Mechanisms underlying sensitivity of nonhuman biota to ionising radiation

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Mechanisms underlying sensitivity of non-human
biota to ionising radiation

• Carmel Mothersill
• McMaster University
• CANADA

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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

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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

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Categories of radiation tolerance suggested in
textbooks

• Archaea and some other bacteria- resistant to
  gt50kGy
• Some insects (e.g. lepidoptera and Diptera) -
  resistant to gt300Gy
• Some lower vertebrates -resistant to gt20Gy
• BUT UNCLEAR
• What of population
• What criteria used to define resistant
• What holding conditions were used

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Pre-Chernobyl
Effects from Short Term Exposures (5 to 60 d)

• minor effects (chromosomal damage changes in
  reproduction and physiology)

• intermediate effects (selective mortality of
  individuals within a population)

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Pre-Chernobyl
Lethal Acute Dose Ranges (Whicker and Schultz,
1982)
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Slide courtesy of Tom Hinton
Factors Influencing the Sensitivity of Plants to
Radiation
(Sparrow, 1961)
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Causes of sensitivity

• Sparrows list but also
• Heterozygous mutations gene
  dosage effects
• Compromised defenses due to
  other stressors
• No conditioning exposure or induced tolerance
• Non-targeted effects such as genomic instability,
  bystander effects and HRS where genetic and
  epigenetic factors play in unknown ways.
  

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Mechanisms and strategies

• Resistance due to efficient DNA repair
• Multiple copies of genome
• Antioxidants/colour/destressing neurochemicals
• Evasion of exposure
• Up-regulation or down-regulation of anti-death
  pathways (depending on whether death is a
  beneficial outcome)

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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 accident-voles, swallows and reindeer
• Hanford site and other uranium mines
• Chalk River, low dose facility and cooling ponds

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Post Chernobyl courtesy of Tom Hinton
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(Kuzubov et al.1990)
Post Chernobyl courtesy of Tom Hinton
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• So relatively high acute or chronic doses appear
• to be needed to markedly affect organisms
• BUT
• What is the role of subtle effects?
• Non-targeted effects
• Signaling effects
• Multiple stressors
• Immune compromising effects

Do we need to worry about these?
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Radiation response data at the other extreme

• 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?

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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
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• BIODIVERSITY IS IMPORTANT BECAUSE WE DONT KNOW
  HOW STRESS AND EVOLUTION COMBINE TO PRODUCE NEW
  ADAPTIONS

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The dose rates known to cause sterility in
different species have a large range0.23 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
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We see effects in the mGy region from egg to adult
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Outline

• Some facts about radiosensitivity of biota
• Some mechanisms
• Extremophiles
• Adaptive responses
• Selection
• Why study radiation response of organisms?
• Evolution of mechanisms
• Environmental protection concerns
• Cancer research/medical uses

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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

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Nature Reviews Microbiology 2006
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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
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DOUBLE STRAND BREAKS FORMED IN D RADIODURANS
COMPARED WITH E. COLI
Average distance Between lesions
Species Genomes
per cell
DNA DSBs At D37
8 - 9 gt275
530,000 bp 10,000 bp
4-5 8-10
E.coli K12 D. radiodurans
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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.
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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-?B STAT3

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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

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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 didnt develop into juvenilles. Adults
appear resistant due to limited regeneration of
adult cells. K. INGEMAR JÖNSSON Astrobiology 7,
757766. 2007
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Adaptation

• Induced resistance to high background within
  individual
• Induced resistance following accidental exposure
  individual or population
• Selection for resistance population
• Ecosystem drift ecosystem

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Leopard frog (Rana pipiens)
Slide courtesy of Marilyn Stuart, AECL, Chalk
River.
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Adaptation Results (in Vitro)
Work conducted using leopard frog primary liver
cell cultures. DDL (1 frog) and TL (1 frog) are
background area sites (can be seen as control
sites). DS (1 frog) is an above background area
site. Green colour Not irradiated in
vitro. Yellow colour Exposed to 100 mGy in
vitro. Red colour Exposed to 4 Gy in vitro. Blue
colour Exposed to 100 mGy prior to being exposed
to 4 Gy in vitro.  
Slide and explanation courtesy of Marilyn Stuart
AECL Canada
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Adaption Results (in Vivo)
All frogs exposed to 4 Gy in the laboratory
(liver cells exposed in vivo). Blue Cells from
frogs from background sites (DDL and TL) Red
Cells from frogs from above background site (DS)
Slide and explanation courtesy of Marilyn Stuart
AECL Canada
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Outline

• Some facts about radiosensitivity of biota
• Some mechanisms
• Extremophiles
• Adaptive responses
• Selection
• Why study radiation response of organisms?
• Evolution of mechanisms
• Environmental protection concerns
• Cancer research/medical uses

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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

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Protection of the environment from ionising
radiation

• IAEA, ICRP, UN, DoE, EU addressing the issue
  politically and scientifically
• ICRP 2007 and UNSCEAR reports plus PROTECT D5
• EU-FREDERIKA, ERICA and PROTECT projects, DoE
  RESRAD-BIOTA developing more ecological
  orientated analysis and computer programmes
  designed to aid regulators in decision making.
• Experimental data needed and mechanistic
  understanding is vital.

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Points about radiation protection studies-1

• Toxicity assays are usually snapshots at best
• We need lifetime studies
• Mechanistic understanding and modeling
• Intra and inter species sensitivity assays
• Validation of endpoints
• Multiple stressor understanding

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Points about radiation protection studies 2

• The new non-targeted effects field suggests that
  low dose effects can fluctuate
• How do we live with and regulate in
• AN ENVIRONMENT OF UNCERTAINTY
• Not
• no effect but what effect?
• and is it important?

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PROTECT D5 recognises thisand acknowledges

• Data gaps
• Assays are mainly based on endpoints in
  individuals
• Different dose ranges for different endpoints
• Assay validation at any level is difficult
• Need more basic information to model accurately
• Need effect driven regulation

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Filling the radiobiological data gaps
experimental approaches

• In vitro studies with explants experimentally
  exposed
• Enable dose response ranges to be obtained
• In vitro culture from in vivo exposed organisms
• Bridge in vivo-in vitro and validate in vitro
  data
• Mesocosm approaches
• Enable fertility/fecundity/hierarchy to be
  examined
• Biomarker approaches
• Enable noninvasive sampling

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Primary Tissue Culture
Rainbow Trout (Oncorhynchus mykiss) Blue
Mussel (Mytilus edulis)
Rainbow Trout Spleen Cells (400x)
Mussel Pallial Mantle (200x)
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Prawn radiation effects

• Apoptosis and cytoplasmic damage following
  exposure of prawn haematopoeitic cells to 5mGy

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In vitro In vivo
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Effect of radiation in vivo on Bystander
signaling in fish tissues
ns






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Combination of metals and radiation
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T. Hinton
A wide spectrum of dose rates are achievable
within the facility, ranging from lt 0.1 mGy / d
to over 500 mGy / d. Dose rates within a
mesocosm are greatest directly under the center
of a 137Cs source, and decrease 70-fold at the
horizontal edge of the mesocosm
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INDUCTION OF THE BYSTANDER EFFECT IN TROUT
(Mothersill et al, 2006) Non invasive biomarker
approach
Waterborne bystander effect
Partner bystander effect
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PROTEOMIC RESPONSES TO THE BYSTANDER EFFECT
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Micronucleus Assayon blood
D Boreham
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Low Dose Effects using cultured lymphocytes
J of Env. Radioactivity Sept 2002 (Bruce Power)
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Genetic Approach using blood

• Cytogenetic Markers
• Chromosome paint libraries for sentinel species
• Molecular Genetic Markers
• Molecular tests for mini-satellite
  microsatellite, or SNPs

D. Boreham
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The challenge

• How to extrapolate
• From effect to harm
• From harm to risk
• From individual risk to population risk
• From population risk to ecosystem risk
• How to protect biodiversity with an SSD approach
• How to regulate with an acceptance of uncertainty

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Acknowledgements
NSERC, COG, Bruce Power, Canada Research Chairs
Programme, EU ERICA, PROTECT and NOTE and all
the animals, plants and environments that made
me think about this!