Effects of prenatal dexamethasone exposure on adult C57BL/6J mouse metabolism and oxidative stress.

Prenatal glucocorticoid exposure has been shown to alter hypothalamic-pituitary-adrenal axis function resulting in altered fetal development that can persist through adulthood. Fetal exposure to excess dexamethasone, a synthetic glucocorticoid, has been shown to alter adult behaviour and metabolism. This study investigated the effects prenatal dexamethasone exposure had on adult offspring cardiac and liver metabolism and oxidative stress. Pregnant C57BL/6 mice received a dose of 0.4 mg/kg dexamethasone on gestational days 15-17. Once pups were approximately 7 months old, glucose uptake was determined using positron emission tomography and insulin resistance (IR) was determined by homeostatic model assessment (HOMA) IR calculation. Oxidative stress was assessed by measuring 4-hydroxynonenal protein adduct formation and total reactive oxygen species. Female dexamethasone group had significantly increased glucose uptake when insulin stimulated compared to vehicle-treated mice. HOMA IR revealed no evidence of IR in either male or female offspring. There was also no change in oxidative stress markers in either cardiac or liver tissues of male or female offspring. These data suggest that prenatal dexamethasone exposure in male mice does not alter oxidative stress or metabolism. However, prenatal dexamethasone exposure increased glucocorticoids, cardiac glucose uptake, and pAkt signaling in female heart tissues in adult mice, suggesting there are sex differences in prenatal dexamethasone exposure.

Transcriptomic Response in the Spleen after Whole-Body Low-Dose X-Ray Irradiation.

As the use of medical radiation procedures continues to rise, it is imperative to further our understanding of the effects of this exposure. The spleen is not known as a particularly radiosensitive organ, although its tolerance to radiation is not well understood. Low-dose radiation exposure has been implicated in beneficial responses, particularly in cell death and DNA damage repair. In this study, adult male rats received 2, 20, 200 mGy or 4 Gy whole-body X-ray irradiation and the transcriptional response in the spleen was analyzed at 0.5, 4 and 24 h postirradiation. We analyzed expression of genes involved in apoptosis, cell cycle progression and DNA damage repair. As expected, 4 Gy irradiated animals demonstrated elevated expression of genes related to apoptosis at 0.5, 4 and 24 h postirradiation in the spleen. These animals also showed upregulation of DNA damage repair genes at 24 h postirradiation. Interestingly, the spleens of 20 mGy irradiated animals showed reduced apoptosis and cell cycle arrest compared to the spleens of sham-irradiated animals. These results further reveal that the cellular response in the spleen to whole-body irradiation differs between low- and high-dose irradiation.

A multi-ingredient dietary supplement abolishes large-scale brain cell loss, improves sensory function, and prevents neuronal atrophy in aging mice.

Transgenic growth hormone mice (TGM) are a recognized model of accelerated aging with characteristics including chronic oxidative stress, reduced longevity, mitochondrial dysfunction, insulin resistance, muscle wasting, and elevated inflammatory processes. Growth hormone/IGF-1 activate the Target of Rapamycin known to promote aging. TGM particularly express severe cognitive decline. We previously reported that a multi-ingredient dietary supplement (MDS) designed to offset five mechanisms associated with aging extended longevity, ameliorated cognitive deterioration and significantly reduced age-related physical deterioration in both normal mice and TGM. Here we report that TGM lose more than 50% of cells in midbrain regions, including the cerebellum and olfactory bulb. This is comparable to severe Alzheimer's disease and likely explains their striking age-related cognitive impairment. We also demonstrate that the MDS completely abrogates this severe brain cell loss, reverses cognitive decline and augments sensory and motor function in aged mice. Additionally, histological examination of retinal structure revealed markers consistent with higher numbers of photoreceptor cells in aging and supplemented mice. We know of no other treatment with such efficacy, highlighting the potential for prevention or amelioration of human neuropathologies that are similarly associated with oxidative stress, inflammation and cellular dysfunction. Environ. Mol. Mutagen. 57:382-404, 2016. © 2016 Wiley Periodicals, Inc.

Synergistic effects of diet and exercise on hippocampal function in chronically stressed mice.

Severe chronic stress can have a profoundly negative impact on the brain, affecting plasticity, neurogenesis, memory and mood. On the other hand, there are factors that upregulate neurogenesis, which include dietary antioxidants and physical activity. These factors are associated with biochemical processes that are also altered in age-related cognitive decline and dementia, such as neurotrophin expression, oxidative stress and inflammation. We exposed mice to an unpredictable series of stressors or left them undisturbed (controls). Subsets of stressed and control mice were concurrently given (1) no additional treatment, (2) a complex dietary supplement (CDS) designed to ameliorate inflammation, oxidative stress, mitochondrial dysfunction, insulin resistance and membrane integrity, (3) a running wheel in each of their home cages that permitted them to exercise, or (4) both the CDS and the running wheel for exercise. Four weeks of unpredictable stress reduced the animals' preference for saccharin, increased their adrenal weights and abolished the exercise-induced upregulation of neurogenesis that was observed in non-stressed animals. Unexpectedly, stress did not reduce hippocampal size, brain-derived neurotrophic factor (BDNF), or neurogenesis. The combination of dietary supplementation and exercise had multiple beneficial effects, as reflected in the number of doublecortin (DCX)-positive immature neurons in the dentate gyrus (DG), the sectional area of the DG and hippocampal CA1, as well as increased hippocampal BDNF messenger ribonucleic acid (mRNA) and serum vascular endothelial growth factor (VEGF) levels. In contrast, these benefits were not observed in chronically stressed animals exposed to either dietary supplementation or exercise alone. These findings could have important clinical implications for those suffering from chronic stress-related disorders such as major depression.

Embryonic development of lake whitefish Coregonus clupeaformis: a staging series, analysis of growth and effects of fixation.

A reference staging series of 18 morphological stages of laboratory reared lake whitefish Coregonus clupeaformis is provided. The developmental processes of blastulation, gastrulation, neurulation as well as development of the eye, circulatory system, chromatophores and mouth are included and accompanied by detailed descriptions and live imaging. Quantitative measurements of embryo size and mass were taken at each developmental stage. Eggs were 3·19 ± 0·16 mm (mean ± s.d.) in diameter at fertilization and embryos reached a total length (LT ) of 14·25 ± 0·41 mm at hatch. Separated yolk and embryo dry mass were 0·25 ± 0·08 mg and 1·39 ± 0·17 mg, respectively, at hatch. The effects of two common preservatives (formalin and ethanol) were examined throughout development and post hatch. Embryo LT significantly decreased following fixation at all points in development. A correction factor to estimate live LT from corresponding fixed LT was determined as live LT = (fixed LT )(1·025) . Eye diameter and yolk area measurements significantly increased in fixed compared with live embryos up to 85-90% development for both measurements. The described developmental stages can be generalized to teleost species, and is particularly relevant for the study of coregonid development due to additionally shared developmental characteristics. The results of this study and staging series are therefore applicable across various research streams encompassing numerous species that require accurate staging of embryos and descriptions of morphological development.

Chromosome Damage Caused by Accidental Chronic Whole-Body Gamma Radiation Exposure in Thailand.

In February 2000, a radiation incident involving a medical (60)Co source occurred in a metal scrapyard in Thailand. Several individuals were suspected to have received chronic or fractionated exposures ranging from a few mGy to a several Gy. Using fluorescence in situ hybridization to paint chromosomes, we determined the frequencies of chromosome aberrations in peripheral blood lymphocytes of 13 people who entered the scrapyard, 3 people who involved in recovering the source, and 9 nearby residents. Aberration frequencies greater than controls were observed in 13 of the donors at 3 months postexposure. The predominant form of aberration observed was simple, complete, symmetrical translocations. An approximate 50% decrease in these aberrations and in total color junctions was observed in 7 donors resampled at 16 months postexposure. Although high, acute exposures are known to have detrimental effects, the biological consequences of chronic, low dose-rate radiation exposures are unclear. Thirteen of the donors had elevated aberration frequencies, and 6 also had symptoms of acute radiation syndrome. If there are any long-term health consequences of this incident, it will most likely occur among this group of individuals. The consequences for the remaining donors, who presumably received lower total doses delivered at lower dose rates, are less clear.

Critical windows in embryonic development: Shifting incubation temperatures alter heart rate and oxygen consumption of Lake Whitefish (Coregonus clupeaformis) embryos and hatchlings.

Critical windows are periods of developmental susceptibility when the phenotype of an embryonic, juvenile or adult animal may be vulnerable to environmental fluctuations. Temperature has pervasive effects on poikilotherm physiology, and embryos are especially vulnerable to temperature shifts. To identify critical windows, we incubated whitefish embryos at control temperatures of 2°C, 5°C, or 8°C, and shifted treatments among temperatures at the end of gastrulation or organogenesis. Heart rate (fH) and oxygen consumption ( [Formula: see text] ) were measured across embryonic development, and [Formula: see text] was measured in 1-day old hatchlings. Thermal shifts, up or down, from initial incubation temperatures caused persistent changes in fH and [Formula: see text] compared to control embryos measured at the same temperature (2°C, 5°C, or 8°C). Most prominently, when embryos were measured at organogenesis, shifting incubation temperature after gastrulation significantly lowered [Formula: see text] or fH. Incubation at 2°C or 5°C through gastrulation significantly lowered [Formula: see text] (42% decrease) and fH (20% decrease) at 8°C, incubation at 2°C significantly lowered [Formula: see text] (40% decrease) and fH (30% decrease) at 5°C, and incubation at 5°C and 8°C significantly lowered [Formula: see text] at 2°C (27% decrease). Through the latter half of development, [Formula: see text] and fH in embryos were not different from control values for thermally shifted treatments. However, in hatchlings measured at 2°C, [Formula: see text] was higher in groups incubated at 5°C or 8°C through organogenesis, compared to 2°C controls (43 or 65% increase, respectively). Collectively, these data suggest that embryonic development through organogenesis represents a critical window of embryonic and hatchling phenotypic plasticity. This study presents an experimental design that identified thermally sensitive periods for fish embryos.

Human Health and the Biological Effects of Tritium in Drinking Water: Prudent Policy Through Science - Addressing the ODWAC New Recommendation.

Tritium is a radioactive form of hydrogen and is a by-product of energy production in Canadian Deuterium Uranium (CANDU) reactors. The release of this radioisotope into the environment is carefully managed at CANDU facilities in order to minimize radiation exposure to the public. However, under some circumstances, small accidental releases to the environment can occur. The radiation doses to humans and non-human biota from these releases are low and orders of magnitude less than doses received from naturally occurring radioisotopes or from manmade activities, such as medical imaging and air travel. There is however a renewed interest in the biological consequences of low dose tritium exposures and a new limit for tritium levels in Ontario drinking water has been proposed. The Ontario Drinking Water Advisory Council (ODWAC) issued a formal report in May 2009 in response to a request by the Minister of the Environment, concluding that the Ontario Drinking Water Quality Standard for tritium should be revised from the current 7,000 Bq/L level to a new, lower 20 Bq/L level. In response to this recommendation, an international scientific symposium was held at McMaster University to address the issues surrounding this change in direction and the validity of a new policy. Scientists, regulators, government officials, and industrial stakeholders were present to discuss the potential health risks associated with low level radiation exposure from tritium. The regulatory, economic, and social implications of the new proposed limit were also considered.The new recommendation assumed a linear-no-threshold model to calculate carcinogenic risk associated with tritium exposure, and considered tritium as a non-threshold chemical carcinogen. Both of these assumptions are highly controversial given that recent research suggests that low dose exposures have thresholds below which there are no observable detrimental effects. Furthermore, mutagenic and carcinogenic risk calculated from tritium exposure at 20 Bq/L would be orders of magnitude less than that from exposure to natural background sources of radiation. The new proposed standard would set the radiation dose limit for drinking water to 0.0003 mSv/year, which is equivalent to approximately three times the dose from naturally occurring tritium in drinking water. This new standard is incongruent with national and international standards for safe levels of radiation exposure, currently set at 1 mSv/year for the general public. Scientific research from leading authorities on the carcinogenic health effects of tritium exposure supports the notion that the current standard of 7,000 Bq/L (annual dose of 0.1 mSv) is a safe standard for human health.Policy-making for the purpose of regulating tritium levels in drinking water is a dynamic multi-stage process that is influenced by more than science alone. Ethics, economics, and public perception also play important roles in policy development; however, these factors sometimes undermine the scientific evidence that should form the basis of informed decision making. Consequently, implementing a new standard without a scientific basis may lead the public to perceive that risks from tritium have been historically underestimated. It was concluded that the new recommendation is not supported by any new scientific insight regarding negative consequences of low dose effects, and may be contrary to new data on the potential benefits of low dose effects. Given the lack of cost versus benefit analysis, this type of dramatic policy change could have detrimental effects to society from an ethical, economical, and public perception perspective.

Single-aliquot EPR dosimetry of wallboard (drywall).

Electron paramagnetic resonance spectra and dose-response curves are presented for a variety of wallboard samples obtained from different manufacturing facilities, as well as for source gypsum and anhydrite. The intensity of the CO(3)(-) paramagnetic centre (G2) is enhanced with gamma radiation. Isothermal decay curves are used to propose annealing methods for the removal of the radiosensitive CO(3)(-) radical without affecting the unirradiated baseline. Post-irradiation annealing of wallboard prevents recuperation of the radiosensitive CO(3)(-) radical with additional irradiation. A single-aliquot additive dose procedure is developed that successfully measures test doses as low as 0.76 Gy.

Elevated DNA damage in a mouse model of oxidative stress: impacts of ionizing radiation and a protective dietary supplement.

Transgenic growth hormone (Tg) mice express elevated free radical processes and a progeroid syndrome of accelerated ageing. We examined bone marrow cells of Tg mice and their normal (Nr) siblings for three markers of DNA damage and assessed the impact of free radical stress using ionizing radiation. We also evaluated the radiation protection afforded by a dietary supplement that we previously demonstrated to extend longevity and reduce cognitive ageing of Nr and Tg mice. Spectral karyotyping revealed few spontaneous chromosomal aberrations in Nr or Tg. Tg mice, however, had significantly greater constitutive levels of both gammaH2AX and 8-hydroxy-deoxyguanosine (8-OHdG) compared to Nr. When exposed to a 2-Gy whole-body dose of ionizing radiation, both Nr and Tg mice showed significant increases in DNA damage. Compared to Nr mice, irradiated Tg mice had dramatically higher levels of gammaH2AX foci and double the levels of chromosomal aberrations. In unirradiated mice, the dietary supplement significantly reduced constitutive gammaH2AX and 8-OHdG in both Nr and Tg mice (normalizing both gammaH2AX and 8-OHdG in Tg), with little difference in gammaH2AX and 8-OHdG over constitutive levels. Induced chromosomal aberrations were also reduced, and in Nr mice, virtually absent. Remarkably, supplemented mice expressed 6-fold lower levels of radiation-induced chromosomal aberrations compared to unsupplemented Nr or Tg mice. Based on our data, the dietary supplement appeared to scavenge free radicals before they could cause damage. This study validates Tg mice as an exemplary model of oxidative stress and radiation hypersensitivity and documents unprecedented radioprotection by a dietary supplement comprised of ingredients available to the general public.

Relative biological effectiveness of 280 keV neutrons for apoptosis in human lymphocytes.

The relative biological effectiveness (RBE) of neutrons varies from unity to greater than ten depending upon neutron energy and the biological endpoint measured. In our study, we examined apoptosis in human lymphocytes to assess the RBE of low energy 280 keV neutrons compared to Cs gamma radiation and found the RBE to be approximately one. Similar results have been observed for high energy neutrons using the same endpoint. As apoptosis is a major process that influences the consequences of radiation exposure, our results indicate that biological effect and the corresponding weighting factors for 280 keV neutrons may be lower in some cell types and tissues.

Radiological emergency response: the National Biological Dosimetry Response Plan.

This presentation will discuss new developments in emergency biological dosimetry and the CRTI (CBRN Research and Technology Initiative) National Biological Dosimetry Response Plan (NBDRP). Biological dosimetry is a technique used to estimate the biological consequences of a radiation exposure and is largely based on chromosome aberration detection. The NBDRP will establish a national network of laboratories to respond to a nuclear event for the purposes of rapid radiation dose estimation for crisis management and for long-term health risk assessment. In the event of a large-scale radiation accident or deliberate act of terrorism this will help guide the actions of emergency officials, emergency responders and health care personnel by providing timely biological dose estimates. The presentation will outline the research initiatives to develop modern techniques used for biological dosimetry. The overall purpose of this presentation is to provide the audience with a brief introduction to radiobiology, radiation-induced DNA damage, the health risks associated with radiation exposure, and the latest cytogenetic techniques used to estimate the risk.

The adaptive response and protection against heritable mutations and fetal malformation.

There are a number of studies that show radiation can cause heritable mutations in the offspring of irradiated organisms. These "germ-line mutations" have been shown to occur in unique sequences of DNA called "minisatellite loci". The high frequencies of spontaneous and induced mutations at minisatellite loci allow mutation induction to be measured at low doses of exposure in a small population, making minisatellite mutation a powerful tool to investigate radiation-induced heritable mutations. However, the biological significance of these mutations is uncertain, and their relationship to health risk or population fitness is unknown. We have adopted this mutation assay to study the role of adaptive response in protecting mice against radiation-induced heritable defects. We have shown that male mice, adapted to radiation with a low dose priming exposure, do not pass on mutations to their offspring caused by a subsequent large radiation exposure to the adapted males. This presentation and paper provide a general overview of radiation-induced mutations in offspring and explain the effect of low dose exposures and the adaptive response on these mutations.It is also known that exposure of pregnant females to high doses of radiation can cause death or malformation (teratogenesis) in developing fetuses. Malformation can only occur during a specialized stage of organ formation known as organogenesis. Studies in rodents show that radiation-induced fetal death and malformation can be significantly reduced when a pregnant female is exposed to a prior low dose of ionizing radiation. The mechanism of this protective effect, through an adaptive response, depends on the stage of organogenesis when the low dose exposures are delivered. To better understand this process, we have investigated the role of an important gene known as p53. Therefore, this report will also discuss fetal effects of ionizing radiation and explain the critical stages of development when fetuses are at risk. Research will be explained that investigates the biological and genetic systems (p53) that protect the developing fetus and discuss the role of low dose radiation adaptive response in these processes.

Retrospective radiation dosimetry using electron paramagnetic resonance in canine dental enamel.

Electron paramagnetic resonance (EPR) biodosimetry of human tooth enamel has been widely used for measuring radiation doses in various scenarios. We have now developed EPR dosimetry in tooth enamel extracted from canines. Molars and incisors from canines were cleaned by processing in supersaturated aqueous potassium hydroxide solution. The dosimetric signal in canine tooth enamel was found to increase linearly as a function of laboratory added dose from 0.44+/-0.02 to 4.42+/-0.22 Gy. The gamma radiation sensitivity of the canine molar enamel was found to be comparable to that of human tooth enamel. The dosimetric signal in canine enamel has been found to be stable up to at least 6 weeks after in vitro irradiation. A dosimetric signal variation of 10-25% was observed for canines ranging from in age 3 years to 16 year old.

Gamma radiation-induced heritable mutations at repetitive DNA loci in out-bred mice.

Recent studies have shown that expanded-simple-tandem-repeat (ESTR) DNA loci are efficient genetic markers for detecting radiation-induced germline mutations in mice. Dose responses following irradiation, however, have only been characterized in a small number of inbred mouse strains, and no studies have applied ESTRs to examine potential modifiers of radiation risk, such as adaptive response. We gamma-irradiated groups of male out-bred Swiss-Webster mice with single acute doses of 0.5 and 1.0 Gy, and compared germline mutation rates at ESTR loci to a sham-irradiated control. To test for evidence of adaptive response we treated a third group with a total dose of 1.1 Gy that was fractionated into a 0.1 Gy adapting dose, followed by a challenge dose of 1.0 Gy 24h later. Paternal mutation rates were significantly elevated above the control in the 0.5 Gy (2.8-fold) and 1.0 Gy (3.0-fold) groups, but were similar to each other despite the difference in radiation dose. The doubling dose for paternal mutation induction was 0.26 Gy (95% CI = 0.14-0.51 Gy). Males adapted with a 0.1 Gy dose prior to a 1.0 Gy challenge dose had mutation rates that were not significantly elevated above the control, and were 43% reduced compared to those receiving single doses. We conclude that pre-meiotic male germ cells in out-bred Swiss-Webster mice are sensitive to ESTR mutations induced by acute doses of ionizing radiation, but mutation induction may become saturated at a lower dose than in some strains of inbred mice. Reduced mutation rates in the adapted group provide intriguing evidence for suppression of ESTR mutations in the male germline through adaptive response. Repetitive DNA markers may be useful tools for exploration of biological factors affecting the probability of heritable mutations caused by low-dose ionizing radiation exposure. The biological significance of ESTR mutations in terms of radiation risk assessment, however, is still undetermined.

Cytogenetic dose-response and adaptive response in cells of ungulate species exposed to ionizing radiation.

In the studies reported here, the micronucleus assay, a common cytogenetic technique, was used to examine the dose-responses in fibroblasts from three ungulate species (white-tailed deer, woodland caribou, and Indian muntjac) exposed to high doses of ionizing radiation (1-4 Gy of (60)Co gamma radiation). This assay was also used to examine the effects of exposure to low doses (1-100 mGy) typical of what these species experience in a year from natural and anthropogenic environmental sources. An adaptive response, defined as the induction of resistance to a stressor by a prior exposure to a small "adapting" stress, was observed after exposure to low doses. This work indicates that very small doses are protective for the endpoint examined. The same level of protection was seen at all adapting doses, including 1 radiation track per cell, the lowest possible cellular dose. These results are consistent with other studies in a wide variety of organisms that demonstrate a protective effect of low doses at both cellular and whole-organism levels. This implies that environmental regulations predicated on the idea that even the smallest dose of radiation carries a quantifiable risk of direct adverse consequences to the exposed organism require further examination. Cytogenetic assays provide affordable and feasible biological effects-based alternatives that are more biologically relevant than traditional contaminant concentration-based radioecological risk assessment.

Biophysical dose measurement using electron paramagnetic resonance in rodent teeth.

Electron paramagnetic resonance (EPR) dosimetry of human tooth enamel has been widely used in measuring radiation doses in various scenarios. However, there are situations that do not involve a human victim (e.g. tests for suspected environmental overexposures, measurements of doses to experimental animals in radiation biology research, or chronology of archaeological deposits). For such cases we have developed an EPR dosimetry technique making use of enamel of teeth extracted from mice. Tooth enamel from both previously irradiated and unirradiated mice was extracted and cleaned by processing in supersaturated KOH aqueous solution. Teeth from mice with no previous irradiation history exhibited a linear EPR response to the dose in the range from 0.8 to 5.5 Gy. The EPR dose reconstruction for a preliminarily irradiated batch resulted in the radiation dose of (1.4+/-0.2) Gy, which was in a good agreement with the estimated exposure of the teeth. The sensitivity of the EPR response of mouse enamel to gamma radiation was found to be half of that of human tooth enamel. The dosimetric EPR signal of mouse enamel is stable up at least to 42 days after exposure to radiation. Dose reconstruction was only possible with the enamel extracted from molars and premolars and could not be performed with incisors. Electron micrographs showed structural variations in the incisor enamel, possibly explaining the large interfering signal in the non-molar teeth.

A dietary supplement abolishes age-related cognitive decline in transgenic mice expressing elevated free radical processes.

We previously found that transgenic mice overexpressing growth hormone (TGM) have elevated and progressively increasing free radical processes in brain that strongly correlates with reduced survivorship. Young mature TGM, however, displayed vastly enhanced learning of an eight-choice cued maze and qualitatively different learning curves than normal controls. Here we document the age-related patterns in learning ability of TGM and normal mice. Learning appeared inferior in both genotypes of very young mice but TGM were confirmed to be superior to normal mice upon maturity. Older TGM, however, showed rapid age-related loss of their exceptional learning, whereas normal mice at 1 year of age showed little change. The cognitive decline of TGM was abolished by a complex "anti-aging" dietary supplement formulated to promote membrane and mitochondrial integrity, increase insulin sensitivity, reduce reactive oxygen and nitrogen species, and ameliorate inflammation. Results are discussed in the context of reactive oxygen and nitrogen species, long-term potentiation, learning, aging and neuropathology, based on known impacts of the growth hormone axis on the brain, and characteristics of TGM.