Mats Harms-Ringdahl

Tardigrades survive exposure to space in low Earth orbit

Summary Vacuum (imposing extreme dehydration) and solar/galactic cosmic radiation prevent survival of most organisms in space [1]. Only anhydrobiotic organisms, which have evolved adaptations to survive more or less complete desiccation, have a potential to survive space vacuum, and few organisms can stand the unfiltered solar radiation in space. Tardigrades, commonly known as water-bears, are among the most desiccation and radiation-tolerant animals and have been shown to survive extreme levels of ionizing radiation [2–4]. Here, we show that tardigrades are also able to survive space vacuum without loss in survival, and that some specimens even recovered after combined exposure to space vacuum and solar radiation. These results add the first animal to the exclusive and short list of organisms that have survived such exposure.

Extracellular 8-oxo-dG as a sensitive parameter for oxidative stress in vivo and in vitro

8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG) is one of the mutagenic base modifications produced in DNA by the reaction of reactive oxygen species. The biological significance of 8-oxo-dG is shown by the existence of repair pathways that are able to recognize and remove this lesion from both DNA and the nucleotide pool. The final outcome of these evolutionarily conserved repair mechanisms in man is excretion of 8-oxo-dG/8-oxo-Gua from the intracellular to extracellular milieu including the blood plasma and urine. The aim of this investigation was to establish dose response relations for radiation-induced appearance of extracellular 8-oxo-dG in cellular model systems. Here we report on excretion of 8-oxo-dG after in vitro irradiation of whole blood and isolated lymphocytes with clinically relevant doses. We find that this excretion is dependent on dose and individual repair capacity, and that it saturates above doses of 0.5–1 Gy of gamma radiation. Our data also suggest that the nucleotide pool is a significant target that contributes to the levels of extracellular 8-oxo-dG; hence the mutagenic target for oxidative stress is not limited to the DNA molecule only. We conclude that extracellular 8-oxo-dG levels after in vitro irradiation have a potential to be used as a sensitive marker for oxidative stress.

Ionizing radiation biomarkers for potential use in epidemiological studies

Ionizing radiation is a known human carcinogen that can induce a variety of biological effects depending on the physical nature, duration, doses and dose-rates of exposure. However, the magnitude of health risks at low doses and dose-rates (below 100mSv and/or 0.1mSvmin(-1)) remains controversial due to a lack of direct human evidence. It is anticipated that significant insights will emerge from the integration of epidemiological and biological research, made possible by molecular epidemiology studies incorporating biomarkers and bioassays. A number of these have been used to investigate exposure, effects and susceptibility to ionizing radiation, albeit often at higher doses and dose rates, with each reflecting time-limited cellular or physiological alterations. This review summarises the multidisciplinary work undertaken in the framework of the European project DoReMi (Low Dose Research towards Multidisciplinary Integration) to identify the most appropriate biomarkers for use in population studies. In addition to logistical and ethical considerations for conducting large-scale epidemiological studies, we discuss the relevance of their use for assessing the effects of low dose ionizing radiation exposure at the cellular and physiological level. We also propose a temporal classification of biomarkers that may be relevant for molecular epidemiology studies which need to take into account the time elapsed since exposure. Finally, the integration of biology with epidemiology requires careful planning and enhanced discussions between the epidemiology, biology and dosimetry communities in order to determine the most important questions to be addressed in light of pragmatic considerations including the appropriate population to be investigated (occupationally, environmentally or medically exposed), and study design. The consideration of the logistics of biological sample collection, processing and storing and the choice of biomarker or bioassay, as well as awareness of potential confounding factors, are also essential.

Radiation tolerance in the eutardigrade Richtersius coronifer

Purpose: Tardigrades have a reputation of being extremely tolerant to extreme environmental conditions including tolerance to ionizing radiation while in a desiccated, anhydrobiotic state. However, the evidence for radio-tolerance in tardigrades is based on only one previous report, and there is an obvious need for complementary studies. In this paper we report an investigation on radio-tolerance in desiccated and hydrated specimens of the eutardigrade Richtersius coronifer. Materials and methods: Groups of 30 – 50 tardigrades were exposed to γ-radiation at doses between 1.0 – 9.0 (anhydrobiotic animals) or 0.5 – 5.0 (hydrated animals) kGy and the animals were followed until all were dead. Radiation tolerance of both desiccated and hydrated tardigrades was studied. Results: Both desiccated and hydrated animals irradiated with 0.5 and 1 kGy did not deviate in survival from the control groups. Animals from all exposed groups underwent their moulting and egg production cycle, but at decreasing frequency for doses above 1 kGy. No eggs laid by irradiated animals hatched, while eggs laid by controls did so. Conclusion: Our study suggests that radiation tolerance in tardigrades is not due to biochemical protectants connected with the desiccated state. Rather, cryptobiotic tardigrades may rely on efficient mechanisms of DNA repair, the nature of which is currently unknown.

Quantitative proteomic analysis reveals induction of premature senescence in human umbilical vein endothelial cells exposed to chronic low-dose rate gamma radiation

Chronic low‐dose ionizing radiation induces cardiovascular disease in human populations but the mechanism is largely unknown. We suggested that chronic radiation exposure may induce endothelial cell senescence that is associated with vascular damage in vivo. We investigated whether chronic radiation exposure is causing a change in the onset of senescence in endothelial cells in vitro. Indeed, when exposed to continuous low‐dose rate gamma radiation (4.1 mGy/h), primary human umbilical vein endothelial cells (HUVECs) initiated senescence much earlier than the nonirradiated control cells. We investigated the changes in the protein expression of HUVECs before and during the onset of radiation‐induced senescence. Cellular proteins were quantified using isotope‐coded protein label technology after 1, 3, and 6 weeks of radiation exposure. Several senescence‐related biological pathways were influenced by radiation, including cytoskeletal organization, cell–cell communication and adhesion, and inflammation. Immunoblot analysis showed an activation of the p53/p21 pathway corresponding to the progressing senescence. Our data suggest that chronic radiation‐induced DNA damage and oxidative stress result in induction of p53/p21 pathway that inhibits the replicative potential of HUVECs and leads to premature senescence. This study contributes to the understanding of the increased risk of cardiovascular diseases seen in populations exposed to chronic low‐dose irradiation.

Stimulating effects of mercuric- and silver ions on the superoxide anion production in human polymorphonuclear leukocytes.

In a survey of a number of heavy metal ions for effects on the oxidative metabolism (respiratory burst) of human polymorphonuclear leukocytes (neutrophils) we have found that mercury(II) and silver ions in micromolar concentration significantly increase the production of superoxide anions in cells, initiated by formyl-methionyl-leucylphenylalanine (fMLP). The stimulation of radical formation induced by a certain ion concentration varied considerably in cells isolated from different blood donors, from a moderate increase to a very large (up to 400% of control values). When the soluble stimulator phorbol myristate acetate (PMA) or the particulate stimulator Zymosan were used to initiate the cell respiratory burst, no additional stimulating effects by the metal ions on superoxide anion formation were observed. This fact might indicate that the effect of the metal ions on the fMLP-dependent initiation of cell activity is a mechanism coupled to the interaction between the chemotactic peptide and its corresponding receptor molecules on the cell surface. By increasing the concentration of silver ions during pre-incubation of resting neutrophils, a spontaneous activation of the cells could be recorded at a concentration exceeding 5 microM. However, the silver ion concentration at which such spontaneous initiation of the respiratory burst occurred varied significantly between blood samples from different donors with a concentration range of 5 to 15 microM. This effect could not be shown for mercuric ions due to the toxicity of the metal above 5 microM. Blood samples from some donors contained neutrophils that could be activated by either mercuric- or silver ions at concentration as low at 1 microM. The spontaneous activation of neutrophils with elevated concentrations of silver ions is kinetically similar to the PMA-induced. The onset of superoxide anion formation is preceded by a lag period whose length varies in time with the concentration of agent applied to the cells. It is a known fact that once the neutrophils have been activated with fMLP it is not possible to reactivate the cells by a second supplementation of fMLP. However, after cessation of the fMLP-induced activation, addition of PMA or silver ions gives rise to renewed production of superoxide anions. We propose two different mechanisms of action of silver ions on oxidative metabolism of neutrophils.(ABSTRACT TRUNCATED AT 400 WORDS)

Osmotic fragility and fluidity of erythrocyte membranes from rats raised on an essential fatty acid deficient diet A spin label study

Erythrocyte membranes from rats raised on a diet with low content of essential fatty acids were studied by osmotic sensitivity tests and spin labeling techniques. This diet induced significant modifications in acylglycerophosphocholine fatty acid composition with regard to 16 : 1, 18 : 1, 18 : 2 (n-6), 20 : 3 (n-9), and 20 : 4 (n-6). No changes in membrane fluidity as monitored by spin label motion were found but the diet caused an increased osmotic sensitivity in essential fatty acid deficient erythrocytes. 50% hemolysis was obtained at a 51.0% dilution of saline with H2O as compared to a 57.0% dilution for the control material. Membrane fluidity was unaffected by gamma-irradiation up to 80 krad.

The PI3K/Akt/mTOR Pathway Is Implicated in the Premature Senescence of Primary Human Endothelial Cells Exposed to Chronic Radiation

The etiology of radiation-induced cardiovascular disease (CVD) after chronic exposure to low doses of ionizing radiation is only marginally understood. We have previously shown that a chronic low-dose rate exposure (4.1 mGy/h) causes human umbilical vein endothelial cells (HUVECs) to prematurely senesce. We now show that a dose rate of 2.4 mGy/h is also able to trigger premature senescence in HUVECs, primarily indicated by a loss of growth potential and the appearance of the senescence-associated markers ß-galactosidase (SA-ß-gal) and p21. In contrast, a lower dose rate of 1.4 mGy/h was not sufficient to inhibit cellular growth or increase SA-ß-gal-staining despite an increased expression of p21. We used reverse phase protein arrays and triplex Isotope Coded Protein Labeling with LC-ESI-MS/MS to study the proteomic changes associated with chronic radiation-induced senescence. Both technologies identified inactivation of the PI3K/Akt/mTOR pathway accompanying premature senescence. In addition, expression of proteins involved in cytoskeletal structure and EIF2 signaling was reduced. Age-related diseases such as CVD have been previously associated with increased endothelial cell senescence. We postulate that a similar endothelial aging may contribute to the increased rate of CVD seen in populations chronically exposed to low-dose-rate radiation.

DNA fragmentation and morphological changes in apoptotic human lymphocytes

Cell suspensions enriched in cells at various stages of apoptosis were obtained by separation of irradiated human peripheral blood lymphocytes on density gradients at different post-irradiation times. The state of DNA fragmentation in the cells was determined by comet assay and pulsed field gel electrophoresis. The morphologically distinguishable features of apoptosis such as chromatin condensation and cell shrinkage correlated with discrete stages of DNA fragmentation. It was found that >/=50 kbp fragmentation of DNA occurs already in cells of normal density whereas the subsequent DNA fragmentation onto fragments <50 kbp occurs in parallel with cell shrinkage and simultaneous increase in cell density. The observed stages of DNA fragmentation seem to be separated in time that could allow in case of abortive apoptosis formation of chromosomal aberrations.

Effects of zero magnetic field on the conformation of chromatin in human cells.

The effects of zero magnetic field on human VH-10 fibroblasts and lymphocytes were studied by the method of anomalous viscosity time dependencies (AVTD). A decrease of about 20% in the AVTD peaks was observed within 40 to 80 min of exposure of fibroblasts. This decrease was transient and disappeared 120 min after beginning of exposure. Similar kinetics for the effect of zero field was observed when cells were exposed 20 min and then kept at an ambient field. A 20% decrease of the AVTD peaks (p < 0.005 to 0.05) 40 to 70 min after 20 min exposure to zero field was reproduced in four independent experiments (out of four) with human lymphocytes from the same healthy donor. Contrary to the effects of zero field, irradiation of lymphocytes or fibroblasts with gamma-rays resulted in significant increase of the AVTD peaks immediately after irradiation. We concluded that zero field and gamma-rays caused hypercondensation and decondensation of chromatin, correspondingly. The effect of ethidium bromide served as a positive control and supported this conclusion. The effects of zero field on human lymphocytes were more significant in the beginning of G1-phase than in G0-phase. Thus, human fibroblasts and lymphocytes were shown to respond to zero magnetic field.