AOP ID and Title:

Short Title: DNA damage and metastatic breast cancer

Authors

Dr Usha S Adiga MD PhD, Professor,Department of Biochemistry,KS Hegde Medical Academy,Nitte Deemed to be University,Mangalore,Karnataka,India

 

Status

Abstract

This adverse outcome pathway details the effect of alcohol as a stressor in metastatic breast cancer. Aim of this AOP is intended to detail the linkage between alcohol and miRNA- SIRT-1 axis induced metastatic breast cancer which represents a knowledge gap as there are not many references available. Consecutive KEs identified are as follows.

Acetaldehyde, which is a metabolite of alcohol is considered a major mutagen which has been determined to induce genotoxic effects on DNA  resulting in increased DNA damage. Inadequate DNA crosslink repair mechanisms leads to accumulation of damaged DNA resulting in impaired DNA synthesis leading to mutations and increased miRNA expression ; leads to disruption of  SIRT-1 signalling . This step is followed by increased acetylation and activity of  NFkB ;  loss of estrogen receptor functions  ; molecular alterations of epithelial cells ; gain of mesenchymal cell features ; eventuating in increased invasion and migration of breast cancer cells resulting in Metastatic breast cancer .

Background

Alcoholic beverages are classified by the International Agency for Research on Cancer(IARC) as Group 1 carcinogens. Studies have reported alcohol consumption to be a  risk factor for breast cancer in women(Room R et al, 2005). A woman drinking an average of two units of alcohol per day has an 8% higher risk of developing breast cancer than a woman who drinks an average of one unit of alcohol per day[2]. Alcohol is metabolized by alcohol dehydrogenase to acetaldehyde which is a mutagen. Various theories have been proposed which explain the mutagenicity of alcohol. Among them, the most relevant one for carcinoma of the breast has been proposed by Purohita et al, suggesting an alcohol-induced inactivation of the tumor suppressor gene BRCA1 and increased estrogen Responsiveness in breast tissues(Purohit V et al, 2005). Boffetta and Hashibe list plausible mechanisms of breast cancer as a result of the genotoxic effect of acetaldehyde-induced increased estrogen concentration(Boffetta P et al 2006). It has also been found that alcohol stimulates the epithelial-mesenchymal transition (EMT), because of which there is distant metastasis (Forsyth C. B. et al 2010). However, this mechanism needs to be elucidated in detail.

MicroRNAs (miRNAs) are non-coding, single-stranded RNA molecules that regulate target gene expression via post-transcriptional modifications [Mohr A. M& Mott J. L 2015 and Lai E. C. 2002). Several studies indicated the promising role of miRNA in the diagnosis and outcome prediction in several cancers (Mirzaei H et al 2018 and Liu, S. Y et al 2017). miRNA-21 is upregulated and promotes metastasis in several cancers (Kunita, A et al 2018 and Liu Z et al 2015). A Chinese study by Kunita et al proved that plasma levels of miRNA were up-regulated in large B-cell lymphoma patients (Kunita, A et al 2018). A study by Wang et al also proved that plasma levels of miR were upregulated in large B-cell lymphoma patients in China (Chen et al 2014). Although miR-21 was indicated to play a crucial role in the metastasis of lung cancer, ovarian cancer, and head and neck cancer through several signaling pathways, the molecular mechanism of how miR-21 regulates the EMT process in breast cancer is not clear (Liu S. Y et al, Lopez-Santillan et al 2018, Panagal M. et al 2018, Zhou, B.et al 2018, Brabletz T et al and Ye, X. et al 2017).There are a number of miRNAs which regulate SIRT 1 expression. The epithelial-mesenchymal transition (EMT) is a process that which epithelial cells lose their cell polarity and cell adhesion ability, which will lead to cancer metastasis (Vaziri H et al 2001 and Luo, J et al 2001). Epithelial cells exhibit the property of regular cell-cell contacts, adhesion to the surrounding cellular fabric, preventing the detachment of individual cells. Whereas mesenchymal cells do not form intercellular contacts. 

Sirtuins are nicotinamide adenine dinucleotide (NAD+)–dependent deacetylases that function as intracellular regulators of transcriptional activity (Blander G & Guarente L 2004 and Roth M & Chen W 2014). It plays important roles in cell survival, signal transduction, and cell apoptosis by deacetylating key cell signaling molecules and apoptotic related proteins, such as NF-kB, p53, Ku70, and HIFs (Zhao, W et al 2008 and Chen W & Bhatia R 2013). Various studies have inconclusive reports on the role of SIRT1 in cancer, because of its opposite effects as both a tumor activator or suppressor in various human cancers, including breast cancer.  Deng et al found that the expression of SIRT1 was lower in prostate cancer, bladder cancer, ovarian cancer, and glioblastoma when compared with normal tissues (Han, L et al 2013).On the contrary, it was found that, in leukemia and lung cancer, SIRT1 was significantly higher(Riggio M et al 2012 and Lee M S et al 2015).

This can be explained as follows: SIRT1-mediated deacetylation suppresses the functions of several tumor suppressors including p53, p73, and HIC1, it has been suggested that SIRT1 has a promoting function in tumor development and progression [Pinton G et al 2016, Pillai VB  et al 2014, Wan G et al 2017 and Hwang B et al 2014]. In contrast, SIRT1 may have a suppressive activity in tumor cell growth by suppressing NF-κB, a transcription factor playing a central role in the regulation of the innate and adaptive immune responses and carcinogenesis, the dysregulation of which leads to the onset of tumorigenesis and tumor malignancy(Yuan J et al 2009, Wang R H et al 2008, Chen L F et al 2004 and Greten F R & Karin M 2004). Here, we aim to further explore the role of the SIRT1-NF kB signaling pathway in tumorigenesis of the breast as well as its associated mechanisms.

The nuclear factor-κB (NF- κB)/REL family of transcription factors is comprised of a RELA/p65,c-REL, RELB, p105/NF- κB1 and p100/NF- κB2 (Van Laere S J et al 2007). The p105 and p100 proteins can be processed by proteolytic cleavage into p50 and p52, respectively. Activation of the NF-κB signaling pathway leads to the induction of target genes that can inhibit apoptosis, interaction with cell cycle regulation, cell invasion, contribute to tumorigenesis and metastatic invasion (Shostak K & Chariot 2011). Activation NF-κB in breast cancer is loss of Estrogen Receptor (ER) expression and Human Epidermal Growth Factor Receptor 2 (HER-2) overexpressed via epidermal growth factor receptor (EGFR) and Mitogen-Activated Protein Kinase (MAPK) pathway (Ali S & Coombes R C 2002). Indeed, the binding of epidermal growth factor (EGF) to its receptor (EGFR) also ultimately activates NF-κB and most likely contributes to the enhanced activity of this transcription factor in ER-negative breast cancer cells (Kalkhoven E et al 1996).

Loss of ER function has been associated with constitutive NFkB activity and hyperactive MAPK, because of constitutive secretion of cytokine and growth factors, which ultimately culminates in aggressive, metastatic, hormone-resistant cancers (Merkhofer E C et al 2010). Activation of the progesterone receptor can lead to inhibition of NF-κB driven gene expression (Sethi G et al 2008) reducing its DNA binding and transcriptional activity. HER-2 activates NF-κB through the canonical pathway which surprisingly, involves IKKα (Ito, T et al 2010). Activation of NF-κB promotes the survival of tumor cells. Several gene products that negatively regulate apoptosis in tumor cells are controlled by NF-κB activation (Lee J et al 2010). Estrogen plays an important role in breast cancer initiation and progression. Breast cancer over time acquires different mutations and the proportion of estrogen receptor-negative cells in tumors increases. This transformation confers aggressive biological characteristics to breast cancer such as rapid growth, poor differentiation, and poor response to hormone therapy. NF-κB pathway plays important role in this pathway (Lee J et al 2010).

Expression of SIRT1 is controlled at multiple levels by transcriptional, post-transcriptional, and post-translational mechanisms under physiological and pathological conditions. Emerging evidence indicates that miRs are important regulators of SIRT1 expression (Lovis P et al 2008, Ortega F J et al 2010, Zovoilis A et al 2011, Yamakuchi M et al 2008 and Mullany L E et al 2017). Studies have shown that miR-34a directly binds to the 3′ untranslated region (UTR) of SIRT1 mRNA and reduces its expression (Ortega F J et al 2010).

Study findings support the hypothesis that alcohol consumption is able to influence miRNA expression. Considerable evidence from rodent and human studies demonstrates that disruption of the hepatic SIRT1 signaling by ethanol plays a central role in the development of AFLD (Yin H et al 2014, Li M et al 2014).Ethanol down-regulates SIRT1 in hepatic cells and in the animal livers. The ethanol-mediated disruption of SIRT1 signaling leads to excess fat accumulation and inflammatory responses in the liver of animals and humans. Treatment with resveratrol, a known SIRT1 agonist, can alleviate liver steatosis . Accumulating evidence demonstrates that ethanol-mediated SIRT1 inhibition leads to the development of AFLD largely through disruption of a signaling network mediated by various transcriptional regulators and co-regulators, including nuclear transcription factor-κB (NF-κB)(Yin H et al 2014, Li M et al 2014).

Summary of the AOP

Events

Molecular Initiating Events (MIE), Key Events (KE), Adverse Outcomes (AO)

Key Event Relationships

Stressors

Overall Assessment of the AOP

Increased DNA damage and mutations [Evidence: high]: DNA damage refers to any modification in the physical and/or chemical structure of DNA resulting in an altered DNA molecule that is different from the original DNA molecule with regard to its physical, chemical, and/or structural properties".External factors to the cell such as environmental or potentially aggressive factors produced by the normal cell metabolism can damage the DNA. The effects caused by the action of endogenous factors may be more serious and/or more extensive than the effect of most of the exogenous DNA damaging factors. Evidence suggested that prolonged alcohol intake is positively associated with an increased risk of cancer.  It can cause changes in the sequence of genomic DNA, which may act as a tumor promoter as well. Alcohol consumption can result in the generation of DNA-damaging molecules such as reactive oxygen species (ROS), lipid peroxidation products, and acetaldehyde. Strand breaks and oxidative base damage in DNA can be produced by hydroxyl radicals which are both mutagenic and cytotoxic. Alcohol is a known inducer of microsomal oxidizing system, which includes a specific ethanol-inducible form of cytochrome P450, referred to as CYP2EL (Lieber C 1992). This effect on the enzyme system has been associated with liver pathology induced by alcohol (Morimoto M et al 1993, French S et al 1993, Nanji A et al 1994, Albano E et al 1996). Again the damaging effects of high levels of CYP2E1 may be mediated by the generation of ROS (Cederbaum 1989, Reinke L et al 1990, Ishii H et al 1989). ROS that is highly reactive, include the oxygen radicals superoxide anion and hydroxyl radicals and can react with lipids, proteins, and DNA and thereby damage them (Knecht K et al 1990).  It has been confirmed in vivo experiments that hydroxyethyl radical formation takes place after ethanol exposure (Albano E et al 1996, Moore D et al 1995, Clot, P et al 1996, Thurman R 1973). Chronic exposure to ethanol also results in increased production of H₂0₂, (Kukieka E et al 1992, Kukielka, E., & Cederbaum, A. I. 1994) which can react with metal ions (such as iron in the Fenton reaction); thus resulting in the production of the highly reactive hydroxyl radicals. DNA is very sensitive to the attack by the hydroxyl radical. A sensitive assay for hydroxyl radical formation from CYP2E1 uses DNA damage (strand breakage) as an endpoint (Breen AP, Murphy JA 1995). Apart from this, more than twenty different types of DNA base damage with diverse biological properties are produced by hydroxyl radical (Moriya M 1993).8-hydroxy-2'-deoxyguanosine, is one such DNA lesion brought about by oxidative stress.  This is mutagenic, due to the tendency of DNA polymerases to misincorporate deoxyadenosine residues opposite this oxidized base (Song B 1996).

 

Inadequate DNA cross-link repair mechanisms [Evidence:high]:

As a result of DNA damage, DNA repair activities change. A variety of genotoxic agents, such as N-nitrosodimethylamine, aflatoxin B1, and 2-acetylaminofluorene induce the protein, O6-Alkylguanine-DNA alkyltransferase (ATase), are responsible for the repair of DNA alkylation damage in rats (O’Connor, 1989; Chinnasamy et al.,1996). Grombacher and Kaina (1996) reported an increased human ATase mRNA expression by alkylating agents like N-methyl-N′-nitro-N-nitrosoguanidine and methyl methanesulphonate and by ionizing radiation via the induction of the ATase promoter. ATase mRNA expression was increased in response to treatment with 2-acetylaminofluorene in rat liver (Potter et al., 1991; Chinnasamy et al., 1996). In another study, it was demonstrated that ATase gene induction is p53 gene-dependent: ATase activity was induced in mouse tissues following γ-irradiation in p53 wild-type mice, but not in p53 null animals (Rafferty et al., 1996). Alkylating agents and X-rays also induce DNA glycosylase, alkylpurine-DNA-N-glycosylase (APNG)  (Lefebvre et al., 1993; Mitra and Kaina, 1993).

 

Increased mutations [Evidence: moderate]: Inadequate repair causes damaged DNA to be retained and used as a template during DNA replication. Incorrect nucleotides may be inserted during the replication of damaged DNA, and these nucleotides become 'fixed' in the cell after replication. The mutation propagates to more cells as a result of further replication. Non-homologous end joining (NHEJ) is one of the repair methods employed in human somatic cells to repair DNA double-strand breaks (DSBs). (Petrini et al., 1997; Mao et al., 2008). However, this mechanism is prone to errors and may result in mutations during the DNA repair process. (Little, 2000). As it does not use a homologous template to repair the DSB, NHEJ is considered error-prone. Many proteins work together in the NHEJ pathway to bridge the DSB gap by overlapping single-strand termini that are typically less than 10 nucleotides long. (Anderson, 1993; Getts & Stamato, 1994; Rathmell & Chu, 1994). Errors are introduced during this process, which can result in mutations like insertions, deletions, inversions, or translocations.

 

Increased micro RNA expression [Evidence: moderate]: DNA damage-responsive transcription factors, such as NF-kB, E2F, and Myc, are also involved in miRNA transcription regulation. The p53 protein also functions as a transcriptional repressor by binding to miRNA promoters and preventing the recruitment of transcriptional activators. For example, p53 prevents the TATA-binding protein from binding to the TAATA site in the promoter of the miR-17-92 cluster gene, suppressing transcription. Under hypoxic conditions, the miR-17-92 cluster is suppressed by a p53-dependent mechanism, making cells more susceptible to hypoxia-induced death (Yan et al.,2009).

Decreased SIRT1 expression [Evidence: moderate]: There are several signaling pathways that establish the role of increased miRNA expression in downregulating the SIRT 1 gene few of which are listed as follows; Butyrate has been demonstrated to cause apoptosis and reduce carcinogenesis in a variety of cancers (Tailor et al.,2014; Rahmani et al.,2002). Although butyrate has been shown to suppress SIRT1 gene expression in various cancers, this has yet to be proven in hepatocellular carcinoma (HCC) (Iglesias et al., 2007). In HCC, miR-22 was found to be downregulated, and its low levels aided carcinogenesis (Zhang et al.,2010). The Huh7 cells' in vitro proliferation was decreased by miR-22 expression, which activated apoptosis. In Huh7 cells, on the other hand, SIRT1 expression was high, which enhanced the expression of antioxidants such as superoxide dismutase (SOD), allowing cell growth to continue (Chen et al.,2012). Butyrate upregulated miR-22 in Huh7 cells, which binds directly to the 3′UTR region of SIRT1 and suppresses its expression.Notch3–SIRT1–LSD1–SOX2 Signaling Pathway in metastasis (Wang et al.,2016; Wu et al .,2017).MiR-486 inhibits HCC invasion and tumorigenicity by directly targeting and suppressing SIRT1 expression. This reduced the tumorigenic and chemo-resistant features of LCSCs and inhibited HCC invasion and tumorigenicity (Yan et al.,2019).

Increased activity of NF kB [Evidence: moderate]: SIRT1 deacetylates  NFkB. In the context of NFkB, all of the evidence so far points to its signaling being inhibited after SIRT1 deacetylation (Morris, 2012). According to Yeung et al, SIRT1 can directly interact with and deacetylate the RelA/p65 component of the NF-B complex (Yeung et al.,2004). NF-B can be activated by cytokines (TNF-, IL-1), growth factors (EGF), bacterial and viral products (lipopolysaccharide (LPS), dsRNA), UV and ionizing radiation, reactive oxygen species (ROS), DNA damage, and oncogenic stress from inside the cells. Almost all stimuli eventually activate a large cytoplasmic protein complex called the inhibitor of B (IB) kinase (IKK) complex via a so-called "canonical pathway." The exact composition of this complex is unknown, however, it has three fundamental components: IKK1/IKK, IKK2/IKK, and NEMO/IKK. IB is phosphorylated by the activated IKK complex, which marks it for destruction by the -transducin repeat-containing protein (-TrCP)-dependent E3 ubiquitin ligase-mediated proteasomal degradation pathway (Liu et al., 2012;Li et al., 2002). As a result, unbound NF-B dimers can go from the cytoplasm to the nucleus, bind to DNA, and control gene transcription.

Antagonism of estrogen receptor [Evidence: moderate]: Activation NF-κB in breast cancer leads to loss of Estrogen Receptor (ER) expression and Human Epidermal Growth Factor Receptor 2 (HER-2) overexpressed via epidermal growth factor receptor (EGFR) and Mitogen-Activated Protein Kinase (MAPK) pathway (Laere et al.,2007). Indeed, the binding of epidermal growth factor (EGF) to its receptor (EGFR) activates NF-B, which most likely contributes to this transcription factor's increased activity in ER-negative breast cancer cells (Shostak et al.,2011). Because of the constitutive production of cytokines and growth factors, loss of ER function has been linked to constitutive NF-kB activity and hyperactive MAPK, resulting in aggressive, metastatic, hormone-resistant malignancies (Ali et al., 2002). Activation of the progesterone receptor can reduce DNA binding and transcriptional activity by inhibiting NF-B-driven gene expression (Kalkhoven et al., 1996). HER-2 stimulates NF-B via the conventional route, which includes IKK (Merkhofer et al., 2010).

Epithelial-mesenchymal transition cell [Evidence: high]: Estrogen/ERa signaling maintains an epithelial phenotype and suppresses EMT.ERa signaling promotes proliferation and epithelial differentiation and opposes EMT. Various studies support this finding (Eeckhoute et al.,2007, Kouros-Mehr et al.,2008, Nakshatri et al., 2009, Taylor et al.,2010). ER-a negative was related to activation of genes implicated in Wnt, Sonic Hedgehog, and TGF-b signaling, indicating epithelial-mesenchymal transition (EMT)(Wik et al.,2013).

Metastatic breast cancer [Evidence: high]: The “epithelial-mesenchymal transition” (EMT), a key developmental regulatory program, has been reported to play critical and intricate roles in promoting tumor invasion and metastasis in epithelium-derived carcinomas in recent years. Some of the cells undergoing EMT have the characteristics of cancer stem cells (CSCs), which are linked to cancer malignancy (Shibue & Weinberg, 2017; Shihori Tanabe, 2015a, 2015b; Tanabe, Aoyagi, Yokozaki, & Sasaki, 2015). Cancer metastasis and cancer therapeutic resistance are linked to the EMT phenomenon (Smith & Bhowmick, 2016; Tanabe, 2013). EMT causes the cell to escape from the basement membrane and metastasize by increasing the production of enzymes that break down extracellular matrix components and decreasing adherence to the basement membrane (Smith & Bhowmick, 2016). 

Overall Assessment:

overall assessment of the AOP was based on the biological domain of the applicability, the essentiality of all KEs, Biological plausibility of each KER, Empirical support for each KER, and Quantitative weight of evidence considerations optional.

	MIE 1669	KE 155	KE 185	KE1980	KE1981	KE 1172	KE 112	KE1457	AO1982
Sex/Life stage /Taxa	Female/Reproductive/Human,human cell line,mice,rat	Female/Reproductive/Rat/rat cel lines/mouse	Female/Reproductive/Mice,yeast,hman cel line	Female/Reproductive/canine,mouse,human cell line	Female/Reproductive/human,human cell ine	Female/Reproductive/human,human cell ine,mice	Female/Reproductive/human,human cell ine,mice	Female/Reproductive/hman,hman cell line	Female/Reproductive/hman,hman cell line,mice
Essentiality of KEs	Direct Evidence	Direct Evidence	Direct Evidence	Direct Evidence	Direct Evidence	Direct Evidence	Direct Evidence	Direct Evidence	Direct Evidence
Empirical Support of KER	High for MIE1669 to KE155	High for KE 155 to KE 185	Moderate for KE 185 to KE1980	Moderate for KE1980 to KE1981	Moderate for KE1981 to KE 1172	Moderate for KE 1172 to KE 112	High for KE 112 to KE1457	High for KE 1457 to AO 1982	-
Biological plausibiity of KER	High for MIE1669 to KE155	High for KE 155 to KE 185	Moderate for KE 185 to KE1980	Moderate for KE1980 to KE1981	Moderate for KE1981 to KE 1172	Moderate for KE 1172 to KE 112	Moderate for KE 112 to KE1457	High for KE 1457 to AO 1982	-
Quantitative assessment	PCR-RFLP OHdG – ELISA &RT- PCR, Western Blot MAPK assay, Immunoprecipitation, Western immunoblotting	Quantification of ATase activity – BSA method APNG assay, OXOG glycosylase activity assay, Western immunoblotting, Immunohistochemical detection of ATase.	Acetaldehyde assay, Extract preparation and Western blotting, N2- Ethyl dGuo quantitation	Western blotting,clonal survival assay,FACs	qRT-PCR,Western blotting,Luciferase reporter assay Micro-array	qRT-PCR,immunohistochemistry	qRT-PCR, immunohistochemistry ()	IHC,micro array,qPCR, SNP array	qRT-PCR,,Luciferase reporter assay ,immunoblotting,immunoprecipitation,cell invasion assay,cell migration assay,
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Domain of Applicability

Life Stage Applicability Taxonomic Applicability Sex Applicability

Sex:The AOP is appicable to women.However study suggests that the relative risk of breast cancer in men is comparable to that in women for alcohol intakes below 60 g per day. It continues to increase at high consumption levels not usually studied in women (Guénel P et al 2004). 

Life stage:There are no research articles which highlight the role alcohol in a particular life stage.In addition, age-related differences in response to alcohol exposure are neither uniform nor linear. The data available is insufficient which direct the construction of a catalog of “appropriate” tests or to define all the factors which influence nonlinear effects (Squeglia LM  et al 2014).

Taxonomic:The evidences for the key events of this AOP are available in various species ike rat,mice and humans.

Essentiality of the Key Events

Direct evidence is available for all the suggested key events. However the strength of weight of evidence varies from moderate to high. however, some inconsistencies are also available. majority of the experimental evidence is available in rats, mice, canine and human cell lines. only a few studies are available on human subjects.

• Human normal hepatocytes (HL-7702) were subjected to escalating doses of N,N-dimethylformamide for 24 hours (C. Wang et al., 2016). At all concentrations, a concentration-dependent increase in ROS was detected; the rise was statistically significant when compared to control (6.4, 16, 40, 100 mM). Until the highest two concentrations (40 and 100 mM), no significant rise in 8-oxodG was seen, indicating inadequate repair at these dosages. Excision repair genes (XRCC2 and XRCC3) were considerably up-regulated at 6.4 and 16 mM, well below the doses that significantly produced 8-oxodG, indicating that adequate DNA repair was possible at these low concentrations. These findings show that repair is competent at low concentrations (removing 8-oxodG quickly), but that repair is swamped (i.e., insufficient) at larger doses, where 8-oxodG greatly increases. AS52 Chinese hamster ovary cells (wild type and OGG1-overexpressing) were exposed to varying doses of ultraviolet A (UVA) radiation (Dahle et al., 2008). Formamidopyrimidine glycosylase (Fpg)-sensitive sites were quantified using alkaline elution after increasing repair times (0, 1, 2, 3, 4 h) following 100 kJ/m² UVA irradiation. OGG1-overexpressing AS52 cells (OGG1+): Fpg-sensitive sites reduced to 71% within half an hour and down to background levels at 4h.Wild type AS52 cells: at 4h, 70% of the Fpg-sensitive sites remained, indicating accumulation of oxidative lesions. Mutations in the Gpt gene was quantified in both wild type and OGG1+ cells by sequencing after 13-15 days following 400 kJ/m² UVA irradiation.G:C→T:A mutations in UVA-irradiated OGG1+ cells were completely eliminated (thus, repair was sufficient when repair overexpressed).G:C→T:A mutation frequency in wild type cells increased from 1.8 mutants/million cells to 3.8 mutants/million cells following irradiation – indicating incorrect repair or lack of repair of accumulated 8-oxo-dG.
• There is evidence from knock-out/knock-down studies indicating there is a strong link between DNA repair adequacy and the frequency of mutations. Defects in proteins involved in DNA repair resulted in altered mutation frequencies in all of the instances studied when compared to wild-type cases. In cell lines deficient in LIG4 (Smith et al., 2003) and Ku80 (Feldmann et al., 2000), there were significant decline in the frequency and accuracy of DNA repair; rescue experiments performed with these two cell lines further confirmed that inadequate DNA repair was the cause of the observed decreases in repair frequency and accuracy (Feldmann et al., 2000; Smith et al., 2003). There was more spontaneous DNA damage in Nibrin-deficient mouse cells than in wild-type controls, implying insufficient DNA repair. In vivo mutation frequencies were also observed to be higher in Nibrin-deficient mice than in wild-type mice using the corresponding Nibrin-deficient and wild-type mice (Wessendorf et al., 2014). Furthermore, depending on the XPC status of cancer patients, mutation densities in certain genomic areas were influenced differentially. In XPC-wild-type patients, mutation frequencies were higher at DHS promoters and 100 bp upstream of TSS than in cancer patients missing functional XPC (Perera et al., 2016). Finally,it was found that radiation exposure caused four times more mutations in WKT1 cells with lower repair capacity than in TK6 cells with normal repair capacity in a research (Amundson and Chen, 1996). 
• There are findings that strongly link the different elements of DNA damage and repair events to the expression of miRNA. Zhang and coworkers examined genome-wide mature miRNA expression in Atm+/+ and Atm-/- littermate mouse embryonic fibroblasts to see how miRNAs are regulated in the DNA damage response (MEFs)(Zhang et al.,2011). MEFs were given neocarzinostatin (NCS), a radiomimetic medication that causes DSBs (Ziv et al., 2006). Mouse miRNA microarray analysis was used to determine miRNA expression profile in each sample, which was done at several time points (0–24 hr). As many as 71 distinct miRNAs were found to be considerably (2-fold) upregulated in the NCS-treated Atm+/+ MEFs, but not in the corresponding Atm-/- MEFs, implying that DNA damage stress causes broad-spectrum changes in miRNA expression. According to Wan et al., regulatory RNA-binding proteins in the Drosha and Dicer complexes, such as DDX5 and KSRP, drive posttranscriptional processing of primary and precursor miRNAs after DNA damage. The findings show that nuclear export of pre-miRNAs is increased in an ATM-dependent manner after DNA damage. The ATM-activated AKT kinase phosphorylates Nup153, a main component of the nucleopore, resulting in enhanced interaction between Nup153 and Exportin-5 (XPO5) and increased nuclear export of pre-miRNAs. These findings demonstrate that DNA damage signalling is important for miRNA transport and maturation. In agreement with previous reports showing that ATM-activated p53 and KSRP promote miRNA expression (Suzuki et al., 2009; Zhang et al., 2011), the study found 61 p53-dependent miRNAs and 29 KSRP-dependent miRNAs among the ATM-induced miRNAs.
• In Jiang et al's study, the cellular function and molecular mechanism of miR2045p in hepatocellular cancer were investigated (HCC)(Jiang et al.,2016). Shen et al showed that downregulation of miR-199b is associated with distant metastasis in colorectal cancer via activation of SIRT1 and inhibition of CREB/KISS1 signalling(Shen et al., 2016). A study by Tian et al found that MicroRNA-133b targets Sirt1 and suppresses hepatocellular carcinoma cell progression(Tian et al., 2016). In liver cancer, Yan et al discovered that MicroRNA 486 5p acts as a tumour suppressor of proliferation and cancer stem-like cell characteristics by targeting Sirt1(Yan et al.,2019). Zhang et al reported that MicroRNA-22 functions as a tumor suppressor by targeting SIRT1 in renal cell carcinoma (Zhang et al., 2016). 
• According to Lu et al, SIRT1 inhibited the growth of gastric cancer through inhibiting the activation of STAT3 and NF-B (Lu et al.,2014). The goal was to look at SIRT1's regulatory effects on gastric cancer (GC) cells (AGS and MKN-45) as well as the links between SIRT1 and STAT3 and NF-B activation in GC cells. The SIRT1 activator (resveratrol RSV) was discovered to contribute to the repression of viability and increase of senescence, which was reversed by SIRT1 inhibitor (nicotinamide NA) and SIRT1 depletion using the CCK-8 and SA-β-gal assays, respectively. SIRT1 activation (RSV supplement) reduced not only STAT3 activation, including STAT3 mRNA level, c-myc mRNA level, phosphorylated STAT3 (pSTAT3) proteins, and acetylizad STAT3 (acSTAT3) proteins, but also pNF-B p65 and acNF-B p65 suppression. The effects of RSV were reversed by NA. Furthermore, when STAT3 or NF-B were knocked down, neither RSV nor NA could affect cellular survival or senescence in MKN-45 cells. Overall, the outcomes of the study revealed that SIRT1 activation could cause GC in vitro to lose viability and senescence. Furthermore, our findings demonstrated that SIRT1 inhibited proliferation in GC cells and was related to deacetylation-mediated suppression of STAT3 and NF-B protein activation. The levels of SIRT1 protein expression in non-small-cell lung cancer (NSCLC) cell lines were examined in a study by Yeung et al.,2004. In comparison to immortalised epithelial human lung NL-20 cells, NSCLC cells exhibit significant quantities of SIRT1 protein, as reported by other researchers (Luo et al, 2001; Vaziri et al, 2001). Pharmacological modulators of Sirtuin activity were employed to see if NF-kB transcription was regulated by Sirtuins (Landry et al, 2000; Bedalov et al, 2001; Howitz et al, 2003). Transient luciferase reporter experiments revealed that cells pretreated with resveratrol had very minimal NF-kB transcription following the presence of TNFa. TNFa-induced NF-kB activity was boosted when cells were pretreated with the Sirtuin inhibitors nicotinamide or splitomicin. NF-kB transcription was also potentiated in cells treated with trichostatin A (TSA), an HDAC class I and class II inhibitor, as expected.
• In specific subclasses of human breast cancer cells and tumour tissue specimens, an enhanced level of activated NF-kB is found, primarily in erbB2-overexpressing ER-negative breast cancer (Biswas et al 2000;2003). Singh et al explored a variety of methods to inhibit NF-kB activation in ER-negative breast cancer cells and looked at the effects on cell proliferation, apoptosis, and tumour growth in xenografts(Singh et al.,2007). In a prospective cohort study, Sampepajung et al used immunohistochemistry (IHC) to examine NF-B expression and intrinsic subtypes of breast cancer tissue and found a significant correlation between negative ER and overexpression of NF-B (p 0.05), with overexpression of NF-B being higher in negative ER (77.3 percent) compared to positive ER (47.4 percent )( Sampepajung et al., 2021). Laere et al suggested that activation of NF-kB in inflammatory breast cancer (IBC) is associated with loss of estrogen receptor (ER) expression, indicating potential crosstalk between NF-kB and ER(Laere et al.,2007). Differential Sensitivity of ER α and ERβ Cells to the NF-kB Inhibitor Go6976 was tested. A differential sensitivity to Go6976 by ER α and ERβ breast cancer cells was observed (Holloway et al.,2004). The ER α cells were more sensitive and less viable after treatment with this NF-kB inhibitor. The IC50 (50% killing) by Go6976 was 1 mM for Era of MDA-MB435 and MDA-MB231 breast cancer cells, whereas it was greater than 10 mM for ERa of MCF-7 and T47D or the normal mammary epithelial H16N  cells. At 10 mM Go6976, about 80% of the ERa cells were killed, whereas only 15–30% of ERa and normal H16N cells were sensitive to this compound. The relative resistance of the H16N normal human mammary cells indicates a possible high therapeutic index of Go6976 against ERa cancer cells.
• Endogenous ER silencing causes EMT in ER-positive breast cancer cells. ER-positive MCF-7 cells were infected with ER shRNA lentiviral particles and stable clones were selected with puromycin (optimal dose of 0.8 g/mL) to knock down ER gene expression (Zheng et al.,2014). When the number of cell passages was increased following infection, the expression of ER was gradually knocked down. Saleh et al. hypothesise that loss of oestrogen receptor function, which causes endocrine resistance in breast cancer, also causes trans-differentiation from an epithelial to a mesenchymal phenotype, which causes enhanced aggressiveness and metastatic tendency(Saleh et al., 2011). 
•  EMT is the most crucial step in initiating metastasis, including metastasis to lymph nodes, because tumour cell movement is a pre-requisite for the metastatic process (Da et al., 2017). Multiple signalling pathways cause cancer cells to lose their cell-to-cell connections and cellular polarity during EMT, increasing their motility and invasive ness (Huang et al., 2017). MMPs cause E-cadherin to be cleaved, which increases tumour cell motility and invasion (Pradella et al., 2017). Chen et al investigated the potential function of MDM2 in ovarian cancer SKOV3 cells' EMT and metastasis(Chen et al.,2015). TGFbeta and Twist induce EMT by upregulating the expression of EMT markers such Snail, Vimentin, N-cadherin, and ABC transporters like ABCA3, ABCC1, ABCC3, and ABCC10 (Saxena et al., 2011).In the treatment with about 0.3, 3, 30 mM of doxorubicin, human mammary epithelial cells (HMLE) stably expressing Twist, FOXC2 or Snail demonstrate increased cell viability compared to control HMLE, dose-dependently (Saxena et al., 2011). 

Weight of Evidence Summary

Increased, DNA damage and mutation leads to Inadequate DNA repair

DNA base excision repair (BER) and, to a lesser extent, nucleotide excision repair (NER)  are used to repair oxidative DNA damage. Previous research has found thresholded dose-response curves in oxidative DNA damage and attributed these findings to a lack of repair capability at the curve's inflection point (Gagne et al., 2012; Seager et al., 2012). Following chemical exposures, in vivo, a rise and buildup of oxidative DNA lesions was seen despite the activation of BER, suggesting poor repair of oxidative DNA lesions beyond a certain level(Ma et al., 2008).

Empirical Evidence (include consideration of temporal concordance ) has been documented in several studies as follows;

 

Compound class	Species	Study type	Dose	KER findings	Reference
N,N-dimethylformamide	Homosapiens hepatocyte cell line	In vitro Experimental	mM	Increased DNA damage leads to decreased DNA cross link repair mechanisms	Wang et al.,2016
UV radiation	Cricetulus griseus(Chinese hamster)	-do-	kJ/m2	-do-	Dahle et al.,2008
X-rays	Human leukemia cell line	-do-	Gy/min	-do-	Li et al.,2013
X rays	Mice	In vivo Experimental	Gy/min	-do-	Li et al.,2013
Aniline	Rat	-do-	Kg/day	-do-	Ma et al.,2008

 

 

 

Inadequate DNA repair leads to Increase, Mutations

 

There will be no increase in mutation frequency if DNA repair is capable of appropriately and efficiently repairing DNA lesions caused by a genotoxic stressor.

For alkylated DNA, for example, efficient AGT removal will result in no increases in mutation frequency. However, once AGT reaches a certain dose, it becomes saturated and can no longer effectively remove alkyl adducts. Mutation occurs when O-alkyl adducts are replicated. The evidence that unrepaired O-alkylated DNA replication induces mutations in somatic cells is vast and has been evaluated. (Basu and Essigmann 1990; Shrivastav et al. 2010).

Empirical Evidence (include consideration of temporal concordance ) has been documented in several studies as follows;

 

Compound class	Species	Study type	Dose	KER findings	Reference
UV radiation	Chinese hamster	In vitro	kJ/m2	inadequate DNA repair leads to increased mutations	Dahle et al.,2008
	Mice	In vivo		-do-	Klungland et al., 1999
X ray	human	In vitro	Gy	-do-(dose-incidence)	Mcmohan et al., 2016

 

Increase, Mutations leads to Increase,miRNA levels

 

Evidences suggest that transcription pathway for miRNAs is regulated in the DNA damage response (DDR).Inadequate repair and mutations increase miRNA expression.DNA damage-responsive transcription factors, such as NF-kB, E2F, and Myc, are also involved in miRNA  transcription regulation.The p53 protein also functions as a transcriptional repressor by binding to miRNA promoters and preventing the recruitment of transcriptional activators.The empirical and dose response evidence for increased mutations inducing miRNA expression has been documented as follows;

Compound class	Species	Study type	Dose	KER findings	Reference
Neocarzinostatin	Mouse Fibroblast	In vitro	Ng/ml	Increased mutation leads to increased microRNA expression	Ziv et al.,2006
Neocarzinostatin	Mouse Fibroblast	In vitro	Ng/ml	-do-	Zhang et al.,2011
Cisplatin and IR	Human mammary epithelial cells	In vitro	mM and Gy	-do-	Jaarsveld et al., 2014

 

Increase,miRNA levels leads to Decrease,SIRT1(sirtuin 1) leves

 

There are several pathways which suggest suppression of SIRT1  expression when miRNA is elevated.SIRT1 was downregulated at the mRNA and protein levels when miR-138 expression was increased. MiR-138 binds to the SIRT1 gene's 3′UTR unique complimentary site and inhibits SIRT1 expression directly, preventing HCC proliferation, migration, and invasion (Luo et al.,2017).When compared to the normal hepatic cell line L02, SIRT1 is overexpressed, while miR-138 levels are lowered in HepG2, SMMC7721, Bel7404, and HCCM3 .

The evidence for this fact has been listed as follows;

Compound class	Species	Study type	KER findings	Reference
	Human HCC Cell lines	In vitro	Increased miRNA leads to Reduced SIRT1	Jiang et al.,2016; Luo et al.,2017; Tian et al., 2016; Yan et al.,2019; Bae et al.,2014;Zhou et al.,2017
	Human CRC cell lines	In vitro	-do-	Shen et al., 2016;Lian et al.,2018
	Human RCC Cell lines	In vitro	-do-	Zhang et al., 2016;Fu et al.,2018
Astragalus Polysachcharide	Prostate cancer cell lines	In vitro	-do-	Guo et al.,2020;Yang et al.,2014
	Lung cancer cell lines	In vitro	-do-	Guan et al.,2017

 

Decrease,SIRT1(sirtuin 1) leves leads to Increase activation, Nuclear factor kappa B (NF-kB)

 

SIRT1 suppresses NF-B signalling either directly by deacetylating the RelA/p65 subunit or indirectly by triggering repressive transcriptional complexes, which frequently involve heterochromatin formation at NF-B promoter regions. SIRT1 expression and signalling are both inhibited by NF-B.

Zhang et al.  found that overexpressing RelA/p65 protein increased SIRT1 expression at both the transcriptional and protein levels (36 h treatment), whereas knocking down RelA/p65 expression decreased TNF-induced SIRT1 expression (8 h treatment)(Zhang et al.,2010). They also discovered that the RelA/p65 protein may bind to the SIRT1 promoter's NF-B motifs. These findings suggest that NF-B may promote SIRT1 expression. Given that SIRT1 induction appeared to occur much later than NF-B activation, it appears that this action could represent a feedback response limiting inflammation and eventually generating endotoxin tolerance.

Evidences supporting this key event is as follows;

Compound class	Species	Study type	KER findings	Reference
nicotinamide	Human gastric cancer cell lines	In vitro	Decreased, SIRT1 leads to increased NF kB activity	Lu et al.,2014
nicotinamide or splitomicin	non-small-cell lung cancer (NSCLC) cell lines	In vitro	Decreased, SIRT1 leads to increased NF kB activity	Yeung et al.,2004; Luo et al, 2001; Vaziri et al, 2001

 

 

Increase activation, Nuclear factor kappa B (NF-kB) leads to Antagonism, Estrogen receptor

 

NF-kB activation in breast cancer has been extensively documented in oestrogen receptor negative (ER) breast tumours and ER breast cancer cell lines, implying a significant inhibitory interaction between both signalling pathways (Biswas et al, 2000, 2001, 2004; Zhou et al, 2005). A rise in both NF-kB DNA-binding activity (Nakshatri et al, 1997) and expression of NF-kB target genes such IL8 coincides with a transition from oestrogen dependence to oestrogen independence in breast cancer, indicating inhibitory cross-talk. The fact that some breast tumours that are resistant to the tumoricidal action of anti-estrogens become sensitised to apoptosis and show a drop in NF-kB activity after treatment with oestrogen supports the inverse relationship between ER and NF-kB activity.

-This shows that oestrogen's proapoptotic actions in these tumours are mediated via NF-kB suppression.

 

Both in vivo and in vitro studies support the finding;

Compound class	Species	Study type	Dose	KER findings	Reference
Bortezomib	Breast cancer cell lines	In vitro		Increased activity of NF kB,  leads to Reduced Estrogen receptor expression	Singh et al.,2017; Holloway et l.,2004
	Human Breast tissue	In vivo		-do-	Biswas et al 2000;2003
	Human Breast tissue	In vivo		-do-	Sampepajung et al., 2021; Laere et al.,2007; Indra et al.,2021;

 

Antagonism, Estrogen receptor leads to EMT

 

E2/ERa signalling, in part through transcriptional activation of luminal/epithelial-related transcription factors, promotes the development of mammary epithelia along a luminal/epithelial lineage. GATA3 and ERa both promote each other (Eeckhoute et al.,2007). In normal breast epithelia, GATA3 is needed for luminal differentiation(Kouros-Mehr et al.,2008) and GATA3 and ERa control many of the same genes (Wilson et al.,2008).  In mice, forcing GATA3 expression in mesenchymal breast cancer cells produces mesenchymal–epithelial transition (MET), a reversible mechanism analogous to EMT, and prevents tumour metastasis (Yan et al.,2010). Another ERa-interacting transcription factor, FOXA1, is essential for luminal lineage in mammary epithelia and stimulates ductal development in mice (Bernardo et al.,2010). FOXA1 enhances ERa gene expression by increasing the accessibility of estrogen-response regions for ERa binding (Nakshatri et al., 2009). In breast cancer cells, on the other hand, E2 appears to increase FOXA1 expression. Importantly, ERa, FOXA1, and GATA3 are all positive breast cancer prognostic factors(Nakshatri et al.,2009).

 

Ye et al.  investigated the impact of ERa overexpression in ERa-negative breast cancer cell lines (MDA-MB-468, MDA-MB-231) or ERa knockdown in ERa-positive cell lines (MCF-7, T47D) on Slug and Snail expression and phenotypes in ERa-positive cell lines (MCF-7, T47D)(Ye et al., 2010). Slug is repressed, E-cadherin is increased, and cells develop as adherent colonies with less invasiveness when ERa is forced to get expressed. ERa knockdown, on the other hand, causes an increase in Slug expression, a decrease in E-cadherin, and spindle-shaped invasive cells.

 

Wik et al used integrated molecular profiling to examine Endometrial cancer samples from a primary investigation cohort and three independent validation cohorts (Wik et al.,2013). Patient survival was closely linked to ER-a immunohistochemical staining and receptor gene (ESR1) mRNA expression. In the study cohort, ER-a negative was related with activation of genes implicated in Wnt, Sonic Hedgehog, and TGF-b signalling, indicating epithelial–mesenchymal transition (EMT)

 

EMT leads to Metastasis, Breast Cancer

 

The “epithelial–mesenchymal transition” (EMT), a key developmental regulatory program, has been reported to play critical and intricate roles in promoting tumor invasion and metastasis in epithelium-derived carcinomas.

EMT is marked by a decrease in E-cadherin and β- -catenin expression and an increase in vimentin, fibronectin, and N-cadherin expression (Irani et al., 2018). EMT is a master mechanism in cancer cells that allows them to lose their epithelial characteristics and gain mesenchymal-like qualities. EMT is the most crucial step in initiating metastasis, including metastasis to lymph nodes, because tumour cell movement is a pre-requisite for the metastatic process (Da et al., 2017). Multiple signalling pathways cause cancer cells to lose their cell-to-cell connections and cellular polarity during EMT, increasing their motility and invasive ness (Huang et al., 2017). MMPs cause E-cadherin to be cleaved, which increases tumour cell motility and invasion (Pradella et al., 2017).

 

 

 

Quantitative Consideration

 

The techniques used for quantifying KE's were reliable with repeatability and reproducibility. Assays were fit for the purpose.

	MIE 1669	KE 155	KE 185	KE1980	KE1981	KE 1172	KE 112	KE1457	AO1982
Human	PCR-RFLP 8-OHdG – ELISA & MDA (Chen CH et al 2011)	-	Acetaldehyde assay, Extract preparation and Western blotting, N2- Ethyl dGuo quantitation Abraham J et al 2011	-	-	qRT-PCR,immunohistochemistry (McGlynn LM et al 2014)	qRT-PCR, immunohistochemistry (Sampepajung E et al 2021, Van Laere SJ et al 2007,)	IHC,micro array,qPCR, SNP array(Wik E et al 2013)	Liang et al., 2013;Liu et al., 2016;Zhang et al.,2015; Chen et al., 2015;Yue et al.,2019;Wang et al.,  2018;Yu et al.,2017
Human Tissues	-	-	-	-	qRT-PCR,Western blotting,Luciferase reporter assay H2,H4,H7,H8,H9 Micro-array (Shen ZL et al 2016)	-	-	-	-
Human Cell lines	RT- PCR, Western Blot MAPK assay, Immunoprecipitation, Western immunoblotting Thymidine uptake ECL-SDS PAGE, RIA Adduct removal measurements, DNA isolation, TLC, LCMS Acetaldehyde estimation, DNA adducts – LC-ESI-MS/ MS-SRM, Western blotting Western blotting, enzymatic assay, LC-ESI-MS/ MS-SRM DNA oxidative damage by ELISA, Immunofluorescence, cell culture, 8-OHdG – ELISA & Ph2Aλ–fociformation assays, P53 luciferase assays, qPCR, Western Blotting (Elise A. Triano et al 2003, Etique.Nicolas etiqu et al 2004, Izevbigie EB et al 2002, Przylipiak A et al 1996, Singletary KW et al 2001, Singletary KW et al 2004, Abraham J et al 2011, Zhao M et al 2017, Jessy Abraham J et al 2011)	-	-	Western blotting,clonal survival assay,FACs(van Jaarsveld MT et al 2014)	Micro-array, qRT-PCR,Western blotting,Luciferase reporter assay (Guo S et al 2020, Bae HJ et al 2014, Zhou J et al 2017, Fu H et al 2018, Lian B et al 2018 Guan Y et al 2017 Yang X et al 2014)	qRT-PCR,,Luciferase reporter assay Cell based HDAC assay(Yeung F et al 2004)	qPCR, western blotting, immunoprecipitation, immunofluorescent microscopy, Luciferase reporter assay EMSA, IHC,Cell viability assay (Singh S et al 2007, Holloway JN  et al 2004, Biswas DK et al 2000, Song RX et al 2005, Scherbakov AM et al 2005, Scherbakov AM et al 2009)	qRT-PCR,cell viability assay, Western blotting,EdU incorporation assay(Bouris P et al 2015, Liu Y et al 2015, Al Saleh S et al 2011, Zeng Q et al 2014, Ye Y et al 2010, Lin, HY et al 2018)	qRT-PCR,,Luciferase reporter assay ,immunoblotting,immunoprecipitation,cell invasion assay,cell migration assay, bioluminesence imaging,wound healing assay,Wound scratch & Transwell assay, Microarray,Immunofluorescence, Immunohistochemistry, Gujral et al.,2014;Cui et al.,2013;Shiota et al.,2012;Gao et al.,2018;Chen et al.,2017;Liu et al.,2020;Casas et al.,2011;Jackstadt et al.,2013;Kong et al.,2016;Zhang et al.,2014;Huang et al.,2014
Rat	Free radical assay GC-MS-SIM  (Hackney JF et al 1992, McDermott EW et al 1992)	Quantification of ATase activity – BSA method APNG assay, OXOG glycosylase activity assay, Western immunoblotting, Immunohistochemical detection of ATase. (Kotova N et al, 2013)	-	Free radicCyQuant cell Proliferation assay (Abdelfattah, N. et al 2018)	-	-	-	-	-
Rat Cell lines	-	Flow cytometric micronucleus assay, Cell cycle analysis, Replication fork elongation assay, Cytotoxicity assay, Recombination assay, (Panida Navasumrit et al, 2001)	-	-	-	-	-	-	-
Mice	Comet assay, ROS generation assay. (Lei  Guo et al 2008)	FISH karyotyping, Invivo point mutation assay, Whole genome sequencing of HSC clones. (Garaycoechea JI et al, 2012)	In vivo point mutation assay Garaycoechea JI et al, 2018	Free radicCyQuant cell Proliferation assay (Abdelfattah, N. et al 2018) RNA sequence analysis,Immuno staining,immunoblotting,Flowcytometry,COMET assay,qRT PCR(Liu Z et al 2017) Microarray (Zhang X et al 2011) qRT PCR,RIP assay,Immunogold EM(Wan G et al 2013)	qRT-PCR,Western blotting,Luciferase reporter assay,ELISA,cell culture Bai XZ et al 2018	qRT-PCR,Southern and northern blotting, reporter gene  assay(Paul T et al 2008)	EMSA,Autoradiography,Immunofluorescent microscopy, Westernblotting (Biswas DK et al 2001)	-	Chen et al.,2017; Gumireddy et al.,2009; Yu et al., 2016; Sarkar et al.,2015
Canine	-	-	-	micro array(Bulkowska M et al 2017)	-	-	-	-	-
Yeast	-	-	Fluctuation assay Voordeckers K et al, 2020	-	-	-	-	-	-

Considerations for Potential Applications of the AOP (optional)

Intended uses of this AOP:

• Helpful for risk assessors, in assessing the risk of alcohol on metastatic breast cancer
• If the causal relationship is established between key events, it may be useful drug targets
• An alternative model to animal model based test methods

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Appendix 1

List of MIEs in this AOP

Event: 1669: Increased, DNA damage and mutation

Short Name: Increased, DNA damage and mutation

Key Event Component

AOPs Including This Key Event

Stressors

Biological Context

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

The DNA damages and mutations can occur in mammals, male or female, and is generally measured in adults.

Key Event Description

DNA damages are alteration of the DNA backbone including abasic site, single or double strand breaks or inter-strand crosslinks. These damages could be recognized and repaired by specialized enzymes. However, if damages persist, mutation in the DNA sequences can occur. Unlike DNA damages, DNA mutations when both strands are modified cannot be repaired and are heritable. Mutations affect the genotype and could affect phenotype.

Different mechanisms are implicated in DNA damage such as oxidative burst, DNA repair dysfunction or centrosome amplification and chromosome instability [1].

How it is Measured or Detected

DNA damages could be measured using different assays, such as micronucleus formation (OECD n°487) [2], comet assay with different protocols for the detection of double and single-strand breaks, DNA-DNA and DNA-protein crosslinks, adduct and oxidized nucleotides (OECD n°489) [3, 4] and γH2AX for the analysis of DNA strand breaks [5].

DNA mutation could be analyzed with Ames test or via the analysis of frequencies of mutations (OECD n°471) [6].

References

1.         Zhang Y. Cell toxicity mechanism and biomarker. 2018;7 1:34; doi: 10.1186/s40169-018-0212-7.

2.         Kato T, Totsuka Y, Ishino K, Matsumoto Y, Tada Y, Nakae D, et al. Genotoxicity of multi-walled carbon nanotubes in both in vitro and in vivo assay systems. Nanotoxicology. 2013;7 4:452-61; doi: 10.3109/17435390.2012.674571.

3.         Pacurari M, Yin XJ, Zhao J, Ding M, Leonard SS, Schwegler-Berry D, et al. Raw single-wall carbon nanotubes induce oxidative stress and activate MAPKs, AP-1, NF-kappaB, and Akt in normal and malignant human mesothelial cells. 2008;116 9:1211-7; doi: 10.1289/ehp.10924.

4.         Hiraku Y, Guo F, Ma N, Yamada T, Wang S, Kawanishi S, et al. Multi-walled carbon nanotube induces nitrative DNA damage in human lung epithelial cells via HMGB1-RAGE interaction and Toll-like receptor 9 activation. Particle and fibre toxicology. 2016;13:16; doi: 10.1186/s12989-016-0127-7.

5.         Catalan J, Siivola KM, Nymark P, Lindberg H, Suhonen S, Jarventaus H, et al. In vitro and in vivo genotoxic effects of straight versus tangled multi-walled carbon nanotubes. Nanotoxicology. 2016;10 6:794-806; doi: 10.3109/17435390.2015.1132345.

           6.         Fukai E, Sato H, Watanabe M, Nakae D, Totsuka Y. Establishment of an in vivo simulating co-culture assay            platform for genotoxicity of multi-walled carbon nanotubes. Cancer science. 2018; doi: 10.1111/cas.13534.

List of Key Events in the AOP

Event: 155: Inadequate DNA repair

Short Name: Inadequate DNA repair

Key Event Component

AOPs Including This Key Event

Stressors

Biological Context

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

The retention of adducts has been directly measured in many different types of eukaryotic somatic cells (in vitro and in vivo). In male germ cells, work has been done on hamsters, rats and mice. The accumulation of mutation and changes in mutation spectrum has been measured in mice and human cells in culture. Theoretically, saturation of DNA repair occurs in every species (prokaryotic and eukaryotic). The principles of this work were established in prokaryotic models. Nagel et al. (2014) have produced an assay that directly measures DNA repair in human cells in culture.

NHEJ is primarily used by vertebrate multicellular eukaryotes, but it also been observed in plants. Furthermore, it has recently been discovered that some bacteria (Matthews et al., 2014) and yeast (Emerson et al., 2016) also use NHEJ. In terms of invertebrates, most lack the core DNA-PK_cs and Artemis proteins; they accomplish end joining by using the RA50:MRE11:NBS1 complex (Chen et al., 2001).  HR occurs naturally in eukaryotes, bacteria, and some viruses (Bhatti et al., 2016).

Key Event Description

DNA lesions may result from the formation of DNA adducts (i.e., covalent modification of DNA by chemicals), or by the action of agents such as radiation that may produce strand breaks or modified nucleotides within the DNA molecule. These DNA lesions are repaired through several mechanistically distinct pathways that can be categorized as follows:

1. Damage reversal acts to reverse the damage without breaking any bonds within the sugar phosphate backbone of the DNA. The most prominent enzymes associated with damage reversal are photolyases (Sancar, 2003) that can repair UV dimers in some organisms, and O6-alkylguanine-DNA alkyltransferase (AGT) (Pegg 2011) and oxidative demethylases (Sundheim et al., 2008), which can repair some types of alkylated bases.
2. Excision repair involves the removal of a damaged nucleotide(s) through cleavage of the sugar phosphate backbone followed by re-synthesis of DNA within the resultant gap. Excision repair of DNA lesions can be mechanistically divided into: 

   a) Base excision repair (BER) (Dianov and Hübscher, 2013), in which the damaged base is removed by a damage-specific glycosylase prior to incision of the phosphodiester backbone at the resulting abasic site.

   b) Nucleotide excision repair (NER) (Schärer, 2013), in which the DNA strand containing the damaged nucleotide is incised at sites several nucleotides 5’ and 3’ to the site of damage, and a polynucleotide containing the damaged nucleotide is removed prior to DNA resynthesis within the resultant gap. 

   c) Mismatch repair (MMR) (Li et al., 2016)  which does not act on DNA lesions but does recognize mispaired bases resulting from replication errors. In MMR the strand containing the misincorporated base is removed prior to DNA resynthesis.

   The major pathway that removes oxidative DNA damage is base excision repair (BER), which can be either monofunctional or bifunctional; in mammals, a specific DNA glycosylase (OGG1: 8-Oxoguanine glycosylase) is responsible for excision of 8-oxoguanine (8-oxoG) and other oxidative lesions (Hu et al., 2005; Scott et al., 2014; Whitaker et al., 2017). We note that long-patch BER is used for the repair of clustered oxidative lesions, which uses several enzymes from DNA replication pathways (Klungland and Lindahl, 1997). These pathways are described in detail in various reviews e.g., (Whitaker et al., 2017). 

3. Single strand break repair (SSBR) involves different proteins and enzymes depending on the origin of the SSB (e.g., produced as an intermediate in excision repair or due to direct chemical insult) but the same general steps of repair are taken for all SSBs: detection, DNA end processing, synthesis, and ligation (Caldecott, 2014). Poly-ADP-ribose polymerase1 (PARP1) detects and binds unscheduled SSBs (i.e., not deliberately induced during excision repair) and synthesizes PAR as a signal to the downstream factors in repair. PARP1 is not required to initiate SSBR of BER intermediates. The XRCC1 protein complex is then recruited to the site of damage and acts as a scaffold for proteins and enzymes required for repair. Depending on the nature of the damaged termini of the DNA strand, different enzymes are required for end processing to generate the substrates that DNA polymerase β (Polβ; short patch repair) or Pol δ/ε (long patch repair) can bind to synthesize over the gap. Synthesis in long-patch repair displaces a single stranded flap which is excised by flap endonuclease 1 (FEN1). In short-patch repair, the XRCC1/Lig3α complex joins the two ends after synthesis. In long-patch repair, the PCNA/Lig1 complex ligates the ends. (Caldecott, 2014). 
4. Double strand break repair (DSBR) is necessary to preserve genomic integrity when breaks occur in both strands of a DNA molecule. There are two major pathways for DSBR: homologous recombination (HR), which operates primarily during S phase in dividing cells, and nonhomologous end joining (NHEJ), which can function in both dividing and non-dividing cells (Teruaki Iyama and David M. Wilson III, 2013). 

In higher eukaryotes such as mammals, NHEJ is usually the preferred pathway for DNA DSBR. Its use, however, is dependent on the cell type, the gene locus, and the nuclease platform (Miyaoka et al., 2016). The use of NHEJ is also dependent on the cell cycle; NHEJ is generally not the pathway of choice when the cell is in the late S or G2 phase of the cell cycle, or in mitotic cells when the sister chromatid is directly adjacent to the double-strand break (DSB) (Lieber et al., 2003). In these cases, the HR pathway is commonly used for repair of DSBs. Despite this, NHEJ is still used more commonly than HR in human cells. Classical NHEJ (C-NHEJ) is the most common NHEJ repair mechanism, but alternative NHEJ (alt-NHEJ) can also occur, especially in the absence of C-NHEJ and HR.

The process of C-NHEJ in humans requires at least seven core proteins: Ku70, Ku86, DNA-dependent protein kinase complex (DNA-PK_cs ), Artemis, X-ray cross-complementing protein 4 (XRCC4), XRCC4-like factor (XLF), and DNA ligase IV (Boboila et al., 2012). When DSBs occur, the Ku proteins, which have a high affinity for DNA ends, will bind to the break site and form a heterodimer. This protects the DNA from exonucleolytic attack and acts to recruit DNA-PK_cs, thus forming a trimeric complex on the ends of the DNA strands. The kinase activity of DNA-PK_cs is then triggered, causing DNA-PK_cs to auto-phosphorylate and thereby lose its kinase activity; the now phosphorylated DNA-PK_cs dissociates from the DNA-bound Ku proteins. The free DNA-PK_cs phosphorylates Artemis, an enzyme that possesses 5’-3’ exonuclease and endonuclease activity in the presence of DNA-PK_cs and ATP. Artemis is responsible for ‘cleaning up’ the ends of the DNA. For 5’ overhangs, Artemis nicks the overhang, generally leaving a blunt duplex end. For 3’ overhangs, Artemis will often leave a four- or five-nucleotide single stranded overhang (Pardo et al., 2009; Fattah et al., 2010; Lieber et al., 2010). Next, the XLF and XRCC4 proteins form a complex which makes a channel to bind DNA and aligns the ends for efficient ligation via DNA ligase IV (Hammel et al., 2011).

The process of alt-NHEJ is less well understood than C-NHEJ.  Alt-NHEJ is known to involve slightly different core proteins than C-NHEJ, but the steps of the pathway are essentially the same between the two processes (reviewed in Chiruvella et al., 2013). It is established, however, that alt-NHEJ is more error-prone in nature than C-NHEJ, which contributes to incorrect DNA repair. Alt-NHEJ is thus considered primarily to be a backup repair mechanism (reviewed in Chiruvella et al., 2013). 

In contrast to NHEJ, HR takes advantage of similar or identical DNA sequences to repair DSBs (Sung and Klein, 2006). The initiating step of HR is the creation of a 3’ single strand DNA (ss-DNA) overhang. Combinases such as RecA and Rad51 then bind to the ss-DNA overhang, and other accessory factors, including Rad54, help recognize and invade the homologous region on another DNA strand. From there, DNA polymerases are able to elongate the 3’ invading single strand and resynthesize the broken DNA strand using the corresponding sequence on the homologous strand.

 

Fidelity of DNA Repair

Most DNA repair pathways are extremely efficient. However, in principal, all DNA repair pathways can be overwhelmed when the DNA lesion burden exceeds the capacity of a given DNA repair pathway to recognize and remove the lesion. Exceeded repair capacity may lead to toxicity or mutagenesis following DNA damage. Apart from extremely high DNA lesion burden, inadequate repair may arise through several different specific mechanisms. For example, during repair of DNA containing O6-alkylguanine adducts, AGT irreversibly binds a single O6-alkylguanine lesion and as a result is inactivated (this is termed suicide inactivation, as its own action causes it to become inactivated). Thus, the capacity of AGT to carry out alkylation repair can become rapidly saturated when the DNA repair rate exceeds the de novo synthesis of AGT (Pegg, 2011).

A second mechanism relates to cell specific differences in the cellular levels or activity of some DNA repair proteins. For example, XPA is an essential component of the NER complex. The level of XPA that is active in NER is low in the testes, which may reduce the efficiency of NER in testes as compared to other tissues (Köberle et al., 1999). Likewise, both NER and BER have been reported to be deficient in cells lacking functional p53 (Adimoolam and Ford, 2003; Hanawalt et al., 2003; Seo and Jung, 2004). A third mechanism relates to the importance of the DNA sequence context of a lesion in its recognition by DNA repair enzymes. For example, 8-oxoguanine (8-oxoG) is repaired primarily by BER; the lesion is initially acted upon by a bifunctional glycosylase, OGG1, which carries out the initial damage recognition and excision steps of 8-oxoG repair. However, the rate of excision of 8-oxoG is modulated strongly by both chromatin components (Menoni et al., 2012) and DNA sequence context (Allgayer et al., 2013) leading to significant differences in the repair of lesions situated in different chromosomal locations.

DNA repair is also remarkably error-free. However, misrepair can arise during repair under some circumstances. DSBR is notably error prone, particularly when breaks are processed through NHEJ, during which partial loss of genome information is common at the site of the double strand break (Iyama and Wilson, 2013). This is because NHEJ rejoins broken DNA ends without the use of extensive homology; instead, it uses the microhomology present between the two ends of the DNA strand break to ligate the strand back into one. When the overhangs are not compatible, however, indels (insertion or deletion events), duplications, translocations, and inversions in the DNA can occur. These changes in the DNA may lead to significant issues within the cell, including alterations in the gene determinants for cellular fatality (Moore et al., 1996).

Activation of mutagenic DNA repair pathways to withstand cellular or replication stress either from endogenous or exogenous sources can promote cellular viability, albeit at a cost of increased genome instability and mutagenesis (Fitzgerald et al., 2017). These salvage DNA repair pathways including, Break-induced Replication (BIR) and Microhomology-mediated Break-induced Replication (MMBIR). BIR repairs one-ended DSBs and has been extensively studied in yeast as well as in mammalian systems. BIR and MMBIR are linked with heightened levels of mutagenesis, chromosomal rearrangements and ensuing genome instability (Deem et al., 2011; Sakofsky et al., 2015; Saini et al., 2017; Kramara et al., 2018). In mammalian genomes BIR-like synthesis has been proposed to be involved in late stage Mitotic DNA Synthesis (MiDAS) that predominantly occurs at so-called Common Fragile Sites (CFSs) and maintains telomere length under s conditions of replication stress that serve to promote cell viability (Minocherhomji et al., 2015; Bhowmick et al., 2016; Dilley et al., 2016).       

Misrepair may also occur through other repair pathways. Excision repair pathways require the resynthesis of DNA and rare DNA polymerase errors during gap resynthesis will result in mutations (Brown et al., 2011). Errors may also arise during gap resynthesis when the strand that is being used as a template for DNA synthesis contains DNA lesions (Kozmin and Jinks-Robertson, 2013). In addition, it has been shown that sequences that contain tandemly repeated sequences, such as CAG triplet repeats, are subject to expansion during gap resynthesis that occurs during BER of 8-oxoG damage (Liu et al., 2009).

How it is Measured or Detected

There is no test guideline for this event. The event is usually inferred from measuring the retention of DNA adducts or the creation of mutations as a measure of lack of repair or incorrect repair. These ‘indirect’ measures of its occurrence are crucial to determining the mechanisms of genotoxic chemicals and for regulatory applications (i.e., determining the best approach for deriving a point of departure). More recently, a fluorescence-based multiplex flow-cytometric host cell reactivation assay (FM-HCR) has been developed to directly measure the ability of human cells to repair plasmid reporters (Nagel et al., 2014).

Indirect Measurement

In somatic and spermatogenic cells, measurement of DNA repair is usually inferred by measuring DNA adduct formation/removal. Insufficient repair is inferred from the retention of adducts and from increasing adduct formation with dose. Insufficient DNA repair is also measured by the formation of increased numbers of mutations and alterations in mutation spectrum. The methods will be specific to the type of DNA adduct that is under study.

Some EXAMPLES are given below for alkylated DNA.

DOSE-RESPONSE CURVE FOR ALKYL ADDUCTS/MUTATIONS: It is important to consider that some adducts are not mutagenic at all because they are very effectively repaired. Others are effectively repaired, but if these repair processes become overwhelmed mutations begin to occur. The relationship between exposure to mutagenic agents and the presence of adducts (determined as adducts per nucleotide) provide an indication of whether the removal of adducts occurs, and whether it is more efficient at low doses. A sub-linear DNA adduct curve suggests that less effective repair occurs at higher doses (i.e., repair processes are becoming saturated). A sub-linear shape for the dose-response curves for mutation induction is also suggestive of repair of adducts at low doses, followed by saturation of repair at higher doses. Measurement of a clear point of inflection in the dose-response curve for mutations suggests that repair does occur, at least to some extent, but reduced repair efficiency arises above the breakpoint. A lack of increase in mutation frequencies (i.e., flat line for dose-response) for a compound showing a dose-dependent increase in adducts would imply that the adducts formed are either not mutagenic or are effectively repaired.

RETENTION OF ALKYL ADDUCTS: Alkylated DNA can be found in cells long after exposure has occurred. This indicates that repair has not effectively removed the adducts. For example, DNA adducts have been measured in hamster and rat spermatogonia several days following exposure to alkylating agents, indicating lack of repair (Seiler et al., 1997; Scherer et al., 1987).

MUTATION SPECTRUM: Shifts in mutation spectrum (i.e., the specific changes in the DNA sequence) following a chemical exposure (relative to non-exposed mutation spectrum) indicates that repair was not operating effectively to remove specific types of lesions. The shift in mutation spectrum is indicative of the types of DNA lesions (target nucleotides and DNA sequence context) that were not repaired. For example, if a greater proportion of mutations occur at guanine nucleotides in exposed cells, it can be assumed that the chemical causes DNA adducts on guanine that are not effectively repaired.

Direct Measurement

Nagel et al. (2014) we developed a fluorescence-based multiplex flow-cytometric host cell reactivation assay (FM-HCR) to measures the ability of human cells to repair plasmid reporters. These reporters contain different types and amounts of DNA damage and can be used to measure repair through by NER, MMR, BER, NHEJ, HR and MGMT.

Please refer to the table below for additional details and methodologies for detecting DNA damage and repair.

Assay Name	References	Description	DNA Damage/Repair Being Measured	OECD Approved Assay
Dose-Response Curve for Alkyl Adducts/ Mutations	Lutz 1991   Clewell 2016	Creation of a curve plotting the stressor dose and the abundance of adducts/mutations; Characteristics of the resulting curve can provide information on the efficiency of DNA repair	Alkylation, oxidative damage, or DSBs	N/A
Retention of Alkyl Adducts	Seiler 1997   Scherer 1987	Examination of DNA for alkylation after exposure to an alkylating agent; Presence of alkylation suggests a lack of repair	Alkylation	N/A
Mutation Spectrum	Wyrick 2015	Shifts in the mutation spectrum after exposure to a chemical/mutagen relative to an unexposed subject can provide an indication of DNA repair efficiency, and can inform as to the type of DNA lesions present	Alkylation, oxidative damage, or DSBs	N/A
DSB Repair Assay (Reporter constructs)	Mao et al., 2011	Transfection of a GFP reporter construct (and DsRed control) where the GFP signal is only detected if the DSB is repaired; GFP signal  is quantified using fluorescence microscopy or flow cytometry	DSBs	N/A
Primary Rat Hepatocyte DNA Repair Assay	Jeffrey and Williams, 2000   Butterworth et al., 1987	Rat primary hepatocytes are cultured with a 3H-thymidine solution in order to measure DNA synthesis in response to a stressor in non-replicating cells; Autoradiography is used to measure the amount of 3H incorporated in the DNA post-repair	Unscheduled DNA synthesis in response to DNA damage	N/A
Repair synthesis measurement by 3H-thymine incorporation	Iyama and Wilson, 2013	Measure DNA synthesis in non-dividing cells as indication of gap filling during excision repair	Excision repair	N/A
Comet Assay with Time-Course	Olive et al., 1990   Trucco et al., 1998	Comet assay is performed with a time-course; Quantity of DNA in the tail should decrease as DNA repair progresses	DSBs	Yes (No. 489)
Pulsed Field Gel Electro-phoresis (PFGE) with Time-Course	Biedermann et al., 1991	PFGE assay with a time-course; Quantity of small DNA fragments should decrease as DNA repair  progresses	DSBs	N/A
Fluorescence -Based Multiplex Flow-Cytometric Host Reactivation Assay (FM-HCR)	Nagel et al., 2014	Measures the ability of human cells to repair plasma reporters, which contain different types and amounts of DNA damage; Used to measure repair processes including HR, NHEJ, BER, NER, MMR, and MGMT	HR, NHEJ, BER, NER, MMR, or MGMT	N/A

 

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Event: 185: Increase, Mutations

Short Name: Increase, Mutations

Key Event Component

AOPs Including This Key Event

Stressors

Biological Context

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Mutations can occur in any organism and in any cell type, and are the fundamental material of evolution. The test guidelines described above range from analysis from prokaryotes, to rodents, to human cells in vitro. Mutations have been measured in virtually every human tissue sampled in vivo.

Key Event Description

A mutation is a change in DNA sequence. Mutations can thus alter the coding sequence of genes, potentially leading to malformed or truncated proteins. Mutations can also occur in promoter regions, splice junctions, non-coding RNA, DNA segments, and other functional locations in the genome. These mutations can lead to various downstream consequences, including alterations in gene expression. There are several different types of mutations including missense, nonsense, insertion, deletion, duplication, and frameshift mutations, all of which can impact the genome and its expression in unique ways.

Mutations can be propagated to daughter cells upon cellular replication. Mutations in stem cells (versus terminally differentiated non-replicating cells) are the most concerning, as these will persist in the organism. The consequence of the mutation, and thus the fate of the cell, depends on the location (e.g., coding versus non-coding) and the type (e.g., nonsense versus silent) of mutation.

Mutations can occur in somatic cells or germ cells (sperm or egg).

How it is Measured or Detected

Mutations can be measured using a variety of both OECD and non-OECD mutagenicity tests. Some examples are given below.

Somatic cells: The Salmonella mutagenicity test (Ames Test) is generally used as part of a first tier screen to determine if a chemical can cause gene mutations. This well-established test has an OECD test guideline (OECD TG 471, 2020). A variety of bacterial strains are used, in the presence and absence of a metabolic activation system (e.g., rat liver microsomal S9 fraction), to determine the mutagenic potency of chemicals by dose-response analysis. A full description is found in Test No. 471: Bacterial Reverse Mutation Test (OECD, 2016).

A variety of in vitro mammalian cell gene mutation tests are described in OECD’s Test Guidelines 476 (2016) and 490 (2015). TG 476 (2016) is used to identify substances that induce gene mutations at the hprt (hypoxanthine-guanine phosphoribosyl transferase) gene, or the transgenic xprt (xanthine-guanine phosphoribosyl transferase) reporter locus. The most commonly used cells for the HPRT test include the CHO, CHL and V79 lines of Chinese hamster cells, L5178Y mouse lymphoma cells, and TK6 human lymphoblastoid cells. The only cells suitable for the XPRT test are AS52 cells containing the bacterial xprt (or gpt) transgene (from which the hprt gene was deleted).

The new OECD TG 490 (2015) describes two distinct in vitro mammalian gene mutation assays using the thymidine kinase (tk) locus and requiring two specific tk heterozygous cells lines: L5178Y tk+/-3.7.2C cells for the mouse lymphoma assay (MLA) and TK6 tk+/- cells for the TK6 assay. The autosomal and heterozygous nature of the thymidine kinase gene in the two cell lines enables the detection of cells deficient in the enzyme thymidine kinase following mutation from tk+/- to tk-/-.

It is important to consider that different mutation spectra are detected by the different mutation endpoints assessed. The non-autosomal location of the hprt gene (X-chromosome) means that the types of mutations detected in this assay are point mutations, including base pair substitutions and frameshift mutations resulting from small insertions and deletions. Whereas, the autosomal location of the transgenic xprt, tk, or gpt locus allows the detection of large deletions not readily detected at the hemizygous hprt locus on X-chromosomes. Genetic events detected using the tk locus include both gene mutations (point mutations, frameshift mutations, small deletions) and large deletions.

The transgenic rodent mutation assay (OECD TG 488, 2020) is the only assay capable of measuring gene mutation in virtually all tissues in vivo. Specific details on the rodent transgenic mutation reporter assays are reviewed in Lambert et al. (2005, 2009). The transgenic reporter genes are used for detection of gene mutations and/or chromosomal deletions and rearrangements resulting in DNA size changes (the latter specifically in the lacZ plasmid and Spi- test models) induced in vivo by test substances (OECD, 2009, OECD, 2011; Lambert et al., 2005). Briefly, transgenic rodents (mouse or rat) are exposed to the chemical agent sub-chronically. Following a manifestation period, genomic DNA is extracted from tissues, transgenes are rescued from genomic DNA, and transfected into bacteria where the mutant frequency is measured using specific selection systems.

The Pig-a (phosphatidylinositol glycan, Class A) gene on the X chromosome codes for a catalytic subunit of the N-acetylglucosamine transferase complex that is involved in glycosylphosphatidyl inositol (GPI) cell surface anchor synthesis. Cells lacking GPI anchors, or GPI-anchored cell surface proteins are predominantly due to mutations in the Pig-a gene. Thus, flow cytometry of red blood cells expressing or not expressing the Pig-a gene has been developed for mutation analysis in blood cells from humans, rats, mice, and monkeys. The assay is described in detail in Dobrovolsky et al. (2010). Development of an OECD guideline for the Pig-a assay is underway. In addition, experiments determining precisely what proportion of cells expressing the Pig-a mutant phenotype have mutations in the Pig-a gene are in progress (e.g., Nicklas et al., 2015, Drobovolsky et al., 2015). A recent paper indicates that the majority of CD48 deficient cells from 7,12-dimethylbenz[a]anthracene-treated rats (78%) are indeed due to mutation in Pig-a (Drobovolsky et al., 2015).

Germ cells: Tandem repeat mutations can be measured in bone marrow, sperm, and other tissues using single-molecule PCR. This approach has been applied most frequently to measure repeat mutations occurring in sperm DNA. Isolation of sperm DNA is as described above for the transgenic rodent mutation assay, and analysis of tandem repeats is done using electrophoresis for size analysis of allele length using single-molecule PCR. For expanded simple tandem repeat this involved agarose gel electrophoresis and Southern blotting, whereas for microsatellites sizing is done by capillary electrophoresis. Detailed methodologies for this approach are found in Yauk et al. (2002) and Beal et al. (2015).

Mutations in rodent sperm can also be measured using the transgenic reporter model (OECD TG 488, 2020). A description of the approach is found within this published TG. Further modifications to this protocol have now been made for the analysis of germ cells. Detailed methodology for detecting mutant frequency arising in spermatogonia is described in Douglas et al. (1995), O'Brien et al. (2013); and O'Brien et al. (2014). Briefly, male mice are exposed to the mutagen and killed at varying times post-exposure to evaluate effects on different phases of spermatogenesis. Sperm are collected from the vas deferens or caudal epididymis (the latter preferred). Modified protocols have been developed for extraction of DNA from sperm.

A similar transgenic assay can be used in transgenic medaka (Norris and Winn, 2010).

Please note, gene mutations that occur in somatic cells in vivo (OECD Test. No. 488, 2020) or in vitro (OECD Test No. 476: In vitro Mammalian Cell Gene Mutation Test, 2016), or in bacterial cells (i.e., OECD Test No. 471, 2020) can be used as an indicator that mutations in male pre-meiotic germ cells may occur for a particular agent (sensitivity and specificity of other assays for male germ cell effects is given in Waters et al., 1994). However, given the very unique biological features of spermatogenesis relative to other cell types, known exceptions to this rule, and the small database on which this is based, inferring results from somatic cell or bacterial tests to male pre-meiotic germ cells must be done with caution. That mutational assays in somatic cells may predict mutations in germ cells has not been rigorously tested empirically (Singer and Yauk, 2010). The IWGT working group on germ cells specifically addressed this gap in knowledge in their report (Yauk et al., 2015) and recommended that additional research address this issue. Mutations can be directly measured in humans (and other species) through the application of next-generation sequencing. Although single-molecule approaches are growing in prevalence, the most robust approach to measure mutation using next-generation sequencing today requires clonal expansion of the mutation to a sizable proportion (e.g., sequencing tumours; Shen et al., 2015), or analysis of families to identify germline derived mutations (reviewed in Campbell and Eichler, 2013; Adewoye et al., 2015).

Please refer to the table below for additional details and methodologies for measuring mutations.

Assay Name	References	Description	OECD Approved Assay
Assorted Gene Loci Mutation Assays	Tindall et al., 1989; Kruger et al., 2015	After exposure to a chemical/mutagen, mutations can be measured by the ability of exposed cells to form colonies in the presence of specific compounds that would normally inhibit colony growth; Usually only cells -/- for the gene of interest are able to form colonies	N/A
TK Mutation Assay	Yamamoto et al., 2017; Liber et al., 1982; Lloyd and Kidd, 2012	After exposure to a chemical/mutagen, mutations are detected at the thymidine kinase (TK) loci of L5178Y wild-type mouse lymphoma TK (+/-) cells by measuring resistance to lethaltriflurothymidine (TFT); Only TK-/- cells are able to form colonies	Yes (No. 490)
HPRT Mutation Assay	Ayres et al., 2006; Parry and Parry, 2012	Similar to TK Mutation Assay above, X-linked HPRT mutations produced in response to chemical/mutagen exposure can be measured through colony formation in the presence of 6-TG or 8-azoguanine; Only HPRT-/- cells are able to form colonies	Yes (No. 476)
Salmonella Mutagenicity Test (Ames Test)	OECD, 1997	After exposure to a chemical/mutagen, point mutations are detected by analyzing the growth capacity of different bacterial strains in the presence and absence of various metabolic activation systems	Yes (No. 471)
PIG-A / PIG-O Assay	Kruger et al., 2015; Nakamura, 2012; Chikura, 2019	After exposure to a chemical/mutagen, mutations  in PIG-A or PIG-O (which decrease the biosynthesis of the glycosylphosphatidylinositol (GPI) anchor protein) are assessed by the colony-forming capabilities of cells after in vitro exposure, or by flow cytometry of blood samples after in vivo exposure	N/A
Single Molecule PCR	Kraytsberg, 2005; Yauk, 2002	This PCR technique uses a single DNA template, and is often employed for detection of mutations in microsatellites, recombination studies, and generation of polonies	N/A
ACB-PCR	Myers et al., 2014 (Textbook, pg 345-363); Banda et al.,  2013; Banda et al.,  2015; Parsons et al., 2017	Using this PCR technique, single base pair substitution mutations within oncogenes or tumour suppressor genes can be detected by selectively amplifying specific point mutations within an allele and selectively blocking amplification of the wild-type allele	N/A
Transgenic Rodent Mutation Assay	OECD 2013; Lambert 2005; Lambert 2009	This in vivo test detects gene mutations using transgenic rodents that possess transgenes and reporter genes; After in vivo exposure to a chemical/mutagen, the transgenes are analyzed by transfecting bacteria with the reporter gene and examining the resulting phenotype	Yes (No. 488)
Conditionally inducible transgenic mouse models	Parsons 2018 (Review)	Inducible mutations linked to fluorescent tags are introduced into transgenic mice; Upon exposure of the transgenic mice to an inducing agent, the presence and functional assessment of the mutations can be easily ascertained due to expression of the linked fluorescent tags	N/A
Error-Corrected Next Generation Sequencing (NGS)	Salk 2018 (Review)	This technique detects rare subclonal mutations within a pool of heterogeneous DNA samples through the application of new error-correction strategies to NGS; At present, few laboratories in the world are capable of doing this, but commercial services are becoming available (e.g., Duplex sequencing at TwinStrand BioSciences)	N/A

 

References

Adewoye, A.B. et al. (2015), "The genome-wide effects of ionizing radiation on mutation induction in the mammalian germline", Nat. Commu., 6:6684. Doi: 10.1038/ncomms7684.

Ayres, M. F. et al. (2006),  “Low doses of gamma ionizing radiation increase hprt mutant frequencies of TK6 cells without triggering the mutator phenotype pathway”,  Genetics and Molecular Biology. 2(3): 558-561. Doi:10.1590/S1415-4757200600030002.

Banda M, Recio L, and Parsons BL. (2013), “ACB-PCR measurement of spontaneous and furan-induced H-ras codon 61 CAA to CTA and CAA to AAA mutation in B6C3F1 mouse liver”, Environ Mol Mutagen. 54(8):659-67. Doi:10.1002/em.21808.

Banda,  M. et al. (2015), “Quantification of Kras mutant fraction in the lung DNA of mice exposed to aerosolized particulate vanadium pentoxide by inhalation”,  Mutat Res Genet Toxicol Environ Mutagen. 789-790:53-60. Doi: 10.1016/j.mrgentox.2015.07.003

Campbell, C.D. & E.E. Eichler (2013), "Properties and rates of germline mutations in humans", Trends Genet., 29(10): 575-84. Doi:  10.1016/j.tig.2013.04.005

Chikura, S. et al. (2019), “Standard protocol for the total red blood cell Pig-a assay used in the interlaboratory trial organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagen Society”,  Genes Environ.  27:41-5. Doi: 10.1186/s41021-019-0121-z.

Dobrovolsky, V.N. et al. (2015), "CD48-deficient T-lymphocytes from DMBA-treated rats have de novo mutations in the endogenous Pig-a gene. CD48-Deficient T-Lymphocytes from DMBA-Treated Rats Have De Novo Mutations in the Endogenous Pig-a Gene", Environ. Mol. Mutagen., (6): 674-683. Doi: 10.1002/em.21959.

Douglas, G.R. et al. (1995), "Temporal and molecular characteristics of mutations induced by ethylnitrosourea in germ cells isolated from seminiferous tubules and in spermatozoa of lacZ transgenic mice", Proceedings of the National Academy of Sciences of the United States of America, 92(16): 7485-7489. Doi: 10.1073/pnas.92.16.7485.

Kraytsberg,Y. &  Khrapko, K. (2005),  “Single-molecule PCR: an artifact-free PCR approach for the analysis of somatic mutations”,  Expert Rev Mol Diagn. 5(5):809-15. Doi: 10.1586/14737159.5.5.809.

Krüger, T. C., Hofmann, M., & Hartwig, A. (2015), “The in vitro PIG-A gene mutation assay: mutagenicity testing via flow cytometry based on the glycosylphosphatidylinositol (GPI) status of TK6 cells”, Arch Toxicol. 89(12), 2429-43. Doi: 10.1007/s00204-014-1413-5.

Lambert, I.B. et al. (2005), "Detailed review of transgenic rodent mutation assays", Mutat Res., 590(1-3):1-280. Doi: 10.1016/j.mrrev.2005.04.002.

Liber, L. H., & Thilly, G. W. (1982),  “Mutation assay at the thymidine kinase locus in diploid human lymphoblasts”,  Mutation Research. 94: 467-485. Doi:10.1016/0027-5107(82)90308-6.

Lloyd, M., & Kidd, D. (2012), “The Mouse Lymphoma Assay. In: Parry J., Parry E. (eds) Genetic Toxicology, Methods in Molecular Biology (Methods and Protocols), 817. Springer, New York, NY.

Myers, M. B. et al., (2014), “ACB-PCR Quantification of Somatic Oncomutation”,  Molecular Toxicology Protocols, Methods in Molecular Biology. DOI: 10.1007/978-1-62703-739-6_27

Nakamura, J. et al., (2012), “Detection of PIGO-deficient cells using proaerolysin: a valuable tool to investigate mechanisms of mutagenesis in the DT40 cell system”, PLoS One.7(3): e33563. Doi:10.1371/journal.pone.0033563.

Nicklas, J.A., E.W. Carter and R.J. Albertini (2015), "Both PIGA and PIGL mutations cause GPI-a deficient isolates in the Tk6 cell line", Environ. Mol. Mutagen., 6(8):663-73. Doi: 10.1002/em.21953.

Norris, M.B. and R.N. Winn (2010), "Isolated spermatozoa as indicators of mutations transmitted to progeny", Mutat Res., 688(1-2): 36–40. Doi: 10.1016/j.mrfmmm.2010.02.008.

O'Brien, J.M. et al.(2013), "No evidence for transgenerational genomic instability in the F1 or F2 descendants of Muta™Mouse males exposed to N-ethyl-N-nitrosourea", Mutat. Res., 741-742:11-7. Doi: 10.1016/j.mrfmmm.2013.02.004.

O'Brien, J.M. et al. (2014), "Transgenic rodent assay for quanitifying male germ cell mutation frequency", Journal of Visual Experimentation, Aug 6;(90). Doi: 10.3791/51576.

O’Brien, J.M. et al. (2015), "Sublinear response in lacZ mutant frequency of Muta™ Mouse spermatogonial stem cells after low dose subchronic exposure to N-ethyl-N-nitrosourea", Environ. Mol. Mutagen., 6(4): 347-355. Doi: 10.1002/em.21932.

OECD (2020), Test No. 471: Bacterial Reverse Mutation Test, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris.

OECD (2016), Test No. 476: In vitro Mammalian Cell Gene Mutation Test, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris.

OECD (2009), Detailed Review Paper on Transgenic Rodent Mutation Assays, Series on Testing and Assessment, N° 103, ENV/JM/MONO 7, OECD, Paris.

OECD (2020), Test No. 488: Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris.

OECD (2016), Test. No. 490: In vitro mammalian cell gene mutation mutation tests using the thymidine kinase gene, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris.

OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris.

Parry MJ, & Parry ME. 2012. Genetic Toxicology Principles and Methods. Humana Press. Springer Protocols.

Parsons BL, McKim KL, Myers MB. 2017. Variation in organ-specific PIK3CA and KRAS mutant levels in normal human tissues correlates with mutation prevalence in corresponding carcinomas. Environ Mol Mutagen. 58(7):466-476. Doi: 10.1002/em.22110.

Parsons BL. Multiclonal tumor origin: Evidence and implications. Mutat Res. 2018. 777:1-18. doi: 10.1016/j.mrrev.2018.05.001.

Salk JJ, Schmitt MW, &Loeb LA. (2018), “Enhancing the accuracy of next-generation sequencing for detecting rare and subclonal mutations”, Nat Rev Genet. 19(5):269-285. Doi: 10.1038/nrg.2017.117.

Shen, T., S.H. Pajaro-Van de Stadt, N.C. Yeat and J.C. Lin (2015), "Clinical applications of next generation sequencing in cancer: from panels, to exomes, to genomes" Front. Genet., 6: 215. Doi: 10.3389/fgene.2015.00215.

Singer, T.M. and C.L. Yauk CL (2010), "Germ cell mutagens: risk assessment challenges in the 21st century", Environ. Mol. Mutagen., 51(8-9): 919-928. Doi: 10.1002/em.20613.

Tindall, R. K., & Stankowski Jr., F. L. (1989),  “Molecular analysis of spontaneous mutations at the GPT locus in Chinese hamster ovary (AS52) cells”, Mutation Research, 220, 241-53. Doi: 10.1016/0165-1110(89)90028-6.

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Yamamoto, A. et al. (2017), “Radioprotective activity of blackcurrant extract evaluated by in vitro micronucleus and gene mutation assays in TK6 human lymphoblastoid cells”, Genes and Environment. 39: 22. Doi: 10.1186/s41021-017-0082-z.

Yauk, C.L. et al. (2002), "A novel single molecule analysis of spontaneous and radiation-induced mutation at a mouse tandem repeat locus", Mutat. Res., 500(1-2): 147-56. Doi: 10.1016/s0027-5107(02)00005-2.

Yauk, C.L. et al. (2015), "Approaches for Identifying Germ Cell Mutagens: Report of the 2013 IWGT Workshop on Germ Cell Assays", Mutat. Res. Genet. Toxicol. Environ. Mutagen., 783: 36-54. Doi: 10.1016/j.mrgentox.2015.01.008.

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Event: 1554: Increase Chromosomal Aberrations

Short Name: Increase chromosomal aberrations

AOPs Including This Key Event

Biological Context

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex 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).

Key Event Description

The term "structural chromosomal aberrations" refers to chromosome damage caused by breaks in the DNA that can result in the deletion, addition, or rearrangement of chromosomal segments. According to whether one or both chromatids are affected, chromosomal aberrations can be classified into two main groups: chromatid-type and chromosome-type. Additionally, they can be divided into rejoined and non-rejoined aberrations. Translocations, insertions, dicentrics, and rings are examples of rejoined aberrations, whereas acentric fragments and breaks are examples of unrejoined aberrations (Savage, 1976). Some of these abnormalities, like reciprocal translocations, are long-lasting and can last for many years (Tucker and Preston, 1996). Others, such as dicentrics and acentric fragments, are unstable and weaken with each cell division due to cell death (Boei et al., 1996). After cell division, these activities might still be visible, and the DNA is irreversibly damaged. The occurrence of chromosomal abnormalities is linked to cancer development and cell death (Mitelman, 1982).

A missing, excess, or asymmetrical part of chromosomal DNA is referred to as a chromosomal aberration (CA). There are various double-strand break (DSB) repair mechanisms that could be responsible for these DNA modifications in the chromosome structure (Obe et al., 2002).

The four basic categories of CAs are inversions, translocations, duplications, and deletions. When a section of a chromosome's genetic material is destroyed, deletions take place. When a chromosome's end portion is cut, terminal deletions result.

When a chromosome splits into two different places and wrongly rejoins, leaving the middle portion out, interstitial deletions result. Duplications occur when excess genetic material is added to or rearranged; they can take the forms of transpositions, tandem duplications, reverse duplications, and misplaced duplications (Griffiths et al., 2000). A segment of one chromosome is transferred to a non-homologous chromosome in translocations (Bunting and Nussenzweig, 2013). A reciprocal translocation occurs when regions of two non-homologous chromosomes are switched. When an inversion occurs, the DNA sequence is effectively reversed because both ends of the chromosome split and are ligated at the opposite ends.

The copy number variant is a fifth type of CA that can exist in the genome (CNV). CNVs are deletions or duplications that can range in size from 50 base pairs (Arlt et al., 2012; Arlt et al., 2014; Liu et al., 2013) up into the megabase pair range and may make up more than 10% of the human genome (Shlien et al., 2009; Zhang et al., 2016; Hastings et al., 2009). (Arlt et al., 2012; Wilson et al., 2015; Arlt et al., 2014; Zhang et al., 2016). According to Wilson et al. (2015), CNV regions are particularly abundant in large active transcription units and genes, and they are especially problematic when they result in the duplication of oncogenes or the loss of tumor suppressor genes (Liu et al., 2013; Curtis et al., 2012). 

 

Recurrent and non-recurrent CNVs are two different types. Non-allelic homologous recombination (NAHR), a recombination process that occurs during meiosis, is hypothesised to be the cause of recurrent CNVs (Arlt et al., 2012; Hastings et al., 2009). These germline CNVs, also known as recurrent CNVs, may be inherited and are hence prevalent in various people (Shlien et al., 2009; Liu et al., 2013). It is thought that non-recurrent CNVs are created in mitotic cells during the replication process. It has been proposed that replication-related stress, particularly stalled replication forks, triggers microhomology-mediated processes to break the replication stall, which frequently leads to duplications or deletions, despite the fact that the mechanism is not well understood.

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).

Depending on whether the aberration affects the chromatid or the chromosome, CAs can be categorized. Chromosome-type aberrations (CSAs) are chromosome breakage and chromatid swaps; ring chromosomes, marker chromosomes, and dicentric chromosomes are examples of chromatid-type aberrations (CTAs) (Bonassi et al., 2008; Hagmar et al., 2004). Micronuclei (MN; small nucleus-like structures that contain a chromosome or a fragment of a chromosome that was lost during mitosis) and nucleoplasmic bridges (NPBs; physical linkages between the two nuclei) are visible in binucleated cells when cells are halted at the cytokinesis step (El-Zein et al., 2014). The DNA sequence can be examined to evaluate other CAs, as it is for identifying copy number variants (CNVs) (Liu et al., 2013).

How it is Measured or Detected

Assay	References	Description
Fluorescent In Situ  Hybridization (FISH)	Beaton et al., 2013; Pathak et al., 2017	Fluorescent assay of metaphase chromosomes that can detect CAs through chromosome painting and microscopic analysis
Cytokinesis Block Micronucleus (CBMN) Assay with Microscopy in vitro	Fenech, 2000; OECD, 2016a	Cells are cultured with cytokinesis blocking agent, fixed to slides, and undergo MN quantification using microscopy.
Micronucleus (MN) Assay by Microscopy in vivo	OECD, 2016b	Cells are fixed on slides and MN are scored using microscopy. Red blood cells can also be scored for MN using flow cytometry (see below)
CBMN with Imaging Flow Cytometry	Rodrigues et al., 2015	Cells are cultured with cytokinesis blocking agent, fixed in solution, and imaged with flow cytometry to quantify MN
Flow cytometry detection of MN	Dertinger et al., 2004; Bryce et al., 2007; OECD 2016a, 2016b	In vivo and in vitro flow cytometry-based, automated micronuclei measurements are also done without cytokinesis block. MN analysis in vivo is performed in peripheral blood cells to detect MN in erythrocytes and reticulocytes.
High-throughput biomarker assays (indirect measures to confirm clastogenicity)	Bryce et al. 2014, 2016, 2018   Khoury et al., 2013, Khoury et al., 2016)     Hendriks et al., 2012, 2016; Wink et al., 2014	Multiplexed biomarkers can be measured by flow cytometry are used to discern clastogenic and aneugenic mechanisms for MN induction. Flow cytometry-based quantification of γH2AX foci and p53 protein expression (Bryce et al., 2016).   Prediscreen Assay– In-Cell Western-based quantification of γH2AX   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.
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
High content imaging	Shahane et al., 2016	DNA can be stained using fluorescent dyes and micronuclei can be scored high-throughput microscopy image analysis.
Chromosomal aberration test	OECD, 2016c; 2016d; 20l16e	In vitro, the cell cycle is arrested at metaphase after 1.5 cell cycle following 3-6 hour exposure   In vivo, the test chemical is administered as a single treatment, bone marrow is collected 18-24 hrs later (TG 475), while testis is collected 24-48 hrs later (TG 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
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 most commonly detected using global DNA microarray technologies; This method, however, is unable to detect balanced CAs, such as inversions
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

References

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 SM, Bemis JC, Avlasevich SL, Dertinger SD. In vitro micronucleus assay scored by flow cytometry provides a comprehensive evaluation of cytogenetic damage and cytotoxicity. Mutat Res. 2007 Jun 15;630(1-2):78-91. doi: 10.1016/j.mrgentox.2007.03.002. Epub 2007 Mar 19. PMID: 17434794; PMCID: PMC1950716.

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.

Bryce SM, Bernacki DT, Smith-Roe SL, Witt KL, Bemis JC, Dertinger SD. Investigating the Generalizability of the MultiFlow ® DNA Damage Assay and Several Companion Machine Learning Models With a Set of 103 Diverse Test Chemicals. Toxicol Sci. 2018 Mar 1;162(1):146-166. doi: 10.1093/toxsci/kfx235. PMID: 29106658; PMCID: PMC6059150.

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.

Shahane SA, Nishihara K, Xia M. High-Throughput and High-Content Micronucleus Assay in CHO-K1 Cells. Methods Mol Biol. 2016;1473:77-85. doi: 10.1007/978-1-4939-6346-1_9. PMID: 27518626.

OECD (2016a), Test No. 487: In Vitro Mammalian Cell Micronucleus Test, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, https://doi.org/10.1787/9789264264861-en.

OECD. (2016b), “Test No. 474: Mammalian Erythrocyte Micronucleus Test. OECD Guideline for the Testing of Chemicals, Section 4.”Paris: OECD Publishing.

OECD. (2016c), “In Vitro Mammalian Chromosomal Aberration Test 473.”

OECD. (2016d). Test No. 475: Mammalian Bone Marrow Chromosomal Aberration Test. OECD Guideline for the Testing of Chemicals, Section 4. Paris: OECD Publishing.

OECD. (2016e). Test No. 483: Mammalian Spermatogonial Chromosomal Aberration 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.

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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.

Event: 1980: Increased micro RNA expression

Short Name: Increase,miRNA levels

AOPs Including This Key Event

Stressors

Biological Context

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Regulation of miRNA expression by DNA replication,damage and repair responses,transcription and translation has been proved in animals like mice,canine and cell line experiments.

Key Event Description

Biological state

Genome integrity must be maintained for the proper functioning and survival of an organism.  There has been an efficient and rapid response developed by the eukaryotic cells to DNA damage  to overcome the harmful effects.  As soon as the DNA damage or replication arrest is detected, the  activation of cell cycle checkpoint and  stopping the progress of the cell cycle thus providing time for the cell to  repair the DNA damage.  The response to  DNA damage  also leads to transcriptional regulation, activation of DNA repair, and, in severe cases, initiation of apoptosis (Harper, J.W., and Elledge, S.J. , 2007). Expression of miRNAs may be regulated by the DNA damage response. A study reported that  that micro RNA expression is a  a partially ATM / ATR-independent manner(Pothof, J. et al , 2009). Subsequent studies have shown that the tumor suppressor p53 promotes PrimeRNA processing via  RNA helicase p68 (Suzuki, H.I et al, 2009).

Han et al evaluated miRNA expression pattern in a DNA damage regulatory protein, DDX1 in controls, as well in DDX1-knockdown U2OS cells with the help of reverse transcription quantitative-PCR (qRT-PCR) and human miRNA array (Han C et al, 2014). The study noticed a significant reduction in the expression levels of a subset of miRNAs -200 family such as miR-200a, miR-200b, miR-200c, miR-141 and miR-429 (cut-off >2-fold).d miR-429). The ovarian cancer genomics study revealed a 8-miRNA signature that defines the mesenchymal subtype of serous ovarian cancer (Yang Y, et al, 2011). Among the eight miRNAs, miR-200a, miR-29c, miR-141 and miR-101 were significantly dependent on DDX1, suggesting that DDX1 may play a role in ovarian tumor progression. Nuclear run-on assays were performed to determine whether DDX1 regulates the miRNA expression at transcriptional or post-transcriptional levels, No notable differences were seen in the transcription of pri-miR-200s from the two miR-200 gene clusters (miR-200a/200b/429 and miR-200c/141) in the control and DDX1-silenced cells . However, in the DDX1-knockdown U2OS cells, the levels of mature DDX1-dependent miRNAs, but not control miR-21, were significantly decreased. Due to the potential inhibition of miRNA processing activity, primary transcripts of the DDX1-dependent miRNAs were accumulated. Conversely, these DDX1-dependent miRNAs were up-regulated in the DDX1- overexpressing cells.  The above findings suggested that expression of specific miRNAs was promoted by DDX1 at the post-transcriptional level.

Biological compartments: 

Cellular, nucleus, cytoplasm and mitochondria

General role in biology: 

MicroRNAs (miRNAs) are endogenous non-coding RNAs that contain approximately 22 nucleotides. They function as major regulators of various biological processes, and their dysregulation is associated with many diseases, including cancer.

Cells trigger a specific cellular responses to preserve the integrity of the genome.  The  DNA damage response (DDR) is one among them along with several distinct DNA repair pathways.Normal cells need to repair DNA damage through various repair mechanisms or induce apoptosis and cell cycle arrest if repair is not possible [Jackson SP and Bartek J, 2009]. Genomic instability and mutagenesis  are brought about by the disruption of repair mechanisms.DNA damage response (DDR) determines the fate of the cell and controls microRNAs expression. This will  in turn  regulate important components of the DNA repair machinery. Various reports suggest the key  role of miRNA  in the regulation of the DDR [d’Adda di Fagagna F, 2014 and WeiW et al 2012].The DDR and DNA damage are known regulators of miRNA expression [Sharma V et al 2013 and Chowdhury D et al 2013]. Several studies have shown that the cellular sensitivity to chemotherapeutic drugs is affected by  DDR- miRNA network.[ van Jaarsveld MT et al 2014].

A bidirectional relationship between miRNAs and the DDR has been suggested by studies. The DDR is a known regulator of miRNA expression at both transcriptional and post-transcriptional levels, and miRNA-mediated gene silencing has been shown to modulate the activity of the DDR [d’Adda di Fagagna F, 2014 ; WeiW et al 2012 and Han C  et al 2012]. A unique set of miRNAs as well as a common core miRNA signature are activated depending on DNA damage type and level,  suggesting that miRNAs regulate the DDR by mechanisms based on the type and/or the intensity of DNA damage [Han C  et al 2012]. miRNAs expression  may be regulated by transcription factors either  binding directly  to miRNA promoters and modulating their transcriptional activity, or by modifying the expression of miRNA processing machinery components.

  Studies have widely explored the TP53-mediated transcriptional pathways regulating miRNA expression following DNA damage. miRNA-34a-c is induced by DNA damage and oncogenic stress, is one of the transcriptional target of the tumor suppressor TP53 [Hermeking H et al 2012]. TP53 directly binds to the promoter of miRNA-34 and activates transcription. Micro  RNA-34 has been reported to repress the mRNA transcripts of several genes involved in the regulation of cell cycle, cell proliferation and survival, such as BCL2, CCND1 CCNE2, MYC, CDK4, CDK6 and SIRT1 [Hermeking H et al 2012]. Activation of miRNA-34a  promotes TP53-mediated apoptosis, cell cycle arrest or senescence [Hermeking H et al 2012].  miRNA-34a may target SIRT1, form a positive feedback loop of the  acetylation of TP53, expression of its transcriptional targets, regulating cell cycle and apoptosis [Hermeking H et al 2012].   The alternative pathway involving p38 MAPK signaling  also induces miR-34c [Cannell IG et al 2010]. Inhibition of miRNA 34 prevents the DNA damage induced cell cycle arrest and results in an increased DNA synthesis [Cannell IG et al 2010].

DNA damage promotes the TP53-dependent upregulation of miRNA-192, miRNA-194 and miRNA-215. The genomic region surrounding the miRNA-194/miRNA-215 cluster contains a putative TP53-binding element, indicating that these miRNAs are transcriptionally activated by TP53 [Hermeking H et al 2012]. The expression of miRNA-192 and miRNA-215 induces cell cycle arrest and targets several transcripts involved in cell cycle checkpoints [Georges SA et al 2008].

MYC and E2F, are the two other transcription factors involved in DNA damage- induced cell cycle checkpoints, that  regulate the expression of several miRNAs. Both factors induce transcription of the miRNA-17-92 cluster that forms a feedback loop by inhibiting E2F expression [Aguda BD et al 2008]. E2F transcription factors are repressed by several other miRNAs, including miRNA-106a-92 and miRNA-106b-25 cluster members, miRNA-210, miRNA-128, miRNA-34 and miRNA-20 [Wan G et al 2011].

 DNA damage upregulates several miRNAs, including miRNA-16-1, miRNA-143 and miRNA-145. [Suzuki HI et al 2009]. Most TP53 mutations found in cancers are located in a domain required for miRNA processing and transcriptional activity [Suzuki HI et al 2009]. Thus, loss of TP53 functions in miRNAs transcription and processing might contribute to cancer progression. Considering that some miRNAs are reduced after DNA damage in an ATM-dependent manner, ATM could be also involved in inhibitory pathways that downregulate miRNA expression [Wang Y et al 2013]. These findings support the existence of a critical link between the DDR and miRNA processing pathway.

 In the DNA damage response, post-transcriptional processing of miRNAs is also regulated. It was reported that DNA damage led to increased levels of some pre-miRNAs and mature miRNAs without significant changes of levels of their primary transcripts, suggesting posttranscriptional mechanisms could contribute to the induction of certain miRNAs under DNA damage stress [Zhang X, et al 2011]. There appears to be functional connections between DNA damage response and miRNA processing and maturation.

Micro RNA - 18a, miR-100, miR-101, miR-181, and miR-421, have been implicated as novel regulators to control the protein level of ATM (Majid S et al 2010). BRCA1, a critical tumor suppressor, BRCA1, is also recruited to DNA damage lesions, where it facilitates DNA repair. The level of BRCA1 is regulated by miR-182, miR-146a, and 146b-5p (Matsui M et al 2013).

The tumor suppressor p53 has a central role in the activation of genes in multiple pathways, including cell cycle regulation, tumor suppression, and apoptosis. Micro RNA-125b and miR-504 have been identified as negative regulators of p53 in several types of human cells (Kreis S et al 2008 and Wang J et al 2012).

The available evidence suggests that DNA damage signaling participates in miRNA biogenesis by regulating both transcriptional and post-transcriptional mechanisms. Further studies can through light on the correlation between DNA damaging signaling and miRNA processing. The majority of the studies have examined the miRNA regulation in response to DNA damage and have focused on events that occur in the nucleus. It is important to extend the investigations in understanding the contribution of cytoplasmic regulation of miRNA biogenesis following DNA damage. It is very interesting to determine whether DNA damage signals can modulate the turnover, stabilization, modification, and degradation of miRNAs.

How it is Measured or Detected

	Method/ measurement reference	Reliability	Strength of evidence	Assay fit for purpose	Repeatability/ reproducibility	Direct measure
Human cell line	Western blotting,clonal survival assay,FACs(van Jaarsveld MT et al 2014)	Yes	Strong	Yes	Yes	Yes
Mice	Free radicCyQuant cell Proliferation assay (Abdelfattah, N. et al 2018)	Yes	Strong	Yes	Yes	Yes
	RNA sequence analysis,Immuno staining,immunoblotting,Flowcytometry,COMET assay,qRT PCR(Liu Z et al 2017)	Yes	Strong	Yes	Yes	Yes
	Microarray (Zhang X et al 2011)	Yes	Strong	Yes	Yes	Yes
	qRT PCR,RIP assay,Immunogold EM(Wan G et al 2013)	Yes	Strong	Yes	Yes	Yes
Canine	micro array(Bulkowska M et al 2017)	Yes	Strong	Yes	Yes	Yes

References

Abdelfattah, N., Rajamanickam, S., Panneerdoss, S., Timilsina, S., Yadav, P., Onyeagucha, B. C., ... & Rao, M. K. (2018). MiR-584-5p potentiates vincristine and radiation response by inducing spindle defects and DNA damage in medulloblastoma. Nature communications, 9(1), 1-19.

Aguda, B. D., Kim, Y., Piper-Hunter, M. G., Friedman, A., & Marsh, C. B. (2008). MicroRNA regulation of a cancer network: consequences of the feedback loops involving miR-17-92, E2F, and Myc. Proceedings of the National Academy of Sciences, 105(50), 19678-19683.

Bulkowska, M., Rybicka, A., Senses, K. M., Ulewicz, K., Witt, K., Szymanska, J., ... & Krol, M. (2017). MicroRNA expression patterns in canine mammary cancer show significant differences between metastatic and non-metastatic tumours. BMC cancer, 17(1), 1-17.

Cannell, I. G., Kong, Y. W., Johnston, S. J., Chen, M. L., Collins, H. M., Dobbyn, H. C., ... & Bushell, M. (2010). p38 MAPK/MK2-mediated induction of miR-34c following DNA damage prevents Myc-dependent DNA replication. Proceedings of the National Academy of Sciences, 107(12), 5375-5380.

Chowdhury, D., Choi, Y. E., & Brault, M. E. (2013). Charity begins at home: non-coding RNA functions in DNA repair. Nature reviews Molecular cell biology, 14(3), 181-189.

di Fagagna, F. D. A. (2014). A direct role for small non-coding RNAs in DNA damage response. Trends in cell biology, 24(3), 171-178.

Georges, S. A., Biery, M. C., Kim, S. Y., Schelter, J. M., Guo, J., Chang, A. N., ... & Chau, B. N. (2008). Coordinated regulation of cell cycle transcripts by p53-Inducible microRNAs, miR-192 and miR-215. Cancer research, 68(24), 10105-10112.

Han, C., Liu, Y., Wan, G., Choi, H. J., Zhao, L., Ivan, C., ... & Lu, X. (2014). The RNA-binding protein DDX1 promotes primary microRNA maturation and inhibits ovarian tumor progression. Cell reports, 8(5), 1447-1460.

Han, C., Wan, G., Langley, R. R., Zhang, X., & Lu, X. (2012). Crosstalk between the DNA damage response pathway and microRNAs. Cellular and molecular life sciences, 69(17), 2895-2906.

Harper, J. W., & Elledge, S. J. (2007). The DNA damage response: ten years after. Molecular cell, 28(5), 739-745.

Hermeking, H. (2012). MicroRNAs in the p53 network: micromanagement of tumour suppression. Nature reviews cancer, 12(9), 613-626.

Jackson SP, Bartek J. (2009). The DNA-damage response in human biology and disease. Nature, 461,1071-8

Kreis, S., Philippidou, D., Margue, C., & Behrmann, I. (2008). IL‐24: a classic cytokine and/or a potential cure for cancer?. Journal of cellular and molecular medicine, 12(6a), 2505-2510.

Liu, Z., Zhang, C., Khodadadi-Jamayran, A., Dang, L., Han, X., Kim, K., ... & Zhao, R. (2017). Canonical microRNAs enable differentiation, protect against DNA damage, and promote cholesterol biosynthesis in neural stem cells. Stem cells and development, 26(3), 177-188.

Majid, S., Dar, A. A., Saini, S., Yamamura, S., Hirata, H., Tanaka, Y., ... & Dahiya, R. (2010). MicroRNA‐205–directed transcriptional activation of tumor suppressor genes in prostate cancer. Cancer, 116(24), 5637-5649.

Matsui, M., Chu, Y., Zhang, H., Gagnon, K. T., Shaikh, S., Kuchimanchi, S., ... & Janowski, B. A. (2013). Promoter RNA links transcriptional regulation of inflammatory pathway genes. Nucleic acids research, 41(22), 10086-10109.

Pothof, J., Verkaik, N. S., Van Ijcken, W., Wiemer, E. A., Ta, V. T., Van Der Horst, G. T., ... & Persengiev, S. P. (2009). MicroRNA‐mediated gene silencing modulates the UV‐induced DNA‐damage response. The EMBO journal, 28(14), 2090-2099.

Sharma, V., & Misteli, T. (2013). Non-coding RNAs in DNA damage and repair. FEBS letters, 587(13), 1832-1839.

Suzuki, H. I., Yamagata, K., Sugimoto, K., Iwamoto, T., Kato, S., & Miyazono, K. (2009). Modulation of microRNA processing by p53. Nature, 460(7254), 529-533.

Suzuki, H. I., Yamagata, K., Sugimoto, K., Iwamoto, T., Kato, S., & Miyazono, K. (2009). Modulation of microRNA processing by p53. Nature, 460(7254), 529-533.

van Jaarsveld, M. T., Wouters, M. D., Boersma, A. W., Smid, M., van IJcken, W. F., Mathijssen, R. H., ... & Pothof, J. (2014). DNA damage responsive microRNAs misexpressed in human cancer modulate therapy sensitivity. Molecular oncology, 8(3), 458-468.

van Jaarsveld MT, Wouters MD, Boersma AW, Smid M, van Ijcken WF, Mathijssen RH, Hoeijmakers JH, Martens JW, van Laere S, Wiemer EA, Pothof J. (2014) .DNA damage responsive microRNAs misexpressed in human cancer modulate therapy sensitivity. Mol Oncol. 8(3), 458-68.

Wan, G., Mathur, R., Hu, X., Zhang, X., & Lu, X. (2011). miRNA response to DNA damage. Trends in biochemical sciences, 36(9), 478-484.

Wan, G., Zhang, X., Langley, R. R., Liu, Y., Hu, X., Han, C., ... & Lu, X. (2013). DNA-damage-induced nuclear export of precursor microRNAs is regulated by the ATM-AKT pathway. Cell reports, 3(6), 2100-2112.

Wang, J., & Li, L. C. (2012). Small RNA and its application in andrology and urology. Translational andrology and urology, 1(1), 33.

Wang, Y., & Taniguchi, T. (2013). MicroRNAs and DNA damage response: implications for cancer therapy. Cell cycle, 12(1), 32-42.

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Yang, Y., Ahn, Y. H., Gibbons, D. L., Zang, Y., Lin, W., Thilaganathan, N., ... & Kurie, J. M. (2011). The Notch ligand Jagged2 promotes lung adenocarcinoma metastasis through a miR-200–dependent pathway in mice. The Journal of clinical investigation, 121(4), 1373-1385.

Zhang, X., Wan, G., Berger, F. G., He, X., & Lu, X. (2011). The ATM kinase induces microRNA biogenesis in the DNA damage response. Molecular cell, 41(4), 371-383.

Zhang, X., Wan, G., Berger, F. G., He, X., & Lu, X. (2011). The ATM kinase induces microRNA biogenesis in the DNA damage response. Molecular cell, 41(4), 371-383.

 

Event: 1981: Decreased SIRT1 expression

Short Name: Decrease,SIRT1(sirtuin 1) levels

AOPs Including This Key Event

Stressors

Biological Context

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Decreased SIRT1 expression  is known to be highly conserved throughout evolution and is present from humans to invertebrates.

Key Event Description

Biological state: 

Mammalian SIRTs include seven proteins (SIRT1-7) with deacetylase activity belonging to the class III histone deacetylase family. SIRTs share homology with the yeast deacetylase Sir2, and have different sequences and lengths in both their N- and C-terminal domains (Carafa, V. et al 2012). Expressed from bacteria to humans (Vaquero, A. 2009), SIRTs target histone and non-histone proteins.

Localization of SIRTs is restricted to mitochondria, cytoplasm and nucleus     The location of SIRT1, SIRT6, and SIRT7  is predominantly in the nucleus,  while SIRT2 in the cytosol, and SIRT3, SIRT4, and SIRT5 are in the mitochondria.  Depending on their role in regulating different pathways,  SIRTs relocalize under different conditions such as  cell cycle phase, tissue type, developmental stage, stress condition, and metabolic status which has been documented in the literature. (McGuinness, D. et al 2011).  As per Mitchishita et al, SIRT1, SIRT2, and SIRT7 are often found in both the nucleus and cytoplasm (Michishita, E et al 2005).

 Cellular pathways  like DNA repair, transcriptional regulation, metabolism, aging, and senescence are modulated by Sirtuins. This has  created  sufficient interest with Sirtuins as target in cancer research as the above mentioned functions are involved in initiation and progression of cancer. Evidence have suggested the association of SIRTs with metabolism-associated TFs, MYC and hypoxia inducible factor-1 (HIF-1),  in terms of  energy metabolic reprogramming.( Zwaans, B. M. et al 2014)

  The biological effect of SIRTs in cancer is either tumor suppression  or tumor promoter (oncogenes) action by altering the cell proliferation, differentiation, and death which in turn depends on cell context and experimental conditions.   These two totally opposite function of SIRTs on cancer cell is remains a highly debated and controversial topic. Whether SIRTs act as tumor suppressors or promoters depends on (i) their  The different expression levels of SIRTS in tumors and its effects on cell cycle, cell growth,  death, their action on specific proto-oncogene and onco-suppressor proteins will determine SIRTs role as  tumor suppressor or tumor promoters  (Deng, C. X. 2009).

Sirtuin Reactions

The NAD+-dependent deacetylation is well known enzymatic reaction catalyzed by SIRTs. Deacetylation reaction begins with amide cleavage from NAD+ with the formation of nicotinamide and an intermediate of reaction, O-ADP-ribose. This intermediate formed is necessary for the deacetylation process by which SIRTs catalyze the transfer of one acetyl group from a lysine to O-ADP-ribose moiety to form O-acetyl-ADP-ribose and the deacetylated lysine product. This reaction requires a mole equivalent of NAD+ per acetyl group removed and is controlled by the cellular [NAD]/[NADH] ratio (Sauve, A. A. 2010 and Shi, Y. et al 2013).

Among the SIRTs family, only SIRT1, SIRT2, and SIRT3 possess a robust deacetylase activity even though SIRT enzymes are primarily known as protein deacetylases. SIRT4, SIRT5, SIRT6, and SIRT7) exhibit a weak or no detectable deacetylation activity at all.Through these reactions, SIRTs are able to regulate several key cellular processes (Jiang, H., et al 2013 and Zhang, S. et al 2017).

 

 

Biological compartments: 

Regulation of gene expression takes place in the cell, subcellular site being nucleus.

 

 

General role in biology: 

Silent Inflammation Regulator 2 (SIR2) proteins belong to the  family of histone deacetylases (HDACs) that catalyze deacetylation of both histone and non- histone lysine residues.

Mammalian sirtuins (SIRT1-7) are involved in  diverse biological processes including energy metabolism,  lifespan and health span regulation (Longo VD et al 2006). Mammalian sirtuins possess will bring about an array of biological functions through its enzymatic activity such as  histone deacetylase, mono-ADP-ribosyltransferase, desuccinylase, demalonylase, demyristoylase, and depalmitoylase activity (Michan S et al 2007). SIRT1 located  in the nucleus play an important role in genomic stability, telomere maintenance, and cell survival (Chen J et al 2011 and, Haigis MC et al 2006).

 

Among the 7 SIRTs, SIRT1 is the largest in terms of total DNA and amino acid sequence studied sirtuin [Fang, Y. and M.B. Nicholl 2011]. SIRT1, a class 3 histone deacetylase, is implicated in the modulation of apoptosis, senescence, proliferation, and aging. It’s actions arebrought about by cellular nicotinamide adenosine dinucleotide (NAD+) which acts as a cofactor for deacetylation reactivity. The liberated nicotinamide from NAD+, generates a  novel metabolite o-acetyl-ADP-ribose . SIRT1 can mediate  the actions at translational level. Various mechanisms have been  proposed to be  involved in dysregulation of SIRT1 in cancer cells  [Yao, C., et al.2016]. In human breast, lung and prostate cancers SIRT1 is significantly elevated . It plays  a role in tumorigenesis by anti-apoptotic activity through oncogene and epigenetic regulator action.[ Saunders, L. and E. Verdin 2007]. SIRT1 deacetylates pro-apoptotic proteins such as p53 and promotes cell survival under genotoxic and oxidative stresses [Kojima, K., et al 2010]. It’s critical role in multiple aspects of resistance to anti-cancer drugs is also well documented [Duan, K., et al 2015]. Therefore, SIRT1 overexpression is associated with the subsequent higher level of tumor cell proliferation, invasion, and migration [Wang, X., et al 2016].

SIRT1 expression is increased in human colon cancer, acute myeloid leukemia, and some skin can- cers (Bradbury, C. A et al 2005, Hida, Y. et al 2007, Huffman, D. M. et al 2007 and Stunkel, W.2007). SIRT1 , by interacting with and inhibiting p53  may act as tumor promoter (van Leeuwen, I., and Lain, S. 2009). Repression of tumor suppression protein expression and DNA repair protein ,are other roles of  SIRT1 in cancer cells.  In colon cancer ,  SIRT1 limits β-catenin signaling while in breast cancer it interacts with  BRCA1 signaling . However it has been observed that  SIRT1 expression is decreased in  ovarian cancer, glioblastoma, and bladder carcinoma (Deng, C. X. 2009).  In these cancers , SIRT1 might serve as a tumor suppressor by blocking oncogenic pathways. Thus SIRT1 can serve as a tumor promoter or tumor suppressor, depending on the oncogenic pathways specific to particular tumors.

In hepatocellular carcinoma , SIRT1 was overexpressed in HCC cells and tissues, and significantly promoted the migration and invasion ability of HCC cells by inducing the epithelial and mesenchymal transition[Hao C et al 2014]. This in vivo study  also supported the oncogenic functions of SIRT1 in enhancing metastasis[Hao C et al 2014]. Bae et al [Bae HJ et al 2014] found that knockdown of SIRT1 inhibited cell growth by transcriptional deregulation of cell cycle proteins, leading to hypophosphorylation of pRb, which inactivated E2F/ DP1 target gene transcription, and thereby caused the G1/S cell cycle arrest. In addition, miR29c was identified as a suppressor of SIRT1 by comprehensive miRNA profiling and ectopic miR29c expression recapitulated SIRT1 knockdown effects in HCC cells [Bae HJ et al 2014].  To contradict the above findings,  Zhang et al [Zhang ZY et al 2015] reported that SIRT1 has anticarcinogenic effects in HCC via the AMPK mammalian target of rapamycin (mTOR) pathway. They evaluated the relationship between p53 mutations and activation of SIRT1 in 252 patients with hepatitis B virus positive HCC and found that activated SIRT1 was associated with a longer recurrence free survival in HCC tissues harbouring mutant p53.  He reported that inhibition of SIRT1 increased cell growth, bearing mutated p53, by suppressing AMPK activity and enhancing mTOR activity.The conflicting results from different published data  indicated that SIRT1 is multifunctional gene and its biological features are left unsolved.

These above evidence indicates the involvement of SIRTs in regulating three important tumor processes: epithelial-to-mesenchymal transition (EMT), invasion, and metastasis. Many SIRTs are responsible for cellular metabolic reprogramming and drug resistance by inactivating cell death pathways and promoting uncontrolled proliferation. These observations are  for the future development of novel tailored SIRT-based cancer therapies.

Wang et al showed that SIRT1 expression was increased in several cancer cell lines, and is generally associated with poor prognosis and overall survival (Wang, C., et al 2017). Vaziri et al reported that SIRT1 interacted  with P53, triggering its deacetylation in Lys382 residue, and determined a block of all P53-dependent pathways, leading to uncontrolled cell cycle and inactivation of the apoptotic process (Vaziri, H., et al 2011).

SIRT1 has a function in metastasis and invasiveness in several cancers that has been reported in several studies. Among them ,the deacetylation of many proteins involved in tumor suppressor processes or DNA damage repair, and the inactivation of specific pathways support the role of SIRT1 as a tumor promoter. The role of  SIRT1  in the initiation, promotion, and progression of several malignant tumors including prostate cancer (Jung-Hynes, B. et al 2009), breast cancer (Jin, X., et al 2018), lung cancer (Han, L. et al 2013) and gastric cancer (Han, L. et al 2013) are well documented. Wilking el al showed in his in vitro experiments that the inhibition of SIRT1 by treatment with small molecule SIRT1 inhibitors determines a significant decrease in cell growth, proliferation and viability (Wilking, M. J., et al 2014).

How it is Measured or Detected

	Method/ measurement reference	Reliability	Strength of evidence	Assay fit for purpose	Repeatability/ reproducibility	Direct measure
Human tissues	qRT-PCR,Western blotting,Luciferase reporter assay H2,H4,H7,H8,H9 Micro-array (Shen ZL et al 2016)	yes	Strong	Yes	Yes	Yes
Human cell lines	Micro-array, qRT-PCR,Western blotting,Luciferase reporter assay (Guo S et al 2020, Bae HJ et al 2014, Zhou J et al 2017, Fu H et al 2018, Lian B et al 2018 Guan Y et al 2017 Yang X et al 2014)	yes	Strong	Yes	Yes	Yes
Mouse	qRT-PCR,Western blotting,Luciferase reporter assay,ELISA,cell culture Bai XZ et al 2018	yes	Moderate	Yes	Yes	Yes

References

Bae, H. J., Noh, J. H., Kim, J. K., Eun, J. W., Jung, K. H., Kim, M. G., ... & Nam, S. W. (2014). MicroRNA-29c functions as a tumor suppressor by direct targeting oncogenic SIRT1 in hepatocellular carcinoma. Oncogene, 33(20), 2557-2567. Bae, H. J., Noh, J. H., Kim, J. K., Eun, J. W., Jung, K. H., Kim, M. G., ... & Nam, S. W. (2014). MicroRNA-29c functions as a tumor suppressor by direct targeting oncogenic SIRT1 in hepatocellular carcinoma. Oncogene, 33(20), 2557-2567. Bai, X. Z., Zhang, J. L., Liu, Y., Zhang, W., Li, X. Q., Wang, K. J., ... & Hu, D. H. (2018). MicroRNA-138 aggravates inflammatory responses of macrophages by targeting SIRT1 and regulating the NF-κB and AKT pathways. Cellular Physiology and Biochemistry, 49(2), 489-500. Bradbury, C. A., Khanim, F. L., Hayden, R., Bunce, C. M., White, D. A., Drayson, M. T., ... & Turner, B. M. (2005). Histone deacetylases in acute myeloid leukaemia show a distinctive pattern of expression that changes selectively in response to deacetylase inhibitors. Leukemia, 19(10), 1751-1759. Carafa, V., Nebbioso, A., & Altucci, L. (2012). Sirtuins and disease: the road ahead. Frontiers in pharmacology, 3, 4. Chen, J., Zhang, B., Wong, N., Lo, A. W., To, K. F., Chan, A. W., ... & Ko, B. C. (2011). Sirtuin 1 is upregulated in a subset of hepatocellular carcinomas where it is essential for telomere maintenance and tumor cell growth. Cancer research, 71(12), 4138-4149.. Deng, C. X. (2009). SIRT1, is it a tumor promoter or tumor suppressor?. International journal of biological sciences, 5(2), 147. Duan, K., Ge, Y. C., Zhang, X. P., Wu, S. Y., Feng, J. S., Chen, S. L., ... & Fu, C. H. (2015). miR-34a inhibits cell proliferation in prostate cancer by downregulation of SIRT1 expression. Oncology letters, 10(5), 3223-3227. Fang, Y., & Nicholl, M. B. (2011). Sirtuin 1 in malignant transformation: friend or foe?. Cancer letters, 306(1), 10-14. Fu, H., Song, W., Chen, X., Guo, T., Duan, B., Wang, X., ... & Zhang, C. (2018). MiRNA-200a induce cell apoptosis in renal cell carcinoma by directly targeting SIRT1. Molecular and cellular biochemistry, 437(1), 143-152. Guan, Y., Rao, Z., & Chen, C. (2018). miR-30a suppresses lung cancer progression by targeting SIRT1. Oncotarget, 9(4), 4924. Guo, S., Ma, B., Jiang, X., Li, X., & Jia, Y. (2020). Astragalus polysaccharides inhibits tumorigenesis and lipid metabolism through miR-138-5p/SIRT1/SREBP1 pathway in prostate cancer. Frontiers in Pharmacology, 11, 598. Haigis, M. C., & Guarente, L. P. (2006). Mammalian sirtuins—emerging roles in physiology, aging, and calorie restriction. Genes & development, 20(21), 2913-2921. Han, L., Liang, X. H., Chen, L. X., Bao, S. M., & Yan, Z. Q. (2013). SIRT1 is highly expressed in brain metastasis tissues of non-small cell lung cancer (NSCLC) and in positive regulation of NSCLC cell migration. International journal of clinical and experimental pathology, 6(11), 2357. Hao, C., Zhu, P. X., Yang, X., Han, Z. P., Jiang, J. H., Zong, C., ... & Wei, L. X. (2014). Overexpression of SIRT1 promotes metastasis through epithelial-mesenchymal transition in hepatocellular carcinoma. BMC cancer, 14(1), 1-10. Hida, Y., Kubo, Y., Murao, K., & Arase, S. (2007). Strong expression of a longevity-related protein, SIRT1, in Bowen’s disease. Archives of dermatological research, 299(2), 103-106. Huffman, D. M., Grizzle, W. E., Bamman, M. M., Kim, J. S., Eltoum, I. A., Elgavish, A., & Nagy, T. R. (2007). SIRT1 is significantly elevated in mouse and human prostate cancer. Cancer research, 67(14), 6612-6618. Jiang, G., Wen, L., Zheng, H., Jian, Z., & Deng, W. (2016). miR‐204‐5p targeting SIRT1 regulates hepatocellular carcinoma progression. Cell biochemistry and function, 34(7), 505-510. Jiang, H., Khan, S., Wang, Y., Charron, G., He, B., Sebastian, C., ... & Lin, H. (2013). SIRT6 regulates TNF-α secretion through hydrolysis of long-chain fatty acyl lysine. Nature, 496(7443), 110-113. Jin, X., Wei, Y., Xu, F., Zhao, M., Dai, K., Shen, R., ... & Zhang, N. (2018). SIRT1 promotes formation of breast cancer through modulating Akt activity. Journal of Cancer, 9(11), 2012. Jung-Hynes, B., Nihal, M., Zhong, W., & Ahmad, N. (2009). Role of sirtuin histone deacetylase SIRT1 in prostate cancer: a target for prostate cancer management via its inhibition?. Journal of Biological Chemistry, 284(6), 3823-3832.. Kojima, K., Fujita, Y., Nozawa, Y., Deguchi, T., & Ito, M. (2010). MiR‐34a attenuates paclitaxel‐resistance of hormone‐refractory prostate cancer PC3 cells through direct and indirect mechanisms. The Prostate, 70(14), 1501-1512. Lian, B., Yang, D., Liu, Y., Shi, G., Li, J., Yan, X., ... & Zhang, R. (2018). miR-128 targets the SIRT1/ROS/DR5 pathway to sensitize colorectal cancer to TRAIL-induced apoptosis. Cellular Physiology and Biochemistry, 49(6), 2151-2162. Longo, V. D., & Kennedy, B. K. (2006). Sirtuins in aging and age-related disease. Cell, 126(2), 257-268. Luo, J., Chen, P., Xie, W., & Wu, F. (2017). MicroRNA-138 inhibits cell proliferation in hepatocellular carcinoma by targeting Sirt1. Oncology reports, 38(2), 1067-1074. McGuinness, D., McGuinness, D. H., McCaul, J. A., & Shiels, P. G. (2011). Sirtuins, bioageing, and cancer. Journal of aging research, 2011. Michan, S., & Sinclair, D. (2007). Sirtuins in mammals: insights into their biological function. Biochemical Journal, 404(1), 1-13. Michishita, E., Park, J. Y., Burneskis, J. M., Barrett, J. C., & Horikawa, I. (2005). Evolutionarily conserved and nonconserved cellular localizations and functions of human SIRT proteins. Molecular biology of the cell, 16(10), 4623-4635. Saunders, L. R., & Verdin, E. (2007). Sirtuins: critical regulators at the crossroads between cancer and aging. Oncogene, 26(37), 5489-5504. Sauve, A. A. (2010). Sirtuin chemical mechanisms. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics, 1804(8), 1591-1603.. Shen, Z. L., Wang, B., Jiang, K. W., Ye, C. X., Cheng, C., Yan, Y. C., ... & Wang, S. (2016). Downregulation of miR-199b is associated with distant metastasis in colorectal cancer via activation of SIRT1 and inhibition of CREB/KISS1 signaling. Oncotarget, 7(23), 35092. Shi, Y., Zhou, Y., Wang, S., & Zhang, Y. (2013). Sirtuin deacetylation mechanism and catalytic role of the dynamic cofactor binding loop. The journal of physical chemistry letters, 4(3), 491-495. Shuang, T., Wang, M., Zhou, Y., & Shi, C. (2015). Over-expression of Sirt1 contributes to chemoresistance and indicates poor prognosis in serous epithelial ovarian cancer (EOC). Medical oncology, 32(12), 1-7. Stünkel, W., Peh, B. K., Tan, Y. C., Nayagam, V. M., Wang, X., Salto‐Tellez, M., ... & Wood, J. (2007). Function of the SIRT1 protein deacetylase in cancer. Biotechnology Journal: Healthcare Nutrition Technology, 2(11), 1360-1368. Tian, Z., Jiang, H., Liu, Y., Huang, Y., Xiong, X., Wu, H., & Dai, X. (2016). MicroRNA-133b inhibits hepatocellular carcinoma cell progression by targeting Sirt1. Experimental cell research, 343(2), 135-147. van Leeuwen, I., & Lain, S. (2009). Sirtuins and p53. Advances in cancer research, 102, 171-195. Vaquero, A. (2009). The conserved role of sirtuins in chromatin regulation. International Journal of Developmental Biology, 53(2-3), 303-322. Vaziri, H., Dessain, S. K., Eaton, E. N., Imai, S. I., Frye, R. A., Pandita, T. K., ... & Weinberg, R. A. (2001). hSIR2SIRT1 functions as an NAD-dependent p53 deacetylase. Cell, 107(2), 149-159. Wang, C., Yang, W., Dong, F., Guo, Y., Tan, J., Ruan, S., & Huang, T. (2017). The prognostic role of Sirt1 expression in solid malignancies: a meta-analysis. Oncotarget, 8(39), 66343. Wang, X., Yang, B., & Ma, B. (2016). The UCA1/miR-204/Sirt1 axis modulates docetaxel sensitivity of prostate cancer cells. Cancer chemotherapy and pharmacology, 78(5), 1025-1031. Wilking, M. J., Singh, C., Nihal, M., Zhong, W., & Ahmad, N. (2014). SIRT1 deacetylase is overexpressed in human melanoma and its small molecule inhibition imparts anti-proliferative response via p53 activation. Archives of biochemistry and biophysics, 563, 94-100. Yan, X., Liu, X., Wang, Z., Cheng, Q., Ji, G., Yang, H., ... & Pei, X. (2019). MicroRNA4865p functions as a tumor suppressor of proliferation and cancer stemlike cell properties by targeting Sirt1 in liver cancer. Oncology reports, 41(3), 1938-1948. Yang, X., Yang, Y., Gan, R., Zhao, L., Li, W., Zhou, H., ... & Meng, Q. H. (2014). Down-regulation of mir-221 and mir-222 restrain prostate cancer cell proliferation and migration that is partly mediated by activation of SIRT1. PloS one, 9(6), e98833. Yao, C., Liu, J., Wu, X., Tai, Z., Gao, Y., Zhu, Q., ... & Gao, S. (2016). Reducible self-assembling cationic polypeptide-based micelles mediate co-delivery of doxorubicin and microRNA-34a for androgen-independent prostate cancer therapy. Journal of Controlled Release, 232, 203-214. Zhang S, Zhang D, Yi C, Wang Y, Wang H, Wang J. (2016). MicroRNA-22 functions as a tumor suppressor by targeting SIRT1 in renal cell carcinoma. Oncol Rep. 35(1), 559-67.  Zhang, Z. Y., Hong, D., Nam, S. H., Kim, J. M., Paik, Y. H., Joh, J. W., ... & Kim, S. J. (2015). SIRT1 regulates oncogenesis via a mutant p53-dependent pathway in hepatocellular carcinoma. Journal of hepatology, 62(1), 121-130. Zhang, S., Huang, S., Deng, C., Cao, Y., Yang, J., Chen, G., ... & Zou, X. (2017). Co-ordinated overexpression of SIRT1 and STAT3 is associated with poor survival outcome in gastric cancer patients. Oncotarget, 8(12), 18848. Zhou, J., Zhou, W., Kong, F., Xiao, X., Kuang, H., & Zhu, Y. (2017). microRNA34a overexpression inhibits cell migration and invasion via regulating SIRT1 in hepatocellular carcinoma Corrigendum in/10.3892/ol. 2019.11048. Oncology letters, 14(6), 6950-6954.           Zwaans, B. M., & Lombard, D. B. (2014). Interplay between sirtuins, MYC and hypoxia-inducible factor in cancer-associated metabolic reprogramming. Disease models & mechanisms, 7(9), 1023-1032.

 

Event: 1172: Increase activation, Nuclear factor kappa B (NF-kB)

Short Name: Increase activation, Nuclear factor kappa B (NF-kB)

Key Event Component

AOPs Including This Key Event

Stressors

Biological Context

Cell term

Organ term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

The ROS directly influences NF-κB signalling, resulting in differential production of cytokines and chemokines (McKay and Cidlowski, 1999; Pernis, 2007). NF-κB regulates pro-inflammatory responses that are transcriptionally mediated by NF‑κB.

Key Event Description

The NF-kB pathway consists of a series of events where the transcription factors of the NF-kB family play a key role. The proinflammatory cytokine (IL-1beta) can be activated by NF-kB , including Reactive Oxygen Species produced by  NADPH oxidase (NOX). Upon pathway activation, the IKK complex will be phosphorylated, which in turn phosphorylates IkBa. There, this transcription factor can express pro-inflammatory and pro-fibrotic genes. This can be achieved by ROS, IKK enhancer or nuclear translocation enhancer. 

How it is Measured or Detected

NF-kB transcriptional activity: Beta lactamase reporter gene assay (Miller et al. 2010). NF-kB transcription: Lentiviral NF-kB GFP reporter with flow cytometry (Moujalled et al. 2012)

NF-κB translocation: RelA-GFP reporter assay (Frederiksson 2012) (Huppelschoten 2017)

IκBa phosphorylation: Western blotting (Miller et al. 2010)

NF-κB p65 (Total/Phospho) ELISA

ELISA for IL-6, IL-8, and Cox

Event: 112: Antagonism, Estrogen receptor

Short Name: Antagonism, Estrogen receptor

Key Event Component

AOPs Including This Key Event

Biological Context

Cell term

Domain of Applicability

Taxonomic applicability: Steroid receptors, including ER are thought to have evolved in the chordate lineage (Baker 1997, 2003; Thornton 2001). An ER ortholog has been isolated from a mollusk species, but no ER orthologs have been detected in arthropods or nematodes (Thornton et al. 2003). Broadly speaking, most vertebrates can be expected to have functional ERs, while most invertebrates do not, although there may be exceptions within the mollusk lineage and evolutionarily-related organisms.

Key Event Description

Site of action: The site of action for the molecular initiating event is the liver (hepatocytes).

Responses at the macromolecular level: Estrogen receptor antagonists have been shown to interact with the ligand binding domain of ERs. However, those interactions occur at different contact sites than those of estrogen agonists, leading to a different conformation in the transactivation domain (Brzozowski et al. 1997; Katzenellenbogen 1996).

Characterization of chemical properties: Two broad categories of ER antagonists have been described. Type I, like tamoxifen act as mixed agonists and antagonists. Type II, like ICI164384 are pure antagonists (Katzenellenbogen 1996). Due to their potential utility for treating estrogen-dependent breast cancers and other estrogen-related disease states as well as concerns regarding endocrine disruption, there is an extensive body of literature on the identification and design of chemical structures that act as ER antagonists (e.g., (Brooks et al. 1987; Brooks and Skafar 2004; Lloyd et al. 2006; Sodero et al. 2012; Vedani et al. 2012; Wang et al. 2006).

How it is Measured or Detected

• The BG1luc estrogen receptor transactivation test method for identifying estrogen receptor agonists and antagonists (OECD Test Guideline 457) has been validated by the National Toxicology Program Interagency Center for Evaluation of Alternative Toxicological Methods (NICEATM) and Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) as an appropriate assay for detecting ER antagonism. (OECD, 2012b).
• Other human ER-based transactivation assays that have been used to detect ERα antagonism include the T47D-Kbluc assay (Wilson et al. 2004); ERα CALUX assay (van der Burg et al. 2010); MELN assay (Witters et al. 2010); and the yeast estrogen screen (YES; (De Boever et al. 2001)). Each of these assays have undergone some level of validation.
• In aquatic ecotoxicology, vitellogenin synthesis in primary fish liver cells and liver slices has also been used to screen for anti-estrogenic activity (e.g., (Bickley et al. 2009; Navas and Segner 2006; Schmieder et al. 2000; Schmieder et al. 2004; Sun et al. 2010). Although these approaches have generally not been subject to as much formal validation as human ER-based transactivation assays, in the case of fish-specific AOPs linked to this key event, these measures of anti-estrogenicity may be more directly relevant to predicting other key events in the pathway.

References

• Brzozowski AM, Pike AC, Dauter Z, Hubbard RE, Bonn T, Engstrom O, et al. 1997. Molecular basis of agonism and antagonism in the oestrogen receptor. Nature 389:753-758.
• Katzenellenbogen B. 1996. Estrogen receptors: Bioactivities and interactions with cell signaling pathways. Biology of Reproduction 54:287-293.
• Brooks SC, Wappler NL, Corombos JD, Doherty LM, Horwitz JP. 1987. Estrogen structure-fuction relationships. Berlin:Walter de Gruyter & Co., 443-466.
• Brooks SC, Skafar DF. 2004. From ligand structure to biological activity: Modified estratrienes and their estrogenic and antiestrogenic effects in mcf-7 cells. Steroids 69:401-418.
• Lloyd DG, Smith HM, O'Sullivan T, Knox AS, Zisterer DM, Meegan MJ. 2006. Antiestrogenically active 2-benzyl-1,1-diarylbut-2-enes: Synthesis, structure-activity relationships and molecular modeling study for flexible estrogen receptor antagonists. Medicinal chemistry 2:147-168.
• Sodero AC, Romeiro NC, da Cunha EF, de Oliveira Magalhaaes U, de Alencastro RB, Rodrigues CR, et al. 2012. Application of 4d-qsar studies to a series of raloxifene analogs and design of potential selective estrogen receptor modulators. Molecules 17:7415-7439.
• Vedani A, Dobler M, Smiesko M. 2012. Virtualtoxlab - a platform for estimating the toxic potential of drugs, chemicals and natural products. Toxicology and applied pharmacology 261:142-153.
• Wang CY, Ai N, Arora S, Erenrich E, Nagarajan K, Zauhar R, et al. 2006. Identification of previously unrecognized antiestrogenic chemicals using a novel virtual screening approach. Chemical research in toxicology 19:1595-1601.
• Denny JS, Tapper MA, Schmieder PK, Hornung MW, Jensen KM, Ankley GT, et al. 2005. Comparison of relative binding affinities of endocrine active compounds to fathead minnow and rainbow trout estrogen receptors. Environmental toxicology and chemistry / SETAC 24:2948-2953.
• Lee HK, Kim TS, Kim CY, Kang IH, Kim MG, Jung KK, et al. 2012. Evaluation of in vitro screening system for estrogenicity: Comparison of stably transfected human estrogen receptor-alpha transcriptional activation (oecd tg455) assay and estrogen receptor (er) binding assay. The Journal of toxicological sciences 37:431-437.
• Rider CV, Hartig PC, Cardon MC, Lambright CR, Bobseine KL, Guillette LJ, Jr., et al. 2010. Differences in sensitivity but not selectivity of xenoestrogen binding to alligator versus human estrogen receptor alpha. Environmental toxicology and chemistry / SETAC 29:2064-2071.
• OECD. 2012b. Test no. 457: Bg1luc estrogen receptor transactivation test method for identifying estrogen receptor agonists and antagonists:OECD Publishing.
• Wilson VS, Bobseine K, Gray LE, Jr. 2004. Development and characterization of a cell line that stably expresses an estrogen-responsive luciferase reporter for the detection of estrogen receptor agonist and antagonists. Toxicological sciences : an official journal of the Society of Toxicology 81:69-77.
• van der Burg B, Winter R, Weimer M, Berckmans P, Suzuki G, Gijsbers L, et al. 2010. Optimization and prevalidation of the in vitro eralpha calux method to test estrogenic and antiestrogenic activity of compounds. Reproductive toxicology 30:73-80.
• Witters H, Freyberger A, Smits K, Vangenechten C, Lofink W, Weimer M, et al. 2010. The assessment of estrogenic or anti-estrogenic activity of chemicals by the human stably transfected estrogen sensitive meln cell line: Results of test performance and transferability. Reproductive toxicology 30:60-72.
• De Boever P, Demare W, Vanderperren E, Cooreman K, Bossier P, Verstraete W. 2001. Optimization of a yeast estrogen screen and its applicability to study the release of estrogenic isoflavones from a soygerm powder. Environmental health perspectives 109:691-697.
• Bickley LK, Lange A, Winter MJ, Tyler CR. 2009. Evaluation of a carp primary hepatocyte culture system for screening chemicals for oestrogenic activity. Aquatic toxicology 94:195-203.
• Navas JM, Segner H. 2006. Vitellogenin synthesis in primary cultures of fish liver cells as endpoint for in vitro screening of the (anti)estrogenic activity of chemical substances. Aquatic toxicology 80:1-22.
• Schmieder P, Tapper M, Linnum A, Denny J, Kolanczyk R, Johnson R. 2000. Optimization of a precision-cut trout liver tissue slice assay as a screen for vitellogenin induction: Comparison of slice incubation techniques. Aquatic toxicology 49:251-268.
• Schmieder PK, Tapper MA, Denny JS, Kolanczyk RC, Sheedy BR, Henry TR, et al. 2004. Use of trout liver slices to enhance mechanistic interpretation of estrogen receptor binding for cost-effective prioritization of chemicals within large inventories. Environmental science & technology 38:6333-6342.
• Sun L, Wen L, Shao X, Qian H, Jin Y, Liu W, et al. 2010. Screening of chemicals with anti-estrogenic activity using in vitro and in vivo vitellogenin induction responses in zebrafish (danio rerio). Chemosphere 78:793-799.
• Baker ME. 1997. Steroid receptor phylogeny and vertebrate origins. Molecular and cellular endocrinology 135:101-107.
• Baker ME. 2003. Evolution of adrenal and sex steroid action in vertebrates: A ligand-based mechanism for complexity. BioEssays : news and reviews in molecular, cellular and developmental biology 25:396-400.
• Thornton JW. 2001. Evolution of vertebrate steroid receptors from an ancestral estrogen receptor by ligand exploitation and serial genome expansions. Proceedings of the National Academy of Sciences of the United States of America 98:5671-5676.
• Thornton JW, Need E, Crews D. 2003. Resurrecting the ancestral steroid receptor: Ancient origin of estrogen signaling. Science 301:1714-1717.

Event: 1457: Induction, Epithelial Mesenchymal Transition

Short Name: EMT

AOPs Including This Key Event

Biological Context

Key Event Description

Inflammatory reactions result in the release of various cytokines which in turn can stimulate the transition of epithelial cells to a mesenchymal phenotype acquiring function characteristics of fibroblasts and myofibroblasts.

How it is Measured or Detected

Loss of E-cadherin and cell polarity is considered to be a fundamental event in epithelial-mesenchymal transition. The simultaneous expression of epithelial (e.g. E-cadherin) and mesenchymal markers (e.g. N-cadherin and vimentin) within the airway epithelium are indicative for ongoing transition (Borthwick et al. 2009, 2010).

References

Borthwick, L. A., Parker, S. M., Brougham, K. A., Johnson, G. E., Gorowiec, M. R., Ward, C., … Fisher, A. J. (2009). Epithelial to mesenchymal transition (EMT) and airway remodelling after human lung transplantation. Thorax, 64(9), 770–777. https://doi.org/10.1136/thx.2008.104133

Borthwick, L. A., McIlroy, E. I., Gorowiec, M. R., Brodlie, M., Johnson, G. E., Ward, C., … Fisher, A. J. (2010). Inflammation and epithelial to mesenchymal transition in lung transplant recipients: Role in dysregulated epithelial wound repair. American Journal of Transplantation, 10(3), 498–509. https://doi.org/10.1111/j.1600-6143.2009.02953.x

List of Adverse Outcomes in this AOP

Event: 1982: metastatic breast cancer

Short Name: Metastasis, Breast Cancer

AOPs Including This Key Event

Stressors

Biological Context

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Increased metastasis of cancerous cells  is known to be highly conserved throughout evolution and is present from humans to invertebrates.

Key Event Description

Biological state: 

Dissemination of the cancer cells from one organ to another which is not directly connected to the primary site is called metastasis. It has a crucial role in the prognosis of cancer patients. In the initial stage of metastasis, cancer cells detach from the primary tumor and disseminate in the tissue. Subsequently cancer cells enter the vascular or lymphatic channels (23-25). The establishment of micro-metastasis mainly depends on the survival of the circulating tumor cells (CTCs) inside the lymphatic or blood channels. Extravasation of cancer cells through the vessel wall takes place resulting in the proliferation of cancer cells in the secondary site.  Various signalling pathways are involved in each of the above mentioned   process. Few theories have been proposed to explain the mechanism of metastasis.  The “organ selection concept” theory suggests that the growth factors establish a successful metastasis in the metastatic site (26,27) whereas the “adhesion theory” proposes the tissue specific adhesion molecules are expressed on endothelial cells of recipient organs  which will anchor the migrating cancer cells,  providing the a pre-metastatic niche. The role of chemokine receptor has been explained in “chemo-attraction theory” while Paget   reported the theory of “seed” for metastatic tumor cells and of “soil” for the secondary site. As per this concept the organ distribution is determined by the site and histopathological type of the primary tumor.  The recent understanding suggested, pre-metastatic niche has been indicated to explain metastasis.  It is interesting to note that prior to co-localization, the primary tumor induces the micro environment of secondary site by CTCs.

 Subsequently, a metastatic niche is generated to support disseminated tumor cells (DTCs) and localize them to develop a metastasis.  The most recent theory describes a bidirectional relationship between the primary and secondary sites. According to this theory, the surviving cancer cells in the metastatic tumor can return to the primary site to promote the primary tumor progression (28,29). Efficient and  direct  blood  flow  can  explain  the  probability  of metastasis  to  the  specific  organs  like  hepatic  metastasis in patients  with colon cancer which  receive direct blood  flow  from  the  primary  site .Vascular  permeability is also the other factor  which  significantly  promotes extravasation  at  the metastatic  site. However at present, understanding of molecular mechanisms of metastasis remains incomplete.

Biological compartment

Organs,Cellular

Role in general biology

Epithelial-  mesenchymal  transition  (EMT) and its reverse  mesenchymal-epithelial  transition (MET)  are characteristics  of cellular  plasticity  during embryogenesis  and  tumor metastasis  (30).  There has been decreased expression of  E-cadherin  and  β-catenin  and  elevated  expression  levels of  vimentin,  fibronectin  and  N-cadherin in EMT   (31).  In cancers, EMT  is a major  process  by which  cancer cells  lose their epithelial  characteristics  to acquire mesenchymal-like properties.  Tumor cell  migration  is a pre-requisite for the metastatic process in which, EMT is  the most critical step to  initiate  metastasis including metastasis to  lymph nodes  (32).  During   EMT, cancer  cells lose their  cell-to-cell junctions and cellular  polarity via multiple  signaling pathways which  increase  the motilities and invasive phenotype of them (33). Cleavage of  E-cadherin mediated by the MMPs  increases  the tumor cell  motility and invasion . Apart from this ,EMT has a  key role  in  drug resistance.  This is supported by the finding that  high levels of vimentin was found  in adriamycin and  vinblastine  resistant  breast  cancer cell  lines  (34). EMT  promotes CSCs  motility, cancer cell invasion, tumor  metastasis and recurrence and drug resistance.   Expression  of  stem cell like  markers  and formation of tumor spheres by CSCs are enhanced by EMT  process.  CSCs acquire mesenchymal  features by undergoing EMT phenomenon. By acquiring mesenchymal features,  CSCs become resistant  to anti-cancer therapies; hence, they can  survive and cause cancer recurrence.  In addition to this ,CSCs invade to the adjacent  stromal  tissues, enter the  vascular channels,  and  finally  reach  the  distant  organs.  In  the target organs, CSCs  cause MET phenomenon  which results in the acquisition of  epithelial  characteristics.  MET  phenomenon also   increases the  cell-to-cell attachment, cancer cells proliferation and differentiation to form  metastatic lesions  (35).  Altogether , EMT induces  CSC properties   and metastatic  activities. On the other hand, EMT  and CSCs collaborate in invasion capacity   hence targeting  the EMT/CSC  phenotype can be a therapeutic  approach for the treatment of metastasis and tumor recurrence (36).

 

EMT programs are regulated by a network of signal- ling pathways that involve components such as growth factors (transforming growth factor-β [TGF-β], epider- mal growth factor [EGF]) and their associated signalling proteins (Wnt, Notch, Hedgehog, nuclear-factor kappa B [NF-κB], extracellular signal-regulated kinase [ERK], and phosphatidylinositol 3-kinase [PI3K]/Akt) in response to stresses involved in tumorigenesis, including hypoxia, oncogenic or metabolic stress, inflammation, and physical constraints [37-41].

These signals activate EMT-inducing transcription factors, including Snail/Slug, ZEB1/δEF1, ZEB2/SIP1, Twist1/ 2, and E12/E47 [42-44]. EMT-inducing transcription factors regulate the expression of proteins involved in cell polarity, cell-cell contact, cytoskeletal structural maintenance, and extracellular matrix (ECM) degradation, and they sup- press key epithelial genes. Loss of E-cadherin is considered a hallmark of EMT; these EMT-inducing transcription factors bind to E-box elements in the E-cadherin gene promoter to repress its transcription. Of particular note, Snail is an early marker of EMT that is involved in the initial cell-migratory phenotype, and it occasionally induces other factors .

 

During EMT, epithelial cells reorganize cytoskeleton and resolve cell–cell junctions, which are accompanied with switching off the expression of epithelial markers and turning on mesenchymal genes. Although changes in epithelial and mesenchymal markers during EMT can vary significantly in different biologic contexts, a network of transcription factors, including TWIST1/2, SNAIL1/2, ZEB1/2, and FOXC2, are consistently required to orchestrate the EMT program (45). The expression of these transcription factors is associated with poor prognosis and distant metastasis in various human cancers has been documented in various studies. (46). Besides its role in promoting tumor cell invasion, EMT is shown to confer tumor cells with resistance to apoptosis  and anoikis (47), thus allowing cell survival in the blood stream after intravasation. EMT could also facilitate tumor cells' escape from the senescence program, especially through TWIST1 and ZEB1 (48,49). Furthermore, EMT has been shown to  cancer cells with cancer stem cell (CSC)–like features, which further aid tumor dormancy and chemo resistance (50,51).Tumor samples or experimental tumor xenograft models have provided convincing evidence for the activation of EMT in various primary epithelial tumors in various studies. . Interestingly, more recent studies reveal a dynamic requirement of EMT in tumor metastasis: activation of EMT promotes local tumor invasion, intravasation, and extravasation of the systemic circulation, whereas reversion of EMT is essential to establish macrometatasis in distant organs (52,53).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

How it is Measured or Detected

	Method/ measurement reference	Reliability	Strength of evidence	Assay fit for purpose	Repeatability/ reproducibility	Direct measure
Cell line,humans,Human cell line studies	qRT-PCR,,Luciferase reporter assay ,immunoblotting,immunoprecipitation,cell invasion assay,cell migration assay, bioluminesence imaging,wound healing assay,Wound scratch & Transwell assay, Microarray,Immunofluorescence, Immunohistochemistry,	+	Strong	Yes	Yes	Yes

References

Anastas JN, Moon RT. (2013) WNT signalling pathways as therapeutic targets in cancer. Nat Rev Cancer. 13(1):11–26.

Ansieau S, Bastid J, Doreau A, Morel A-P, Bouchet BP, Thomas C, et al. (2008) Induction of EMT by TWIST proteins as a collateral effect of tumor- promoting inactivation of premature senescence. Cancer Cell. 14: 79–89.

Da  C,  Wu  K,  Yue  C,  Bai  P,  Wang  R,  Wang  G,  et al. (2017) N-cadherin promotes thyroid  tumorigenesis  through modulating  major signaling pathways. Oncotarget. 8:8131-8142.

Huang  R,  Zong X.  (2017) Aberrant  cancer metabolism  in epithelialmesenchymal  transition and cancer metastasis: Mechanisms in cancer progression.  Crit Rev Oncol Hematol.115:1322.

Irani S,  Dehghan  A.  (2018) The expression  and  functional significance  of  vascular  endothelial-cadherin,  CD44,  and vimentin in oral  squamous cell  carcinoma. J Int  Soc Prev Community Dent. 8:408-417.

Ishiwata  T.(2016) Cancer  stem cells and epithelial-mesenchymal transition:  Novel therapeutic targets for  cancer. Pathol Int.66:601-608.

Jolly MK,  Tripathi SC, Jia D,  Mooney SM,  Celiktas  M,  Hanash SM,  et al. (2016) Stability  of the hybrid epithelial/mesenchymal phenotype. Oncotarget.7:27067-27084.

Lamouille S, Xu J, Derynck R.(2014) Molecular mechanisms of epithelial-mesenchymal transition. Nat Rev Mol Cell Biol. 15(3):178–96.

Liu  S,  Ye  D,  Guo  W,  Yu  W,  He  Y,  Hu  J,  et al. (2015) G9a is essential for  EMT-mediated  metastasis and maintenance of  cancer  stem cell-like characters  in head and neck  squamous cell  carcinoma. Oncotarget . 6:6887-6901.

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Appendix 2

List of Key Event Relationships in the AOP