55-18-5WBNQDOYYEUMPFS-UHFFFAOYSA-NWBNQDOYYEUMPFS-UHFFFAOYSA-N
N-NitrosodiethylamineDEN
Ethanamine, N-ethyl-N-nitroso-
Diethylamine, N-nitroso-
Diethylnitrosamide
Diethylnitrosoamin
Diethylnitrosoamine
dietilnitrosoamina
N,N-Diethylnitrosoamine
N-Ethyl-N-nitrosoethanamine
Nitrosodiethylamine
N-NITROSODIAETHYLAMIN
N-Nitroso-N,N-diethylamine
NSC 132
diethylnitrosamine
DTXSID202102864-67-5DENRZWYUOJLTMF-UHFFFAOYSA-NDENRZWYUOJLTMF-UHFFFAOYSA-N
Diethyl sulfateSulfuric acid, diethyl ester
DIAETHYLSULFAT
diethyl sulphate
Diethylsulfat
NSC 56380
Sulfate de diethyle
sulfato de dietilo
Sulfuric acid diethyl ester
UN 1594
DTXSID102404562-75-9UMFJAHHVKNCGLG-UHFFFAOYSA-NUMFJAHHVKNCGLG-UHFFFAOYSA-N
N-NitrosodimethylamineDMN
Methanamine, N-methyl-N-nitroso-
DIMETHYLAMINE, N-NITROSO-
Dimethylnitrosoamin
Dimethylnitrosoamine
dimetilnitrosoamina
Nitrosodimethylamine
Nitrosodimetilamina
N-Methyl-N-nitrosomethanamine
N-NITROSODIMETHYLAMIN
N-Nitroso-N,N-dimethylamine
NSC 23226
Dimethylnitrosamine
DTXSID702102977-78-1VAYGXNSJCAHWJZ-UHFFFAOYSA-NVAYGXNSJCAHWJZ-UHFFFAOYSA-N
Dimethyl sulfateSulfuric acid, dimethyl ester
Dimethyl monosulfate
dimethyl sulphate
Dimethylsulfat
DIMETHYLSULFATE
NSC 56194
Sulfate de dimethyle
sulfato de dimetilo
Sulfuric acid dimethyl ester
SULFURIC ACID DIMETHYLESTER
UN 1595
DTXSID502405562-50-0PLUBXMRUUVWRLT-UHFFFAOYSA-NPLUBXMRUUVWRLT-UHFFFAOYSA-N
Ethyl methanesulfonateEMS
Methanesulfonic acid, ethyl ester
Ethyl mesylate
Ethyl methane sulfonate
ethyl methanesulphonate
Ethylmethansulfonat
metanosulfonato de etilo
Methanesulfonate d'ethyle
Methanesulfonic acid ethyl ester
METHANSULFONSAEURE-AETHYLESTER
METHYLSULFONATE, ETHYL
NSC 26805
O-Ethyl methylsulfonate
DTXSID6025309759-73-9FUSGACRLAFQQRL-UHFFFAOYSA-NFUSGACRLAFQQRL-UHFFFAOYSA-N
1-Ethyl-1-nitrosoureaENU
Urea, N-ethyl-N-nitroso-
N-AETHYL-N-NITROSO-HARNSTOFF
N-Ethyl-N-nitrosoharnstoff
N-ethyl-N-nitrosourea
N-Ethyl-N-nitrosouree
N-etil-N-nitrosourea
N-Nitroso-N-ethylurea
NSC 45403
Urea, 1-ethyl-1-nitroso-
DTXSID802059363885-23-4ZGONASGBWOJHDD-UHFFFAOYSA-NZGONASGBWOJHDD-UHFFFAOYSA-N
N′-Ethyl-N-nitro-N-nitrosoguanidineDTXSID3020592926-06-7SWWHCQCMVCPLEQ-UHFFFAOYSA-NSWWHCQCMVCPLEQ-UHFFFAOYSA-N
Isopropyl methanesulfonateDTXSID803149766-27-3MBABOKRGFJTBAE-UHFFFAOYSA-NMBABOKRGFJTBAE-UHFFFAOYSA-N
Methyl methanesulfonateMMS
Methanesulfonic acid, methyl ester
metanosulfonato de metilo
Methanesulfonate de methyle
methyl methanesulphonate
Methyl methylsulfonate
Methylmethansulfonat
METHYLSULFONATE, METHYL
NSC 50256
DTXSID702084570-25-7VZUNGTLZRAYYDE-UHFFFAOYSA-NVZUNGTLZRAYYDE-UHFFFAOYSA-N
Methylnitronitrosoguanidine1-Methyl-3-nitro-1-nitroso-guanidine
Guanidine, N-methyl-N'-nitro-N-nitroso-
1-Methyl-1-nitroso-2-nitroguanidine
1-Methyl-1-nitroso-3-nitroguanidine
1-Methyl-3-nitro-1-nitrosoguanidin
1-METHYL-3-NITRO-1-NITROSO-GUANIDIN
1-Methyl-3-nitro-1-nitrosoguanidine
1-metil-3-nitro-1-nitrosoguanidina
1-Nitroso-3-nitro-1-methylguanidine
Guanidine, 1-methyl-3-nitro-1-nitroso-
Methylnitronitrosoguanidine
N-Methyl-N1-nitro-N-nitrosoguanidine
N-Methyl-nitroso-N'-nitroguanidine
N-Methyl-N'-nitro-N-nitrosoguanadine
N-Methyl-N'-nitro-N-nitrosoquanidine
N-Methyl-N-nitroso-N'-nitroguanidine
N-Nitroso-N-methylnitroguanidine
N-Nitroso-N-methyl-N'-nitroguanidine
N-Nitroso-N'-nitro-N-methylguanidine
NSC 9369
DTXSID2020846CHEBI:16991deoxyribonucleic acidD009369NeoplasmsGO:0006281DNA repairD009154mutationGO:0006305DNA alkylation7functional change1increasedIonizing Radiation<p>Ionizing radiation can vary in energy, dose, charge, and in the spatial distributions of energy transferred to other matter (linear energy transfer per unit length or LET) (ICRU 1970). At the same dose, low and high LET both generate energy deposition events, including many higher energy events (Goodhead and Nikjoo 1989). However, they differ in the spatial distribution and upper range of intensity of energy deposited. Lower LET such as gamma rays sparsely deposit many individual excitations or small clusters of excitations of low energy (Goodhead 1988). In contrast, high LET such as alpha particles have fewer tracks but readily transfer their energy to matter and therefore deposit their energy over a much smaller area (Goodhead 1994). Consequently, alpha and other high LET particles penetrate less deeply into tissue, interactions are densely focused on a narrow track, and individual energy depositions can be large (Goodhead 1988). These different energy deposition patterns can lead to differences in radiation effects including the pattern of DNA damage.</p>
<p>Exposure to ionizing radiation can come from natural and industrial sources. Space and terrestrial radiation includes a range of LET particles, while diagnostic radiation methods such as X-ray imaging, mammography and CT scans use low LET X-rays. Radiation therapy can use an external beam to direct radiation on a focused tissue area, or deposit solid or liquid radioactive materials in the body that release (mostly gamma) radiation internally. External radiotherapy typically uses X-rays but is moving towards higher LET charged particles such as protons and heavy ions (Durante, Orecchia et al. 2017).</p>
2019-05-03T12:36:362019-05-07T12:12:13Diethyl nitrosamine2016-11-29T18:42:092016-11-29T18:42:09Diethyl sulfate2016-11-29T18:42:112016-11-29T18:42:11Dimethyl nitrosamine2016-11-29T18:42:112016-11-29T21:19:02Dimethyl sulfate2016-11-29T18:42:132016-11-29T18:42:13Ethyl methanesulfonate2016-11-29T18:42:142016-11-29T18:42:14Ethyl nitrosourea2016-11-29T18:42:142016-11-29T18:42:14Ethyl-N'-nitro-N-nitrosoguanidine2016-11-29T18:42:152016-11-29T18:42:15Isopropyl methanesulfonate2016-11-29T18:42:172016-11-29T18:42:17Methyl methanesulfonate2016-11-29T18:42:192016-11-29T18:42:19Methyl-l-N'-nitro-N-nitroguanidine2016-11-29T18:42:192016-11-29T18:42:1910090mouse10116rat10036Syrian golden hamster9606Homo sapiens9913cow10090Mus musculus8090medaka10116Rattus norvegicusWCS_9606humanInadequate DNA repairInadequate DNA repairCellular<p>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:</p>
<ol>
<li><strong>Damage reversal</strong> 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.</li>
<li><strong>Excision repair</strong> 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:
<p style="margin-left:40px"><strong>a) Base excision repair (BER)</strong><span style="font-size:1rem"> (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. </span>This leads to an intermediate that contains a DNA strand break, whereby DNA ligase is then recruited to seal the ends of the DNA.</p>
<p style="margin-left:40px"><strong>b) Nucleotide excision repair (NER)</strong> (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 and sealing of the ends by DNA ligase. </p>
<p style="margin-left:40px"><strong>c) Mismatch repair (MMR)</strong> (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.</p>
<p style="margin-left:40px">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). </p>
</li>
<li><strong>Single strand break repair (SSBR) </strong>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 where a common DNA intermediate as BER was generated, 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, although end processing is generally done by polynucleotide kinase. 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). </li>
<li><strong>Double strand break repair (DSBR)</strong> 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 the S phase of dividing cell types, and nonhomologous end joining (NHEJ), which can function in both dividing and non-dividing cell types. No repair occurs in the M phase (Teruaki Iyama and David M. Wilson III, 2013). DNA repair in mitosis is controversial (Mladenov et al., 2023).</li>
</ol>
<p style="margin-left:40px">Complex lesions can be created by a single mutagen and can be more difficult to repair, as the mechanism behind how different repair pathways cooperate to address this is still unclear (Aleksandrov et al., 2018). 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.</p>
<p style="margin-left:40px">The process of C-NHEJ in humans requires at least seven core proteins: Ku70, Ku86, DNA-dependent protein kinase complex (DNA-PK<sub>cs </sub>), 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<sub>cs</sub>, the catalytic subunit, thus forming a trimeric complex on the ends of the DNA strands. Alternative NHEJ, or alt NHEJ, uses small similar sequences in two broken DNA ends to join them together. Unlike the usual repair method (cNHEJ), aNHEJ doesn't need specific proteins like LIG4 and KU. Instead, it relies on the MRN complex to process the breaks. However, alt NHEJ tends to cause mutations by adding or removing bits of DNA during the repair (Chaudhuri and Nussenzweig, 2017). The kinase activity of DNA-PK<sub>cs </sub>is then triggered, causing DNA-PK<sub>cs </sub>to auto-phosphorylate and thereby lose its kinase activity; the now phosphorylated DNA-PK<sub>cs</sub> dissociates from the DNA-bound Ku proteins. The free DNA-PK<sub>cs</sub> phosphorylates Artemis, an enzyme that possesses 5’-3’ exonuclease and endonuclease activity in the presence of DNA-PK<sub>cs</sub> 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).</p>
<p style="margin-left:40px">The process of alt-NHEJ is less well understood than C-NHEJ and is a lower fidelity mechanism. Alt-NHEJ is known to involve slightly different core proteins than C-NHEJ and required microhomology repeats, 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). </p>
<p style="margin-left:40px">In contrast to NHEJ, HR takes advantage of similar or identical DNA sequences to repair DSBs and is not error-prone (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.</p>
<p> </p>
<p><strong><u>Fidelity of DNA Repair</u></strong></p>
<p><br />
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).</p>
<p>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.</p>
<p>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).</p>
<p>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). </p>
<p>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).</p>
<p>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).</p>
<p><u><strong>Indirect Measurement</strong></u></p>
<p>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.</p>
<p>Some EXAMPLES are given below for alkylated DNA.</p>
<p>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 (shape of dose-response curve) between exposure to mutagenic agents and mutations provide an indication of whether the removal of adducts occurs, and whether it is more efficient at low doses. Sub-linear dose-response curves (hockey stick or j-shape curves) for mutation induction indicates that adducts are not converted to mutations at low doses. This suggests the effective repair of adducts at low doses, followed by saturation of repair at higher doses (Clewell et al., 2019). Thus, measurement of a clear point of inflection in the dose-response curve for mutations suggests that repair does occur, at least to some extent, at low dosees but that reduced repair efficiency arises above the inflection point. 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.</p>
<p>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).</p>
<p>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.</p>
<p><br />
<u><strong>Direct Measurement</strong></u></p>
<p>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.</p>
<p><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:12px">Please refer to the table below for additional details and methodologies for detecting DNA damage and repair.</span></span></p>
<table border="1" cellpadding="1" cellspacing="1" style="height:2082px; width:629px">
<tbody>
<tr>
<td style="background-color:#eeeeee; text-align:center"><span style="font-size:14px"><strong>Assay Name</strong></span></td>
<td style="background-color:#eeeeee; text-align:center"><span style="font-size:14px"><strong>References</strong></span></td>
<td style="background-color:#eeeeee; text-align:center"><span style="font-size:14px"><strong>Description</strong></span></td>
<td style="background-color:#eeeeee; text-align:center"><span style="font-size:14px"><strong>DNA Damage/Repair Being Measured</strong></span></td>
<td style="background-color:#eeeeee; text-align:center"><span style="font-size:14px"><strong>OECD Approved Assay</strong></span></td>
</tr>
<tr>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Dose-Response Curve for Alkyl Adducts/ Mutations</span></span></td>
<td>
<p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Lutz 1991</span></span></p>
<p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif"> </span></span></p>
<p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Clewell 2016</span></span></p>
</td>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">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</span></span></td>
<td>
<p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Alkylation,</span></span></p>
<p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">oxidative damage, or DSBs</span></span></p>
</td>
<td style="text-align:center"><span style="font-size:14px">N/A</span></td>
</tr>
<tr>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Retention of Alkyl Adducts</span></span></td>
<td>
<p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Seiler 1997</span></span></p>
<p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif"> </span></span></p>
<p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Scherer 1987</span></span></p>
</td>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Examination of DNA for alkylation after exposure to an alkylating agent; Presence of alkylation suggests a lack of repair</span></span></td>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Alkylation</span></span></td>
<td style="text-align:center"><span style="font-size:14px">N/A</span></td>
</tr>
<tr>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Mutation Spectrum</span></span></td>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Wyrick 2015</span></span></td>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">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</span></span></td>
<td>
<p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Alkylation,</span></span></p>
<p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">oxidative damage, or DSBs</span></span></p>
</td>
<td style="text-align:center"><span style="font-size:14px">N/A</span></td>
</tr>
<tr>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">DSB Repair Assay (Reporter constructs)</span></span></td>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:times new roman,serif"><span style="font-family:arial,sans-serif">Mao</span></span><span style="font-family:arial,sans-serif"> et al., 2011</span></span></td>
<td style="text-align:center"><span style="font-size:14px">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</span></td>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">DSBs</span></span></td>
<td style="text-align:center"><span style="font-size:14px">N/A</span></td>
</tr>
<tr>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Primary Rat Hepatocyte DNA Repair Assay</span></span></td>
<td>
<p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Jeffrey and Williams, 2000</span></span></p>
<p style="text-align:center"><span style="font-size:14px"><u><span style="font-family:arial,sans-serif"> </span></u></span></p>
<p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Butterworth et al., 1987</span></span></p>
</td>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Rat primary hepatocytes are cultured with a <sup>3</sup>H-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 <sup>3</sup>H incorporated in the DNA post-repair</span></span></td>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Unscheduled DNA synthesis in response to DNA damage</span></span></td>
<td style="text-align:center"><span style="font-size:14px">N/A</span></td>
</tr>
<tr>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Repair synthesis measurement by </span><sup><span style="font-family:arial,sans-serif">3</span></sup><span style="font-family:arial,sans-serif">H-thymine incorporation</span></span></td>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Iyama and Wilson, 2013</span></span></td>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Measure DNA synthesis in non-dividing cells as indication of gap filling during excision repair</span></span></td>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Excision repair</span></span></td>
<td style="text-align:center"><span style="font-size:14px">N/A</span></td>
</tr>
<tr>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Comet Assay with Time-Course</span></span></td>
<td>
<p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Olive et al., 1990</span></span></p>
<p style="text-align:center"><span style="font-size:14px"><u><span style="font-family:arial,sans-serif"> </span></u></span></p>
<p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Trucco et al., 1998</span></span></p>
<p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">-</span></span></p>
<p style="text-align:center">Dunkenberger et al., 2022 </p>
</td>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Comet assay is performed with a time-course </span></span>under alkaline conditions to detect SSBs and DSBs.<span style="font-size:14px"><span style="font-family:arial,sans-serif"> Quantity of DNA in the tail should decrease as DNA repair progresses</span></span></td>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">DSBs</span></span></td>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif"> </span><span style="font-family:times new roman,serif"><a href="https://read.oecd-ilibrary.org/environment/test-no-489-in-vivo-mammalian-alkaline-comet-assay_9789264264885-en"><span style="font-family:arial,sans-serif">Yes</span></a></span><u><span style="font-family:arial,sans-serif"> (No. 489)</span></u></span></td>
</tr>
<tr>
<td style="text-align:center">Flow Cytometry </td>
<td>Corneo et al., 2007 </td>
<td style="text-align:center">The alt-NHEJ flow cytometer method involves utilizing an extrachromosomal substrate. Green fluorescent protein (GFP) expression is indicative of successful alt-NHEJ activity, contingent on the removal of 10 nucleotides from each end of the DNA and subsequent rejoining within a 9-nucleotide microhomology region. This approach provides a quantitative and visual means to measure the efficiency of alternative non-homologous end joining in cellular processes. </td>
<td style="text-align:center">Alt NHEJ</td>
<td style="text-align:center">No</td>
</tr>
<tr>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Pulsed Field Gel Electro-phoresis (PFGE) with Time-Course</span></span></td>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:times new roman,serif"><span style="font-family:arial,sans-serif">Biedermann</span></span><u><span style="font-family:arial,sans-serif"> </span></u><span style="font-family:arial,sans-serif">et al., 1991</span></span></td>
<td style="text-align:center"><span style="font-size:14px">PFGE assay with a time-course; Quantity of small DNA fragments should decrease as DNA repair progresses</span></td>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">DSBs</span></span></td>
<td style="text-align:center"><span style="font-size:14px">N/A</span></td>
</tr>
<tr>
<td>
<p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Fluorescence -Based Multiplex Flow-Cytometric Host Reactivation Assay </span></span></p>
<p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">(FM-HCR)</span></span></p>
</td>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Nagel et al., 2014</span></span></td>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">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</span></span></td>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">HR, NHEJ, BER, NER, MMR, or MGMT</span></span></td>
<td style="text-align:center"><span style="font-size:14px">N/A</span></td>
</tr>
<tr>
<td><span style="font-size:14px">Alkaline Unwinding Assay with Time Course </span></td>
<td style="text-align:center"><span style="font-size:14px">Nacci et al. 1991 </span></td>
<td style="text-align:center"><span style="font-size:14px">DNA is stored in alkaline solutions with DNA-specific dye and allowed to unwind following removal from tissue, increased strand damage associated with increased unwinding. Samples analyzed at different time points to compare remaining damage following repair opportunities </span></td>
<td style="text-align:center"><span style="font-size:14px">DSBs </span></td>
<td style="text-align:center"><span style="font-size:14px">Yes (<u><span style="font-family:arial,sans-serif">No. 489)</span></u> </span></td>
</tr>
<tr>
<td><span style="font-size:14px">Sucrose Density Gradient Centrifugation with Time Course </span></td>
<td style="text-align:center"><span style="font-size:14px">Larsen et al. 1982 </span></td>
<td style="text-align:center"><span style="font-size:14px">Strand breaks alter the molecular weight of the DNA piece. DNA in alkaline solution centrifuged into sugar density gradient, repeated set time apart. The less DNA breaks identified in the assay repeats, the more repair occurred </span></td>
<td style="text-align:center"><span style="font-size:14px">SSBs </span></td>
<td style="text-align:center"><span style="font-size:14px">N/A</span></td>
</tr>
<tr>
<td><span style="font-size:14px">y-H2AX Foci Staining with Time Course </span></td>
<td style="text-align:center">
<p><span style="font-size:14px">Mariotti et al. 2013 </span></p>
<p><span style="font-size:14px">Penninckx et al. 2021 </span></p>
</td>
<td style="text-align:center"><span style="font-size:14px">Histone H2AX is phosphorylated in the presence of DNA strand breaks, the rate of its disappearance over time is used as a measure of DNA repair </span></td>
<td style="text-align:center"><span style="font-size:14px">DSBs </span></td>
<td style="text-align:center"><span style="font-size:14px">N/A</span></td>
</tr>
<tr>
<td><span style="font-size:14px">Alkaline Elution Assay with Time Course </span></td>
<td style="text-align:center"><span style="font-size:14px">Larsen et al. 1982 </span></td>
<td style="text-align:center"><span style="font-size:14px">DNA with strand breaks elute faster than DNA without, plotted against time intervals to determine the rate at which strand breaks repair </span></td>
<td style="text-align:center"><span style="font-size:14px">SSBs </span></td>
<td style="text-align:center"><span style="font-size:14px">N/A</span></td>
</tr>
<tr>
<td><span style="font-size:14px">53BP1 foci Detection with Time Course </span></td>
<td style="text-align:center"><span style="font-size:14px">Penninckx et al. 2021 </span></td>
<td style="text-align:center"><span style="font-size:14px">53BP1 is recruited to the site of DNA damage, the rate at which its level decreases over time is used to measure DNA repair </span></td>
<td style="text-align:center"><span style="font-size:14px">DSBs</span></td>
<td style="text-align:center"><span style="font-size:14px">N/A </span></td>
</tr>
</tbody>
</table>
<p> </p>
<p>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.</p>
<p>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<sub>cs</sub> 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).</p>
<p><strong>Taxonomic applicability:</strong> Inadequate DNA repair is applicable to all species, as they all contain DNA (White & Vijg, 2016). </p>
<p><strong>Life stage applicability:</strong> This key event is not life stage specific as any life stage can have poor repair, though as individuals age their repair process become less effective (Gorbunova & Seluanov, 2016). </p>
<p><strong>Sex applicability: </strong>There is no evidence of sex-specificity for this key event, with initial rate of DNA repair not significantly different between sexes (Trzeciak et al., 2008). </p>
<p><strong>Evidence for perturbation by a stressor: </strong>Multiple studies demonstrate that inadequate DNA repair can occur as a result of stressors such as ionizing and non-ionizing radiation, as well as chemical agents (Kuhne et al., 2005; Rydberg et al., 2005; Dahle et al., 2008; Seager et al., 2012; Wilhelm, 2014; O’Brien et al., 2015). </p>
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2016-11-29T18:41:232024-03-08T12:15:51Increase, MutationsIncrease, MutationsMolecular<p><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">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. </span></span></p>
<p><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">Missense mutations are the substitution of one base in the codon with another. This change is significant because the three bases in a codon code for a specific amino acid and the new combination may signal for a different amino acid to be formed. Nonsense mutations also result from changes to the codon bases, but in this case, they cause the generation of a stop codon in the DNA strand where there previously was not one. This stop codon takes the place of a normal coding triplet, preventing its translation into an amino acid. This will cause the translation of the strand to prematurely stop. Both missense and nonsense mutations can result from substitutions, insertions, or deletions of bases (Chakarov et al. 2014). </span></span></p>
<p><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">Insertion and deletion mutations are the addition and removal of bases from the strand, respectively. These often accompany a frameshift mutation, as the alteration in the number of bases in the strand causes the frame of the base reader to shift by the added or reduced number, altering the amino acids that are produced if that number is not devisable by three. Codons come in specific orders, sectioned into groups of three. When the boundaries of which three bases are included in one group are changed, this can change the whole transcriptional output of the strand (Chakaroy et al. 2014). </span></span></p>
<p><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">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.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">Mutations can occur in somatic cells or germ cells (sperm or egg).</span></span></p>
<p>Mutations can be measured using a variety of both OECD and non-OECD mutagenicity tests. Listed below are common methods for detecting the KE, however there may be other comparable methods that are not listed.</p>
<p><strong>Somatic cells:</strong> 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).</p>
<p>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).</p>
<p>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-/-.</p>
<p>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.</p>
<p>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.</p>
<p>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).</p>
<p><br />
<strong>Germ cells:</strong> 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).</p>
<p>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 been made as of 2022 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.</p>
<p>A similar transgenic assay can be used in transgenic medaka (Norris and Winn, 2010).</p>
<p><br />
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).</p>
<p><span style="font-size:14px"><span style="font-family:arial,sans-serif">Please refer to the table below for additional details and methodologies for measuring mutations. </span></span></p>
<table border="1" cellpadding="1" cellspacing="1" style="height:2351px; width:633px">
<tbody>
<tr>
<td style="background-color:#eeeeee; text-align:center">A<strong>ssay Name</strong></td>
<td style="background-color:#eeeeee; text-align:center"><strong>References </strong></td>
<td style="background-color:#eeeeee; text-align:center"><strong>Description </strong></td>
<td style="background-color:#eeeeee; text-align:center"><strong>OECD Approved Assay</strong></td>
</tr>
<tr>
<td><span style="font-family:arial,sans-serif; font-size:11pt">Assorted Gene Loci Mutation Assays</span></td>
<td>
<p style="text-align:center"><span style="font-family:arial,sans-serif; font-size:11pt">Tindall et al., 1989; <span style="font-family:arial,sans-serif; font-size:11pt"><span style="font-family:times new roman,serif; font-size:12pt"><span style="font-family:arial,sans-serif; font-size:11pt">Kruger</span></span><span style="font-family:arial,sans-serif; font-size:11pt"> et al., 2015</span></span></span></p>
</td>
<td><span style="font-family:arial,sans-serif; font-size:11pt">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</span></td>
<td>N/A</td>
</tr>
<tr>
<td><span style="font-family:arial,sans-serif; font-size:11pt">TK Mutation Assay</span></td>
<td>
<p style="text-align:center"><span style="font-family:arial,sans-serif; font-size:11pt">Yamamoto et al., 2017; <span style="font-family:arial,sans-serif; font-size:11pt"><span style="font-family:arial,sans-serif; font-size:11pt">Liber et al., 1982; <span style="font-family:arial,sans-serif; font-size:11pt"><span style="font-family:arial,sans-serif; font-size:11pt">Lloyd and Kidd, 2012</span></span></span></span></span></p>
</td>
<td><span style="font-family:arial,sans-serif; font-size:11pt">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</span></td>
<td><span style="font-family:arial,sans-serif; font-size:11pt">Yes (No. 490)</span></td>
</tr>
<tr>
<td><span style="font-family:arial,sans-serif; font-size:11pt">HPRT Mutation Assay</span></td>
<td>
<p style="text-align:center"><span style="font-family:arial,sans-serif; font-size:11pt">Ayres et al., 2006; <span style="font-family:arial,sans-serif; font-size:11pt"><span style="font-family:arial,sans-serif; font-size:11pt">Parry and Parry, 2012</span></span></span></p>
</td>
<td><span style="font-family:arial,sans-serif; font-size:11pt">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</span></td>
<td><span style="font-family:arial,sans-serif; font-size:11pt">Yes (No. 476)</span></td>
</tr>
<tr>
<td><span style="font-family:arial,sans-serif; font-size:11pt">Salmonella Mutagenicity Test (Ames Test)</span></td>
<td><span style="font-family:arial,sans-serif; font-size:11pt">OECD, 1997</span></td>
<td>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 </td>
<td><span style="font-family:arial,sans-serif; font-size:11pt">Yes (No. 471)</span></td>
</tr>
<tr>
<td><span style="font-family:arial,sans-serif; font-size:11pt">PIG-A / PIG-O Assay</span></td>
<td>
<p style="text-align:center"><span style="font-family:arial,sans-serif; font-size:11pt">Kruger et al., 2015; <span style="font-family:arial,sans-serif; font-size:11pt"><span style="font-family:arial,sans-serif; font-size:11pt">Nakamura, 2012; <span style="font-family:arial,sans-serif; font-size:11pt"><span style="font-family:arial,sans-serif; font-size:11pt">Chikura, 2019</span></span></span></span></span></p>
</td>
<td>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 <em>in vitro</em> exposure, or by flow cytometry of blood samples after <em>in vivo </em>exposure</td>
<td><span style="font-family:arial,sans-serif; font-size:11pt">N/A</span></td>
</tr>
<tr>
<td><span style="font-family:arial,sans-serif; font-size:11pt">Single Molecule PCR</span></td>
<td>
<p style="text-align:center"><span style="font-family:arial,sans-serif; font-size:11pt">Kraytsberg & Khrapko, 2005; <span style="font-family:arial,sans-serif; font-size:11pt"><span style="font-family:arial,sans-serif; font-size:11pt">Yauk, 2002</span></span></span></p>
</td>
<td><span style="font-family:arial,sans-serif; font-size:11pt">This PCR technique uses a single DNA template, and is often employed for detection of mutations in microsatellites, recombination studies, and generation of polonies</span></td>
<td><span style="font-family:arial,sans-serif; font-size:11pt">N/A</span></td>
</tr>
<tr>
<td><span style="font-family:arial,sans-serif; font-size:11pt">ACB-PCR</span></td>
<td>
<p>Myers et al., 2014 (Textbook, pg 345-363); Banda et al., 2013; Banda et al., 2015; Parsons et al., 2017</p>
</td>
<td><span style="font-family:arial,sans-serif; font-size:11pt">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 </span></td>
<td><span style="font-family:arial,sans-serif; font-size:11pt">N/A</span></td>
</tr>
<tr>
<td><span style="font-family:arial,sans-serif; font-size:11pt">Transgenic Rodent Mutation Assay </span></td>
<td>
<p style="text-align:center"><span style="font-family:arial,sans-serif; font-size:11pt">OECD 2013; <span style="font-family:arial,sans-serif; font-size:11pt"><span style="font-family:arial,sans-serif; font-size:11pt">Lambert 2005; <span style="font-family:arial,sans-serif; font-size:11pt"><span style="font-family:arial,sans-serif; font-size:11pt">Lambert 2009</span></span></span></span></span></p>
</td>
<td>This <em>in vivo</em> test detects gene mutations using transgenic rodents that possess transgenes and reporter genes; After<em> in vivo</em> exposure to a chemical/mutagen, the transgenes are analyzed by transfecting bacteria with the reporter gene and examining the resulting phenotype</td>
<td><span style="font-family:arial,sans-serif; font-size:11pt">Yes (No. 488)</span></td>
</tr>
<tr>
<td><span style="font-family:arial,sans-serif; font-size:11pt">Conditionally inducible transgenic mouse models</span></td>
<td><span style="font-family:arial,sans-serif; font-size:11pt">Parsons 2018 (Review)</span></td>
<td><span style="font-family:arial,sans-serif; font-size:11pt">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 </span></td>
<td>N/A</td>
</tr>
<tr>
<td><span style="font-family:arial,sans-serif; font-size:11pt">Error</span><span style="font-family:arial,sans-serif; font-size:12pt">-</span><span style="font-family:arial,sans-serif; font-size:11pt">Corrected Next Generation Sequencing (NGS)</span></td>
<td><span style="font-family:arial,sans-serif; font-size:11pt">Salk 2018 (Review)</span></td>
<td><span style="font-family:arial,sans-serif; font-size:11pt">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) </span></td>
<td>N/A </td>
</tr>
</tbody>
</table>
<p> </p>
<p><strong>Taxonomic applicability:</strong> 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.</p>
<p><strong>Life stage applicability:</strong> This key event is not life stage specific as all stages of life have DNA that can be mutated; however, baseline levels of mutations are seen to increase with age (Slebos et al., 2004; Kirkwood, 1989). </p>
<p><strong>Sex applicability:</strong> This key event is not sex specific as both sexes undergo mutations. Males have a higher mutation rate than females (Hedrick, 2007). </p>
<p><strong>Evidence for perturbation by a stressor:</strong> Many studies demonstrate that increased mutations can occur as a result of ionizing radiation (Sankaranarayanan & Nikjoo, 2015; Russell et al., 1957; Winegar et al., 1994; Gossen et al., 1995). </p>
HighUnspecificHighAll life stagesHighModerateHighModerate<p>Adewoye, A.B. et al. (2015), "The genome-wide effects of ionizing radiation on mutation induction in the mammalian germline", <em>Nat. Commu.</em>, 6:6684. Doi: 10.1038/ncomms7684.</p>
<p>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”, <em>Genetics and Molecular Biology</em>. 2(3): 558-561. Doi:10.1590/S1415-4757200600030002.</p>
<p>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”, <em>Environ Mol Mutagen</em>. 54(8):659-67. Doi:10.1002/em.21808.</p>
<p>Banda, M. et al. (2015), “Quantification of Kras mutant fraction in the lung DNA of mice exposed to aerosolized particulate vanadium pentoxide by inhalation”, <em>Mutat Res Genet Toxicol Environ Mutagen</em>. 789-790:53-60. Doi: 10.1016/j.mrgentox.2015.07.003</p>
<p>Campbell, C.D. & E.E. Eichler (2013), "Properties and rates of germline mutations in humans", <em>Trends Genet</em>., 29(10): 575-84. Doi: 10.1016/j.tig.2013.04.005</p>
<p>Chakarov, S. et al. (2014), “DNA damage and mutation. Types of DNA damage”, BioDiscovery, Vol.11, Pensoft Publishers, Sofia, https://doi.org/10.7750/BIODISCOVERY.2014.11.1.</p>
<p>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”, <em>Genes Environ</em>. 27:41-5. Doi: 10.1186/s41021-019-0121-z.</p>
<p>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.</p>
<p>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", <em>Proceedings of the National Academy of Sciences of the United States of America</em>, 92(16): 7485-7489. Doi: 10.1073/pnas.92.16.7485.</p>
<p>Gossen, J.A. et al. (1995), "Spontaneous and X-ray-induced deletion mutations in a LacZ plasmid-based transgenic mouse model", Mutation Research, 331/1, Elsevier, Amsterdam, https://doi.org/10.1016/0027-5107(95)00055-N. </p>
<p>Hedrick, P.W. (2007), “Sex: Differences In Mutation, Recombination, Selection, Gene Flow, And Genetic Drift”, Evolution, Vol.61/12, Wiley, Hoboken, https://doi.org/10.1111/j.1558-5646.2007.00250.x. </p>
<p>Kirkwood, T.B.L. (1989), “DNA, mutations and aging”, Mutation Research, Vol.219/1, Elsevier B.V., Amsterdam, https://doi.org/10.1016/0921-8734(89)90035-0</p>
<p>Kraytsberg,Y. & Khrapko, K. (2005), “Single-molecule PCR: an artifact-free PCR approach for the analysis of somatic mutations”, <em>Expert Rev Mol Diagn</em>. 5(5):809-15. Doi: 10.1586/14737159.5.5.809.</p>
<p>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”, <em>Arch Toxicol</em>. 89(12), 2429-43. Doi: 10.1007/s00204-014-1413-5.</p>
<p>Lambert, I.B. et al. (2005), "Detailed review of transgenic rodent mutation assays", <em>Mutat Res.</em>, 590(1-3):1-280. Doi: 10.1016/j.mrrev.2005.04.002.</p>
<p>Liber, L. H., & Thilly, G. W. (1982), “Mutation assay at the thymidine kinase locus in diploid human lymphoblasts”, <em>Mutation Research</em>. 94: 467-485. Doi:10.1016/0027-5107(82)90308-6.</p>
<p>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.</p>
<p>Myers, M. B. et al., (2014), “ACB-PCR Quantification of Somatic Oncomutation”, <em>Molecular Toxicology Protocols, Methods in Molecular Biology</em>. DOI: 10.1007/978-1-62703-739-6_27</p>
<p>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”, <em>PLoS One</em>.7(3): e33563. Doi:10.1371/journal.pone.0033563.</p>
<p>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.</p>
<p>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.</p>
<p>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", <em>Mutat. Res</em>., 741-742:11-7. Doi: 10.1016/j.mrfmmm.2013.02.004.</p>
<p>O'Brien, J.M. et al. (2014), "Transgenic rodent assay for quanitifying male germ cell mutation frequency", <em>Journal of Visual Experimentation</em>, Aug 6;(90). Doi: 10.3791/51576.</p>
<p>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", <em>Environ. Mol. Mutagen.</em>, 6(4): 347-355. Doi: 10.1002/em.21932.</p>
<p>OECD (2020), Test No. 471: Bacterial Reverse Mutation Test, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris.</p>
<p>OECD (2016), Test No. 476: In vitro Mammalian Cell Gene Mutation Test, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris.</p>
<p>OECD (2009), Detailed Review Paper on Transgenic Rodent Mutation Assays, Series on Testing and Assessment, N° 103, ENV/JM/MONO 7, OECD, Paris.</p>
<p>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.</p>
<p>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.</p>
<p>OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris.</p>
<p>Parry MJ, & Parry ME. 2012. Genetic Toxicology Principles and Methods. Humana Press. Springer Protocols.</p>
<p>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.</p>
<p>Parsons BL. Multiclonal tumor origin: Evidence and implications<em>. Mutat Res</em>. 2018. 777:1-18. doi: 10.1016/j.mrrev.2018.05.001.</p>
<p>Russell, W.L. et al. (1957), "Radiation Dose Rate and Mutation Frequency.", Science, Vol.128/3338, American Association for the Advancement of Science, Washington, https://doi.org/10.1126/science.128.3338.1546.</p>
<p>Salk JJ, Schmitt MW, &Loeb LA. (2018), “Enhancing the accuracy of next-generation sequencing for detecting rare and subclonal mutations”, <em>Nat Rev Genet</em>. 19(5):269-285. Doi: 10.1038/nrg.2017.117.</p>
<p>Sankaranarayanan, K. & H. Nikjoo (2015), "Genome-based, mechanism-driven computational modeling of risks of ionizing radiation: The next frontier in genetic risk estimation?", Mutation Research, Vol.764, Elsevier, Amsterdam, https://doi.org/10.1016/j.mrrev.2014.12.003. </p>
<p>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" <em>Front. Genet.</em>, 6: 215. Doi: 10.3389/fgene.2015.00215.</p>
<p>Singer, T.M. and C.L. Yauk CL (2010), "Germ cell mutagens: risk assessment challenges in the 21st century", <em>Environ. Mol. Mutagen.</em>, 51(8-9): 919-928. Doi: 10.1002/em.20613.</p>
<p>Slebos, R.J.C. et al. (2004), “Mini-and microsatellite mutations in children from Chernobyl accident cleanup workers”, Mutation Research/Genetic Toxicology and Environmental Mutagenesis, Vol.559/1-2, Elsevier, Amsterdam, https://doi.org/10.1016/j.mrgentox.2004.01.003. </p>
<p>Tindall, R. K., & Stankowski Jr., F. L. (1989), “Molecular analysis of spontaneous mutations at the GPT locus in Chinese hamster ovary (AS52) cells”, <em>Mutation Research</em>, 220, 241-53. Doi: 10.1016/0165-1110(89)90028-6.</p>
<p>Waters, M.D. et al. (1994), "The performance of short-term tests in identifying potential germ cell mutagens: a qualitative and quantitative analysis", <em>Mutat. Res.</em>, 341(2): 109-31. Doi: 10.1016/0165-1218(94)90093-0.</p>
<p>Winegar, R.A. et al. (1994), "Radiation-induced point mutations, deletions and micronuclei in lacI transgenic mice", Mutation Research, Vol.307/2, Elsevier, Amsterdam, https://doi.org/10.1016/0027-5107(94)90258-5. </p>
<p>Yamamoto, A. et al. (2017), “Radioprotective activity of blackcurrant extract evaluated by in vitro micronucleus and gene mutation assays in TK6 human lymphoblastoid cells”,<em> Genes and Environment. </em>39: 22. Doi: 10.1186/s41021-017-0082-z.</p>
<p>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.</p>
<p>Yauk, C.L. et al. (2015), "Approaches for Identifying Germ Cell Mutagens: Report of the 2013 IWGT Workshop on Germ Cell Assays", <em>Mutat. Res. Genet. Toxicol. Environ. Mutagen.</em>, 783: 36-54. Doi: 10.1016/j.mrgentox.2015.01.008.</p>
<p>Yeat and J.C. Lin. 2015. Clinical applications of next generation sequencing in cancer: from panels, to exomes, to genomes. <em>Front. Genet</em>., 6: 215. Doi: 10.3389/fgene.2015.00215.</p>
2016-11-29T18:41:232023-05-15T08:47:43Increase, CancerIncrease, CancerTissue<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Cancer is a general key event for related diseases each exhibiting uncontrolled proliferation of abnormal cells (for review see Hanahan and Weinberg 2011). A cancer often is initially associated with a specific organ, with malignant tumors developing ability to metastasize, or travel to other areas of the body. Most cancers develop from genetic mutations in normal cells, although a minority of cancers are hereditary. Exposure to chemical stressors, radiation, tobacco smoke, or viruses can increase the likelihood that cancer will develop.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Cancer cells proliferate due to capabilities summarized by Hanahan and Weinberg (2011): </span></span></p>
<ol>
<li><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Sustained proliferation signaling – by deregulating normal cell signals, cancer cells can sustain chronic proliferation.</span></span></li>
<li><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Evading growth suppressors – by evading activities of tumor suppressor genes, cancer cells continue to proliferate.</span></span></li>
<li><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Activating invasion and metastasis – by altering shape and attachment to cells in the extracellular matrix, cancer cells gain ability to move to other locations.</span></span></li>
<li><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Enabling replicative immortality – by disabling senescence pathways, cancer cells have extended lifespans.</span></span></li>
<li><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Inducing angiogenesis – by enabling neovasculature, cancer cells receive nutrients and oxygen and get rid of waste products.</span></span></li>
<li><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Resisting cell death – by evading apotosis and necrosis defense pathways, cancer cells avoid elimination.</span></span></li>
</ol>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Most carcinogenicity studies are conducted with rodents (see OECD 2018; Zhou et al. 2023 for methods) or in-vitro with mammalian cell lines (see OECD 2023 for methods). Cancer is usually detected by biopsy or histopathological examination of tissue. Gene expression levels can also be assessed, as increased transcription of known genes have been associated with specific cancers (ex. Tumor Necrosis Factor (Pavet et al. 2014); Heat Shock Factors (Vihervaara and Sistonen 2014; Androgen Receptor (Heinlein and Chang 2004)).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt">Life Stage:</span> All life stages. Older individuals are more likely to manifest this key event (adults > juveniles > embryos).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt">Sex: A</span>pplies to both males and females.</span></span></p>
<p><span style="font-size:12.0pt"><span style="font-family:"Calibri",sans-serif">Taxonomic:</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"> Appears to be present broadly, with representative studies including mammals (humans, lab mice, lab rats), teleost fish, and invertebrates (cladocerans, mussels).</span></span></p>
HighUnspecificHighAll life stagesHighHighHigh<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Abraha, A.M. and Ketema, E.B. 2016. Apoptotic pathways as a therapeutic target for colorectal cancer treatment. World Journal of Gastrointestinal Oncology 8 (8): 583-491</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">European Parliament. 2022. Directive 2004/37/EC of the European Parliament on the protection of workers from the risks related to exposure to carcinogens, mutagens or reprotoxic substances at work. Retrieved 3 August 2023 from http://data.europa.eu/eli/dir/2004/37/2022-04-05</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Hanahan, D. and Weinberg, R.A. 2011. Hallmarks of cancer: the next generation. Cell 144(5): 646-674.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Heinlein, C.A. and Chang, C. 2004. Androgen receptor in prostate cancer. Endocrine Reviews 25: 276-308.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">OECD. 2018. Test no. 451: OECD Guideline for the Testing of Chemicals: Carcinogenicity Studies. OECD Publishing, Paris. Retrieved 3 August 2023 from <a href="https://www.oecd.org/env/test-no-451-carcinogenicity-studies-9789264071186-en.htm" style="color:#0563c1; text-decoration:underline">https://www.oecd.org/env/test-no-451-carcinogenicity-studies-9789264071186-en.htm</a></span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">OECD. 2023. Test No. 487: In Vitro Mammalian Cell Micronucleus Test, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris. Retrieved 3 August 2023 from <a href="https://doi.org/10.1787/9789264264861-en.htm" style="color:#0563c1; text-decoration:underline">https://doi.org/10.1787/9789264264861-en.htm</a></span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">OSHA. 2023. Carcinogens. Retrieved 3 August 2023 from <a href="https://www.osha.gov/carcinogens/standards" style="color:#0563c1; text-decoration:underline">https://www.osha.gov/carcinogens/standards</a></span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Pavet, V., Shlyakhtina, Y., He, T., Ceschin, D.G., Kohonen, P., Perala, M., Kallioniemi, O., and Gronemeyer, H. 2014. Plasminogen activator urokinase expression reveals TRAIL responsiveness and support fractional survival of cancer cells. Cell Death and Disease 5: e1043.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Vihervaara, A. and Sistonen, L. 2014. HSF1 at a glance. Journal of Cell Scientce 127: 261-266.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Zhou, Y., Xia, J., Xu, S., She, T., Zhang, Y., Sun, Y., Wen, M., Jiang, T., Xiong, Y., and Lei, J. 2023. Experimental mouse models for translational human cancer research. Frontiers in Immunology 14: 1095388.</span></span></p>
2016-11-29T18:41:272023-08-22T14:32:34Alkylation, DNAAlkylation, DNAMolecular<p>The event involves DNA alkylation to form a variety of different DNA adducts (i.e., alkylated nucleotides). Alkylation occurs at various sites in DNA and can include alkylation of adenine- Nl, - N3, - N7, guanine- N3, - O6, - N7, thymine-O2, - N3, - O4, cytosine- O2, -N3, and the phosphate (diester) group (reviewed in detail in Beranek 1990). In addition, alkylation can involve modification with different sizes of alkylation groups (e.g., methyl, ethyl, propyl). It should be noted that many of these adducts are not stable or are readily repaired (discussed in more detail below). A small proportion of adducts are stable and remain bound to DNA for long periods of time.
</p><p>There is no OECD guideline for measurement of alkylated DNA, although technologies for their detection are established. Reviews of modern methods to measure DNA adducts include Himmelstein et al,. 2009 and Philips et al., 2000.
</p><p>High performance liquid chromatography (HPLC) methods can be used to measure whether an agent is capable of alkylating DNA in somatic cells. Alkyl adducts in somatic cells can be measured using immunological methods (described in Nehls et al. 1984), as well as HPLC (methods in de Groot et al. 1994) or a combination of 32P post-labeling, HPLC and immunologic detection (Kang et al. 1992). We note that mass spectrometry provides structural specificity and confirmation of the structure of DNA adducts.
</p><p>DNA alkylation can also be measured using a modified comet assay. This method involves the digestion of alkylated DNA bases with 3–methyladenine DNA glycosylase (Collins et al., 2001; Hasplova et al., 2012) followed by the standard comet assay to detect where alkyl adducts occur. The advantage of this method is that the alkaline version of the comet assay, as a core method, has an in vivo OECD guideline.
</p><p>Finally, structure-activity relationships (SARs) have been developed to predict the possibility that a chemical will alkylate DNA (e.g., Vogel and Ashby, 1994; Benigni, 2005; Dai et al., 1989; Lewis and Griffith, 1987).
</p><p><br />
Measurement of alkylation in male germ cells:
</p><p>In rodent testes, studies have detected adducts in situ by immuhistocytological staining. For example, fixed testes are incubated with O6-EtGua -specific mouse monoclonal antibody and subsequently with a labeled anti-mouse IgG F antibody. Nuclear DNA is counterstained with DAPI 4,6-diamidino- 2-phenylindole. Fluorescence signals from immunostained O6-EtGua residues in DNA are visualized by fluorescence microscopy and quantitated using an image analysis system. Methods are described in (Seiler et al. 1997). An immunoslot blot assay for detection of O6-EtGua has been described previously in (Mientjes et al. 1996).
</p><p>Alternatively, rodents have also been exposed to radio-labeled alkylating agents. Examples from the literature include [2-3H] ENU, [1-3H]di-ethyl sulfate, or [1-3H]ethyl-methane sulfonate. Following treatment with the labeled chemical, testis and other tissues of interest are removed. Germ cells are released from tubuli by pushing out the contents with forceps. Using this procedure all germ-cell stages are liberated from the tubuli, with the possible exception of part of the population of stem-cell spermatogonia that remain attached to the walls of the tubuli. DNA is then extracted from germ cells, empty testis tubuli and other tissues of interest. DNA adduct formation is determined after neutral and acid hydrolysis of DNA followed by separation of the various ethylation products using HPLC (described in van Zeeland et al. 1990).
</p><p>Alkylated DNA has been measured in somatic cells in a variety of species, from prokaryotic organisms, to rodents in vivo, to human cells in culture. Theoretically, DNA alkylation can occur in any cell type in any organism.
</p>CL:0000255eukaryotic cellHighMixedHighHighHighHigh<p><br />
Benigni, R. (2005), "Structure-activity relationship studies of chemical mutagens and carcinogens: mechanistic investigations and prediction and approaches", <i>Chem. Rev.</i>, 105: 1767-1800.
</p><p>Beranek, D.T. (1990), "Distribution of methyl and ethyl adducts following alkylation with monofunctional alkylating agents", <i>Mutation Res.</i>, 231: 11-30.
</p><p>Collins, A.R., M. Dusinská and A. Horská (2001), "A Detection of alkylation damage in human lymphocyte DNA with the comet assay". <i>Acta Biochim Pol.</i>, 48: 611-4.
</p><p>Dai, Q.H. and R.G. Zhong (1989), "Quantitative pattern recognition for structure-carcinogenic activity relationship of N-nitroso compounds based upon Di-region theory", <i>Sci China B.</i>, 32:776-790.
</p><p>de Groot, A.J., J.G. Jansen, C.F. van Valkenburg and A.A. van Zeeland (1994), "Molecular dosimetry of 7-alkyl- and O6-alkylguanine in DNA by electrochemical detection", <i>Mutat Res.</i>, 307: 61-6.
</p><p>Hašplová, K., A. Hudecová, Z. Magdolénová, M. Bjøras, E. Gálová, E. Miadoková and M. Dušinská (2012), "DNA alkylation lesions and their repair in human cells: modification of the comet assay with 3-methyladenine DNA glycosylase (AlkD)", <i>Toxicol Lett.</i>, 208: 76-81.
</p><p>Himmelstein, M.W., P.J. Boogaard, J. Cadet, P.B. Farmer, J.J. Kim, E.A. Martin, R. Persaud and D.E. Shuker (2009), "Creating context for the use of DNA adduct data in cancer risk assessment: II. Overview of methods of identification and quantitation of DNA damage", <i>Crit. Rev. Toxicol.</i>, 39: 679-94.
</p><p>Kamino, K., F. Seiler, M. Emura, J. Thomale, M.F. Rajewsky and U. Mohr (1995), "Formation of O6-ethylguanine in spermatogonial DNA of adult Syrian golden hamster by intraperitoneal injection of diethylnitrosamine", <i>Exp. Toxicol. Pathol.</i>, 47: 443-445.
</p><p>Kang, H.I., C. Konishi, G. Eberle, M.F. Rajewsky, T. Kuroki and N.H. Huh (1992), "Highly sensitive, specific detection of O6-methylguanine, O4-methylthymine, and O4-ethylthymine by the combination of high-performance liquid chromatography prefractionation, 32P postlabeling, and immunoprecipitation", <i>Cancer Res.</i>, 52: 5307-5312.
</p><p>Lewis, D.F. and V.S. Griffiths (1987), "Molecular electrostatic potential energies and methylation of DNA bases: a molecular orbital-generated quantitative structure-activity relationship", <i>Xenobiotica</i>, 17: 769-776.
</p><p>Mientjes, E.J., K. Hochleitner, A. Luiten-Schuite, J.H. van Delft, J. Thomale, F. Berends, M.F. Rajewsky, P.H. Lohman and R.A. Baan (1996), "Formation and persistence of O6-ethylguanine in genomic and transgene DNA in liver and brain of lambda(lacZ) transgenic mice treated with N-ethyl-N-nitrosourea", <i>Carcinogenesis</i>, 17: 2449-2454.
</p><p>Nehls, P., M.F. Rajewsky, E. Spiess, D. Werner (1984), "Highly sensitive sites for guanine-O6 ethylation in rat brain DNA exposed to N-ethyl-N-nitrosourea in vivo", <i>EMBO J.</i>, 3:327-332.
</p><p>Phillips, D.H., P.B. Farmer, F.A. Beland, R.G. Nath, M.C. Poirier, M.V. Reddy and K.W. Turteltaub (2000), "Methods of DNA adduct determination and their application to testing compounds for genotoxicity", <i>Environ. Mol. Mutagen.</i>, 35: 222-233.
</p><p>Scherer, E., A.A. Jenner and L. den Engelse (1987), "Immunocytochemical studies on the formation and repair of O6-alkylguanine in rat tissues", <i>IARC Sci. Publ.</i>, 84: 55-58.
</p><p>Sega, G.A., C.R. Rohrer, H.R. Harvey and A.E. Jetton (1986), "Chemical dosimetry of ethyl nitrosourea in the mouse testis", <i>Mutat. Res.</i>, 159: 65-74.
</p><p>Seiler, F., K. Kamino, M. Emura, U. Mohr and J. Thomale (1997), "Formation and persistence of the miscoding DNA alkylation product O6-ethylguanine in male germ cells of the hamster", <i>Mutat. Res.</i>, 385: 205-211.
</p><p>van Zeeland, A.A., A. de Groot and A. Neuhäuser-Klaus (1990), "DNA adduct formation in mouse testis by ethylating agents: a comparison with germ-cell mutagenesis", <i>Mutat. Res.</i> 231: 55-62.
</p><p>Vogel, E.W., Ashby, J. (1994), "Structure-activity relationships: experimental approaches." In: Methods to asses DNA Damage and repair: Interspecies comparisons. Edited by R.T. Tardiff, P.H.M. Lohman and G.N. Wogan, SCOPE, Wiley and Sons LTD.
</p>2016-11-29T18:41:222017-09-16T10:14:297560153a-2d20-4223-b67a-bbb6c4cfb01d9559ebbc-3602-499e-87fe-86b750161fe3<p>The described Key Event Relationship (KER) outlines a sequence of events related to DNA repair and its consequences. The upstream event is characterized by "Inadequate DNA repair," indicating that the cellular mechanisms responsible for repairing DNA damage are compromised or insufficient. This could result from various factors, such as genetic mutations, environmental exposures, or other cellular processes.</p>
<p>The downstream event in this KER is an "Increase in Mutations." As a consequence of inadequate DNA repair, the accumulation of unrepaired or incorrectly repaired DNA damage can lead to an elevated rate of mutations in the genome. These mutations can involve changes in the DNA sequence, structure, or arrangement, which may have various implications for cellular function, including potential disruptions to normal processes and pathways.</p>
<p>This KER highlights the critical role of DNA repair mechanisms in maintaining genomic stability and preventing the buildup of mutations that can contribute to various biological outcomes, including disease development and other adverse effects.</p>
<p><span style="font-size:12px">Insufficient repair results in the retention of damaged DNA that is then used as a template during DNA replication. During replication of damaged DNA, incorrect nucleotides may be inserted, and upon replication these become ‘fixed’ in the cell. Further replication propagates the mutation to additional cells.</span></p>
<p><span style="font-size:12px">For example, it is well established that replication of alkylated DNA can cause insertion of an incorrect base in the DNA duplex (i.e., mutation). Replication of non-repaired O4 thymine alkylation leads primarily to A:T→G:C transitions. Retained O6 guanine alkylation causes primarily G:C→A:T transitions.</span></p>
<p><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">For repairing DNA double strand breaks (DSBs), non-homologous end joining (NHEJ) is one of the repair mechanisms used in human somatic cells (Petrini et al., 1997; Mao et al., 2008). However, this mechanism is error-prone and may create mutations during the process of DNA repair (Little, 2000). NHEJ is considered error-prone because it does not use a homologous template to repair the DSB. The NHEJ mechanism involves many proteins that work together to bridge the DSB gap by overlapping single-strand termini that are usually less than 10 nucleotides long (Anderson, 1993; Getts & Stamato, 1994; Rathmell & Chu, 1994). Inherent in this process is the introduction of errors that may result in mutations such as insertions, deletions, inversions, or translocations.</span></span></p>
<p>Furthermore, other repair mechanisms such as a loss in the mismatch repair (MMR) system can lead to a buildup of errors such as base-base mismatches and insertion-deletion errors in repetitive DNA sequences which are known as microsatellites. This could occur if an MMR gene (e.g. MLH1, PMS2) is inactivated through mutations or epigenetic silencing (Wang et al., 2022). </p>
<p><span style="font-size:12px">Overall Weight of Evidence: High </span></p>
<p><span style="font-size:12px">If DNA repair is able to correctly and efficiently repair DNA lesions introduced by a genotoxic stressor, then no increase in mutation frequency will occur.</span></p>
<p><span style="font-size:12px">For example, for alkylated DNA, efficient removal by O<sup>6</sup>-alkylguanine DNA alkyltransferase will result in no increases in mutation frequency. However, above a certain dose AGT becomes saturated and is no longer able to efficiently remove the alkyl adducts. Replication of O-alkyl adducts leads to mutation. The evidence demonstrating that replication of unrepaired O-alkylated DNA causes mutations is extensive in somatic cells and has been reviewed (Basu and Essigmann 1990; Shrivastav et al. 2010); specific examples are given below.</span></p>
<p><span style="font-size:12px">It is important to note that not all DNA lesions will cause mutations. It is well documented that many are bypassed error-free. For example, N-alkyl adducts can quite readily be bypassed error-free with no increase in mutations (Philippin et al., 2014).</span></p>
<p><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif"><strong>Inadequate repair of DSB</strong></span></span></p>
<p><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Collective data from tumors and tumor cell lines has emerged that suggests that DNA repair mechanisms may be error-prone (reviewed in Sishc et al., 2017) (Sishc & Davis, 2017). NHEJ, the most common pathway used to repair DSBs, has been described as error-prone. The error-prone nature of NHEJ, however, is thought to be dependent on the structure of the DSB ends being repaired, and not necessarily dependent on the NHEJ mechanism itself (Bétermier et al., 2014). Usually when perfectly cohesive ends are formed as a result of a DSB event, ligase 4 (LIG4) will have limited end processing to perform, thereby keeping ligation errors to a minimum (Waters et al., 2014). When the ends are difficult to ligate, however, the resulting repair may not be completed properly; this often leads to point mutations and other chromosomal rearrangements. It has been shown that approximately 25 - 50% of DSBs are misrejoined after exposure to ionizing radiation (Löbrich et al., 1998; Kuhne et al., 2000; Lobrich et al., 2000). Defective repair mechanisms can increase sensitivity to agents that induce DSBs and lead eventually to genomic instability (reviewed in Sishc et al., (2017)).</span></span></p>
<p><span style="font-size:12px">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). </span> </p>
<p><strong>I<span style="font-size:12px">NSUFFICIENT REPAIR OF ALKYLATED DNA</span></strong></p>
<p><span style="font-size:12px">Evidence in somatic cells</span></p>
<p><span style="font-size:12px">Empirical evidence to support this KER is primarily from studies in which synthetic oligonucleotides containing well-characterized DNA lesions were genetically engineered in viral or plasmid genomes and subsequently introduced into bacterial or mammalian cells. Mutagenicity of each lesion is ascertained by sequencing, confirming that replication of alkylated DNA (i.e., unrepaired DNA) causes mutations in addition to revealing the important DNA repair pathways and polymerases involved in the process. For example, plasmids containing O6-methyl or O6-ethylguanine were introduced into AGT deficient or normal Chinese hamster ovary cells (Ellison et al. 1989). Following replication, an increase in mutant fraction to 19% for O6-methylguanine and 11% for O6-ethylguanine adducts was observed in AGT deficient cells versus undetectable levels for control plasmids. The relationship between input of alkylated DNA versus recovered mutant fractions revealed that a large proportion of alkyl adducts were converted to mutations in the AGT deficient cells (relationship slightly sublinear, with more adducts than mutations). The primary mutation occurring was G:C-A:T transitions. The results indicate that replication of the adducted DNA caused mutations and that this was more prevalent with reduced repair capacity. The number of mutations measured is less than the unrepaired alkyl adducts transfected into cells, supporting that insufficient repair occurs prior to mutation. Moreover, the alkyl adducts occur prior to mutation formation, demonstrating temporal concordance.</span></p>
<p><span style="font-size:12px">Various studies in cultured cells and microorganisms have shown that the expression of O<sup>6</sup>-methylguanine DNA methyltransferase (AGT/MGMT) (repair machinery – i.e., decrease in DNA strand breaks) greatly reduces the incidence of mutations caused by exposure to methylating agents such as MNU and MNNG (reviewed in Kaina et al. 2007; Pegg 2011). Thomas et al. (2013) used O6-benzylguanine to specifically inhibit MGMT activity in AHH-1 cells. Inhibition was carried out for one hour prior to exposure to MNU, a potent alkylating agent. Inactivation of MGMT resulted in increased MNU-induced HPRT (hypoxanthine-guanine phosphoribosyltransferase) mutagenesis and shifted the concentrations at which induced mutations occurred to the left on the dose axis (10 fold reduction of the lowest observed genotoxic effect level from 0.01 to 0.001 µg/ml). The ratio of mutants recovered in DNA repair deficient cells was 3-5 fold higher than repair competent cells at concentrations below 0.01 µg/ml, but was approximately equal at higher concentrations, indicating that repair operated effectively to a certain concentration. Only at this concentration (above 0.01 µg/ml when repair machinery is overwhelmed and repair becomes deficient) do the induced mutations in the repair competent cells approach those of repair deficient. Thus, induced mutation frequencies in wild type cells are suppressed until repair is overwhelmed for this alkylating agent. The mutations prevented by MGMT are predominantly G:C-A:T transitions caused by O6-methylguanine.</span></p>
<p><br />
<span style="font-size:12px">Evidence in germ cells</span></p>
<p><span style="font-size:12px">That saturation of repair leads to mutation in spermatogonial cells is supported by work using the OECD TG488 rodent mutation reporter assay in sperm. A sub-linear dose-response was found using the lacZ MutaMouse assay in sperm exposed as spermatogonial stem cells, though the number of doses was limited (van Delft and Baan 1995). This is indirect evidence that repair occurs efficiently at low doses and that saturation of repair causes mutations at high doses. Lack of additional data motivated a dose-response study using the MutaMouse model following both acute and sub-chronic ENU exposure by oral gavage (O’Brien et al. 2015). The results indicate a linear dose-response for single acute exposures, but a sub-linear dose-response occurs for lower dose sub-chronic (28 day) exposures, during which mutation was only observed to occur at the highest dose. This is consistent with the expected pattern for dose-response based on the hypothetical AOP. Thus, this sub-linear curve for mutation at low doses following sub-chronic ENU exposure suggests that DNA repair in spermatogonia is effective in preventing mutations until the process becomes overwhelmed at higher doses.</span></p>
<p><span style="font-size:12px">Mutation spectrum: Following exposure to alkylating agents, the most mutagenic adducts to DNA in pre-meiotic male germ cells include O6-ethylguanine, O4-ethylthymine and O2-ethylthymine (Beranek 1990; Shelby and Tindall 1997). Studies on sperm samples collected post-ENU exposure in transgenic rodents have shown that 70% of the observed mutations are at A:T sites (Douglas et al. 1995). The mutations observed at G:C base pairs are almost exclusively G:C-A:T transitions, presumably resulting from O6-ethylguanine. It is proposed that the prevalence of mutations at A:T basepairs is the result of efficient removal of O6-alkylguanine by AGT in spermatogonia, which is consistent with observation in human somatic cells (Bronstein et al. 1991; Bronstein et al. 1992). This results in the majority of O6-ethylguanine adducts being removed, leaving O4- and O2-ethylthymine lesions to mispair during replication. Thus, lack of repair predominantly at thymines and guanines at increasing doses leads to mutations in these nucleotides, consistent with the concordance expected between diminished repair capabilities at these adducts and mutation induction (i.e., concordance relates to seeing these patterns across multiple studies, species and across the data in germ cells and offspring).</span></p>
<p> </p>
<p><span style="font-size:12px"><u>Inadequate repair of oxidative DNA lesions: In vitro studies</u></span></p>
<ul>
<li><span style="font-size:12px">AS52 Chinese hamster ovary cells (wild type and OGG1-overexpressing) were exposed to kJ/m<sup>2 </sup>UVA radiation (Dahle et al., 2008).</span>
<ul style="list-style-type:circle">
<li><span style="font-size:12px">Mutations in the gpt gene were quantified in both wild type and OGG1+ cells by sequencing after 13-15 days following 400 kJ/m<sup>2 </sup>UVA irradiation</span>
<ul>
<li><span style="font-size:12px">G:C-A:T mutations in UVA-irradiated OGG1+ cells were completely eliminated</span></li>
<li><span style="font-size:12px">G:C-A:T 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</span></li>
<li><span style="font-size:12px">Elevated levels of OGG1 was able to prevent G:C-A:T mutations, while the OGG1 levels in wild type cells was insufficient, leading to an increase in mutants (demonstrates inadequate repair leading to mutations)</span></li>
</ul>
</li>
</ul>
</li>
<li><span style="font-size:12px">Xeroderma pigmentosum complementation group A (XPA) knockout (KO) and wild type TSCER122 human lymphoblastoid cells were transfected with TK gene-containing vectors with no adduct, a single 8-oxo-dG, or two 8-oxo-dG adducts in tandem (Sassa et al., 2015).</span>
<ul style="list-style-type:circle">
<li><span style="font-size:12px">XPA is a key protein in nucleotide excision repair (NER) that acts as a scaffold in the assembly the repair complex.</span></li>
<li><span style="font-size:12px">Mutation frequency was determined by the number of TK-revertant colonies</span></li>
<li><span style="font-size:12px">Control vector induced a mutation frequency of 1.3% in both WT and XPA KO</span></li>
<li><span style="font-size:12px">Two 8-oxo-dG in tandem on the transcribed strand were most mutagenic in XPA KO, inducing 12% mutant frequency compared to 7% in WT</span></li>
<li><span style="font-size:12px">For both XPA KO and WT, G:C-A:T transversion due to 8-oxo-dG was the most predominant point mutation in the mutants </span></li>
<li><span style="font-size:12px">The lack of a key factor in NER leading to increased 8-oxo-dG-induced transversions demonstrates insufficient repair leading to increase in mutations </span></li>
</ul>
</li>
</ul>
<p> </p>
<p><span style="font-size:12px"><u>Inadequate repair of oxidative DNA lesions: In vivo studies in mice</u></span></p>
<ul>
<li><span style="font-size:12px">Spontaneous mutation frequencies in the liver of Ogg1-deficient (-/-) Big Blue mice was measured at 10 weeks of age (Klungland et al., 1999).</span>
<ul style="list-style-type:circle">
<li><span style="font-size:12px">Mutation frequencies were 2- to 3-fold higher in the <em>Ogg1</em>-/- mice than in wild type</span></li>
<li><span style="font-size:12px">Of the 16 base substitutions detected in <em>Ogg1</em> -/- mutant plaques analyzed by sequencing, 10 indicated G:C-A:T transversions consistent with the known spectrum of mutation</span></li>
<li><span style="font-size:12px">The results support that insufficient repair of oxidized bases leads to mutation.</span></li>
</ul>
</li>
<li><span style="font-size:12px"><em>Ogg1 </em>knockout (<em>Ogg1</em>-/-) in C57BL/6J mice resulted in 4.2-fold and 12-fold increases in the amount of 8-oxo-dG in the liver compared to wild type at 9 and 14 weeks of age, respectively (Minowa et al., 2000).</span>
<ul style="list-style-type:circle">
<li><span style="font-size:12px">In these mice, there was an average of 2.3-fold increase in mutation frequencies in the liver (measured between 16-20 weeks)</span>
<ul>
<li><span style="font-size:12px">57% of the observed base substitutions were G:C-A:T transversions, while 35% in wild type mice corresponded to this transversion.</span></li>
<li><span style="font-size:12px">Approximately 70% of the increase in mutation frequency was due to G to T transversions.</span></li>
</ul>
</li>
<li><span style="font-size:12px">Concordantly, KBrO3 treatment resulted in a 2.9-fold increase in mutation frequency in the kidney of <em>Ogg1 </em>-/- mice compared to KBrO3-treated wild type (Arai et al., 2002).</span>
<ul>
<li><span style="font-size:12px">G:C-A:T transversions made up 50% of the base substitutions in the <em>Ogg1-/- </em>mice.</span></li>
</ul>
</li>
<li><span style="font-size:12px">Heterozygous <em>Ogg1 </em>mutants (<em>Ogg1</em>+/-) retained the original repair capacity, where no increase in 8-oxo-dG lesions was observed in the liver at 9 and 14 weeks (Minowa et al., 2000).</span>
<ul>
<li><span style="font-size:12px">This observation was consistent even after KBrO3 treatment of the mice (Arai et al., 2002).</span></li>
</ul>
</li>
<li><span style="font-size:12px">From these results, we can infer that OGG1 proteins are present in excess and that one functional copy of the gene is sufficient in addressing endogenous and, to a certain degree, chemical-induced oxidative DNA lesions.</span></li>
</ul>
</li>
</ul>
<p><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif"><strong><u>Inadequate Repair of </u><u>DSB</u></strong></span></span></p>
<p><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Empirical data obtained for this KER moderately supports the idea that inadequate DNA repair increases the frequency of mutations. The evidence presented below related to the inadequate repair of DSBs is summarized in table 5, <a href="https://docs.google.com/spreadsheets/d/1iehBBqhFFSOhgis-0U3tasQwJ50bZJPVmenWUiR4vmA/edit?usp=sharing" target="_blank">here (click link)</a>. The review article by Sishc & Davis (2017) provides an overview of NHEJ mechanisms with a focus on the inherently error-prone nature of DSB repair mechanisms, particularly when core proteins of NHEJ are knocked-out. </span>Although NHEJ is predominantly the preferred repair mechanism throughout the cell cycle, homologous recombination (HR) and single-stranded annealing (SSA) are favored during the S and G2 phases in scenarios where the NHEJ repair pathway is inhibited. The absence of HR leading to an increase in SSA activity is still a matter to debate (Ceccaldi et al., 2016).</span> <span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Another review also provides an overview of DSB induction, the repair process and how mutations may result, as well as the biological relevance of misrepaired or non-repaired DNA damage (Sage & Shikazono, 2017).</span></span></p>
<p><br />
<span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif"><u><strong>Dose and Incidence Concordance</strong></u></span></span></p>
<p><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">There is evidence in the literature suggesting a dose/incidence concordance between inadequate DNA repair and increases in mutation frequencies. Evidence presented below related to the dose-response of mutation frequencies is summarized in table 2, <a href="https://docs.google.com/spreadsheets/d/1iehBBqhFFSOhgis-0U3tasQwJ50bZJPVmenWUiR4vmA/edit?usp=sharing" target="_blank">here (click link)</a>. In response to increasing doses from a radiation stressor, dose-dependent increases in both measures of inadequate DNA repair and mutation frequency have been found. In an analysis that amalgamated results from several different studies conducted using in vitro cell-lines, the rate of DSB misrepair was revealed to increase in a dose-dependent fashion from 0 - 80 Gy, with the mutation rate also similarly increasing from 0 - 6 Gy (Mcmahon et al., 2016). Additionally, using a plant model, it was shown that increasing radiation dose from 0 - 10 Gy resulted in increased DNA damage as a consequence of inadequate repair. Mutations were observed 2 - 3 weeks post-irradiation (Ptácek et al., 2001). Moreover, increases in mutation densities were found in specific genomic regions of cancer samples (namely promoter DNAse I-hypersensitive sites (DHS) and 100 bp upstream of transcription start sites (TSS)) that were also found to have decreased DNA repair rates attributable to inadequate nucleotide excision repair (NER) (Perera et al., 2016).</span></span><br />
</p>
<p><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Interestingly, mutation rates have been shown to increase as the required DNA repair becomes more complex. Upon completion of DSB repair in response to radiation and treatment with restriction enzymes, more mutations were found in cases where the ends were non-complementary and thus required more complex DNA repair (1 - 4% error-free) relative to cases where ends were complementary (34 - 38% error-free) (Smith et al., 2001).</span></span></p>
<p><span style="font-size:12px"><u><span style="font-family:arial,helvetica,sans-serif"><strong>Temporal Concordance</strong></span></u></span></p>
<p><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">There is evidence in the literature suggesting a time concordance between the initiation of DNA repair and the occurrence of mutations. For simple ligation events, mutations were not evident until 12 - 24 hours, whereas DSB repair was evident at 6 -12 hours. For complex ligation events, however, mutations and DSB repair were both evident at 12 - 24 hours. As the relative percent of DNA repair increased over time, the corresponding percent of error-free rejoining decreased over time in both ligation cases, suggesting that overall DNA repair fidelity decreases with time ((Smith et al., 2001).</span></span></p>
<p><span style="font-size:12px"><u><span style="font-family:arial,helvetica,sans-serif"><strong>Essentiality</strong></span></u></span></p>
<p><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Inadequate DNA repair has been found to increase mutations above background levels. There is evidence from knock-out/knock-down studies suggesting that there is a strong relationship between the adequacy of DNA repair and mutation frequency. In all examined cases, deficiencies in proteins involved in DNA repair resulted in altered mutation frequencies relative to wild-type cases. There were significant decreases in the frequency and accuracy of DNA repair in cell lines deficient in LIG4 (DNA ligase 4, a DNA repair protein) (Smith et al., 2003) and Ku80 (Feldmann et al., 2000). 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). In primary Nibrin-deficient mouse fibroblasts, there was increased spontaneous DNA damage relative to wild-type controls, suggestive of inadequate DNA repair. Using the corresponding Nibrin-deficient and wild-type mice, in vivo mutation frequencies were also found to be elevated in the Nibrin-deficient animals (Wessendorf et al., 2014). Furthermore, mutation densities were differentially affected in specific genomic regions in cancer patients depending on their Xeroderma pigmentosum group C (XPC) gene status. Specifically, mutation frequencies were increased in XPC-wild-type patients at DNase I-hypersensitive site (DHS) promoters and 100 bp upstream of TSS relative to cancer patients lacking functional XPC (Perera et al., 2016). Lastly, in a study using WKT1 cells with less repair capacity, radiation exposure induced four times more mutations in these cells than in TK6 cell, which had a normal repair capacity (Amundson and Chen, 1996). </span></span></p>
<p><span style="font-size:12px">Repair of alkylated DNA</span></p>
<p><span style="font-size:12px">There were no inconsistencies in the empirical data reviewed or in the literature relating to biological plausibility. Much of the support for this KER comes predominantly from data in somatic cells and in prokaryotic organisms. We note that all of the data in germ cells used in this KER are produced exclusively from ENU exposure. Data on other chemicals are required. We consider the overall weight of evidence of this KER to be strong because of the obvious biological plausibility of the KER, and documented temporal association and incidence concordance based on studies over-expressing and repressing DNA repair in somatic cells.</span></p>
<p><span style="font-size:12px">Repair of oxidative lesions</span></p>
<ul>
<li><span style="font-size:12px">Thresholded concentration-response curve of mutation frequency was observed in AHH-1 human lymphoblastoid cells after treatment with pro-oxidants (H<sub>2</sub>O<sub>2 </sub>and KBrO<sub>2</sub>) known to cause oxidative DNA damage (Seager et al., 2012), suggesting that cells are able to tolerate low levels of DNA damage using basal repair. However, increase in 8-oxo-dG lesions and up-regulation of DNA repair proteins were not observed under the same experimental condition.</span></li>
<li><span style="font-size:12px">Mutagenicity of oxidative DNA lesions other than 8-oxo-dG, such as FaPydG and thymidine glycol, has not been as extensively studied and there are mixed results regarding the mutagenic outcome of these lesions.</span></li>
</ul>
<p><span style="font-size:12px">Repair of double strand breaks </span></p>
<ul>
<li><span style="font-size:12px">One review paper found that DNA DSBs are repaired more efficiently at low dose (≤0.1 Gy) compared to high dose (>1 Gy) X-rays, but delayed mutation induction and genomic instability have also been demonstrated to occur at low doses (<1 cGy) of ionizing radiation (Preston et al., 2013). </span></li>
</ul>
<p><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Overall</span></span></p>
<ul>
<li><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:12px">Mutation induction is stochastic, spontaneous, and dependent on the cell type as well as the individual’s capability to repair efficiently (NRC, 1990; Pouget & Mather, 2001).</span></span></li>
</ul>
<p><span style="font-size:12px">Thresholds for mutagenicity indicate that the response at low doses is modulated by the DNA repair machinery, which is effectively able to remove alkylated DNA at low doses [Gocke and Muller 2009; Lutz and Lutz 2009; Pozniak et al. 2009]. Kinetics of DNA repair saturation in somatic cells is described in Muller et al. [Muller et al. 2009].</span></p>
<p><span style="font-size:12px">For O-methyl adducts, once the primary repair process is saturated, in vitro data suggest that misreplication occurs almost every time a polymerase encounters a methylated guanine [Ellison et al. 1989; Singer et al. 1989]; however, it should be noted that this process can be modulated by flanking sequence. This conversion of adducts to mutations also appears to be reduced substantially in vivo [Ellison et al. 1989]. The probability of mutation will also depend on the type of adduct (e.g., O-alkyl adducts are more mutagenic than N-alkyl adducts; larger alkyl groups are generally more mutagenic, etc.). Overall, a substantive number of factors must be considered in developing a quantitative model.</span></p>
<p><span style="font-size:12px"><u>Inadequate repair of oxidative </u><u>lesions</u></span></p>
<p><span style="font-size:12px">The relationship between the quantity/activity of repair enzymes such as OGG1 in the cell and the quantity of oxidative lesions need to be better understood to define a threshold on the quantity of oxidative lesions exceeding basal repair capacity. Moreover, the proportion of oxidative lesions formed that lead to mutation versus strand breaks is not clearly understood.</span></p>
<p><span style="font-size:12px">Mutations resulting from oxidative DNA damage can occur via replicative polymerases and translesion synthesis (TLS) polymerases during replication, and during attempted repair. However, an in vitro study on TLS in yeast has shown that bypass of 8-oxo-dG by TLS polymerases during replication is approximately 94-95% accurate. Therefore, the mutagenicity of 8-oxo-dG and other oxidative lesions may depend on their abundance, not on a single lesion (Rodriguez et al., 2013). Applicability of this observation in mammalian cells needs further investigation. Information on the accuracy of 8-oxo-dG bypass in mammalian cells is limited. </span></p>
<p><span style="font-size:12px">The most notable example of mutation arising from inadequate repair of DNA oxidation is G to T transversion due to 8-oxo-dG lesions. Previous studies have demonstrated higher mutation frequency of this lesion compared to other oxidative lesions; for example, Tan et al. (1999) compared the mutation rate of 8-oxo-dG and 8-oxo-dA in COS-7 monkey kidney cells and reported that under similar conditions, 8-oxo-dG was observed to be four times more likely to cause base substitution (Tan et al., 1999). </span></p>
<p><span style="font-size:12px"><strong><u>Inadequate Repair of DSB</u></strong></span></p>
<p><span style="font-size:12px">Quantitative understanding of this linkage is derived from the studies that examined DSB misrepair rates or mutation rates in response to a radiation stressor. In general, combining results from these studies suggests that increased mutations can be predicted when DNA repair is inadequate. At a radiation dose of 10 Gy, the rate of DSB misrepair was found to be approximately 10 - 15% (Lobrich et al., 2000); this rate increased to 50 - 60% at a radiation exposure of 80 Gy (Kuhne et al., 2000; Lobrich et al., 2000; McMahon et al., 2016). For mutation rates in response to radiation across a variety of models and radiation doses, please refer to the example table below.</span></p>
<table border="1" cellpadding="1" cellspacing="1" style="height:158px; width:645px">
<tbody>
<tr>
<td style="text-align:center; width:150px"><span style="font-size:12px"><strong>Reference</strong></span></td>
<td style="text-align:center"><span style="font-size:12px"><strong>Summary</strong></span></td>
</tr>
<tr>
<td style="text-align:center"><span style="font-size:12px">Matuo et al., 2018</span></td>
<td><span style="font-size:12px">Yeast cells (saccharomyces cerevisiae) exposed to high LET cardbon ions (25 keV/um) and low LET carbon ions (13 keV/um) between 0-200 Gy induces a 24-fold increase overbaseline of mutations (high LET) and 11-fold increase over baseline mutations (low LET).</span></td>
</tr>
<tr>
<td style="text-align:center"><span style="font-size:12px">Nagashima et al., 2018</span></td>
<td><span style="font-size:12px">Hamster cells (GM06318-10) exposed to x-rays in the 0-1 Gy. Response of 19.0 ± 6.1 mutants per 10<sup>9</sup> survivors.</span></td>
</tr>
<tr>
<td style="text-align:center"><span style="font-size:12px">Albertini et al., 1997</span></td>
<td><span style="font-size:12px">T-lymphcytes isolated from human peripheral blood exposed to low LET gamma-rays (0.5-5 Gy) and high LET radon gas (0-1 Gy). Response of 7.0x10<sup>-6</sup> mutants/Gy (Gamma-rays 0-2 Gy), 54x10<sup>-6</sup> mutants/Gy (Gamma-rays 2-4 Gy) and 63x10<sup>-6</sup> mutants/Gy (0-1 Gy).</span></td>
</tr>
<tr>
<td style="text-align:center"><span style="font-size:12px">Dubrova et al., 2002</span></td>
<td><span style="font-size:12px">Observation of paternal ESTR mutation rates in CBAH mice following exposure to acute low LET X-rays (0-1 Gy), chronic low LET gamma-rays (0-1 Gy) and chronic high LET neutrons (0-0.5 Gy). Modelled response of y = mx + C, values of (m,C): X-rays: (0.338, 0.111), Gamma-rays: (0.373±0.082, 0.110), Neutrons: (1.135±0.202, 0.136).</span></td>
</tr>
<tr>
<td style="text-align:center"><span style="font-size:12px">McMahon et al., 2016</span></td>
<td><span style="font-size:12px">Study of HPRT gene in Chinese hamster cells following exposure to radiation of 1-6 Gy. Observation of 0.2 mutations in HPRT gene per 10<sup>4</sup> cells and 0.1 point mutations per 10<sup>4</sup> cells (1 Gy). At 6 Gy, observation of 1.5 mutations in the HPRT gene per 10<sup>4</sup> cells and 0.4 point mutations per 10<sup>4</sup> cells.</span></td>
</tr>
</tbody>
</table>
<p> </p>
HighUnspecificHighAll life stagesHighHighHigh<p><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">This KER is plausible in all life stages, sexes, and organisms with DNA. The majority of the evidence is from in vivo adult mice and male human, and mice in vitro models. </span></span></p>
<p><span style="font-size:12px">All organisms, from prokaryotes to eukaryotes, have DNA repair systems. Indeed, much of the empirical evidence on the fundamental principles described in this KER are derived from prokaryotic models. DNA adducts can occur in any cell type with DNA, and may or may not be repaired, leading to mutation. While there are differences among DNA repair systems across eukaryotic taxa, all species develop mutations following excessive burdens of DNA lesions like DNA adducts. Theoretically, any sexually reproducing organism (i.e., producing gametes) can also acquire DNA lesions that may or may not be repaired, leading to mutations in gametes.</span></p>
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<p><span style="font-size:12px">Thomas, A.D., G.J. Jenkins, B. Kaina, O.G. Bodger, K.H. Tomaszowski, P.D. Lewis, S.H. Doak and G.E. Johnson (2013), "Influence of DNA repair on nonlinear dose-responses for mutation", <em>Toxicol. Sci.</em>, 132(1): 87-95.</span></p>
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<p><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Wilson, T.E. & M.R. Lieber (1999), "Efficient Processing of DNA Ends during Yeast Nonhomologous End Joining.", J. Biol. Chem. 274(33):23599–23609. doi:10.1074/jbc.274.33.23599.</span></span></p>
2016-11-29T18:41:332024-03-08T15:00:389559ebbc-3602-499e-87fe-86b750161fe336a85ffe-ec65-4c35-b590-84cb4320bddf2016-11-29T18:41:352016-12-03T16:38:01Alkylation of DNA leading to cancer 2DNA alkylation -> cancer 2Not under active developmentArchived<p>Alkylating agents are prototypical DNA-reactive compounds and have been extensively studied for decades (reviewed in Beranek 1990). The chemicals can be direct-acting electrophiles, or can be converted from non-reactive substances to reactive metabolites via metabolism. A prototypical alkylating agent is N-ethyl-N-nitrosourea (chemical formula C3H7N3O2) (ENU). ENU is rapidly absorbed following oral exposure and intraperitoneal injections and distributed widely across the tissues. ENU is unstable and readily reacts with somatic and germ cell DNA in mice, rats, flies and hamsters, to alkylate DNA. Very generally, mono-functional (referring to the transfer of a single alkyl group) alkylating agents include:
1. Alkyl sulfates: e.g., diethyl (DES) and dimethyl sulfate (DMS);
2. Alkyl alkanesulfonates: e.g., methyl (MMS) and ethyl methanesulfonate (EMS);
3. Nitrosamides: e.g., methyl (MNU) and ethyl nitrosourea (ENU), methyl- (MNNG) and ethyl-N'-nitro-N-nitrosoguanidine (ENNG), and the indirect-acting (i.e., requiring metabolic activation) dimethyl (DMN) and diethyl nitrosamines (DEN).
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ENU is the most widely studied and understood alkylating agent and as such has been instrumental in contributing to the knowledgebase in this field. Immunohistochemistry studies clearly indicate the presence of alkylated DNA following exposure to ENU in both somatic cells and spermatogonia (Kamino et al. 1995; Seiler et al. 1997; van Zeeland et al. 1990).
</p><p><span style="font-size:16px"><span style="font-family:Calibri,sans-serif">Cancer is a critical endpoint in human health risk assessment. It is embedded in regulatory frameworks for human health protection in many countries (see OSHA 2023 for examples of US regulations and European Parliament 2022 for examples of regulations in Europe).</span></span></p>
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