RESUMO
BACKGROUND: Attention-deficit/hyperactivity disorder (ADHD) is a prevalent and highly heritable neurodevelopmental disorder of major societal concern. Diagnosis can be challenging and there are large knowledge gaps regarding its etiology, though studies suggest an interplay of genetic and environmental factors involving epigenetic mechanisms. MicroRNAs (miRNAs) show promise as biomarkers of human pathology and novel therapies, and here we aimed to identify blood miRNAs associated with traits of ADHD as possible biomarker candidates and further explore their biological relevance. METHODS: Our study population consisted of 1126 children (aged 5-12 years, 46% female) from the Human Early Life Exposome study, a study spanning six ongoing population-based European birth cohorts. Expression profiles of miRNAs in whole blood samples were quantified by microarray and tested for association with ADHD-related measures of behavior and neuropsychological functions from questionnaires (Conner's Rating Scale and Child Behavior Checklist) and computer-based tests (the N-back task and Attention Network Test). RESULTS: We identified 29 miRNAs significantly associated (false discovery rate < .05) with the Conner's questionnaire-rated trait hyperactivity, 15 of which have been linked to ADHD in previous studies. Investigation into their biological relevance revealed involvement in several pathways related to neurodevelopment and function, as well as being linked with other neurodevelopmental or psychiatric disorders known to overlap with ADHD both in symptomology, genetic risk, and co-occurrence, such as autism spectrum disorder or schizophrenia. An additional three miRNAs were significantly associated with Conner's-rated inattention. No associations were found with questionnaire-rated total ADHD index or with computer-based tests. CONCLUSIONS: The large overlap of our hyperactivity-associated miRNAs with previous studies on ADHD is intriguing and warrant further investigation. Though this study should be considered explorative and preliminary, these findings contribute towards identifying a set of miRNAs for use as blood-based biomarkers to aid in earlier and easier ADHD diagnosis.
Assuntos
Transtorno do Deficit de Atenção com Hiperatividade , Transtorno do Espectro Autista , MicroRNAs , Humanos , Criança , Feminino , Masculino , Transtorno do Deficit de Atenção com Hiperatividade/diagnóstico , Transtorno do Deficit de Atenção com Hiperatividade/genética , Transtorno do Deficit de Atenção com Hiperatividade/epidemiologia , MicroRNAs/genética , Transtorno do Espectro Autista/psicologia , Coorte de Nascimento , Biomarcadores , Agitação Psicomotora/complicaçõesRESUMO
Mechanistic toxicology is gaining weight for human health risk assessment. Different mechanistic assays are available, such as the comet assay, which detects DNA damage at the level of individual cells. However, the conventional alkaline version only detects strand breaks and alkali-labile sites. We have validated two modifications of the in vitro assay to generate mechanistic information: (1) use of DNA-repair enzymes (i.e., formamidopyrimidine DNA glycosylase, endonuclease III, human 8-oxoguanine DNA glycosylase I and human alkyladenine DNA glycosylase) for detection of oxidized and alkylated bases as well as (2) a modification for detecting cross-links. Seven genotoxicants with different mechanisms of action (potassium bromate, methyl methanesulfonate, ethyl methanesulfonate, hydrogen peroxide, cisplatin, mitomycin C, and benzo[a]pyrene diol epoxide), as well as a non-genotoxic compound (dimethyl sulfoxide) and a cytotoxic compound (Triton X-100) were tested on TK-6 cells. We were able to detect with high sensitivity and clearly differentiate oxidizing, alkylating and cross-linking agents. These modifications of the comet assay significantly increase its sensitivity and its specificity towards DNA lesions, providing mechanistic information regarding the type of damage.
Assuntos
Ensaio Cometa/métodos , Dano ao DNA/efeitos dos fármacos , Enzimas Reparadoras do DNA/metabolismo , Mutagênicos/toxicidade , Alquilação , Linhagem Celular , Humanos , Oxirredução , Sensibilidade e EspecificidadeRESUMO
Reliable data on the effects of chronic prenatal exposure to low dose (LD) of ionizing radiation in humans are missing. There are concerns about adverse long-term effects that may persist throughout postnatal life of the offspring. Due to their slow cell cycle kinetics and life-long residence time in the organism, mesenchymal stem cells (MSCs) are more susceptible to low level genotoxic stress caused by extrinsic multiple LD events. The aim of this study was to investigate the effect of chronic, prenatal LD gamma irradiation to the biology of MSCs later in life. C3H mice were exposed in utero to chronic prenatal irradiation of 10 mGy/day over a period of 3 weeks. Two years later, MSCs were isolated from the bone marrow and analyzed in vitro for their radiosensitivity, for cellular senescence and for DNA double-strand break recognition after a second acute gamma-irradiation. In addition to these cellular assays, changes in protein expression were measured using HPLC-MS/MS and dysregulated molecular signaling pathways identified using bioinformatics. We observed radiation-induced proteomic changes in MSCs from the offspring of in utero irradiated mice (leading to ~ 9.4% of all detected proteins being either up- or downregulated) as compared to non-irradiated controls. The proteomic changes map to regulation pathways involved in the extracellular matrix, the response to oxidative stress, and the Wnt signaling pathway. In addition, chronic prenatal LD irradiation lead to an increased rate of in vitro radiation-induced senescence later in life and to an increased number of residual DNA double-strand breaks after 4 Gy irradiation, indicating a remarkable interaction of in vivo radiation in combination with a second acute dose of in vitro radiation. This study provides the first insight into a molecular mechanism of persistent MSC damage response by ionizing radiation exposure during prenatal time and will help to predict therapeutic safety and efficacy with respect to a clinical application of stem cells.
Assuntos
Raios gama/efeitos adversos , Células-Tronco Mesenquimais/efeitos da radiação , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Proteoma/efeitos da radiação , Animais , Bioensaio , Células Cultivadas , Senescência Celular/efeitos da radiação , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Reparo do DNA , Desenvolvimento Embrionário , Feminino , Masculino , Troca Materno-Fetal , Células-Tronco Mesenquimais/metabolismo , Camundongos Mutantes , Gravidez , Via de Sinalização WntRESUMO
The alkaline comet assay, in vivo and in vitro, is currently used in several areas of research and in regulatory genotoxicity testing. Several efforts have been made in order to decrease the inter-experimental and inter-laboratory variability and increase the reliability of the assay. In this regard, lysis conditions are considered as one of the critical variables and need to be further studied. Here, we tested different times of lysis (from no lysis to 1 week) and two different lysis solutions in human lymphoblast (TK6) cells unexposed or exposed to X-rays. Similar % tail DNA values were obtained independently of the time of lysis employed for every X-ray dose tested and both lysis solutions. These results, taken together with our previous ones with methyl methanesulfonate and H2O2, which showed clear lysis-time dependence, support that the influence of the lysis time in the comet assay results depends on the type of lesion being detected; some DNA lesions may spontaneously give rise to apurinic or apyrimidinic (AP) sites during the lysis period, which can be converted into strand breaks detectable with the comet assay. Testing different times of lysis would be useful to increase the sensitivity of the comet assay and to ensure the detection of DNA lesions of an unknown compound, thereby providing some insight into the chemical nature of the lesions induced. However, the same lysis conditions (i.e. lysis time and lysis solution) should be used when comparing results between different experiments or laboratories.
Assuntos
Ensaio Cometa/métodos , Ensaio Cometa/normas , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Relação Dose-Resposta à Radiação , Humanos , Peróxido de Hidrogênio/farmacologia , Concentração de Íons de Hidrogênio , Padrões de Referência , Reprodutibilidade dos Testes , Soluções , Fatores de Tempo , Raios X/efeitos adversosRESUMO
The alkaline comet assay and a cell-free system were used to characterise DNA lesions induced by treatment with glycidamide (GA), a metabolite of the food contaminant acrylamide. DNA lesions induced by GA were sensitively detected when the formamidopyrimidine-DNA-glycosylase (Fpg) enzyme was included in the comet assay. We used LC-MS to characterise modified bases from GA-treated naked DNA with and without subsequent Fpg treatment. N7-GA-Guanine and N3-GA-Adenine aglycons were detected in the supernatant showing some depurination of adducted bases; treatment of naked DNA with Fpg revealed no further increase in the adduct yield nor occurrence of other adducted nucleobases. We treated human lymphocytes with GA and found large differences in DNA lesion levels detected with Fpg, depending on the duration and the pH of the lysis step. These lysis-dependent variations in GA-induced Fpg sensitive sites paralleled those observed after treatment of cells with methyl methane sulfonate (MMS). On the other hand, oxidative lesions (8-oxoGuanine) induced by a photoactive compound (Ro 12-9786) plus light, and also DNA strand breaks induced by X-rays, were detected largely independently of the lysis conditions. The results suggest that the GA-induced lesions are predominantly N7-GA-dG adducts slowly undergoing imidazole ring opening at pH 10 as in the standard lysis procedure; such structures are substrate for Fpg leading to strand breaks. The data suggest that the characteristic alkaline lysis dependence of some DNA lesions may be used to study specific types of DNA modifications. The comet assay is increasingly used in regulatory testing of chemicals; in this context, lysis-dependent variations represent a novel approach to obtain insight in the molecular nature of a genotoxic insult.
Assuntos
Ensaio Cometa , Dano ao DNA/efeitos dos fármacos , Compostos de Epóxi/toxicidade , Acrilamida/toxicidade , Animais , Bovinos , Cromatografia Líquida , Ensaio Cometa/métodos , DNA , Adutos de DNA , Reparo do DNA , DNA-Formamidopirimidina Glicosilase/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Espectrometria de Massas , Mutagênicos/toxicidadeRESUMO
Gamma radiation represents a potential health risk to aquatic and terrestrial biota, due to its ability to ionize atoms and molecules in living tissues. The effects of exposure to 60Co gamma radiation in zebrafish (Danio rerio) were studied during two sensitive life stages: gametogenesis (F0: 53 and 8.7mGy/h for 27 days, total doses 31 and 5.2Gy) and embryogenesis (9.6mGy/h for 65h; total dose 0.62Gy). Progeny of F0 exposed to 53mGy/h showed 100% mortality occurring at the gastrulation stage corresponding to 8h post fertilization (hpf). Control and F0 fish exposed to 8.7mGy/h were used to create four lines in the first filial generation (F1): control, G line (irradiated during parental gametogenesis), E line (irradiated during embryogenesis) and GE line (irradiated during parental gametogenesis and embryogenesis). A statistically significant cumulative mortality of GE larva (9.3%) compared to controls was found at 96 hpf. E line embryos hatched significantly earlier compared to controls, G and GE (48-72 hpf). The deformity frequency was higher in G and GE, but not E line compared to controls at 72 hpf. One month after parental irradiation, the formation of reactive oxygen species (ROS) was increased in the G line, but did not significantly differ from controls one year after parental irradiation, while at the same time point it was significantly increased in the directly exposed E and GE lines from 60 to 120 hpf. Lipid peroxidation (LPO) was significantly increased in the G line one year after parental irradiation, while significant increase in DNA damage was detected in both the G and GE compared to controls and E line at 72 hpf. Radiation-induced bystander effects, triggered by culture media from tissue explants and observed as influx of Ca2+ ions through the cellular membrane of the reporter cells, were significantly increased in 72 hpf G line progeny one month after irradiation of the parents. One year after parental irradiation, the bystander effects were increased in the E line compared to controls, but not in progeny of irradiated parents (G and GE lines). Overall, this study showed that irradiation of parents can result in multigenerational oxidative stress and genomic instability in irradiated (GE) and non-irradiated (G) progeny of irradiated parents, including increases in ROS formation, LPO, DNA damage and bystander effects. The results therefore highlight the necessity for multi- and transgenerational studies to assess the environmental impact of gamma radiation.
Assuntos
Gametogênese/efeitos da radiação , Raios gama/efeitos adversos , Instabilidade Genômica/efeitos da radiação , Reprodução/efeitos da radiação , Peixe-Zebra/fisiologia , Animais , Embrião não Mamífero/efeitos da radiação , Peixe-Zebra/genéticaRESUMO
Genome instability is a prerequisite for the development of cancer. It occurs when genome maintenance systems fail to safeguard the genome's integrity, whether as a consequence of inherited defects or induced via exposure to environmental agents (chemicals, biological agents and radiation). Thus, genome instability can be defined as an enhanced tendency for the genome to acquire mutations; ranging from changes to the nucleotide sequence to chromosomal gain, rearrangements or loss. This review raises the hypothesis that in addition to known human carcinogens, exposure to low dose of other chemicals present in our modern society could contribute to carcinogenesis by indirectly affecting genome stability. The selected chemicals with their mechanisms of action proposed to indirectly contribute to genome instability are: heavy metals (DNA repair, epigenetic modification, DNA damage signaling, telomere length), acrylamide (DNA repair, chromosome segregation), bisphenol A (epigenetic modification, DNA damage signaling, mitochondrial function, chromosome segregation), benomyl (chromosome segregation), quinones (epigenetic modification) and nano-sized particles (epigenetic pathways, mitochondrial function, chromosome segregation, telomere length). The purpose of this review is to describe the crucial aspects of genome instability, to outline the ways in which environmental chemicals can affect this cancer hallmark and to identify candidate chemicals for further study. The overall aim is to make scientists aware of the increasing need to unravel the underlying mechanisms via which chemicals at low doses can induce genome instability and thus promote carcinogenesis.
Assuntos
Carcinogênese/induzido quimicamente , Carcinógenos Ambientais/efeitos adversos , Exposição Ambiental/efeitos adversos , Instabilidade Genômica/efeitos dos fármacos , Substâncias Perigosas/efeitos adversos , Neoplasias/induzido quimicamente , Neoplasias/etiologia , Animais , HumanosRESUMO
Lifestyle factors are responsible for a considerable portion of cancer incidence worldwide, but credible estimates from the World Health Organization and the International Agency for Research on Cancer (IARC) suggest that the fraction of cancers attributable to toxic environmental exposures is between 7% and 19%. To explore the hypothesis that low-dose exposures to mixtures of chemicals in the environment may be combining to contribute to environmental carcinogenesis, we reviewed 11 hallmark phenotypes of cancer, multiple priority target sites for disruption in each area and prototypical chemical disruptors for all targets, this included dose-response characterizations, evidence of low-dose effects and cross-hallmark effects for all targets and chemicals. In total, 85 examples of chemicals were reviewed for actions on key pathways/mechanisms related to carcinogenesis. Only 15% (13/85) were found to have evidence of a dose-response threshold, whereas 59% (50/85) exerted low-dose effects. No dose-response information was found for the remaining 26% (22/85). Our analysis suggests that the cumulative effects of individual (non-carcinogenic) chemicals acting on different pathways, and a variety of related systems, organs, tissues and cells could plausibly conspire to produce carcinogenic synergies. Additional basic research on carcinogenesis and research focused on low-dose effects of chemical mixtures needs to be rigorously pursued before the merits of this hypothesis can be further advanced. However, the structure of the World Health Organization International Programme on Chemical Safety 'Mode of Action' framework should be revisited as it has inherent weaknesses that are not fully aligned with our current understanding of cancer biology.
Assuntos
Carcinogênese/induzido quimicamente , Carcinógenos Ambientais/efeitos adversos , Exposição Ambiental/efeitos adversos , Substâncias Perigosas/efeitos adversos , Neoplasias/induzido quimicamente , Neoplasias/etiologia , Animais , HumanosRESUMO
Many studies have investigated genotoxic effects of high Se diets but very few have addressed the genotoxicity of Se deprivation and its consequences in germ cells and none in somatic cells. To address these data gaps, C57BL/6 male mice were subjected to Se deprivation starting in the parental generation, i.e. before conception. Mice were given a diet of either low (0.01mg Se/kg diet) or normal (0.23mg Se/kg diet) Se content. Ogg1-deficient (Ogg1 (-/-) ) mice were used as a sensitive model towards oxidative stress due to their reduced capacity to repair oxidised purines. Ogg1 (-/-) mice also mimic the repair characteristics of human post-meiotic male germ cells which have a reduced ability to repair such lesions. The genotoxicity of Se deficiency was addressed by measuring DNA lesions with the alkaline single cell gel electrophoresis (+ Fpg to detect oxidised DNA lesions) in somatic cells (nucleated blood cells and lung cells) and male germ cells (testicular cells). Total Se concentration in liver and GPx activity in plasma and testicular cells were measured. Gene mutation was evaluated by an erythrocyte-based Pig-a assay. We found that Se deprivation of F1 from their conception and until early adulthood led to the induction of DNA lesions in testicular and lung cells expressed as significantly increased levels of DNA lesions, irrespective of the mouse genotype. In blood cells, Se levels did not appear to affect DNA lesions or mutant cell frequencies. The results suggest that the testis was the most sensitive tissue. Thus, genotoxicity induced by the low Se diet in the spermatozoal genome has potential implications for the offspring.
Assuntos
Dano ao DNA , Estresse Oxidativo , Selênio/deficiência , Espermatozoides , Animais , DNA Glicosilases/genética , Reparo do DNA/genética , Glutationa Peroxidase/análise , Leucócitos , Pulmão/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Selênio/análiseRESUMO
Paternal exposure to high levels of radioactivity causes heritable germline minisatellite mutations. However, the effect of more general paternal exposures, such as cigarette smoking, on germline mutations remains unexplored. We analyzed two of the most commonly used minisatellite loci (CEB1 and B6.7) to identify germline mutations in blood samples of complete mother-father-child triads from the Norwegian Mother and Child Cohort Study (MoBa). The presence of mutations was subsequently related to general lifestyle factors, including paternal smoking before the partner became pregnant. Paternally derived mutations at the B6.7 locus (mutation frequency 0.07) were not affected by lifestyle. In contrast, high gross yearly income as a general measure of a healthy lifestyle coincided with low-mutation frequencies at the CEB1 locus (P=0.047). Income was inversely related to smoking behavior, and paternally derived CEB1 mutations were dose dependently increased when the father smoked in the 6 mo before pregnancy, 0.21 vs. 0.05 in smoking and nonsmoking fathers, respectively (P=0.061). These results suggest that paternal lifestyle can affect the chance of heritable mutations in unstable repetitive DNA sequences. To our knowledge, this is the first study reporting an effect of lifestyle on germline minisatellite mutation frequencies in a human population with moderate paternal exposures.
Assuntos
Mutação em Linhagem Germinativa , Peptídeos e Proteínas de Sinalização Intracelular/genética , Repetições Minissatélites/genética , Fumar , Adulto , Alelos , Criança , Análise Mutacional de DNA , Feminino , Frequência do Gene , Humanos , Renda , Estilo de Vida , Masculino , Taxa de Mutação , Núcleo Familiar , Comportamento Paterno , Gravidez , Estudos Prospectivos , Inquéritos e QuestionáriosRESUMO
Selenium is an essential trace element in humans, critical to the normal physiology in all animal species. The main form of selenium in food is selenomethionine, selenocysteine and a variety of organic compounds, while inorganic salts mainly occur in food supplements. In animals and humans, selenium occurs as selenocysteine in selenoproteins encoded by 25 genes (specific selenium pool). Several selenoproteins are part of the antioxidant enzyme system and serve as oxido-reductases and in thyroid hormone regulation. SelenoproteinP (SELENOP) transports selenium to peripheral tissues, is the main plasma selenoprotein, and has been used as biomarker of selenium status and intake. SELENOP in plasma represents a saturable pool of selenium and is maximised at a selenium concentration in plasma of about 110 µg/L or an intake of selenomethionine at about 1.2 µg/kg body weight in adults. In Finland, with an estimated selenium intake of 88 µg/day in men and 68 µg/day in women, the average selenium concentration in plasma is about 110 µg/L. Imported wheat from selenium rich areas is an important dietary source in Norway. Dietary intakes in the Nordic and Baltic area vary from 39 to 88 µg/day in men and 22 to 68 µg/day in women, the highest levels were from Finland. Most intervention trials on the effect of selenium supplementation on health outcomes have been carried out in 'selenium-replete'-populations and show no beneficial effect, which from a nutritional point of view would rather not be expected. Some intervention studies conducted in populations low in selenium have showed a beneficial effect. Observational studies suggest an inverse relationship between selenium status and risk of cardiovascular diseases (CVDs), cancer and all-cause mortality, and some other outcomes at low levels of intake (<55 µg/day) or in plasma or serum (<100 µg/L). However, a lack of quantitative data and inconsistencies between studies precludes these studies to be used to derive dietary reference values. At high intakes above 330 to 450 µg/day selenium may cause toxic effects affecting liver, peripheral nerves, skin, nails, and hair. An upper tolerable level (UL) of 255 µg selenium/day in adults was established by EFSA.
RESUMO
Ionizing radiation (IR) impact cellular and molecular processes that require chromatin remodelling relevant for cellular integrity. However, the cellular implications of ionizing radiation (IR) delivered per time unit (dose rate) are still debated. This study investigates whether the dose rate is relevant for inflicting changes to the epigenome, represented by chromatin accessibility, or whether it is the total dose that is decisive. CBA/CaOlaHsd mice were whole-body exposed to either chronic low dose rate (2.5 mGy/h for 54 d) or the higher dose rates (10 mGy/h for 14 d and 100 mGy/h for 30 h) of gamma radiation (60Co, total dose: 3 Gy). Chromatin accessibility was analysed in liver tissue samples using Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-Seq), both one day after and over three months post-radiation (>100 d). The results show that the dose rate contributes to radiation-induced epigenomic changes in the liver at both sampling timepoints. Interestingly, chronic low dose rate exposure to a high total dose (3 Gy) did not inflict long-term changes to the epigenome. In contrast to the acute high dose rate given to the same total dose, reduced accessibility at transcriptional start sites (TSS) was identified in genes relevant for the DNA damage response and transcriptional activity. Our findings link dose rate to essential biological mechanisms that could be relevant for understanding long-term changes after ionizing radiation exposure. However, future studies are needed to comprehend the biological consequence of these findings.
Assuntos
Cromatina , Metilação de DNA , Animais , Camundongos , Cromatina/genética , Raios gama/efeitos adversos , Camundongos Endogâmicos CBA , Radiação IonizanteRESUMO
The in vivo comet assay is widely used to measure genotoxicity; however, the current OECD test guideline (TG 489) does not recommend using the assay to assess testicular germ cells, due to the presence of testicular somatic cells. An adapted approach to specifically assess testicular germ cells within the comet assay is certainly warranted, considering regulatory needs for germ cell-specific genotoxicity data in relation to the increasing global production of and exposure to potentially hazardous chemicals. Here, we provide a proof-of-concept to selectively analyze round spermatids and primary spermatocytes, distinguishing them from other cells of the testicle. Utilizing the comet assay recordings of DNA content (total fluorescence intensity) and DNA damage (% tail intensity) of individual comets, we developed a framework to distinguish testicular cell populations based on differences in DNA content/ploidy and appearance. Haploid round spermatid comets are identified through (1) visual inspection of DNA content distributions, (2) setting DNA content thresholds, and (3) modeling DNA content distributions using a normal mixture distribution function. We also describe an approach to distinguish primary spermatocytes during comet scoring, based on their high DNA content and large physical size. Our concept allows both somatic and germ cells to be analyzed in the same animal, adding a versatile, sensitive, rapid, and resource-efficient assay to the limited genotoxicity assessment toolbox for germ cells. An adaptation of TG 489 facilitates accumulation of valuable information regarding distribution of substances to germ cells and their potential for inducing germ cell gene mutations and structural chromosomal aberrations.
Assuntos
Espermatozoides , Testículo , Masculino , Animais , Ensaio Cometa , Dano ao DNA , Células Germinativas , DNARESUMO
Adverse health outcomes of ionizing radiation given chronically at low dose rates are highly debated, a controversy also relevant for other stressors. Increased knowledge is needed for a more comprehensive understanding of the damaging potential of ionizing radiation from all dose rates and doses. There is a lack of relevant low dose rate data that is partly ascribed to the rarity of exposure facilities allowing chronic low dose rate exposures. Using the FIGARO facility, we assessed early (one day post-radiation) and late (recovery time of 100-200 days) hepatic genome-wide transcriptional profiles in male mice of two strains (CBA/CaOlaHsd and C57BL/6NHsd) exposed chronically to a low dose rate (2.5 mGy/h; 1200h, LDR), a mid-dose rate (10 mGy/h; 300h, MDR) and acutely to a high dose rate (100 mGy/h; 30h, HDR) of gamma irradiation, given to an equivalent total dose of 3 Gy. Dose-rate and strain-specific transcriptional responses were identified. Differently modulated transcriptional responses across all dose rate exposure groups were evident by the representation of functional biological pathways. Evidence of changed epigenetic regulation (global DNA methylation) was not detected. A period of recovery markedly reduced the number of differentially expressed genes. Using enrichment analysis to identify the functional significance of the modulated genes, perturbed signaling pathways associated with both cancer and non-cancer effects were observed, such as lipid metabolism and inflammation. These pathways were seen after chronic low dose rate and were not restricted to the acute high dose rate exposure. The transcriptional response induced by chronic low dose rate ionizing radiation suggests contribution to conditions such as cardiovascular diseases. We contribute with novel genome wide transcriptional data highlighting dose-rate-specific radiation responses and emphasize the importance of considering both dose rate, duration of exposure, and variability in susceptibility when assessing risks from ionizing radiation.
Assuntos
Raios gama , Radiação Ionizante , Transcrição Gênica/efeitos dos fármacos , Animais , Metilação de DNA/efeitos da radiação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Estresse Oxidativo/efeitos da radiação , Doses de RadiaçãoRESUMO
BACKGROUND: Pregnant women and their fetuses are exposed to multiple toxic metals that together with variations in essential element levels may alter epigenetic regulation, such as DNA methylation. OBJECTIVES: The aim of the study was to investigate the associations between gestational levels of toxic metals and essential elements and mixtures thereof, with global DNA methylation levels in pregnant women and their newborn children. METHODS: Using 631 mother-child pairs from a prospective birth cohort (The Norwegian Mother, Father and Child Cohort Study), we measured maternal blood concentration (gestation week ~18) of five toxic metals and seven essential elements. We investigated associations as individual exposures and two-way interactions, using elastic net regression, and total mixture, using quantile g-computation, with blood levels of 5-methylcytocine (5mC) and 5-hydroxymethylcytosine (5hmC) in mothers during pregnancy and their newborn children (cord blood). Multiple testing was adjusted for using the Benjamini and Hochberg false discovery rate (FDR) approach. RESULTS: The most sensitive marker of DNA methylation appeared to be 5mC levels. In pregnant mothers, elastic net regression indicated associations between 5mC and selenium and lead (non-linear), while in newborns results indicated relationships between maternal selenium, cobalt (non-linear) and mercury and 5mC, as well as copper (non-linear) and 5hmC levels. Several possible two-way interactions were identified (e.g. arsenic and mercury, and selenium and maternal smoking in newborns). None of these findings met the FDR threshold for multiple testing. No net effect was observed in the joint (mixture) exposure-approach using quantile g-computation. CONCLUSION: We identified few associations between gestational levels of several toxic metals and essential elements and global DNA methylation in pregnant mothers and their newborn children. As DNA methylation dysregulation might be a key mechanism in disease development and thus of high importance for public health, our results should be considered as important candidates to investigate in future studies.
Assuntos
Metilação de DNA , Gestantes , Estudos de Coortes , Epigênese Genética , Feminino , Sangue Fetal , Humanos , Lactente , Recém-Nascido , Exposição Materna/efeitos adversos , Noruega , Gravidez , Estudos ProspectivosRESUMO
Oxidative DNA damage in the brain has been implicated in neurodegeneration and cognitive decline. DNA glycosylases initiate base excision repair (BER), the main pathway for oxidative DNA base lesion repair. NEIL1 and NEIL3 DNA glycosylases affect cognition in mice, while the role of NEIL2 remains unclear. Here, we investigate the impact of NEIL2 and its potential overlap with NEIL1 on behavior in knockout mouse models. Neil1-/-Neil2-/- mice display hyperactivity, reduced anxiety and improved learning. Hippocampal oxidative DNA base lesion levels are comparable between genotypes and no mutator phenotype is found. Thus, impaired canonical repair is not likely to explain the altered behavior. Electrophysiology suggests reduced axonal activation in the hippocampal CA1 region in Neil1-/-Neil2-/- mice and lack of NEIL1 and NEIL2 causes dysregulation of genes in CA1 relevant for synaptic function. We postulate a cooperative function of NEIL1 and NEIL2 in genome regulation, beyond canonical BER, modulating behavior in mice.
Assuntos
Ansiedade/genética , DNA Glicosilases/genética , Aprendizagem , Camundongos/psicologia , Animais , DNA Glicosilases/metabolismo , Regulação da Expressão Gênica , Hipocampo/fisiologia , Masculino , Camundongos/genética , Camundongos Knockout , Estresse Oxidativo/fisiologiaRESUMO
The industrial compound and food contaminant acrylamide (AA) is a probable human carcinogen, also known to induce male-mediated reproductive effects in animals. Most data suggest that its metabolite glycidamide (GA) is involved in the observed toxicity. We have investigated in vitro effects of AA/GA in mouse male germ cells (prior to spermatid elongation) and human and mouse peripheral blood lymphocytes, to assess inter-species and cell-type differences in susceptibility, and to explore the nature of the DNA lesion(s) as well as their potential repair. The comet assay was used in combination with the DNA-repair enzymes Fpg and hOGG1 to measure specific DNA lesions. In contrast to AA, GA induced significant levels of DNA lesions (strand breaks and alkali-labile sites) at millimolar concentrations in mouse testicular cells and human peripheral blood lymphocytes (hPBL). Using Fpg, the GA-induced DNA damage was measured at 20-50-fold higher sensitivity, in all cell types investigated. GA-induced DNA damage could not be recognised by hOGG1, suggesting that, based on the known affinities of these repair enzymes, alkylation of guanine is involved, rather than oxidation. Human lymphocytes appeared to be more susceptible to GA-induced lesions than both types of mouse cells. Mouse testicular cells and lymphocytes seemed to respond similarly to GA-induced Fpg-sensitive DNA lesions. The persistence of lesions was explored with cells from mice either proficient or deficient in Ogg1 (mouse 8-oxoguanine DNA glycosylase). Low in vitro repair of GA-induced Fpg-sensitive lesions was observed in primary male germ cells and lymphocytes from both Ogg1(+/+) and Ogg1(-/-) mice. We conclude that there may be differences between mice and humans in AA/GA-induced genotoxicity, and DNA from mouse male germ cells does not appear to be more sensitive to GA than DNA from peripheral blood lymphocytes in vitro. The usefulness of the comet assay in combination with DNA-repair enzymes is demonstrated.
Assuntos
Acrilamida/toxicidade , Dano ao DNA , Compostos de Epóxi/toxicidade , Linfócitos/efeitos dos fármacos , Mutagênicos/toxicidade , Espermátides/efeitos dos fármacos , Animais , Reparo do DNA , Humanos , Masculino , CamundongosRESUMO
Mitochondria are vulnerable to the effects of ionizing radiation; damage to mitochondrial DNA (mtDNA) may be more extensive and persistent than damage to nuclear DNA (nDNA). Variation in mtDNA copy number has been proposed as a marker for mitochondrial dysfunction in response to ionizing radiation. We have developed a precise and sensitive duplex droplet digital PCR (ddPCR) method for quantitation of the mtDNA/nDNA ratio in the model organism Caenorhabditis elegans. The effect on this ratio was investigated over a wide range of doses (0.03-72 Gy) of chronic gamma irradiation. Five mitochondrial targets and two nuclear reference genes were amplified pairwise in duplex PCR format (one mitochondrial and one nuclear target per PCR) by both ddPCR and quantitative PCR (qPCR). The results showed that ddPCR but not qPCR enabled detection of a significant increase in mtDNA copy number (1.6 ± 0.1-fold) for nematodes exposed to high doses (≥24 Gy). Thus, ddPCR provided higher precision and greater sensitivity than qPCR for detection of mtDNA copy number variation. The variation followed a Hill-type dose response with threshold 10.3 ± 1 Gy. This strongly suggests that chronic genotoxic stress affects mtDNA replication. The duplex ddPCR method is a novel, high-precision, sensitive tool for determination of mitochondrial DNA copy number variation and function in C. elegans.