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Terminal nucleotide end labeling of DNA nicks (TUNEL) in parasagittal sections, similar to the boxed regions shown in Figure 4. The forebrain of wild-type (a-f) or Atm-null (g-l) embryos reveals delayed cell death in irradiated Atm-null embryos compared with wild-type controls. Unirradiated E10.5 embryos (a, d, g, j) and E10.5 embryos at 6 h (b, e, h, k) or 48 h (c, f, i, l) postirradiation are shown. Images in d-f and j-l are higher magnification views of those shown in a-c and j-l. a) Scale bar represents 100 µm and applies to a-c and g-i. d) Scale bar represents 100 µm and applies to d-f and j-l.
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ATM (ataxia-telangiectasia mutated) is a genotoxic stress transducer. In this first report of Atm-dependent birth defects, Atm-null embryos were uniquely susceptible to low-dose (0.5 Gy) radiation, exhibiting severe runting, tail anomalies, and lethality, independent of cell cycle arrest or insulin-like growth factor 1. This treatment enhanced leve...
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Citations
... Previous studies of ROS-mediated developmental disorders have consistently shown that models deficient in proteins involved in DNA damage recognition and repair signaling [37,38], DNA repair [13,15,30,39] or antioxidative activity [22,35] were more susceptible to developmental disorders caused by several different ROS-initiating teratogens, and in some cases even caused by physiological levels of ROS formation in untreated animals [14,30,[34][35][36]40,41]. This study's findings align with these previous observations, as Brca1 direct KO +/embryos exhibit enhanced γH2AX following both saline and EtOH exposures, with the latter showing an additive effect. ...
Although widely known as a tumor suppressor, the breast cancer 1 susceptibility protein (BRCA1) is also important in development, where it regulates fetal DNA repair pathways that protect against DNA damage caused by physiological and drug-enhanced levels of reactive oxygen species (ROS). We previously showed that conditional heterozygous (+/-) knockout (cKO) mouse embryos with a minor 28% BRCA1 deficiency developed normally in culture, but when exposed to the ROS-initiating drug, alcohol (ethanol, EtOH), exhibited embryopathies not evident in wild-type (+/+) littermates. Herein, we characterized a directBrca1 +/- knockout (KO) model with a 2-fold greater (58%) reduction in BRCA1 protein vs. the cKO model. We also characterized and compared learning & memory deficits in both the cKO and KO models. Even saline-exposed Brca1 +/- vs. +/+ KO progeny exhibited enhanced oxidative DNA damage and embryopathies in embryo culture and learning & memory deficits in females in vivo, which were not observed in the cKO model, revealing the potential pathogenicity of physiological ROS levels. The embryopathic EtOH concentration for cultured direct KO embryos was half that for cKO embryos, and EtOH affected Brca1 +/+ embryos only in the direct KO model. The spectrum and severity of EtOH embryopathies in culture were greater in both Brca1 +/- vs. +/+ embryos, and direct KO vs. cKO +/- embryos. Motor coordination deficits were evident in both male and female Brca1 +/- KO progeny exposed in utero to EtOH. The results in our direct KO model with a greater BRCA1 deficiency vs. cKO mice provide the first evidence for BRCA1 protein dose-dependent susceptibility to developmental disorders caused by physiological and drug-enhanced oxidative stress.
... ATM is necessary to initiate double-strand break (DSB) repair by homologous recombination (HR) [77,78], which acts as a genotoxic stress transducer and teratological suppressor tole to protect the embryo from pathological cell death and teratogenesis initiated by DNA damage. Consequently, ATM may have a significant developmental role in protecting the embryo from more subtle oxidative DNA damage caused by endogenous and xenobiotic-enhanced ROS [80][81][82]. Our study showed that the ATM gene was more highly expressed in EBs cultured in low-oxygen conditions. ...
The formation of embryoid bodies (EBs) from human pluripotent stem cells resembles the early stages of human embryo development, mimicking the organization of three germ layers. In our study, EBs were tested for their vulnerability to chronic exposure to low doses of MeHgCl (1 nM) under atmospheric (21%O2) and physioxia (5%O2) conditions. Significant differences were observed in the relative expression of genes associated with DNA repair and mitophagy between the tested oxygen conditions in nontreated EBs. When compared to physioxia conditions, the significant differences recorded in EBs cultured at 21% O2 included: (1) lower expression of genes associated with DNA repair (ATM, OGG1, PARP1, POLG1) and mitophagy (PARK2); (2) higher level of mtDNA copy number; and (3) higher expression of the neuroectodermal gene (NES). Chronic exposure to a low dose of MeHgCl (1 nM) disrupted the development of EBs under both oxygen conditions. However, only EBs exposed to MeHgCl at 21% O2 revealed downregulation of mtDNA copy number, increased oxidative DNA damage and DNA fragmentation, as well as disturbances in SOX17 (endoderm) and TBXT (mesoderm) genes expression. Our data revealed that physioxia conditions protected EBs genome integrity and their further differentiation.
... While the discussion below is limited to DNA repair proteins that have been examined in developmental studies using EtOH (OGG1, BRCA1), it would not be surprising if other DNA repair proteins like p53, ATM and CSB that are developmentally protective against other ROS-initiating agents like benzo[a]pyrene, gamma radiation, phenytoin and methamphetamine (Bhuller & Wells, 2006;Laposa, Henderson, Xu, & Wells, 2004;McCallum, Wong, & Wells, 2011;Nicol, Harrison, Laposa, Gimelshtein, & Wells, 1995) prove to be similarly protective against EtOH. ...
This review covers molecular mechanisms involving oxidative stress and DNA damage that may contribute to morphological and functional developmental disorders in animal models resulting from exposure to alcohol (ethanol, EtOH) in utero or in embryo culture. Components covered include: (a) a brief overview of EtOH metabolism and embryopathic mechanisms other than oxidative stress; (b) mechanisms within the embryo and fetal brain by which EtOH increases the formation of reactive oxygen species (ROS); (c) critical embryonic/fetal antioxidative enzymes and substrates that detoxify ROS; (d) mechanisms by which ROS can alter development, including ROS‐mediated signal transduction and oxidative DNA damage, the latter of which leads to pathogenic genetic (mutations) and epigenetic changes; (e) pathways of DNA repair that mitigate the pathogenic effects of DNA damage; (f) related indirect mechanisms by which EtOH enhances risk, for example by enhancing the degradation of some DNA repair proteins; and, (g) embryonic/fetal pathways like NRF2 that regulate the levels of many of the above components. Particular attention is paid to studies in which chemical and/or genetic manipulation of the above mechanisms has been shown to alter the ability of EtOH to adversely affect development. Alterations in the above components are also discussed in terms of: (a) individual embryonic and fetal determinants of risk and (b) potential risk biomarkers and mitigating strategies. FASD risk is likely increased in progeny which/who are biochemically predisposed via genetic and/or environmental mechanisms, including enhanced pathways for ROS formation and/or deficient pathways for ROS detoxification or DNA repair.
... Our results point to the notion that changes in the epigenetic state of the genome can be induced early in development by environmental conditions, and that these changes can have consequences for both gene expression in adulthood [35,36] and the inheritance of epigenetic phenotypes. We are unaware of the reason why DFS-ICSI renders a kinkier tail phenotype, though a kinked tail has been described as an embryopathy produced by oxidative DNA damage due to ROS [37]. One of the targets for oxidative DNA damage is the methylated base m 5 C found in mammalian DNA. ...
Unlabelled:
Intracytoplasmic sperm injection (ICSI) in mice using DNA-fragmented sperm (DFS) has been linked to an increased risk of genetic and epigenetic abnormalities both in embryos and offspring. This study examines: whether embryonic stem cells (ESCs) derived from DFS-ICSI embryos reflect the abnormalities observed in the DFS-ICSI progeny; the effect of DFS-ICSI on male fertility; and whether DFS-ICSI induces epigenetic changes that lead to a modified heritable phenotype. DFS-ICSI-produced embryos showed a low potential to generate ESC lines. However, these lines had normal karyotype accompanied by early gene expression alterations, though a normal expression pattern was observed after several passages. The fertility of males in the DFS-ICSI and control groups was compared by mating test. Sperm quantity, vaginal plug and pregnancy rates were significantly lower for the DFS-ICSI-produced males compared to in vivo-produced mice, while the number of females showing resorptions was higher. The epigenetic effects of DFS-ICSI were assessed by analyzing the phenotype rendered by the Axin1Fu allele, a locus that is highly sensitive to epigenetic perturbations. Oocytes were injected with spermatozoa from Axin1Fu/+ mice and the DFS-ICSI-generated embryos were transferred to females. A significantly higher proportion of pups expressed the active kinky-tail epiallele in the DFS-ICSI group than the controls.
In conclusion:
1) ESCs cannot be used as a model of DFS-ICSI; 2) DFS-ICSI reduces sperm production and fertility in the male progeny; and 3) DFS-ICSI affects the postnatal expression of a defined epigenetically sensitive allele and this modification may be inherited across generations.
... Interestingly, when the same dose of BrdU (50-100 mg/kg) was administered to an adult rat or mouse, no toxic effects were seen in the adult brain, indicating that the fetal brain and adult brain have different sensitivities for BrdUinduced toxicity. Other genotoxic compounds, including 1-b-darabinofuranosylcytosine, ethylnitrosourea, 5-azacytidine, and X-rays, have been reported to cause cell death and cell cycle arrest in the fetal brain (Ueno et al. 2002;Laposa et al. 2004;Yamauchi et al. 2004;Katayama et al. 2005) and to induce abnormal behavior in rodents (Artlich et al. 1994;Londei et al. 1995;Keays and Nolan 2003;Bacon et al. 2004). These results suggest that the fetal brain, in which tissues are actively proliferating, should be highly susceptible to genotoxic agents, and that embryonic neural stem cells should be toxicologically heterogeneous (Ogawa et al. 2005;Kuwagata et al. 2007). ...
Developmental neurotoxicity (DNT) tests usually focus on postnatal indicators, such as behavior and neuropathology, for the detection of chemically induced neurodevelopmental defects in the central nervous system (CNS). However, low reliability, especially low reproducibility, of behavioral results often causes concern among scientists and the scientific community in general. Guidance of neurohistopathological examination in the DNT guideline also has some shortcomings, especially relating to the methodological aspects. Ongoing international trends in DNT tests have shifted from the use of original in vivo animal (mammalian) studies to in vitro experiments using cell cultures and/or non-mammalian species, such as fish. In vitro systems might initially be useful to screen test chemicals for their DNT potential. Although in vitro systems are employed as alternative approaches for DNT studies, the use of in vivo studies based on animal models remains an important factor when data are to be extrapolated to the human case. In this review, a new in vivo approach that focuses on histopathological observation of each developmental step of the CNS, such as proliferation of neural stem cells, migration of immature neurons, and formation of neural networks, using fetal and neonatal brains after chemical exposure is introduced, and some queries and arguments for current DNT experimental guidelines are discussed.
... Following DNA damage, H2AX becomes rapidly phosphorylated by members of the phosphatidylinositol-3-kinase family (Bonner et al., 2008; Rogakou et al., 1998), which includes ATM, AT and Rad3-related protein (ATR), and DNA-dependent protein kinase (DNA-PK; Fernandez-Capetillo et al., 2004; Stiff et al., 2004 Stiff et al., , 2006). Of these three kinases, ATM is considered to play a prominent role in γH2AX induction following DSB damage (Falck et al., 2005; Redon et al., 2002; Stiff et al., 2004 Stiff et al., , 2006), and ATM-deficient progeny are more susceptible to in utero death and birth defects caused by in utero exposure to low-dose ionizing radiation (Laposa et al., 2004). Exposure to ionizing radiation, such as x-rays, γ-radiation, α-particles, and heavy ions, causes the direct formation of DNA DSBs (Cebulska-Wasilewska et al., 2005; Desai et al., 2005; Hanasoge and Ljungman, 2007; Usami et al., 2006), which leads to a rapid rise in γH2AX levels that are maximal at 30–60 min after irradiation (Keogh et al., 2006; Markova et al., 2007; Sedelnikova and Bonner, 2006; Sedelnikova et al., 2003). ...
Methylmercury (MeHg) is a potent neurotoxin, teratogen, and probable carcinogen, but the underlying mechanisms of its actions remain unclear. Although MeHg causes several types of DNA damage, the toxicological consequences of this macromolecular damage are unknown. MeHg enhances oxidative stress, which can cause various oxidative DNA lesions that are primarily repaired by oxoguanine glycosylase 1 (OGG1). Herein, we compared the response of wild-type and OGG1 null (Ogg1(-/-)) murine embryonic fibroblasts to environmentally relevant, low micromolar concentrations of MeHg by measuring clonogenic efficiency, cell cycle arrest, DNA double-strand breaks (DSBs), and activation of the DNA damage response pathway.Ogg1(-/-) cells exhibited greater sensitivity to MeHg than wild-type controls, as measured by the clonogenic assay, and showed a greater propensity for MeHg-initiated apoptosis. Both wild-type and Ogg1(-/-) cells underwent cell cycle arrest when exposed to micromolar concentrations of MeHg; however, the extent of DSBs was exacerbated in Ogg1(-/-) cells compared with that in wild-type controls. Pretreatment with the antioxidative enzyme catalase reduced levels of DSBs in both wild-type and Ogg1(-/-) cells but failed to block MeHg-initiated apoptosis at micromolar concentrations. Our findings implicate reactive oxygen species mediated DNA damage in the mechanism of MeHg toxicity; and demonstrate for the first time that impaired DNA repair capacity enhances cellular sensitivity to MeHg. Accordingly, the genotoxic properties of MeHg may contribute to its neurotoxic and teratogenic effects, and an individual's response to oxidative stress and DNA damage may constitute an important determinant of risk.
... The importance of DNA damage detection is supported by the enhanced teratogenicity of B[a]P in p53 knockout mice (Nicol et al, 1995). We noted that primary sensors of DNA damage and stress appear to be the phosphotidyl-inostitol-3-(PI-3) kinases such as DNA-dependent protein kinase (DNA-PK), ATM (ataxia-telangiectasiamutated), and/or ATR(ATM related), with activation of p53 involving phosphorylation on serine 15 (Laposa et al, 2004;Bhuller and Wells, 2006).We previously found that PI-3K pathway was implicated in activator factor 1 (AP-1) transactivation induced by B[a]P (Gao, et al., 2007). C-Jun, a primary member components of AP-1 families, have been proved to be required for B[a]P-induced cell cycle alternation (Jiao et al, 2008). ...
... Conversely, p53 knockout mice are protected against the ocular teratogenic effects of 2chloro-2 0 -deoxyadenosine, presumably due to a reduced signal for apoptosis (Wubah et al., 1996), illustrating the variable role of DNA damage detection pathways in teratogenesis. A similar protective role is observed with ATM, evidenced by enhanced, gene dose-dependent teratogenicity in Atm knockout mice exposed to ionizing radiation in vivo (Laposa et al., 2004) or to phenytoin in embryo culture . The protective effect of ATM against phenytoin teratogenicity in vivo was less comprehensive than that in embryo culture. ...
Several teratogenic agents, including ionizing radiation and xenobiotics such as phenytoin, benzo[a]pyrene, thalidomide, and methamphetamine, can initiate the formation of reactive oxygen species (ROS) that oxidatively damage cellular macromolecules including DNA. Oxidative DNA damage, and particularly the most prevalent 8-oxoguanine lesion, may adversely affect development, likely via alterations in gene transcription rather than via a mutational mechanism. Contributions from oxidative DNA damage do not exclude roles for alternative mechanisms of initiation like receptor-mediated processes or the formation of covalent xenobiotic-macromolecular adducts, damage to other macromolecular targets like proteins and lipids, and other effects of ROS like altered signal transduction. Even in the absence of teratogen exposure, endogenous developmental oxidative stress can have embryopathic consequences in the absence of key pathways for detoxifying ROS or repairing DNA damage. Critical proteins in pathways for DNA damage detection/repair signaling, like p53 and ataxia telangiectasia mutated, and DNA repair itself, like oxoguanine glycosylase 1 and Cockayne syndrome B, can often, but not always, protect the embryo from ROS-initiating teratogens. Protection may be variably dependent upon such factors as the nature of the teratogen and its concentration within the embryo, the stage of development, the species, strain, gender, target tissue and cell type, among other factors.
... A similar atm gene dose-dependent increase in embryopathies was observed in þ/À and À/À ATM-deficient fetuses in vivo for some but not all parameters following maternal treatment with only the saline vehicle . Similarly in another in vivo study, the untreated À/À ATM-deficient control mice had a lower fetal body weight than their þ/À and þ/þ littermates (Laposa et al., 2004). The observed enhanced embryopathies in ATMdeficient mice in the absence of teratogen exposure may, similar to deficiencies in antioxidative enzymes, reveal the pathogenic potential of constitutive oxidative stress, in this case allowing an accumulation of oxidative DNA damage, possibly in selective tissues and/or cell types. ...
... When ROS formation and oxidative DNA damage are enhanced by xenobiotics, the full pathogenic potential of oxidative DNA damage, and perhaps 8-oxoG, is revealed, along with the developmental importance of activities of DNA damage response and repair pathways as risk factors that determine individual susceptibility. With respect to ROS sensing and DNA damage response (Fig. 7), when pregnant atm knockout mice are exposed to a low, normally nonteratogenic single dose of gamma radiation (0.5 Gy), À/À ATM-deficient fetuses exhibit a significant increase in fetal and neonatal death and structural tail anomalies (Laposa et al., 2004). If the maternal radiation dose is raised to 2.0 Gy, even the þ/À ATM-deficient fetuses are affected, with an overall atm gene dose-dependent pattern of severity. ...
In the developing embryo and fetus, endogenous or xenobiotic-enhanced formation of reactive oxygen species (ROS) like hydroxyl radicals may adversely alter development by oxidatively damaging cellular lipids, proteins and DNA, and/or by altering signal transduction. The postnatal consequences may include an array of birth defects (teratogenesis), postnatal functional deficits, and diseases. In animal models, the adverse developmental consequences of in utero exposure to agents like thalidomide, methamphetamine, phenytoin, benzo[a]pyrene, and ionizing radiation can be modulated by altering pathways that control the embryonic ROS balance, including enzymes that bioactivate endogenous substrates and xenobiotics to free radical intermediates, antioxidative enzymes that detoxify ROS, and enzymes that repair oxidative DNA damage. ROS-mediated signaling via Ras, nuclear factor kappa B and related transducers also may contribute to altered development. Embryopathies can be reduced by free radical spin trapping agents and antioxidants, and enhanced by glutathione depletion. Further modulatory approaches to evaluate such mechanisms in vivo and/or in embryo culture have included the use of knockout mice, transgenic knock-ins and mutant deficient mice with altered enzyme activities, as well as antisense oligonucleotides, protein therapy with antioxidative enzymes, dietary depletion of essential cofactors and chemical enzyme inhibitors. In a few cases, measures anticipated to be protective have conversely enhanced the risk of adverse developmental outcomes, indicating the complexity of development and need for caution in testing therapeutic strategies in humans. A better understanding of the developmental effects of ROS may provide insights for risk assessment and the reduction of adverse postnatal consequences.
... Less direct evidence implicating oxidative DNA damage and repair in the mechanism of teratogenesis includes the enhanced susceptibility of p53 and atm knock-out mice, which lack key proteins for detecting and/or repairing oxidative DNA damage, to the embryopathic and teratogenic effects of various ROSinitiating agents (Moallem and Hales, 1998;Laposa et al., 2004;. In humans, it has recently been suggested that polymorphisms in DNA repair genes, including ogg1, may be involved in teratogenesis (Olshan et al., 2005). ...
In utero methamphetamine (METH) exposure enhances the oxidative DNA lesion 7,8-dihydro-8-oxoguanine (8-oxoG) in CD-1 fetal mouse brain, and causes long-term postnatal motor coordination deficits. Herein we used oxoguanine glycosylase 1 (ogg1) knock-out mice to determine the pathogenic roles of 8-oxoG and OGG1, which repairs 8-oxoG, in METH-initiated neurodevelopmental anomalies. Administration of METH (20 or 40 mg/kg) on gestational day 17 to pregnant +/- OGG1-deficient females caused a drug dose- and gene dose-dependent increase in 8-oxoG levels in OGG1-deficient fetal brains (p < 0.05). Female ogg1 knock-out offspring exposed in utero to high-dose METH exhibited gene dose-dependent enhanced motor coordination deficits for at least 12 weeks postnatally (p < 0.05). Contrary to METH-treated adult mice, METH-exposed CD-1 fetal brains did not exhibit altered apoptosis or DNA synthesis, and OGG1-deficient offspring exposed in utero to METH did not exhibit postnatal dopaminergic nerve terminal degeneration, suggesting different mechanisms. Enhanced 8-oxoG repair activity in fetal relative to adult organs suggests an important developmental protective role of OGG1 against in utero genotoxic stress. These observations provide the most direct evidence to date that 8-oxoG constitutes an embryopathic molecular lesion, and that functional fetal DNA repair protects against METH teratogenicity.
