No abstract

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.

The breast cancer 1 (brca1) gene is associated with breast and ovarian cancers, and heterozygous (+/−) brca1 knockout progeny develop normally, suggesting a negligible developmental impact. However, our results show BRCA1 plays a broader biological role in protecting the embryo from oxidative stress. Sox2-promoted Cre-expressing hemizygous males were mated with floxed brca1 females, and gestational day 8 +/− brca1 conditional knockout embryos with a 28% reduction in protein expression were exposed in culture to the reactive oxygen species (ROS)-initiating drug ethanol (EtOH). Untreated +/− brca1-deficient embryos developed normally, but when exposed to EtOH exhibited increased levels of oxidatively damaged DNA, measured as 8-oxo-2'-deoxyguanosine, γH2AX, which is a marker of DNA double strand breaks that can result from 8-oxo-2'-deoxyguanosine, formation, and embryopathies at EtOH concentrations that did not affect their brca1-normal littermates. These results reveal that even modest BRCA1 deficiencies render the embryo more susceptible to drug-enhanced ROS formation, and corroborate a role for DNA oxidation in the mechanism of EtOH teratogenesis.

Reactive oxygen species (ROS) have been implicated in the teratogenicity of alcohol (ethanol, EtOH). To determine the involvement of embryonic oxidative DNA damage, DNA repair-deficient oxoguanine glycosylase 1 (ogg1) knockout embryos were exposed in culture to EtOH (2 or 4 mg/ml), with or without pretreatment with the free radical spin trap phenylbutylnitrone (PBN) (0.125 mM). Visceral yolk sacs were used to genotype embryos for DNA repair status and gender. EtOH caused a concentration-dependent decrease in anterior neuropore closure (ANPC), somite development, turning, crown-rump length (CRL), yolk sac diameter (YSD) and head length (HL) (p < 0.001) in all 3 ogg1 genotypes. There was a further ogg1 gene dose-dependent decrease from +/+ to -/- embryos in ANPC, somite development, turning, CRL and HL (p < 0.05), and a gene-dependent correlation between HL and ANPC (p < 0.01). Female embryos exhibited lesser ANPC and turning than males (p < 0.05), suggesting underlying gender-dependent target-specific determinants. PBN pretreatment increased ANPC, somite development, turning, CRL, YSD and HL (p < 0.001), although this protection against EtOH was slightly less effective in -/- embryos. Oxidatively damaged DNA determined as 8-oxo-2'-deoxyguanosine (8-oxodGuo), which is repaired by OGG1, was measured in single embryos in vivo after maternal EtOH treatment (4 g/kg i.p). EtOH increased embryonic 8-oxodGuo in an ogg1 gene-dependent fashion, with the highest levels in -/- embryos. These results show that embryonic DNA repair status and gender are determinants of risk. ROS-initiated embryonic DNA oxidation is involved in EtOH embryopathies.