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Radiobiological mechanisms underlying sensitivity of nonhuman biota to ionising radiation Outline • Some facts about radiosensitivity of biota • Some mechanisms – – – – Extremophiles avoidence Adaptive responses Selection • Why study radiation response of organisms? – Evolution of mechanisms – Environmental protection concerns – Cancer research/medical uses Take home messages • Radioresistance/ssensitivity must be considered in relation to the endpoint e.g. reproductive fertility, death, enzyme activity, individual organ sensitivity, ability to compete. • Resistance to high doses of radiation is often the result of evolutionary adaptation to a different environmental extreme. • Sensitivity can be due to genetic OR epigenetic/environmental causes, complicating determination of species sensitivity • Determining the link between effect – harm – risk can be challenging even at the level of the individual Slide courtesy of Tom Hinton Factors Influencing the Sensitivity of Plants to Radiation Increasing Sensitivity Decreasing Sensitivity Large nucleus Large chromosomes Acrocentric chromosomes Small nucleus Small chromosomes Metacentric chromosomes Low chromosome number Diploid or haploid Sexual reproduction Long intermitotic time High chromosome number High polyploid Asexual reproduction Short intermitotic time Long dormant period Short or no dormant period (Sparrow, 1961) Taxonomic and Developmental Aspects of Radiosensitivity F.L. Harrison S.L. Anderson This paper was prepared for submittal to the Symposia on Ionizing Radiation Stockholm, Sweden May 20-24, 1996 November 1996 Table 1. Summary showing ranges of LD50s obtained from acute 1 irradiation of organisms from different taxonomic groups. Group Dose, Gy Protista 30 - 30,000 Invertebrates 2.1 - 1,100 Vertebrates Fishes Amphibians Reptiles Birds Mammals 10 -> 600 7 - >22 3 - 40 5 - 20 2.5 - 150 Plants 1.5 - > 130 1 The radiation units in references were converted to grays for comparative purposes and for some values are approximations. LD50S FROM ACUTE IRRADIATION OF MAMMALS AND 1 FISHES. Dose, Gy Mammals Humans Monkey Dog Swine Hamster Mouse Rabbit Bat Pisces Goldfish Mummichog Tench Guppy Chinook salmon Mosquitofish Pinfish 3 6 2.5 2.5 6 6.4 7.5 150 3.75 - 100 10 -20 12 - 55 23.5 25 37 50 The dose rates known to cause sterility in different species have a large range—0.23 to 1400 mGy/h. Differences occur because the processes of gametogenesis are not the same from species to species, and for a given species, the response of male and female reproductive tissues may differ. In general, the testis is more radioresistant than the ovary Reproductive success for a given species may be related not only to its sensitivity to radiation during gametogenesis and early development but also its reproductive strategy. For example, in a highly fecund species, the survival of early life stages may be very low, and the loss of abnormal embryos induced from radiation exposure may be masked completely by those lost from other ecological factors, such as food limitation and predation. CHANGES IN THE RADIOSENSITIVITY OF RAINBOW TROUT SALMO GAIRDNERII EXPOSED TO ACUTE IRRADIATION DURING DEVELOPMENT STAGE IN LIFE CYCLE LD50 (GY) GAMETE 0.5 - 1.0 CELL 0.58 2-32 CELL 3.1 GERM RING 4.5 - 4.6 EYED 4.1 - 9.0 ADULT 15 REF: HARRISON AND ANDERSON 1996 Table 5. S ensitivity of different endpoints in the polychaete worm Neanthes arenaceodentata. Dose, Gy Endpoint >0.3 >0.5 DNA-strand breakage Reduced fertility Increased sister chromatid exchanges >2 Increased chromosomal aberrations >50 Sterility >100 Lifespan reduction >500 Mortality Figure 1. For the same LD50 value, the shape of species response curves from radiation may differ significantly. developing gametes or in the size of the gonad. They may be quantified also by observing changes in the number of fertilized eggs produced and in the morphology and physiology of the developing embryos. When early life stages are irradiated, the effects quantified include the induction of abnormalities in the embryos and increases in mortality. Although the database is far from complete, sufficient information is available to permit some comparisons to be made. An example of changes in sensitivity among developmental stages is Causes of sensitivity • • • • UNSCEAR list but also Heterozygous mutations – gene dosage effects Compromised defenses due to other stressors Lack of conditioning exposure or induced tolerance Some examples of data from the “experimental” field • Field irradiators from Colorado • Exp. aquatic mesocosms from Savannah River plus work of Hingston et al using woodlice in a mesocosm. • Chernobyl and Fukushima accidents-, plants, voles, swallows, butterflies and reindeer • Hanford site and other uranium mines • Chalk River, low dose facility and cooling ponds Data on radiation effects for nonhuman species Wildlife Group Morbidity Mortality Amphibians Aquatic invertebrates Aquatic plants Bacteria Birds Crustaceans Fish Fungi Insects Mammals Molluscs Moss/Lichens Plants Reptiles Soil fauna Zooplankton No data To few to draw conclusions www.ceh.ac.uk/PROTECT Some data Reproductive capacity Mutation www.ceh.ac.uk/PROTECT Fixed assessment factor method PNEV = minimal Effect Concentration / Safety Factor Main underlying assumptions In the frame of this approach, extrapolations are made from: •The ecosystem response depends on the most sensitive species •Acute to chronic •One life stage to the whole life cycle •Individual effects to effects at the population level •One species to many species •One exposure route to another •Direct to indirect effects •One ecosystem to another •Different time and spatial scales •Protecting ecosystem structure protects community function www.ceh.ac.uk/PROTECT Background radiation exposure for ICRP RAPs (weighted dose rates) Freshwater organisms – 0.4 – 0.5 μGy/h (Hosseini et al., 2010) Marine organisms – 0.6 - 0.9 μGy/h (Hosseini et al., 2010) Terrestrial animals and plants – 0.07-0.6 μGy/h (Beresford et al., 2008) www.ceh.ac.uk/PROTECT Background radiation exposure for ICRP RAPs Freshwater organisms – 0.4 – 0.5 μGy/h (Hosseini et al., 2010) Derived screening dose rate (10organisms μGy/h)–is more than 10 Marine times these background 0.6 - 0.9 μGy/h (Hosseinivalues et al., 2010) Terrestrial animals and plants – 0.07-0.6 μGy/h (Beresford et al., 2008) www.ceh.ac.uk/PROTECT Zone and Classification External Gamma Dose (Gy) Conifer Death (4 km2) Complete death of pines; partial damage to deciduous trees over 80 - 100 Sublethal (38 km2) Death of most growth points, partial death of coniferous trees, morphological changes to deciduous trees Medium Damage (120 km2) Suppressed reproductive ability, dried needles, morphological changes Minor Damage Disturbances in growth, reproduction and morphology of coniferous trees Post Chernobyl courtesy of Tom Hinton 10 – 20 4–5 0.5 – 1.2 (Kuzubov et al.1990) Low dose data – a reminder! – Reports of effects of less than 5 mGy on • Tandem mutation frequency (microGy, Sykes in mice), • Adaptive response using micronucleus endpoint (Stuart, Redpath in frogs in vivo (microGy)and rat cell lines) • Microsatellite instability (Dubrova in humans and mice) • Oxidative stress response (Einset in plant root hairs ) • Calcium flux and bystander effect ( in rainbow trout, zebrafish, prawns and various cell lines and communication of bystander signals between fish- our group Is an effect important even if not “harmful”? Mechanisms and strategies • • • • • Resistance due to DNA repair Multiple copies of genome Antioxidants/colour Evasion of exposure Up-regulation or down-regulation of antideath pathways (depending on whether death is a beneficial outcome) The tunicate story Tunicate LD50 is about 3KGy BUT budding is inhibited in the mGy region and rescue by normal tunicate grafts is similarly affected. Allorecognition processes are very sensitive to low radiation doses Refs by Rinkevitch and Weissmann et al 1970-2008 Suggested mechanisms - extreme radioresistance • Multiple copies of the genome – Cockroaches and many other insects – Thermophilic bacteria • Anti-oxidant colours/enzymes – e.g. Rubrobacter radiotolerans • DNA breaks protected – D. radiodurans family Nature Reviews – Microbiology 2007 Deinococcus radiodurans • Part of a family including some of the most radiation-resistant organisms known • Survives 5000 Gy of gamma radiation • Genome is 4 circular molecules, 2 chromosomes, 1 megaplasmid, and 1 small plasmid • Multiploid • Genome lacks genes for RecB and RecC (it has recD) • Lots of interesting proteins Courtesy of Michael Daly Uniformed Services University of the Health Sciences DOUBLE STRAND BREAKS FORMED IN D RADIODURANS COMPARED WITH E. COLI Species Genomes per cell E.coli K12 D. radiodurans DNA DSB’s At D37 Average distance Between lesions 4-5 8-9 530,000 bp 8-10 >275 10,000 bp RADICAL SCAVANGING AS AN ADDITIONAL MECHANISM? Red pigment anti oxidant activity?? Rubrobacter radiotolerans red pigmented highly radioresistant Deinococcus radiodurans also red pigmented 1. E. Asgarani, H. Terato, K. Asagoshi, H.R. Shahmohammadi, Y. Ohyama, T. Saito, O. Ymamoto and H. Ide (2000) J. Radiat. Res. 41, 19-34. 2. E. Asgarani, H. Funamizu, T. Saito, H. Terato, Y. Ohyama, O. Yamamoto and H. Ide (1999) Microbiol. Res. 154, 185-190. Other biochemical mechanisms • Hypoxia • Protective sugars (tetrahalose) in the exoskeleton • Use of neurotransmitter antagonists such as L-DOPA which can modulate stress responses • Sensitization by plant polyphenols • ROS mediates both pro-apoptotic and anti-apoptotic signaling, but the precise mechanisms that lead to these polar outcomes are not yet clear. • Growth factor pathways (e.g., EGFR, PDGFR) Bcl-2 Survivin Protein kinase B/Akt MDR proteins ROI COX-2 NF-κB STAT3 Multiple copies of chromosomes? • Kira S. Makarova et al, Genome of the Extremely Radiation-Resistant Bacterium Deinococcus radiodurans Viewed from the Perspective of Comparative Genomics Microbiol Mol Biol Rev. 2001 March; 65(1): 44–79. “copy number NOT correlated with radioresistance” • Alexander T. Smith et al HIP1 mediated excisions in Escherichia Coli. “Data not consistent with a 'copy choice' mechanism of DNA rearrangement ---underlying extreme resistance to DNA damage” • The genome sequence of the extreme thermophile Thermus thermophilus Anke Henne, Nature Biotechnology 22, 547 - 553 (2004) Cross resistance? • Anke Henne, Nature Biotechnology 22, 547 - 553 (2004) The genome sequence of the extreme thermophile Thermus thermophilus – enzymes of thermophilic and radioresistant organisms are not only more thermostable, but also more resistant to chemical agents than their mesophilic homologs. • In D. radiodurans, it is likely that the radioresistance is due to mechanisms evolved to cope with dessication » Michael Cox p.comm Several studies have shown that tardigrades can survive -irradiation well above 1 kilogray, and desiccated and hydrated (active) tardigrades respond similarly to irradiation. Thus, tolerance is not restricted to the dry anhydrobiotic state suggesting possible involvement of an efficient, but yet undocumented, mechanism for DNA repair. Other anhydrobiotic animals (Artemia, Polypedium), when dessicated, show a higher tolerance to irradiation than hydrated animals, possibly due to the presence of high levels of the protective disaccharide trehalose in the dry state. But even though eggs were laid after 1kGy, they didn’t develop into juvenilles. Adults appear resistant due to limited regeneration of adult cells. K. INGEMAR JÖNSSON Astrobiology 7, 757–766. 2007 Evolution of mechanisms • Primitive mechanisms which evolved for other purposes can be harnessed eg bystander signalling, extremophiles • Polymorphisms in enzymes can be selected for leading to population drift • Genomic instability can result in increased diversity available for selection VALUABLE MATERIAL FOR EVOLUTIONARY BIOLOGISTS Summary • Theoretical basis for radiosensitivity not based on biology • Very little data for most species/classes of organisms • Actual studies from experimental and accident situations show huge variation within and between species depending on many factors • Conservative generic screening values to try to take account of variability • Biological strategies to cope with radiation cross evolved • Very low dose effects seen but relationship ti harm/risk not established