Chakravarti et al. 2006; Yager 2015).
Estrogen can also increase double strand breaks through a transcription and replication-dependent mechanism (Stork, Bocek et al. 2016). Estradiol increases double strand breaks and rearrangements at R-loops (RNA-DNA hybrids with an associated single-stranded DNA) formed at ERa-mediated transcription sites. This damage is dependent on Transcription-Coupled Nucleotide Excision Repair and occurs after a delay compared with the ER-independent breaks. This mechanism is a major contributor to overall double strand break formation after estrogen treatment (Stork, Bocek et al. 2016).
Estrogen also affects DNA damage less directly through effects on cell cycle checkpoint regulation and DNA repair mechanisms (Caldon 2014; Li, Chen et al. 2014; Schiewer and Knudsen 2016). It enhances some aspects of the cellular response to DNA damage including enhancing Rad51 recruitment of repair machinery but inhibits others aspects of the response including suppressing multiple regulators of cell cycle checkpoints and delaying complete repair of DSBs (Caldon 2014; Li, Chen et al. 2014). Estrogen also promotes relatively error-prone NHEJ repair mechanisms (Caldon 2014). The net effect promotes survival and replication at the expense of genomic integrity. This effect of estrogen on cell cycle and repair also serves to promote the more direct DNA damaging effects of estrogen, since several of the mechanisms by which estrogen damages DNA require replication before repair is complete (Savage, Matchett et al. 2014; Stork, Bocek et al. 2016). Interestingly, the protection against breast cancer afforded by early parity may at least partially be mediated by a change in the response to estrogen signaling to promote p53 activity and genomic integrity at the cost of proliferation (Jerry, Dunphy et al. 2010).
The effect of estrogen on cell cycle machinery is closely linked with the canonical proliferative effect of estrogen. Since replication can create DNA damage through collapse of replicative forks encountering unrepaired sites to form DSBs, the proliferative effect itself promotes DNA damage even in the absence of other mechanisms.
Regulatory Significance of the Adverse Outcome
DNA damage increases the susceptibility to and probability of subsequent mutations, described in the key event ‘Increase in Mutation’. Mutations can impair the functional capacity of the cell and are an endpoint of regulator significance in their own right.
Multiple guideline toxicity tests exist for DNA damage. The OECD test guideline for DNA synthesis Test No. 486 (OECD 1997) detects nucleotide excision repair, so it will reflect the formation of bulky DNA adducts but not the majority of oxidative damage to nucleotides, which is typically repaired via the Base Excision Repair pathway. The OECD test guideline alkaline comet assay Test No. 489 (OECD 2016) detects single and double strand breaks, including those arising from repair as well as some (alkali sensitive) nucleotide lesions including some lesions from oxidative damage. OECD tests for chromosomal damage and micronuclei Test No. 473, 475, 483, and 487 measure longer term effects of DNA damage but these tests require the damaged cell to subsequently undergo replication (OECD 2016; OECD 2016; OECD 2016; OECD 2016). They can therefore reflect a wider range of sources of DNA damage including changes in mitosis.
References
OECD (1997). Test No. 486: Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo.
OECD (2016). Test No. 473: In Vitro Mammalian Chromosomal Aberration Test.
OECD (2016). Test No. 475: Mammalian Bone Marrow Chromosomal Aberration Test.
OECD (2016). Test No. 483: Mammalian Spermatogonial Chromosomal Aberration Test.
OECD (2016). Test No. 487: In Vitro Mammalian Cell Micronucleus Test.
OECD (2016). Test No. 489: In Vivo Mammalian Alkaline Comet Assay.