Search

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 CO3− paramagnetic centre (G2) is enhanced with gamma radiation. Isothermal decay curves are used to propose annealing methods for the removal of the radiosensitive CO3− radical without affecting the unirradiated baseline. Post-irradiation annealing of wallboard prevents recuperation of the radiosensitive CO3− radical with additional irradiation. A single-aliquot additive dose procedure is developed that successfully measures test doses as low as 0.76 Gy. [ABSTRACT FROM PUBLISHER]

Information on several papers discussed at the 15th Pacific Basic Nuclear Conference, held on October 18, 2006 in Sydney, Australia is presented. Topics include the different biological responses to radiation predominate at doses and dose rates that are substantially lower than those at which risks have been observed. It also highlights the benefits of zero dose and to low dose rates model.

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. [ABSTRACT FROM AUTHOR]

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 radiationinduced 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. [ABSTRACT FROM AUTHOR]

The article discusses the study on the effectiveness of 280 keV neurons for apoptosis in human lymphocytes. In this study, apoptosis in human lymphocytes was examined to assess the RBE of low energy keV neutrons compared to (1,3,7)C gamma radiation using three flow cytometry assays such as Annexin V, 3, 3′ -dihexaoxacarbocyanine iodine and Caspase-3. The result shows that 280 keV neutrons are equally effective as gamma radiation at inducing apoptosis in lymphocytes. It indicates that these two radiation qualities may produce similar level of biological effect in some cell types. Moreover, the finding that radiation quality and dose rate do not seem to affect the apoptotic response further adds to the potential utility of this endpoint for biolgical dosimetry.

Purpose: To determine whether yeast cells (Saccharomyces cerevisiae) defective in damage-inducible cell-cycle arrest can invoke an adaptive response and become resistant to normally lethal doses of ionizing radiation. Materials and methods: Wild-type yeast cells, cells defective for DNA-damage-responsive G1 and G2 cell-cycle arrest (rad9 δ), and cells defective for recombinational repair of DNA damage (rad50, 51, 52) were subjected to adapting treatments of heat or radiation and subsequently exposed to normally lethal doses of radiation. Survival, as measured by colony-forming ability, was compared with non-adapted, control cells. Results: Wild-type and rad9 δ cells became more resistant to potentially lethal doses of radiation after exposure to conditions that are known to elicit the adaptive response. Further, the relative magnitude of resistance developed by the normal, wildtype and rad9 δ yeast cells was similar, with a dose modifying factor (at D[sub 1]) for radiation-induced radiation resistance of 1.3 for both strains. Dose modifying factors (at D[sub 1]) for heat-induced radiation resistance were 1.7 and 1.6 for wild-type and rad9 δ cells, respectively. In contrast, none of the recombinational repair-defective cells exhibited radiation resistance after an adapting treatment. Conclusions: The ability of yeast cells to arrest in cell-cycle gap phases did not appear to contribute significantly to radiation resistance induced by radiation or heat. Instead, it is suggested that the adaptive response was due mainly to the existence and enhancement of cellular recombinational repair capacity, which was sufficient to repair any DNA damage without the requirement of a detectable cell-cycle delay. [ABSTRACT FROM AUTHOR]

Purpose: To investigate the repair of ionizing radiation-induced DNA lesions in human skin fibroblasts in the presence of cisplatin-DNA adducts and to determine the persistence of DNA repair inhibition by cisplatin. Materials and methods: Normal human fibroblasts (AG1522) treated with cisplatin were exposed to 4 Gy 60Co gamma-radiation and assayed for repair of radiation-induced damage under growth-permissive conditions. DNA damage was measured by the fluorescence analysis of DNA unwinding (FADU) and cytokinesis-blocked micronucleus assays. Results: Rejoining of strand breaks caused by 4 Gy radiation in cells without cisplatin pre-treatment appeared to be biphasic with an initial fast component (up to 15 min of repair time) followed by a slower component, and was completed by 90 min. Cisplatin treatment (10 mu g/ml, 30 min) immediately before irradiation had no effect on the fast rejoining component, but inhibited the slow component ( p 0.01). The same cisplatin treatment 24 h prior to irradiation inhibited both slow and fast components ( p 0.01). In contrast, decreasing the cisplatin exposure to 1.0 mu g/ml for 30 min, 24 h prior to irradiation, resulted in an increased amount of strand break repair at each time point measured compared with irradiated control cells. This mild cisplatin treatment (95% survival) also resulted in a reduction of radiation-generated micronuclei indicating an adaptive response. Conclusions: Cisplatin used in combination with ionizing radiation can produce differential cellular responses depending upon the severity of the cisplatin treatment and the time interval between cisplatin and radiation exposures. [ABSTRACT FROM AUTHOR]

Abstract. Chromosomes are located within the interphase nucleus in regions called domains. Using fluorescence in situ hybridization with whole chromosome paints, a pair of homologous chromosomes can be visualized as two discrete domains and their relative spatial location determined. This study examines the effects of an ionizing radiation exposure on the relative spatial location of chromosome 7 and 21 domains in human skin fibroblasts and lung endothelial cells. The distance between homologous chromosome domains was assessed for each nucleus, before and after exposure to ionizing radiation, using conventional epifluorescence and confocal laser scanning microscopy. Results from conventional microscopy indicated that homologous chromosome domains were re-positioned closer to each other within interphase nuclei after exposure to radiation. Analysis of three-dimensional data obtained from confocal microscopy confirmed these results. In control cells, and in cells examined immediately after irradiation, 66.2% 2.1% of the homologous chromosome 21 domains within endothelial cell nuclei were located greater than 4.0 mu m apart (33.8% 1.9% were less than 4.0 mu m apart). However, when cells were examined 2 h after a 4.0 Gy gamma-ray exposure, only 30.5% 2.1% of the homologous chromosome domains were greater than 4.0 mu m apart (69.5% 2.1% were less than 4.0 mu m apart). Similar results were obtained for chromosomes 7 and 21 in skin fibroblast nuclei. The results indicate that homologous chromosome domains rearranged and became closer together within the interphase nuclei in response to ionizing radiation. The exact mechanism of this response is unknown, but it may be related to DNA repair processes. It is speculated that chromosome domains are re-positioned to permit repair of radiation-induced DNA damage. [ABSTRACT FROM AUTHOR]

Gas chromatography mass spectrometry of a number of steroidal spirolactones and their TMS ethers is reported. Open tubular column gas chromatography provides useful structural information for these compounds as shown by shifts in retention index values. The electron impact fragmentation pathways have been established for a number of the compounds. The results of the study indicate that open tubular column gas chromatography mass spectrometry will be of value in studies of the metabolism of this class of steroids in animals and man. [ABSTRACT FROM AUTHOR]

In February 2000, a radiation incident involving a medical 60Co 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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., Competing Interests: The authors declare there are no competing interests.

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., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

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., (Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.)

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.

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.

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.

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.

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.

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.

For radiation exposures below 100 mGy, the dosimetric signal in tooth enamel is too small to be measured by using the traditional dose reconstruction procedure. This is because low amplitude zero-added-dose signal can not be identified in an EPR spectrometer. A technique is presented wherein, zero-added-dose signal. when amplified by a proper known dose, can be measured in the EPR spectrometer. Mathematically, the accidental dose x is modified by a known amount of exposure, y (large enough so that the signal is now visible), and total exposure becomes x' = x + y, which is the modified-zero-added dose. The exposure x' is then quantified using the conventional backward extrapolation method and the accidental dose can be measured. In a laboratory controlled experiment, the feasibility of dose reconstruction in the 100 mGy range has been demonstrated. This may enable measurements of dose even due to suspected low exposure in tooth enamel.

Cis-diamminedichloroplatinum II (cisplatin) is a DNA inter- and intrastrand crosslinking agent which can sensitize prokaryotic and eukaryotic cells to killing by ionizing radiation. The mechanism of radiosensitization is unknown but may involve cisplatin inhibition of repair of DNA damage caused by radiation. Repair proficient wild type and repair deficient (rad52, recombinational repair or rad3, excision repair) strains of the yeast Saccharomyces cerevisiae were used to determine whether defects in DNA repair mechanisms would modify the radiosensitizing effect of cisplatin. We report that cisplatin exposure could sensitize yeast cells with a competent recombinational repair mechanism (wild type or rad3), but could not sensitize cells defective in recombinational repair (rad52), indicating that the radiosensitizing effect of cisplatin was due to inhibition of DNA repair processes involving error free RAD52-dependent recombinational repair. The presence or absence of oxygen during irradiation did not alter this radiosensitization. Consistent with this result, cisplatin did not sensitize cells to mutation that results from lesion processing by an error prone DNA repair system. However, under certain circumstances, cisplatin exposure did not cause radiosensitization to killing by radiation in repair competent wild type cells. Within 2 h after a sublethal cisplatin treatment, wild type yeast cells became both thermally tolerant and radiation resistant. Cisplatin pretreatment also suppressed mutations caused by exposure to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a response previously shown in wild type yeast cells following radiation pretreatment. Like radiation, the cisplatin-induced stress response did not confer radiation resistance or suppress MNNG mutations in a recombinational repair deficient mutant (rad52), although thermal tolerance was still induced. These results support the idea that cisplatin adducts in DNA interfere with RAD52-dependent recombinational repair and thereby sensitize cells to killing by radiation. However, the lesions can subsequently induce a general stress response, part of which is induction of RAD52-dependent error free recombinational repair. This stress response confers radiation resistance, thermal tolerance, and mutation resistance in yeast.

We have tested the possibility of using apoptosis (programmed cell death) in human peripheral blood lymphocytes as a short-term biological dosimeter. Lymphocytes isolated from whole blood were irradiated in culture with 250 kVp x-rays or 60Co gamma rays. Two assays were used to measure apoptosis in lymphocytes after irradiation: in situ terminal deoxynucleotidyl transferase assay and fluorescence analysis of DNA unwinding assay. Similar qualitative and quantitative results were produced by the assays, supporting the notion that the fluorescence analysis of DNA unwinding assay measured DNA fragmentation associated with apoptosis. Induction of apoptosis in lymphocytes irradiated in vitro was proportional to dose and could be detected following exposures as low as 0.05 Gy. Lymphocytes from individual donors had reproducible dose responses. There was, however, variation between donors. X-ray and gamma-ray exposures induced similar levels of apoptosis at similar doses. The induction kinetics of apoptosis in vitro indicate a maximum is reached about 72 h after irradiation. In conclusion, the in vitro experimental evidence indicates that radiation-induced apoptosis in human lymphocytes has the kinetics, sensitivity, and reproducibility to be a potential biological dosimeter.

Unrepaired or misrepaired radiation damage in mammalian chromosomes can result in micronucleus formation at the first cell division. This represents loss of genomic information which may cause cell death. To improve our understanding of the mechanism of radiation-induced micronucleus formation, we characterized micronucleus ultrastructure and identified the origin of micronucleus DNA. Immunofluorescence microscopy showed that micronuclei were structurally similar to main nuclei since they contained nuclear lamins A and C and were encapsulated by a network of vimentin intermediate filaments. The contents of radiation-induced micronuclei were characterized using fluorescence in situ hybridization to probe for DNA originating from chromosomes 2, 7, 11 and 16. We postulated that if incorporation of DNA into micronuclei were random, then the probability of chromosomal DNA in micronuclei would be related to the target, i.e. chromosome size. Our results demonstrated that incorporation of DNA from smaller chromosomes (11 and 16) was not different from expected values but incorporation of DNA from the larger chromosomes (2 and 7) was significantly greater than expected. Not all chromosomes in the human genome, therefore, were equally susceptible to genomic loss by micronucleus encapsulation. In conclusion, radiation-induced micronuclei have similar structural characteristics to main nuclei, chromosome damage and/or repair after ionizing radiation may be non-random, and micronucleus formation may reflect this variability.

The matrix metalloproteinase, matrilysin, is thought to play an important role in the early steps of tumor progression. We determined the chromosome location of the matrilysin gene (MMP7) by Southern and PCR analysis of two different panels of somatic cell hybrids and in situ hybridization of metaphase chromosomes. Matrilysin maps to the region, 11q21-->q22, adding MMP7 to the cluster of matrix metalloproteinase genes that have already mapped to this region.

We have investigated the influence of the cellular adaptive response to ionizing radiation on radiation-induced apoptosis in human cells. The adaptive response is believed to be a protective mechanism that confers resistance to the detrimental effects of ionizing radiation and that can be induced by different agents, including hyperthermia and radiation. We have used fluorescence analysis of DNA unwinding (FADU) to assay the induction of apoptosis in human peripheral blood lymphocytes by ionizing radiation. Using the FADU assay, we have observed the initial radiation-induced DNA damage, its subsequent disappearance due to enzymatic repair, and its time- and dose-dependent reappearance. We believe this reappearance of DNA damage to be indicative of the DNA fragmentation event associated with apoptosis. This interpretation has been supported at the individual cell level using an in situ terminal deoxynucleotidyl transferase (TDT) assay (Apoptag, Oncor Inc.), which detects the 3'-hydroxyl ends of fragmented DNA, and by fluorescence analysis of nuclear morphology in Hoechst 33258 stained cells. Pretreatment of cells with low-dose gamma-radiation (0.1 Gy) or mild hyperthermia (40 degrees C for 30 min) altered the extent of radiation-induced (3 Gy) apoptosis. Both pretreatments sensitized lymphocytes to become apoptotic after the 3-Gy radiation exposure. This sensitization may represent an adaptive response mechanism that reduces the risk that genetically damaged cells will proliferate. The ability to modify the probability of radiation-induced apoptosis may lower the cancer risk from a radiation exposure.

A single oral dose (200 mg) of an equimolar mixture of potassium canrenoate and its 20,20,21,21-tetradeutero analogue was administered to three healthy men. The steroids in urine collected for 24 hr after dosage were isolated on XAD-2 resin, and purified and fractionated into groups by lipophilic gel chromatography before and after hydrolysis of conjugates. GC/MS analysis of these fractions allowed the detection and identification of canrenone, canrenoic acid and its ester glucuronide, 3 beta-hydroxy-3-deoxocanrenone, 3beta-hydroxy-4,5alpha-dihydro-3-deoxocanrenone and a 3epsilon-hydroxy-4,5,6,7-tetrahydro-3-deoxocanrenone. In addition a number of di- and trihydroxy compounds formed by reduction and hydroxylation were partially identified from their E1 and C1 mass spectra. The results provide information on the metabolism of oral potassium canrenoate in man, and demonstrate the utility of combining stable isotope labeling, lipophilic gel chromatography, and GC/MS in studies of steroidal spirolactones.

Electron impact and chemical ionization mass spectra are reported for several steroidal spirolactones and their TMS ethers. The electron impact spectra were characterized generally by low abundance molecular ions and large numbers of fragment ions. Methane chemical ionization spectra exhibited high intensity [M+H]+ and/or [M+H-H2O]+ or [M+H-TMSOH]+ ions with few other fragment ions. Ammonia chemical ionization spectra had intense [M+H]+ and/or [M+NH4]+ ions with a few fragment ions generally formed by loss of H2O or TMSOH from these parent ions. Ammonia chemical ionization gave intense parent ions even for polyhydroxy compounds and their TMS ethers in contrast to methane chemical ionization. The results of this study suggest that a combination of electron impact with ammonia chemical ionization mass spectrometry would offer the best techniques for detection and identification of these compounds in biological fluids.

Chromatography of steroidal spirolactones on DEAE-Sephadex A-25 under selected pH conditions allowed efficient separation of these compounds from other steroids and many of the endogenous components of human urine. The spirolactones were recovered in high yield, mostly over 90%. Lipophilic-gel chromatography provided a useful method for group fractionation of mixtures of these spirolactones with high recoveries (generally over 90%), unaffected by the presence of endogenous material from normal human urine.

Metabolism of spironolactone in man is extensive and complex. For many years the dethioacetylated metabolite, canrenone, was assumed to be the major metabolite. However, recent studies using specific high performance liquid chromatography (HPLC) have demonstrated the presence of spironolactone and the sulfur-containing metabolites 7 alpha-thiomethylspirolactone (IV) and 6 beta-hydroxy-7 alpha-thiomethylspirolactone (V), in addition to canrenone, in the serum after a single oral dose of spironolactone. The importance of spironolactone and metabolites IV and V relative to canrenone at steady state remains unknown and was the subject of the present investigation. Twelve healthy males received 100 mg spironolactone, once daily, for 15 days. Repeated blood samples were taken on days 1, 8 and 15 for estimation of spironolactone and its metabolites. Peak serum levels [mean (SD)] of spironolactone, canrenone, and sulfur-containing metabolites IV and V were 72 (45), 155 (43), 359 (106) and 101 (26) ng/ml, respectively on day 1 and 80 (20), 181 (39), 391 (118) and 125 (24) ng/ml, respectively on day 15. The AUC (0-24) values of these compounds on day 15 were 231 (50), 2173 (312), 2804 (777) and 1727 (367) ng.hr/ml, respectively and the post-steady state elimination half-life (t1/2) values were 1.4 (0.5), 16.5 (6.3), 13.8 (6.4), and 15.0 (4.0) hours, respectively. It was concluded that unmetabolized spironolactone is present in the serum and that the sulfur-containing metabolite IV rather than canrenone is the major metabolite in serum following single or repeated doses of spironolactone.