Are non-targeted effects important in radiation protection? Carmel Mothersill and Colin Seymour McMaster University Canada

Are non-targeted effects important in radiation protection? Carmel Mothersill and Colin Seymour McMaster University Canada

Non-targeted radiation effects Inter-animal/plant signaling Effects in neighbouring animals now established in rodents, fish And frogs/tadpoles, yeast and aribidopsis Long-term effects on innate immune Response function may occur

Outline What are non-targeted effects of radiation What are the mechanisms? Do they happen in vivo? Do they always happen or are they dose/genotype/phenotype dependent? Can we shut them off? Human in vitro data Mouse in vivo and in vitro data Fish model and data Conclusions

Modern ideas leading to new paradigm Induced repair/response Hormesis IR can promote not just induce carcinogenic processes Non-targeted effects occur Very low doses may be below a response activation threshold and therefore more dangerous

Cancer incidence Old paradigm: The LNT hypothesis is the basis of human radiation protection dose

Old Paradigm: Cytogenetic damage in cells following exposure can indicate risk

Tikvah Alper (1909-1995) Published with us one of the first demonstrations of lethal mutations (delayed cell death) in distant progeny of irradiated cells. Our current work focuses on the examination of these delayed effects in vivo High yields of lethal mutations in somatic mammalian cells that survive ionizing radiation.seymour CB, Mothersill C, Alper T. Int J Radiat Biol Relat Stud Phys Chem Med. 1986 Jul;50(1):167-79.

Lethal mutations/delayed reproductive death Seed cells then irradiate 7-10 days 6-7 PD v Heritable lethal defects manifested as reduced colony-forming efficiencies Control cells CE 2 and CE3 = CE 1 Irradiated cells CE 2 and CE 3 < CE1 Count colonies = CE 1 2. Grow cells to confluence 15-18 PD 3. Grow cells to confluence 30-40PD 1. Pick off individual Colonies and Grow to confluence CE=cloning efficiency PD=population doublings Test CE = CE 2 Test CE = CE 3

Irradiated haemopoietic stem cellderived clones Expected Clonal abnormalities Kadhim et.al., 1992, Nature 355, 738 Kadhim et.al., 1995, IJRB, 67, 287 Harper et.al., 1997, Exp. Hematol., 25, 263 ROS Unexpected Radiation-induced genomic instability

The bystander effect Ionizing radiation, UVA, UVB, ELF-EMF and heavy metals induce affected cell to signal to others. Responses to the signals include apoptosis, micronucleus formation, transformation, mutation, induction of stress and adaptive pathways. Serotonin (5HT), L-type calcium channels (which are 5HT-3 receptors) and Calcium ions known to be involved in signal production Ca 2+ UV photons GJIC connexins 1 o and 2 o response 5HT exosomes ROS/Nitric oxide/cytokines Biogenic amines,tgfb, p53???? response Ca 2+ response Ca 2+ Amplification/ Cascade effects? response Ca 2+

The link between bystander effects and genomic instability twin pillars of the new paradigm Old view- clonal outcome Hit Progeny are all i.e. identical and mutation is passed to all progeny New view-non-clonal, population-determined outcome Hit? Cells continue to be produced with non-clonal changes Progeny cells are non-clonal and give rise to a variety of mutations or die

Published stuff we know! Bystander mechanism perpetuates genomic instabiity Calcium pulse occurs within 30 secs after 5mGy acute dose and up Serotonin binding to cell membrane receptor which is a voltage gated calcium, triggers calcium flux Excitation decay UV photon emission appears to initiate signal production pathway P53 and cytokines involved in transducing response to signals Response depends on genotype and p53 status, signal production independent of these Proteome reveals changes in energy, oxidative stress and structural proteins

Dose response and ROS Relative contribution of Direct and BE cell death ROS stable with increasing dose and persistent over 35 generations BOTTOM LINE: BE major contributor to cell death at high as well as low doses % cells affected 80 70 60 50 40 30 20 10 0 0 Gy 0.5 Gy 5 Gy Dose to progenitors Initial Passage 1 Passage 3 Passage 5 Passage 7

Signal after exposure to ICCM from 5mGy irradiated cells 0.005 Gy medium 0 Gy medium 1.2 1 Fluo 3 / Fura Red 0.8 0.6 0.4 0.2 0 0 8 16 24 32 40 48 56 64 72 80 88 96 104 112 120 128 136 144 152 160 168 176 184 192 time (s) 1.2 1 0.8 0.6 0.4 0.2 0 0 14 28 42 56 70 84 98 112 126 140 154 168 182 196 210 224 238 252 266 280 294 308 322 336 350 364 378 392 Fluo 3 / Fura Red time (s)

Dose Response in vitro 140 120 Surviving fraction 100 80 60 40 No Ca signal Ca signal ICCM Irradiation 20 0 0.01 0.1 1 10 100 1000 10000 Calcium signal and bystander effect only triggered after dose exceeds 2mGy gamma rays Dose (mgy) Z Liu, C Mothersill, F McNeill, SH Byun, C Seymour W Prestwich, A dose threshold for a medium transfer bystander effect in a skin cell line, Radiat. Res.(2006) 166, 19-23

No RIBE in TCC and associated normal tissue ex-vivo explants and in PC3 tumour cell line No BE in reporter cells exposed to medium from TCC explants OR surrounding normal tissue No BE in radioresistant PC3 carcinoma cell line % Cloning efficiency 100 90 80 70 60 50 40 30 20 10 0 bystander Direct Total 0 0.01 0.03 0.05 0.07 0.1 0.3 0.5 1 5 Dose to donors (Gy)

PE PE P53 and TGFb important in response HCT -/- Reporters 120 100 80 60 40 20 0 1 Treatments Direct 0Gy "Direct 0.1Gy" "Direct 0.5Gy" got -/- 0Gy got -/- 0.1Gy got -/- 0.5 Gy got +/+ 0Gy got +/+ 0.1Gy 120 100 HCT +/+ Reporters 80 60 40 20 0 1 Treatments Direct 0Gy Direct 0.1Gy Direct 0.5Gy got -/- 0Gy got -/- 0.1Gy got -/- 0.5Gy got +/+ 0Gy got +/+ 0.1Gy P53 knockouts generate bystander signals but do not respond so the -/- reporters give no BSE even if +/+ medium is applied. +/+ reporters respond even to -/- medium p53 mutated in many tumours; maybe this explains loss of BE response in some tumours and tumour cell lines?

Individual variation in the cytotoxic properties of bystander medium 35 Number of patients 30 25 20 15 10 5 0 0-20 20-40 40-60 60-80 80-100 100-120 % of Control Keratinocyte P.E. Human CBA mouse C57 mouse

Apoptosis data for mouse strains CBA is a radioresistant mouse C57 is a radiosensitive mouse

Calcium ratios in reporters receiving signals from control or 0.5Gy TBI CBA/Ca and C57BL/6 mice Medium from unirradiated tissues from both strains Calcium pulse associated with apoptosis prone C57 strain only CBA/Ca 1 0.9 CBA H 0Gy ITCM C57BL/6 0.8 CBA H 0.5Gy ITCM 1.6 0.7 1.4 C57Bl 6 0Gy ITCM C57Bl 6 0.5Gy ITCM 0.6 1.2 Fluo 3 / Fura Red 0.5 0.4 0.3 Fluo 3 / Fura Red 1 0.8 0.6 0.2 0.4 0.1 0.2 0 0 8 16 24 32 40 48 56 64 72 80 88 96 104 112 120 128 136 144 152 160 168 176 184 192 0 0 8 16 24 32 40 48 56 64 72 80 88 96 104 112 120 128 136 144 152 160 168 176 184 192 time (sec) time (sec)

Measuring bystander response to radiation in vivo adapted from Mothersill et al 2006 Irradiated fish Partner fish Irradiate or sham irradiate fish, allow to swim with unexposed partner for 2hrs Unexposed fish introduced into water from irradiated or sham fish After 2hrs. Dissect tissues Do proteomics Explant pieces taken from skin, fin, gill, spleen and kidney Do tissue culture Culture of explants for 2 days Grow up culture examine explant outgrowth do immunocytochemistry Harvest culture medium for calcium flux, ELISA and clonogenic assays Add medium to unirradiated clonogenic cell line determine surviving fraction by counting colonies after 10 days

Molecular size (kda) Molecular size (kda) Gill proteomics in two species Rainbow trout Medaka 76.0 66.2 Hemopexinlike protein Pyruvate dehydrogenase (PDH) 76.0 66.2 Complement component C3 43.0 43.0 Chromosome 5 SCAF protein Warm temperature acclimation related 65 kda protein (Wap65) 36.0 31.0 21.5 Annexin II Chromosome 1 SCAF protein RhoGDP dissociation inhibitor (RhoGDI) 36.0 31.0 21.5 Annexin A4 Annexin A1 Creatine kinase Lactate dehydrogenase Direct irradiation 17.5 17.5 Bystander effect 4.5 5.1 5.4 5.6 6.0 7.0 8.5 Isoelectric point (ph units) Trout bystander proteome protective Smith RW, et al 2007 Evidence for a protective bystander response in rainbow trout gills exposed to x-irradiation. Proteomics. 7(22):4171-80. Proteomic changes in the gills of DNA repair proficient and DNA repair deficient medaka following exposure to direct irradiation and to X-ray induced bystander signals. R Smith et al BBA 2011 Feb;1814(2):290-8. 4.5 5.15.4 5.6 6.0 7.0 8.5 Isoelectric point (ph units) Both Medaka bystander proteome may indicate protective and adaptive response

Low dose effects are different Adaptive effects not only strict radiobiological adaptive response but long-term evolutionary acclimation Stuart and Boreham labs Hormetic effects low dose of radiation is beneficial leading to non-linear dose responses for a variety of endpoints Calabrese reviews, Boreham lab Homeostatic effects- systems accommodate and adjust to low dose induced perturbations Seymour, Mothersill proteomics data, Tapio Lab Genetic and environmental factors more important than dose Oughton/Salbu, Mosse/Marozik, Ullrich, Wright, many others

Danger of taking a framework from one field in radiobiology and applying it elsewhere High dose issues Cellular Response Low dose issues Cancer Whole Organism Population Ecosystem Transgenerational Micro-environment Therapeutic Advantage Consistency Individual

Chronic v Acute effects Not simply related complicated by low dose responses such as adaptation Depend on assimilation which varies between individuals and tissues Depends on the isotope and its chemical function, speciation and abundance of competing elements DDREF (relating acute to chronic outcome) of 2 is simplistic

Chronic Medaka low LET data suggests protective/adaptive responses Direct x-ray Bystander to direct x-ray

Example of chronic high LET data Fed 226 Ra for 1 month Fed 226 Ra for 6 months ID Activity (Bq kg -1 wet) Annual dose (mgy y -1 ) Approx 50mBq per Bq assimilated in 6 months Control Fish 36 ± 22 0,9 ± 0,5 Control Fish 28 ± 28 0,7 ± 0,7 Fed 10 mbq g -1 39 ± 15 1,0 ± 0,7 Fed 10 mbq g -1 23 ± 8 0,6 ± 0,2 Fed 100 mbq g -1 11 ± 12 0,2 ± 0,2 Fed 100 mbq g -1 9 ± 12 0,2 ± 0,3 Fed 1 Bq g -1 26 ± 11 0,7 ± 0,3 Fed 1 Bq g -1 33 ± 13 0,8 ± 0,3 Fed 10 Bq g -1 100 ± 18 2,5 ± 0,4 Fed 10 Bq g -1 124 ± 16 3,0 ± 0,4

Comparison of 6 and 18 months showing loss of accumulated Ra-226 at 18 months

So there is non-linearity so what? Need to re-consider linear dose response relationships based on dose (target theory) Need to find a way to incorporate adaptive and hormetic responses into risk models Need to do mechanistic experiments to try to see if these effects are relevant in real life situations (i.e. in the field, in epi studies, in radiotherapy) Need to get away from dose based risk assessment and move to response based methods.

What do bystander effects do to radiation protection? Dissociate Dose from effect Effect from harm Harm from risk Enables the concept of a zone of uncertainty where outcome can be assessed relative to the context in which the dose is delivered

Proposed dose response relationship for radiation-induced effects Yellow arrows indicate mechanistic break points where new, more appropriate, response pathways emerge Zone of linearity New coping mechanism Zone of uncertainty Dose tolerance saturation 0.5Gy Natural background

Factors influencing outcome in the zone of uncertainty Existing stress Lifestyle Zone of uncertainty Genetic background Innate immune response Dose Natural background

Possible model for expression of bystander effects in biota With intervention points for dectection or protective strategies Radiation or other environmental stressor 5HT Ca 2+ 5HT No signal Targeted cell 1 Genotype and environment dependent 1-4 = potential intervention points signal Genomic instability phenotype Anti-apoptotic proteins Recipient cell 3 4 ROS Chance of Life ± mutation Ca 2+ Genotype and environment. dependent 2 Chance of death pro-apoptotic proteins Apoptosis phenotype

The problems posed by the new radiobiology Effects of radiation not determined by DSB s A mechanistic difference between high and low dose effects leading to a threshold or breakpoint Interactions between radiation and chemicals which can both induce delayed effects Cell communication related amplification of dose Cellular response as the key determinant of effect Genetic background as a key determinant of response Assessment of harm at multiple levels of organisation How do we quantify the extent and relevance of epigenetic risk factors which operate at the population level? The ability of organisms to adapt

Summary points to consider Horizontal and vertical transmission of radiation effects mean the target is not confined to the cell or organism receiving the dose Need to consider the hierarchical level at which damage (effect/response) is being assessed or is of concern May need to define new critical endpoints including emergent properties of systems (both tissue and ecosystems) Need to be careful about interpretation of effects data at levels lower than the individual organism

Conclusions At low doses and following chronic exposure, LNTH does not apply NTE mean that the system response is as important as the dose Very long term effects may be transmitted and become fixed in the transgenome Probabilistic approaches to risk/benefit assessment are needed

What is science? Science alone of all the subjects contains within itself the lesson of the danger of belief in the infallibility of the greatest teachers of the preceding generation. FEYNMAN

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