BRCA1 protein dose-dependent risk for embryonic oxidative DNA damage, embryopathies and neurodevelopmental disorders with and without ethanol exposure.

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.

The UN Joint Group of Experts on the Scientific Aspects of Marine Environmental Protection (GESAMP) - An ocean science-policy interface standing the test of time.

Addressing the wide range of marine pollution problems facing the global ocean requires a continual transfer of credible, relevant and timely scientific information to policy and decision makers in coastal and ocean management. The United Nations GESAMP (Joint Group of Experts on the Scientific Aspects of Marine Environmental Protection) is a long-standing scientific advisory group providing such information on a wide range of marine topics and emerging issues of concern to ten UN Sponsoring Organizations. This paper presents an overview of GESAMPs operation and examples of its current work. The group's scientific output is often cited by national governments, inter-governmental groups, and a range of non-governmental groups. Given the growing concerns about ocean health and the impacts of many stressors in an era of climate change, the development of timely and effective ocean policy and decision making would benefit from wider recognition and application of GESAMPs work.

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.

The leatherback turtle (Dermochelys coriacea) and plastics in the Northwest Atlantic ocean: A hazard assessment.

Atlantic leatherback turtles are faced with multiple threats, such as ship strikes, pollution and predation, throughout their annual migratory routes in the Northwest (NW) Atlantic. The risks associated with encounters with floating and submerged plastic debris are currently unknown. This study is a hazard assessment of plastics for this turtle's sub-population, using 2010-2019 data from the national Great Canadian Shoreline Cleanup (GSCS) program, therefore potential exposure, and published evidence on the interactions of plastics and leatherbacks, hence potential effects. The type of plastic items and their abundance along shorelines of three Atlantic Provinces - Nova Scotia (NS), Prince Edward Island (PEI), Newfoundland and Labrador (NL) - were evaluated and compared to plastic items known to interact with leatherbacks. During the 2010-2019 period, a total of 220,590 plastic items were collected from 578 sites, representing 1264 km of shoreline. Plastic bags and rope are in the top ten most common items found on shorelines of NS, PEI, and NL. Pot gear and trap nets are in the top ten for PEI and are the 14th most common plastic item found on all shorelines. Cigarette debris is also commonly found. From the literature, plastic bags, pot gear and trap nets, and rope are known to adversely affect leatherbacks. Assuming that a large proportion of the shoreline debris comes in from the sea, after being in coastal waters for unknown periods, the study shows that such items pose a hazard to leatherbacks through ingestion and entanglement, based on published studies. Evidence is now needed on actual exposure at sea to the most common items to establish the ecological risk of plastics to these turtles in NW Atlantic waters.

Novel mechanisms in alcohol neurodevelopmental disorders via BRCA1 depletion and BRCA1-dependent NADPH oxidase regulation.

The breast cancer 1 protein (BRCA1) facilitates DNA repair, preventing embryolethality and protecting the fetus from reactive oxygen species (ROS)-induced developmental disorders mediated by oxidatively damaged DNA. Alcohol (ethanol, EtOH) exposure during pregnancy causes fetal alcohol spectrum disorders (FASD), characterized by aberrant behaviour and enhanced ROS formation and proteasomal protein degradation. Herein, ROS-producing NADPH oxidase (NOX) activity was higher in Brca1 +/- vs. +/+ fetal and adult brains, and further enhanced by a single EtOH exposure. EtOH also enhanced catalase and proteasomal activities, while conversely reducing BRCA1 protein levels without affecting Brca1 gene expression. EtOH-initiated adaptive postnatal freezing behaviour was lost in Brca1 +/- progeny. Pretreatment with the free radical spin trap and ROS inhibitor phenylbutylnitrone blocked all EtOH effects, suggesting ROS-dependent mechanisms. This is the first in vivo evidence of NOX regulation by BRCA1, and of EtOH-induced, ROS-mediated depletion of BRCA1, revealing novel mechanisms of BRCA1 protection in FASD.

Reflecting on an anniversary. The 1970 SS Arrow oil spill in Chedabucto Bay, Nova Scotia, Canada.

Fifty years ago, the tanker SS Arrow ran aground in Chedabucto Bay on the east coast of Canada, causing a massive spill of Bunker C oil in winter conditions. This article reflects on this anniversary, briefly describing the spill response and the considerable follow-up research over five decades on the fate and effects of oils spilled in northern cold marine waters. Importantly, the spill led to considerable work in Canada on spill control and bioremediation measures, and advice about oil spills as a marine pollutant of global importance.

Monitoring chemical contaminants in the Gulf of Maine, using sediments and mussels (Mytilus edulis): An evaluation.

The objective of this paper is to determine whether contaminant data on mussels and sediments can be used interchangeably, or not, when assessing the degree of anthropogenic contamination of a water body. To obtain adequate coverage of the entire Gulf of Maine, Bay of Fundy sediment samples were collected, analyzed and combined with similar data from four coastal monitoring programs. This required careful interpretation but provided robust results consistent with published literature. A strong correspondence was found between sediment and mussel concentrations for polycyclic aromatic hydrocarbons, moderate to weak correspondence for polychlorinated biphenyls, and except for mercury and zinc, little to no correspondence was found for metals. We conclude that mussel contaminant data are likely sufficient for providing information on the spatial and temporal distribution of chemical contaminants, in coastal waters, under a broad range of environmental conditions and contaminant levels, and unlike sediments, provide direct information on contaminant bioavailability.

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.

Measurement of the Oxidative DNA Lesion 8-Oxoguanine (8-oxoG) by ELISA or by High-Performance Liquid Chromatography (HPLC) with Electrochemical Detection.

Reactive oxygen species (ROS) can oxidize cellular macromolecules like DNA, causing DNA damage. The most common form of DNA damage is the 8-oxoguanine (8-oxoG) lesion, typically repaired by the base excision repair (BER) pathway, which is initiated by the enzyme oxoguanine glycosylase 1 (OGG1). ROS are produced endogenously and can be enhanced by environmental factors, such as xenobiotics, radiation, and microbial pathogens. As a commonly used biomarker of oxidative damage, 8-oxoG can be measured in two different ways described herein. Commercially available ELISA kits allow for easy detection of the 8-oxoG lesion, while more difficult HPLC assays with UV and electrochemical detection allow for a more definitive identification and quantification of 8-oxoG.

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.

Western Analysis of Breast Cancer 1 Protein (BRCA1).

Known for its tumor suppressor activity in breast and ovarian cancers, the breast cancer 1 susceptibility gene (Brca1) is involved in a variety of cellular pathways including DNA repair, antioxidant signaling, apoptosis, and cell cycle regulation. BRCA1 can translocate between the cytoplasm and nucleus to perform its various roles. Herein is a procedure for measuring BRCA1 protein levels in the whole cell lysate (WCL), as well as in the nuclear (N) and cytoplasmic (C) fractions of mouse tissues at different gestational ages. The method employs multiple loading controls to ensure proper separation of fractions and a total protein stain for more consistent comparisons of dissimilar samples. This method is useful for identifying BRCA1 deficiencies and localization in a variety of research fields, including development, neurodegeneration, and cancer.

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.

Hyperthermia-mediated drug delivery induces biological effects at the tumor and molecular levels that improve cisplatin efficacy in triple negative breast cancer.

Triple negative breast cancer is an aggressive disease that accounts for at least 15% of breast cancer diagnoses, and a disproportionately high percentage of breast cancer related morbidity. Intensive research efforts are focused on the development of more efficacious treatments for this disease, for which therapeutic options remain limited. The high incidence of mutations in key DNA repair pathways in triple negative breast cancer results in increased sensitivity to DNA damaging agents, such as platinum-based chemotherapies. Hyperthermia has been successfully used in breast cancer treatment to sensitize tumors to radiation therapy and chemotherapy. It has also been used as a mechanism to trigger drug release from thermosensitive liposomes. In this study, mild hyperthermia is used to trigger release of cisplatin from thermosensitive liposomes in the vasculature of human triple negative breast cancer tumors implanted orthotopically in mice. This heat-triggered liposomal formulation of cisplatin resulted in significantly delayed tumor growth and improved overall survival compared to treatment with either non-thermosensitive liposomes containing cisplatin or free cisplatin, as was observed in two independent tumor models (i.e. MDA-MB-231 and MDA-MB-436). The in vitro sensitivity of the cell lines to cisplatin and hyperthermia alone and in combination was characterized extensively using enzymatic assays, clonogenic assays, and spheroid growth assays. Evaluation of correlations between the in vitro and in vivo results served to identify the in vitro approach that is most predictive of the effects of hyperthermia in vivo. Relative expression of several heat shock proteins and the DNA damage repair protein BRCA1 were assayed at baseline and in response to hyperthermia both in vitro and in vivo. Interestingly, delivery of cisplatin in thermosensitive liposomes in combination with hyperthermia resulted in the most significant tumor growth delay, relative to free cisplatin, in the less cisplatin-sensitive cell line (i.e. MDA-MB-231). This work demonstrates that thermosensitive cisplatin liposomes used in combination with hyperthermia offer a novel method for effective treatment of triple negative breast cancer.

The Gulfwatch contaminants monitoring program in the Gulf of Maine: Are its data being used for ocean protection, with special reference to Nova Scotia, Canada?

The Gulfwatch Contaminants Monitoring Program is part of the Canada-US, Gulf of Maine Council on the Marine Environment (GOMC). Programs monitoring legacy toxic substances, i.e., chemical contaminants, such as Gulfwatch, collect and analyse environmental samples (e.g., blue mussels), interpret the data, and report on chemical levels and trends (spatial and temporal) in coastal waters. This study explored the extent to which its extensive information (data, reports, papers) has been used broadly and by Nova Scotia, a GOMC member. A mixed-methods study was conducted, using quantitative and qualitative metrics. Citations to some Gulfwatch papers and analysis of use of the Gulfwatch website showed that its data and information were accessed, mostly by government departments. However, interviews revealed that the departments were not using the data to inform Nova Scotia provincial coastal policy or practices. Recommendations are presented to improve the visibility and use of information provided by long-term, environmental monitoring programs.

The iconic Torrey Canyon oil spill of 1967 - Marking its legacy.

March 2017 marks the 50th anniversary of the SS Torrey Canyon oil spill and cleanup, off the Cornwall coast in the English Channel. It was the world's first major supertanker disaster. It was a signature event in the marine pollution field, especially related to oil spill response and the initiation of scientific studies of monitoring and researching the fate and effects of oil in the sea. This paper recalls this event, notes our growing understanding of marine pollution and global efforts for cleaner seas, and encourages further work on both oil and the many emerging environmental issues affecting the marine environment.