Modifying the Basicity of the dNTP Leaving Group Modulates Precatalytic Conformational Changes of DNA Polymerase ß.

The catalytic function of DNA polymerase ß (pol ß) fulfills the gap-filling requirement of the base excision DNA repair pathway by incorporating a single nucleotide into a gapped DNA substrate resulting from the removal of damaged DNA bases. Most importantly, pol ß can select the correct nucleotide from a pool of similarly structured nucleotides to incorporate into DNA in order to prevent the accumulation of mutations in the genome. Pol ß is likely to employ various mechanisms for substrate selection. Here, we use dCTP analogues that have been modified at the ß,γ-bridging group of the triphosphate moiety to monitor the effect of leaving group basicity of the incoming nucleotide on precatalytic conformational changes, which are important for catalysis and selectivity. It has been previously shown that there is a linear free energy relationship between leaving group pKa and the chemical transition state. Our results indicate that there is a similar relationship with the rate of a precatalytic conformational change, specifically, the closing of the fingers subdomain of pol ß. In addition, by utilizing analogue ß,γ-CHX stereoisomers, we identified that the orientation of the ß,γ-bridging group relative to R183 is important for the rate of fingers closing, which directly influences chemistry.

Temporal coordination of the transcription factor response to H2O2 stress.

Oxidative stress from excess H2O2 activates transcription factors that restore redox balance and repair oxidative damage. Although many transcription factors are activated by H2O2, it is unclear whether they are activated at the same H2O2 concentration, or time. Dose-dependent activation is likely as oxidative stress is not a singular state and exhibits dose-dependent outcomes including cell-cycle arrest and cell death. Here, we show that transcription factor activation is both dose-dependent and coordinated over time. Low levels of H2O2 activate p53, NRF2 and JUN. Yet under high H2O2, these transcription factors are repressed, and FOXO1, NF-κB, and NFAT1 are activated. Time-lapse imaging revealed that the order in which these two groups of transcription factors are activated depends on whether H2O2 is administered acutely by bolus addition, or continuously through the glucose oxidase enzyme. Finally, we provide evidence that 2-Cys peroxiredoxins control which group of transcription factors are activated.

Transcriptomic signaling in zebrafish (Danio rerio) embryos exposed to environmental concentrations of glyphosate.

Glyphosate [N-(phosphonomethyl)glycine] is one of the most popular herbicides worldwide. Globally, the use of glyphosate is increasing, and its residues have been found in drinking water and food products. The data regarding the possible toxic effects of this herbicide are controversial. Therefore, the aim of this study was to evaluate the effects of glyphosate at environmental concentrations in zebrafish (Danio rerio) embryos. Embryos were exposed to 0, 1, 100, and 1,000 µg/L glyphosate for 96 h, and mortality, heart rate, and hatching rate were evaluated. After the experiment, RNA was extracted from the embryos for transcriptional analysis. No mortality was recorded, and exposure to 100 µg/L and 1,000 µg/L of glyphosate resulted in lower heart rates at 48 h. In addition, RNA-seq analysis revealed that glyphosate exposure induced subtle changes in gene transcription profiles. We found 30 differentially expressed genes; however, the highest glyphosate concentration (1,000 µg/L) induced the greatest number of differentially expressed genes involved in oocyte maturation, metabolic processes, histone deacetylation, and nervous system development.

Epigenetic Transgenerational Modifications Induced by Xenobiotic Exposure in Zebrafish.

Zebrafish (Danio rerio) is a well-established vertebrate model in ecotoxicology research that responds to a wide range of xenobiotics such as pesticides, drugs, and endocrine-disrupting compounds. The epigenome can interact with the environment and transform internal and/or external signals into phenotypic responses through changes in gene transcription. Environmental exposures can also generate epigenetic variations in offspring even by indirect exposure. In this review, we address the advantages of using zebrafish as an experimental animal model to study transgenerational epigenetic processes upon exposure to xenobiotics. We focused mostly on DNA methylation, although studies on post-translational modifications of histones, and non-coding RNAs related to xenobiotic exposure in zebrafish are also discussed. A revision of the methods used to study epigenetic changes in zebrafish revealed the relevance and reproducibility for epigenetics-related research. PubMed and Google Scholar databases were consulted for original research articles published from 2013 to date, by using six keywords: zebrafish, epigenetics, exposure, parental, transgenerational, and F2. From 499 articles identified, 92 were considered, of which 14 were selected as included F2 and epigenetic mechanisms. Current knowledge regarding the effect of xenobiotics on DNA methylation, histone modifications, and changes in non-coding RNAs expressed in F2 is summarized, along with key experimental design considerations to characterize transgenerational effects.

DNA glycosylase deficiency leads to decreased severity of lupus in the Polb-Y265C mouse model.

The Polb gene encodes DNA polymerase beta (Pol ß), a DNA polymerase that functions in base excision repair (BER) and microhomology-mediated end-joining. The Pol ß-Y265C protein exhibits low catalytic activity and fidelity, and is also deficient in microhomology-mediated end-joining. We have previously shown that the PolbY265C/+ and PolbY265C/C mice develop lupus. These mice exhibit high levels of antinuclear antibodies and severe glomerulonephritis. We also demonstrated that the low catalytic activity of the Pol ß-Y265C protein resulted in accumulation of BER intermediates that lead to cell death. Debris released from dying cells in our mice could drive development of lupus. We hypothesized that deletion of the Neil1 and Ogg1 DNA glycosylases that act upstream of Pol ß during BER would result in accumulation of fewer BER intermediates, resulting in less severe lupus. We found that high levels of antinuclear antibodies are present in the sera of PolbY265C/+ mice deleted of Ogg1 and Neil1 DNA glycosylases. However, these mice develop significantly less severe renal disease, most likely due to high levels of IgM in their sera.

Differential immunomodulatory effect of PARP inhibition in BRCA1 deficient and competent tumor cells.

Poly-ADP-ribose polymerase (PARP) inhibitors are active against cells and tumors with defects in homology-directed repair as a result of synthetic lethality. PARP inhibitors (PARPi) have been suggested to act by either catalytic inhibition or by PARP localization in chromatin. In this study, we treat BRCA1 mutant cells derived from a patient with triple negative breast cancer and control cells for three weeks with veliparib, a PARPi, to determine if treatment with this drug induces increased levels of mutations and/or an inflammatory response. We show that long-term treatment with PARPi induces an inflammatory response in HCC1937 BRCA1 mutant cells. The levels of chromatin-bound PARP1 in the BRCA1 mutant cells correlate with significant upregulation of inflammatory genes and activation of the cyclic GMP-AMP synthase (cGAS)/signaling effector stimulator of interferon genes (STING pathway). In contrast, an increased mutational load is induced in BRCA1-complemented cells treated with a PARPi. Our results suggest that long-term PARP inhibitor treatment may prime both BRCA1 mutant and wild-type tumors for positive responses to immune checkpoint blockade, but by different underlying mechanisms.

The double-edged sword of cancer mutations: exploiting neoepitopes for the fight against cancer.

Defects in DNA repair have been linked to the accumulation of somatic mutations in tumours. These mutations can promote oncogenesis; however, recent developments have indicated that they may also lead to a targeted immune response against the tumour. This response is initiated by the development of new antigenic epitopes (neoepitopes) arising from mutations in protein-coding genes that are processed and then presented on the surface of tumour cells. These neoepitopes are unique to the tumour, thus enabling lymphocytes to launch an immune response against the cancer cells. Immunotherapies, such as checkpoint inhibitors (CPIs) and tumour-derived vaccines, have been shown to enhance the immunogenic response to cancers and have led to complete remission in some cancer patients. There are tumours that are not responsive to immunotherapy or conventional tumour therapeutics; therefore, there is a push for new treatments to combat these unresponsive cancers. Recently, combinatorial treatments have been developed to further utilise the immune system in the fight against cancer. These treatments have the potential to exploit the defects in DNA repair by inducing more DNA damage and mutations. This can potentially lead to the expression of high levels of neoepitopes on the surface of tumour cells that will stimulate an immunological response. Overall, exploiting DNA repair defects in tumours may provide an edge in this long fight against cancer.

Early-life Pb exposure as a potential risk factor for Alzheimer's disease: are there hazards for the Mexican population?

Alzheimer's disease (AD) is the main cause of dementia in elderly. Increasing life expectancy is behind the growing prevalence of AD worldwide with approximately 45 million cases currently documented and projection studies suggesting a triplication of this number by 2050. Mexico does not have an accurate AD registry, but 860,000 cases were reported in 2014 and the prediction reaches 3.5 million cases by 2050. Amyloid plaques and neurofibrillary tangles represent the main hallmarks of AD, being constituted of amyloid beta (Aß) peptide and phosphorylated tau, respectively. The risk factors for AD include genetic mutations, lifestyle and environmental pollution. Particularly, lead (Pb) has attracted attention due to its ability to target multiple pathways involved in the pathophysiology of AD. Although the epidemiological data are limiting, animal and in vitro studies show growing evidence of causal effects of Pb exposure on AD-linked features including Aß aggregation and tau phosphorylation. Interestingly, many Pb effects occur selectively following early-life exposure to the metal, suggesting an epigenetic mechanism. This hypothesis is supported by changes in DNA methylation and microRNA expression patterns inflicted by early-life Pb exposure. Pb pollution in Mexico represents a significant problem because past and current mining activities, historical use of Pb as fuel additive and culturally rooted use of Pb in glazed ceramics, contribute to high levels of Pb pollution in Mexico. In this review we will discuss potential risks of AD development in Mexican populations chronically exposed to Pb in their childhood.

Modified CDKN2B (p15) and CDKN2A (p16) DNA methylation profiles in urban pesticide applicators.

Gene-specific changes in DNA methylation by pesticides in occupationally exposed populations have not been studied extensively. Of particular concern are changes in the methylation profile of tumor-suppressor, such as CDKN2B and CDKN2A, genes involved in oncogenesis. The aim of this study was to evaluate the methylation profiles of CDKN2B and CDKN2A genes in urban pesticide applicators and their relationship with occupational exposure to pesticides. A cross-sectional study was conducted in 186 urban pesticide applicators (categorized as high or moderate exposures) and 102 participants without documented occupational exposures to pesticides. Acute and chronic pesticide exposures were evaluated by direct measurement of urinary dialkylphosphates, organophosphate metabolites, and a structured questionnaire, respectively. Anthropometric characteristics, diet, clinical histories, and other variables were estimated through a validated self-reported survey. DNA methylation was determined by pyrosequencing of bisulfite-treated DNA. Decreased DNA methylation of the CDKN2B gene was observed in pesticide-exposed groups compared to the non-exposed group. In addition, increased methylation of the CDKN2A promoter was observed in the moderate-exposure group compared to the non-exposed group. Bivariate analysis showed an association between CDKN2B methylation and pesticide exposure, general characteristics, smoking status, and micronutrients, while changes in CDKN2A methylation were associated with pesticide exposure, sex, educational level, body mass index, smoking status, supplement intake, clinical parameters, and caffeine consumption. These data suggest that pesticide exposure modifies the methylation pattern of CDKN2B and CDKN2A genes and raise important questions about the role that these changes may play in the regulation of cell cycle activities, senescence, and aging.

In utero exposure to ultrafine particles promotes placental stress-induced programming of renin-angiotensin system-related elements in the offspring results in altered blood pressure in adult mice.

BACKGROUND: Exposure to particulate matter (PM) is associated with an adverse intrauterine environment, which can promote adult cardiovascular disease (CVD) risk. Ultrafine particles (UFP) (small size and large surface area/mass ratio) are systemically distributed, induce inflammation and oxidative stress, and have been associated with vascular endothelial dysfunction and arterial vasoconstriction, increasing hypertension risk. Placental stress and alterations in methylation of promoter regions of renin-angiotensin system (RAS)-related elements could be involved in UFP exposure-related programming of hypertension. We investigated whether in utero UFP exposure promotes placental stress by inflammation and oxidative stress, alterations in hydroxysteroid dehydrogenase 11b-type 2 (HSD11B2) and programming of RAS-related elements, and result in altered blood pressure in adult offspring. UFP were collected from ambient air using an aerosol concentrator and physicochemically characterized. Pregnant C57BL/6J pun/pun female mice were exposed to collected UFP (400 µg/kg accumulated dose) by intratracheal instillation and compared to control (nonexposed) and sterile H2O (vehicle) exposed mice. Embryo reabsorption and placental stress by measurement of the uterus, placental and fetal weights, dam serum and fetal cortisol, placental HSD11B2 DNA methylation and protein levels, were evaluated. Polycyclic aromatic hydrocarbon (PAH) biotransformation (CYP1A1 and NQO1 (NAD(P)H dehydrogenase (quinone)1)) enzymes, inflammation and oxidative stress in placentas and fetuses were measured. Postnatal day (PND) 50 in male offspring blood pressure was measured. Methylation and protein expression of (RAS)-related elements, angiotensin II receptor type 1 (AT1R) and angiotensin I-converting enzyme (ACE) in fetuses and lungs of PND 50 male offspring were also assessed. RESULTS: In utero UFP exposure induced placental stress as indicated by an increase in embryo reabsorption, decreases in the uterus, placental, and fetal weights, and HSD11B2 hypermethylation and protein downregulation. In utero UFP exposure induced increases in the PAH-biotransforming enzymes, intrauterine oxidative damage and inflammation and stimulated programming and activation of AT1R and ACE, which resulted in increased blood pressure in the PND 50 male offspring. CONCLUSIONS: In utero UFP exposure promotes placental stress through inflammation and oxidative stress, and programs RAS-related elements that result in altered blood pressure in the offspring. Exposure to UFP during fetal development could influence susceptibility to CVD in adulthood.

Relationship between LINE-1 methylation pattern and pesticide exposure in urban sprayers.

Recently a relationship has been reported between pesticide exposure and changes in global DNA methylation patterns. Urban sprayers are a particularly vulnerable population because of the high risk of pesticide exposure that their work implies. Therefore, the aim of this study was to estimate the changes in the Long Interspersed Nucleotide Element (LINE-1) in urban sprayers and its relationship with pesticide exposure. The study population consisted of 190 individuals stratified into three study groups: no occupational pesticide exposure; moderate exposure, and high exposure. Pesticide exposure and other external factors such as diet, lifestyle, and others were evaluated through a validated questionnaire, and the butyrylcholinesterase enzyme activity was evaluated spectrophotometrically and used as exposure biomarker. DNA methylation was evaluated by pyrosequencing on bisulfite-treated DNA. The results showed a significant decrease of %5mC in both the moderate- and high-exposure groups with respect to the non-exposed group (p < 0.05). In addition, alcohol intake was associated with a higher percentage of LINE- 1 methylation. In conclusion, our results suggest that occupational pesticide exposure and external factors appears to modify the DNA methylation pattern measured through LINE-1.

Environmental Epigenetic Changes, as Risk Factors for the Development of Diseases in Children: A Systematic Review.

BACKGROUND: Children are susceptible to environmental contaminants and are at risk of developing diseases, more so if the exposure begins at an early age. Epidemiological studies have postulated the hypothesis of the fetal origin of disease, which is mediated by epigenetic changes. Epigenetic marks are inheritable; they modulate the gene expression and can affect human health due to the presence of environmental factors. OBJECTIVE: This review focuses on DNA-methylation and its association with environmental-related diseases in children. METHODS: A search for studies related to DNA-methylation in children by pre- or post-natal environmental exposures was conducted, and those studies with appropriate designs and statistical analyses and evaluations of the exposure were selected. FINDINGS: Prenatal and early life environmental factors, from diet to exposure to pollutants, have been associated with epigenetic changes, specifically DNA-methylation. Thus, maternal nutrition and smoking and exposure to air particulate matter, polycyclic aromatic hydrocarbons, arsenic, heavy metals, persistent organic pollutants, and some endocrine disrupters during pregnancy have been associated with genomic and gene-specific newborns' DNA-methylation changes that have shown in some cases sex-specific patterns. In addition, these maternal factors may deregulate the placental DNA-methylation balance and could induce a fetal reprogramming and later-in-life diseases. CONCLUSIONS: Exposure to environmental pollutants during prenatal and early life can trigger epigenetic imbalances and eventually the development of diseases in children. The integration of epigenetic data should be considered in future risk assessments.

Inorganic mercury prevents the differentiation of SH-SY5Y cells: Amyloid precursor protein, microtubule associated proteins and ROS as potential targets.

Exposure to mercury (Hg) occurs through different pathways and forms including methylmecury (MeHg) from seafood and rice, ethylmercury (EtHg), and elemental Hg (Hg0) from dental amalgams and artisanal gold mining. Once in the brain all these forms are transformed to inorganic Hg (I-Hg), where it bioaccumulates and remains for long periods. Hg is a well-known neurotoxicant, with its most damaging effects reported during brain development, when cellular key events, such as cell differentiation take place. A considerable number of studies report an impairment of neuronal differentiation due to MeHg exposure, however the effects of I-Hg, an important form of Hg found in brain, have received less attention. In this study, we decided to examine the effects of I-Hg exposure (5, 10 and 20µM) on the differentiation of SH-SY5Y cells induced by retinoic acid (RA, 10µM). We observed extension of neuritic processes and increased expression of neuronal markers (MAP2, tubulin-ßIII, and Tau) after RA stimulation, all these effects were decreased by the co-exposure to I-Hg. Interestingly, I-Hg increased the levels of reactive oxygen species (ROS) and nitric oxide (NO) accompanied with increased levels of inducible nitric oxide synthase (iNOS) and, dimethylarginine dimethylaminohydrolase 1 (DDHA1). Remarkably I-Hg decreased levels of nitric oxide synthase neuronal (nNOS). Moreover I-Hg reduced the levels of tyrosine hydroxylase (TH) and amyloid precursor protein (APP) a protein recently involved in neuronal differentiation. These data suggest that the exposure to I-Hg impairs cell differentiation, and point to new potential targets of Hg toxicity such as APP and NO signaling.

Increased methylation of repetitive elements and DNA repair genes is associated with higher DNA oxidation in children in an urbanized, industrial environment.

DNA methylation in DNA repair genes participates in the DNA damage regulation. Particulate matter (PM), which has metals and polycyclic aromatic hydrocarbons (PAHs) adsorbed, among others has been linked to adverse health outcomes and may modify DNA methylation. To evaluate PM exposure impact on repetitive elements and gene-specific DNA methylation and DNA damage, we conducted a cross-sectional study in 150 schoolchildren (7-10 years old) from an urbanized, industrial area of the metropolitan area of Mexico City (MAMC), which frequently exhibits PM concentrations above safety standards. Methylation (5mC) of long interspersed nuclear element-1 (LINE1) and DNA repair gene (OGG1, APEX, and PARP1) was assessed by pyrosequencing in peripheral mononuclear cells, DNA damage by comet assay and DNA oxidation by 8-OHdG content. PAH and metal contents in PM10 (≤10µm aerodynamic diameter) were determined by HPLC-MS and ICP-AES, respectively. Multiple regression analysis between DNA methylation, DNA damage, and PM10 exposure showed that PM10 was significantly associated with oxidative DNA damage; a 1% increase in 5mC at all CpG sites in PARP1 promoter was associated with a 35% increase in 8-OHdG, while a 1% increase at 1, 2, and 3 CpG sites resulted in 38, 9, and 56% increments, respectively. An increase of 10pg/m3 in benzo[b]fluoranthene content of PM10 was associated with a 6% increase in LINE1 methylation. Acenaphthene, indene [1,2,3-cd] pyrene, and pyrene concentrations correlated with higher dinucleotide methylation in OGG1, APEX and PARP1 genes, respectively. Vanadium concentration correlated with increased methylation at selected APEX and PARP1 CpG sites. DNA repair gene methylation was significantly correlated with DNA damage and with specific PM10-associated PAHs and Vanadium. Data suggest that exposure to PM and its components are associated with differences in DNA methylation of repair genes in children, which may contribute to DNA damage.