Altered Epigenetic Marks and Gene Expression in Fetal Brain, and Postnatal Behavioural Disorders, Following Prenatal Exposure of Ogg1 Knockout Mice to Saline or Ethanol.

Oxoguanine glycosylase 1 (OGG1) is widely known to repair the reactive oxygen species (ROS)-initiated DNA lesion 8-oxoguanine (8-oxoG), and more recently was shown to act as an epigenetic modifier. We have previously shown that saline-exposed Ogg1 -/- knockout progeny exhibited learning and memory deficits, which were enhanced by in utero exposure to a single low dose of ethanol (EtOH) in both Ogg1 +/+ and -/- progeny, but more so in Ogg1 -/- progeny. Herein, OGG1-deficient progeny exposed in utero to a single low dose of EtOH or its saline vehicle exhibited OGG1- and/or EtOH-dependent alterations in global histone methylation and acetylation, DNA methylation and gene expression (Tet1 (Tet Methylcytosine Dioxygenase 1), Nlgn3 (Neuroligin 3), Hdac2 (Histone Deacetylase 2), Reln (Reelin) and Esr1 (Estrogen Receptor 1)) in fetal brains, and behavioural changes in open field activity, social interaction and ultrasonic vocalization, but not prepulse inhibition. OGG1- and EtOH-dependent changes in Esr1 and Esr2 mRNA and protein levels were sex-dependent, as was the association of Esr1 gene expression with gene activation mark histone H3 lysine 4 trimethylation (H3K4me3) and gene repression mark histone H3 lysine 27 trimethylation (H3K27me3) measured via ChIP-qPCR. The OGG1-dependent changes in global epigenetic marks and gene/protein expression in fetal brains, and postnatal behavioural changes, observed in both saline- and EtOH-exposed progeny, suggest the involvement of epigenetic mechanisms in developmental disorders mediated by 8-oxoG and/or OGG1. Epigenetic effects of OGG1 may be involved in ESR1-mediated gene regulation, which may be altered by physiological and EtOH-enhanced levels of ROS formation, possibly contributing to sex-dependent developmental disorders observed in Ogg1 knockout mice. The OGG1- and EtOH-dependent associations provide a basis for more comprehensive mechanistic studies to determine the causal involvement of oxidative DNA damage and epigenetic changes in ROS-mediated neurodevelopmental disorders.

Breast cancer 1 (BRCA1) protection in altered gene expression and neurodevelopmental disorders due to physiological and ethanol-enhanced reactive oxygen species formation.

The breast cancer 1 (Brca1) susceptibility gene regulates the repair of reactive oxygen species (ROS)-mediated DNA damage, which is implicated in neurodevelopmental disorders. Alcohol (ethanol, EtOH) exposure during pregnancy causes fetal alcohol spectrum disorders (FASD), including abnormal brain function, associated with enhanced ROS-initiated DNA damage. Herein, oxidative DNA damage in fetal brains and neurodevelopmental disorders were enhanced in saline-exposed +/- vs. +/+ Brca1 littermates. A single EtOH exposure during gestation further enhanced oxidative DNA damage, altered the expression of developmental/DNA damage response genes in fetal brains, and resulted in neurodevelopmental disorders, all of which were BRCA1-dependent. Pretreatment with the ROS inhibitor phenylbutylnitrone (PBN) blocked DNA damage and some neurodevelopmental disorders in both saline- and EtOH-exposed progeny, corroborating a ROS-dependent mechanism. Fetal BRCA1 protects against altered gene expression and neurodevelopmental disorders caused by both physiological and EtOH-enhanced levels of ROS formation. BRCA1 deficiencies may enhance the risk for FASD.

DNA Damage and Repair and Epigenetic Modification in the Role of Oxoguanine Glycosylase 1 in Brain Development.

Oxoguanine glycosylase 1 (OGG1) repairs the predominant reactive oxygen species-initiated DNA lesion 8-oxoguanine. Human OGG1 polymorphisms resulting in reduced DNA repair associate with an increased risk for disorders like cancer and diabetes, but the role of OGG1 in brain development is unclear. Herein, we show that Ogg1 knockout mice at 2-3 months of age exhibit enhanced gene- and sex-dependent DNA damage (strand breaks) and decreased epigenetic DNA methylation marks (5-methylcytosine, 5-hydroxymethylcytosine), both of which were associated with increased cerebellar calbindin levels, reduced hippocampal postsynaptic function, altered body weight with age and disorders of brain function reflected in behavioral tests for goal-directed repetitive behavior, anxiety and fear, object recognition and spatial memory, motor coordination and startle response. These results suggest that OGG1 plays an important role in normal brain development, possibly via both its DNA repair activity and its role as an epigenetic modifier, with OGG1 deficiencies potentially contributing to neurodevelopmental disorders.

Sex- and OGG1-dependent reversal of in utero ethanol-initiated changes in postnatal behaviour by neonatal treatment with the histone deacetylase inhibitor trichostatin A (TSA) in oxoguanine glycosylase 1 (Ogg1) knockout mice.

Oxoguanine glycosylase 1 (OGG1) is both a DNA repair enzyme and an epigenetic modifier. We assessed behavioural abnormalities in OGG1-deficient progeny exposed once in utero to a low dose of ethanol (EtOH) and treated postnatally with a global histone deacetylase inhibitor, trichostatin A (TSA). The goal of this study was to determine if neurodevelopmental disorders initiated in the fetal brain by in utero exposure to EtOH could be mitigated by postnatal treatment with TSA. EtOH and TSA alone improved preference for novel location (short-term, 90 min) and novel object (long-term, 24 h) sex- and OGG1-dependently. Combined EtOH/TSA treatment reversed these effects in the short-term novel location test sex- and OGG1-dependently. In females but not males, the incidence of high shredders of nesting material was not altered by either TSA or EtOH alone, but was reduced by combined EtOH/TSA treatment in +/+ progeny. Similar but non-significant effects were observed in Ogg1 -/- females. Accelerated rotarod performance was enhanced by both EtOH and TSA alone in only male Ogg1 +/+ but not -/- progeny, and was not altered by combined EtOH/TSA exposure. The OGG1-dependent effects of EtOH and TSA particularly on novel location and the incidence of high shredders, and the reversal of EtOH effects on these parameters by combined EtOH/TSA treatment, suggests both xenobiotics may alter behaviour via a mechanism involving OGG1 acting as an epigenetic modifier, in addition to repairing DNA damage. These preliminary results suggest that the postnatal use of more selective epigenetic modifying agents may constitute a novel strategy for mitigating some components of ROS-initiated neurodevelopmental disorders.

DNA damage and synaptic and behavioural disorders in glucose-6-phosphate dehydrogenase-deficient mice.

Mice deficient in glucose-6-phosphate dehydrogenase (G6PD) cannot replenish the cellular antioxidant glutathione, which detoxifies neurodegenerative reactive oxygen species (ROS). To determine the functional consequences of G6PD deficiency, young and aging G6PD-deficient mice were evaluated for brain G6PD activity, DNA damage (comets, γH2AX), Purkinje cell loss, brain function (electrophysiology, behaviour) and lifespan. DNA comet formation was increased and Purkinje cell counts were decreased in a G6pd gene dose-dependent fashion. γH2AX formation varied by age, sex and brain region, with increased levels in G6PD-deficient young and aging females, and in aging males. Aging male G6PD-deficient mice exhibited synaptic dysfunction in hippocampal slices. G6PD-deficient young and aging females exhibited deficits in executive function, and young deficient mice exhibited deficits in social dominance. Conversely, median lifespan in G6PD-deficient females and males was enhanced. Enhanced ROS-initiated brain damage in G6PD deficiency has functional consequences, suggesting that G6PD protects against ROS-mediated neurodegenerative disorders.

Quantifying Activity for Repair of the DNA Lesion 8-Oxoguanine by Oxoguanine Glycosylase 1 (OGG1) in Mouse Adult and Fetal Brain Nuclear Extracts Using Biotin-Labeled DNA.

The reactive oxygen species (ROS)-initiated DNA lesion 8-oxoguanine (8-oxoG) is commonly used as a biomarker to measure oxidative stress levels in tissue samples from animals and humans. This lesion also can play a pathogenic role in cancer, birth defects, and neurodegeneration, among other disorders. The level of 8-oxoG may be enhanced due to ROS-initiating environmental factors (e.g., drugs, gamma radiation, microbial infection) or due to a decrease in the activity of oxoguanine glycosylase 1 (OGG1), an enzyme that repairs this lesion. Measurement of the activity of OGG1 can be useful in elucidating mechanisms and complements measurements of 8-oxoG levels in tissues of interest. This protocol describes an assay for measuring the activity of 8-oxoG in mouse adult and fetal brain tissues. Briefly, a synthetic duplex containing the 8-oxoG residue in one of the nucleotides (49-mer), labeled with biotin at the 3'-end, is incubated with protein extract from the tissue of interest containing OGG1, which cleaves the 8-oxoG residue producing a cleavage product of ~27-mer. The percent cleavage quantifies the activity of OGG1 in that tissue. The biotin tag allows rapid and sensitive detection of the cleavage product via chemiluminescence, avoiding the problems of safety and short half-lives of radionuclides encountered in assays employing a radioactively-labeled substrate.

Characterization of Epigenetic Histone Activation/Repression Marks in Sequences of Genes by Chromatin Immunoprecipitation-Quantitative Polymerase Chain Reaction (ChIP-qPCR).

Chromatin immunoprecipitation (ChIP) is widely used to measure protein-DNA interactions. This protocol outlines a ChIP method used to identify the association of a protein or protein modification (such as a specific histone modification-methylation, acetylation, etc.) of interest with a specific DNA sequence in a target gene in fetal mouse brains on gestational day (GD) 17. Briefly, DNA and proteins are cross-linked (via formaldehyde), and chromatin is sonicated into fragments between 200 and 1000 base pair (bp) long, with an average length of 500 bp. The DNA-protein complexes are captured using antibodies directed toward the protein or protein modification of interest. These immunoprecipitated complexes are retrieved using agarose beads. The DNA-protein cross-links are reversed (via heat and via presence of high salt concentrations), and the ChIP DNA is purified and measured via a quantitative polymerase chain (qPCR) reaction. The results show the association of histone modifications at unknown sites of specific genes of interest, indicating which epigenetic modifications of specific genes may be responsible for the outcome of interest.

Oxidative stress and DNA damage in the mechanism of fetal alcohol spectrum disorders.

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.

Fetal oxidative stress mechanisms of neurodevelopmental deficits and exacerbation by ethanol and methamphetamine.

In utero exposure of mouse progeny to alcohol (ethanol, EtOH) and methamphetamine (METH) causes substantial postnatal neurodevelopmental deficits. One emerging pathogenic mechanism underlying these deficits involves fetal brain production of reactive oxygen species (ROS) that alter signal transduction, and/or oxidatively damage cellular macromolecules like lipids, proteins, and DNA, the latter leading to altered gene expression, likely via non-mutagenic mechanisms. Even physiological levels of fetal ROS production can be pathogenic in biochemically predisposed progeny, and ROS formation can be enhanced by drugs like EtOH and METH, via activation/induction of ROS-producing NADPH oxidases (NOX), drug bioactivation to free radical intermediates by prostaglandin H synthases (PHS), and other mechanisms. Antioxidative enzymes, like catalase in the fetal brain, while low, provide critical protection. Oxidatively damaged DNA is normally rapidly repaired, and fetal deficiencies in several DNA repair proteins, including oxoguanine glycosylase 1 (OGG1) and breast cancer protein 1 (BRCA1), enhance the risk of drug-initiated postnatal neurodevelopmental deficits, and in some cases deficits in untreated progeny, the latter of which may be relevant to conditions like autism spectrum disorders (ASD). Risk is further regulated by fetal nuclear factor erythroid 2-related factor 2 (Nrf2), a ROS-sensing protein that upregulates an array of proteins, including antioxidative enzymes and DNA repair proteins. Imbalances between conceptal pathways for ROS formation, versus those for ROS detoxification and DNA repair, are important determinants of risk. Birth Defects Research (Part C) 108:108-130, 2016. © 2016 Wiley Periodicals, Inc.