The Role of Excitation Events in radiobiology

Carmel Mothersill and Colin Seymour McMaster University 1

Background • Non-targeted effects predominate after low doses of radiation • These include bystander effects, delayed lethal mutations and delayed

genomic instability also • Low dose hyper-radiosensitivity/induced radioresistance (HRS/IRR) and

adaptive/hormetic responses • Characterised by the appearance of effects in parts of the system which

did not receive a direct dose (energy deposition in Joules/Kg) • Mechanisms of generation and communication unclear • Seem to coordinate system level responses to individual level damage • Potentially very important novel targets for therapy and radiation

protection

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Mechanisms for investigating RIBE communication in vitro

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Microbeam based Insert well based Medium transfer based

All assume a signal contained in liquid medium which induces response in recipients

(can also show inter-organism signaling in rodents, fish, tadpoles and plants)

Lots of data BUT…….. • Little thought given to how ionising radiation leads to

bystander signal generation • Ionising radiation involves energy deposition. Leads to

ionisation and excitation. • Excitation important after low doses and low radiation

energy exposures – seldom considered. • Possibility of a physical component to the actual signal? • First suggested by Irma Mosse in 2006 because melanin

was found to prevent the bystander effect (Marozik et al )

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Mothersill C, Moran G, McNeill F, et al. A Role for Bioelectric Effects in the Induction of Bystander Signals by Ionizing Radiation? Dose-Response. 2007;5(3):214-229. doi:10.2203/dose-response.06-011.Mothersill.

Mothersill, C., Smith, R. W., Fazzari, J., McNeill, F., Prestwich, W., & Seymour, C. B. (2012). Evidence for a physical component to the radiation-induced bystander effect?. International journal of radiation biology, 88(8), 583-591.

Assessing photon emission and bystander cell survival

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1. Irradiation of HaCaT cells with tritium (3H) while reporter cells sit

<1.5 cm superior to irradiated culture for 24 hours

2. Incubate reporter cells at 37˚C, 5% CO2 for an additional 7-8 days

3. Assess clonogenic survival using assay developed by Puck and Marcus (1956)

Photon Quantification Bystander cell survival

Interference filter centered at 340 ± 5 nm

Strong relationship between cell death and photon flux

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Effect is abolished following use of an UV absorption filter

9 Without filter With filter

Effect magnitude can be modulated by photosensitizers and photoprotectors

10 Photosensitizer Photoprotector

Response to UV signaling is dependent upon p53 status

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(p53 Mutated but

part functionall )

(p53 mutated)

(p53 Mutated and no function)

(p53 wild type) (p53 wild type)

(p53 null)

UV does not cause a reduction in survival if p53 is non functional or absent

Around this time….. • Reports in the literature claimed the

bystander “factor” in medium was exosomes! • Papers by Al-Mayah et al 2012, 2014 and

Kumar Jella et al in 2013 independently found this

• So what about our electromagnetic signal?

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140 000x

160 000x

Scale bar: 100 nm

UV signaling and soluble signaling factors • Objective: to investigate whether the UV bystander signal and the

soluble factor bystander signal are mutually exclusive mechanisms or are related

• Exosomes:

– Membrane-derived extracellular vesicles – 50-150 nm in diameter – Present in various biological fluids – Carry diverse array of cargo (RNA, protein receptors, ligands, DNA) – Mediator of intercellular signaling

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Exosome Work: experimental methods

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UV-exposed bystander cells

Tritium-irradiated cells

Harvest and filter

UV-exposed medium

Transfer UV-exposed medium directly onto clonogenic reporter cells

Ultracentrifuge UV-exposed medium for

90 minutes at 4oC

Transfer exosome fraction onto JC-1 incubated cells for detection

of mitochondrial membrane depolarization

Transfer exosome fraction onto clonogenic reporter

cells

Resuspend pellet containing

exosomes in PBS

Incubate for 24 hours

Cell death is induced following exosome transfer to bystanders

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Bystanders receiving UV-ICCM Bystanders receiving exosomes extracted from UV-ICCM

p=0.493

No significant difference between bystander medium or exosomes extracted fro the medium

Mitochondrial membrane depolarization is induced following exosome transfer to bystander cells

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Exosomes from UV-

ICCM

Exosomes from control

CCM

Quantification of exosomes in control and UV conditioned medium

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+ control cell lysate

UV-derived exosomes

UV-exposed cell lysate

Control exosomes

Control cell lysate

Practical relevance of the UV-mediated bystander effect

• Cellular communication can be accomplished without direct contact or exchange of biological fluids

• Can estimate the distribution of bystander responses using knowledge of UV’s optical properties in tissue

• Introduces additional considerations that must be taken when balancing risks and benefits associated with prescribing procedures using radiation in concert with drugs that may be photosensitizing or photoprotective

• Suggests new potential targets for radiation protection and the possibility of a unique physical radiation signature

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Biophotons emitted from excitation decay In directly irradiated cells

Biophotons trigger exosome release in cells that receive photon energy

Exosomes contain information leading to system level response

Exosomes delivered to other cells in the system

The future • Paper with unifying theory published last

month on line in Plos 1 • Now we need to find out what is in the

exosomes and what the impacts of dose, dose rate and radiation quality are on the biophoton emission and exosome content

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Acknowledgements • Michelle Le, (did the experiments) Dr. Fiona McNeill, Dr. Andrew Rainbow • Dr. Cristian Fernandez-Palomo (University of Bern; contributions to exosome work) • Dr. Marta Princz (McMaster Biointerfaces Institute) • Marcia West (McMaster HSC Electron Microscopy Centre) • Dr. Kevin Diamond (spectrometry) • Dr. Hayley Furlong & Dr. Warren Foster (western blot protocol) • Dr. Robert Bristow & Ramya Kumareswaren (UofT; HCT116 cells) • Lisa Mutschelknaus (Helmholtz Institute; exosome profiling)

• Contributor institution: McMaster University unless otherwise stated

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Funding

QUESTIONS?