United Nations Scientific Committee on the Effects of Atomic Radiation

S O U R C E S , E F F E C T S A N D R I S K S O F I O N I Z I N G R A D I AT I O N

UNSCEAR 2020/2021 Report

Volume III
SCIENTIFIC ANNEX C:
Biological mechanisms relevant for the inference of cancer risks
from low-dose and low-dose-rate radiation

SOURCES, EFFECTS AND RISKSOF IONIZING RADIATION
United Nations Scientific Committee on the
Effects of Atomic Radiation
UNSCEAR 2020/2021
Report to the General Assembly,
with Scientific Annexes
VOLUME III
Scientific Annex C

UNITED NATIONSNew York, 2021

NOTE
The report of the Committee without its annexes appears as Official Records of theGeneral Assembly, Seventy-sixth Session, Supplement No. 46 (A/76/46).
The designations employed and the presentation of material in this publication do not imply
the expression of any opinion whatsoever on the part of the Secretariat of the United Nations
con-cerning the legal status of any country, territory, city or area, or of its authorities, orconcerning the delimitation of its frontiers or boundaries.
The country names used in this document are, in most cases, those that were in use at the time thedata were collected or the text prepared. In other cases, however, the names have been updated,
where this was possible and appropriate, to reflect political changes.

UNITED NATIONS PUBLICATION
Sales No. E.22.IX.3
ISBN: 978-92-1-139208-1
eISBN: 978-92-1-001005-4© United Nations, December 2021. All rights reserved, worldwide.
This publication has not been formally edited.
Information on uniform resource locators and links to Internet sites contained in the present
publication are provided for the convenience of the reader and are correct at the time of issue.
The United Nations takes no responsibility for the continued accuracy of that information or forthe content of any external website.

Corrigendum to Sales No. E.22.IX.3

16 September 2022

Sources, Effects and Risks of Ionizing Radiation: United
Nations Scientific Committee on the Effects of Atomic
Radiation 2020/2021 Report to the General Assembly, with
Scientific Annexes, Volume III – Scientific Annex C
Corrigendum
Glossary, page 226
Low dose rate
The definition should read
The Committee has defined “low dose rate” as 0.1 mGy per minute or less, averaged
over about one hour, for radiations such as external X-rays and gamma rays.

V.22-10461 (E)*2210461*

ANNEX C
BIOLOGICAL MECHANISMS RELEVANT FOR THE
INFERENCE OF CANCER RISKS FROM LOW-DOSE
AND LOW-DOSE-RATE RADIATIONCONTENTS
LIST OF ABBREVIATIONS......................................................................................................................................... 3
I.

INTRODUCTION................................................................................................................................................. 5II.

PREVIOUS FINDINGS OF THE COMMITTEE UP TO 2012 AND THE BROADER CONTEXT ............... 7
A. Previous findings of the Committee................................................................................................... 7
B.

Broader context on current understanding of mechanisms of carcinogenesis.................. 10

III. BIOLOGICAL MECHANISMS RELEVANT TO DOSE–RESPONSE RELATIONSHIPS........................... 12
A. DNA damage .......................................................................................................................................... 14
B.

DNA damage signalling, chromatin remodelling and epigenetics ........................................ 17

C. Effects on other signal transduction pathways ............................................................................ 27
D. Gene and protein expression ............................................................................................................ 30
E.

DNA repair and effects on somatic cells ......................................................................................... 53

F.

Genomic instability, bystander effects, damage/effects on non-nuclear
cellular components, adaptive response and hyper-radiosensitivity .................................... 64

G. Stem cells and target cell populations for radiation carcinogenesis ..................................... 81
H. Effects at the whole organism level ................................................................................................. 90
IV. INTEGRATION OF DATA AT DIFFERENT LEVELS OF ORGANIZATION AND MODELLING OFCANCER MECHANISMS ...................................................................................................................................... 138
A. Biologically-based mechanistic models of carcinogenesis ..................................................... 138
B.

Bronchial airway deposition of inhaled radon progeny and cellular and tissue
effects ..................................................................................................................................................... 151

C. Intercellular communication ........................................................................................................... 154

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V. IMPLICATIONS FOR CANCER RISK INFERENCE..................................................................................... 155
A. DNA damage ........................................................................................................................................ 155
B.

DNA damage signalling, chromatin remodelling and epigenetics ...................................... 156

C. Effects on signal transduction pathways ...................................................................................... 157
D. Gene and protein expression........................................................................................................... 157
E.

DNA repair and effects on somatic cells ....................................................................................... 158

F.

Genomic instability, bystander effects, damage/effects on non-nuclear
cellular components, adaptive response and hyper-radiosensitivity .................................. 159

G. Stem cells and target cell populations for radiation carcinogenesis.................................... 160
H. Effects at the whole organism level ............................................................................................... 161
I.

Integration of data at different levels of organization and modelling ofcancer mechanisms ............................................................................................................................ 163

VI. DIRECTIONS FOR FUTURE RESEARCH .................................................................................................... 163
VII. CONCLUSIONS .............................................................................................................................................. 166
ACKNOWLEDGEMENTS...................................................................................................................................... 170
Members of the Expert Group ................................................................................................................. 170
APPENDIX A. PRINCIPLES AND CRITERIA FOR ENSURING THE QUALITY OF THE COMMITTEE’S
REVIEWS OF EXPERIMENTAL STUDIES OF RADIATION EXPOSURE ........................................................ 171
REFERENCES .......................................................................................................................................................... 185
GLOSSARY.............................................................................................................................................................. 221
LIST OF GENES AND PROTEINS ........................................................................................................................ 231

ANNEX C: BIOLOGICAL MECHANISMS RELEVANT FOR THE INFERENCE OF CANCER RISKS ...

LIST OF ABBREVIATIONS
BrdU

Bromodeoxyuridine

CpG

CpG dinucleotide, cytosine-phosphate-guanineCT

Computed tomography

DNA

Deoxyribonucleic acid

DNMTDNA methyltransferase

DoReMiLow Dose Research towards Multidisciplinary Integration[European Commission funded project]

DSB

Double-strand break

dsDNA

Double-stranded DNA

ELISA

Enzyme-linked immunosorbent assay

EMT

Epithelial-to-mesenchymal transition

G0

Quiescent phase during cell-division cycle

G1/S

Stage in the cell cycle at transition point between G1 and S stage

G2/M

Transition between G2 and M phases of the cell cycle

GSEA

Gene-set-enrichment-analysis

hCG

Human celera genome

HBRA

High-level natural background radiation area

HRR

Homologous recombination repair

HZE

High atomic-number elementkb

Kilo-base pair

ICR

Institute of Cancer Research

LET

Linear energy transfer

LINAC

Linear accelerator

lncRNALong non-coding RNA

LOH

Loss of heterozygosity

miRNA

MicroRNAmRNA

Messenger RNA

NBS

Nijmegen Breakage Syndrome caused by NBS1 (nibrin) genemutation

NHEJ

Non-homologous end joining

NHF

Normal human fibroblast

NK cellNatural killer cell

NKT cellNatural killer T-cell

PCR

Polymerase chain reaction

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UNSCEAR 2020/2021 REPORT

phospho

Phosphorylated

Ku (e.g. Ku70, Ku80)

ATP-dependent DNA helicase II subunit 1

PMA

Phorbol-12-myristate-13-acetate

PRISMA

Preferred Reporting Items for Systematic Reviews and MetaAnalyses

PTC

Papillary thyroid cancer

RBE

Relative biological effectiveness

RIDDLE syndrome

Radiosensitivity, ImmunoDeficiency Dysmorphic features andLEarning difficulties syndrome

RIFs

Radiation-induced fociRNA

Ribonucleic acid

RNS

Reactive nitrogen species

ROS

Reactive oxygen species

RT-PCR

Reverse transcription-polymerase chain reaction

SCID

Severe combined immunodeficiency

siRNA

Small interfering RNA

ssDNA

Single-stranded DNA

sSPE

Simulated solar particles event

Tconv

Conventional T-cells

TCR

T-cell receptor

TF

Transcription factor

Th (e.g. Th1, Th2, etc.)

T-helper cells (1, 2, etc.)

TRECs

T-cell receptor excision circles

Treg

Regulatory T-cells

TSCE

Two-stage clonal expansion

TUNEL

Terminal deoxynucleotidyl transferase assay

WLM

Working level monthANNEX C: BIOLOGICAL MECHANISMS RELEVANT FOR THE INFERENCE OF CANCER RISKS ...

5

I. INTRODUCTION
1.
In 1955, the General Assembly of the United Nations established the Scientific Committee on the
Effects of Atomic Radiation (UNSCEAR) to assess and report levels and effects of exposure to ionizing
radiation. The Scientific Committee issues detailed reports and reviews which are widely regarded as
authoritative. Governments and organizations throughout the world rely on the Committee’s estimates as
the scientific basis for evaluating radiation risk and for establishing protective measures.
2.
Since the establishment of the Scientific Committee, its mandate has been to undertake broad
estimates of the sources of ionizing radiation and its effects on human health and the environment. In
1973, the mandate expanded to include scientific estimates of radiation risk. These assessments of the
Scientific Committee provide the scientific foundation used, inter alia, by the relevant agencies of theUnited Nations system in formulating international standards for the protection of the general public andworkers against ionizing radiation; those standards, in turn, are linked to important legal and regulatory
instruments. In its UNSCEAR 2012 Report, the Committee considered the attribution of health effects
and the inference of risks from radiation exposure, as well as on the uncertainties in risk estimates [U10].
The understanding of the biological mechanisms by which radiation-induced effects such as cancer may
occur is a relevant element for the inference of radiation risk. This annex is intended to synthesize thecurrent knowledge on biological mechanisms of radiation actions at doses mostly in the low to moderate
range relevant for cancer risk inference. It is emphasized that this is not a report on radiation effects; inparticular, it is not a report on cancers that can be attributed to radiation exposure situations.
3.
The Committee’s strategic objectives adopted in 2019 included giving priority to “Health andenvironmental effects, and inferred risks”. This included a commitment that “The Committee will collate
and evaluate information such as epidemiological data, in particular for the low dose range and for longterm low-dose-rate exposure and analyse data from research on mechanisms of biological reactions atsuch exposures. The Committee will pursue innovative approaches to integrate radiobiological andepidemiological research, to enhance the understanding of (a) radiation-induced health effects andassociated inferred risks related to the induction of cancer, non-cancer effects, and hereditary disease;and (b) environmental effects. Attention will be given to individual-related factors affecting the incidenceof such effects, including age, sex, lifestyle, genetic or familial predisposition and health status.”
4.
This evaluation has been undertaken to fulfil a commitment made by the Committee in the
UNSCEAR 2012 White Paper, “Biological mechanisms of radiation actions at low doses” to further
review the biological mechanisms that influence the health risks from low-dose radiation exposure [U9].
5.
A number of relevant evaluations have been carried out by the Committee over many years. These
include annex B of the UNSCEAR 1986 Report [U2], annexes E and F of the UNSCEAR 1993 Report[U3], annex B of the UNSCEAR 1994 Report [U4], annexes F, G and H of the UNSCEAR 2000 Report[U5], annex of the UNSCEAR 2001 Report [U6], and annexes C and D of the UNSCEAR 2006 Report[U7]. Additionally, the Committee has provided a summary of low-dose radiation effects on health in the
UNSCEAR 2010 Report [U8]. The rationale for the judgements on the mechanisms relevant to the healtheffects of low-dose and dose-rate radiation exposures were summarized in the UNSCEAR 2012 White
Paper [U9] that also presents a brief review of the post-2006 literature on this topic.
6.
This evaluation is intended to synthesize the current knowledge on biological mechanisms ofradiation actions at low doses (i.e. those less than or equal to 100 milli-Gray (mGy) low-linear energy
transfer (LET) exposure, or less than or equal to one track traversal per cell of high-LET exposure) and6

UNSCEAR 2020/2021 REPORT

low dose rates (i.e. those of 0.1 mGy/min or less low-LET exposure, or no more than one high-LET track
traversal per cell per hour), assess their implications for understanding the processes of cancer
development after exposure to ionizing radiation, and to explore the implications for dose–response
relationships of radiation-induced cancers. By comparison, the Committee takes moderate doses to be
those of 0.1 to about 1 Gy for low-LET radiations [U10]. This annex addresses the following questions:(a) For which biological mechanisms and pathways is there evidence that indicates that they canaffect the frequency of cancers following exposure to ionizing radiation, including at low doses andlow dose rates? What are the differences in utilization and/or activation of these pathways andmechanisms at low doses compared with moderate doses? What evidence is available on the form of
the dose–response relationships for these mechanisms?
(b) Considering the mechanisms identified above, can any conclusions be drawn as to their overall
influence on the dose–response relationships for cancers associated with radiation exposure at low
doses compared with moderate doses?
(c) Are there ways to link information on the biological processes and mechanisms found to be
relevant to human cancer and existing epidemiological data on incidence of disease in exposed
populations?
(d) Is there evidence for tissue-specific variation in the mechanisms of response to ionizing radiationthat relate to the differing sensitivity of tissues to radiation-induced cancers?
(e) Are the mechanisms that can be associated with cancer development similar following low- andhigh-LET exposures?
7.
To address these questions, the evaluation focuses on particular topic areas that include theestablished mechanisms of targeted DNA damage/mutation and additionally a range of mechanisms that
may contribute to radiation carcinogenesis at low doses and low dose rates. The areas are as follows:(a) DNA damage;(b) DNA damage signalling, chromatin remodelling and epigenetics;(c) Effects on other signal transduction pathways;(d) Gene and protein expression;(e) DNA repair and effects on somatic cells;(f) Genomic instability, bystander effects, damage/effects on non-nuclear cellular components,
adaptive response and hyper-radiosensitivity;(g) Stem cells and target cell populations for radiation carcinogenesis;(h) Effects at the whole organism level.
8.
Relevant literature was identified using searches of PubMed, current review articles, from theknowledge of members of the Expert Group, and suggestions from delegations to the Committee. The
evaluation considers that peer-reviewed primary scientific papers provide the best evidence on which tobase conclusions. A limited number of review articles and reports were also taken into account. Given
that the most recent published substantial work of the Committee considered the literature up to 2006,
this evaluation draws mainly on publications from 2006 to 2020; some earlier publications were included
which had not previously been considered by the Committee or are of particular relevance to themechanistic arguments presented. Papers were evaluated against the following criteria:

ANNEX C: BIOLOGICAL MECHANISMS RELEVANT FOR THE INFERENCE OF CANCER RISKS ...

7(a) Is this an original publication (and not a review, editorial, commentary or correspondence)?
(b) Is there experimental evidence indicating that the endpoints described can be linked directly or
indirectly to radiation carcinogenesis?
(c) Is the experimental design adequate and free of substantial flaws, including in dosimetry?
(d) Are the results statistically sound?
(e) Have the results been replicated or otherwise substantiated?
9.
In gathering and reviewing the literature, the focus has been on reports that consider low-dose andlow-dose-rate effects. However, for context, and particularly for consideration of implications forapproaches to risk extrapolation, it is necessary to consider studies that use higher doses. Where apparent,
qualitative and/or qualitative differences in response to low and moderate doses are taken into account.

II. PREVIOUS FINDINGS OF THE COMMITTEE UP TO 2012 AND
THE BROADER CONTEXT
A. Previous findings of the Committee
10. In its summary of low-dose radiation effects on health in the UNSCEAR 2010 Report [U8], theCommittee noted that the health concerns at low doses and low dose rates were (a) the risk of cancer inseveral tissues (for which there was substantial evidence of an increased incidence of cancer at moderate
and high levels of exposure) and (b) the risk of heritable effects (which are assumed to occur in humanpopulations, although they have not been observed directly). In terms of the possible mechanisms
underlying these health effects, the Committee noted the importance placed upon mutational
mechanisms. The paradigm of radiation-induced damage to DNA, which may be converted to a DNA
sequence mutation, provided important underpinning of the use of a linear no-threshold model for
estimating risk of disease at low doses and low dose rates. The rationale for the judgements on themechanisms relevant to the health effects of low-dose and low-dose-rate radiation exposures made inprevious evaluations of the Committee were summarized in the UNSCEAR 2012 White Paper [U9] toguide the Committee’s future programme of work.
11. In addition to direct mutational mechanisms, the Committee has considered “non-targeted” effectsof radiation, that is, cellular action of radiation exposure that occur in cells neighbouring the cell that was
actually “hit”, or in the descendents of “hit” cells (UNSCEAR 2006 Report, annex C [U7]). The
judgement of the Committee in its 2006 report was that non-targeted and delayed effects of radiation may
be associated with radiation-induced disease but no evidence for disease causation had been found. It wasnoted that any contribution of non-targeted and delayed effects would be implicitly incorporated in theestimates of risk of radiation-induced health effects derived epidemiologically.
12. The Committee also provided an assessment of the effects of ionizing radiation on the immune
system (UNSCEAR 2006 Report, annex D [U7]). The immune system could act to modify the cancer
risk if radiation exposure served to enhance or diminish the capacity of the body to mount an immune
response against developing cancers, whatever their cause. While many studies were examined, it
remained impossible to judge clearly whether the effects of radiation at low doses on the immune system
served to stimulate or suppress or have no effect on immune responses.

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13. In developing the UNSCEAR 2012 White Paper [U9], the Committee additionally undertook alimited literature review to identify any major advances in understanding the biological mechanisms
acting at low doses. The Committee concluded that:(a) Since 2006, significantly more data had become available on the biological consequences of lowdose radiation exposure and non-targeted effects. The White Paper [U9] considered advances in theareas of genomic instability, bystander and abscopal effects, adaptive response, reactive oxygen
metabolism and mitochondrial function, DNA sequence analysis and genetic polymorphisms, gene andprotein expression, cellular interactions and tissue-level phenomena and systems biology approaches;(b) While mechanistic understanding of non-targeted effects was improving, many studies remained
observational;(c) There were reports of differential gene and protein expression responses at high and low
radiation doses and dose rates, but these reports were mixed in outcome, and there was little of thecoherence required of robust data that could be used confidently for risk assessment;(d) There was at the time no indication of a causal association of non-targeted phenomena withradiation-related disease and indeed, some may not operate at low doses in vivo;(e) A systems-level framework should provide a useful guide for future integration of mechanistic
data into risk estimation methods;(f) In the case of radiation-induced perturbation of immune function or inflammatory reactions,
there was a clearer association with disease, but the impact of radiation was less well understood.
14. The Committee agreed to: (a) continue to encourage research into the mechanistic understandingof low-dose radiation action that may contribute to improved understanding of disease risk in humans
and into the factors that can modulate risk, and (b) consider developing further biologically-based risk
models and the systems-level framework to integrate mechanistic data into a risk-assessment framework.
The overall schema for radiation carcinogenesis proposed in the UNSCEAR 2012 White Paper [U9] isreproduced in figure I. In this schema, carcinogenesis progresses through a series of steps, or key events
that persist in the target cell population (caused by DNA mutation, epigenetic alteration or other process),that convert a normal cell into a fully malignant cancer cell; this central path can be modified by a range
of influencing factors including signalling from the surrounding micro-environmental cells, rates of cell
death, immune surveillance and immunoediting of cancer cells. This schema and the general approach issimilar to the use of adverse outcome pathways in toxicology (see [L17, O7]). The use of adverse outcome
pathways is increasingly advocated for improving the assessment of the risks of radiation health effects[C12, P23], and recently the National Council on Radiation Protection and Measurements has publisheda report on the topic and more generally the integration of radiobiological and epidemiological data forradiation risk assessment [N11]. Thus, there is growing consensus internationally that such approaches
are promising.
15. Additionally, to the above considerations, the Committee notes that there are reports of effects ofionizing radiation on other cellular processes and systems. For example, effects of radiation on cell
membrane systems have been reported. High dose exposure effects have been summarized by Huber et
al. [H40], notably effects on ion channels, with effects on membrane permeability associated withthymocyte apoptosis also reported [P2], along with similar permeability and integrity effects in yeast[C5]. Damage to membranes as assessed by levels of lipid peroxidation has also been suggested to becaused by low dose occupational exposures [D21]. Further, the Committee is aware of effects on otherbio-molecules, e.g. RNA and proteins that are not considered in detail here as most information relates
to high dose exposures.

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9

Figure I. The systems view of radiation carcinogenesis modified from [U9]The systems view takes account of the tissue context in which target stem and early progenitor cells reside, andof intercellular signalling and bystander signalling of responses to radiation exposure between cells. DNA
damage may be repaired or converted to mutations; additionally, radiation may affect gene expression, protein
modification and epigenetic status. Cells may die through apoptosis or other pathways, enter terminal
differentiation or senescence (or otherwise be selected against – unshaded, crossed cells) thereby removing
them from cancer-development pathways. Radiation may affect these processes at any stage of cancer
development. The induced alterations to genome, epigenome, transcriptome and proteome together affectcellular differentiation (phenotype) – some of these altered states of differentiation may lead to cells having
growth or survival advantage and thus are linked to cancer development. Genomic instability may be anotheroutcome. Cells with altered phenotype may be subject to detection and killing by the immune system(immunoediting). Effects of radiation on all the processes noted above have been observed; however, it is only
mutations in DNA sequences sustaining radiation damage that have an established role in radiation
carcinogenesis. The availability of robust quantitative data on radiation-induced changes associated with diseasedevelopment will provide the way to integrate mechanistic findings into risk estimation. Superimposed onto thesomatic pathway(s) of cancer development are influences from the individual’s inherited genome (e.g. diseasesusceptibilities, variations in target/stem cell numbers), history of exposure to other environmental agents(e.g. the known interaction between tobacco smoke and radon in lung carcinogenesis) and developmental/agerelated factors (e.g. stem-cell development and numbers, life expectancy). Large grey cells – target cell and its
descendents, large white cells – cells removed due to apoptosis, immuno-editing or other process, smaller oval
cells – surrounding tissue cells, two-way arrow – bystander signalling and inter-cellular communication,”Y” shaped symbols – representative of immune responses, including both humoral and cellular immunity.

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B. Broader context on current understanding of mechanisms ofcarcinogenesis
16. Cancer is prevalent in the population. Current estimates indicate that in the United Kingdom, whichis representative of Western European and North American populations, some 50 per cent of the
population will develop cancer during life and around 30 per cent will die from cancer [C2]. About halfof cancers are diagnosed in those over 70 years of age, and cancer incidence is highest in those over
85 years of age [C2]. There is considerable global variation in cancer incidence and mortality and thisinformation is collated by the International Agency for Research on Cancer [I1], global cancer incidence
rates are highest in the 75+ year age group. Therefore, in general, cancer is a disease of older ages, though
there are cases of cancer and leukaemia among children and young adults. The observation that humancancers are more common later in life is interpreted as suggesting that multiple events have to accumulate
in cells over a long latency period before they become fully cancerous. Cancers are also multifactorial inorigin with contributions from “spontaneous” events in DNA replication, exposures to occupational andenvironmental carcinogens, and lifestyle factors such as diet, exercise levels, alcohol consumption andtobacco consumption having an impact.
17. Cancer, which we use here to include both solid cancers and leukaemias, can be defined as theuncontrolled proliferation of abnormal cells in a body tissue; these abnormal cells may spread to otherparts of the body to form metastases. Research efforts over decades have yielded a good understandingof the characteristics of cancers and the differences between normal cells and cancer cells. These are bestsummarized in the hallmarks of cancer as defined by Hanahan and Weinberg [H9, H10]. The hallmarks(figure II) are defined as the biological capabilities acquired through the multistep development ofcancers in humans. The original hallmark capabilities [H9] that are distinctive for cancers are:(a) Sustained proliferative signalling;(b) Evading growth suppressors;(c) Resisting cell death;(d) Enabling replicative immortality;(e) Inducing angiogenesis;(f) Activating invasion and metastasis.
18. These hallmarks were extended in the 2011 publication [H10] to include two “enabling”
characteristics: (a) genome instability and mutation, and (b) tumour-promoting inflammation; and two“emerging” hallmarks: (i) deregulating cellular energetics and (ii) avoiding immune destruction.
19. Not all malignancies necessarily have all hallmarks; for example, angiogenesis is not acharacteristic of leukaemia. It is generally the case that fewer alterations are required for leukaemogenesis
than for the development of solid cancers. This is reflected in the relatively short latent periods between
exposure to radiation and excess leukaemia presentation in the survivors of the Japanese atomic
bombings, for example.

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Figure II. The hallmarks of cancer: a framework summarizing the capabilities of cancer cells and themechanisms contributing to carcinogenesis (adapted from [H10])

20. Understanding the underlying drivers of the acquisition of hallmarks has changed over the years asa widening range of contributory processes have been discovered [H11]. Clearly though, cancer cells
acquire specific phenotypic traits, as defined by the hallmarks; as such, cancer can be viewed as a disease
where the normal patterns of differentiation in specific tissues are altered (or reprogrammed) allowing
cancer cells to escape the normal tight regulation of proliferation in tissues.
21. The acquisition of multiple biological capabilities as set out in the hallmarks depends on theaccumulation of changes in several pathways. There is a long-held understanding that these changes canbe caused by somatic mutation of the genomes of cancer cells. Recent large DNA sequencing studies
bear this out. For example, Martincorena et al. [M19] have analysed exome sequences for over 7,500
cancer cases representing 29 different tumour types. This analysis concluded that on average there are
four (range 1–10) coding substitution mutations driving the cancer process that are recurrent in specifictumour types, and these are under positive selection. Whole genome sequencing has additionally revealed
that many genotoxic agents produce distinct damage-related mutational signatures in DNA, and in somecases similar damage-related mutational signatures are observed in cancers – for example a simulated
solar radiation signature in UV-associated skin cancers, and a polyaromatic hydrocarbon signature insmoking-related lung cancers [K46]. It has also become increasingly clear that there are additional nonmutational routes to modify cellular phenotypes; collectively these are known as epigenetic pathways.
The epigenetic pathways lead to long-lasting alterations in gene expression, and these include alterations
to DNA methylation of cytosine residues in gene upstream regulatory elements, modification of histones
and chromatin status through acetylation, phosphorylation, ubiquitination plus a range of other chromatin
modifications, and expression of non-coding RNAs that modify transcript stability and abundance [D5].
Given that targeting of epigenetic regulators in cancer therapy holds promise, it would appear that
epigenetic processes play an important role in carcinogenesis [D5]. Epigenetic alterations and non-coding
RNA (largely microRNA) alterations have also been observed as recurrent changes in cancers [V9].

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Inflammation can also contribute to the acquisition of hallmark capabilities through modification of thetumour microenvironment, and in some cases, can lead to the release of reactive oxygen species (ROS)
that can actively contribute to mutagenesis during tumour pathogenesis [H10].
22. It is also important to consider which of the cells within the body represent the target cells for
carcinogenesis. Most cells in the body are terminally differentiated, many with rather short existence
before loss, skin and intestinal epithelia being good examples. Given the observation that cancers arepresent predominantly in older age groups, the cells of origin must necessarily be long lived. Thus,
attention tends to focus on the stem and early progenitor cells that serve to maintain tissues throughout
life. It is also possible that the elderly are more vulnerable to cancer due to impaired repair/defence
mechanisms; however for induced cancers from instantaneous exposures, such as experienced by thesurvivors of the atomic bombings in Japan, effects take from a few to many years to manifest.
23. The hallmarks provide a current overview of the pathways that need to be considered in order tounderstand the mechanisms involved in the development of cancer. Consideration of the enabling
characteristics (i.e. somatic mutation, epigenetic modification and inflammation) is clearly important inorder to understand how radiation contributes to carcinogenesis. Thus, the hallmarks framework and thebroader understanding of cancer mechanisms in general are of considerable value in directing judgementson the mechanisms associated with carcinogenesis following low-dose and low-dose-rate radiation
exposure. In this annex, the Committee considers the recognized mechanisms of carcinogenesis and theobserved impacts of low-dose and/or low-dose-rate radiation on these mechanisms, including someconsideration of moderate/high-dose effects for context and potential implications for risk extrapolation.
This evaluation aims to inform judgements on the pathways relevant to low-dose and low-dose-rateradiation carcinogenesis and further inform judgements on the best justified models for extrapolation ofradiation cancer risk for exposures in the 10–100 mGy range.

III. BIOLOGICAL MECHANISMS RELEVANT TO DOSE–RESPONSE
RELATIONSHIPS
24. In order to identify papers relevant to the evaluation of biological mechanisms relating to theinference of cancer risks from low-dose and low-dose-rate radiation exposure, literature databases weresearched with defined search terms as set out below. Most papers were identified through searching of
PubMed, and the papers identified were supplemented by papers identified by the expert group as ofrelevance during review and discussion of the evaluation. All identified papers, irrespective of source,
underwent a standard evaluation as described in appendix A. The search terms employed were not always
sufficiently specific to exclude papers using for example UV radiation, such papers were deemed out of
scope by the expert group and thus not included among the papers reviewed. Table 1 provides a summaryof the information on search terms used for each section of this chapter, the number of papers reviewed,
and the number positively evaluated.
25. A wide range of potentially relevant literature is available that utilizes a wide range of experimental
approaches, including cells, tissue and animal models. All experimental systems have limitations in theirapplicability to cancer risk inference in humans. Cell models are unable to fully recapitulate the processof carcinogenesis, but human material can be utilized. While able to follow entire disease development,
experimental animals may differ in response by comparison with humans but whole organism responses
can be examined. The use of genetically or otherwise modified animal models can provide useful insights
into the pathways of disease pathogenesis, but the nature of genetic alterations can differ from those foundANNEX C: BIOLOGICAL MECHANISMS RELEVANT FOR THE INFERENCE OF CANCER RISKS ...

13

in human populations naturally. In terms of cell models, greater weight is placed on non-transformed
human cells studies, and particularly those cells that are targets for radiation carcinogenesis (stem andearly progenitor cells). As transformed cells carry cancer-related alterations they are not considered to be
the best models. Most experimental animal work relates to mice, but a wide range of organisms havebeen used. More generally there is the issue of the robustness of findings in terms of reproducibility andfrom a statistical point of view; all experimental systems and assays have limitations of sensitivity andreproducibility, these factors are considered in greater detail in appendix A, “Principles and criteria for
ensuring the quality of the Committee’s reviews of experimental studies of radiation exposure”. The aim
here has been to consider evidence from all systems and search for responses that are consistent among
a range of models that credibly relate to human cancer.
26. Most papers identified relate to studies of external low-LET radiation exposures. Where available
papers concerning internal exposure and high-LET exposures were also considered (see table 1).
Table 1. Summary of literature search terms utilized, number of papers identified and positively
evaluated
DSB: double-strand break, ROS: reactive oxygen species

Search terms utilized

Number of
papersreviewed

Number of
paperspositively
evaluated a

III.A. DNA damage

Radiation, low-dose radiation, DSB, basedamage, ROS, complex DNA lesions

25

19

III.B. DNA damage signalling,
chromatin remodelling andepigenetics

High- and/or low-dose radiation, DNAdamage response, post-translational
modification, chromatin remodellers,
histone modifiers, DNA methylation,
microRNAs, transcription

132

82

III.C. Effects on other signaltransduction pathways

Same as “Immune system and cytokine
responses”

19

18

III.D. Gene and protein expression

Radiation, ionizing, low dose, NOT
therapy, gene expression, protein
expression

164

95

III.E. DNA repair and effects onsomatic cells

High- and/or low-dose radiation, repair of
DSB, repair of complex DNA lesions,repair of base damage, induction ofgenetic alterations, repair mutants andradiation-induced cancer

112

74

III.F. Genomic instability, bystander
effects, damage/effects on nonnuclear cellular components,
adaptive response and hyperradiosensitivity

Genomic instability/ionizing
radiation/low dose

183

11

III.G. Stem cells and target cell
populations for radiation
carcinogenesis

Stem cell/ionizing radiation/low dose

334

35

Section

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Number of
paperspositively
evaluated a

SectionSearch terms utilized

Number of
papersreviewed

III.H. Effects at the whole organism
level

Immune system and cytokine responses:– radiation, ionizing, irradiation, low,
low dose, cancer, immune*b, inflamm*,
excluding therapy, radiotherapy,
review;– low-dose radiotherapy.

117

101

III.H.5. Effects on vascularization, cell
migration/invasion and epithelialmesenchymal transformation

Cell migration/invasion AND ionizing
radiation AND low dose

92

5

67

45

IV. Integration of data at different
levels of organization andmodelling of cancer mechanisms

Radiation, carcinogenesis, modelling,
NOT therapy
Radiation, modelling, ionizing, NOT
therapya

A positive evaluation indicates that a paper is considered suitable for consideration in this annex.

b

The asterisk indicates that the word stem (e.g. Immun-) can be followed by any string of letters.

A. DNA damage
27. The important role of radiation damage to DNA in radiation carcinogenesis was made clear in the
UNSCEAR 1993 Report, annex E, “Mechanisms of radiation oncogenesis” [U3] and the UNSCEAR
2000 Report, annex F, “DNA repair and mutagenesis” and annex G, “Biological effects at low radiation
doses” [U5]. These reports indicated that radiation acts mainly through the induction of damage to nuclear
DNA in somatic cells, and that DNA double-strand breaks (DSBs) and complex lesions are most likely
of greatest importance in causing long-lived mutations. Carcinogenesis was described as a multistage
process originating from single cells that have sustained mutations to critical growth-regulating genes.
28. The DNA damaging effects of radiation on somatic cells have been investigated over many decades
and there is a rich literature available. Many studies, both historically and currently, utilize in vitro cell
culture systems where it is common that cells are proliferating, often exponentially. This is unlike thesituation for a substantial proportion of somatic cells in the body, where a majority reside in a G0 state.
As discussed in section III.G, it is generally considered that stem and early progenitor cells are the main
target cell populations for radiation carcinogenesis. These cell populations can reside in a quiescent G0
or be actively cycling. The responses of G0-phase cells to radiation exposures are known to differ insubstantial respects from the responses of actively cycling cells, and this has to be borne in mind whentranslating findings from cell culture systems to the in vivo situation.
29. As noted previously biophysical arguments support the observations that damage to DNA is
induced as a simple linear function of dose. The Committee has placed emphasis on the importance of
mis-repaired and un-repaired DNA double-strand breaks and complex damage sites in determining thecellular effects of radiation exposure. Double-strand breaks are relatively rare (around 40 DSBs per cell
per Gy low-LET), at doses of a few mGy and below and so DSBs will be present in only a fraction ofirradiated cells. The use of immunostaining and fluorescence tagging of DNA damage response-related
chromosomal proteins (see section III.B), γH2AX and 53BP1 [R18] in particular, have brought some

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new information on the quantitative aspects of DSB formation and their repair following radiation
exposure. These experimental approaches use knowledge of DNA damage signalling, i.e. the early andrapid assembly of protein foci at sites of DNA double-strand breaks, rather than direct physicalassessment of strand breakage to estimate frequencies of DSBs at low dose.
30. Neumaier et al. [N12] assessed 53BP1 focus formation (termed radiation-induced foci (RIF)) using
live cell imaging of immortalized mammary epithelial cells transiently transfected with fluorescently
tagged 53BP1 (53BP1-GFP). They observed a nonlinear dose response in human cells (0.1–2 Gy
X-rays). A greater number of smaller RIFs than expected (35 RIF/Gy) were formed per unit dose
following low-dose X-ray exposure (100 mGy) by comparison with high-dose exposure, with time thesmall foci were observed to aggregate into larger foci. This phenomenon appears to be cell type
dependent, i.e. normal human diploid fibroblasts displayed the expected RIF frequency (30 RIF/Gy).
Similar observations of supra-linear induction of foci have been made using immune-fluorescencemethods [B26]. These observations may reflect the aggregation of DNA breaks into “repair complexes”
following higher dose exposures. Besides cell type specificity, there remain some technical issues thatmight be relevant to these findings and their interpretation [B21]; therefore, the findings of higher focus
numbers at low by comparison with high doses require further substantiation.
31. With conventional staining for γH2AX in confluent human fibroblasts exposed to X-rays, a linear
relationship has been reported between the number of foci per cell and the ionizing radiation dose ranging
from 1.2 mGy to 2 Gy [R17]. Also in vivo (human lymphocytes, mouse retinae) linear relationships for
γH2AX foci formation within the dose range 3–30 mGy have been observed [L39, M34, R20]. In
contrast, evidence for low-dose supra-linear induction of foci (<10 mGy) after in vivo (paediatric patientswith congenital heart disease) and in vitro X-ray radiation of human lymphocytes has been reported[B26]. The earlier step of ataxia-telangiectasia-mutated (ATM) protein activation (phospho-ATM)
precedes 53BP1 focus formation. Induction of phospho-ATM foci has a linear dose–response relationshipwith doses ranging for 10 mGy to 1 Gy, with an average 50 foci per unit dose (Gy); furthermore, the sizeof foci was independent of dose [S70]. These findings are further considered in Mullenders et al. [M45].
32. In order to study the DNA damage induction from diagnostic procedures such as computed
tomography (CT), angiography etc., blood samples from 45 patients who have been subjected to CT or
angiography and 15 controls were stained for γH2AX [K23]. The exposed cohort was further subgrouped
into patients undergoing one CT scan, multiple scans and angiography groups. For the exposed group,
blood was collected 1 hour after the diagnostic exposure. γH2AX staining was carried out and10,000 cells per sample were analysed for γH2AX positivity using a flow-cytometer. Increased numbersof γH2AX positive cells were identified in exposed samples compared to control samples. Multiple CT
exposures produced the highest increase in γH2AX positive cells. It was concluded that multiple CT
scans produce more DSBs compared to the single scans as observed in the form of γH2AX positive cellsin blood cells. Yoon et al. [Y11] measured two established radiation biomarkers, γH2AX and pCHK2(activated checkpoint kinase 2), in individuals exposed to X-rays during dental examinations. Exfoliatedoral epithelial cells were collected from 100 healthy individuals undergoing routine dental radiographic
examination (23.4 mGy) before and after the radiographic examinations. Both biomarkers displayed
statistically significant up-regulated expression after exposure.
33. The spectrum of damage to DNA caused by ionizing radiation exposure, particularly high-LET
radiation, also includes complex lesions/clustered damage sites (two or more oxidized bases, abasic sites
or strand breaks on opposing DNA strands within a few helical turns). These complex lesions/clustereddamages are more difficult to repair than isolated lesions and are thought to trigger carcinogenic events
via formation of complex and persistent genomic alterations. The formation and repair of clustereddamage and the impact of type of radiation have been assessed in modelling studies, in vitro studies
employing plasmid templates with engineered damage clusters, and rarely in in vivo studies with

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mammalian cells. A study with tumour and immortalized epithelial human cells [A22] compared theformation of clustered damage at single cell level by low- and high-LET irradiations (X-rays, Si ions andFe ions) and H2O2 (control). Complex lesions/clustered damage sites were identified in single-cells by
3D-analysis of three types of repair foci: DSBs, single-strand breaks and base damage using specificmolecular probes for DSBs (53BP1), ssDNA (XRCC1) and 8-oxoGuanine (OGG1), respectively. The
spatial distribution of these repair foci tagged by different colours, was monitored in the same cell andcolocalization of three colours was scored as clustered damage. The result indicated colocalization
30 minutes after irradiation (1 Gy) by Si ions (40%), Fe ions (75%), X-rays (4%) but not after H2O2 (0%).
These results are supported by unified stochastic modelling based on radiation tracks, that predicts boththe induction, spatial distribution and complexity of DNA damage. This modelling approach is coupled
to experimental data (γH2AX foci) from 2D fluorescence microscopy of human cells exposed to X-rays
and neutron beams (broad-energy neutron beam with an average energy of 2.4 MeV) [B17]. In the dose
range of 1–5 Gy, DSB multiplicity (average number of DSBs/focus) is found to be 1.20 and 11.09 for
X-rays and neutrons, respectively.
34. Ionizing radiation induces ROS, i.e. highly reactive metabolites of oxygen, such as superoxide (O2−),hydrogen peroxide (H2O2), Their lifetime in biological systems ranges from nanoseconds to seconds
depending on their reactivity [D17]. ROS detection therefore requires probes that very rapidly react with
ROS and produce stable quantifiable products such as chemiluminescent and fluorescent probes.
35. The ROS production as function of the X-ray dose was examined in human endothelial cells.
Fluorescent measurements of ROS induced by doses of 0.03, 0.1 and 0.5 Gy and profiled three hours
after irradiation, showed a linear dose response [K37]. Analysis of ROS (using 20,70-dichlorofluorescin
diacetate (DCFDA)) in chronically low dose X-rays (0.010 or 0.05 Gy/d for 31 days) treatedimmortalized human fibroblasts revealed persistent ROS production when measured 24 hours after thelast radiation. Surprisingly, radiosensitive ATM and NBS (Nijmegen breakage syndrome gene) deficient
cells lacked induction of ROS [S36]. In view of the short lifespan of ROS, these data reflect radiationinduced perturbations in cellular redox homeostasis rather than induced ROS per se.
36. The induction of various types of base damage (DNA single-strand breaks, apurinic/apyrimidinicsites, alkali labile sites) was assessed by the alkaline comet assay in human peripheral blood mononuclear
cells obtained from 20 individuals exposed to gamma radiation and processed immediately. These data
reveal a linear dose response (100 mGy–2 Gy) [T20]. Alkaline comet assay measurements on whole
blood exposed to X-rays (29 kV, 220 kV) and gamma irradiation (137Cs, 60Co) (0.5–3.0 Gy) also reveal
for all types of radiation a linear dose–response relationship with the same slope when analysed for“% DNA in the tail” or “tail moment” [R15]. Of note, the induction frequency of DNA base modifications
per unit dose (Gy) of X-rays is close to the steady-state level of oxidized bases in fibroblasts andlymphocytes cultured under common (high) oxygen conditions, i.e. approximately one modification per
107 normal bases measured in various studies using different methodologies, including HPLC-EC, cometassay, enzymatic assays and DNA repair labelling [C29, P16]. On a population level, basal amounts of
DNA damage (DNA strand breaks, alkali-labile sites and oxidative DNA damage (Endo III and hOGG1
sensitive sites)) were determined using comet assay in peripheral blood mononuclear cells of 67 healthy
adult male individuals from normal and high-level natural background radiation areas (HBRAs) of theKerala coast [K47]. The study included 45 individuals from HBRA (>1.50 mGy/y) and 22 individuals
from a normal level natural background radiation areas (≤1.50 mGy/y). Overall frequencies of DNA
strand breaks and oxidative damage observed in HBRA individuals were not significantly different fromnormal level natural background radiation area individuals. In the same cohort, a lack of increase of DNA
double-strand break in the HBRA individuals when compared to normal level natural backgroundradiation area individuals was observed as well [J9]. In contrast, after in vitro irradiation (2 or 4 Gy) ofperipheral blood mononuclear cells initial levels of DNA strand breaks were lower in subjects from
HBRAs compared to subjects of normal level natural background radiation areas; an opposite effect was

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observed when DSBs were measured by premature chromosome condensation [K48, K49]. A preliminary
study of γH2AX foci in blood of residents living in the HBRA of Mamuju, West Sulawesi, showed atrend towards higher (albeit not significant) average values relative to the control area [B22].
37. In summary, ionizing radiation induced DNA damage has been quantified in mammalian cells
exposed to low and moderate doses, exploiting various methodologies, i.e. direct measurement of DNA
breaks by single cell electrophoresis and by indirect measurements using immunostaining andfluorescence tagging of DNA damage response-related proteins. In almost all studies the induction ofdifferent types of DNA lesions are in accordance with linear dose response. Experimental data on dose
dependence of clustered damage induction are sparse; the data available from modelling and experimental
studies suggest a low induction of clustered damage by photon radiation (X-rays) compared to high-LET
alpha particles.

B. DNA damage signalling, chromatin remodelling andepigenetics
38. The UNSCEAR 2000 Report, annex F, “DNA repair and mutagenesis” [U5] recognized that DNA
damage, particularly DSBs, was responsible for initiating a signalling process that resulted in repair orother responses to the damage. Ionizing radiation activates base excision repair, single-strand break repair
and DSB repair as major responses to the types of DNA damage that it induces. Only DSB repair has aseparation of damage recognition, signalling and repair. It was recognized that these signalling pathways
could impact on individual sensitivity and response to radiation exposure. The potential importance of
epigenetic effects in carcinogenesis was acknowledged in the UNSCEAR 2000 Report, annex G,“Biological effects at low radiation doses” [U5] but the rudimentary understanding of this topic precluded
any substantive conclusions being drawn.

1.

DNA damage signalling

39. Cellular surveillance mechanisms are essential to maintain genome integrity as DNA in cells is
continually being damaged in many ways. To counteract threats posed by DNA damage, the cell has
evolved a capability to detect DNA damage as well as aberrant DNA structures generated by DNA
damage (chromatin perturbations, stalled transcription and replication) and to elicit cellular responses(i.e. the DNA damage response. The DNA damage response is initiated by a cellular signalling cascadeto promote DNA damage removal through activation of DNA repair, alteration of chromatin, pausing ofcell-cycle progression (checkpoint activation), modulation of transcription and replication, and inductionof apoptosis. Cells defective in these protective mechanisms often manifest enhanced sensitivity towards
DNA-damaging agents including ionizing radiation, and many such defects cause human disease [J1].
40. Experiments dealing with the biological effects of deficiencies in DNA damage response factors on
signalling, repair, cell cycle, genetic alterations etc. in low-dose irradiated cells are limited. On thecontrary, mechanistic studies often exploit cell-based assays that utilize single-copy fluorescent reporters
to measure repair of an enzymatically-induced single DSB. These assays have been applied to assess theimpact of DNA damage response deficiencies on repair of a single DSB by non-homologous end joining(NHEJ) and homologous recombination repair (HRR) [P18]. These assays measure a cellular response
to low-level DNA damage. While enzymatically-generated breaks are not structurally identical toradiation-induced DSBs, they represent a somewhat simplified model; nonetheless the simpler enzymatic

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induced breaks, even when only one is present, do trigger a standard DNA damage response as
demonstrated by foci formation [D19].
41. The main molecular mechanism governing DNA damage response activation consists of posttranslational modification of specific proteins including phosphorylation, ubiquitylation and acetylation.
One of the most frequent modifications is the reversible and dynamic phosphorylation of proteins at
specific serine, threonine and tyrosine residues [O16] by protein kinases catalysing the transfer ofphosphate to their substrates. The key kinases that are considered to orchestrate DNA double-strand breakdamage signalling, are ATM kinase, ATR (ataxia telangiectasia and Rad3-related) kinase and DNA-PKcs[B36]; each kinase is activated by a distinct DNA structure. Recruitment to DNA damage requires a
specific co-factor for each kinase: NBS1 for ATM, ATRIP (ATR-interacting protein) for ATR and Ku80
for DNA-PKcs. The DSB is considered to be the main DNA lesion that activates ATM. ATR is activatedby single-stranded DNA at ssDNA/dsDNA junctions and cells defective in ATR signalling are sensitive
to ultraviolet radiation and mitomycin C, but not to low-LET ionizing radiation [O2]. However, sustained
resection at sites of DNA breaks due to complex types of damage by high-LET radiation might lead to aswitch from ATM to ATR signalling [S2]. DNA-PKcs is recruited to DSBs by interaction with Ku80 andactivated by autophosphorylation thereby promoting repair by NHEJ. DNA-PKcs kinase activity isimportant for repair of DSBs by NHEJ; moreover, DNA-PKcs mutants are sensitive to low-dose ionizing
radiation [L31]. It is important to note that processes other than DNA damage response can be regulated
by post-translational modification following low-dose radiation. For instance, radiation differentiallyregulates protein acetylation in human coronary artery endothelial cells. Notably, ionizing radiation mightalter the acetylation status of proteins belonging to several pathways including protein synthesis,
cytoskeleton-related processes, protein folding and calcium signalling, by differentially regulating
histone acetylase/deacetylase activities in 0.5 Gy irradiated human endothelial cells [B20].
42. Mutations identified in ataxia teleangiectasia patients occur throughout the ATM gene with no “hotspots” and generally lead to protein instability. Autophosphorylation of ATM at multiple sites (the mainsite S1981, and additional sites such as pS367 and pS1893) following ionizing radiation exposure [K42]activates ATM and is functionally important because mutant forms of ATM refractory to phosphorylation
are defective in correcting radiosensitivity of ataxia-telangiectasia cells [K41, K42]. Activation of ATM
after DSB induction orchestrates the DNA damage response by initiating phosphorylation of multiple
downstream targets involving DNA repair factors (such as H2AX, 53BP1), checkpoint kinases,
chromatin remodellers and transcription factors [S32]. Phosphorylation of ATM is detectable as discrete
foci over a 10 mGy to 1 Gy dose range and appears to be linear with dose. Moreover, ATM foci displayeda similar average size at 20 mGy and 1 Gy [S70] indicating a sensitive and effective induction of DNA
damage response at doses as low as 20 mGy of ionizing radiation. Also, phosphorylation of ATM’s direct
downstream target, H2AX, is linear with dose [L39].
43. Ubiquitin modifications are another type of signalling post-translational modifications within the
DNA damage response that regulate the repair of DSBs (and other types of DNA damage) within
chromatin [S18, S49]. Formation of γH2AX by ATM initiates binding of the MDC1 protein that
thereafter recruits a number of factors to DSBs including ubiquitin ligases (E3 ubiquitin ligases RNF8,
RNF168 and others). These ligases generate specific ubiquitin marks on histones near the breaks,
providing a platform for recruitment of DSB repair factors. The signalling cascade thus switches from
phosphorylation to ubiquitylation events. Defects in ubiquitylation may underlie some human diseases
associated with radiosensitivity. For example, mutation in ubiquitin ligase RNF168 leads to RIDDLE
syndrome, a disorder associated with radiosensitivity (1 Gy) and immunodeficiency [P19, S64]; theunderlying cause for the disease phenotype might be a deficiency of DSB repair by homologous
recombination [L50].

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44. The ATM and ATR kinases play key roles in cell-cycle regulation after DNA damage. Cell-cycle
checkpoints allow the integrity of genetic material to be monitored before progression through key stages
such as replication and mitosis. Checkpoints at G1/S and G2/M as well as intra-S-phase stage have been
described. Checkpoints are initially activated by phosphorylation of ATM and its downstream checkpoint
kinase Chk2 (phosphorylation of Chk2 is linear in the dose range 0.2–2 Gy) and ATR and its downstream
checkpoint kinase Chk1 (phosphorylation of Chk1 is linear in the dose range 0.2–2 Gy) in mammaliancells following ionizing radiation exposure [B36, S41]. In addition, experiments with X-irradiatedDrosophila flies revealed a radiation sensitive spindle checkpoint [R19]. This checkpoint only affects
entry into anaphase at high doses (3–6 Gy X-rays). In contrast, low doses of X-irradiation only activate
the DNA damage Chk1 checkpoint at metaphase. In spite of sensitive Chk1/Chk2 activation, radiation
doses less than 200 mGy fail to activate the G2/M checkpoint, due to a G2/M checkpoint activationthreshold of 10–20 DSBs per cell [D12, K43].
45. In clonal cell survival assays, mammalian cells often exhibit hyper-radiosensitivity (relatively highcell killing) to low-dose (<100–200 mGy low-LET radiation) radiation followed by increased
radioresistance (induced radioresistance, relatively low cell killing) at higher doses (see also
section III.F.6). The low dose hypersensitivity described in several cell types following exposure to low
doses [K44, M16] is likely attributable at least in part to the lack of G2/M checkpoint induction. ATM(and ATM activation) correlates with the development of increased radioresistance (the level of cellkilling at higher dose), but not to the higher levels of cell killing at low dose, i.e. low dose hyperradiosensitivity [E16]. ATR function is required for the IR-induced G2/M checkpoint activation andcooperates with ATM in the mechanism of low dose hyper-radiosensitivity induced by C ion beam [X2].
On the basis of information gathered from studies using short-term endpoints, doses of less than 200 mGy
may be predicted to be more effective in terms of carcinogenic potential than higher doses where G2/M
checkpoints are effectively induced [E16]. The low dose hyper-radiosensitivity is associated withinduction of apoptosis when cells in G2 are irradiated [M17]. Thus, low-dose radiation damaged cells
may survive and progress through aG2/M-phase transition with a high frequency of chromosome
misalignments and anaphase bridges, subsequently enter apoptosis and so are eliminated from the poolof damaged and therefore potentially pre-cancerous cells. Low-dose irradiation of non-transformed cells(as low as 2 mGy gamma rays and 0.29 mGy alpha particles) can increase the apoptosis of transformed
pre-cancer cells through an intercellular process; however, this process also saturates at very low doses(50 mGy for gamma rays and 25 mGy for alpha particles) [B23, P22]. The proposed mechanism is, that
non-transformed cells selectively remove transformed cells from co-culture via cytokine and reactive
oxygen/nitrogen species (ROS/RNS) signalling. However, it is obvious, that low doses of ionizing
radiation (10–100 mGy) can induce apoptosis in normal (stem) cells as shown for mouse spermatogonia[G11]. The relevance of apoptosis induced in either damaged normal or pre-cancer cells for cancer is notentirely clear. The killing of pre-cancer cells would be expected to reduce cancer risk. Likewise, thekilling of cells with directly induced damage and/or mutations would be expected to reduce risk. In bothcases, cell killing would lead to stimulation of proliferation with risk of induced genomic instability. In
addition, low dose-damaged cells might undergo chromothripsis-like rearrangements in the subsequentcell cycle by incorrect reassembly of DNA fragments through erroneous DNA repair (see section III.E)
thereby increasing the low-dose cancer risk.
46. Mouse experiments revealed the importance of ATM and DNAPKcs for X-ray induced DNA
damage signalling in whole organisms. Homozygous SCID−/−, ATM−/− knockout and ATM+/−
heterozygotes were compared to C57BL/6 wild-type mice in a publication from Flockerzi et al. [F15].
Animals were given 100 mGy X-rays daily for 2–10 weeks locally to the lungs at dose rate of 2 Gy/min
once or five days a week for 2, 4, 6, 8 or 10 weeks. At the conclusion of irradiation, animals weresacrificed at 0.5, 24, or 48 hours post-irradiation. Bronchiolar and alveolar cells were screened for
γH2AX foci as a sign of DNA damage. In repair-proficient C57BL/6 mice that received a single dose of

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100 mGy or doses in the range of 1–4 Gy (over 2–8 weeks) the numbers of foci decreased with time up
to 72 hours after termination of irradiation. In mice that received a dose of 5 Gy (10 weeks exposure)differences in numbers of foci at 24 and 72 hours after termination of exposure were not significant. In
ATM knockout mice, numbers of foci increased with dose and passage of time (up to 72 hours aftertermination of irradiation) did not modulate that. In SCID mice, some decrease in the number of foci wasseen when 0.5 hours post-irradiation was compared to longer timepoints. Finally, in ATM heterozygotes,
differences in the number of foci were notable for the post-irradiation time of 0.5 hours compared withlonger timepoints, but the differences between post-irradiation times of 24 and 72 hours were negligible.
Overall, the numbers of foci in SCID mice were comparable with those in C57BL/6 mice. Hence, thegenetic background relating to DNA repair determined the extent of cumulative low-dose radiation
damage. Moreover, increased DNA damage during fractionated exposures affected DNA replication andapoptosis in the lung parenchyma, which may influence overall lung function.
47. A similar study from the same group explored persistence of DNA damage foci in cells of irradiatedanimals [L44]. Mouse strains used in this work were ATM wild-type mice C57BL/6, ATM heterozygotes,
ATM null and SCID mice. An X-ray irradiator was used to deliver doses of 100 or 6,000 mGy at dose
rate of 2,000 mGy/min. Animals were sacrificed at 0.1, 0.5, 5, 24 or 72 hours after exposure to 6,000 mGy
or 0.5, 24 or 72 hours after exposure to a single dose of 100 mGy. In addition, fractionated exposures
were carried out, where animals received 100 mGy per day, five days a week for 2, 4 or 8 weeks (10, 20
or 40 fractions accumulating to total doses of 1,000, 2,000 or 4,000 mGy). DNA damage foci stained for
53BP1 were imaged in nuclei of cortical neurons – in all exposure scenarios SCID mice had the mostfoci, and of all timepoints, 0.5 hours after exposure had the most foci in all strains of mice. In mice
exposed to multiple fractions, ATM knockouts had the least foci after a single fraction dose of 100 mGy
and early post-irradiation timepoints but after multiple fractions cortical neurons of these mice had many
more foci than wild-type or ATM heterozygotes. Median diameter of foci was not the same in all micestrains – ATM knockout mice had the largest foci. Finally, this study also included transmission electron
microscopy of cells with developed foci; antibodies against pKu70 repair protein were labelled with goldand scored. Once again, the morphology of DNA damage was different in cells from different genotypes.
In their subsequent work, this laboratory perfected these methods and expanded studies to skin andintestine [S12]. In this work C57BL/6 (C57BL/6NCrl) mice received whole-body irradiation with 10 or
100 mGy 6 MV photons (linear accelerator (LINAC) X-rays) at dose rate: 2 Gy/min. Single exposure to
100 mGy dose caused DNA damage that lasted 24 hours in keratinocytes, but only 30 minutes inenterocytes. Moreover, enterocytes showed 72 hours lasting foci only when 50 fractions of 100 mGy
were applied, while the same was true for keratinocytes exposed to ten 100 mGy fractions.
48. To summarize, cellular surveillance mechanisms (DNA damage response) are initiated by a cellularsignalling cascade to promote DNA damage removal, alteration of chromatin, checkpoint activation,
modulation of transcription and replication, and induction of apoptosis. Cells defective in these protective
mechanisms are often sensitized towards ionizing radiation, most notably in dose ranges above 0.5 Gy;in humans, these defects may underlie disease including cancer. The main molecular mechanism
governing DNA damage response activation consists of post-translational modification of specificproteins including phosphorylation, ubiquitylation and acetylation. One of the most frequent
modifications is the reversible and dynamic phosphorylation of proteins at specific serine, threonine andtyrosine residues by protein kinases catalysing the transfer of phosphate to their substrates and byphosphatases removing phosphate. The principal kinases involved in DNA damage response are ATM,ATR and DNA-PKcs. Mutations in enzymes involved in post-translational modification of specificproteins underlie human diseases associated with radiosensitivity (i.e. ataxia telangiectasia ATM, Seckel
Syndrome ATR, SCID DNA-PKcs, Riddle syndrome ubiquitin ligase RNF168) and often confer
radiosensitivity in mouse models carrying these specific mutations or gene deletions.

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2.

21

Chromatin remodelling

49. All DNA metabolizing processes in living cells including repair of DNA damage, are complicatedby the packaging of genomic DNA into chromatin. In addition to DNA damage signalling, efficient repairof DSBs in the chromatin context of the cell requires post-translational histone modifications and ATPdependent chromatin remodelling to allow access to the repair machinery [J14]. Numerous covalentmodifications of core histones in chromatin are linked to DNA repair including phosphorylation,ubiquitylation, acetylation and methylation of several different histone tails. The archetypical example ofγH2AX is a prerequisite for the binding of MDC1 and the subsequent accumulation of DNA damage
response factors at DSBs to promote NHEJ and HRR. Moreover, cells lacking H2AX phosphorylation
capacity manifest a G2/M checkpoint defect after doses of ionizing radiation <1 Gy [F11]. Chromatin
flanking DSBs is modified by histone acetyltransferases which acetylate histones H3 and H4. Histone
acetyltransferases bind to γH2AX leading to further acetylation, while recruiting acetyl-dependent
chromatin remodellers. This creates a chromatin micro-environment that is rich in γH2AX and acetylated
H3 and that promotes DSB repair by HRR. Besides HRR, histone acetylation also significantly stimulates
NHEJ [L48]. Low/moderate-dose radiation (doses of less than 1 Gy in this context) differentiallyregulates protein acetylation in human cells as described in section III.B.1. In addition, human primarycells exposed to chronic (7 days) 137Cs gamma radiation delivered at 6–20 mGy/h revealed dosedependent reduction in cell proliferation and increased cellular senescence and concomitantly up to 40%
reduction of core histone protein levels due to reduced histone gene expression. In contrast and consistent
with senescence induction, chronic gamma radiation increased the expression of the senescence related
histone variant H2AJ [L45].
50. Enzymes involved in the above-mentioned core histone modifications are often involved inradiosensitivity and cancer. For example, it is becoming clear that acetylation, deacetylation, methylationand demethylation of histone tails are all tightly controlled at sites of DSBs by histone acetyltransferases,
histone deacetylases, methyltransferases and demethylases [L48]. The spermidine/spermine-N1acetyltransferase 1 (SAT1) promotes acetylation of histone H3, regulates HRR and is overexpressed inradioresistant glioblastoma tumours. SAT1 depletion leads to reduction in BRCA1 expression, explaining
decreased HRR capacity and radiosensitization [B56]. Histone acetylase Tip60 binds to chromatinsurrounding DSBs and its depletion impairs both DNA damage-induced histone H4 hyperacetylation andaccumulation of repair molecules at sites of DSBs resulting in defective HRR and radiosensitization.
Activation of the Tip60 acetyltransferase by DSB, however requires interaction of Tip60 with methylated
histone H3 (H3K9me3); the latter modification is generated by the methyltransferase Suv39h1. Cells
lacking Suv39h1 display defective activation of Tip60 and ATM, decreased HRR and increased
radiosensitivity [A28]. This illustrates the intricate process required to repair DSBs in the context ofchromatin. Importantly, the impaired HRR (and NHEJ) of DSBs as a consequence of defects in chromatin
modifications are amenable to study at the level of a single DSB in model systems. Tables 2 and 3summarize the various enzymes involved in chromatin modification and DSB repair.
51. Along with ATM-dependent signalling that promotes post-translational modifications of chromatinin the vicinity of DSBs, ATP-dependent chromatin remodelling is essential to overcome the chromatinbarrier and facilitate access of proteins and enzymes to damaged DNA. Emerging evidence shows thatATP-dependent chromatin remodellers are not only involved in DSB repair, but also key in preventing
cancer and other human health disorders [O12]. Chromatin remodelling enzymes are large protein
complexes that use ATP hydrolysis to alter the nature of the interaction between DNA and histones byvirtue of nucleosome sliding, nucleosome disruption, histone eviction or histone exchange. They areclassified for ease into 4 families: SWI/SNF (BAF in mammalian cells), ISWI, CHD and INO80 (table 3).
All modellers except for INO80, bind to γH2AX and modified histones at the vicinity of DSBs [R16].

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Table 2. Histone alterations and enzymatic activities known to be modulated after ionizing radiation
exposure (adapted from [L48])
DSB: double-strand break, HRR: homologous recombination repair, NHEJ: non-homologous end joining
Histone modification

EnzymeH2AX phosphorylation(γH2AX, S139P)

ATM, ATR, DNA-Pkcs

Checkpoint activation, stimulation of HRR and
NHEJ

[B36,
F11]

H2AX acetylation(K5ac)

Tip60

Required for chromatin ubiquitylation and H2AX
release[I5, J17]

P300/CBPDSB repair pathway

Reference

Promotion of ionizing radiation survival
independently of γH2AX

H2A ubiquitylation
K13,15ub

RNF8 (mono/di-ub),
RNF168 (poly-ub)

Accumulation of BRCA1 and 53BP1 after ionizing
radiation, checkpoint activation and repair of
DSBs in heterochromatin[S49]

H2B ubiquitylation
K120ub

RNF20 and RNF40

Accumulation of Ku, BRCA1 and RAD51; repair by
NHEJ and HRR[N7]

H3 phosphorylation
S10P

CHK1

H3 acetylation K56ac

GCN5, SAT1

Recruitment of SWI/SNF complex, promotion of
HRR

[D2,
M22]

H3 methylation K4me
and K9me

Suv39H1/Suv39H2

Stimulation of Tip60 activation and recruitmentof HP1 proteins to DSBs to promote repair

[D18]

SET1

H4 acetylation K16ac

MOF1 (reversed by
HDAC1/2)

Regulation of HRR and NHEJ byacetylation/deacetylation[L25]

H4 ubiquitylation
K91ub

BBAP

53BP1 recruitment to DSBs

[C10]

H4 methylation
K20me2

Suv4-20H1/Suv4-20H2

Recruitment of 53BP1. The 53BP1-H4meinteraction suppresses HRR, but promotes NHEJ

[P10]

[S33]

52. In mammalian cells, BAF family members are also known as SMARCA. Among these members,
SMARCA2 and SMARCA4 have been implicated in DSB repair by facilitating NHEJ and likely HRR
by facilitating acetylation of histones and spreading of γH2AX around the DSB. Somatic mutations in
SMARCA2 and SMARCA4 predispose to cancer and depletion of BAF subunits results inradiosensitivity in a dose range 0.5–4 Gy [W7]. Also, SMARCA5 affects DSB repair by HRR and NHEJ
by a joined action with histone deacetylase SIRT6. Depletion of SIRT prevents the local deacetylation ofchromatin and inhibits recruitment of SMARCA5 to DSBs and sensitizes mouse embryonic stem cells to
X-rays (1–8 Gy) [T15]. CHD remodellers have common domains that allow the binding to methylated
histones and DNA and five members have been classified as being involved in the DSB response [R16].
Among these, CHD1-like or ALC1 (amplified in liver cancer) and CHD2 are recruited to DSBs in a
PARP1-dependent fashion, ALC1 interacts with the NHEJ core proteins Ku70 and DNA-PKcs, and cells
depleted of ALC1 show distinct sensitivity to ionizing radiation (1–2 Gy) [S22]. In contrast, CHD2
involves a different mechanism namely insertion of histone variant H3.3 in DSB flanking chromatin
thereby regulating the assembly of NHEJ core proteins to promote repair. The remodeller CHD3 has beenshown to act on DSBs in heterochromatin by binding to SUMOlyated KAP-1 (KRAB (Krüppel
associated box)-associated protein-1, which keeps the chromatin compact), thereby facilitating the releaseof KAP-1. Subsequent release of CHD3 enables chromatin relaxation and allows the recruitment of
SMARCA5. These findings showed that a two-step release and recruitment system modulates opposing

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chromatin remodelling activities during DSB repair in heterochromatin. Finally, CHD4 has been shown
to affect both DNA damage signalling and DSB repair. The signalling of DSBs involves the ubiquitin
ligase RNF8-dependent recruitment of CHD4 to DSBs which promotes further decondensation ofchromatin to facilitate ubiquitylation of chromatin and subsequent recruitment of BRCA1. The repair of
DSBs by HRR is largely dependent on CHD4 and CHD4-depleted cells are sensitive for DSB inducing
agents [P1].
53. The exposures of cells depleted of chromatin remodellers or of histone modifiers, have been carriedout only at doses of ionizing radiation greater than 0.5 Gy, and hence, no information is available for theeffects at low doses. However, in almost all studies survival experiments were complemented withmeasurement of DSB repair in cellular systems where a single DSB per cell can be induced by enzymatic
cutting of the DNA and fluorescent single-cell visualization of repair. The general conclusion is that lossof functional chromatin remodelling and modification generally impairs the repair of DSBs by either
HRR or NHEJ or both and confers radiosensitivity of cells to high doses of ionizing radiation. Recent
work has explored an alternative NHEJ pathway that involves polymerase theta and requires
microhomology repeats as being important when classical NHEJ or HRR are compromised. Tables 2 and3 provide a summary of information on the functions of chromatin modifiers and chromatin remodellers,
respectively. In addition to post-translational modification and chromatin remodelling, chronic low-doserate exposures (6–20 mGy/h 137Cs gamma irradiation over 7 days, total doses 1.008–3.36 Gy) of humanprimary fibroblasts have been observed to deplete histone proteins by up to 40% and this reduction wascorrelated with histone gene expression changes. Furthermore, cells displayed enlarged nuclear size withincreased global transcription including that of pro-inflammatory genes [L45].
Table 3. Chromatin remodellers involved in DSB repair (adapted from [L48])
DSB: double-strand break, HRR: homologous recombination repair, NHEJ: non-homologous end joining
Remodeller

DSB repair
pathway

Function

Reference

SWI/SNF (PBAF or BAF)
SMARCA2 and
SMARCA4

HRR and NHEJ

H3 acetylation, Rad51 loading, Ku loading

SMARCA5

HRR and NHEJ

RPA and Rad51 loading, BRCA1 loading, Ku loading[L12, P11]
[N7]

ISWI
ACF/CHRAC

HRR, NHEJ

Ku loading

[L5]

RSF1

HRR

RPA and Rad51 loading

[P15]

[K14]

CHD
CHD1

HRR

CtIP loading

ALC1

NHEJ

Association with Ku and DNA-PKcs

[A7]

CHD2

NHEJ

Chromatin expansion, H3.3 deposition, Ligase 4 loading

[L49]

CHD3

NHEJ

Repair of DSBs in heterochromatin

[K9]

CHD4

HRR, possible
NHEJ

RNF8 response (signalling), BRCA1 loading, RPA andRad51 loading

[L6]

INO80

HRR and NHEJ

Histone H2A.Z, end-resection, Histone H3 removal[V1]

SMARCAD1

HRR

End-resection[C27]

p400/Tip60

HRR and NHEJ

Prevents resection by H2A.Z deposition at DSBs

[C28]

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54. To summarize, multiple genes have been recently identified that remodel chromatin to facilitate
repair of ionizing radiation induced DSB. Chromatin remodelling is achieved by post-translational
modification of histones or eviction of histones. ATP-dependent chromatin remodelling is essential to
overcome the chromatin barrier and facilitate access of proteins and enzymes to damaged DNA. The
exposure of cells depleted of chromatin remodellers (or of histone modifiers) to ionizing radiation confers
radiosensitivity, but these experiments have been carried out only at doses of greater than 0.5 Gy.
However, DSB repair has been measured in cellular systems in which a single DSB per cell can be
induced by enzymatic cutting of the DNA. These experiments reveal that loss of functional chromatin
remodelling and modification generally impairs the repair of a single DSB by either HRR or NHEJ or
both. Somatic mutations in chromatin remodelling genes (germline mutations and copy number
alterations) have been shown to predispose to cancer and radiosensitivity (at moderate/high dose).

3.

Epigenetic mechanisms (radiation-induced methylation and others)

55. The concept of epigenetics implies that non-genetic events could generate stable phenotypic
differences [S77]. Epigenetic changes include DNA methylation, miRNA expression and chromatin(histone) modifications and are known to play an important role in regulation of cellular processes, mostnotably gene expression and genomic instability. Epigenetic aberrations can lead to human disease
including cancer [J23]. The level of DNA methylation is regulated by DNA methyltransferases (DNMTs)a family of enzymes that catalyse the transfer of a methyl group to DNA. Three active DNA
methyltransferases have been identified in mammals named DNMT1, DNMT3A and DNMT3B. DNMT1
is the most abundant DNA methyltransferase in mammalian cells and the key maintenancemethyltransferase in mammals. It predominantly methylates hemimethylated CpG di-nucleotides in the
mammalian genome.
56. To make firm conclusions on the impact of DNA methylation on phenotype, changes in gene
methylation status need to be presented in the context of gene expression patterns. As a paradigm, arsenic,a non-mutagenic human carcinogen, induces tumours through epigenetic changes, particularly in DNA
methylation [B45, R4]. Comparison of genome-wide changes in DNA methylation and gene expression
revealed several key aspects related to arsenic toxicity and carcinogenicity namely that (a) the overallchanges in methylation are complex depending on the baseline methylation level and the gene positionand (b) little overall correlation between arsenic related changes in methylation and gene expression by
microarray measurements. Despite the low correlation, a subset of genes that play a role in arsenic-related
cancer, showed a relationship between methylation and expression.
57. Since ionizing radiation is known to alter gene expression patterns and to induce several DNA
damage response genes, it may alter transcription-associated chromatin marks, including histone
modifications, but also DNA methylation status. Indeed, a single DSB can induce DNA methylation in
the vicinity of the break. This result was achieved in a model system exploiting breast cancer cells withan exogenous promotor CpG island and expression of a nuclease to induce a defined DSB [O3]. So far,studies on the effects of ionizing radiation on DNA methylation have yielded conflicting results. A recent
review summarized the current knowledge of the effects of ionizing radiation on DNA methylation, andis cautious on the implications of the currently available data [M33].
58. Using breast cancer cells irradiated with 2 and 6 Gy of X-rays at 0.86 Gy/min, Antwih et al. [A14]analysed DNA methylation changes over a period of 72 hours after radiation treatment using high density
Illumina 450K DNA methylation arrays. Significantly differentially methylated genes related to radiation
response pathways, were found without a clear dose or time response. Irradiation of colorectal cancer
cells (2 and 5 Gy) and application of the same 450K arrays revealed global DNA methylation changes

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and reduced methylation at specific gene loci [B1]. To investigate the relation between radiation-induced
methylation, radiosensitivity and genomic instability, mouse embryonic stem cells have been studied
containing different levels of methylation due to the presence and absence of DNA methyltransferases[A19]. Functional absence of DNMT1 as well as both DNMT3A and DNMT3B led to a decrease ingenomic methylation relative to wild-type mouse embryonic stem cells. None of the mouse embryonicstem cells displayed significant differences in cytosine methylation measured up to 10 days postirradiation with 3 Gy of X-rays and global methylation levels did not determine radiosensitivity norradiation-induced (delayed) chromosomal instability. In vivo, phenotypic alterations have been linked tolocus specific methylation in the agouti mouse strain. Low doses delivered during gestation by a MicroCT(80 kVp X-rays) caused significantly increased DNA methylation at the viable yellow agouti locus in a
sex-specific manner. Average DNA methylation was significantly increased in male offspring exposed
to doses between 7 and 76 mGy. Maternal dietary antioxidant supplementation mitigated both the DNA
methylation changes and coat colour shift in the irradiated offspring indicating that low-dose exposure
during gestation elicits epigenetic alterations particularly by oxidative stress [B31].
59. Examination of normal human fibroblasts and bronchial epithelial cells seven days after gamma
irradiation (0.1–1 Gy), did not reveal an appreciable direct effect on DNA cytosine methylation patterns
in exposed cells using a methylated CpG island recovery assay combined with microarrays [L3]. Also,at the early phase after irradiation of primary human fibroblasts (6 and 24 hours after 2 or 4 Gy)
employing Illumina 450K methylation arrays, there were no detectable methylation differences in genic(promotor and gene body) and intergenic regions between irradiated and control fibroblasts [M6] nor ininterspersed ALU and LINE-1 repeats and centromeric alpha satellite DNA. In contrast, peripheral blood
cells of occupationally radiation-exposed power plant workers (mean accumulated effective dose 157.74+/− 85.4 mSv (standard error), approximately 20 years average exposure) displayed increased LINE-1
methylation after radiation exposure following assessment of global DNA methylation which was lower
than in control, unexposed subjects [L14]. Low- and high-dose (0.1, 1.0 and 2.5 Gy) radiation of rats
however decreased CpG methylation in the LINE-1 promoter region in the mammary glands followed by
up-regulation of LINE-1 RNA levels and synthesis of LINE-1 protein without a specific radiation dosedependent response [L54].
60. Methylation changes after radiation have been assessed in whole organisms. Examination of DNA
methylation in F1 embryos (5.5 hours post-fertilization) of zebrafish with whole genome bisulfite
sequencing following parental exposure to 8.7 mGy/h for 27 days revealed 5,658 differentiallymethylated regions, predominantly gene promoters and enhancers [K11]. The results also indicate thationizing radiation-related effects in offspring can be linked to DNA methylation changes that partly canpersist over generations. BALB/c mice exposed to moderate dose radiation (X-rays, acute exposure,
0.5 Gy, or exposure 0.05 Gy/d for 10 days) revealed global hypomethylation and tissue-specific promoter
hypermethylation of particular genes. Notably, promoter hypermethylation, rather than global
hypomethylation, was relatively stable [W2]. The key DNA methyltransferase DNMT1 was downregulated in a tissue-specific manner but down-regulation did not persist in time. Exposure of C3H/HeN
mice to doses of high-LET 56Fe ion radiation between 0.1 and 1.0 Gy demonstrated tissue and locus
specific as well as dose- and time-dependent DNA methylation changes. However, these changes in DNA
methylation did not correlate with significant changes in mRNA level at the same timepoints, nor were
they observed for all loci studied [L29].
61. Newman et al. [N13] found that methylation of L1 repeat elements after 1 Gy X irradiation in
spleens of C57BL/6, CBA and BALB/c mice always increased at day 1, at day 6 only in BALB/c and at
day 12 decreased in C57BL/6 and CBA mice. However, when detailed sequences of CpG islands were
evaluated, changes at day 1 post-irradiation were found to be driven by differences in females in C57BL/6
animals or mostly in males in CBA and BALB/c mice.

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62. miRNAs are pleiotropic epigenetic modulators of gene expression. Their modulation in response toradiation is more varied than many other radiation responses. For example, radiation dose responses are
rarely observed in in vitro miRNA expression studies, on the contrary, miRNAs are often observed torespond distinctly to different radiation doses [C11]. This is even more noticeable for exposures to lowdose fractions of radiation, though total doses may be moderate to high. A recent study by Liang et al.
[L28] focused on expression of 11 miRNAs in male 12 weeks old FVB/NJ mice exposed every threedays to 28 mGy of gamma rays for four months (for a total dose of 1.12 Gy). miRNAs, including
miR-34a, miR-375, miR-185, miR-21, miR-421, miR-193a, miR-199a, miR-146a, miR-155, miR-221,
and miR-222, all play significant roles in cancer initiation, promotion or suppression. For example, miR155 prevents apoptosis and promotes proliferation, invasion and migration in hepatocellular carcinoma[F21]; presence of miR-21 in serum of liver cancer patients is an indicator of poor clinical prognosis[W6], while miR-375 suppresses autophagy in liver cancer [Z16]. Changes in expression for some ofthese 11 miRNAs were noted either at conclusion of exposure, two months later or, occasionally, at both
timepoints. In addition, miRNA expression patterns were different in liver, testis and heart, and few oforgan specific miRNA expression changes were statistically significant. These included, for example,
increased expression of miR-21 in liver at conclusion of treatment, decreased expression of miR-375 atconclusion of treatment and decreased expression of miR-155 at both timepoints [L28]. miRNA sex
differences were studied in 45-day-old C57BL/6 mice exposed to radiation either to whole body or head
only (body was shielded) with 1 Gy of X-rays (dose rate 50 mGy/s) [K39]. To simulate scatter dose, someof the “control” animals were exposed to 10 mGy. No changes in miRNA expression were recorded inthis group of animals, regardless of sex.
63. Long non-coding RNAs (lncRNAs) are abundant in genomes of higher organisms and regulatetranscription fidelity. Exposure to moderate-dose irradiation (0.25 Gy) in human cells causes transiently
elevated expression of the lncRNA PARTICLE that regulates the expression of tumour suppressor
MAT2A. This altered expression of lncRNA PARTICLE results in repression of MAT2A via methylationand the formation of transcription-repressive complexes [O4]. PARTICLE levels were significantly
elevated in exosomes isolated from medium of cells exposed to 0.25 Gy implicating a role of this lncRNAin intercellular communication and transcription-silencing in response to irradiation. A follow-up study
demonstrated that elevated expression of PARTICLE operates as a trans-acting mediator of DNA andhistone lysine methylation [O5]. Alterations in both miRNA and lncRNA levels following moderate-doseradiation have been also reported in human blood, with dose and time dependence of responses for certain
RNAs [A21, K2]. Whole genome expression analysis revealed lncRNA profiles changing in response toradiation (1 to 12 Gy of X-rays) in blood of mice 16−48 hours post-irradiation. Radiation-induction oflncRNAs known for their involvement in DNA damage response has been observed, including lncRNA
targets of P53 as well as lncRNAs regulating immune response. The profiles of lncRNAs (and mRNAs)
from peripheral blood mononuclear cells of BALB/c mice at 24 hours after gamma radiation (60Co) withrepeated 0.05 Gy (for 10 times) and single 0.5 Gy exposures revealed lncRNAs induced by these radiation
conditions, that exert a crucial role in the regulation of immune response related gene expression [Q1].
64. To summarize, the data suggest that radiation-induced effects (dose range 0.1–6 Gy) on DNA
methylation depend on tissue/cell type, sex, and species. Moreover, radiation type, dose, and time after
exposure have to be taken into consideration as well as the relative sensitivity of the technologies used todetermine DNA methylation. The data also suggest that dose–response relationships are complex. The
evidence from the small number of studies suggests that low-dose exposure is capable of causing changes
to DNA methylation and that the methylation changes might differ between low and high dose. The
methylation data are inconclusive if related to transcriptional responses and radiation-mediated changes
of DNA methyltransferases. Dose–response relationships for miRNA are not yet well characterized.

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C. Effects on other signal transduction pathways
65. The recognition of cell signal transduction pathways beyond those involved in DNA damage
signalling has been somewhat indirect in prior UNSCEAR reports. Annexes relating to cancer
mechanisms acknowledge that signal transduction pathways can be disrupted in cancer development, but
no judgements are made in the direct effects of radiation exposures on signal transduction pathways.
66. The physical effects of ionizing radiation (damage to the different cellular and tissue components)
are translated into biological effects by the activation or modulation of various intracellular signalling
pathways. These events are generally mediated by post-translational modification of proteins andculminate in changes in gene expression to dictate cell fate in response to radiation exposure. Radiationinduced changes in gene expression are discussed in section III.D. In this section, only articles
documenting the modulation of these signalling networks at the biochemical level or demonstrating theiractivity in functional assays will be considered. The UNSCEAR 2006 Report, annex D, “Effects ofionizing radiation on the immune system [U7] did not specifically address the issue of effects on signaltransduction pathways and contains few, references to modulation of signal transduction pathways byionizing radiation.
67. The expression of immune checkpoint proteins such as the Programmed cell Death Ligand (PDL1) on tumour cells inhibits the activity of tumour-specific cytotoxic T lymphocytes by engaging theirinhibitory Programmed Death (PD)-1 receptor. This pathway has received a lot of attention in recent
years due to the efficiency of the association of immune checkpoint inhibition with radiotherapy in cancer
treatment [B18]. High-dose radiation exposure induces DSBs that can up-regulate the expression of
PD-L1 on tumour cells [S11]. The presence of DNA in the cytosol activates the cGAS/STING
inflammatory pathway that culminates with the production of interferons (IFNs) [L24]. IFN-γ signalling
can also induce up-regulation of PD-L1 expression. However, it seems that radiation exposure does not
induce PD-L1 expression on non-transformed cells (primary human fibroblasts) [H2]. Whether cells
newly elicited or transformed by radiation exposure use PD-L1 up-regulation to escape immune
surveillance and by which pathway and whether this phenomenon occurs after low-dose and low-doserate exposure has not been addressed yet.
68. The exposure in vitro of human purified primary monocytes to 0.05, 0.1 and 1 Gy of X-ray radiation
dose-dependently modulates the expression of several genes involved in innate immunity signalling. Hence,
expression of Toll-like receptor (TLR) 4 and TLR9 genes, and of adaptor signalling proteins MyD88 and
IRAK1 is increased after low but not high-dose exposure. The quantification by enzyme-linked
immunosorbent assay (ELISA) of the activated (phosphorylated) form of several molecules involved in key
signalling pathways showed that low-dose exposure (0.05 and/or 0.1 Gy) specifically resulted in an increasein phospho-IκB, phospho-p38MAPK, phospho-ERK1/2 and phospho-JNK. In contrast, increase inphospho-p53 was found only in 1 Gy irradiated cells [E15]. Thus, low-dose exposure in vitro activatesspecific signalling pathways involved in signal transduction from innate immune receptors.
69. Musculoskeletal disorders are associated with a destruction of cartilage and bone tissue due tochronic inflammation. Hong et al. [H34] investigated the mechanisms underlying the inhibition by lowdose radiation of chondrocyte de-differentiation in inflammatory situations [H34]. Exposure of humanprimary chondrocytes to 5–20 mGy (137Cs, 6.7 mGy/min) has no effects on the expression of chondrocyte
markers (type II collagen, Sox9) and chondrocyte activation (NF-κB-induced) Cox2 signalling.
Treatment with IL-1β induced de-differentiation of chondrocytes over 24 to 48 hours, with loss of
specific markers and activation of an NF-κB-mediated inflammation. These events can be inhibited orreversed by exposure of IL-1β treated chondrocytes to 5 or 10 mGy. IL-1β effects are mediated throughprotein kinase B (Akt) phosphorylation, which results in increase in α, β and γ catenin expression. Lowdose-rate exposure reduces phospho-Akt accumulation and catenin expression. Of note, low-dose-rate

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exposure has no effects on catenin expression in un-stimulated chondrocytes. Low dose rate similarly
inhibits up-regulation of catenin proteins induced by other stimuli such as Phorbol-12-myristate-13acetate, EGF or retinoic acid. This showed that moderate dose rate is able to inhibit/attenuate the effects
of inflammation in primary chondrocytes, and that the effects of moderate dose rate on Akt-signalling
pathways depend on the cellular context.
70. Several immortalized cell lines were used to investigate the mechanisms promoting cell survival or
apoptosis following radiation exposure, without and with prior low-dose exposure. The level of phosphoAKT was found to correlate with radiosensitivity of the different cell lines. Transfection of IM-9 (EBVtransformed B-lymphoblastoid cell line) cells with a constitutive active form of AKT provides
radioresistance. Prior exposure to 0.01 Gy at a dose rate of 0.01 Gy/h protected cells against apoptosis
induced by a subsequent exposure to 2 Gy, with reduced levels of cleaved PARP-1, Casp-3 and Casp-9.
Low-dose priming induces in IM-9 cells a transient increase in phospho-ERK1/2, phospho-p38MAPK,
phospho-JNK and others over the following 48 hours. Exposure to 2 Gy decreases the level of phosphoERK, but this effect is abolished by prior low-dose and low-dose-rate exposure. Furthermore,
pharmacological or siRNA-mediated inhibition of ERK significantly increase IM-9 death following 2 Gy
exposure [P3]. These results showed that AKT and ERK pathways modulate cell fate following low- andhigh-dose exposures.
71. In a follow-up study, the same laboratory compared the early effects (4 hours) of low dose (0.1 Gy)and high dose (2 Gy) gamma-radiation exposure on protein phosphorylation in IM-9 cells by using
phospho-antibody arrays [E17]. As expected, high-dose radiation induced cell cycle arrest, more γH2AX
foci, which lasted longer, and some apoptosis as detected by terminal deoxynucleotidyl transferase assay(TUNEL) and the cleavage of Casp-3 and PARP-1. The phosphorylation of ATM and Chk2 was alsostronger after exposure to 2 Gy. Low-dose radiation nonetheless induced changes in the phosphorylationof 95 proteins (67 with increased and 28 with decreased phosphorylation). High-dose radiation resulted
in the modification of 109 proteins (68 with increased and 41 with decreased phosphorylation). Only
21 proteins with increased and 8 with decreased phosphorylation level were common in the two exposure
conditions. While common regulated proteins are mainly involved in cell growth, proliferation, survival
and death, low-dose exposure seems to activate cell maintenance and immune cell activation and highdose exposure activates several cancer-related pathways. Thus, the early responses to low- and high-doseradiation are largely different and affect different signalling pathways.
72. Low to moderate dose radiotherapy is used clinically to control some inflammatory conditions; forexample, treatment of benign inflammatory, hyperproliferative and degenerative diseases, including
painful elbow syndrome, painful shoulder syndrome, calcaneodynia, achillodynia and osteoarthritis of
the joints. The mechanisms underlying the anti-inflammatory effects of low/moderate dose radiotherapy
observed around 0.3 to 0.5 Gy were analysed in TNF-α activated EA.hy.926 endothelial cells exposed todoses ranging from 0.1 to 3 Gy. The transcriptional activity of AP-1 was selectively induced at 0.3 Gy[R12]. The induction of X-linked inhibitor of apoptosis protein (XIAP), a protein involved in themodulation of apoptosis and NF-κB activation, was also induced in a bi-phasic pattern, peaking at 0.5 Gy,
when apoptosis is minimal. Transfection with a siRNA targeting XIAP expression suppressed thedecrease in apoptosis and prevented the increase in radiation-induced NF-κB transcriptional activity
observed at 0.5 Gy [R13]. Thus, it appears that the anti-inflammatory effects of low-dose radiotherapy,
used to treat chronic inflammatory syndromes, are mediated in part by AP-1 induction of XIAP, whichthen prevents apoptosis and modulates NF-κB activation in endothelial cells.
73. The effects of radiation exposure on activated immune cells/immune cell activation were also
addressed in IgE-mediated allergic responses using either the rat mast cell line, RBL-2H3 [J24], humanmast cell lines or an in vivo mouse model [J25]. These responses are induced after the cross-linking ofcell surface expressed FcεRI receptor, which initiates a cascade of phosphorylation events that culminate

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in the release of mediators (histamine, beta hexosaminidase) and the production of inflammatory
cytokine. Exposure (0.01–0.5 Gy, 137Cs source, 0.8 Gy/min) of the different mast cell models before
FcεRI cross-linking prevents phosphorylation of proximal (Lyn, Syck, PKC, PLC gamma) and distal(ERK, JNK, p38) kinases and reduces the release of mediators and cytokines. The expression of the FcεRI
is also reduced by radiation exposure, in absence of cell death. The same decrease in FcεRI and incytokine production was observed in vivo in mice exposed to 0.05, 0.1 and 0.5 Gy. Thus, low-dose
exposure can inhibit immune cell activation by decreasing the cell surface expression of FcεRI receptors
and the phosphorylation of signalling kinases activated by these receptors in mast cells.
74. In a further study, the mechanisms of attenuation of mast cell activation by low-dose gamma
radiation were investigated in the RBL-2H3 rat mast cell line [S56]. Exposure to 0.01 to 0.1 Gy, but not
to 0.5 Gy (137Cs source, 0.6 Gy/min) was found to reduce FcεRI-activated mast cell migration. The
phosphorylation of PI3K and BTK kinases and activation of Rho GTPases involved in intracellular actin
dynamics were also reduced in these conditions, as was expression of the orphan nuclear receptor Nr4a2,
induced during mast cell activation. Inhibition of Nr4a2 inhibits PI3K/BTK activation, migration ofactivated mast cells, and their secretion of MCP-1. Anti-MCP-1 neutralizing antibodies inhibit activatedmast cell migration to about the same extent as exposure to 0.05 Gy. Thus, low-dose radiation could
modulate mast cell activation by attenuation of Nr4a2-induced MCP-1 production.
75. The putative mechanisms underlying radiation-induced cytokine secretion were also analysed inmurine macrophages following whole body X-irradiation [S26]. Peritoneal macrophages were harvested
from non-irradiated mice and mice exposed to 0.05–6 Gy at 12.5 mGy/min (0.05–0.2 Gy) or
0.287 Gy/min (0.5–6 Gy), from 4 to 48 hours post-exposure. Radiation exposure results in a dosedependent increase of IL-18 and IL-1β secretion ex vivo until 4 Gy, with a decline at 6 Gy. This increase
lasted for 48 hours after exposure to 0.075 and 2 Gy. In these conditions, the nuclear translocation of
p65/p50 and p50/p50 NF-κB dimers increase sharply for up to 8 hours post-exposure and then declines
for the next 40 hours. Expression of the TLR4 receptor and associated MD2 and CD14 co-receptors andof MyD88 followed mostly similar patterns, except that maximum induction was observed after 2 Gy. It
therefore appears that the whole signal transduction pathway is activated, from expression of membrane
receptors able to sense radiation-induced danger signals to adaptor proteins and transcription factors.
76. The level of Nrf2 (redox signalling) and phospho-IκBα (i.e. activation of NF-κB signalling) wereanalysed by Western blot in spleen cells at day 2 and 7 after exposure of C57BL/6 mice to 0.001, 0.01,
0.1 Gy gamma radiation (total body irradiation) in a single dose or in three fractions delivered onconsecutive days. There is an early (day 2) increase in Nrf2 at very low dose (1 mGy) and phospho-IκBαat all doses in mice exposed to three fractions, but no change in mice exposed to a single dose, whereas
the pattern is inverted at day 7: increase in Nrf2 for 0.001 and 0.01 Gy and phospho-IκBα at all doses inmice exposed in a single dose, but no changes in those mice exposed to three fractions. Thus, very low
and low doses do elicit a response in vivo. The kinetics of this response depends on the dose and dose
fractionation [S57].
77. The effects of low-dose radiation fractionation on the activation of the Akt and Nrf2 signalling
pathways was also analysed in the kidneys of mice with experimentally induced diabetes exposed to
25 mGy, 75 mGy or 3 times 25 mGy in 3 consecutive days of X-rays delivered at 25 mGy/min, 1 to
12 hours post-exposure. The level of p-Akt and p-Akt2 were increased for 12 hours after 75 mGy, but
only for 6 hours after 1 or 3 fractions of 25 mGy, Nrf2 induction was observed transiently, from 1 to6 hours after 75 mGy, and 3 to 6 hours after 3 fractions of 25 mGy. The expression of Nrf2 target genes
NADPH quinone oxidoreductase and heme oxigenase 1 was up-regulated from 3 to 6 and 3 to 9 hours,
respectively after exposure to 75 mGy delivered in one or three fractions [X1].

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78. To analyse the effects of low-dose gamma radiation and its eventual ability to modulate the response
to a further high-dose exposure, either proton or gamma radiation, Rizvi et al. [R9] quantified the levelof total and activated proteins belonging to different signalling pathways: NF-κB, JNK, p38MAPK, Lckand ZAP70 in CD4+ T-cells purified from irradiated mice 4 and 21 days post-exposure and activated invitro. Importantly, the modulation of the total and activated proteins was not always concordant, showing
different effects of radiation exposure on protein expression and activation of the signalling pathways.
Only activated proteins will be discussed below. At day 4, low-dose radiation induced only a downregulation of JNK and p38MAPK whereas at day 21, it induced an increase in NF-κB. Thus, different
signalling pathways are affected at different times during the response to low-dose radiation [R9]. Whenmice were exposed to a simulated solar particles event (sSPE; 2 Gy protons, 30 to 201 MeV) after low
dose and low-dose-rate exposure, the overall pattern resembled more the one evoked by low dose andlow dose rate alone than by sSPE alone, suggesting an imprint of prior low dose and low-dose-rate
response, broadly similar to an adaptive response. These observations were largely confirmed in afollowing study and extended to an exposure of 2 Gy of gamma radiation instead of sSPE after low doseand low dose rate, and from 21 to 56 days post-exposure [G14].
79. In summary, low-dose radiation modulates the activation of intra-cellular signalling pathways in all
types of human and animal immune, and non-immune cell types analysed, after in vivo or ex vivo
exposure. These effects may be visible after exposure to as low as 1 mGy in murine splenocytes. A given
pathway can be activated or inhibited according to the activated or resting status of the cell type analysed.
Low dose priming radiation exposure may modify the response to a subsequent high dose challenge. The
effects of radiation may follow non-linear dose–response relationships.

D. Gene and protein expression
80. That radiation exposure can lead to altered expression of genes was recognized in the UNSCEAR
1994 Report, annex B “Adaptive responses to radiation in cells and organisms” [U4]. The UNSCEAR
2012 White Paper on “Biological mechanisms of radiation actions at low doses” [U9] noted that gene
expression responses at high and low doses had been reported but were inconsistent. The Committee has
not previously undertaken a comprehensive review of the area.
81. Over the last several years studies have considered the impact of radiation exposures on gene andprotein expression. There are now data available from exposures at high, moderate and low doses as well
as more limited information from studies carried out at low dose rate. Here, gene expression is the main
focus as methods to assess transcriptional response are better developed and more comprehensive thanthose for protein expression. It should be noted, however, that gene expression changes per se will notalter cell phenotypes, that requires changes in protein expression.
82. Global changes in gene expression are probably one of the key components of biological responses
to radiation exposures. This is especially important in the case of low-dose exposures and their possible
long-term consequences, such as cancer. However, almost all work on radiation alteration of gene
expression considers only rather short post-exposure time points; as cancer develops only after protracted
lag periods of years, relating short term (up to 1 or 2 days) post-irradiation alterations to long term (several
years) development of cancer is very challenging. More work on expression changes following exposureand their evolution over time is required. Exposure to ionizing radiation regulates multiple genes as wellas signalling pathways in cells. Radiation response at the gene level may depend on radiation type or
quality, dose rate and dose. When compared to high-dose radiation exposures, responses to low-doseradiation exposures may differ both qualitatively and quantitatively. Consequently, it is an important

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issue to determine how robustly low- and high-dose responses can be considered as different and leading
to different long-term consequences. Dose–response relationships have been obtained for some genes
over dose ranges of 1 to 3 Gy and 0.5 to 4 Gy and down to doses as low as 20 mGy. Most of the studies
have used human blood lymphocytes, and this is often carried out with a view to the development of
biodosimetry methods. There are studies conducted on cells in culture as well as 3D skin models that
more closely mimic human tissue exposure situations. Transcriptional responses to radiation exposures
can be determined by global gene expression profiling or by reverse transcription (RT)-polymerase chain
reaction (PCR) for specific genes in question.
83. A number of studies focused on particular pools of genes and types of pathways. Selected genes
were generally validated by RT-PCR methods to confirm the differential expression by microarray
analysis. Transcriptomic analysis compared well with other modes of validation of specific genes. Morerobust analysis is required to strengthen the importance of such gene-expression studies and associatethem with cancer induction. Research articles published from 2006 to 2019 were reviewed and the results
are summarized in table 4.
84. Transcriptomic responses of peripheral blood cells from healthy individuals exposed chronically to
low doses of 0.2 to 49 mSv ranging from natural background levels to elevated levels due tocontamination accumulated over a period of 11 to 13 years as a result of the Chernobyl accident were
determined by complementary DNA cytokine and cytokine receptor microarrays, which revealed specificdifferential expressions of TGF receptor, CD40 and EB13 in more than 50% of the volunteers evaluated.
Though such direct studies are rather scarce, in vitro and experimental analysis of gene expression
profiling provided crucial information on the genetic pathways involved in tumour development
following low doses of ionizing radiation. Some of the most common gene products which were shown
to be differentially expressed following low-dose radiation exposure are: CDKN1A, GADD45, DDB2,
BAX and PCNA [K1, S54], i.e. genes known to be involved in carcinogenesis.
85. The majority of the research reviewed here on radiation-induced gene expression changes has been
conducted with an objective of developing biomarkers of radiation exposure. It is evident from theresearch covered in this annex that multiple signalling pathways are modulated by low-dose radiation
exposure. Genes that fall under the following pathways are important in maintaining the genome toprevent tumour development – cell death, cellular development, DNA replication, recombination andrepair, cell growth and proliferation, cell cycle, cell-mediated immune response. Canonical pathways thatwere affected by radiation exposure within the range of low to high doses include: DNA damage
response, apoptosis and, in general, molecular mechanisms of cancer. Some evidence suggests that lowdose radiation exposure may primarily modulate immune response, whereas high-dose exposure activates
the recognized radiation responses of p53 signalling pathway, DNA damage response and apoptosis
pathways at transcriptional levels. Genes such as CCR4, GNG11, and which belong to the cytokine andchemokine signalling pathways, immune-related and inflammation processes were shown to bedifferentially expressed at doses of 0.05 Gy [S54]. There is convincing evidence that low-dose, moderatedose and high-dose exposures lead to changes in biological processes within human blood lymphocytes.
Detailed analysis of the published data using a bioinformatics approach may help in understanding thedifferent signalling pathways altered after low-dose exposure. The data analysed so far, reveal thecomplexity of transcriptional changes in human blood cells at low levels of ionizing radiation. Some ofthese changes appear to be transient. However, long-term consequences of more persistent changes
cannot be ruled out.

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1. Gene expression changes in cells from occupationally/environmentally
exposed individuals and irradiated human blood and other cell types in vitro
86. The city of Ramsar, Islamic Republic of Iran, has one of the highest levels of natural backgroundradiation in the world (doses from radon ranging from 200 Bq/m3 to 3,700 Bq/m3). In the study by
Bakhtiari et al. [B5] blood samples from 60 individuals from a high background radiation area and from30 individuals from a normal background radiation area were analysed for two mismatch repair genes,
MLH1 and MSH2. There was a significant up-regulation of the MLH1 gene in residents of highbackground radiation areas. The association between MLH1 and MSH2 gene expression was seen in boththe sexes. The increased MLH1 expression was detected in people aged above 50 years and a decreased
expression of MSH2 was observed in ages under 50 years. The results suggest the activation of themismatch repair system in individuals living in high natural background radiation areas.
87. Albanese et al. [A9] generated gene expression profiles for mononuclear cells derived from 19
apparently healthy individuals (9 females and 10 males) exposed for 11–13 years to low-level ionizing
radiation (doses from 0.18 to 49 mSv, including elevated levels due to exposure as a consequence ofcontamination of the environment with 137Cs, 90Sr and Pu isotopes) in Belarus following the accident at
the Chernobyl nuclear power plant. These individuals did not show any signs of malignancy at the timeof analysis. Expression changes were recorded for specific cytokine and cytokine receptor pathways.
Multivariate statistical methods – hierarchical clustering analysis, principal component analysis andprojection pursuit – were employed in the analysis. The results showed distinct changes in the gene
expression pattern in cells from individuals exposed to >10 mSv and <10 mSv. Serine/threonine protein
kinase receptor, TGF receptor, EB13 and CD40 ligands were commonly expressed in irradiated samples.
The authors reported that they have identified differences in the genes involved in the pathways such as
growth factors, cytokine receptors and their cognate ligands as well as apoptosis-modulating proteins insamples from individuals that experienced exposures >10 mSv compared with those of <10 mSv. The
authors highlighted the variability in the analytical tools and complexity in the microarray analysis whilehandling the gene expression data. This is an important consideration and possible limitation of all suchstudies, the low sample size and potential for confounding due to infection or stress probably also limit
the interpretation of this study.
88. A study by Detours et al. [D16] involved a search for the gene signatures in papillary thyroid cancers(PTC) using the sporadic cases as well as samples from thyroid cancers in patients who were exposed toradiation as a consequence of the Chernobyl accident (i.e. samples from the Chernobyl Tissue Bank).
Tumours from 14 patients with no history of exposure to radiation and 12 patients from the Chernobyl
Tissue Bank were used in the analysis. Gene expression profile data from PTC from adult French patientswith no history of exposure to radiation and from Ukrainian patients exposed to radiation from the
Chernobyl accident during childhood were obtained in order to identify possible gene signatures forradiation carcinogenesis. The authors confirm their earlier finding that French and Chernobyl Tissue Bank
tumours have the same overall expression profiles and have indistinguishable B-Raf proto-oncogene
serine/threonine kinase mutation and RET/PTC frequencies. Globally transcriptional responses of humancells to H2O2 and gamma radiation (a dose of 2.5 Gy) were also similar. However, on a finer scale, a 118
gene signature discriminated the gamma radiation and H2O2 responses. They were able to classify tumours(such as those from France and the Chernobyl Tissue Bank) with an error of 15–27%. Similar results wereobtained with an independent signature of 13 genes involved in HRR. Even though both sporadic andradiation-induced PTC represent the same disease, they can be distinguished with molecular signaturesreflecting specific responses to gamma radiation and H2O2. Such signatures may reflect the susceptibility
profiles of the patients. Unlike most other studies described in this section, this study relates to gene
expression changes in tumours that are sampled long after radiation exposure, it is unclear if the alterations
observed are a direct or indirect consequence of the exposure.

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89. Samples from thyroid cancer tissues from 11 post-Chernobyl PTC and 41 sporadic PTC wereanalysed for gene signatures for radiation-induced thyroid cancers. Chernobyl radiation exposure inchildren ranged from 0.15 to 9 Gy. Microarray screening criteria included a change of about 5-folddifferences in the gene expression among the groups studied. About 646 up-regulated and 677 downregulated genes were identified in the tumour samples which discriminate the two PTCs. Seven genes(SFRP1, MMP1, ESM1, KRTAP2-1, COL13A1, BAALC and PAGE1) were shown to distinctively
separated in the two types of PTCs. Gene expression patterns which are reminiscent of aggressive tumour
type in older individuals were more pronounced in post-Chernobyl PTCs [P21].
90. Occupationally-exposed individuals from the Mayak Production Association, Ozeorsk, RussianFederation employed since 1949 were selected in the study by Abend et al. [A3]. Plutonium workers were
exposed to densely ionizing alpha particles along with external exposure to gamma radiation whereas thereactor workers were exposed to external gamma radiation only. A control group without any exposures
was chosen for comparison. This is a study of 146 individuals from the cohort of which 17 were exposed
to external gamma rays only (~0.5 Gy), 81 were exposed to both plutonium (~0.7 kBq) and gamma
radiation (~1.01 Gy) during their occupation from 1949 onwards and 48 unexposed controls. Blood
samples were taken from 150 individuals in 2011 with stringent exclusion criteria such as individuals
dying within 6 months of sampling or developing malignant cancer. Individual dose estimations weremade through records as well as by biophysical dosimetry. Peripheral blood was taken from workers
older than 70 years of age. Using transcriptional (microarray, mRNA) and post-transcriptional analysis(microRNA), the authors identified a set of 376 candidate genes (80 up-regulated and 296 downregulated) and all appeared to be associated with internal 239Pu exposure and to a lesser extent wereassociated with external gamma-ray exposure. A similar trend was observed for microRNA expressions
as well. Down-regulated genes predominantly associated with 239Pu exposure while up-regulated genes
after external gamma-ray exposure. However, no enrichment of genes related to certain biologicalprocesses could be found in this study.
91. Fachin et al. [F2] used gene expression profiling on lymphocytes derived from individuals who
were occupationally exposed to different doses of ionizing radiation and from unexposed individuals.
Peripheral blood samples were taken from 14 donors with a record of occupational exposure to radiation
and 9 donors with no radiation exposure history. Physical dosimetry records of these workers revealed
that the cumulative doses ranged from around 0.7 to 39 mSv. There were five males and nine females in
the occupational group, three males and six females in the control group. The workers included
physicians, nurses and radiological technicians. They were exposed during their work for an average of
9.32 ± 5.97 (mean ± SD) years. In addition, the workers were exposed to mixed radiation sources suchas X-rays, gamma rays and beta rays. Based on analysis of the “exposed” and “unexposed” groups, gene
expression data identified a total of 78 genes (21 up-regulated and 57 down-regulated) involved in several
biological processes such as ubiquitin cycles (UHRF2 and PIAS1), DNA repair (LIG3, XPA, ERCC5,
RAD52, DCLRE1C), and cell cycle regulation/proliferation (GSTP1, PPP2R5A, DUSP22). Some of thesegene expression changes were validated by an RT-PCR technique. Fachin et al. [F2] speculate that someof these gene markers might be suitable as biomarkers of radiation exposure and as such may be relevant
for risk estimation for workers exposed to radiation.
92. Visweswaran et al. [V13] investigated the quantification of entrance surface dose of X-rays fromneuro-interventional procedures to various regions (forehead, eyes, shoulders and thyroid) and correlated
the doses with the levels of early DNA damage biomarkers measured in the peripheral blood lymphocytesof patients. Fifty-one patients underwent a clinically indicated neuro-interventional procedures(diagnostic – cerebral angiography or therapeutic – embolization, stenting, flow diverter placement,
coiling, sclerotherapy and percutaneous transhepatic biliary drainage procedures) and doses received
were calculated to be in the range of 4.9–273 mGy (forehead), 14–398 mGy (eyes), 8–433.3 mGy(shoulders) and 4.7–242.5 mGy (thyroid). Blood samples were collected at different time points – pre-34

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exposure, and post-exposure (within 30 minutes). Phosphorylation of γH2AX and p53ser-15 wasincreased in samples obtained post-exposure compared to pre-exposure, but the increase was statisticallysignificant only for p53. Selected genes such as CDKN1A, FDXR, BAX, DDB2, SESN1, BCL2, MDM2,
TNFSF10B and PCNA showed statistically non-significantly reduced expression while GADD45A, ATM
and TNFSF9 showed elevated expression. The data suggest that neuro-interventional procedures as
explained above produced higher DNA damage and altered gene expression in patients after receiving
low doses of X-radiation.
93. Azimian et al. [A29] undertook a study to assess the effects of low doses of ionizing radiation on
the expression of specific apoptotic genes such as BCL2 and BAX. Blood lymphocytes from four healthy
donors were exposed ex vivo to 20, 50 and 100 mGy of 60Co gamma rays at a dose rate of 13 mGy/min.
Expression analysis was carried out at 4, 24, 48, 72 and 168 hours post-irradiation by real-time PCR.
They reported that the low doses used in the study can induce early down-regulation of BAX with normal
levels restored at 168 hours. BCL2, the anti-apoptotic gene, was up-regulated. They reasoned that, in viewof the transient nature of the observed effects, gene regulation/modification as early as four hours postirradiation may be due to adaptive response [A29].
94. El-Saghire et al. [E15] exposed blood samples obtained from 10 healthy donors to doses of 0.05 or
1 Gy of X-rays ex vivo at a dose rate of 30 mGy/min. Gene expression profiling was carried out on thesesamples and gene-set-enrichment-analysis (GSEA) was performed on differentially expressed genes.
Functional analysis on these genes exposed to a dose of 0.05 Gy displayed enrichment of chemokine andcytokine signalling. GSEA produced a higher ranking of inflammatory and immune-related gene sets thatare responsible for response and secretion of growth factors, chemokines and cytokines. At a dose of
1 Gy, GSEA revealed enrichment of established radiation-response pathways such as p53 signalling,
apoptosis, DNA damage response and repair. Subsequent validation with RT-PCR showed the significant
induction of specific chemokine-related gene alterations (PF4, GNG11 and CCR4) at a dose of 0.05 Gy
and DNA damage/repair genes such as DDB2, AEN and CDKN1A at a dose of 1 Gy. Thus, this study
suggests a differential response in gene signalling pathways at low and moderate doses of radiation which
may be relevant for assessment of risk to health based on the dose of radiation.
95. Fachin et al. [F1] exposed blood samples taken from four healthy individuals to doses of 20, 50,
100, 500 and 1,000 mGy of 60Co gamma rays for cytogenetic analysis. The dose rates used were
47 mGy/min for the first three dose levels (100 mGy and below) and 910 mGy/min in the last two. For
gene expression experiments, blood lymphocytes obtained from the four donors were exposed to 60Co
gamma rays at doses of 0, 100, 250 and 500 mGy (at a dose rate of 1.18 Gy min−1) and the cells were
cultured for 48 hours before conducting gene expression profiling. The study identified 86, 130 and 142
differentially expressed genes of which 25, 35 and 33 genes were exclusively modulated, at doses of 100,
250 and 500 mGy, respectively. Dose-dependent changes, up-regulation (↑) or down-regulation (↓), wereseen in some of the genes such as (a) CYP4X1(↑), MAPK10 (↓) and ATF 6 (↑) (100 mGy), (b) DUSP16(↑) and RAD51L1 (↑) (250 mGy), and (c) RAD50 (↑), REV3L (↓) and DCLRE1A (↓) (500 mGy). A set of
34 genes significantly up/down-regulated by radiation was common for all doses. In comparison,chromosome damage was seen at doses >100 mGy for total aberrations and >500 mGy for dicentrics.
The biological processes affected based on the transcriptional changes in genes were metabolism, stressresponse/DNA repair, cell growth/differentiation and transcription regulation. No information wasprovided on potential confounding factors for the described radiation responses.
96. Ghandhi et al. [G4] exposed human blood to doses of 0.56, 2.23 or 4.45 Gy of X-rays at a dose rateof 1,030 mGy/min (high dose rate) or 3.1 mGy/min (moderate dose rate). Even though the dose range
used in this study is from moderate to high doses, the relatively low dose rate study of 3.1 mGy/min is ofimportance to identify gene expression changes. The cells were subjected to gene expression profiling
24 hours after irradiation. The data showed that 454 genes were differentially expressed after irradiation

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with the 3.1 mGy/min dose rate at all doses. However, after acute or high-dose-rate exposure, 598 genes
were differentially expressed in cells at all doses. Gene ontology revealed genes related to immune
processes and B cell-mediated immunity was enriched in both sets of cells. However, acute exposure
produced enrichment of genes with functions related to natural killer (NK) cell activation and cell-to-cellsignalling. Further, significant enrichment of the p53 pathway was seen at all doses and both dose rates.
97. Gruel et al. [G18] studied the responses of different lymphocyte subtypes, namely CD4+, CD8+ andCD56+ cells, to ionizing radiation. Human blood lymphocytes from five healthy donors were exposed
ex vivo to doses of 0.05 and 0.5 Gy of 60Co (1.25 MeV) gamma rays at a dose rate of 0.45 Gy/min. The
different subsets of lymphocytes were subjected to microarray analysis at 3 and 24 hours post-exposure.
The data indicated that there was no change in the percentage of the different subpopulations oflymphocytes following irradiation. BAX, PCNA, GADD45, DDB2 and CDKN1A were induced at
24 hours following a dose of 0.5 Gy, but not 0.05 Gy. The number of modulated genes differed between
cell types. The number of down-regulated genes was 10 times higher for the CD4+ population than forother cell types three hours after exposure to a dose of 0.05 Gy. Pathway analysis has indicated that mostof the down-regulated genes participate in the cellular processes of protein biosynthesis and oxidative
phosphorylation. Several biological pathways in CD4+ cells seem to be sensitive to radiation exposure.
98. Knops et al. [K29] exposed human peripheral blood lymphocytes from six healthy donors to low(0.02 and 0.1 Gy), moderate (0.5 and 1.0 Gy) and high (2 and 4 Gy) doses of 137Cs gamma rays. Gene
expression analysis using whole human genome DNA microarrays was used on lymphocytes at 6, 24 and48 hours (with those lymphocytes exposed to moderate and high doses) and 24 and 48 hours (with thoselymphocytes exposed to low doses) after irradiation. The authors identified nine genes that could be used
to determine low-dose radiation exposure with 96% sensitivity with doses in the range of 0.02 and 0.1 Gyused in the study. Expression alterations increased with increasing dose and time after exposure. Alteredgenes were assigned to biological processes such as nucleosome assembly, apoptosis and DNA repair
response. The authors concluded that it is possible to use gene expression profiling data to determine
radiation exposures as low as 20 mGy. At all doses, genes involved in proteolysis and in the positive andnegative regulation of apoptosis were significantly altered as determined by one-way analysis of variance(p<0.05). This study has identified 9 genes (MKL2, FDXR, C10orf39, PFKFB3, FLJ35379, LY6G5C and3 other genes with no gene symbols available (Agilent ID A-24_P506680, A_32_P138939,
A_32_P20997)) as signature genes which discriminate low-dose exposures.
99. The objective of a study by Manning et al. [M10] was to develop a dose–response relationship forgene expression profiling following low-, moderate- and high-dose ex vivo irradiation of blood
lymphocytes. Peripheral blood samples were collected from 26 healthy human donors (17 females and9 males). Moderate to high doses (0.5, 1, 2 and 4 Gy) and low doses (5, 10, 20, 50, 75 and 100 mGy) of
X-rays (250 kV and 0.2 mA) were used in the study at a dose rate of 0.5 Gy/min (for the moderate- tohigh-dose study) or 4.9 mGy/min (for the low-dose study). Blood aliquots were irradiated, and thesamples were studied for cell viability. At 2 and 24 hours after irradiation, multiplex RT-quantitative(q)PCR was used to determine the expression profiles of selected genes. The results indicated thatmoderate- to high-dose response curves for individual genes (from doses of 0.5 to 4 Gy) were best
constructed using a polynomial fit while low-dose response curves used a linear fit. The authors also
observed individual variations in the gene expression data. The experiments were conducted more froma biodosimetric point of view. However, the data were obtained for certain genes such as CDKN1A,
PCNA, MYC, DDB2, MDM2 etc. that may have roles in carcinogenesis [M10].
100. Paul and Amundson [P6] studied gene signatures that developed in human blood lymphocytes
following ex vivo exposure to doses (moderate to high) of gamma radiation of 0.5, 2, 5 and 8 Gy using
whole genome microarray expression profiling. Peripheral blood samples were taken from 10 donors(with five independent experiments each at 6 hours post-irradiation and 24 hours post-irradiation using

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different donors for each experiment and time-point) and gene expression profiling was generated forbiodosimetry purposes. On the basis of a pooled analysis from all donors, the study identified a 74 genesignature which was distinguishable between the four different radiation doses used. More than one thirdof the genes were regulated by p53. Five selected genes (CDKN1A, FDXR, SESN1, BBC3 and PHPT1)
were also validated by RT-PCR.
101. Turtoi et al. [T23] exposed blood from a healthy donor to radiation doses of 1, 2 and 4 Gy (137Cs
gamma rays, 0.8 Gy/min) and performed gene expression analysis to identify early radiation-response
genes. Blood lymphocytes from another two healthy donors were irradiated with similar doses as above
and the identified gene markers were validated in these samples. This study was conducted with anobjective to develop biomarkers of exposure for use in biodosimetry. Twenty-four of 102 genes identified
as markers showed a dose-response pattern. Fifteen genes were up-regulated (CD69, CDK1A, EGR1,
EGR4, FLJ35725, hCG2041177, hCG16434662, IFNG, ISG20L, C-JUN, MDM2, MUC5B, PLK2,
RND1, TNFSF9). However, the other nine genes were down-regulated (GRIK3, OCLN, RPL10A,
SERHL2, SGK3, STARD13, hCG2038519, hCG1998530, hCG1985174). Genes such as EGR1, EGR4,
IFNG, C-JUN and TNFF9 produced significant correlations with absorbed radiation dose over the range
of 1–4 Gy.
102. Turtoi and Schneeweiss [T24] exposed human lymphocytes obtained from a healthy donor to doses
of 1, 2 and 4 Gy (137Cs gamma rays, dose rate of 0.8 Gy/min). Quantitative evaluation of early response
proteins and genes was carried out at two hours post-irradiation. A set of early response proteins andgenes was identified based on the response at two hours post-irradiation. Most of the proteins and genes
indicated alterations of cellular structure (BACTIN, TALIN 1, TALIN 2, ZYXIN-2), immune and defence
reactions (MBP2, IL17E and IFNG), cell-cycle control (CDKN1A, MDM2, ANXA6, GADD45A, PCNA,
DUSP) as well as detoxification processes (PEX1) and apoptosis (BBC3). The main aim of this study was
to identify biomarkers of exposure for biodosimetry purposes.
103. McDonald et al. [M26] exposed primary human prostate fibroblast cells and a prostate epithelial
cell line (RPWE-1, human papilloma virus 18 (HPV-18) immortalized) to acute and chronic gamma
radiation. Acute radiation exposures were performed using a 137Cs source that gave a dose rate of
0.48 Gy/min. The total doses were 0.1 and 2 Gy. Chronic irradiation employed a 57Co source giving adose rate of about 24.5 mGy/d (approx. 0.017 mGy/min). The exposure times were about 7,000 and
1,500 minutes. Samples were subjected to clonogenicity assays and gene expression profiling 24 hours
after irradiation. A total of 396 genes were shown to be significantly differentially regulated by both acuteand chronic doses compared to non-irradiated samples. There was no measurable effect at doses of
100 mGy. There were 106 genes which are common between the samples that had been given an acutedose of 2 Gy compared to those that had been subjected to chronic doses. These genes were downregulated and were involved in cell cycle or chromosome movement in M-phase. Pathway analysis
revealed that the genes which were differentially expressed map to the biological processes such as cell
cycle, chromosomal movement, cell survival and DNA replication, recombination and repair as well as
predicted activation of transcriptional regulators p53 and Cdkn2a. Most of them showed decreased
expression following irradiation. Prostate epithelial cells displayed functional decreases in proliferationand mitotic cells following exposure to a dose of 2 Gy or after chronic irradiation.
104. Zhou et al. [Z20] used normal human fibroblasts (NHF1, NHF3, NHF10) and their hTERTimmortalized versions in an experiment where the cells were exposed to doses of 0.5, 1.5, 4.5 Gy of 137Cs
gamma rays at a dose rate of 0.84 Gy/min. A dose of 1.5 Gy reduced the colony-forming ability of thecells, thereby affecting their proliferation. This dose was therefore used in the experiments on gene
expression profiling. Fibroblasts displayed severe cell-cycle arrest from G2 to M at two hours postirradiation and G1 to S at 6 and 12 hours post-irradiation. Global gene expression profiling revealed nineradiation-responsive genes which were commonly affected in all the three different cell types including

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a dominant p53-dependent G1 checkpoint response. Many genes regulated by the E2F familytranscription factors (CDK2, CCNE1, CDC6, CDC2, MCM2) were significantly down-regulated at
24 hours after irradiation. Several affected genes fell under processes such as DNA metabolism. The
results revealed a highly stereotypic pattern of response to ionizing radiation in human fibroblasts, which
could be the result of synchronization of cell cycle following radiation exposure.
105. Mezentsev and Amundson [M31] exposed EPI-200, a 3D tissue model which mimics theconstruction and function of human epidermis, to high (2.5 Gy) and low (0.1 Gy) doses of low-LET
protons. Samples were exposed to 0, 0.1 or 2.5 Gy 4.5 MeV protons (5.5 MV singletron accelerator) andthe samples were cultured for 4, 16 or 24 hours before gene expression analysis. Gene ontology analysis
revealed that the genes involved in the recovery and tissue repair were altered at the low dose while thehigh dose affected the genes involved in structural integrity and terminal differentiation. Network analysisof the significantly responding genes indicated that the dose of 2.5 Gy produced a p53-response while
Hnf4A, a transcription factor not previously associated with radiation response, was prominent following
the low-dose exposure. The authors found that Hnf4A protein levels and its phosphorylation were
elevated in tissues and cells after low- but not high-dose irradiation.
106. Tilton et al. [T13] used the reconstituted human skin model EpiDermFT in their experiments. The
3D skin model was exposed to doses of 0.1, 2 and 10 Gy over periods of 3.0, 3.4 and 6 minutes at dose
rates of 0.033, 0.6 and 1.6 Gy/min, respectively. Using a systems biology approach, dose- and timedependent responses in transcripts were determined using the direct RNA sequencing (RNAseq) method.
A total of 1,701 genes were significantly affected by high-dose radiation exposure (2 or 10 Gy) with amore pronounced effect at 10 Gy. About 29 genes, including BBC3, PPM1D, FDXR, GADD45A, MDM2,
CDKN1A, TP53inP1, CYCSP27, SeSN1, SESN2, PCNA and AEN were differentially expressed at dosesof 2 and 10 Gy but not at 0.1 Gy at both 3 and 8 hours timepoints. A higher effect was observed at 10 Gy
with a larger set of up-regulated genes. At higher doses, down-regulated genes exhibited a linear dose
response and these genes regulate the cell cycle. Very few genes were altered by exposure to a dose of
0.1 Gy. It was seen that genes regulating cell cycle progression and inflammatory responses consistently
displayed opposite trends in their regulation compared to the high-dose irradiated groups. The study
highlighted the fact that certain biomarkers identified could be useful in predicting future disease
outcomes in individuals.
107. A study of the temporal response to radiation exposure of a 3D human skin tissue model usingtranscriptional profiling by microarray analysis was carried out by von Neubeck et al. [V14]. Skin tissue
was exposed to a dose of 100 mGy from X-rays at a dose rate of 6.3 mGy/min. Radiation-induced cell
type specific changes in the genes, with >1,400 genes differentially expressed in the dermis while >400
genes were regulated in the epidermis. There were no overlapping genes which showed differential
response in both tissue compartments. Pathway analysis showed changes in the processes such as cellcycle regulation, immune response, hypoxia, reactive oxygen signalling and DNA damage repair. The
paper discusses the role of cell proliferation and emphasizes how dysregulation might lead to radiationinduced diseases including cancer. The data from this study are not comparable with Tilton et al. [T13]though both have used similar experimental model. The X-ray dose and dose rate and machine parameters
were different in the studies.
108. Skin keratinocytes were exposed to low doses of ionizing radiation (Varian 2100 C linear
accelerator) (doses 10 or 100 mGy) at 800 mGy/min [B30]. Expression of selected proteins was analysed
1 or 4 days post- exposure. Proteomic analysis revealed that seven of the 11 proteins (epidermal-fatty
acid binding protein (EFABP), alpha-enolase, histidine triad nucleotide-binding protein (HINT-1), heatshock protein (HSP27), lactate dehydrogenase A (LDH-A), protein disulphide isomerase precursor (PDI)and S100A9) showed significant differences for radiation exposure alone. In combination with arsenic,

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these effects might have influence on the long-term consequences in the form of skin carcinogenesis fromcombined exposures.
109. Normal human dermal fibroblasts were exposed to low-dose radiation at 0.1 Gy gamma radiationin the study by Bae et al. [B2] and cellular responses were analysed at 6 and 24 hours post-irradiation.
Differential cell viability and cell-cycle status were observed at the two timepoints studied. Collagen typeI alpha 1 (COL1A1) mRNA expression was up-regulated at 24 hours while matrix metalloproteinase-1(MMP1) mRNA was down-regulated at 24 hours post-irradiation. The authors speculated anti-ageing
effects of low-dose radiation based on the regulation of collagen synthesis-related genes. microRNA
analysis revealed that some of the deregulated miRNAs are associated with early or late radio-adaptive
response. However, the authors did not provide any functional studies to prove their speculation onreverse ageing or adaptive response.
110. Recently, Lowe et al. [L45] published a study on chronic irradiation experiments with primary
human cells. Primary cells from human neonatal foreskin, adult facial skin and RPE-1 cells were used in
the study. Cells were chronically irradiated by exposing them to 137Cs gamma rays at the following dose
rates for 7 days: 6 mGy/h (accumulated dose 1.0 Gy), 8 mGy/h (accumulated 1.3 Gy), 12 mGy/h(accumulated 2.0 Gy) and 20 mGy/h (accumulated 3.4 Gy). Acute exposures were carried out with Xrays at 0.5 Gy/min. Chronic radiation exposure resulted in the reduction of histone levels. Protein analysis
revealed specific reductions in the levels of non-histone chromatin associated proteins such as HMGB
and SMC as well as levels of certain proteins involved in DNA replication and cell-cycle progression(MCM2-7, SMC1A/3, POLD1). Pathway analysis of proteins indicated that the altered proteinsparticipate in DNA replication, cell cycle, DNA mismatch repair, base excision repair and nucleotide
excision repair pathways. In addition, the levels of HMGB2, Lamin B1 and TMPO were reduced in
irradiated along with elevated expression of p16 (CDKN2A) and p21 (CDKN1A) – influencing theactivation of cellular senescence in the irradiated cells. Histone reductions were also accompanied by anincrease in global transcription including that of pro-inflammatory genes. This study concluded thatchronic irradiation, at low dose rates, can promote cellular senescence indirectly possibly via anepigenetic route, limiting the tumorigenic events.
111. Sokolov and Neumann [S54] exposed four different lines of human embryonic stem cells to dosesof 0.05 and 1 Gy from 60Co gamma rays (a teletherapy unit was used but no dose-rate information wasprovided). The authors used one low-dose and one high-dose exposure in their experiments to determine
the changes in gene expression at 2 or 16 hours post-irradiation. The data showed that the gene expression
profiles are dose-, time- and cell type-dependent. About 50 genes were identified as potential early
response signatures of high-dose radiation exposure across all the cell types studied. However, the authors
identified substantial differences in the signalling pathways affected by low- and high-dose radiation.
CDKN1A was shown to be up-regulated in all the cell types and after low-dose exposure (0.05 Gy).
Commonly affected pathways are cell-cycle arrest, and p53 signalling in all the cell types.
112. Blood samples were collected from 8 healthy adult volunteers and peripheral blood mononuclear
cells were exposed ex vivo to 300 mGy and 1 Gy of gamma rays (60Co) at a dose rate of 0.4 Gy/min.
Proteomic analysis was carried out by mass spectrometry after 1 and 4 hours after irradiation. Alterationsof 23 proteins were observed in the irradiated cells compared to the control peripheral blood mononuclear
cells with +/−1.5-fold change whereas three proteins showed >2.5-fold change. Redox sensor protein(chloride intracellular channel protein 1- CLIC-1), antioxidant protein (peroxiredoxin-6) and pro-survival
molecular chaperone 78kDA (glucose regulated protein GRP78) were among the 23 modulated proteinsafter radiation exposure in peripheral blood mononuclear cells. The response seemed dose dependent
where coefficient of variation calculated to be at 33.7% for 300 mGy and 48.3% for 1 Gy. Proteomic
responses may help in understanding the cell protective mechanisms following irradiation [N16].

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113. The human lymphoblastoid cell line AHH-1 was irradiated with 0.05, 0.2, 0.5, 2.0 and 10 Gy(60Co gamma rays) at 1.7 Gy/min (for doses >0.5 Gy) or 9.6 mGy/min (for 0.2 and 0.05 Gy). Global
transcriptional changes were analysed 4 hours after irradiation. It was found that doses as low as 0.05 Gy
produced efficient induction of transcriptional changes including up-regulation of 25 genes, some ofwhich are involved in signal transduction pathways (BMPR2, GPR124, MAPK8IP2, AGGF1) and thedown-regulation of 18 genes. The dose range of 0.05 to 10 Gy induced changes in a number of radiation
response genes, i.e. DNA repair gene XPC, TP53I3 and immediate early response 5 gene in a dose
dependent manner. The identification of such early response genes provides opportunity to develop
biomarkers of radiation injury as well as to understand the mechanisms of their biological effects [L41].
114. Sanzari et al. [S9] exposed human thyroid epithelial cells (Htori-3) to high-atomic-number andenergy (HZE) particles at a dose of 100 mGy from 1.0 GeV/nucleon Fe ions. The experiments were also
conducted on cells treated with 5 µM selenomethionine to determine the protective effects of thisantioxidant. Two hours after exposure to Fe ions, gene expression profiling was carried out on cells withor without selenomethionine treatment. Radiation exposure up-regulated the expression of certain classes
of genes, namely secreted proteins of the chemokine/cytokine gene cluster (CXCL1, CXCL2, IL6, IL11,
IL8, IL24 and TGFB2). Selenomethionine treatment significantly decreased the expression of the above
up-regulated genes. Selenomethionine counteracted the radiation-induced expression of several specificgenes, PLAU, IGFBP3, FOLR1, B4GALT1 and COL1A1. Validation of the altered expression of certain
genes was carried out by qPCR. While the study specifically highlighted up-regulation of the genes
belonging to selected pathways (chemokine-cytokine pathways), selenomethionine regulated thesealtered expressions as a countermeasure for some of the acute inflammatory/immune responses induced
by low-dose HZE-particle irradiation.
115. It is clear that one of the key responses to ionizing radiation is the activation of cells to reprogramme
gene expression. Previous studies have not distinguished between changes in RNA synthesis and RNA
turnover. A recently developed BruDRB-seq (Bru-seq) methodology by Veloso et al. [V6] determines
the contribution of synthesis as well as degradation of RNA to transcriptional responses. The techniqueallows the measurement of rapid changes in transcription rates alone without the influence of pre-existing,steady-state RNA and assessment of transcription originating from different promoters of multi-promoter
genes. Exposure to human HF1 and HCT116 cells to 2 Gy X-rays (acute) revealed that ATM and p53
regulate both RNA synthesis and stability. Moreover, many genes in the p53-signalling pathway werecoordinately up-regulated by both increased synthesis and RNA stability while down-regulated genes
were suppressed either by reduced synthesis or stability. To distinguish between DNA damage anddamage to other biomolecules (e.g. proteins) transcriptional responses were measured in cells in which
DSBs were induced by restriction enzyme (AsiSI) or alternatively treated with Nutlin-3 to activate p53[N10]. Comparing the top 200 genes showing induced transcription revealed 39 genes common between
the three treatment groups (ionizing radiation, restriction enzyme, p53 activator) many of which were
known as p53 target genes. A functional annotation analysis of the database for annotation, visualizationand integrated discovery revealed that DSBs by restriction enzyme and p53 induction by Nutlin-3 lead totranscriptional changes of genes belonging to similar groups as 2 Gy ionizing radiation. This also suggeststhat it is p53 activation in response to DSBs that drives the transcriptional reprogramming after radiation
exposure at moderate doses. This technique has the potential to define more precisely the effects of lowdose exposures on de novo gene transcription.
116. There are several reports of gene expression changes which have used moderate to high doses ofradiation in their studies; these are summarized in table 4 below. Although high doses are used in thesestudies, the changes observed in the genes in response to radiation exposure may be relevant for lowdose exposures and are important in terms of understanding dose–response relationships. Currently there
is no agreed gene expression pattern unique to either high- or low-dose exposures. Many of the genes

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belong to the signalling pathways which have major implications in cancer development. Data obtained
from radiotherapy patients are summarized as well.

2. Gene expression changes in vivo in experimental organisms and in cells
from radiotherapy and cancer patients
117. Work with animals exposed to whole-body irradiation has revealed numerous gene expression
changes, with many groups of genes affected by radiation in ways unique for specific animal genotype,tissues and organ(s), doses and dose rate. Similar to in vitro cell assays – even within the same tissue, nogene was found to be universally modulated by same or similar low-dose or low-dose-rate radiation
exposures in all cases.
118. For example, the study conducted by Lee et al. [L13] used gamma rays at total dose of 0.2 Gydelivered at dose rate of 3.81 Gy/min for whole-body irradiation of 7-week-old female C57BL/6 mice.
Several different tissues (brain, heart, lung, spleen and intestine) were collected one or three days after
exposure and evaluated by immunohistochemistry for presence of p53 and p21 proteins and for numbersof apoptotic cells (TUNEL assay). At day 3, all tissues showed increased staining for p53 and p21
including those tissues (brain and heart) where apoptosis was not evident. Expression of 23 genes was
explored by RT-PCR in all organs at day 3; the genes neogenine, Apo1, nuclease-sensitive elementbinding protein 1, syntaxin, cyclin G1, hNOP56, paraoxonase and glutathione peroxidase were found to
be increased in all tissues, albeit less than twofold in most cases.
119. A study by Meadows et al. [M28] sought to determine gene signatures in peripheral blood of miceand humans and to determine the specificity of these signatures under different conditions. Mice of
C57BL and BALB/c strains (5–10 mice per group 10–11-week old) were exposed whole body to 137Cs
gamma rays at doses of 0.5, 2 and 10 Gy. Human blood samples were obtained from healthy volunteers,
patients who were exposed to 1.5 to 2 Gy (as part of pre-transplantation screening for stem celltransplantation and patients prior to chemotherapy – bloods from 18 healthy donors, 47 patients prior tohaematopoietic stem cell transplantation, and 41 patients who had undergone alkylator-based
chemotherapy only). DNA microarray analysis was carried out on these samples at specific timepoints.
A clear pattern of gene expression was identified in mice without any influence of strains used in the
study. However, sex did influence the accuracy of the assessment of radiation exposure at the lowest levelof dose of 0.5 Gy. Gene signatures were able to distinguish the radiation exposure and sepsis conditions.
Comparison of lymphocyte gene expression profiles of radiation-exposed individuals (1.5–2 Gy) andchemotherapy patients distinguished radiation exposure from chemical exposure. The study was more
focused on distinguishing gene signatures than predicting cancer risk using such data [M28].
120. Meadows et al. [M29] identified gene signatures in peripheral blood lymphocytes following partialbody irradiation of mice. C57BL/6 mice were exposed to doses of X-rays from 0.5, 2 to 10 Gy (hardened
by 0.1 mm Cu/2.5 mm Al filtration). The dose rates used were 1.4 Gy/min (anterior and posterior region)and 1.25 Gy/min (hindlimb region). Gene expression analysis indicated the radiation status with a highlevel of accuracy compared to the non-irradiated mice. The differential gene expression profiles forwhole-body and partial-body irradiation were different – different sets of genes were affected. Since thegene profile changes are different for partial-body and total-body irradiation, partial-body signatures fromtotal-body irradiated mice failed to completely predict the radiation status of partially irradiated animals
or non-irradiated controls. However, the study can be applied in biomarker identification andbiodosimetry in the case of partial-body exposure during therapy or accidents as they produce uniquegene signatures, but authors suggest the use of multiple signatures [M29].

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121. In a different study, 8-week-old male C57BL/6J mice were exposed to chronic whole-body gammaray radiation in two separate experiments. In the first one, dose rates used were 16.6, 0.858 or
0.042 mGy/d for 485 days, accumulating to total doses of 8, 0.4 and 0.02 Gy, respectively. In the second
experiment, dose rates were 20.0, 1.00 or 0.050 mGy/d for 401 days accumulating to total doses of 8, 0.4
and 0.02 Gy, respectively [U1]. Gene expression profiles in the liver of six animals from each exposuregroup were compared individually with pooled sham-irradiated animals using microarrays; findings wereconfirmed by RT-PCRs. Numbers of genes modulated more than 1.5-fold in mice irradiated with total
dose of 8, 0.4 and 0.02 Gy were 20, 11 (nine of them shared with 8 Gy mice) and 3 (all of them sharedwith 0.4 Gy and 8 Gy mice), respectively. Most of these genes were down-regulated (Usp2, Dbp, St3gal5,
Zbtb16, Lcn13, Herpud1, Rapgef4, Slc22a5, Hspa1b, Serpina4-ps1, Angptl4, Slc25a30, Socs2, Alas1,
Pim3, Lpin2, Gne, Acot1) and only two in the 8 Gy group (Camk1d and D9wsu90e) and one (Pde4b) in
the 0.4 Gy group were increased compared to controls. The reported bioinformatics analysis suggested
that these genes may be involved in obesity and tumorigenesis.
122. Seven-week-old C57BL/6 mice were X-ray irradiated at high-dose rate of 1.03 Gy/min orcontinuously exposed with a dose rate of 3.09 mGy/min to total doses of 1.1, 2.2 or 4.4 Gy and theirblood drawn 24 hours later for RNA isolation [P8]. After microarray analysis and RT-PCR confirmationof select transcripts, mRNAs with similar or opposite patterns of expression dependent on dose rate wereidentified; a group of 164 genes could be used as a classifier for different exposures.
123. Six- to seven-week-old female Institute of Cancer Research (ICR) mice were exposed to 0.25, 0.5
or 1 Gy of gamma rays delivered at dose rate of 500 mGy/h or 500 mGy/min and sacrificed after fourhours [M13]. Skin samples were collected, and RNA used for RT-PCR analysis. A group of 80 genes
participating in regulation of extracellular matrix or acting as adhesion molecule genes was tested; inmost cases gene expressions did not follow dose rate or dose differences. MMP2 and MMP15 showedmost change in response to 500 mGy/h dose rate and decreased with the increase of dose, while MMP1
and MMP9 responded best to 500 mGy/min dose rate and increased with total dose. This study suggeststhat exposure to high-dose-rate photon radiation induced oxidative stress and extracellular matrixassociated alterations in gene expression profiles.
124. Expression of DNA repair genes in thymi of low dose rate irradiated AKR/J mice was the focus ofa study by Bong et al. [B50]. This work compared AKR/J mice housed in a low-dose-rate/high-total-dose(137Cs, 0.7 mGy/h, 2.1 Gy) gamma irradiation facility with sham or high dose rate (137Cs, 0.8 Gy/min,
4.5 Gy) irradiated mice. The thymi were isolated, at the conclusion of irradiation in low dose rate exposed
mice, or 130 days after acute irradiation in high dose rate mice. Genes including MSH2, MSH3, ERCC8,ERCC6, GTF2H2, OGG1, LIG4, NEIL3, UNG, LIG1, RAD51L1, MBD4, TERF2, H2AX, ATM, TFRC,
FTH1, RRM1, RRM2, RRM2B, P53, BAX and PARP1 were up-regulated according to RT-PCR evaluationin both groups of mice (albeit to different degrees, and with most of the genes over- or under-expressedby less than threefold. Low dose rate exposed animals developed fewer thymic lymphomas and lived
longer; moreover, an increased expression of DNA repair-associated genes, LIG4, RRM2, H2AX and
ATM, was noted at 130 days after the start of low-dose-rate irradiation, although no functional DNA
repair data were presented.
125. Mammary tissue differences, both up-regulation and down-regulation in expression of lncRNAs
were studied in BALB/c and SPRET/EiJ mice, known to have different susceptibilities to breast cancer
due to non-homologous-end-joining of DSBs differences between the strains, were studied by Tang et al.
[T10] using microarray analyses. Baseline inter-strain differences between non-irradiated controls in thiswork were significant with nearly 300 lncRNAs and 600 mRNA differentially expressed. X-ray
irradiation to a single dose of 100 mGy was carried out with animals aged 8 to 9 weeks and mammary
tissues were collected for analyses 2, 4 and 8 weeks after exposure for BALB/c mice and at four weeks
for SPRET/EiJ mice. Several interesting considerations were employed in this work – for example,

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lncRNA expression was internally verified by correlating it to expression of a dependent mRNA (1,337 of
1,338 lncRNAs identified to be differentially expressed in response to 100 mGy exposure in either strain
were verified in this manner); gene groups associated with changes of expression compared to controls
were “subtracted” for genes regulated by the oestrus cycle. Importantly, more than 3,000 genes are
modulated by oestrus cycle, so after this subtraction groups of about 300 lncRNAs and mRNAs were
found to be “specific” outcomes of radiation exposure. Several gene expression pathways were associatedwith low-dose exposure in each strain; interestingly, enrichment for inflammatory response genes wasnoted in both strains [T10].
126. A study by Cassie et al. [C7] used C57BL/6 mice divided into controls, acutely exposed group(500 mGy at a dose rate of 2 mGy/s), and a group subjected to protracted exposure (50 mGy/d for
10 days). At the start of the experiment, animals were 45 days old and they were sacrificed three hours
after the final exposure given to the mice exposed to protracted radiation. Spleens from irradiated animals
showed signs of both apoptosis and cell division with some differences between two sexes. Apoptotic
index of spleen cells was greater in males regardless of the means of exposure, and as a consequence cellproliferation was greater in males as well. Differences between the two types of exposure were noted aswell. The most pronounced sex differences were found in expression of Ki67 in spleen of mice acutely
exposed to 500 mGy and expression of PCNA in spleens of mice exposed to 500 mGy over 10 days.
127. Snijders et al. [S50] compared BALB/c mice (breast cancer prone) with C57BL/6 mice (resistant bycomparison), for gene expression in mammary tissue and blood cells. Exposures of 75 mGy and 1.8 Gy
were given weekly, for 4 weeks (total doses of 300 mGy and 7.2 Gy). Strain and exposure differences werenumerous but with substantial overlaps; the more resistant mice showed down-regulation of SOX9, whichis associated with breast cancer. The sensitive strain showed early transcriptional responses in pathways
involving immune response (diminished), cell stress (increased) and altered TGFβ signalling and abnormal
expression of developmental genes. The study provides evidence for cancer-risk prediction based on theappropriate gene expression changes following radiation exposure.
128. Yi et al. [Y8] studied proteins by 2D gel electrophoresis in samples taken from chronically exposed
C57BL/6J male mice (6 weeks of age, 40 mice in 4 groups). The exposures took place over 180 days
(22 hours per day); the dose rates used were <50, 50–500 and 500–1,000 µGy/h from 137Cs gamma rays.
The higher dose rates produced pathological changes in the liver. 2D analysis showed that with increasing
dose rates 20, 21 and 28 proteins had 1.5-fold changes in their expression compared to control. Morestringent analysis showed that about 22 proteins had >2.5-fold changes in their expression. These proteins
belong to the pathways such as cytoskeleton, cell metabolism, biological defence, mitochondrial damage,detoxification and tumorigenesis. The authors validated the expression of one protein, calreticulin, whichis involved in stress reactions in the mouse liver as a marker of low-dose-rate exposure. They concluded
that calreticulin may be a marker of exposure and may be used to predict the risk of radiation-induced
liver tumorigenesis in mice.
129. Hurem et al. [H41] used the vertebrate model of zebrafish to determine the toxic effects as well as
the transcriptomic changes of radiation exposure. Fish embryos were exposed to gamma rays over 3 to
92 hours beginning with 2.5 hours post-fertilization, with dose rates varying between 0.4 and 38 mGy/hfor toxicity assays and 0.54, 5.4 and 10.9 mGy/h for transcriptomic analysis. Total doses ranged between
1.6 to 3,496 mGy after 43.8, 92 and 3 hours exposures. The survival of fish following irradiation at adose rate of 38 mGy/h was significantly reduced, and viable embryos showed developmental changes
following exposures at all dose rates. RNA sequencing analysis was carried out at 5.5 hours postfertilization. There was a dose–response relationship in the total number of differentially expressed genes
in the exposed groups starting at a dose rate of 0.54 mGy/h. Pathway analysis indicated retinoic acidreceptor activation, apoptosis and glutathione mediated detoxification signalling as the most affected
pathways at low dose rates, while eif2 and mTOR involved in the modulation of angiogenesis were

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affected at high dose rates. Altered expression of Myc, Tp53, Tnf, Hnf4a, Tgfb1 and Cebpa were identified
as the most radiation responsive genes that also affect the developmental processes in zebrafish.
130. Zhikrevetskaya et al. [Z18] exposed fruit flies to low and moderate doses of gamma rays (50 to
400 mGy) at a dose rate of 36 mGy/h. Flies were exposed for 1 hour 23 minutes, 2 hours 47 minutes,
5 hours 34 minutes and 11 hours 8 minutes, resulting in doses of 50, 100, 200 and 400 mGy, respectively.
Changes in lifespan and expression of stress-sensitive genes were identified. The authors found extended
lifespan for males exposed to 50 mGy and in females exposed to 400 mGy but a lifespan decrease infemales at lower doses. Changes in stress gene expression were not dose-dependent. All in all, the study
showed a non-linear response to low-dose radiation exposure and sex-specific radiation resistance inDrosophila melanogaster.
131. In a study by Berglund et al. [B29] skin biopsies from five prostate cancer patients were taken fromthe patients before and over a 24-hour period after radiotherapy to determine the radiation response after
a single exposure ex vivo to a dose of 100 mGy. Previously known gene groups and pathways wereselected in the expression analysis. Significant transient transcriptional changes in the human tissue
samples were identified in nine gene groups which returned to baseline by 24 hours post-exposure. Genes
involved in DNA repair and tissue remodelling, cell-cycle transition and inflammation show significant
changes in expression with variability between patients. The authors concluded that the data may serve
as a reference for the temporal response dynamics following low-dose radiation exposure.
132. Albrecht et al. [A10] carried out ex vivo irradiation of skin from two adult female patients
undergoing abdominoplasty. Skin samples were viable for six weeks in culture under appropriate
conditions. Skin was exposed to low (0.05 Gy) or high (5 Gy) doses of X-rays at a dose rate of 4 Gy/min.
Microarray analysis was carried out on irradiated skin samples at 0, 2, 8 and 30 hours post-exposure andalso on non-irradiated skin samples. The study revealed qualitative and quantitative differences in the
responses to low- and high-dose radiation. Three different categories of changes were identified:(a) unique genes responsive to doses of either 0.05 or 5 Gy but not both, and also showing dependenceon time in culture; (b) specific genes that respond to doses of either 0.05 or 5 Gy but not both and notdependent on time in culture; and (c) dose-dependent responsive genes. While exposure to a dose of
0.05 Gy induced transient changes in gene expression, the higher dose at 5 Gy resulted in persistent gene
expression changes. Of all the changes, the authors highlighted that neither p53- nor TGFβ-targeted genes
were modified at low dose and this claim suggests a p53-dependent DNA damage response.
133. Using the whole genome gene exposure approach, Abend et al. [A4] have identified and selected
genes for further disease association in the cohorts from Chernobyl and Mayak. Dose–response
relationships (dose-to-gene) and clinical outcome relationships (gene-to-effect) were analysed todetermine whether such information can be utilized to understand mechanism of cancer progression, todevelop radiation protection measures or for clinical purposes. The authors report that performing
molecular radiation epidemiological studies is more challenging and expensive than doing conventional
epidemiological studies, however, such studies have the potential for improving our understanding of
biological mechanisms underlying health effects of radiation and identification of new and clinically
relevant molecular targets for diagnosis and/or therapeutic intervention.
134. Pernot et al. [P13] highlight the importance of using established radiation biomarkers to identify
health risks. This approach will be helpful in determining the magnitude of long-term healthconsequences of low dose and low dose rates (below 100 mSv and/or 0.1 mSv/min). Most of thesebiomarkers have been in use to investigate exposure, effects and susceptibility to ionizing radiation. The
authors under the framework of DoReMi (Low Dose Research towards Multidisciplinary Integration)
propose a classification of biomarkers that are relevant for molecular epidemiology with special reference
to disease manifestation late in life.

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UNSCEAR 2020/2021 REPORT135. The literature considered in this section and the table 4 below provide strong evidence that ionizing
radiation at high and low doses, chronic or acute exposure and of varying qualities provide changes ingene expression in many cell types. These studies have yielded some reliable quantitative biomarkers ofradiation exposure that have been applied for biodosimetry. While some studies provide evidence ofspecific responses to low and high doses, and low and high dose rate, there is no consensus on a gene
expression profile to distinguish low- from high-dose exposure nor low-dose-rate from high-dose-rate
exposure. Cancers have gene expression patterns different from those of normal cells, and many of thegenes, the expression of which can be modulated by ionizing radiation, are among those dysregulated incancers. Therefore, some of the gene expression changes following low-dose radiation exposure may
relate to carcinogenesis; however, as noted, many studies consider only 24–48 hours post-exposure andthis makes it difficult to relate the observed effects directly to cancer development. It should be notedthat a range of analytical methods have been used in these studies, and the methods vary in sensitivity
and range of genes assessed. There continue to be developments and refinements in the available methods
to quantify gene expression, and it may be that these refined methods are able to define expression profiles
characteristic of specific exposure scenarios. This will require consideration of inter-individual variations
in response and cell type dependence of responses.

Table 4. Summary of gene/protein expression literature considered in this evaluation
Cell type/model

Definitive findings

RNAs, proteins, metabolite, epigenetics

Reference

X-radiation
Dose: 2, 4 Gy
Dose rate: 0.7 Gy/min

Gene expression analysis
at 2 and 24 hours
post-exposure

The study has identified genes which areconsistently up-regulated following exposure to 2
or 4 Gy at different timepoints. Down-regulated
genes included cyclins, centromeric and mitotic
checkpoint with direct relevance to chromosome
instability and cancer

Sestrin 1 (SESN1), growth arrest and DNA
damage inducible 45 alpha (GADD45A),
cyclin dependent kinase inhibitor 1A(CDKN1A), cyclin G1 (CCNG1), ferredoxin
reductase (FDXR), p53 up-regulated
mediator of apoptosis (BBC3) and Mdm2
p53-binding protein homolog (MDM2)[K1]

Human whole blood

Natural backgroundradiation(200 to 3 700 Bq/m3)

Changes in mismatch
repair genes

Activation of mismatch repair system

MLH1 and MSH2

[B5]

Human

Chernobyl Nuclear Power
plant area
Exposed to 0.18 to 49 mSv
over 11–13 years

Cytokine and receptor
arraysVariable expression patterns for genes andcytokines in the low-dose-irradiated individuals.
More frequent expression of serine/threonine
protein kinase receptor, TGF receptor, EB13 andCD40 ligands in the exposed individuals. Authors
suggest appropriate use of statistical tools toevaluate the data generated

Cytokines, mRNA, receptor arrays[A9]

Patients with sporadic
thyroid cancers

Gene expression

Molecular signatures reflecting specific responses
to gamma radiation in patients were identified;
about 118 gene signature discriminated radiation
exposure

Thyroid cancer patientsfrom Chernobyl area
Dose: 0.15–9 Gy

Gene expression analysis

646 up-regulated and 677 down-regulated genes
were identified in discriminating thyroid cancers
which have history of radiation exposure

SFRP1, MMP1, ESM1, KRTAP2-1, COL13A1,
BAALC and PAGE1[P21]

Occupational exposure atMayak Product
Association
Gamma radiation
Dose: ~0.5–1.01 Gy

Gene expression
profiling – microarray as
well as microRNA

A set of 376 candidate genes – 80 up-regulated and296 down-regulated – associated with 239Pu
exposure

mRNA and microRNA[A3]

Blood lymphocytes

Mononuclear cells
from blood

Human
Thyroid cancer
samples
Human
Thyroid cancer
tissues
Human whole blood

mRNA expression
especially TGF receptor,
EB13 and CD40 ligands

Patients who were exposed
to radiation in Chernobyl

239

Pu: ~0.7 kBq

Down-regulation in 239Pu exposure;
up-regulation in gamma ray exposed[D16]

ANNEX C: BIOLOGICAL MECHANISMS RELEVANT FOR THE INFERENCE OF CANCER RISKS ...

Outcomes recorded

Human

Irradiation conditions

45

46

Outcomes recorded

Definitive findings

Occupational exposure
Absorbed dose:
0.696–39.088 mSv

Gene expression profiling

A total of 78 genes (21 up-regulated and 57 downregulated) involved in several biological processes
such as ubiquitin cycle, cell cycle
regulation/proliferation and stress response.
Alterations in these gene products may have
implication on long-term genetic effects on theseworkers

UHRF2, PIAS1, LIG3, XPA, ERCC5, Rad52,
DCLRE1C, RHOA, CABLES2, TGFB2, IL16,
GSTP1, PPP2R5A, DUSP22[F2]

X-radiation
Dose: 23.4 mGy during
dental examination

Selected markers H2AX
and pCHK2Both the markers were up-regulated after dental Xray exposure

H2AX and pCHK2[Y11]

Human peripheral
blood lymphocytes

Diagnostic procedures –computed tomography,
angiography

Selected marker γH2AX

Significant γH2AX positivity in exposed individuals

γH2AX

[K23]

Human peripheral
blood lymphocytes

Diagnostic neurointerventional procedures
Dose:– forehead 4.9 to 273 mGy– eyes 14–98 mGy– shoulders 8–433.3 mGy– thyroid 4.7–242.5 mGy)γH2AX and p53

CDKN1A, FDXR, BAX, DDB2, SESN1, BCL2,
MDM2, TNFSF10B, PCNA GADD45A, ATM
and TNFSF9[V13]

Gamma radiation (60Co)
Dose: 20, 50, 100 mGy
Dose rate: 13.7 mGy/min

Expression of apoptotic
genes at 4, 24, 48, 72
and 168 hours following
irradiation

Low doses of gamma radiation induced downregulation of Bax immediately after irradiation
which is restored to normal level by 168 hours.
Up-regulation of Bcl-2 was seen in most of thedoses

BAX (pro-apoptotic)[A29]

X-radiation
Dose: 0, 0.5, 1 Gy
Dose rate: 30 mGy/min

Global gene expression
profiling

p53 signalling and DNA damage response wasevident at high doses, leading to the activation ofcell-cycle arrest and apoptosis processes.
However, at low doses there appears to be amixed response – in addition to DNA damage, theinnate and adaptive immune responses are
induced through the activation of MAPK, NFĸB,chemokines and cytokines

DNA damage, p53 signalling, apoptosis

Human
Blood lymphocytes
from radiation
workers

Human
Exfoliated oral
epithelial cells

Human
Mononuclear cells

Human
Peripheral blood
lymphocytes

Irradiation conditions

RNAs, proteins, metabolite, epigenetics

Reference

Multiple CT scans produced higher DNA damage

Selected gene expression
profiling

Increased phosphorylation of γH2AX and p53 in
exposed samples
Differential regulation of selected genes

Bcl-2 (anti-apoptotic) Bcl[E15]

UNSCEAR 2020/2021 REPORT

Cell type/model

Cell type/model
Human
Blood lymphocytes

Human
Blood lymphocytes

Blood lymphocytes

Human
Blood lymphocytes

Human
Blood lymphocytes

Outcomes recorded

Definitive findings

Gamma radiation
Dose: 100, 250, 500 mGy

Gene expression profiling

Dose specific changes in specific gene pools –CYP4X1, MAPK10 and ATF6 (100 mGy) DUSP1 andRAD51L1 (250 mGy) and RAD50, REV3L and
DCLRE1A (500 mGy) The authors report that they
have identified a set of 34 significant genes which
are common for all the doses – some upregulated and some down-regulated

SERPINB2 and C14orf104 – up-regulated

X-radiation
Dose: 0.56, 2.23, 4.45 Gy
Dose rates:– acute: 1.03 Gy/min– low-dose-rate:
3.1 mGy/min

Gene expression at
24 hours post-exposure

There were 454 genes which were differentiallyexpressed for low-dose-rate exposure whereas
598 genes were differentially expressed after
acute exposure. Genes related to immune
processes and B cell-mediated immunity wereshown to be affected. Study concluded that lowdose-rate exposure produced distinctive gene
expression patterns compared to high dose rate

Immune processes and B cell-mediatedimmunity; NK cell activation and cell-tocell signalling; p53 pathway was enrichedat both low- and high-dose-rate
exposures[G4]

Gamma radiation (60Co)
Dose: 0.0, 0.05, 0.5 Gy
Dose rate: 0.45 Gy/min

Gene expression analysis
at 3 and 24 hours
post-exposure

Genes BAX, PCNA, GADD45, DDB2 and CDKN1A were
induced at 24 hours. CD4+ cells displayed downregulation of genes even at 0.05 Gy – genes
involved in protein biosynthesis and oxidative
phosphorylation. Conclusion: CD4+ cells may
provide useful information in the biologicalprocesses affected after irradiation

Different subgroups of lymphocytes suchas CD4+, CD8+, CD56+ cells[G18]

Gamma radiation (137Cs)
Dose: 0.02, 0.1, 0.5, 1.0, 2.0,
4.0 GyDose rate: 0.7 Gy/min

Gene expression at 24 and48 hours (0.02, 0.1 Gy)
and 6, 24 or 48 hours(0.5, 1.0, 2.0 and 4.0 Gy)

Expression alterations increased with increasing
dose and time after exposure. The authors
concluded that it is possible to use gene
expression profiling data to determine radiation
exposures as low as 20 mGy

Differentially (positively or negatively)
expressed genes were assigned topathways such as nucleosome assembly,
apoptosis and DNA repair response; nine
signature genes were identified (MKL2,
FDXR, C10orf39, PFKFB3, FLJ35379, LY6G5C
and 3 other genes with no gene symbols
available (Agilent ID A-24_P506680,_A_32_P138939, A_32_P20997))[K29]

X-radiation
Low dose: 5, 10, 20 50, 75,
100 mGy exposed at dose
rate of 4.9 mGy/min

Dose response analysis forgene expression
changes

Dose response curves for high doses(0.5 to 4 Gy) were constructed for gene expression
changes. Individual variation was noticed in both
high- and low-dose exposures

FDXr, DDB2 and CCNG1[M10]

Moderate to high dose: 0.5,
1, 2, 4 Gy exposed at dose
rate of 0.5 Gy/min

RNAs, proteins, metabolite, epigenetics

Reference[F1]

CREB3L2, DDX49, STK25 and XAB2 – downregulated

ANNEX C: BIOLOGICAL MECHANISMS RELEVANT FOR THE INFERENCE OF CANCER RISKS ...

Human

Irradiation conditions

47

48

Human
Blood lymphocytes

Human
Blood lymphocytes

Human
Blood lymphocytes

Human
Fibroblasts (primary)and a prostateepithelial cell line

Irradiation conditions

Outcomes recorded

Definitive findings

Gene expression profilingat 6 and 24 hours
post-irradiation

The study identified 74 gene signature which was
distinguishable between the four different
radiation doses – where more than ⅓ genes wereregulated by p53

Gamma radiation (137Cs)
Dose: 1, 2, 4 Gy
Dose rate: 0.8 Gy/min

Gene expression profiling

24 of 102 genes identified as markers of radiation
showed dose response pattern. 15 genes (CD69,
CDK1A, EGR1, EGR4, FLJ35725, hCG2041177,
hCG16434662, IFNG, ISG20L, CJUN, MDM2, MUC5B,
PLK2, RND1, TNFSF9) up-regulated, and 9 genes(GRIK3, OCLN, RPL10A, SERHL2, SGK3, STARD13,
hCG2038519, hCG1998530, hCG1985174) downregulated

Gamma radiation (137Cs)
Dose: 1, 2, 4 Gy
Dose rate: 0.8 Gy/min

Gene expression profilingat 2 hours postirradiation

Pathways affected – alterations in cellular structure(Bactin, Talin 1, Talin 2, Zyxin-2), immune anddefence reactions (MBP2, IL17E and IFNG), cell
cycle control (CDKN1A, MDM2, ANXA6), GADD45A,
PCNA, DUSP), detoxification process (per1) andapoptosis (BBC3)

Acute: Gamma radiation(137Cs)
Dose: 0.1 and 2 Gy
Dose rate: 0.48 Gy/min

Clonogenicity assay andgene expression
profiling at 24 hours

No measurable effects at 100 mGy

Gamma radiation
Dose: 0.5, 2, 5, 8 Gy

396 genes were differentially expressed for bothacute and chronic irradiation. 106 genes were
down-regulated at acute 2 Gy exposure

RNAs, proteins, metabolite, epigenetics
Validated genes – CDKN1A, FDXR, SESN1,
BBC3, PHPT1

Reference[P6]

Full list of 74 genes (see table 1 in [P6])
EGR1, EGR4, IFNG, CJUN and TNFF9 –correlated with absorbed dose of 1 to
4 Gy

[T23]

[T24]

Pathways affected were cell cycle,
chromosomal movement, cell survival
and DNA replication, recombination andrepair as well as CDKN2A and P53[M26]

Chronic: Gamma radiation(57Co)
Dose: 10–24.5 mGy
Dose rate: 17.0 µGy/min
Human
Fibroblasts andimmortalized cells
Human
3D tissue model
mimicking humanepidermis

Gamma radiation (137Cs)
Dose: 0.5, 1.5, 4.5 Gy
Dose rate: 0.84 Gy/min

Colony formation andgene expression
profiling

Inactivation of colony forming ability at 1.5 Gy. DNA
metabolism affected; p53-dependent G1
checkpoint response altered

CDK2, CCNE1, CDC6, CDC2, MCM2 - downregulated at 24 hours[Z20]

Low-LET protons
Dose: 2.5, 0.1 Gy

Gene expression

The genes involved in the recovery and tissue
repair were altered at the low dose while the highdose affected the genes involved in structural
integrity and terminal differentiation

TP53 for high dose and HNF4A after lowdose exposure[M31]

UNSCEAR 2020/2021 REPORT

Cell type/model

Cell type/model

Irradiation conditions

Outcomes recorded

X-radiation
Dose: 0.1, 2, 10 Gy
Dose rate: 0.033, 0.6,
1.6 Gy/min, respectively

3 or 8 hours postexposures transcript
analysis by RNA
sequencing

1 701 genes were significantly affected by highdoses and 29 genes at doses 2 and 10 Gy but not
at 0.1 Gy at both 3 or 8 hour timepoints. Very few
genes were altered at 0.1 Gy

X-radiation
Dose: 100 mGy
Dose rate: 6.3 mGy/min

Gene expression profling>1 400 genes differentially expressed in the dermis
while >400 genes in the epidermis. Processes
such as cell cycle regulation, immune response,
hypoxia, reactive oxygen signalling and DNA
damage repair were affected. Points out to therole of dysregulated cell proliferation in inducing
cancer by radiation

Human skinkeratinocytes

Varian 2100 C linear
accelerator
Dose: 1 cGy or 10 cGy
Dose rate: 80 cGy/min

Proteomic analysis of
selected proteins on 1 or
4 days after exposure

Seven proteins showed significant changes afterradiation exposure

EFABP, Alpha-enolase, HINT-1, HSP27, LDH1, PDI and S100A9[B30]

Human dermal
fibroblasts

Gamma radiation (137Cs)
Dose: 0.1 Gy

microRNA analysis

De-regulated microRNAs are associated with earlyor late radioadaptive response

COL1A1, MMP1

[B2]

Human
Skin model

Human
3D skin tissue model

Definitive findings

RNAs, proteins, metabolite, epigenetics
BBC3, PPM1D, FDXR, GADD45A, MDM2,
CDKN1A, TP53inP1, CYCSP27, SeSN1,
SESN2, PCNA and AEN

Reference
[T13]

[V14]

Human neonatal
foreskin, adult facialskin cells,
RPE-1 cells

Gamma radiation (137Cs) or
X-radiation
Dose: 1–3.4 Gy
Dose rate was 6–20 mGy

Histone analysis, protein
expression studies

Reduction of histone levels, reduction in the levelsof non-histone chromatin associated proteins, cell
cycle, DNA replication, proinflammatory genes

HMGB, SMC, MCM2-7, CDKN2A (p16),
CDKN1A (p21)

[L45]

Human

Gene expression profilingat 2 or 16 hours postexposure

Gene expression profiles were dose, time and cell
type-dependent. 50 early responsive genes forhigh dose were identified. Substantial differencesin the pathways affected at low- and high-dose
exposures

CDKN1A increased at low-dose exposure;common pathways – cell-cycle arrest, p53
signalling[S54]

Embryonic stem cells

Gamma radiation (60Co)
Dose: 0.05 and 1 Gy

Human peripheral
blood mononuclear
cells

Gamma radiation (60Co)
Dose: 300 mGy and 1 Gy
Dose rate: 0.4 Gy/min

Proteomic analysis

Alterations in 23 proteins in irradiated samples with
1.5-fold change and 3 proteins showing 2.5-foldchange

GRP78, Peroxiredoxn-6, CLIC-1

[N16]

Human
lymphoblastoid cellline

Gamma radiation (60Co)
Dose: 0.05, 0.2, 0.5, 2,
10 Gy
Dose rate: 1.7 Gy/min

Gene expression profilingat 4 hours after
irradiation

Transcriptional changes in 25 genes at 0.05 Gy

BMPR2, GPR124, MAPK8P2, AGGF1, XPC,TP53I3[L41]

ANNEX C: BIOLOGICAL MECHANISMS RELEVANT FOR THE INFERENCE OF CANCER RISKS ...

Regulation of collagen-synthesis related genes

49

50

Mouse
Brain, heart, lung,spleen andintestine
Human and mouse
Blood lymphocytes

Irradiation conditions

Outcomes recorded

Gamma radiation
Dose: 0.2 Gy
Dose rate: 3.81 Gy/min

Immunohistochemistry
for selected genes andgene expression
profiling

Increased expression of p53 and p21 at day 3

Gamma radiation (137Cs)
Dose: 0.5, 2, 10 Gy forwhole-body irradiation

Gene expression profiling

A clear pattern on gene specific changes without
any influence of mouse strains used but sex of theanimals did influence the outcome of radiation
exposure in mice. Though the goal of the human
lymphocyte experiments is to distinguish genesignatures rather than predicting cancer risk

[M28]

X-radiation
Dose: 0.5, 2.0, 10 Gy
Dose rate:– 1.4 Gy/min foranterior/posterior region– 1.25 Gy/min for
hindlimb region

Gene expression forpartial-body and wholebody irradiation

Gene expression analysis was useful in comparing
irradiated and non-irradiated mice but not helpful
in discriminating whole-body and partial-body
irradiation in mice

[M29]

Chronic whole-body
gamma irradiation
Dose: 8 030, 416, 20.6 mGy
Dose rate: 16.6, 0.858,
0.042 mGy/day for
485 days

Gene expression profilingin liverBioinformatics analysis revealed that the genes
which are differentially expressed may be
involved in obesity and tumorigenesis

Gene expression profilingin blood after 24 hours

Same genes were differentially expressed in bothlow- and high-dose-rate exposures

Dose: 1.5–2.0 Gy for pretransplantation screeningor patients prior tochemotherapy (humansamples)
Mouse
Peripheral blood
lymphocytes

Mouse

Definitive findings
Out of 23 genes tested, 8 genes were found to be
increased in all tissues at day 3

RNAs, proteins, metabolite, epigenetics

Reference

p53 and p21; neogenine, Apo1, nucleasesensitive element binding protein 1,
syntaxin, cyclin G1, hNOP56, paraoxonaseand glutathione peroxidase[L13]

Usp2, Dbp, St3gal5, Zbtb16, Lcn13, Herpud1,
Rapgef4, Slc22a5, Hspa1b, Serpina4-ps1,
Angptl4, Slc25a30, Socs2, Alas1, Pim3,
Lpin2, Gne, Acot1, Camk1d, D9Wsu90e and
Pde4b

[U1]

Dose: 8 015, 401, 20 mGy
Dose rate: 20, 1,
0.05 mGy/day for 401 days
Mouse

Gamma radiation
Dose: 1.1, 2.2 or 4.4 Gy
High dose rate:
1.03 Gy/min
Low dose rate:
3.09 mGy/min[P8]

UNSCEAR 2020/2021 REPORT

Cell type/model

Cell type/model

Irradiation conditions

Outcomes recorded

Definitive findings

RNAs, proteins, metabolite, epigenetics

Reference
[M13]

Mouse

Gamma radiation
Dose: 0.25, 0.5, 1 Gy
Dose rate: 500 mGy/h,
500 mGy/min

Gene expression on skinsamples at 4 hours postexposure

A group of 80 genes in regulation of extracellularmatrix (adhesion molecule genes) were tested. No
dose rate or dose differences were observed.
MMP2 and MMP15 displayed most changes in
expression with a decrease at 500 mGy/h in adose-dependent manner. MMP1 and MMP9
increased with dose at 500 mGy/min

MouseGamma radiation
Dose: 2.1, 4.5 Gy
Dose rate: 0.7 mGy/h,
0.8 Gy/min

Gene expression inthymusGenes involved in repair in thymus - Lig4 and RRM2
are overexpressed 130 days after low-dose-rate
exposure. Many additional genes including MSH2,
MSH3, ERCC8, ERCC6, GTF2H2, OGG1, LIG4, NEIL3,
UNG, LIG1, RAD51L1, MBD4, TERF2, H2AX, ATM,
TFRC, FTH1, RRM1, RRM2, RRM2B, P53, BAX and
PARP1 were up-regulated according to RT-PCR

Mouse

X-rays
Dose: 10 cGymRNA and lncRNA
2, 4, and 8 weeks
post-exposure

Genes involved in tissue injury inflammatory
responses and mammary gland development.
Influence of genetic background in the response

Mouse

X-rays acutely exposed
group

Selected gene/protein
expression

Gender differences in the expression of genes
important in cell proliferation

PCNA, RB, RBBP9[C7]

X-radiation
Dose: 75 mGy, 1.8 Gy for
4 weeks
Dose rate: 196,
783 mGy/min

Gene expression profiling

Differential expression of genes involved in breast
cancer tumorigenesis in these strains; study
provides evidence to predict breast cancer risk
following changes in gene signatures

SOX9 down-regulation[S50]

Chronic gamma radiation(137Cs)Protein expression

69 proteins – 1.5-fold changes and 22 proteins with
>2.5-fold changes were identified. Pathways
affected are cytoskeleton, cell metabolism,
biological defence, mitochondrial damage,detoxification and tumorigenesis. They propose
calreticulin as may predict radiation-induced liver
tumorigenesis in mice

Calreticulin[Y8]

Including MSH2, MSH3, ERCC8, ERCC6,GTF2H2, OGG1, LIG4, NEIL3, UNG, LIG1,
RAD51L1, MBD4, TERF2, H2AX, ATM, TFRC,
FTH1, RRM1, RRM2, RRM2B, P53, BAX and
PARP1

[B50]

Acute:
Dose: 500 mGy
Dose rate: 2 mGy/s
protracted exposure(50 mGy/d for 10 days)
Mouse
Mice with differential
susceptibility tobreast cancer
development
Mouse

Dose rate: 50–1 000 µGy/h(22 hours per day for
180 days)

ANNEX C: BIOLOGICAL MECHANISMS RELEVANT FOR THE INFERENCE OF CANCER RISKS ...

[T10]

51

52

Irradiation conditions

Outcomes recorded

Definitive findings

RNAs, proteins, metabolite, epigenetics
myc, tp53, TNF, hnf4a, TGFB1 and cebpα –most relevant downstream targets
affected

Reference[H41]

Gamma radiation (60Co)
Dose: 1.62–3 496 mGy
Dose rate: 0.4, 3.9, 15,
38 mGy/hSurvival and RNA
sequencing

Survival reduced at 2 to >10 mGy/hFruit flyGamma radiation (226Ra)
Dose: 50, 100, 200,
400 mGy
Dose rate: 36 mGy/hLifespan and gene
expression profiling

Changes in lifespan and stress sensitive genes wereidentified. They report “hormesis” after exposure
to doses of 50, 100 and 400 mGy

Human

X-radiation
Dose: 100 mGy
Dose rate: 6.0 Gy/min
before therapy

Gene response to
100 mGy as well as afterradiotherapy(3, 8 and 24 hours)

Transient transcriptional changes in previously
known radioresponsive genes

X-radiation
Dose: 0.05, 5 Gy
Dose rate: 4 Gy/min

Gene expression at
0, 2, 8, and 30 hours

Transient changes in gene expressions at low-doseand persistent changes at high dose. No changes
in TGFB or TP53 at low dose[A10]

Fe ions
Dose: 100 mGy from
1.0 GeV/n

Radiation protection by
selenomethionine andgene expression
profiling

Up-regulation of genes in the chemokine/cytokine
gene cluster (CXCL1, CXCL2, IL6, IL11, IL8, IL24 andTGFB2)[S9]

Radiation type not given in
paper, but priorpublications suggest 11
and 15 MV photon fields)
Dose: 0.015 to 1.10 Gy
fractionated radiotherapy
treatments for 7 days

γH2AX and 53BP1 staining
before radiation
exposure and at 30 min,2 to 72 hours postexposure

Dose-dependent activation of γH2AX and 53BP1 inskin biopsies

Zebrafish

Skin biopsies fromprostate cancer
patients
Human
Skin from patients
undergoing
abdominoplasty

Human
Thyroid epithelial
cells
Skin biopsies fromprostate cancer
patients

Dose–response relationship in the number ofdifferentially expressed genes in the exposedgroup starting at a total dose of 1.62 mGy; lowdose rates affected retinoic acid receptor
activation, apoptosis and glutathione mediateddetoxification signalling whereas modulation ofangiogenesis (eif2 and mTOR) at high dose rates

Persistence of hyper-radiosensitivity[Z18]

DNA repair, tissue remodelling, cell-cycletransition and inflammation pathways

γH2AX and 53BP1

[B29]

[Q2]

UNSCEAR 2020/2021 REPORT

Cell type/modelANNEX C: BIOLOGICAL MECHANISMS RELEVANT FOR THE INFERENCE OF CANCER RISKS ...

53

E. DNA repair and effects on somatic cells
136. The repair of radiation damage to DNA has been considered in detail in the UNSCEAR 2000
Report, annex F, “DNA repair and mutagenesis” [U5]. This report concluded that DNA repair and related
damage response functions were of crucial importance for radiation carcinogenesis, and that mutations
in DNA repair/response genes play an important role in the maintenance of genome stability. In
particular, the importance of the response to DNA double-strand breaks and complex lesions indetermining dose, dose rate and radiation quality effects was noted. No evidence was found that DNA
repair functions were entirely error-free at low exposure levels, and thus any exposure, however low,
carries a risk, albeit low, of mutation in genes critical for the development of cancers. The ability ofradiation to lead to cellular consequences such as mutation and chromosomal rearrangements was noted,
and the importance of these for carcinogenesis.
137. Ionizing radiation-induced DSBs are repaired by two distinct pathways: NHEJ and HRR. The
principal difference between these pathways is that HRR occurs in late S and G2 phase of the cell cycle
and requires an undamaged template to guide relatively error-free repair, whereas NHEJ is capable torepair DSBs throughout the cell cycle, with the exception of the mitotic phase, as no undamaged template
copy is required. DSB repair by HRR is dependent on sequence and generates stretches of 3′-overhanging
single-stranded DNA (end resection) as a priming end that copies the undamaged template. End resection
is mainly carried out by the nuclease activity of the MRE11, RAD50, and NBS1 complex (MRN
complex). NHEJ is a sequence independent process driven by DSB end binding and protection by theheterodimer Ku70-Ku80 as well as their ligation by the XRCC4-DNA ligase IV complex. However,alternative endjoining pathways exist as residual end-joining activity was observed in NHEJ deficient
cells. These alternative noncanonical endjoining pathways are characterized by their dependence on small
stretches of repetitive DNA sequences up- and downstream of DSBs (so-called microhomology) and aretermed alternative end joining (Alt-EJ) and microhomology-mediated end joining [S14, S20].
Microhomology-mediated end joining is driven by DNA polymerase theta and may occur in S-phasewhen an undamaged template is not available.
138. Ionizing radiation induced oxidative base damage repair is carried out by base excision repair that
operates throughout the cell cycle. Base excision repair is initiated by DNA glycosylases, which
recognize and remove damaged bases, forming so called apurinic/apyrimidinic sites. These are thencleaved by an AP endonuclease. The resulting single-strand break can then be processed by either shortpatch (where a single nucleotide is replaced) or long-patch base excision repair (where 2–10 new
nucleotides are synthesized, often additionally associated with single-strand break repair).
139. The main molecular mechanism that initiates repair of radiation-induced DSBs is signalling, i.e.
post-translational modification of specific proteins. This includes the modification of chromatin flanking
the DSB and core repair proteins. Antibodies raised against these proteins have been successfully
employed to measure induction and removal of DSB in cultured cells, human primary lymphocytes andin vivo in mouse tissues. These assays also revealed inter-individual variation in DSB foci parameters inprimary fibroblasts exposed to doses of ionizing radiation (50–250 mGy, linear induction with dose) in asmall survey (25 persons) of apparently normal people suggesting that hypomorphic genetic variants in
DNA damage response may contribute to differential susceptibility to cancer after exposure to ionizing
radiation [W12]. In vitro systems with reporter genes have been applied to measure repair of single
enzymatically engineered DSB in mammalian cells. These systems in combination with defined repair
deficient cells have allowed to elucidate the role of NHEJ and HRR after X-rays, including some lowdose exposures or enzymatically-induced single DSB. The consequences of incomplete repair (ormisrepair) have been assessed by measuring induction of cancer-related genetic damage such as loss of54

UNSCEAR 2020/2021 REPORT

heterozygosity (LOH), chromosomal aberrations and micronuclei as well as cancer induction employing
irradiated normal and (genetically modified) repair deficient organisms.
140. The number and complexity of chromosomal aberrations is critically related to the structure of
DSBs. Whereas the majority of DSBs induced by low-LET radiation of mammalian cells are rejoined inless than 60 minutes, a small (variable) fraction of DSBs (<20%) requires much more time for repair (upto >24 hours) or are left unrepaired [D3, L40]. The proportion of the slowly repaired DSBs rises withincreased LET of radiation and largely reflects the increasing proportion of complex/clustered damage
sites, i.e. DSBs that have a high frequency of oxidized base modifications and abasic (AP) sites directly
adjacent their ends. The base excision repair pathway is compromised close to DSB ends. This poor repairof base lesions and apurinic/apyrimidinic sites close to ends of DSB drastically reduces the repair of DSB
by inhibition of ligation of ends [D4]. A minority of low-LET radiation induced DNA damage represents
clustered damage sites of varying structural and chemical complexity, whereas densely ionizing radiation
generates primarily clustered damage sites including DSBs. The complexity of the clusters, reflecting thenumber of lesions present in non-DSB clusters (oxidative bases) and DSB clusters (DSB and oxidative
bases), increases with the LET of the radiation. Although dose–response relationships for the inductionof complex DNA lesions are not well characterized, it is highly likely that complex lesions are induced
by low dose (<100 mGy) photon radiation as oxidative base damages and DSBs are induced linearly by
X-rays down to 100 and 1.5 mGy, respectively (see section III.A). Kinetics of repair of DSBs after lowor high-LET irradiation revealed that rejoining of DSBs consists of a fast and a slow component and that
free radical scavenging by dimethyl sulfoxide reduces mainly the fast component of DSB-repair, whileslow repair is related to repair of complex DSBs, particularly by HRR. The relative involvement of NHEJ,
HRR and base excision repair in repair of complex DNA lesions induced by low- or high-LET radiation(0.05–18 Gy) has been studied with normal and NHEJ, HRR and base excision repair deficient hamster
cells. The data revealed that DSBs induced by the indirect effect of radiation (sensitive to dimethyl
sulfoxide), are more efficiently repaired by NHEJ and base excision repair compared to HRR [B3].
141. Repair of DSBs can be followed over time by quantifying DNA damage repair focus numbers(γH2AX and phospho-ATM), and these measurements also indicate some possible differences between
high- and low-dose exposure. Although the number of DSBs induced may be low and less than one percell, at low doses of the order of a few milligray, repair as monitored by loss of repair foci has been
observed to be slower or even incomplete, leading to long-term persistence of residual foci over time after
irradiation [G17, O14, R17]. Similar observations of slower and/or incomplete repair have been made instem cells (see section III.F). Assessment of DSB repair following CT scans however leads to a different
conclusion, that repair following the lowest dose exposures can be complete within 24 hours [L39].
142. The findings of incomplete repair following low-dose exposure are suggestive of an inducible
component to DSB repair processes. Grudzenski et al. [G17] showed following a dose of 10 mGy, that cells
failed to show any foci loss. However, pre-treatment with 10 μM H2O2 led to efficient removal of γH2AX
foci induced by 10 mGy. Moreover, 10 μM H2O2 up-regulated a set of genes that was also up-regulated
after moderate (200 mGy) but not after low (10 mGy) radiation doses. This suggests that low levels ofoxidative damage induce an oxidative response required for the repair of radiation-induced DSBs. The dataalso suggested that a threshold for endogenous stress may be required to activate the cellular response toradiation-induced DSBs. A notable issue is that persistence of DSB at low dose solely depends on focianalysis and lacks physical quantification of DSB and that other explanations might be possible.
143. Some studies of γH2AX foci and chromosome aberration induction by radiation suggest that DNA
repair activity changes with age. Higher yields of dicentric chromosome aberrations have been found in
adult blood samples by comparison with umbilical cord blood and blood from under 5-year old children,
this difference is observed at only 1 Gy and not at the low dose of 41 mGy [G7]. The chromosome
aberration results were not reflected in γH2AX foci assays, i.e. γH2AX foci frequencies did not differ

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significantly among age groups. Automatic scoring of dicentric chromosomes improved the sensitivityof similar assays. It was found that under 5-year-old children and newborns have lower background
dicentric frequencies and higher yields of induced dicentrics were observed in the younger groups thanadults at both 41 mGy and 1 Gy X-rays delivered to blood in vitro in a clinical CT scanner [O8].
144. Hernández et al. [H26] explored the DNA damage response of normal human mammary epithelial
cells obtained from women 28, 50 and 58 years of age. Cells were exposed once, twice or ten times to
10 mGy X-rays delivered by a CT scanner (X-rays molybdenum-anode tube with 28 keV voltage at adose rate of 0.67 Gy/min). Two 10 mGy exposures led to delayed resolution and accumulation of doublestranded DNA breaks and a significant increase in micronuclei frequency in human mammary epithelial
cells from old but not young donors. In addition, cells of all three donors went both through 24–
29 population doublings or through 32–38 doublings for “aging in cell culture”. Accumulation of γH2AX
foci was greater in cells from later passages in all three cases, but the increase of the number of foci wasalways the most prominent in cells from the 58-year-old. Note that this study included only one donor ofeach of the given ages and this limits the ability to generalize from the conclusions drawn.
145. Sakane et al. [S8] investigated DNA and chromosomal damage in lymphocytes from 209 patients
undergoing chest CT examinations. Significantly elevated γH2AX foci and unstable chromosomal
aberrations were observed in those having standard dose CT (5 mSv effective dose) but not in thosereceiving low dose (1.5 mSv effective dose) CT examinations. The latter result may be a reflection of thelimited statistical power of the study, especially in light of the modest effect observed at the higher
exposure (5 mSv); this issue is discussed in more detail by Brenner [B55].
146. Impaired DSB repair has been suggested as a cause of aging of human oocytes based on theobservation that DSBs (γH2AX foci) accumulate in primordial follicles (2–28 years) and oocytes (23–24 years) with age [T14]. In parallel, expression of key DSB repair genes BRCA1, MRE11, Rad51, andATM decline in single human oocytes. Also, ovarian reserve was impaired in young women with germline
BRCA1 mutations compared to controls. In line, (eyelid) fibroblast cell cultures (in total 50) isolated fromhealthy donors at the age of 16–75 years, revealed that the efficiency of NHEJ and HRR declines withage leading to increased ionizing radiosensitivity in cells (0–8 Gy X-rays) isolated from old donors [L26].
Whereas decreased expression of NHEJ factors with age was observed, the key HRR-related factor Rad51
did not change expression with age but exhibited slow kinetics of recruitment to DNA damage sites in aged
fibroblasts. In contrast to the above-mentioned results, 20 (eyelid) adipose-derived stem cell cultures fromhealthy individuals (ages ranging 17–59 years) revealed that the efficiency of base excision repair but not
NHEJ or HRR is impaired in aged human stem cells [Z11].
147. The role of HRR and NHEJ in X-ray induced tumorigenesis (medulloblastoma) has been assessedin heterozygous Patched1 (Ptc1+/−) mice; exposure of newborn mice to X-rays increases the frequency
and shortens the latency of medulloblastoma. In Ptc1+/− mice, medulloblastoma is characterized by lossof the normal remaining Ptc1 allele. To test the role of defective HRR or NHEJ in tumorigenesis, control
and irradiated (2 Gy) Ptc1+/− mice with two, one or no functional Rad54 (HRR) or DNA-PKcs (NHEJ)
alleles were monitored for medulloblastoma development [T11]. It was found that although HRR and
NHEJ collaborate in protecting cells from DNA damage and apoptosis, they have opposite roles inmedulloblastoma tumorigenesis. In fact, although Rad54 deficiency increased both spontaneous andradiation-induced medulloblastoma development, DNA-PKcs disruption suppressed medulloblastoma
tumorigenesis. The data provided the evidence that Rad54-mediated HRR in vivo is important for
suppressing tumorigenesis by maintaining genomic stability. In radiation-induced medulloblastoma
tumours the pattern of Ptc1 LOH was distinct from spontaneous tumours: radiogenic medulloblastomas
showed large interstitial chromosome 13 deletions, suggesting different molecular mechanisms for

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spontaneous or radiation-induced Ptc1 loss. Within the sample size analysed, no obvious difference inthe pattern of Ptc1 LOH was observed following genetic inactivation of Rad54 or DNA–PKcs.
148. Exploring the same mouse genotypes as described in the previous paragraph, different types of repair
were found to be engaged differently in vivo dependent on stage of development of embryonal mice. Ptc1+/−
mice lacking one Ptc1 allele were crossed with Rad54−/− (lacking HRR) and DNA-PKcs−/− (lacking NHEJ)
mice on C57BL/6 background [T12]. F1 mice used for experiments came from further crosses of genotypes
DNA-PKcs+/−/Ptc1+/− × DNA-PKcs+/−/Ptc1+/+ and Rad54+/−/Ptc1+/− × Rad54+/−/Ptc1+/+. F2 mice were
X-ray irradiated in utero at embryonic day E13.5 or E16.5 with 0.25 or 3 Gy and followed up until
development of medulloblastoma, which depends on loss or mutation of remaining wild-type allele of Ptc1.
An exposure of 3 Gy induced medulloblastoma sooner in mice irradiated at E13.5 while 0.25 Gy exposure
caused medulloblastoma development sooner in mice irradiated at E16.5. In addition, apoptosis wasfollowed in sacrificed embryonal animals and this study noted that loss of HRR (absence of Rad54) drives
apoptosis in mice irradiated at E13.5, while compromised NHEJ (absence of DNA-PKcs) leads to more
apoptosis in mice irradiated at E16.6. Thus, a more efficient if erroneous repair may be more important forcell survival at higher doses, while at the lower dose HRR, more accurate but less efficient, ensuresdevelopment of fewer medulloblastomas. Medulloblastoma incidence pattern changes at different ages fordifferent doses.
149. Tsuruoka et al. [T22] exposed Ptch1 heterozygotes to a dose of 100 mGy and this resulted in LOH
following the same deletion pattern as LOH mutations in non-irradiated mice. A portion of thechromosome arm from the telomere to beyond the Ptch1 gene occurred most often as a result of faulty
recombination which was different from the LOH pattern that followed a dose of 500 mGy. In a study
that used newborn one day old Ptc1+/− mice (Ptc1neo67/+ with disrupted exons 6 and 7 on C57BL/6JCrl
background were crossed with C3H/He females) [I10] total-body irradiation was carried out by X-rays
and with radiation doses ≤100 mGy (at 100 mGy/min) or >100 mGy (at 650 mGy/min). Life shortening
followed increase of dose, beginning with 50 mGy. As in most other studies using heterozygotes fortumour suppressor genes, one of the endpoints in this work was LOH. Importantly (and again, similarly
to studies with p53 heterozygotes), LOH patterns elicited by radiation were different than LOH patterns
that occurred spontaneously in the absence of radiation and LOH due to faulty recombination wasobserved at 50 mGy. In the case of the latter, loss of the entire chromosome arm carrying Ptc1 gene
predominated, while “local” DNA loss with the remainder of the chromosome present was most frequent
for doses above 1 Gy. Low doses showed a mix of the two LOH patterns.
150. An animal model of accelerated aging was used by Nomura et al. [N17] to evaluate lifespanradiation effects of radiation exposures. Four-week-old homozygous mutant female klotho knockout
mice and wild-type littermates were exposed to 137Cs gamma rays at dose rates of 0.35, 0.7 or 1.5 mGy/hover a period of 23 hours a day. While non-irradiated knockout mice lived 80 days at most, daily
irradiation with either 0.35, 0.7 mGy/h dose rate (but not with 1.5 mGy/h dose rate) extended the lifespanin more than 10% of the animals; the eldest klotho knockout animal died at 119 days (it belonged to
0.70 mGy/h group). Very few differences between irradiated and non-irradiated wild-type littermates
were registered in this study. With regard to antioxidant enzymes, catalase activity in klotho knockout
animals irradiated for 21 days was the most significantly increased.
151. Penninckx et al. [P12] examined ex vivo repair of radiation-induced damage in non-immortalizedskin fibroblasts from 15 strains of mice (5 inbred reference strains and 10 collaborative-cross strains) of
both sexes. Radiation exposure was low-LET (X-rays, (0.1, 1 and 4 Gy)) and high-LET (1.1 or 3 particles
per 100 µm2 of 350 MeV/nucleon 40Ar or 600 MeV/nucleon 56Fe ions). As the doses increased, strain tostrain differences became more apparent suggesting that the lowest doses of exposure may not trigger
DNA damage repair programmes for which these animals had genetic differences as effectively. Thisphenomenon was especially pronounced for high-LET exposures.

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152. Transgenic mice with repair deficiency mutations were used to study the effects of low- andmoderate dose radiation on embryonic brain. Animals carrying a mutation for DNA ligase IV (Lig),
coding for an enzyme involved in NHEJ, were crossed with animals with a knockdown of the ATM genewhich is critical for all DNA double-strand break repair. Animals of all three genotypes (Lig4 mutants,
ATM knockouts and double-mutants) were exposed to radiation as embryos at E13.5 as pregnant mice
were exposed to X-rays between 50 and 200 mGy. DNA double-strand breaks in embryos were studied
as γH2AX or 53BP1 foci up to 60 minutes post-exposure. More foci and less ongoing repair-associated
DNA synthesis were found in mutant embryos, and more apoptosis triggered by DNA double-strand
breaks. Overall the numbers of spontaneous DNA damage lesions and frequency of apoptosis inembryonic mutant animals matched those in wild-type embryonic mice exposed to 100 mGy. After birth,
in 2–3 month-old mice endogenous DNA damage in Lig4 mutants is reduced and irradiation of agematched wild-type mice with dose of 50 mGy causes a twofold increased number of DNA damage fociand TUNEL staining when compared to the spontaneous foci and TUNEL staining in Lig4 mutants [B15].
153. If radiation carcinogenesis is largely driven by the induction of gene mutations and chromosome
aberrations, then DNA repair deficiencies should modify radiation cancer risk. There is evidence thatgenes involved in the two main DSB repair pathways, NHEJ and HRR, modify radiation cancer risk inmice with indications for some tissue specificity [D13, H5, H6, T12]. Also, a single nucleotide changeor polymorphism in CtIP, a factor that affects repair pathway selection, has been demonstrated to lead toincreased radiation leukaemogenic sensitivity in mice [P5]. These studies have however involved
moderate to high doses (0.5–3 Gy) and therefore relate only to situations where DSBs and theirconsequences are of prime importance. Functional assays similarly indicated that DSB repair after
chronic gamma-ray exposure correlated with lung cancer risk in mice [O6].
154. Several studies explored chromosomal changes in bone marrow cells from wild-type and mutant
animals. One such study using fluorescence in situ hybridization analysis on 400–1,000 scored bonemarrow cells isolated from severe combined immunodeficiency (SCID) mice (loss of NHEJ) irradiatedin groups of 20 [R8] found that exposures to 50 and 100 mGy (unlike exposure to 1,000 mGy) did notincrease the numbers of altered chromosomes one or six months after irradiation, although earlier
timepoints one and four hours did show chromosomal changes. At most timepoints the complexity ofchromosomal alterations was smallest in 50 mGy exposed animals. At the time of exposure to 137Cs
gamma rays (dose rate 750 mGy/min) mice were 10 to 12-weeks old. Similarly, 10 to 12-weeks old male
BALB/cJ and C57BL/6J mice were irradiated and screened in the same way [R6]. Chromosomal
aberrations were increased at late timepoints only in mice exposed to 1,000 mGy. Exposure to 50 mGy
was found to be “protective” where animals had fewer than control numbers of chromatid andchromosome breaks one and six months after radiation exposure.
155. In another study, DSB foci were scored in four different genotypes of mice exposed to 10 mGy per
day for 10 or 20 days, sacrificed four hours after final exposure [S13]. Animals used in this study included
repair competent C57BL/6 mice, repair deficient SCID mice (loss of NHEJ) and ATM−/− mice(checkpoint and HRR deficient) as well as their repair proficient ATM+/− littermates (both generated bycrosses between ATMtm1Awb/+ animals). Exposures of 10 mGy per day, five days a week for 2, 4, 6, 8 or
10 weeks were carried out; mice exposed only once were sacrificed at 0.5, 24 and 72 hours and animals
exposed to higher doses were sacrificed at 24 or 72 hours after final irradiation. DSBs were detected byimmunofluorescence with antibodies against 53BP1. In lung, heart and brain of repair proficient micemodest but statistically significant increase in number of foci was found (this was even more pronounced
at 0.5 hours after single irradiation). When all four genotypes were compared, SCID mice had the greatest
number of foci regardless of tissue, while ATM−/− mice showed more foci than repair proficient animals
only in brain cortical neurons but not other tissues.

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UNSCEAR 2020/2021 REPORT156. While development of cataracts was the primary focus of a study by Dalke et al., many otherendpoints relevant for cancer were also explored [D1]. Both F1 mice from the cross between C3H wildtype males with C57BL/6J wild-type females and mutant F1 heterozygotes from a cross between malehomozygous ERCC2 knockout on C3H background and C57BL/6J wild-type females were used;
10 week-old mice were irradiated with doses of 63, 125 and 500 mGy delivered at a dose rate of
63 mGy/min from a 60Co gamma irradiator. At 4 and 24 hours after irradiation and at 12 and 18 months
after irradiation bone marrow cells were isolated from three mice of each group and examined forchromosomal aberrations and telomere length. No statistically significant differences were found forchromosomal aberrations or telomere length. Moreover, cumulative survival of mice exposed to 63 mGy
was the longest, while 125 and 500 mGy exposed mice showed life shortening. No tumour type wasfound to have increased frequency in the 63 mGy irradiation category, ovarian and pituitary adenoma
increased in 125 and 500 mGy groups, and other tumours and inflammation also increased in 500 mGy
mice. Heterozygotes had slightly higher rates of ovarian cancers but lower rates for all other cancer types.
Radiation had different effects on the two genotypes: a higher risk of chromosomal aberrations,particularly dicentrics, in the heterozygous mutants was observed 12 and 18 months particularly after
500 mGy irradiation. Because non-symmetric cytogenetic aberrations such as dicentric chromosomes
show a loss of 50% per cell division, with increasing time since irradiation the number of such radiationinduced cytogenetic defects decreased.
157. Studies with zebrafish treated with anti-Ku80 antibody (inducing loss of NHEJ) support the notionthat functional repair is necessary as protection from even lowest doses of radiation. TUNEL staining
was used to identify apoptotic cells in wild-type zebrafish (Danio rerio) [B37]; to modulate repair
capacity Ku80 gene expression was suppressed by injecting an antisense phosphorodiamidate morpholino
oligonucleotide into the yolk of one-cell embryos. Irradiations used two approaches: control embryos
were divided into 12 groups and received total doses of 0, 50, 100, 200, 300 etc. (increasing by 100 mGy)
to 1,000 mGy, delivered in 10 mGy increments at dose rate of 1 Gy/min produced by a 137Cs gamma-ray
source. Also, 6 MeV X-rays were used to deliver total doses of 1, 3, 8, 20 and 50 mGy to anti-Ku80
injected fish embryos. Fish were exposed as shield-stage embryos and allowed to develop for additional
18 hours before harvesting for TUNEL assay. Anti-Ku80 injected fish were irradiated six hours after
fertilization and fixed at 24 hours for TUNEL processing. In both groups of animals, apoptotic cellnumbers increased linearly with dose; in zebrafish without functional Ku80 apoptotic cells were 34-foldmore numerous than in control injected embryos and dose response could be seen already at total doses
between 1–50 mGy.
158. Gene mutation studies have been carried out in mammalian cells, in fruit flies and mice. However,mutation assays at low doses are challenging due to the low frequency of induction, unless mutant animals
are used as described in the case of Ptch−/− mice [T22]. LOH mutations are particularly relevant in termsof cancer as studies of mutations in both spontaneously arising and radiation-associated cancers haveshown that LOH is a common feature, and furthermore, LOH can be generated through a variety ofmechanisms including chromosomal translocation events that are known to be induced by radiation
exposure. Studies using an HLA-A2 gene loss system in human TK6 or derived p53−/− WI-L2-NS cells
reported a linear dose–response for gene loss by LOH in the 50–500 mGy range [B44], p53 status did notaffect mutation yields. Using the human TK6 cell thymidine kinase locus mutation assay, Manesh et al.
[M9] observed no dose-rate dependence of mutation induction at 0.5 or 1 Gy doses accumulated at 1.4,
5, 15 mGy/min or 24 Gy/h. These results from TK6 cells and derivatives suggest dose rates above
1.4 mGy/min are broadly similar with respect to LOH induction.
159. Studies using the fruit fly Drosophila melanogaster, argue against a similarity in mutational effectof the dose level, as 200 mGy X-ray radiation reduced the somatic mutation frequency below“spontaneous” levels [K33]. This reduction of somatic mutation frequency by X-rays is supported byprevious studies such as the 2007 study by Koana et al. [K31] that used fruit flies with a sex-linked

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recessive lethal mutation; irradiation of immature spermatocytes and spermatogonia (achieved byirradiation of fly embryos) was carried out at dose rates of 0.05 or 0.5 Gy/min, for total doses of 0.2 or
10 Gy. While mutation frequencies at 10 Gy were increased in both cases, exposure to 0.2 Gy at higher
dose rate had no effect on mutation frequency. Exposure to 0.2 Gy at lower dose rate (0.05 Gy/min) had
a protective effect and decreased mutation frequency and lethality in fruit fly progeny. In other words –spontaneous mutation frequency was decreased at the lower-dose and low-dose-rate exposure because it
increased innate error-free DNA repair functions. In subsequent studies [K32, O11] experiments wereextended to include irradiation of immature wild-type flies. When irradiation was carried out with a total
dose of 0.2 Gy either at a dose rate of 0.0224 Gy/h [O11] or at a dose rate of 0.05 Gy/min [K32] mutation
frequency was reduced compared to the sham-irradiated groups. It is important to note that when total
dose was dropped to 40 µGy delivered at dose rate of 1.5 mGy/h, the mutation frequency was once again
approaching value found in the control, though the data were not shown in the publication just cited as“data not shown” [O11]. Finally, repetition of some of the same experiments (exposure to 0.2 Gy total
dose at dose rate of 0.5 Gy/min) in the presence of anti-apoptotic p35 gene introduced by baculovirus,
reconfirmed that the mutation frequency is reduced in irradiated flies [K33]. However, this was not true
for Drosophila melanogaster mutants with deficient single-strand DNA repair.
160. Okudaira et al. [O15] investigated deletions of a red+gam+ bacteriophage lambda transgene in the
spleen and liver of gamma irradiated SWR mice. Deletion induction was assessed at dose rates of
920 mGy/min, 1 mGy/min and 12.5 μGy/min with total accumulated doses of 2–8 Gy. Mutation rates
were found to be about fivefold elevated at the high dose rate compared with the low dose rate. Dose–
response relationships at all dose rates and in both tissues were linear; however, mutation induction at the12.5 µGy/min dose rate was fivefold lower than for 920 mGy/min for equal total doses and for the dose
rate of 1 mGy/min spleen and liver had different responses. Larger, multi-locus deletions appeared lessfrequently in low-dose-rate irradiated tissue.
161. The induction of micronuclei is a sensitive approach to measure genetic damage in vitro and in vivo
at the few milligray range. Results of recent studies are not fully consistent with a complete loss of cells
harbouring micronuclei. Moreover, pulverized DNA from micronuclei might be reintegrated into themain genome thereby inducing locally clustered rearrangements in single or a few chromosomes. Usinga sensitive system to evaluate the induction of micronuclei in primary human skin fibroblasts, a linear
dose–response was reported in the 0–100 mGy range with statistical significance at 20 mGy and above[B44]. S-phase cells were more sensitive to the induction of micronuclei by ionizing radiation than thosein other cell-cycle phases. Induction of micronuclei in reticulocytes after in vivo irradiation of mice wasalso reported to be linear over the 10–100 mGy X-ray range with no evidence for a threshold above
10 mGy [M10]. These studies demonstrate the induction of micronuclei at doses in the region around
10 mGy both in vitro and in vivo. The in vitro studies with fibroblasts do not appear consistent with thefinding from γH2AX staining studies of DSB repair that suggest breaks in non-dividing cells are not
repaired in the mGy region and cells may be lost to apoptosis [R17]. The reticulocyte assay results would
seem particularly relevant as they relate to in vivo exposure and the cells, in which micronuclei werescored, are derived from exposed haematopoietic stem/progenitor cells. Analysis of micronuclei
induction in human blood lymphocytes irradiated in vitro indicated that at a dose rate of approximately
0.3 Gy/min induction is linear with dose, while at an approximately 1 Gy/min dose rate a linear quadratic
relationship applies [B32]. Reduced micronuclei yields in human fibroblasts were observed following
low-dose-rate exposure (100 mGy delivered over 48 hours) and this reduction could be attributed to upregulation of antioxidant defences [D10]. Consistently, in vivo under conditions of chronic exposure to
low doses and dose rates (0.88–35 mGy/y) no increase of micronucleus by natural background radiation
was observed [K16]. A plausible mechanism for the linear non-threshold induction of micronuclei by
X-rays is based on the recent observation that in repair proficient cells mitosis inhibits DNA damage
checkpoint functions and DSB repair. Shut down of DSB repair is proposed to be a guarding mechanism

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against telomere fusions [C9, L25, O18]. Neither the DNA damage checkpoint nor DSB repair occur byvirtue of blockage of recruitment of two crucial repair factors RNF8 and PB531 to DSB due to theirphosphorylation that prevents checkpoint activation and DSB repair. As a consequence, DNA fragments
generated by ionizing radiation linearly with dose, will be processed into micronucleus ultimately leading
to a linear dose–response relationship for micronucleus.
162. Another recent study focused on reticulocytes was carried out with human and animal samples[K24]. Six to eight-week-old male C57BL/6N mice were exposed to X-rays at dose rates equivalent toCT scans and their blood was collected 48 hours later and processed to evaluate percentages of
micronucleated CD71+ reticulocytes. Doses of 8.3 to 1,333 mGy were tested, and a statisticallysignificant increase in micronuclei was noted for all doses, although it should be mentioned that a doseof 243 mGy was necessary for doubling the frequency of micronuclei.
163. A different study was carried out by Manning et al. [M11]; their objective was to evaluate effects
in vivo caused by exposure to Fluorodeoxyglucose-radiolabelled with 18F, a positron emission
tomography imaging agent. In this work wild-type mice 47–48 weeks of age were injected with different
quantities of imaging agent, corresponding to bone marrow doses between 7 and 370 mGy. To provide
external beam controls 18 weeks old mice were irradiated with X-ray doses between 5 and 300 mGy.
Curves describing the frequency of micronucleated reticulocytes had a similar slope although fewer
micronuclei were found in the external beam exposure cohort of mice. BBC3 and Cdkn1a mRNA
expression was checked in these animals and found to be congruous for internal emitter and externalbeam exposures. A notable increase in gene expression was found only in samples exposed to 300 mGy
or equivalent.
164. In vivo irradiated spleen cells from female C3H/HeN mice eight weeks of age exposed for 5 to
400 days to low dose rate of 20 mGy/d or high dose rate of 200 mGy/d of 137Cs gamma rays were screened
for chromosomal aberrations by Giemsa staining [T5]. These included, dicentric chromosomes, centricring chromosomes, acentric ring chromosomes and fragments, as well as minutes, deletions, hyperploids
and chromatid-type aberrations such as gaps, breaks and exchanges. In both dose rate groups seriallysacrificed mice that received increasing doses showed linear increases in numbers of dicentrics.
Moreover, micronuclei in bi-nucleated cells were counted in splenocytes, while centromere probes ofchromosomes 5, 15 and 18 were used in bone marrow cells for interphase fluorescence in situ
hybridization. Again, dicentrics and ring chromosomes increased linearly with total dose, reaching
maximum for a total dose of 8 Gy. That maximum was 4 chromosomal aberrations per 100 cells for
20 mGy/d and 12 chromosomal aberrations per 100 cells for 200 mGy/d dose-rate exposures. The
maximal numbers of aberrations per cell did not go above 1.6 per cell but this maximum was reachedpromptly, already at total dose of 800 mGy for low dose rate exposed mice. Numbers of micronuclei did
not significantly differ from controls, possibly due to loss of micronucleus carrying cells.
165. A recently developed approach to study the effects of radiation on haematopoietic cells is to use“humanized mice”. Such animals were used in a study by Hoehn et al., who engrafted 6–8 weeks old
NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice with commercially available human cord blood CD34+
cells. Host mice were irradiated (2 Gy) before transplantation of the human cells. Such humanized mice
were irradiated with 0.4 Gy 350 MeV/nucleon 28Si ions (with a calculated 1.2 particles traversing eachcell of the body) or 1 Gy X-rays (250 kVp) [H30]. In this work, heavy ion exposure (at doses used)consistently had fewer effects on human and mouse cell populations isolated from exposed mice,
regardless of the cell surface marker examined; moreover, human cells from X-ray exposed mice had twotimes more dicentrics compared to Si ion exposed mice. It will be interesting to use humanized animals
for low-dose irradiation studies.

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166. Exposure of actively cycling cells to ionizing radiation activates DNA damage signalling cascades
and complex cellular responses including activation of apoptosis. Reduction in cell numbers throughapoptosis might be compensated by repopulation through stem cell proliferation and differentiation.
Recent studies suggest that a majority of mutations observed in human cancers may have their origin in
DNA replication errors [T17, T18], and thus any factor that leads to increased stem cell replication could
enhance cancer risk through replication-associated mutation. However, some stem cell populations
appear to have properties that serve to reduce the burden of replication-associated mutations (see [I3] forreview (chapter II)). Additionally, it should be noted that the views of Tomasetti and Vogelstein havebeen challenged by Little et al. [L37] who failed to find a correlation between stem cell proliferation andrisk of radiation-associated cancer. Further challenge to the proposition of Tomasetti and Vogelsteincomes from the experimental evaluation of stem cell mutation rates in organoid cultures from humancolon, liver and small intestine. Blokzijl et al. [B42] found that mutations accumulate at similar frequency
in colon, liver and small intestine organoid cultures, despite differences in cancer rates between thesetissues. The mutational spectra observed suggest that different mechanisms may act in liver as opposed
to colon and small intestine, but spectra are similar to those observed in cancer driver genes, and thus
intrinsic mutational processes can contribute to carcinogenesis in humans; furthermore, little
interindividual variation in mutational frequency or spectra were observed. Thus, the relationshipbetween stem cell division and cancer risk remains uncertain and environmental carcinogenic exposures
are still likely to be a major source of mutagenesis.
167. One in vivo study that ties in spontaneous generation of cancer and the subsequent role that lowdose radiation exposure may play in cancer progression is the following: extremely low doses and dose
rates were used in work with B-cell lymphoma prone SJL/J mice [L1, L2]. The dose rate delivered was
40 mGy/month by gamma irradiation from thorium nitrate (or 1.3 mGy/d) and exposures were life-long.
This type of exposure did not hasten or delay onset of lymphoma, however in mice that did develop
lymphoma chronic low-dose radiation exposures slowed down disease progression and diminishedproliferation index in lymphomatous lymph nodes as measured by Ki67 [L2], potentially indicating an
association of cell proliferation and cancer progression.
168. It has long been held that chromosomal aberrations provide a mechanistic link between direct
cellular effects of radiation and cancers, which are frequently characterized by chromosomal aberrations,
yet the specific type of aberration(s) induced by radiation leading to cancer is generally unknown.
Translocations are frequently observed in leukaemias and deletions in solid cancers but making direct
links is challenging. In a mouse model of radiation-induced acute myeloid leukaemia there is strong
evidence for aberrations directly induced by radiation to be causally linked to disease [V7, V8]. Also,
several human population studies have suggested that chromosomal aberrations in peripheral blood act
as an indicator for cancer risk [B47, B48]. So, while there is evidence for an association between
chromosome aberrations and cancer, it is likely that a range of aberration types will be involved.
Transmissibility of aberrations through cell division is of course essential and likely to be more common
for genuinely simple aberrations.
169. Chromosomal aberrations following chronic irradiation (at low dose rate and accumulating tomoderate or high total dose), were studied in normal tissues in lymphomas. Some of the chromosomal
aberrations were radiation-induced. A systematic investigation of DNA damage caused by chronic
radiation exposure at different dose rates and with different total doses of radiation was reported in detailby Tanaka et al. [T6]. In this work, chromosomal changes in splenocytes were counted as numbers ofdicentric and ring chromosomes following pan-centromere staining. These chromosomal aberrations canbe seen only as proxy for cancer induction. Cells with dicentric and ring chromosomes are expected togo through mitotic catastrophe while cells with chromosomal translocations (not included in this study)
are the ones with a significant likelihood to develop into neoplasia. Details of this study are the following:
8-week-old female C3H/HeN mice were exposed to low-dose-rate (1 mGy/22 h each day or 20 mGy/22 h

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each day) or high-dose-rate (200 mGy/22 h each day or 400 mGy/22 h each day) chronic gamma ray
137
Cs radiation. Fine gradation of low-dose-rate exposures between 100 and 8,000 mGy was delivered
over 5–615 days. High-dose-rate chronic exposures varied between 200 and 8,000 mGy for 1–40 days.
At the conclusion of the final irradiation, spleens were harvested and splenocytes cultured for 48 hours
before chromosomes were imaged and scored; dicentric chromosomes, centric ring chromosomes,
acentric ring chromosomes and fragments were scored in 500–1,000 metaphases after Giemsa staining,
dicentrics were also evaluated by fluorescence in situ hybridization using a centromeric probe. For dose
rates of 20, 200 and 400 mGy/d numbers of aberrant chromosomes increased linearly with total dose,
however, in mice exposed to 20 mGy/d total number of Giemsa evaluated aberrant chromosomes neverexceeded a total of four dicentrics and rings per 100 cells analysed. Evaluation of dicentrics byfluorescence in situ hybridization was also used for 1 and 20 mGy/d comparisons; in this case themaximal total dose for lowest dose rate was 615 mGy and at that dose numbers of dicentrics had a valueof less than one per 100 cells analysed. Using fluorescence in situ hybridization, less than six dicentrics
per 100 cells were found at total dose of 8,000 mGy for dose rate of 20 mGy/d. A similar study using
8-week-old C3H/HeN mice for 1 mGy/d daily exposures with total doses of 125–700 mGy and 20 mGy/dchronic exposures with total doses of 500–8,000 mGy were evaluated lymphocyte translocations using
multiplex fluorescence in situ hybridization [T7]. Translocation frequencies showed similar results as
dicentric chromosome counts: Overall linear increase with increase of total dose was noted, with lessthan two translocations per 100 cells for total dose of up to 800 mGy regardless of dose rate, increasing
to 10 translocations per 100 cells for 20 mGy/d chronically exposed mice for total dose of 8,000 mGy.
Finally, chromosomes from lymphomas from B6C3F1 mice chronically exposed at dose rates of
21 mGy/d to a total dose of 8,000 mGy were characterized by Takabatake et al. [T1]. Chromosomal
aberrations in 82 malignant lymphomas from irradiated and non-irradiated mice were compared by
microarray-based comparative genomic hybridization. In both groups, loss of the Igh region on
chromosome 12 suggested B cell origin lymphomas. Gain of whole chromosome 15 occurred
predominantly in lymphomas from the irradiated group. Partial gains on chromosomes 12, 14 and X were
found in tumours from non-irradiated animals, while irradiation induced lymphomas showed partial
losses on chromosomes 4 and 14. These findings suggest that lymphomagenesis under the effects ofcontinuous low-dose-rate irradiation is accelerated by a mechanism different from spontaneous
lymphomagenesis that is characterized by the unique spectrum of chromosomal aberrations.
170. The prevailing hypothesis regarding cancer evolution is that driver mutations are acquired overyears to decades. However, accumulating evidence suggests that somatic mutations and genomic
rearrangements may in fact accrue in relatively short periods of time through several mechanisms thatmight be relevant for ionizing radiations of different kinds and levels of dose. Next-generation sequencing
of tumours revealed a novel form of genomic instability in cancer cells characterized by thousands of
locally clustered rearrangements affecting one or a few chromosomes [S62]. This phenomenon, termed“chromothripsis”, is likely to emerge from a single catastrophic event inducing locally clustered multiple
DSBs, which are repaired by error-prone mechanisms. These tumours include osteosarcoma for which
the only established exogenous carcinogen is ionizing radiation [G6]. While detected in a wide range oftumour entities [R10], the prevalence of chromothripsis in tumours remains unclear due to different
methodologies and definitions used in the various studies.
171. Mechanisms underlying the formation of chromothripsis are now starting to be explored, but adetailed picture of the process remains elusive. One likely model to explain how complex chromosomal
rearrangements involve only certain chromosomal regions, points to the formation of micronuclei as key
intermediates in this process. Micronuclei are markers of genomic instability [K45] and can arise indamaged cells during mitosis either from lagging chromosomes or chromosomal fragments. Crasta et al.
[C30] showed that mis-segregated chromosomes are entrapped in micronuclei and that the DNA content

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of micronuclei can be pulverized (highly fragmented) due to aberrant (reduced) replication during theS-phase of the cell cycle.
172. A key step forward for understanding relevance of chromothripsis in carcinogenesis was thedemonstration of re-integration of pulverized DNA into the main genome. Zhang et al. [Z10] used a newtechnique combining live-cell imaging and single-cell sequencing to demonstrate that chromosomes missegregated to micronuclei, frequently undergo chromothripsis-like rearrangements in the subsequent cell
cycle by incorrect reassembly of fragments through erroneous DNA repair. How generally thismechanism operates over all cell types, both normal and immortalized, requires clarification.
173. This mechanism may be relevant for low-dose radiation exposure, but also other dose levels.
Clastogenic chemicals and ionizing radiation are very potent inducers of micronuclei in cultured cells
and animals [H29]. Cultured human cells exposed to X-rays and mice treated with 18F in the form offluorodeoxyglucose, display the formation of micronuclei even at doses below 50 mGy [B44, M11].
Moreover, ionizing radiation promotes recombination and integration of plasmid or viral DNA in themain genome dependent on dose but independently of dose rate (low 0.95–1.0 Gy/h and high 1.60–
1.78 Gy/min) at doses >1 Gy [S63]. Importantly, recent studies show an efficient genomic integration of
extrachromosomal DNA by low and moderate doses of ionizing radiation. An investigation by Zelensky
et al. [Z4] provided evidence that low doses of ionizing radiation (10–100 mGy) stimulated random
integration of transfected and viral episomal DNA in human and mouse cells with an extremely highefficiency. Likewise, moderate doses of 400 mGy enhanced DNA integration (gene targeting) throughHRR in human induced pluripotent and embryonic stem cells by 30-fold [H12]. Microbeam irradiationof cancer cells [M41] combined with sequence analysis of single-cell clones revealed chromothripsislike complex chromosomal alterations. Furthermore, induction of clustered DSBs by meganuclease in
Chinese hamster ovary cells [S15] in concert with sequence analysis of cell clones generated evidence
for multiple clustered DNA alterations. Currently, studies relating chromothripsis to ionizing radiation
are scarce and key experiments that would demonstrate the re-incorporation of radiation-induced
micronuclear DNA in the main genome (such as was carried out by Zhang et al. [Z10] for micronucleatedchromosomes) are lacking.
174. The lack of a threshold in micronuclei formation raises the question on repair efficiencies, althoughvariation in background spontaneous micronucleus levels can present difficulties for the quantificationof low dose effects [V15]. As pointed out in paragraph 161 mitosis inhibits DSB repair and DNA damage
checkpoints as a mechanism protecting against telomere fusions [O18] and explaining the linear nonthreshold induction of micronuclei by X-rays. This demonstrates that DNA damage response is not active
in all phases of the cell cycle. Another important question is whether or not DNA damage response isequally efficient after low and high doses of ionizing radiation. At low doses (<100 mGy), repair of DSBs
and base damage by end-joining mechanisms and excision repair, respectively, might require a thresholdof cellular stress to trigger activation [G17, S6]. If this is the case, low-dose radiation might generate anexcess of chromosomal damage/micronuclei and oxidative-induced mutagenesis. Indeed, oxidative
radicals generated by low-dose radiation might induce micronuclei [D10], but the observations of linear
no threshold dose-responses for radiation-induced micronuclei, in vitro and in vivo, suggests that theexcess or the impact of low dose induced oxidative damage on chromosomal damage/micronuclei isminor. The situation is different for base damage mediated mutagenesis.
175. It is unclear if DNA repair is equally efficient after low and high doses. Cellular stress mediated byendogenous processes poses a major source of mutations and genomic instability. Saintigny et al. [S6]showed that incorporation of [3H]-thymidine into DNA provides a model to analyse cellular responses toendogenous oxidative species. Incorporation of [3H]-thymidine in mammalian cells generates oxidationinduced mutagenesis (mostly base substitutions) but with a peak at low to moderate doses (20–250 mGy)
even though the initial exposure was high (1.85–185 kBq/ml). The results point to involvement of redox

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homeostasis and DNA damage signalling pathways and reveal dose-dependent induction of base excision
repair. Collectively, the data reveal that a threshold of endogenous stress must be reached to trigger
cellular detoxification and DNA repair pathways and, as previously shown, repair foci and HRR [S5].
The authors suggest that below this threshold, the consequences of endogenous stress escape cellularsurveillance, leading to high levels of mutagenesis and genomic instability. However, the peak ofmutagenesis at 20–250 mGy was not observed when using external radiation exposure.
176. In summary, antibodies raised against signalling proteins (γH2AX, ATM, 53BP1, etc.) have been
employed to measure induction and removal of DSB in cultured cells and in vivo human tissue at low
doses (<100 mGy). In vitro systems with reporter genes allow the measurement of repair of single
enzymatically engineered DSB in mammalian cells by the principle DSB repair pathways NHEJ and
HRR. Defined repair deficient cells elucidated the role of NHEJ and HRR in DSB repair after low dose
X-rays or enzymatic induced single DSB. In general, repair of DSB in mammalian cells is efficient andcomplete. Only in case of DSB induced in confluent human fibroblasts, a threshold of repair has been
observed by lack of activation of repair due to insufficient ROS. For low dose induced base damage,evidence points also to a threshold of repair due to insufficient ROS to up-regulate the repair. The genetic
consequences of ionizing radiation induced DNA damage have been assessed by measuring induction ofcancer related genetic damage such as LOH, chromosomal aberrations and micronuclei. Slowly repaired
complex DSBs are particular good inducers of chromosomal damage. No threshold is found for inductionof micronuclei at low dose (10–100 mGy) in cultured cells and mice. Reduced micronuclei yields
observed following low-dose-rate exposure (100 mGy delivered over 48 hours), can be attributed to upregulation of antioxidant defences indicating a role for ROS. Induction of tumours in high dose
X-irradiated NHEJ and HRR deficient mice revealed the importance of HRR and NHEJ for suppressing
radiation-induced cancer but their relative roles depend on tumour types. There is good evidence fromtumours induced (at high dose) in irradiated mice, that the spectrum of radiation-induced mutations isdifferent from the spontaneous mutation spectrum.

F. Genomic instability, bystander effects, damage/effects on
non-nuclear cellular components, adaptive response andhyper-radiosensitivity
177. A range of different approaches and strategies have been used to explore how low-dose and lowdose-rate radiation could cause effects through damage to non-nuclear components of the cell and also
trigger either “non-targeted” effects, beneficial or harmful responses in adjacent cells or daughter cellsof cells exposed to radiation, at organ level or on the level of the whole organism. The Committee
published a comprehensive review of non-targeted effects of radiation exposure in 2006 [U7] that
concluded that no specific roles for genomic instabilities or bystander phenomena in radiogenic diseasecausation had been identified. The UNSCEAR 2012 White Paper [U9] to consider the impact of the nontargeted effects, including genomic instability, bystander phenomena and adaptive responses [U9] noted(a) the apparent inter-relationships between these phenomena, including possible mechanistic
similarities; (b) the expressed concerns on the inherent variability and on reproducibility; (c) the
increased body of relevant literature; (d) an emerging consensus on a lack of observations of transmissible
instability following low-LET exposures below 0.5 Gy; and (e) the absence of a coherent body ofknowledge that can be applied to assessment of low dose health risk. An earlier report on adaptive
responses is also available [U4].

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Effects on mitochondria

178. The possible outcomes of low-dose radiation exposure include changes in non-nuclear
compartments of the cell. For example, changed performance of mitochondria may be an outcome of adirect impact on the function of these organelles, or a mutation in genomically-encoded or
mitochondrially-encoded genes that encode proteins important for mitochondrial function.
179. There is considerable evidence that mitochondria can be an important target for radiation effects.
Respiratory complexes I and III of the mitochondria of haematopoietic progenitor cells have been
identified as a non-nuclear target of high doses (10 Gy and above) [P9]. Belikova et al. [B28] identified
a role for mitochondrial cytochrome c in mediating radiation-induced apoptosis at high doses (>5 Gy).
Furthermore, inhibition of mitochondrial cytochrome c peroxidase reduces radiation-induced cell deathby apoptosis at 10 Gy [A24]. Using cells derived from mice deficient in the FancD repair factor it has
been possible to demonstrate that radioprotectors targeting mitochondria can ameliorate the effects ofhigh doses even where DNA repair is defective [G10]. FancD2 and its related and interacting proteinsare clearly multifunctional having both nuclear (DNA repair) and mitochondrial roles [J13, S69, Z14].
180. At lower dose levels mitochondrial fusion and increased expression of complexes I and III has been
observed in 0.2 Gy 6 MeV X-ray-irradiated rat hippocampal neurons [C23]. This was interpreted toindicate that neuronal cells modify energy metabolism in response to 0.2 Gy irradiation; no effects on
survival, ROS levels, mitochondrial DNA levels or synaptic function were observed. By contrast, afractionated X-irradiation protocol (31 twice daily exposures of 0.01 or 0.05 Gy) has been found toincrease mitochondrial mass and mitochondrial ROS levels followed by nuclear accumulation of cyclin
D1 and consequent growth retardation and senescence in normal human lung fibroblasts (MRC5 andTIG3) [S36]. However, in ATM- and NBS1-deficient cells, similar fractionated exposures led toaccumulation of abnormal mitochondria; this was determined by mitochondrial fragmentation anddecreased mitochondrial membrane potential [S35]. Differential effects of X-rays and C ions have beenreported in mitochondria of HeLa cells; lower doses (0.2 and 0.5 Gy) of X-rays lead to mitochondrial
fusion, with higher doses leading to mitochondrial fission while C ion irradiation at all doses examined(0.2–3 Gy) caused mitochondrial fission [J20]. The role of mitochondria in ROS production following
low-dose irradiation has been reviewed recently [K20].
181. The importance of the mitochondrial target has been demonstrated with low-dose alpha particle
exposure also. For example, the bystander induction of γH2AX foci by 10 mGy alpha particle exposure
in medium transfer experiments is reduced in cells with fewer mitochondria or inhibited respiratory chain
function [C16]. Similarly, radon-induced apoptosis is reduced in bronchial airway cells with fewer
mitochondria [L20]. Direct damage to mitochondria and mitochondrial function has been shown
following precisely targeted microbeam alpha irradiation (2 or 10 alpha particles per cell, a high dose ofhigh-LET radiation) [Z6]. Cytoplasmic irradiation with alpha particles can lead to nuclear damage asassessed by γH2AX foci and micronucleus formation [Z7]. Additionally, cytoplasmic gamma irradiation
leads to a metabolic shift to a more glycolytic energy metabolism mediated by Pim1 expression, notably
Pim1 was not observed to be induced by nuclear irradiation [W15].
182. Persistent induction of ROS as a consequence of low-dose (20 mGy gamma ray) radiation exposure
have been observed to lead to autophagy and mitochondrial dysfunction in haematopoietic stem cells[R14] with the effects apparently restricted to irradiation in vitro and having effects on cell survival.
183. Direct connections to cancer are difficult to ascertain from differences in mitochondrial function,
protein expression or metabolism over extended periods of time. Nevertheless, it is conceivable that theseeffects can be associated with modulation of cancer incidence, for example through persistent elevationof ROS. When wild-type animals were used for low-dose-rate experiments, some lifespan experiments

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revealed protein expression differences to be more noticeable than the DNA/RNA differences. For
example, at ages of six months and two years, cardiac tissue from mice exposed to radiation in utero
presented a number of protein expression differences. The differences between controls and mice exposed
to a dose of 1 Gy were only two times higher than the difference between controls and mice exposed toa dose of 100 mGy [B9]. More importantly, of all possible broad cellular functions, the most substantial
and consistent differences were those for mitochondrial proteins. However, the relevance of thesechanges to cancer are not clear but they provide evidence of low-dose radiation effects.
184. The literature outlined above clearly demonstrate an effect of radiation at high and low dose on
mitochondria. Not all studies are entirely consistent in their reported effects, but one can conclude thatmitochondria represent an important non-nuclear target for the effects of radiation.

2.

Genomic instability

185. In the context of studies of radiation effects, genomic instability is defined as a persisting andtransmissible (through cell generations) increase in observed frequencies of mutations, chromosome
aberrations or other similar endpoint following exposure to radiation. It has been described largely in in
vitro studies where cells are exposed to radiation, grown for several cell generations and then assessed
for chromosome aberrations, mutation, etc.
186. Zyuzikov et al. [Z23] used C57BL/6 mice, relatively resistant to development of radiation-induced
myeloid leukaemia, as well as CBA/Ca mice, susceptible to radiation-induced myeloid leukaemia andchromosomal instability in the bone marrow [Z23], to explore potentially adverse bystander effects oflow doses of radiation. At 8–12 weeks of age mice were either sham-irradiated or exposed to X-rays at adose rate of 1 mGy/s to total doses of 1.7, 5, 12.1, 25, 50 or 100 mGy; dose rate of 7.5 mGy/s was used
for delivery of high doses of 0.5, 1 and 3 Gy. Regardless of dose rate, doses below 1 Gy did not cause
genomic instability in bone morrow cells of either strain. Percentage of p53+ and p21+ cells in C57BL/6
and C57BL/6 mice stayed below 7.5 and 2, respectively, and no evidence of bystander effect via p53
signalling was detected at low-dose exposures up to 100 mGy.
187. Rithidech et al. [R6] similarly reported that doses of 50 and 100 mGy gamma rays were incapableof inducing genomic instability as assessed by late forming chromosome aberrations after in vivo
exposures of BALB/c mice. By contrast, a 1 Gy dose did induce instability in the BALB/c strain, noneof the doses led to persisting instability in C57BL/6. However, 50 mGy led to a reduced frequency ofaberrations in C57BL/6 mice at 1 month after irradiation. Similar levels of instability were reported to be
induced in BALB/c mice irradiated in vivo with 1 Gy 100 MeV protons and 137Cs gamma rays [R7], no
dose dependence between 0.5 and 1 Gy was observed for proton exposures.
188. Cho et al. [C24] reported that doses of 200, 500 and 1,000 mGy X-rays did not induce persistentelevation of micronucleus frequencies up to 10 days (5 passages) in normal human fibroblasts (hTERT
immortalized) but level of aneuploidy of chromosomes 1 and 4 were elevated at 10 days post-exposure.
189. Genomic instability specifically investigated in the T-cell receptor rearrangements of irradiated
CBA mice were not observed following 0.1 and 1 Gy in vivo exposures [C4]. High-dose-rate exposures
to X-rays at 0.5 Gy/min and lower dose-rate exposures to gamma rays at 0.43 or 2.1 Gy/h 137Cs wereused, and no difference between dose rates was reported.
190. Using microsatellite instability as a readout of persistent genomic instability it has been possible toexamine mouse tumours induced by radiation and compare to spontaneously arising tumours to gauge
the role of induced instability in radiation carcinogenesis [H4]. A mixture of acute myeloid leukaemias,ANNEX C: BIOLOGICAL MECHANISMS RELEVANT FOR THE INFERENCE OF CANCER RISKS ...

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intestinal tumours in Apcmin mice, medulloblastomas and basal cell carcinomas in Ptch mice wereconsidered. This study found that 16% of spontaneously arising tumours had the microsatellite instability
phenotype, while 23% of tumours arising in X-irradiated mice were microsatellite instability-positive,
and 83% of tumours in neutron-irradiated (fast neutrons with a mean energy of 1 MeV) mice weremicrosatellite instability-positive. It was concluded that microsatellite instability may play a role inradiation tumorigenesis, particularly after high-LET exposures.
191. In breast cancers arising among those irradiated for haemangioma, a dose-dependent elevation in
DNA copy number variants has been described [B34]. While this finding may indicate transmissible
instability as having a role in radiation-associated carcinogenesis in human, the paper does not make clear
if the DNA copy number variants have increased in number over time since exposure. A higher level ofgenomic re-arrangement would be anticipated in tumours exposed to higher doses.
192. The more recent genomic instability studies considered in the preceding paragraphs do notsubstantially alter previous conclusions drawn on the role of genomic instability in radiation
carcinogenesis. There is more evidence indicating the existence of a threshold of around 100 mGy lowLET radiation being required to trigger instabilities.

3.

Bystander and abscopal effects(a) In vitro studies
193. Functional studies of different types of bystander and abscopal effects have focused on different
cell lines, cell densities and qualities of radiation using in vitro models. Abscopal effects operated oversubstantial distance between organs while bystander phenomena relate to effects observed over the range
of a few cell diameters. A range of endpoints have been utilized to assess bystander effects, someinvolving ‘omics methods. The relatively slow advance of computational analyses for ‘omics studies islikely to have limited progress in bystander effect studies.
194. Furlong et al. [F25] explored directly irradiated and bystander human keratinocyte HaCaT cellswith regard to gene expression in radiation-induced bystander effects. Direct irradiation or conditioned
media from cells exposed to 0.05 and 0.5 Gy gamma rays was used to treat HaCaT cells and induction ofgene expression was frequently found to include the same genes (p53, Bax, Bcl-2, JNK etc.) regardlessof direct or “indirect” exposure. Exceptions were ERK and Caspase 9 – induced only by direct irradiation
and Caspase 3 – induced only by exposure to conditioned medium.
195. Fournier et al. [F17] irradiated several different normal skin fibroblast cell lines using microbeams
with C and Ar ions (4.8 MeV nucleon; LET 250 keV/µm and 1,950 keV/µm, respectively). 53BP1 fociwere detected only in cells hit with the beam while p21 expression was found in surrounding cells
including cells relatively remote from the site of microbeam impact. Ion energies were not found to beimportant for induction of response. While increased p21 expression was documented, no indication ofterminal differentiation of cells was found. Subsequently, the same research team [F18] continued to use
different ion beams to explore bystander effects in normal fibroblast cells. In this work, primary focus
was on γH2AX and other markers of DNA damage. The authors found no increase in DNA damage in
bystander cells either by evaluating γH2AX foci, numbers of sister chromatid exchanges or developmentof micronuclei when microbeams were used. In control experiments, use of X-rays or ion broad beamcaused DNA damage as extensive as expected. A study by Groesser et al. [G16] corroborates thesefindings – 20 MeV/nucleon nitrogen ions (LET 150 keV/µm) or 968 MeV/nucleon (LET 151 keV/µm)

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or 575 MeV/nucleon (LET 176 keV/µm) used to irradiate cells in culture carrying repair interferingmutations caused no detectable DNA alterations in bystander cells.
196. Yeles et al. [Y6] conducted bioinformatics analysis of molecular pathways associated withradiation-induced bystander effects – GEO datasets [N15], and of many endpoints most applicable to alldatasets (universal for radiation-induced bystander effects) were the following: the negative regulationof growth, cellular response to Zn2+-Cd2+, and Wnt and NIK/NF-κB signalling.
197. Sekihara et al. [S21] exposed U937 cells mimicking skin-infiltrating monocytes and human primary
keratinocytes taken from newborn humans to a 0.1 Gy dose of 4 MeV X-rays delivered at a dose rate of
2.0 Gy/min. Conditioned media were harvested at 24 hours and used to produce bystander cells. Gene
expression and protein expression were checked in directly irradiated and bystander cells and potential
transcription regulators explored by pathway analyses. Of these, only prolactin was surmised to be a gene
expression regulator both in directly irradiated and bystander cells. A similar situation was encounteredin directly irradiated and bystander human primary keratinocytes cells. Only few (e.g. c-Myc) genes were
found to be activated in both cell lines following the same irradiated cell-bystander cell pattern.
198. Le et al. [L9] described a role for p53 in bystander responses in five cell lines with different p53
status (HaCaT: p53 mutated; SW48 p53 wild-type; HT29 p53 mutated; HCT116+/+ p53 wildtype;
HCT116–/– p53 null). Cells were irradiated with beta particles from tritium and UV-mediated bystander
response evaluated and found to be associated with p53 status such that presence of wild-type p53
correlated with the presence of a radiation-induced bystander effects.
199. Hagelstrom et al. [H1] found that DNA dependent protein kinase catalytic subunit (DNA-PKcs)
and ATM are necessary for a bystander signal production but not cell signal reception in normal humancells (with wild-type p53) following 1 Gy gamma-ray exposure. In addition, this study found that directly
irradiated cells do not respond to simultaneously experienced bystander signals. Sister chromatid
exchange was the endpoint of the study; normal human fibroblasts and normal mouse kidney cells wereused for the analyses.
200. Studies focusing on ROS and enzymes involved in their production include work from Abdelrazzak
et al. [A1] where 208F rat fibroblast cell lines were exposed to less than one to a few hundred mGy ofalpha particles (3.0 MeV, LET of 127 keV/µm produced from a 238Pu source), gamma rays (60Co) or
ultrasoft X-rays (1.49 keV AlK characteristic X-rays) while non-irradiated bystander cells were v-srctransformed 208Fsrc3 cells. The successful induction of a bystander effect depended on at least 10% of
208F cells being irradiated (i.e. traversed by one or more alpha particles, with one particle traversal
equating to a mean dose of 26 mGy). Radiation caused production of enzymes such as peroxidase andthis effect could be replaced by exogenous addition of TGF-β [A2]. The importance of TGF-β in
bystander effects was also explored in [M25]. In this study, human mammary epithelial cells wereclonally analysed to evaluate genomic instability. A threshold for instability was >100 mGy but it could
be reduced by exposure to TGF-β1. Work by Wang et al. [W4] on the other hand, indicated that TGF-β1
given on its own or when it is induced by heavy ion irradiation (e.g. delivery of 0.4 Gy of Si or Fe nuclei
to human oesophageal epithelial cells (EPC2-hTERT)) causes epithelial-to-mesenchymal transition(EMT). EMT process has been implicated in wound healing, fibrosis, tumour invasion and metastasis, all
included under the concept of “stemness”. In addition, EMT can be caused by mutations, epigenetic
events, and inappropriate signalling.
201. Yang et al. [Y4] found that the frequency of DNA break foci in irradiated and bystander AG01522
human cell fibroblasts doubled after exposure to 0.47 mGy of Fe ions (about 0.02 Fe ions/cell,
1 GeV/nucleon, LET 151 keV/µm) and that delivery of higher doses to targeted cells does not increase
damage to bystander cells. The situation was similar with DNA damage doubling both in irradiated andANNEX C: BIOLOGICAL MECHANISMS RELEVANT FOR THE INFERENCE OF CANCER RISKS ...

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bystander cells at 70 µGy protons (about 2 protons/cell, 1 GeV; LET 0.24 keV/µm). With further increaseof dose of either type of radiation DNA damage did not change in bystander cells. In the irradiatedpopulation, irrespective of radiation type, the fraction of damaged cells was constant from the lowest
damaging fluence (0.02 Fe ions/cell or 2 protons/cell) to about a 10 mGy dose and then the damage
increased linearly with dose. Overall, the bystander effect was less variable across doses and dominatedeffects at low doses.
202. Lam et al. [L4] observed a “reversed bystander effect” where protection from radiation damage wasachieved by use of media harvested from non-irradiated HeLa and NIH/3T3 cells that were previously
temporarily sharing media with the irradiated cells. Irradiations were carried out with 50 mGy by alphaparticles using an 241Am irradiation source (diameter of active area = 12 mm, average alpha particle
energy = 5.16 MeV, activity = 5.02 µCi, dose rate = 180 mGy/min). Incubation of designated bystander
cells with irradiated cells that was necessary to trigger the bystander effect was prevented in the presenceof NF-κB activation inhibitor BAY-11-7082.
203. Maeda et al. [M5] reported that synchrotron-generated low energy photons (5.35 keV, X-ray
microbeam focused to 10 × 10 µm) were used to irradiate the nuclei of, or (5.35 keV, X-ray microbeam
focused to 50 × 50 µm) was used to irradiate the whole of, V79 Chinese hamster lung cells. The endpoint
was clonogenic survival of the bystander cells. In both cases, NO mediated increased cell death since useof carboxy-PTIO as a scavenger obviated this response. It should be noted that cells were not touchingeach other in this experiment.
204. Klammer et al. [K27] explored the adaptive response and its induction in bystander cells as well as
the ability of cells to respond better to high doses after exposure to low doses. This work used plasmids
to monitor DNA repair by NHEJ and HRR in mouse embryo fibroblasts and human fibroblast cell lines
HF12 and HF19 for paired exposures to 10 mGy and 5 Gy X-rays. Bystander effects were induced bymedium-transfer technique in mouse embryo fibroblasts but not human cells.
205. Heuskin et al. [H28] studied lung adenocarcinoma A549 cells irradiated with 100 keV/µm alphaparticles or 5 keV/µm protons at a dose rate of 0.1 Gy/min and showed hyper-radiosensitivity below
0.1 Gy. Because of the low fluence of radiation, it is possible to attribute this response to a bystander
effect. Addition of Lindane at a concentration of 40 µM abolished gap junctions and increased cell
survival after low- dose exposures.
206. Much work on gap junctions comes from the laboratory of Edouard Azzam. For example,
Autsavapromporn et al. [A26] evaluated bystander mechanisms in AG1522 normal human diploid skinfibroblast cell cultures exposed to 137Cs gamma rays, 3.7 MeV alpha particles, 1,000 MeV protons or
1,000 MeV/nucleon Fe ions. Mean adsorbed doses ranged between 0.25 and 2 Gy and numbers of
bystander cells with micronuclei were higher in dishes where cells were grown to confluency. Radiation
exposure of HeLa cells engineered to express connexin 26 or connexin 32 showed more bystander cellswith micronuclei when connexin 26 was expressed.
207. Autsavapromporn et al. [A27] exposed confluent normal human skin fibroblast NB1RGB cells to
0.05 to 0.37 Gy adsorbed dose to a target cell from monochromatic 5.35 keV X-rays (LET, 6 keV/µm),
18.3 MeV/µm C ion (LET, 103 keV/µm), 13 MeV/nucleon Ne ion (LET, 380 keV/µm) or
11.5 MeV/nucleon Ar ion (LET, 1,260 keV/µm) microbeams in the presence or absence of 18aglycyrrhetinic acid, an inhibitor of gap junction intracellular communication. Variable effects wereobtained – in the case of X-ray exposures in the presence or absence of 18-aglycyrrhetinic acid equal
numbers of micronuclei were found in bystander cells suggesting a role for secreted factors (not gap
junctions) in signal mediation. In cells exposed to heavy ions bystander cells had fewer micronuclei inthe presence of 18-aglycyrrhetinic acid suggesting that gap junctions in this case lead to stress increase.

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Similar results were reported by [D11] who worked on apparently normal AG1522 human diploid skinfibroblasts expressing connexins 26, 32 and 43, and communicating by gap junctions and secreted factors.
These cells were exposed to 3.7 MeV alpha particles, originating from 7.4 MBq 241Am-collimated source(corresponding to a dose rate of 20 mGy/min), 1,000 MeV/nucleon Fe ions, 600 MeV/nucleon Si ions,or 137Cs gamma rays. Experiments included 18-aglycyrrhetinic acid – a reversible inhibitor of connexin
channels and hemi-channels, or LaCl3, an inhibitor of hemi-channels. Bystander effects evaluated as anumber of cells with micronuclei were prominent after all three types of radiation and they weredecreased by application of 18-aglycyrrhetinic acid inhibitor.
208. Buonanno et al. [B58] used C3H 10TK mouse embryo fibroblasts to evaluate modulation of the
spontaneous frequency of neoplastic transformation in the progeny of bystander mouse embryo
fibroblasts that had been in co-culture 10 population doublings earlier with mouse embryo fibroblasts
exposed to moderate doses (0.25 Gy) of densely ionizing Fe ions (1 GeV/nucleon) or sparsely ionizing
protons (1 GeV). An increase (p<0.05) in neoplastic transformation frequency, likely mediated byintercellular communication through gap junctions, was observed in the progeny of bystander cells that
had been in co-culture with cells irradiated with Fe ions, but not with protons.
209. A different focus on gap junctions comes from Ramadan et al. [R2]. Two human endothelial cell lines
immortalized to permit telomere maintenance were used: hTERT telomerase immortalized human coronary
artery endothelial cells and telomerase immortalized human dermal microvascular endothelial cells.
Exposure to single 0.1 and 5 Gy doses and three fractions (0.033 or 1.67 Gy/fraction/d) of X-ray irradiation
modulated gene expression of atheroprotective connexins 37, 40 and proatherogenic connexin 43. Further,
irradiations with acute but not fractionated 0.1 Gy caused increase in gap junctional communication at
72 hours as indicated by exchange of 6-CF dye. This effect was abolished by carbenoxolone.
210. Ojima et al. [O13] used shielding and a change in mAmps to modulate X-ray delivery from total
doses of 1.2 to 200 mGy. Gap junctions were inhibited by Lindane, an inhibitor of gap junction
intercellular communication. Phosphorylated ATM foci in cells were imaged and quantified. Foci were
found at 1.2 mGy and increased supralinearly or, after addition of Lindane linearly. Primary normal
human lung fibroblasts MRC-5 were used in this study.
211. Jelonek et al. [J16] carried out a review of exosomes indicating that radiation induces increased
exosome release in a dose- and time-dependent manner due to the activation of stress-inducible pathwaysof exosome secretion: increased expression of TSAP6 (transmembrane protein tumour suppressoractivated pathway 6), which is stimulated by p53 transcription factor as noted in human epithelial lungH460 cell line and aneuploid immortal keratinocyte HaCaT, human breast adenocarcinoma MCF7, andhuman prostate cancer cell lines, where this effect was additionally related to senescence. In humanglioblastoma cell lines increase in exosome release following irradiation was observed in p53mutated/overexpressed cell lines (LN18 and U251) in comparison to U87 cell line with wild-type p53.
212. Freudenmann et al. [F19] studied exosomes from PC3 (prostate adenocarcinoma) or A549 (lungadenocarcinoma) cells released into conditioned medium following radiation. Conditioned medium fromeither cell line when no irradiation was carried out induced proliferation of self, one another or HSF7
(normal skin fibroblasts). This effect was decreased when conditioned media was harvested from cells
previously exposed to 2 Gy. Exosomes from all cells had no effect on proliferation of any of the threecell lines. While irradiated exosomes from PC3 cells did not have the capacity to induce cell proliferation,
conditioned media from non-irradiated cells did. Exosomes of non-irradiated PC3 cells contain L-Plastin:
this protein was absent after 2 Gy irradiation.
213. Jella et al. [J15] irradiated HaCaT cells with 0.005, 0.05 and 0.5 Gy gamma rays from a 60Co
instrument and harvested exosomes isolated after 1 hour. While complete conditioned media had

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extensive effects on non-irradiated cells, exosome free conditioned media did not cause any reduction in
viability, calcium influx or production of ROS.
214. Zhang et al. [Z13] explored skin tissue secretome – protein secretion in a reconstituted 3dimensional skin tissue model (human skin tissues, EpiDerm Full Thickness 400) that was evaluated 48 h
after exposure to 30 or 100 mGy (both at a dose rate of 600 mGy/min) and 2 Gy (dose rate 600 mGy/min)of X-rays using a targeted irradiation of mitochondria. Twelve replicates each were carried out; different
proteins showed a variety of responses to radiation including dose response and biphasic response.

(b) In vivo bystander studies
215. Blyth et al. [B43] employed transplantation of sham or tritium loaded or X-ray irradiatedsplenocytes into animals not exposed to radiation (genotypes in this work were C57BL/6J mice donors
and pKZ−/− and pKZ−/+ recipients). Donor splenocytes were exposed to total doses of 0.1 or 1 Gy (doserate 5 Gy/min) or labelled with similar amounts of [methyl-3H]-thymidine (specific activity
3.18 TBq/mM), about 0.3 mBq per cell. Regardless of the approach, however, adjacent splenocytes fromrecipient mice showed no changes for endpoints under consideration such as apoptosis or proliferation at
22 and 72-hour timepoints post-transplantation.
216. A very different study design was used to investigate effects of cell-free serum from gamma ray
irradiated mice [S67]. Eight-week-old female C3H/HeN and B6C3F1 mice were either irradiated,
22 hours a day by a dose rate of 0.0181 Gy/h for 10 or 20 days (to a total accumulated dose of 4 or 8 Gy),or acutely exposed to equal doses at high dose rate of radiation 0.9 Gy/min. Serum was collected andadded to primary cells in culture (mouse embryonic fibroblast cells or hepatocytes) that did not encounter
radiation. After 24 hours gene expression in these cells was checked and increased expression of LCN2
protein was found in mouse embryo fibroblasts exposed to sera from acutely exposed mice. This effect
was removed when antibodies against interleukin (IL)-1α were added to the mix. At the same time, LCN2
was not altered in primary hepatocytes cultured in the presence of the irradiated serum of the mice. A
different LCN2 expression status was discovered in liver tissue isolated from irradiated mice: While
LCN2 expression increased in livers of acutely exposed animals, it was unchanged in mice exposed for
10 days and significantly decreased in animals irradiated for 20 days.
217. Mancuso et al. [M7] irradiated neonatal Ptch +/− mice with whole- or partial-body exposure to study
abscopal effects. Control animals had the highest frequency of medulloblastoma and lowest numbers of
apoptotic cells in the cerebellum. Test mice were irradiated with whole body X-ray irradiation (doses
0.036 to 3 Gy) or with partial body irradiation of 3 Gy delivered to different parts of the body. The
greatest number of apoptotic cells in cerebellum was found in whole body or partially irradiated mice
where 3 Gy was delivered. The same experimental groups of mice had the highest incidence and the
speediest onset of medulloblastoma as well. Conversely – mice exposed to 0.1 Gy had fewer
medulloblastomas than the control mice. Importantly, connexin 43 induction was the greatest in situations
where abscopal apoptosis in non-irradiated cerebellum was the greatest (i.e. 3 Gy partial body exposure).
218. Szatmari et al. [S74] found that extracellular vesicles mediate systemic bystander effects at 24 hours
post-injection when they are isolated from BM and spleen of 9–14-week-old male C57BL/6 mice at
24 hours after exposure to 0.1, 0.25, 2 Gy (THX-250 therapeutic X-ray source). While DNA damage
found as chromosomal aberrations and γH2AX increases with dose in irradiated animals (maximum at
2 Gy), bystander mice exposed to extracellular vesicles from irradiated mice show more DNA damage
after injection with extracellular vesicles from mice exposed to 0.25 Gy.

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UNSCEAR 2020/2021 REPORT219. Szatmari et al. [S75] continued exploration of directly irradiated (0.1 or 2 Gy) and bystander miceand screened both the miRNA expression in extracellular vesicles and proteins from whole blood
focusing on cytokines. A complex situation arose. Among increased proteins the only dose-dependent
direct radiation and bystander radiation effect that overlapped was an increase in lipocalin 2 at 2 Gy.
M-CSF and pentraxin 3 were increased in all cases: i.e. directly irradiated and bystander samples after
either 0.1 or 2 Gy exposure. miRNAs isolated from extracellular vesicles from directly irradiated animals
suggested activation of acute myeloid leukaemia and TGF-β regulatory pathways.
220. The work considered in this section continues to highlight the variability of bystander phenomena.
There is no consistently identified mechanism for bystander signalling, and thus the effects may represent
a range of effects at the cellular or tissue level, the importance of which for carcinogenesis at low doses
is not yet clear. Tomita and Maeda [T19] provide a detailed and extensive review of bystander phenomena
and similarly note the mixed results reported. They conclude by concurring with the position on bystander
phenomena stated in the UNSCEAR 2012 White Paper [U9], that there is little of the coherence requiredof robust data among bystander studies that can be used confidently for risk assessment.

4.

Adaptive response

221. A number of studies relating to adaptive responses have been published in recent years. Thesestudies include in vitro studies, in vivo studies usually utilizing rodent models and a few studies relating
to human populations. Some studies concern the optimal adapting dose (also referred to variously as
priming dose, adaptive dose or pre-exposure in certain studies) to adapt cells to respond differently to thesubsequent, higher, challenge dose.
222. Azimian et al. [A29] studied the effects of low doses of ionizing radiation on the expression of
specific apoptotic genes such as Bcl-2 and Bax. Blood lymphocytes from four healthy donors were
exposed to 20, 50 and 100 mGy of 60Co gamma rays at a dose rate of 13 mGy/min. Expression profiling
analysis was carried out at 4, 24, 48, 72 and 168 hours post-irradiation by real-time PCR. It is reported
that low doses used in the study can induce early down-regulation of Bax with normal levels restored at
168 hours. Bcl-2, the anti-apoptotic gene, was up-regulated. They argue that gene regulation/modification
as early as 4 hours post-irradiation may underlie adaptive responses, due to their transient nature. All
expression levels returned to normal within one week after exposure.
223. A study by Saini et al. [S4] combined both acute exposures to low doses of gamma radiation andadaptive response of human lymphocytes by measuring expression levels of selected DNA damage
response markers. Blood samples were collected from 24 healthy human volunteers and exposed to dosesof 0.1, 0.3, 0.6, 1.0 and 2.0 Gy from 60Co gamma rays at a dose rate of 0.7 Gy/min. Both DNA damage
measured by comet assay and γH2AX expression showed dose-dependent increases. There was dosedependent up-regulation of GADD45A, CDKN1A and TP53 from doses up to 1 Gy at five hours postirradiation. Adaptive responses (adapting doses of 0.1–0.6 Gy, challenge dose of 2 Gy) at the level ofmRNA expression were seen for CDK2, CYCLIN E and TP53 (i.e. these transcripts showed higher levelsof expression 1 and 5 hours after exposure to adapting doses followed by a 2 Gy challenge than a single
2 Gy exposure), whereas other genes such as ATM, ATR, GADD45A and MDM2, did not display any
adaptive response (i.e. transcript levels were similar following a 2 Gy exposure with or without anadapting dose) [S4]. In a further study of gene expression modifications correlating with adaptive
response in G0 human lymphocytes, Shelke and Das [S29] found that Ku70, Ku80, XLF and Ligase IV
were significantly up-regulated at 4 hours after exposure to an adapting dose of 0.1 Gy, suggesting the
involvement of the NHEJ pathway of repair in adaptive response.

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224. Kumar et al. [K48] investigated DNA breakage assessed by alkaline comet assays in peripheral
blood lymphocytes drawn from subjects living in the Kerala province of southern India. Blood samples
were from one of three groups: (a) 43 from a “low dose group” living in an area of elevated natural
background radiation receiving an average annual effective dose of 2.69 mSv, (b) 34 from a “high dosegroup” living in areas of elevated natural background radiation receiving on average a 9.62 mSv annual
effective dose and (c) 37 from those living in areas of normal background radiation levels, <1 mSv per
year average. The initial induction of breakage by 2 and 4 Gy 60Co irradiation was found to be lower inthe high dose group samples that either of the other groups. Repair of breakage over a 30 minute timecourse was marginally more rapid initially in the high dose group samples, but all samples had repaired
the same proportion of breaks at 30 minutes post-exposure. The lower initial level of breakage observedin the high dose group is contrary to the usually observed equal initial damage induction irrespective ofcell type and may suggest some inconsistent dosimetry or sample handling. Nonetheless, the authors
suggest that the study may indicate that exposure to elevated levels of background radiation induces anadaptive response in blood lymphocytes. In broadly similarly exposure groups, a reduced initialfrequency of micronuclei were observed in those samples from persons aged 40 years or greater, but notin those of less than 40 years of age; basal micronucleus frequencies were statistically indistinguishable
among groups though somewhat higher in the samples from those living in the higher backgroundradiation areas [R1]. Assessment of stable (translocations, inversions) and unstable (dicentrics, rings)
chromosomal aberrations among the Kerala populations using standard Giemsa stained metaphases of
peripheral blood lymphocyte revealed no differences in the background frequencies of these aberrations
between high and normal natural background residents by Karuppasamy et al. [K17]. Prematurechromosome condensation assays, again on samples from broadly similar groups, though notdistinguishing between the two elevated levels of natural background exposure, found similar basals and2 Gy 60Co irradiation induced premature chromosome condensation fragments, though initial (1 hourpost-irradiation) repair of premature chromosome condensation fragments was observed to be greater insamples from those living in areas of elevated natural background radiation [K49]. Nishad and Ghosh[N16] conducted a proteomic analysis of peripheral blood lymphocytes identified some differences inprotein levels between samples collected from high natural background residents and normal natural
background area residents, and increased responsiveness of some pro-survival proteins following
challenge with 2 Gy gamma irradiation in samples from high natural background area residents.
Collectively these results have been interpreted in the cited papers as providing evidence of an adaptive
response having been induced in blood cells of individuals inhabiting areas of substantially elevatednatural background radiation.
225. In a study by Jain and Das [J10], gene expression profiling was carried out on individuals living inhigh natural background radiation areas in Kerala, India. Peripheral blood lymphocytes were taken from36 individuals. The natural background radiation dose ranges from <1.0 to 45 mGy in a year. Global
transcriptomic analysis was carried out on the blood lymphocytes to find out the significance of chronic
exposure. The results point to a dose-dependent increase in the number of differentially expressed genes
with respect to different natural background radiation dose levels. Gene ontology analysis indicated thatmajority of differentially expressed genes fall under pathways responsible for DNA damage response,
DNA repair, cell-cycle arrest, apoptosis, histone/chromatin modification and immune response. The
authors report the identification of 64 genes which could be considered as biological signatures of
exposure to natural background radiation. They link differential expression of genes in DNA damage
response and DNA repair pathways to radiation adaptive response and normal or reduced cancer
incidence reported [N4] in the population in the high-natural background radiation area. There are,however, no adaptive response data presented in the publication, but the findings may explain theobserved efficient repair of DNA damage among persons inhabiting high natural background radiation
areas of Kerala using alkaline comet or γH2AX assays [J11, K48].

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UNSCEAR 2020/2021 REPORT226. Studies have also been carried out on blood samples drawn from the inhabitants of areas in the
Ramsar region of the Islamic Republic of Iran living in elevated natural background radiation regions.
Alkaline comet assays are reported to show elevated basal levels of DNA damage in those living in areas
with average 10.2 mSv per year effective doses. The induction of damage by 0.3–4 Gy 60Co irradiation
was reduced in control (normal background radiation level) groups by comparison with samples fromdonors living in areas of high natural background radiation. Breaking the samples from those inhabiting
elevated natural background radiation areas into <10.2 mSv per year average effective dose and>10.2 mSv per year groups revealed that incomplete repair was observed in the highest backgroundexposed groups but in the <10.2 mSv per year group repair was elevated compared to control exposuregroups [M20]. Zakeri et al. [Z3] observed elevated levels of unstable (chromatid-type and chromosometype) chromosomal aberrations in those inhabiting areas of elevated natural background radiation in the
Ramsar area. Mohammadi et al. [M38] report that blood lymphocytes drawn from those living in areasof Ramsar with elevated natural background radiation show reduced micronucleus induction, increased
apoptosis induction and increased DNA damage repair assessed in alkaline comet assays, all after 4 Gy
60
Co irradiation. Unlike in the Kerala groups described in the previous paragraph, comet assays revealed
an elevated level of basal damage and elevated induction of damage by 4 Gy 60Co irradiation in thesamples from those living in elevated natural background radiation conditions.
227. Basri et al. [B22] carried out a small study of DNA damage among those living under elevatednatural background radiation conditions in a region of Sulawesi, Indonesia. Elevated background levels
were an average of 7 mSv per year effective dose and normal background regions 2 mSv per year. Though
not statistically significant due to small sample size (8 from high background areas, 29 from normal
background areas), an elevated level of γH2AX foci was observed among lymphocyte samples from thosein the high background regions. Looking at mitotic index in cultured peripheral blood lymphocytes frompersons living in high (n=35, average 2.52 mSv/y, range 1.99–3.17 mSv/y) or normal (n=25) backgroundradiation areas of Botteng village, Indonesia, Ramadhani et al. [R3] found no difference in proliferation
characteristics of lymphocytes taken from elevated or normal background radiation areas.
228. To explore the effects of low- and high-dose radiation in human immune cell lines, cell sensitivity
was tested following irradiation with 0.05, 0.1 or 2 Gy of 137Cs gamma rays (dose rate – 0.0173 Gy/s) innormal cell lines as well as in tumour cell lines [P3]. The survival after exposure of different normal andtumour human immune cell lines correlated AKT expression, which seems to be under the control of
PP2A. Exposure to a low dose of 0.01 or 0.05 Gy 4 hours before a challenge dose of 2 Gy protects IM9(B lymphoblast) from cell death, and decreases cleavage of PARP-1, caspase-3 and caspase-9. These
effects seem to be mediated by ERK activation which may have a role in the adaptive response toradiation at low doses.
229. The study by Hou et al. [H36] investigated the adaptive response induced in human fibroblasts(AG01522) exposed to a 50 mGy of adapting dose (X-rays, 60 kV at 105 mGy/min) and then to 2 Gy of
X-rays as a challenge dose (220 kV, 2 Gy/min). Transcriptomic analysis revealed that low-dose X-ray
exposure produced an alert, triggering and altering cellular responses (particularly p53-mediatedresponses) potentially to protect against high dose-induced damage. Changes in gene expression levels
were associated with reduced chromosomal changes (micronuclei formation) in these cells throughinduction of adaptive response. Also using human fibroblasts in culture, Yim et al. [Y10] studied theexpression of phosphoproteins following radiation exposure. Cells were exposed to 0.05, 0.1, 0.2, 0.5, 1,
2 or 4 Gy of 137Cs gamma rays at a dose rate of 1.03 Gy/min. Cell proliferation was not affected and cells
continued to proliferate without genomic instability (γH2AX foci formation and micronuclei induction)
at low dose of gamma irradiation (50 mGy). Phospho-antibody microarray analysis and western blotting
revealed the elevated expression of two phosphoproteins - phospho-NFĸB (Ser536) and phosphoP70S6K (Ser418) at 8 hours after irradiation with 50 mGy. It is concluded that activation of phosphoNFĸB (Ser536) and phospoho-P70S6K (Ser418) and absence of genomic instability after low-doseANNEX C: BIOLOGICAL MECHANISMS RELEVANT FOR THE INFERENCE OF CANCER RISKS ...

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radiation exposure (50 mGy) may indicate their involvement in subsequent DNA damage response andrepair processes. The up-regulation may contribute to adaptive responses.
230. Vares et al. [V4] demonstrated that adapting doses of heavy ion radiation (0.01 Gy,
290 MeV/nucleon C ion and 400 MeV/nucleon Ne ion beams, with LET values of 20, 40 (C ion) or
150 keV/µm (Ne ion)) induce an adaptive response to a subsequent 6 hours later challenge dose (1–4 Gy)of high-LET radiation (C ion: 20 and 40 keV/µm, Ne ion: 150 keV/µm) in TK6, AHH-1 and NH32 cells.
A significant decrease of micronuclei was found in the first two cell lines. Similarly, γH2AX foci repair
was more rapid when adapting exposures were applied only in TK6 and AHH-1 cells.
231. Effects of low-dose radiation on the proteome in both human fibroblasts and stem cells were studied
by Hauptmann et al. [H13]. Cells were irradiated with a 137Cs source with a dose rate of 26,400 and
50 mGy/h. Cells were exposed to a 40 mGy adapting dose followed by challenge dose of 100 mGy after
two-hour incubation. Challenge doses of 40, 100 and 140 mGy were also given. Non-linear dose–
response relationships were observed for the up- and down-regulated proteins. Primarily, the structuraland stress-response proteins were affected. The responses observed could be on/off effects involving
different proteins, which depended on dose and dose rate. Exposure to an adapting dose significantly
altered the proteome regulation by a challenge dose later.
232. The existence of an adaptive response was also investigated in human aortic endothelial cells(HAoECs) pre-exposed to high-dose gamma radiation (2 Gy) delivered at high (1 Gy/min) or low(6 mGy/h) dose rate as an adapting dose before receiving a challenge dose of 2 Gy delivered at 1 Gy/min.
Overall, low-dose-rate priming increased the transcription of genes involved in anti-oxidative stressresponse, vascular growth factors and cytokines when compared to 2 Gy challenge dose only, while highdose-rate priming by comparison either reduced the expression of these genes or had no effect [V10]. In
this case, it appears that radiation promotes a protective response of irradiated endothelial cells.
233. In addition to the human cell studies, two relevant non-human cell studies have been published. Fanet al. [F7] exposed mouse epidermal cell line JB6P+ to low doses of 50 or 100 mGy of X-rays and achallenge dose of 2 Gy gamma rays (60Co gamma radiation, 2.3 Gy/min). The low doses activated stresssensitive genes NF-ĸB, MnSOD, pERK, 14-3-3eta and cyclin B1; activation of these genes increased
radioresistance to higher radiation dose (2 Gy). In order to study the immune adaptive response, Chenget al. [C19] investigated expression levels of selected DNA repair proteins in murine lymphoma cells(EL-4) following irradiation. Adaptive low-dose exposure to X-rays (0.075 Gy at 0.0125 Gy/min;
200 kVp 10 mA) significantly prevented cell death and cell-cycle arrest in EL-4 cells exposed to highdose/high dose rate (1 to 2 Gy, 0.287 Gy/min). Low-dose radiation exposure prevented the increase of
p53 expression in response to high-dose radiation. It is observed that these effects require PARP-1 activity
in the cells. Therefore, PARP-1 and p53 might play important roles in adaptive response induced by lowdose irradiation.
234. Beneficial effects of an adaptive dose of 100 mGy to X-rays (0.028 Gy/min) of BALB/c mice werestudied with the mice irradiated at 6–8 weeks to a challenge dose of 5 Gy alone or to the adaptive dose
followed by the challenge dose [J19]. Pre-exposure was found to be beneficial for mouse skin in animals
exposed to 100 mGy. MnSOD activity was higher, as well as oxygen consumption and ATP production,suggesting improved mitochondrial resilience.
235. Another study used in utero exposures to low doses of gamma radiation [H38] followed by acuteexposure as a challenge dose. Pregnant BALB/c mice were sham-irradiated or exposed to an adaptive
dose of 10–13 mSv/d for 10 days during organogenesis and weaned pups, 16–18 days old, were given an
acute challenge dose of 2.4 Sv. Comparisons of DNA damage, white blood cell counts, and gene
expression suggest that an adaptive response was elicited in pups that experienced in utero exposure.

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UNSCEAR 2020/2021 REPORTWith regard to gene expression, RT-PCR of liver mRNAs showed that genes involved in DNA damage
response (Brca1, Egr1, Ercc6, Gjc1, Gpx1, Hif1a, Hist3h2a, Hspa1b, Mlh1, Mos, Parp1, Polb, Rad51,
Raf1, Sod1, Tnf, Trp53 and Xpa) had a more moderate suppression of expression in mice exposed to an
adaptive dose prior to challenge dose than animals exposed to challenge dose only.
236. The induction of an adaptive response by low-dose and low-dose-rate exposure on the modulationof immune parameters by high dose low- or high-LET radiation was also investigated in C57BL/6 mice
irradiated in vivo and ex vivo. In particular, Gridley et al. [G13] showed that protracted exposure to0.01 Gy of gamma radiation delivered at 0.18 mGy/h did not modulate the response of mice, andespecially their CD4+ T-lymphocytes, to a subsequent proton exposure representing a simulated solar
particle event (sSPE, 1.7 Gy at 30 to 210 MeV delivered in 36 hours). A following study showed that inthis model, low dose and low dose rate and sSPE exposures independently activate different intracellularsignalling pathways in CD4+ T-cells purified and activated in vitro 4 and 21 days post-exposure, as
revealed by phosphorylation of NF-κB, p38 MAPK, JNK, and T-cell receptor-associated signalling
adaptors Lck and ZAP-70. When low-dose and low-dose-rate exposure was applied before sSPE
exposure, most of the effects of proton exposure were inhibited, demonstrating that low-dose and lowdose-rate gamma radiation induces radio-adaptation able to modulate intracellular signalling induced byproton exposure [R9]. Despite this modulation of intracellular signalling pathways in activated CD4+ Tcells, protracted low-dose and low-dose-rate exposure was found to have only a marginal effect on the
homeostasis of leukocyte populations, platelets and erythrocytes in mice 4 and 17 days post-proton
exposure (2 or 3 Gy, 210 MeV) [L51].
237. In line with these findings, the response of mice and their CD4+ T-cells to 2 Gy gamma or proton
radiation exposure was largely unaffected by prior low-dose and low-dose-rate exposure to gamma rays
(0.01 Gy delivered at 0.3 mGy/h). Only a limited number of subtle changes were observed at day 21 and
56 (higher frequency of splenic CD4+CD25+Foxp3+ regulatory T-cells (Tregs) in low-dose and low-doserate exposure + gamma radiation compared with gamma radiation only at day 21; inhibition of increasein the number of splenic Tregs and of the increase in vascular endothelial growth factor (VEGF) secretionin low-dose and low-dose-rate exposure + protons compared with protons only at day 56). Thus, theradio-adaptive ability of low-dose and low-dose-rate exposure to a challenge dose of high-dose gammaor protons radiation is quite limited at the level of cellular homeostasis [G15]. The expression of
apoptosis-related genes was analysed in the liver of mice exposed in similar conditions. Low-dose andlow-dose-rate exposure alone had no effects, but pre-exposure to low dose and low dose rate adapting
doses modulates gene expression in mice exposed to 2 Gy gamma rays and protons. While only two genes
are down-regulated in 2 Gy gamma-irradiated mice, 11 are modulated (up- and down-regulated) in micepreviously exposed to low-dose and low-dose-rate exposure. Similarly, six and 12 genes are modulated(up or down) in proton compared with low-dose and low-dose-rate exposure + protons exposed mice.
This study does not allow a conclusion as to whether this radio-adaptation is beneficial (increased
apoptosis of exposed cells) or potentially detrimental (increased survival of possibly exposed damaged
cells) [G15].
238. The main focus of the study by Lopez-Nieva et al. [L43] was to investigate the adaptive response
in mouse thymocytes. C57BL/6J mice were exposed to an adaptive dose of 0.075 Gy of X-rays(100 kV/15 mA). This adaptive dose was known to induce an adaptive response in mouse thymocytes.
After 6 hours, mice were subjected to a challenge dose of 1.75 Gy of gamma rays (137Cs source). Levelsof caspase-3 mediated apoptosis and global transcriptional expression in isolated thymocytes were
determined in both male and female mice. Appropriate controls of mice exposed to a dose of 0.075 Gy
alone, or a dose of 1.75 Gy alone were used for comparison. Male mice displayed lower levels of caspase3 mediated apoptosis than females: a differential sex effect on adaptive response was observed. In thetranscriptomic profiles of radioadapted thymocytes, 17 transcripts among the 1,944 genes involved in
apoptosis signalling exhibited differential expression among both sexes. Dlc1 and Fis1 closely related

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genes in p53 mediated apoptosis response were up-regulated and this up-regulation correlated with theincreased accumulation of phosphoserine-18-TRP53 and caspase-3 in radio-adapted thymocytes offemale mice. In contrast, down-regulation of Fis1 and phosphoserine-18-TRP53 was found to be
associated with the protection from thymocyte apoptosis mediated by caspase-3 in males. It is concluded
that transcriptomic analysis has revealed for the first time a sex-specific differences in gene expression
influenced by radio-adaptive response in mouse thymocytes.
239. A series of publications have utilized the pKZTg/+ transgenic mouse that allows assessment ofchromosomal inversions. pKZ1Tg/+ heterozygotes maintained by backcrosses with C57BL/6J mice wereused for evaluation of chromosomal rearrangements in prostate tissue [D6]. Exposure to a priming doseof 0.001 mGy X-rays (250 kV, 15 mA at 0.0014 mGy/min), reduced frequency of the inversions thatwould have been induced by a single 1,000 mGy dose (dose rate 180 mGy/min) as well as the portion of
inversions for spontaneous background frequencies. The study shows that adaptive responses produced
from priming doses of X-rays protected against the formation of inversions induced by high doses of
X-rays (1,000 mGy). This adaptive response has been shown to require only a single ATM copy. Day etal. [D7] investigated the influence of heterozygous mutation of ATM on radiation-induced adaptive
response. Furthermore, in prostate an adapting dose of 0.001 mGy delivered several hours after achallenge dose of 1,000 mGy was observed to lead to a similar adaptive response as when the priming
doses are delivered before challenge doses [D8].
240. Bannister et al. [B13] tested 20 mGy low dose (0.5 mGy/min), 100 mGy dose (dose rate
10 mGy/min) and 500 mGy dose (dose rate 10 mGy/min) as priming doses. When priming doses weredelivered 4 or 24 hours ahead of a challenge dose of 2 Gy, there were no effects on micronucleus
frequencies in splenocytes following in vivo or in vitro irradiation in C57BL/6 or BALB/c mice. A
subsequent report confirmed and extended these findings [B14].
241. Collectively, these studies illustrate the ability of low-dose and low-dose-rate gamma radiation tomodulate the response of immune cells in mice to a subsequent high dose of gamma or proton radiation.
This modulation has apparently no discernible effects of the homeostasis and/or phenotype of immune
cells, but is able to affect intracellular signalling pathways and gene expression. Potential differential
response between males and females has been reported.
242. To conclude, there continue to be adaptive-response studies reported using both in vivo and in vitro
approaches. There are both positive and negative findings reported, some indication of potential sexdependent differences, and some endpoints showing adaptive response while others do not. A range ofadapting doses have been reported, and different time schedules for the adapting and challenge exposures,
including delivery of the adapting dose after the challenge dose. The gene expression studies have yet toprovide a consensus on the pathways potentially up-regulated by adapting dose exposures, though several
studies suggest p53 and/or NF-κB-dependence while ATM is apparently not involved. These reported
findings are not consistent with current knowledge of DNA damage response signalling, which shows
linear response consistently.

5.

Examples of DNA repair modulation in cellular and animal models

243. Some animal models, with an innately high rate of spontaneous mutations showed decreased
mutation burden after low-dose radiation exposures. These studies may be considered as providing
evidence for the concept of an adaptive response, though there is no coherence between the animal andcellular data considered to date.

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UNSCEAR 2020/2021 REPORT244. Low-dose-rate and/or low-dose exposures were found to act as deterrents for cancer development
in animal models that harbour mutations making them predisposed to cancer development. Examples ofsuch mouse models include (a) AKR/J mice that carry murine leukaemia viral oncogenes, known todevelop thymic lymphoma during later periods of life [M43, S38]; (b) different strains and strain
combinations of mice with knocked-out or mutated p53 gene and their heterozygous counterparts,
developing different cancers from lymphomas (e.g. [M12]) to a broad spectrum of solid tumours (e.g.
p53 delta proline mice described by Adams et al. [A6]); (c) animals with mutated or knocked-out
transcription factor gene, Ptch1, regulator of the hedgehog pathway and acting as a tumour suppressor
leading to skin cancers and medulloblastomas [A23].
245. AKR/J mice are a well-known model for thymic leukaemia; these animals are viraemic from birthand express the ecotropic retrovirus AKV in all tissues. Shin et al. [S38] exposed female AKR/J mice togamma-ray radiation beginning with eight weeks of age for a total of 4.5 Gy delivered at low dose rate
(0.7 mGy/h) or high dose rate (800 mGy/h). Thymic lymphoma incidence was lowest in low-dose-rateirradiated mice compared to either non-irradiated or high-dose-rate irradiated mice by 10 and 20%,
respectively (P<0.01). Correspondingly, low-dose-rate irradiated animals lived 243 days, compared with
230 days for non-irradiated mice and 208 days for high-dose-rate irradiated animals. This study also
included gene expression analysis. Thymic gene expression studies used animals 130 days after
irradiation, 8–9 per group. Six hundred ninety-five genes increased and 1,726 decreased twofold inexpression in high-dose-rate irradiated mice. In low-dose-rate irradiated mice 410 genes increased and
198 decreased twofold. Considering that low-dose-rate exposure extended life expectancy of these miceand high-dose-rate exposure decreased it, it is not surprising that only few genes had the same expression
pattern in both irradiated groups of mice. These genes were Cd5l, Pycard, Fcgr3, Lilrb3, Igh6, Fcgr2band Mgc60843 and are involved in apoptosis and T- and B-cell activation.
246. Additional studies in AKR/J mice were carried out by the same group [B49, B50, B51]. Eightweek-old female AKR/J mice, carrying murine leukaemia viral oncogenes that lead to spontaneousdevelopment of thymic lymphoma, were exposed either to gamma rays produced by 137Cs sources, onedelivering dose rate of 800 mGy/min for an acute exposure, or 0.7 mGy/h for 24 hours per day low-doserate exposure. In one of these studies, animals were sacrificed and their thymi extracted 130 days after
acute 4.4 Gy exposure or after cumulative dose of 2.1 Gy delivered at low dose rate [B49]. In this case,thymus weights were lowest and apoptosis most frequent in low-dose-rate exposed mice. Up-regulated
and down-regulated genes numbered 55 and 186 in acutely irradiated or 29 and 8 in low-dose-rate
exposed mice. Among these, Cds1 (CDP-diacylglycerol synthase 1) was increased only in low-dose-rate
mice, while ItgA4 (integrin-α4), ItgB1 (integrin-β1) and Myc showed increased expression in both groupsof irradiated mice.
247. A further study used both AKR/J (inbred) and ICR (outbred) strains mice with slightly shorter
exposures to 0.7 mGy/h 137Cs gamma rays reaching a total dose of 1.7 Gy, but with a post-irradiation
period of 100 days before animal sacrifice and thymus extraction [B51]. This work showed after pathway
analyses that three pathways overlapped between two different animal genotypes: PPAR signalling
pathway, calcium signalling pathway and systemic lupus erythematosus. Unique to ICR mice were
pathways for: cancer, neuroactive ligand-receptor interaction, insulin signalling pathway, extracellularmatrix receptor interaction and adipocytokine signalling pathway. Unique to AKR/J mice were pathways
for: T-cell receptor signalling pathway, haematopoietic cell lineage, chronic myeloid leukaemia, Notch
signalling pathway, primary immunodeficiency and circadian rhythm. Similarly, as before, low dose rate
exposed AKR/J mice showed the most thymic apoptosis. This was an important finding considering thatin this case low-dose-rate exposure was concluded 100 days prior to animal sacrifice and thymus harvest.
TUNEL staining of thymi of ICR mice showed that the highest apoptosis (though not nearly as great inextent as that in AKR/J animals) was evident in high dose acutely exposed animals.

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248. A long-term low-dose-rate exposure of p53+/− and wild-type mice was carried out by Mitchel et al.
[M36]. The exposure periods lasted 30, 60 and 90 weeks. Animals were irradiated for five days a week
and the daily dose was 0.33 mGy delivered at a low dose rate (0.7 mGy/h). Thus, the total doses fell into
low and moderate dose ranges: 48, 97 or 146 mGy, respectively. Life shortening, and increased cancer
incidences were found in wild-type mice exposed for 30 or 60 weeks, but not in those exposed for
90 weeks. At the same time, no differences in cancer incidence were found between non-irradiated or anyof the irradiated heterozygote animals.
249. To summarize, the animal model used is important for exploration of effects of irradiation at low
doses and low dose rates. In many cases, animals with genetic alterations that lead to development ofdifferent diseases benefited from exposures to low doses of radiation. While a direct connection to cancer
outcomes is difficult to make in such cases, majority of these studies indicate that low-doses or low-doserate exposures to radiation lead to mild stress responses such as increased expression of MnSOD [T21]
while in mice heterozygous for p53 the quantity of p53 protein increased after low-dose radiation
exposures [L18, L19]. In other words, low doses of radiation mobilize animals’ gene expression resourcesin ways that may be, in the context of the underlying genetic status, beneficial.

6.

Hyper-radiosensitivity

250. Low-dose hyper-radiosensitivity describes an effect of extreme cell death due to excessive
sensitivity to small single doses of ionizing radiation but a more resistant phenotype (per unit dose) tolarger single doses. The underlying molecular mechanism for hyper-radiosensitivity is linked to a dosedependent pre-mitotic cell-cycle checkpoint that is specific to cells irradiated in the G2 phase and thelack of activation of this checkpoint at very low doses [M17].
251. The adaptive effects of low-dose and/or low-dose-rate exposure on the modulation of the hyperradiosensitive response to low/intermediate dose gamma radiation, i.e. the proportionally increased cell
death after low-/intermediate-dose exposure at high dose rate (0.1–0.5 Gy at 40 Gy/h) was addressed ina series of articles by Edin et al. [E3, E4, E5, E6, E7, E8]. Hyper-radiosensitive response of T47D humanbreast cancer cells or T98G human glioblastoma cells is abrogated by a first exposure to a low/moderate
dose (adapting dose) (0.06–0.3 Gy) delivered at moderate dose rate (0.06–0.3 Gy/h) [E5, E6]. The effectsof this adapting dose could be replicated by culturing non-irradiated cells in a conditioned medium
harvested from adapted cells, or even in conditioned medium irradiated after cell removal. This effect
was attributed to a soluble factor present in culture medium, most probably secreted by the cells even in
absence of irradiation, which could then be activated by low-dose and low-dose-rate exposure. iNOS
activity was required for the effects of the adapting irradiation. In subsequent work [E7], anti-TGF-β3
neutralizing antibodies were found to prevent inhibition of hyper-radiosensitive response by low-dose
and/or low-dose-rate exposure, and conversely, the addition of purified TGF-β3 to un-adapted T47D or
T98G cells could abrogate hyper-radiosensitive response. The effects of TGF-β3 required peroxinitrites,and “activation” of the adapted cells by IL-13 [E3]. These results were confirmed by in vivo experiments.
The addition of serum collected from DBA/2 mice exposed to 0.3 or 0.03 Gy at a dose rate of 0.3 or
0.03 Gy/h, respectively, into the culture medium abrogates hyper-radiosensitive response in T47D and
T98G cells. This effect is prevented by pre-treatment of the mouse-serum containing culture medium
with a neutralizing anti-TGF-β3 antibody, or if the mice were injected with an iNOS inhibitor for six days
before collecting their serum, starting one week after exposure [E8]. The pre-exposure of mice to 0.3 Gyover one-hour results in a better survival than for un-adapted mice after a subsequent exposure to 9.0 or
9.5 Gy. However, adapted mice do not survive longer than control animals if they are not further exposed
to challenge doses [E4].

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UNSCEAR 2020/2021 REPORT252. In a study by Qvarnström et al. [Q2], persistence of hyper-radiosensitivity was investigated in
prostate cancer patients undergoing radiotherapy. Skin biopsies (452) from 42 prostate cancer patients
who received 7-week fractionated radiotherapy treatment with daily dose fractions of 0.015–1.10 Gy to
the skin were collected at regular time intervals before and after treatment (30 minutes, 2 to 72 hours
post-irradiation). Epidermal skin keratinocytes were stained by immunofluorescence andimmunohistochemical staining for γH2AX and 53BP1. There was a dose–response relationship for DSB
foci (γH2AX and 53BP1 foci) observed in the skin keratinocytes throughout the treatment procedure.
Presence of foci 72 hours after damage induction (irradiation) demonstrated the considerable persistenceof DSBs produced. The authors conclude that this is the first evidence of preserved hyper-radiosensitivity
as assessed by γH2AX and 53BP1 throughout the 7-week treatment.
253. Mechanistically, hyper-radiosensitivity is a response specific to G2-phase and is attributed toevasion of an ATM-dependent G2-phase cell-cycle checkpoint [M17], however, hyper-radiosensitivity
is not caused by a failure to recognize DSBs. Exploiting three pairs of transformed isogenic cell lines
with known and distinct differences in radiosensitivity and DNA repair (ATM, NHEJ), no relationshipwas evident between the initial numbers of DSBs monitored immediately after radiation exposure todoses less than 0.2 Gy or cell survival for any of the cell lines indicating that the prevalence of hyperradiosensitivity is not related to recognition of DSBs. Instead, persistence of γH2AX foci (4 hours after
exposure) was significantly correlated with cell survival [W17]. In agreement, primary AT fibroblasts
displayed a similar hyper-radiosensitivity positive response to normal fibroblasts [E16]. Assessment ofearly G2-checkpoint activity after irradiation in 4 normal hyper-radiosensitivity positive and 4 hyperradiosensitivity negative fibroblasts of cancer patients showed that in hyper-radiosensitivity positive
fibroblasts the checkpoint was not triggered at doses lower than 0.2 Gy X-rays [S47]. In another study[S48], residual phospho-ATM and γH2AX foci were examined 24 h after 0.1–4 Gy and were found at
higher frequency after exposure to low dose/intermediate dose (0.1–0.5 Gy) radiation in hyperradiosensitivity positive compared with hyper-radiosensitivity negative fibroblasts. In doses exceeding
1 Gy, no difference appeared between positive and negative hyper-radiosensitivity fibroblasts.
254. A recent study addressed the response to low-dose radiation with 220 kV X-rays, 90 keV/μ C ionsand broad beam neutrons with average energy of 5.8 MeV in rat and human stem cells and showed thatmurine and human glandular tissue stem cells exhibit a dose threshold (<1 Gy) in DNA damage response
activation, resulting in intermediate-dose hyper-radiosensitivity with implication for salivary glandsecretory function [N3]. Furthermore, inhibition of ATM led to impaired DNA repair in human salivary
gland organoid cells after 1 Gy, but not after 0.25 Gy. Whereas (as expected) the initial number of γH2AX
foci per cell increased with increasing doses indicating an efficient DNA signalling by DNA damage
response, similar levels of residual damage were observed at 24 hours following both 0.25 and 1 Gy.
These data might suggest an efficient DNA damage signalling, but poor repair at relative low doses ofionizing radiation in the organoid cells.
255. To investigate the mechanism of hyper-radiosensitivity and induced radioresistance after irradiation
with medium- and high-LET particles, normal and two ATM deficient/mutant human skin fibroblast lines
were irradiated by C ion beam and the involvement of ATR pathway in hyper-radiosensitivity/induced
radioresistance response was detected with pre-treatment of specific inhibitors [X2]. The early G2/M
arrest was observed in ATM deficient/mutant cells with an effective ATR signalling, even as low as
0.2 Gy for C ion radiation. Inhibition of the ATR pathway in normal cells affected the hyperradiosensitivity/induced radioresistance occurrence similarly as ATM inhibitor. These data demonstratethat ATR pathway may cooperate with ATM in the mechanism of intermediate dose hypersensitivity
induced by C ion beams. Pre-treatment of AT fibroblasts with ATR inhibitor fully recovered mitotic entry
after C ion beam radiation (0.2 Gy).

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256. To summarize, some experiments suggest that in response to low- or intermediate-dose exposure
delivered in one hour, cells and animals up-regulate their production of TGF-β3 to provide increased
protection from hyper-radiosensitivity against a wide range of radiation doses delivered at high dose rate.
The production of TGF-β3 seems to require IL-13 and iNOS activity. Thus, inflammatory factors may
participate in radio-adaptive effects in relation to hyper-radiosensitivity. Hyper-radiosensitivity occurs inprimary differentiated cells, stem cells and tumour cells after low- and high-LET radiation independent
from ATM and ATR. Most evidence suggests that the efficiency of DSB repair based on γH2AX fociafter intermediate dose (<0.2 Gy) is not affected early after treatment. Evidence indicates that persistenceof DSB (i.e. γH2AX foci) correlates with cell survival.

G. Stem cells and target cell populations for radiation
carcinogenesis
257. While not specifically mentioning stem cells, the UNSCEAR 1993 Report, annex E, “Mechanismsof radiation oncogenesis” [U3] recognized the likely single cell origin of cancers, and the accumulationof genetic and other alterations in stem and progenitor cell populations. Similar observations have been
made in the UNSCEAR 2000 Report, annex G, “Biological effects at low radiation doses” [U5]. Previous
work of the Committee has not undertaken a comprehensive evaluation of the effects of radiation on stemand progenitor cell populations.
258. Stem and early progenitor cells are widely held to be the main target cells from which cancers arise(e.g. [P24]). This is because of the inherent properties of such cell types, notably including long lifespanin the organism, thus allowing sufficient time for the accumulation of multiple alterations to the cellular
phenotype, required to convert normal cells into malignant cells. Substantial evidence underpins the stem
cell origin of cancers in general [L42], and experimental animal studies indicate stem and early progenitor
cell origin for some radiation-induced cancers, notably acute myeloid leukaemias in the mouse [V8].
There are lines of evidence, however, that more mature differentiated cells can undergo a process of dedifferentiation and thereby contribute to carcinogenesis [W1]. The relative contributions of the stem celland de-differentiation routes is not known, but clearly any de-differentiation route would require theaccumulation of larger numbers of cell phenotype alterations before cancers present clinically. Studies
modelling cancer incidence over time indicate that for leukaemias one or two genetic alterations are
required and while a number in the range of 5–7 is required for solid cancers. These estimates find support
in more recent whole genome sequencing analyses of cancers [M19] where solid cancers were found tocarry on average four driver mutations with a range of 1–10.
259. There remains a debate on the origin of mutations in cancers. Some recent studies suggest that in
the majority of cancers errors in DNA replication in stem cells is the major source of mutations [T17,
T18]. Therefore, the numbers of stem cells in a tissue and their frequency of division would be a major
contributor to carcinogenesis. This view has been challenged as underestimating the role of mutations
induced by environmental carcinogens [L37]. Mutation frequencies determined by whole genome
sequencing in three adult stem cell populations with varying proliferation rates and cancer risk (colon,
small intestine and liver) have been found to be similar [B42], a finding that also contrasts with theproposals from Tomasetti and colleagues. Further work on whole genome sequencing of radiotherapyassociated cancers that identifies putative genome sequence markers specific to radiation-induced cancers[B27] lends support to the view that a significant contribution of induced as opposed to replicative
mutations is important for radiation carcinogenesis.

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UNSCEAR 2020/2021 REPORT260. Clonal haematopoiesis refers to the presence of clones derived from single stem cells in the mature
haematopoietic system of humans, identified by the presence of chromosome aberrations inhaematopoietic cells and is a risk factor for haematological malignancy. It has been found recently bymeans of whole genome sequencing that clonal haematopoiesis is more common in elderly people. Ten
per cent of over 65 year olds compared with 1% of under 50 year olds, carry clones with leukaemiaassociated mutations [G3]. The fact that clonal haematopoiesis can be detected in the healthy before
disease presentation suggests it may be a useful early marker of subsequent haematological malignancy.
Similar age-related increase in clonal haematopoiesis with clones carrying leukaemia-associatedtranslocations has been confirmed independently [J12]. These studies confirm the importance of earlyclonal expansion of leukaemia mutation carrying clones as a precursor to clinical presentation of
haematological malignancy. Furthermore, the occurrence of clonal haematopoiesis has been found to be
associated with prior exposure to radiation in the course of radiotherapy (but not chemotherapy) andtobacco usage [C26]. Recently, it has been shown in a mouse model of radiation acute myloid leukaemia
that expanding clones with leukaemia-associated mutations can be detected prior to disease presentationand further indicate that the stem/progenitor cells of origin can differ between irradiated male and female
mice [V8]. Such studies and approaches provide a link between experimental and modelling studies (seealso chapter IV).
261. Notwithstanding the above discussions, a consideration of the effects of radiation at low doses instem cells is of relevance to understanding mechanisms driving low-dose radiation carcinogenesis. It is
important to establish if the responses of stem cells to radiation exposures are similar to those of the more
widely studied differentiated cell types, and if responses vary between stem cell populations.
262. While there are not large numbers of relevant papers available (table 5), many different stem cell
types have been examined, including embryonic, epithelial, neural and mesenchymal stem cells, someincluding low-dose and low-dose-rate studies. A number of key points can be derived from the studies
reported to date. These are summarized in the following paragraphs, note that not all papers listed intable 5 are discussed in further detail below.

Table 5. Stem cell and radiation response literatureSpecies/cell type

Irradiation conditions

Effect reported at low dose

Other effects/comments

Reference

1. EMBRYONIC STEM CELLS
Mouse
Embryonic stem cell

Mouse

Human
Embryonic stem cell/ induced
pluripotent stem cell

Human
Chord blood-derived
endothelial colony forming
cells
Human
Embryonic stem cell

Human
Embryonic stem cell

Human
Embryonic stem cell/ induced
pluripotent stem cell

25 mGy reduced effect of 1.5 Gy(5 hours later) on micronuclei and cytotoxicity

X-radiation
Dose: 0.1–1 Gy
Dose rate: 0.7 Gy/min

Increase in apoptotic cells in irradiated embryos and reduced normal
full-term pregnancies

Gamma radiation, Xradiation
Dose: 0.1–4 Gy
Dose rate: most at 0.4–
~0.8 Gy/min

0.4 Gy increased targeted vector integration by homologous
recombination (about 30-fold) with no effect on viability or HPRT
mutation, or point mutations/indels in exome, some effects on gene
expression 24 hours after 0.4 Gy. γH2AX foci repair observed andapoptosis at 0.4 Gy[H12]X-radiation
Dose: 60–380 mGy
Dose rate: 7–
35 mGy/min

Reduced proliferation, increased doubling times, cytotoxicity[K26]

Gamma radiation
Dose: 0.1–1 Gy
Dose rate: not given,assumed to be acute

Gene expression at 2 and 16 hours after exposure by Taq-man assays –wide range of dose–responses observed and variation between
embryonic stem cell lines

Gamma radiation
Dose: 0.05–1 Gy
Dose rate: not given,assumed to be acute

Agilent oligo microarrays – transient expression changes at 0.05, but
fewer genes respond than at 1 Gy, significant cell line variation,
especially at 0.05 Gy in responsive transcripts

Gamma radiation (LINAC)
Dose: 0.25–15 Gy
Dose rate: not given,assumed to be acute

Only very modest alterations in gene expression (P53, RAD51, PRKDC,XRCC4, BRCA2)[K10]

p21, Smad2, Foxo2 expression increased byirradiation but no effect of stem cell factors

Considerable variation in results presented

[H14]

[S54]

[S55]

Most response observed at 2 Gy and above,
probably quite crude PCR method
employed[S65]

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Embryonic stem cells ex vivo
irradiated 8-cell stageembryos

Gamma radiation
Adaptive dose: 25–
50 mGyChallenge Dose: 1.5 Gy
Dose rate: 130–
136 mGy/min

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Human
Embryonic stem cell

Human
Embryonic stem cell

Irradiation conditions

Effect reported at low dose

Gamma radiation
Dose: 0.4–2 Gy
Dose rate: not given,assumed to be acute

No reported effects on core stem cell transcription factors and no effecton pluripotency; cytotoxicity observed but proliferation continues

Gamma radiation
Dose: 0–2 Gy
Dose rate: 1.16 Gy/min

Chromosome aberrations, micronuclei and γH2AX foci induction at
0.5 Gy. Differentiated derivatives of human embryonic stem cells
displayed higher induction of aberrations, micronuclei and foci thanundifferentiated human embryonic stem cell

Other effects/comments

Reference[W13]

Patterns of gene expression changes inducedby 1 Gy varied between differentiated andundifferentiated human embryonic stem
cells, and peripheral blood lymphocytes[V11]

Review paper[F14]

2. HAEMATOPOIETIC STEM CELLS
Human and mouse
Various
Mouse
Purified primitive
haematopoietic stem cells
and more committed
lineages
Mouse
Irf5−/− and wild-type mice on
C57BL/6 backgroundGamma radiation
Dose: 20 mGy–2 Gy
Dose rate: acute

Hyper-radiosensitivity of haematopoietic stem cells (only) seen in vitro,
but not in vivo (four months after exposure), sensitivity driven by ROS

Concludes that low doses have long-term
detrimental effects that could lead toleukaemias[R14]

Gamma radiation (137Cs)
Dose: 4 weekly doses of
1.75 Gy from 5 weeks ofage in vivo
Dose rate: not given

Reduced lymphoma yields in −/− mice attributed to reduced apoptosis
in haematopoietic stem cells leading to less compensatory
proliferation

Proposes Irf5 as potential target foramelioration of bone marrow injury
following radiation[B33]

3. NEURAL STEM CELLS
Mouse
Embryonic brain ventricularzone/subventricular zone
Mouse
Adult and embryonic brain
sub-ventricular zone,
ventricular zone and subgranular zone regions
Human
Neuronal stem cell in culture

X-radiation
Dose: 10–20 mGy in vivo
Dose rate: 4.9 mGy/min

Linear dose–response for 53BP1 foci, apoptosis especially at E13.5, linear
induction. Delayed repair if doses below 50 mGy

No response to 50 Hz magnetic fields[S3]

X-radiation
Dose: 50–200 mGy in
vivo
Dose rate: acute

Embryonic brain highly sensitive to DSBs and apoptosis induction; adulthighly sensitive to apoptosis also

Summarizes work of Jeggo laboratory[B15]

Various charged particles
Dose: 50 mGy–1 Gy
Dose rate: 100–
500 mGy/min

Elevated ROS and RNS (reactive nitrogen species) over one-week postirradiation[B25]

UNSCEAR 2020/2021 REPORT

Species/cell typeSpecies/cell type
Human
Neuronal stem cells,
differentiated neurons andfibroblasts
Mouse
Adult brain
subventricular zone

Irradiation conditions

Effect reported at low dose

Other effects/comments

Reference

X-radiation
Dose: 0.46 or 2.3 Gy
Dose rate: 0.01 or
0.05 Gy twice daily,
5 days per week

Elevated mitochondrial numbers and function in differentiated cells but
not Neuronal stem cells[S37]

X-radiation
Dose: 50 mGy–2 Gy
Dose rate: acute,
0.5 Gy/min

Apoptosis induced linearly with dose, 50–500 mGy, within this range[B16]

Proliferation arrest has threshold around 200 mGy and induction ofdifferentiation a threshold around 500 mGy

4. MESENCHYMAL STEM CELLS
Human

Human
Myc/hTERT immortalized
mesenchymal stem cell line
Rat
Primary bone marrow derived
mesenchymal stem cells
Human
4 primary oral mucosa
derived mesenchymal stem
cell lines
Human
Primary bone marrow derived
mesenchymal stem cells(one donor)
Human
Primary fat-tissue derived
mesenchymal stem cells
MiscellaneousSome proliferation delay induced by 100 mGy in 3/5 lines. One linedifferentiation affected. Co-culture with BM haematopoietic stem cells
increase differentiation of CD34/38 positive cells (more mature), again
only in some

Authors conclude that 100 mGy can affectmesenchymal stem cell function, butvariation is a concern. Accurate dosedelivery problematic[F23]

Gamma radiation
Dose: 0.01–1 Gy
Dose rate: 0.79 Gy/min

Global gene expression assessed at 1–48 hours post-exposure. Somelow dose specific responsive genes identified and most with nonlinear D-R. Most doses statistically very similar response for individualtranscripts

Accurate dose delivery problematic,
particularly for 10 mGy. Questionable ifanything of significance in this study[J21]

X-radiation
Dose: 20 - 100mGy
Dose rate: 100 mGy/min

Proliferation stimulated and increase S-phase at 50 and 75 mGy;
stimulation of MEK, ERK, Raf phosphorylation also

[L27]

X-radiation
Dose: 25 - 250 mGy
Dose rate: 40 mGy/min

Linear accumulation of γH2AX and pATM foci, slower repair at 20–
80 mGy[O19]

X-radiation
Dose: 80/1 000 mGy
Dose rate: 40 mGy/min

Delayed H2AX foci repair at 80 as opposed to 1 000 mGy, residuals notwith pATM and in proliferating cells. 1 000 mGy exposure leads tosenescence at passage 11 post-irradiation and reduced proliferation

X-radiation
Dose: 100 mGy only
Dose rate: 100 mGy/min

Transient apoptosis (2 hours) with 100 mGy and release of oxidized
DNA. Propose release of oxidized DNA may be signalling molecule foradaptive response

With one donor, no information on variability
which is widely reported elsewhere

[P25]

[S25]

Review paper, including Alessio et al. results
below [A11][S60]

ANNEX C: BIOLOGICAL MECHANISMS RELEVANT FOR THE INFERENCE OF CANCER RISKS ...

Primary bone marrow derived
mesenchymal stem cells

Gamma radiation
Dose: 100 mGy
Dose rate: 800 mGy/min

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Human
Primary bone marrow derived
mesenchymal stem cells
Human
Primary bone marrow derived
mesenchymal stem cells

Effect reported at low dose

Other effects/comments

Reference

X-radiation
Dose: 75 mGy
Dose rate: not given

Slight and transient increase in mesenchymal stem cell proliferation
observed, and increase in S-phase cells. Increased secretion of IL-11.
Mild reduction in ability of mesenchymal stem cells to support T-cellproliferation. Claimed to be mediated by Rb, CDK1 and CDC25b

Only modest effects reported, and probably
from a single donor[Y5]

X-radiation
Dose: 40–2 000 mGy

40 mGy reduced proliferation and induced senescence and autophagy;
impaired DSB repair at 40 mGy. Reduced stemness at 40 mGy, also at
higher doses

Irradiation conditions[A11]

5. MISCELLANEOUS
Mouse
Spermatogonial stem cell in
vivo
Mouse
Muscle spindle (stem) cells
Mouse
Skin, bulge and sebaceous
gland stem cells
Human
Induced pluripotent stem
cells

Mouse
Keratinocyte and melanocyte
stem cells

Human induced pluripotent
stem cell line ND41658H
differentiated tochondrocytes

X-radiation
Dose: 100 mGy
Dose rate:
1 000 mGy/min

Increase in 53BP1 at 45 minutes but not caspase or γH2AX, no apoptosis
observed

More mature cells show γH2AX and its repair.
Conclude repair in spermatogonial stem
cells may differ from that in more mature
cells

Gamma radiation
Dose: 60–7 500 mGy

Reduced satellite cell numbers over time (50 mGy and above), noevidence of hormetic effects[M21]

Gamma radiation
Dose: 50 mGy/5 Gy
Dose rate: 10 mGy/min
or 2.34 Gy/min

50 mGy leads to apoptosis in sebaceous gland stem cells, but not Bulge
stem cells. Survival of Bulge stem cells due to Hif1α expression andmetabolic shift to glycolosis. Surviving bulge stem cells lead to Basalcell carcinoma development at 50 mGy in Ptch +/− mice

[R5]

Gamma radiation
Dose: 2–6 Gy
Dose rate: not given

Find enhanced DNA damage response, especially HRR in stem as
opposed to NO-induced differentiated cells

Gamma radiation
Dose: 0.5/1/2.5 Gy in
vivo
Dose rate: 0.3 mGy/min
and 0.3 Gy/min;

Low-dose-rate exposure reduced hair follicle density and pigmentation,
no significant reduction in melanocyte stem cell number, so assume
functional change in skin stem cell compartments

Gamma radiation
Dose: 1–5 Gy
Dose rate: 2.5Gy/min

DNA damage response and repair observed, along with relatively low
ROS levels but high induction of senescence

Concludes that pluripotent stem cells haveenhanced DNA damage response bycomparison with more differentiated cells,
notable higher homologous recombination
but variation in response with different
tissue stem cell types

[L10]

[M44]

[S66]

Concludes induced pluripotent stem cell
derived chondrocytes have greater repair
activity than standard chondrocyte cell lines[S61]

UNSCEAR 2020/2021 REPORT

Species/cell type

Irradiation conditions

Effect reported at low dose

Human dental pulp stem cells
from deciduous teeth

X-radiation (Cone-beamCT)
Dose: 5.4–107.7 mGy

Transient induction of phospho-H2AX and full repair even at 5.4 mGy.
Increased release of cytokines IL-8, IL-1α and TNF-α over 24 hours

Concludes that cone-beam CT doses may
carry risk

[V12]

Mouse

Gamma radiation (137Cs)
Dose: 1–5 fractions of
50 mGyDose rate: 16 mGy/min

Single or multiple 50 mGy exposures prompted wild-type progenitors to
reduce proliferation and increase differentiation. P53-mutation
carrying progenitors do not differentiate but continued to expand intoclones overgrowing the normal epithelium

The overgrowth of p53-mutation carrying
cells was abrogated by treatments withantioxidants[F10]

Reduced fitness of 1 Gy irradiated cells[F22]

Oesophageal epithelium
progenitor cell
Mouse
Lgr5 intestinal stem cells inorganoid culture+

Lgr5 intestinal epithelial
stem cells+

High dose-rate reduced stem cell replenishment of epithelium, lowdose-rate exposure did not affect stem cell replenishment of theepithelium[O20]

X-radiation (LINAC)
Dose: 10–50 fractions of
100 mGy

Exposures caused accumulation of 53BP1 foci in nuclear
heterochromatin followed by ATM dependent senescence but not
apoptosis. Highly damaged stem cells differentiate promoting stem
cell proliferation[S17]

X-radiation (260 keV)
Dose: 0.1–4 Gy
Dose rate: 0.5 Gy/min

In the small intestine, 53BP1 foci detected immediately after irradiation,
but rapidly disappeared thereafter, especially in Lgr5+ stem cells.
Cellular growth was temporarily arrested; however, cell numbers andmitotic cell numbers in the crypt did not change. The kinetics of DNA
damage repair in Lgr5+ stem cells were similar to those in the small
intestines, while the colon was more susceptible to radiation-induced
damage[O21]

Gamma radiation (137Cs)
Dose: 1 Gy
Dose rate: 0.003 Gy/h
X-radiation
Dose: 1 Gy
Dose rate: 30 Gy/h

Mouse
Hair follicle stem cells

Mouse
Lgr5 intestinal stem cells+

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Mouse

X-radiation
Dose: 1 000 mGy(Operated at 150 kV with
20 mA and a 0.5 mm Al
plus 0.3 mm Cu filter)
Dose rate: 600 mGy/min,Other effects/comments

ReferenceSpecies/cell type

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UNSCEAR 2020/2021 REPORT263. Stem cells are responsive to low-dose as well as moderate-/high-dose exposures to ionizing
radiation, and such responses are observed under both in vitro and in vivo exposure conditions. DNA
damage responses are broadly similar to those observed in differentiated cells but with some indications
that responses in terms of DNA repair, notably homologous recombinational repair are greater in stem
cells as opposed to their more differentiated counterparts, at least at high doses of 2–6 Gy gamma
radiation [M44]. Moderate doses of 400 mGy gamma radiation can enhance DNA integration throughhomologous recombination in human induced pluripotent and embryonic stem cells [H12], supporting anenhanced HRR function in stem cells. This would suggest that repair of double-stranded DNA damage
in stem cells being generally of a higher fidelity than in differentiated cell types. Support for this is foundin a comparative study of chromosome aberration, micronucleus and γH2AX foci induction where
undifferentiated human embryonic stem cells had reduced yields of these endpoints compared todifferentiated counterparts [V11]. By contrast, mouse spermatogonial stem cells appear to have reduced
HRR [L10, L11].
264. In common with findings in differentiated cells, the induction of DNA damage foci in stem cells(γH2AX, 53BP1 and others) is linear with dose, even in the low-dose area, down to 10 mGy [O19, P25,
S3]. Furthermore, slower repair of DNA damage foci after low-dose exposures has been observed inneural [S3], and mesenchymal [A11, O19, P25, S3] stem cells. As with results from studies employing
more differentiated cells, the long-term consequences of this delayed repair are not clear.
265. The induction of apoptosis by low doses in stem cells is variable according to stem cell type [R5]and developmental stage [B15] and this may be influencing the induction of cancers by low doses [R5].
The Revenco et al. [R5] study used a transgenic mouse model based on Ptch1 [R5]. In this study, work
was carried out on wild-type mice and effects followed in epidermal stem cell populations in thesebaceous gland and hair follicle bulge region. DNA damage induced by a dose of 50 mGy gammaradiation exposure led to death of the sebaceous gland stem cells, while the bulge epidermal stem cells
survived and underwent a metabolic switch with activation of Hif1α and overexpression of many cellularpathways (with endocytosis as the only pathway that was decreased). In Ptch1 heterozygous mice, lowdose irradiation led to the development of skin cancers that look similar to human basal cell carcinoma.
In this model system, moderate doses of radiation had the same but a more pronounced effect – moreskin cancer foci per animal developed. Nevertheless, regardless of dose, 100% of these animals developed
skin cancer.
266. In adult mouse neural stem cells irradiated in vivo with 50–500 mGy, a linear dose-dependence forthe induction of apoptosis was observed, with no apparent threshold and with statistical significanceachieved at 50 mGy [B16]. By contrast, the induction of reduced proliferation in the stem cellcompartment required a dose of 200 mGy or above, and induction of stem cell differentiation required
doses of 500 mGy or above. Barazzoul et al. [B16] argue that because quiescent stem cells accumulate
fewer chromosomal translocations, the threshold of around 200 mGy for the induction of proliferationarrest may serve to protect stem cells from the consequences of irradiation, and that this may be relevant
for low dose risk assessment [B16].
267. Fractionated exposure to X-rays (100 mGy fractions, 5 fractions per week at daily intervals, 2–
10 weeks total) in mouse hair follicle stem cells is reported to lead to accumulation of DNA damage(53BP1) foci and the induction of senescence in highly damaged cells that promotes proliferation in thestem cell pool [S17].
268. Fernandez-Antoran et al. [F10] have demonstrated the sensitivity of oesophageal epithelial
progenitor cells to 50 mGy exposure to gamma radiation in vivo that leads to a reduction in proliferationand increase in differentiation of wild-type progenitors. Progenitors carrying mutations in the p53 gene
respond differently to low-dose exposure and out-compete wild-type progenitors in the epithelium.

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Treatment with antioxidants can reverse the competitive advantage afforded to pre-malignant p53
mutation carrying cells/clones, allowing proliferation of wild-type cells and loss of mutation carrying
cells through differentiation. This study suggests that after low-dose exposures the tissue can remove
damaged cells through differentiation, but the exposure can promote the growth of pre-existing premalignant cells, depending on the oxidative environment. This effect can be seen as a pro-carcinogenic
effect of low-dose exposure through promotion. By contrast, in the intestinal epithelium it has beenreported that while high dose-rate exposure (1 Gy dose) reduced replenishment of the epithelium, thesame dose delivered at low dose-rate (0.003 Gy/h) had no impact on replenishment rates [O20].
269. In the mouse haematopoietic system, primitive stem cells respond to in vitro low-dose exposure
with a specific radiation hypersensitivity which is not observed in more differentiated haematopoietic
cells [R14]. Such hypersensitivity is not seen in vivo indicating an important role of the stem cell
microenvironment (or niche) in modulating responses. Low-dose responses were found to be driven byalterations in reactive oxygen metabolism, and the niche may play an important role in controlling theoxygen tension in vivo [R14]. Elevated ROS and reactive nitrogen species (RNS) have been observed inhuman neural stem cells at least one week post-exposure of haematopoietic stem cells [B25].
270. A number of analyses of gene expression at up to 48 hours after exposure to low and moderate
doses have been reported. There are indications that the responsive genes may differ between dose levels
but there is no clear consensus on the nature of low- as opposed to moderate-dose responsive genes;
however, these studies are indicative of non-linear responses operating at the level of gene transcription.
Adaptive response has been reported in embryonic stem cells [K10] but by contrast hormetic effects were
not observed in mouse muscle stem cells (spindle cells) after gamma irradiation [M21].
271. A review by Manda et al. [M8] was unable to conclude whether low-dose radiation is likely to cause
transformation of stem cells into cancer stem cells due to insufficient evidence being available. This
review mentioned that exposure of neural stem cells to a moderate dose of 300 mGy low-LET radiation
resulted in increased protein expression of Wnt1, Wnt3a, Wnt5a and β catenin leading to proliferation as
well as differentiation, while exposure of diabetic rats to repeated doses of 75 mGy (total accumulated
doses, 375–825 mGy) increased the number of bone marrow and circulating stem cells and improved
wound healing. Similarly, regenerative neovascularization was also associated with moderate-dose
exposures of four times 0.3 Gy doses [M32]. Thus, moderate to low doses radiation seem to increaseproliferation followed by differentiation that can only indirectly support cancer development.
272. The International Commission on Radiological Protection (ICRP) published a report on “Stem cellbiology with respect to carcinogenesis aspects of radiological protection” [I3], which presented
arguments that low dose rate effects in vivo may differ from acute low dose effects in stem cell
populations due to the ability of stem cells to repopulate tissues, based on studies with haematopoietic
stem cell systems. The competition for occupancy/space in stem cell niches between undamaged anddamaged stem cells is likely to be of relevance here in that damaged cells generally appear to be less“fit”, i.e. less able to occupy a niche when undamaged cells are also present. Similar loss of fitness of
1 Gy X-irradiated cells has also been shown in an in vitro organoid culture system based on intestinal
cells [F22].

Summary273. Stem and early progenitor cells are viewed as main target cell populations for radiation cancer due
to their long lifespan in the organism. Stem cell populations in several tissues have been shown to respondto low doses of radiation, and in terms of DNA damage response they appear to respond in a similar

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fashion to other somatic cells, and this holds true for mutation frequencies that do not strictly correlatewith replication cycles traversed.
274. However, some stem cells, most notably embryonic stem cells, but also induced pluripotent stem
cells, depend more on homologous recombinational repair of DNA damage than NHEJ. This repair
pathway preference likely serves to preserve genome integrity and avoid mutation in the presence ofDNA damage given the higher fidelity of recombinational repair compared to NHEJ. This is likely
therefore to act to protect stem cells from mutation, much as the retention of parental DNA strands
through DNA replication and cell division that has been observed in some stem cell populations.
275. Some stem cell populations enter apoptosis readily following radiation exposure, and this likely
serves to protect from cancer. Loss of cells through apoptosis though may lead to substantial proliferationof stem cell populations to restore tissue that brings risk of replication-associated mutation, at low doses
this is expected to be a minor consideration.
276. A recent study has demonstrated how low (50 mGy) radiation exposures can lead to the preferential
expansion of cells and clones carrying mutations in the Trp53 gene [F10]. This supports a role of low
doses in promoting the growth of pre-existing pre-neoplastic clones.

H. Effects at the whole organism level277. In this section, the effects of radiation exposure at the level of the whole organism, and how these
relate to the induction of cancer or the modulation of cancer risk are considered. The studies considered
include those investigating the effects on the immune system that might modulate cancer risk, animal
carcinogenesis studies and a range of studies that investigate specific aspects of carcinogenesis.

1.

Immune system and cytokine responses

278. The Committee published a comprehensive evaluation of the effects of radiation on the immune
system as its UNSCEAR 2006 Report, annex D, “Effects of ionizing radiation on the immune system”[U7]. This report noted the well-documented effects of high radiation exposures and concluded that itremained uncertain if low dose exposures stimulated or suppressed responses.
279. The impact of radiation over a range of doses on the immune system has been examined in a numberof exposure contexts, including environmental, medical and accidental exposures. The UNSCEAR 2006
Report [U7] summarized general features of the immune system, the effects of radiation on immune
functions observed in experimental systems, the possible mechanisms underlying these effects, andepidemiological studies on the effects of radiation on the immune system in humans. The review
concluded that low-dose and low-dose-rate exposures to radiation could stimulate or suppress the immune
system, while exposures to higher doses radiation was clearly immunosuppressive. In this annex, more
recent studies on the effects of exposure to radiation on the immune system and function are considered.
The impact of radiation over a range of doses has been examined in a number of exposure contexts,
including human exposure (survivors of the atomic bombings, environmental, medical and accidental
exposures) and experimental exposure of cell lines and animals.

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91(a) Immune system and cancer
280. Sigurdson et al. [S45] investigated the relationship between polymorphisms in a series of eightgenes involved in apoptosis, inflammation and proliferation with breast cancer risk in a cohort ofradiological technologists, considering both occupational and medical breast exposure. Occupationaldose to the breast was categorized as <0.05 Gy (77%), <0.05–0.1 Gy (13%), >0.1–0.2 Gy (8%) and>0.2 Gy (2%). The distribution of personal medical diagnostic X-ray breast dose was similar. Theirresults showed that a minor allele of the gene coding for Casp-8, involved in apoptosis, had a significant
protective effect, while a minor allele of the gene coding for IL-1α by contrast increased the risk of breast
cancer development. Schonfeld et al. [S16] investigated the role of oxidative stress and chronic
inflammation genes as modifiers of the risk of radiation-induced breast cancer in the same cohort. Theirresults suggest that a polymorphism in the PTGS2 gene, coding for the COX-2 enzyme, is associatedwith an increased risk of breast cancer following occupational exposure. Together, these results showthat genes coding for proteins involved in immune or immune-related processes can modify the risk ofcancer development associated with low-dose radiation exposure.
281. Snijders et al. [S51] reported on the existence in mice of an oestrus cycle-dependent gene expression
signature in mammary gland that includes 117 genes linked to interferon signalling. Human breast cancer
patients with high expression of this signature had increased chances of metastasis-free survival,suggesting here again that the expression of factors associated with immune response can influence theoutcome of cancer development. In breast-cancer- and radiation-sensitive BALB/c mice, but not incancer- and radiation-resistant C57BL/6 mice, oestrus cycle-dependent variations of leukocytes andinflammatory cytokine levels in the serum were also observed. Although low-dose radiation (100 mGy
X-rays) did not affect the expression of the interferon-dependent gene signature in any of the mousestrains, it resulted in low blood lymphocyte counts and circulatory inflammatory cytokine levels in
BALB/c, but not C57BL/6 mice, suggesting that it may participate in the radiosensitivity of these animals.
282. In the study of Shin et al. [S40], AKR/J mice, prone to the development of thymic lymphomas,
received 4.5 Gy of gamma rays, either at 0.8 Gy/min or at 0.7 mGy/h. The lower-dose-rate exposed micesurvived longer and developed fewer thymomas. Gene expression analysis at 130 days post-exposure
showed that in the thymus of lower-dose-rate exposed mice, most of the changes were the increase in
apoptosis-related and B- and T-lymphocyte activation-related genes. In contrast, in high-dose-rate
exposed mice most of these changes were down-regulation not only of genes involved in apoptosis andimmune activation but also of genes involved in DNA repair and genome stability, DNA damage
signalling, cell cycle, cancer and p53 signalling pathways. This difference might explain why, in contrast
to high-dose-rate delivery, lower-dose-rate radiation better promotes a thymoma-free survival in response
to high-dose radiation [S40]. More recently, Gao et al. [G2] analysed the modulation of long non-coding
RNA (lncRNA) and their target genes in the thymus of ICR mice exposed to 0.075 or 4 Gy. Thymi werecollected 12 hours post-exposure. Gene ontology classification of the mRNA potentially regulated byradiation-modulated lncRNA in mice exposed to 4 Gy identified cell death and cell-cycle regulation
processes while those down-regulated are rather associated with nuclear organization. Thus, some of theeffects observed in irradiated AKR mice 130 days post-exposure in [S40] may have already been initiated
12 hours post-exposure.
283. Radiation exposure is a recognized risk factor for the development of sarcomas. Injection of micewith the beta-emitting radionuclide 45Ca (1 µCi/37 kBq per gram, intra-peritoneal) results in a highincidence of osteosarcomas. In this model, Kansara et al. [K13] showed that the onset of osteosarcoma
development is accelerated in mice heterozygous for the tumour suppressor gene, Rb1. Rb1 inactivationin primary human osteoblasts leads to a reduction of radiation-induced senescence in these cells,
accompanied by a reduction in the expression of genes coding for inflammatory factors of the senescenceassociated secretory pattern. In a cohort of patients, high RB1 and high-senescence-associated secretory

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pattern levels are associated with metastasis-free survival. IL-6 was shown to be required for tumour
suppression in vivo. The number of NKT cells infiltrating tumours was found to be reduced in the absenceof IL-6 or Rb1 expression, and the development of radiation-induced osteosarcoma was accelerated in
NKT cell-deficient mice, without affecting senescence induction. A model was proposed in which RB1dependent senescence and senescence-associated secretory pattern induction leads to the recruitment of
NKT cells able to clear pre-malignant cells.
284. A series of articles addressed the role of NK cells and macrophages in the reduction of pulmonary
engraftment of (intravenous) injected L1 sarcoma cells in low-dose exposed BALB/c mice. Whole-body
exposure to 0.1 or 0.2 Gy X-rays at 2.2 Gy/h, reduces lung colony formation by half. Radiation exposure
results in an increased cytotoxic activity of activated peritoneal macrophages and NK cells. The blockadeof these activities abrogates the radiation-induced reduction in L1 cell engraftment [C13]. The productionof NO by activated macrophages appears to be required for both in vitro cytotoxic activity against L1
cells and reduction of engraftment after low-dose exposure [N19]. Dose fractionation (10 times 0.01 Gy,
0.02 Gy and 0.1 Gy X-rays) appears inefficient at reducing the number of pulmonary L1 sarcoma cellcolonies, but still induces an increase in the cytotoxic activity of “NK-enriched” splenocytes and activatedmacrophages purified from irradiated mice, albeit with different kinetics and amplitude than in mice
acutely irradiated to the same total doses [N21]. An increase in cytokine secretion is observed in activatedmacrophages (IL-1β, TNF-α, IL-12), PHA-stimulated splenocytes (IL-2) and “NK-enriched” splenocytes(IFN-γ) purified from mice exposed to 0.1 and 0.2 Gy. The kinetics and/or amplitude of secretion in thenine days following exposure differed according to the dose delivery (acute compared with fractionation)[C14]. In a new series of experiments, using more mice, exposure to 0.1, 0.2 and 1 Gy in 10 fractions
was this time found to induce a reduction of pulmonary L1 cell engraftment [N20]. All exposures induce
a biphasic increase in the cytotoxicity and NO production of activated macrophages ex vivo. These effects
of fractionated radiation exposure (increased cytotoxic activity of “NK-enriched” splenocytes andactivated macrophages, production of NO by activated macrophages, reduction of pulmonary sarcoma
cell engraftment) have been largely confirmed in a different mouse strain (C57BL/6) [N20].

(b) Studies of the Japanese atomic bombing survivors
285. Past studies, reviewed in the UNSCEAR 2006 Report, annex D, “Effects of ionizing radiation on the
immune system” [U7] addressed the late effects of atomic radiation on immune cells in the atomic bombing
survivors. A reduced frequency of blood T-lymphocytes with an imbalance in Th1/Th2 responses was foundin survivors, with a reduced proportion of naïve T-cells, while on the contrary memory T-cells wereincreased. The proportion of B-lymphocytes was increased, as was the concentration of serum IgA infemales and IgM in both sexes. No consistent changes were reported in circulating innate immune cells,
although radiation-associated inflammatory responses were observed. Some of these qualitative andquantitative changes suggested that radiation exposure could accelerate the normal ageing of immune
system, characterized by a functional decline with age, a process known as immunosenescence.
286. T-lymphocytes are generated in the thymus from circulating BM-derived progenitors. Thymic
activity (its ability to support T-lymphocyte differentiation) can be evaluated by measuring in the
peripheral blood the number and frequency of T-cells containing TRECs (T-cell receptor excision
circles), circular DNA fragments generated during the somatic rearrangement process required for T-cell
receptor gene expression. As TRECs do not replicate, they are more abundant in populations enriched inrecently produced naïve T-cells. TRECs were quantified in the blood of 1,073 aging atomic bombing
survivors who donated blood samples between 2003 and 2009 (median radiation dose 0.17 Gy, range 0–
2.1 Gy, median age 77 years, range 62–90 years), in relation with lifestyle/obesity indicators [Y12].
TREC numbers showed a strong positive association with naïve T-cell frequency, were inversely

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correlated with age at examination and more abundant in females, but no significant association withradiation dose was found, neither in CD4 nor in CD8 population. However, TRECs level was inversely
correlated with obesity (fatty liver, type-2 diabetes) and inflammatory (serum C reactive protein
concentration) markers.
287. The late effects of radiation exposure on the morphology and function of the thymus were analysedfrom thymic tissue samples collected from deceased atomic bombing radiation exposed individuals during
autopsy (N=165, radiation dose <5 mGy (non-exposed), 5–200 mGy, >200 mGy, age 20–91 years) andstored at Radiation Effects Research Foundation [I14]. Immunohistochemistry and immunofluorescencestaining of tissue slides allowed determination of the size and respective proportions of epithelial andlymphoid areas, and the presence of immature thymocytes. These data were analysed in correlation with theage at the time of death and the dose of radiation received and compared with results obtained from tissues(N=11) and slides (N=25) archived at Duke University. As expected, thymic atrophy increased andthymopoiesis decreased with age. This decline was evident earlier in males before the age of 60 years, but
was similar in both sexes thereafter. Thymic measurements were also decreased in tissues from exposed
individuals in a relation depending both on the dose of radiation and the age at the time of bombing. A
decline in thymic measurement is more likely to be observed in individuals exposed at older age. The effectsof radiation exposure were stronger than the effects of aging, suggesting that any radiation-dose canaccelerate thymic aging, and these effects persist for years after exposure.
288. Radiation-induced T-cell immunosenescence was evaluated by fluorescence-activated cell sorting
analysis of various T-cell populations in peripheral blood of aging atomic bombing survivors. Changes
in the frequency of total conventional T-cells (Tconv), Tregs, and naïve, effector, central memory andeffector memory CD4+ T-cells were measured in relation to radiation dose (0.005–4.0 Gy), inflammation,
sex and age at time of blood collection [K54]. The frequency of Tconv but not Tregs, declined with age.
There was a radiation-dose dependent increase in Tregs and central memory CD4+ T-lymphocytes.
Radiation also induced a reduction in the frequency of naïve CD4+ T-cells. The concentration of TNF-α
was inversely associated with the modulation of naïve CD4+ T-lymphocytes but not with changes in
Tregs. Thus, radiation exposure seems to accelerate immunosenescence by a shift towards
immunosuppressive Tregs and inflammation-related decrease in naïve T-cells.
289. The association between changes in T-lymphocytes homeostasis, obesity/metabolic status andtelomere length was measured in a group of 620 atomic bombing survivors (median radiation dose
0.102 Gy, range 0–1.7 Gy) [Y13]. Telomere length decreases with age in T-lymphocytes andgranulocytes. Surprisingly, when analysed in relation with dose, it was observed that there is first an
increase in telomere length until 0.5–1 Gy, and then a decrease such that there was no difference intelomere length between the 0.005 and ≥1 Gy categories. Overall, radiation-associated telomere length
shortening was observed in individuals exposed to ≥0.5 Gy in lymphocytes, but not granulocytes. Thisdecrease was modulated (up or down) by different metabolic indicators, suggesting that metabolism may
modify the long-term effects of radiation exposure.
290. In blood samples donated 11 years apart by a different group of 415 atomic bombing survivors
categorized by the study authors into low (median radiation dose 0.23 Gy, range 0.005–0.69 Gy) and high(median radiation dose 1.3 Gy, range 0.70–3.8 Gy) dose exposure groups, telomere length was found to besignificantly shorter in the high dose group than in control group (<0.005 Gy) [L52]. When all groups wereclassified according to the age at time of bombing, telomere length shortening in the high dose group wasstill significant in younger (age at time of bombing <12 years old) but no longer in older (age at time ofbombing >12 years old) individuals. The rate of telomere length shortening with aging over 11 years was
similar in all groups, irrespective of age at time of bombing. Telomere length was positively or negatively
associated with different metabolic and/or cellular indicators in individuals exposed to <0.005 Gy, but most

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UNSCEAR 2020/2021 REPORTof these associations disappeared in the exposed groups, suggesting that radiation exposure modifies themetabolic status and/or immune parameters, resulting in the loss of their correlation with telomere length.
291. The association of metabolic markers with the homeostasis of peripheral lymphocyte populations
was also analysed in 3,113 atomic bombing survivors (median radiation dose 0.08 Gy, range 0–1.8 Gy)[Y14]. Several indicators of obesity and related diseases (fatty liver, diabetes) were associated withincreases in total T, Th1, CD8+ T- and B-cell populations, while the association was negative for naïve
CD4+ T and NK cells. In addition, age and radiation exposure were found to correlate with decreased
total T, CD4+ T and naïve CD4+ T-cell populations, while proportion of Th1 and Th2 cells increased.
However, the only significant interaction between radiation dose and metabolic/disease indicators wasthat of C reactive protein and radiation for B-lymphocytes.
292. The number of circulating white blood cells is an indicator of inflammation. The effects of radiation
exposure on age-associated changes in total and differential (neutrophils, monocytes, lymphocytes) whiteblood cells were investigated in a longitudinal study involving 7,652 atomic bombing survivors who
donated blood every two years between 1964 and 2004 [H39]. Both total and differential white blood
cells declined with age in males and females, but with different patterns. Smoking increased the numbersof all cell types. Radiation exposure (>2 Gy) also results in increased white blood cell counts, but to alower level than smoking. When age at time of bombing was considered, a significant radiation dosedependent increase in white blood cells counts was identified in men exposed before the age of 20 years,
but not in women. Thus, irradiation can result in persistent elevation of total and differential white blood
cells for decades after exposure.
293. More recently, the effects of radiation exposure on white blood cells were re-assessed in a larger
study (N=14,346) with a longer follow-up period (until 2010), different endpoints (frequency of whiteblood cells in addition to counts) and different statistical methods [Y15]. Baseline frequencies oflymphocytes, neutrophils and monocytes were similar at the study entry in never exposed not-in-city
residents, individuals exposed to ≥1 Gy (median dose 1.5 Gy) and individuals exposed to <1 Gy (mediandose 0.038 Gy). The different cell populations evolve differently, but for each of them the trend isdifferent before and after 60 years of age for both men and women. The frequency and counts ofmonocytes were found to be dose-dependently increased by radiation exposure, and this increase is more
pronounced after 60 years. This radiation-induced persistent elevation in monocytes could indicate
ongoing inflammation and might be related to the acceleration of age-dependent clonal haematopoiesis.
294. The effects of radiation exposure and aging on inflammation have been compared between a groupof 260 atomic bombing survivors exposed to more than 5 mGy and a group of 182 non-exposed(<0.005 Gy) individuals [H15]. The amount of several cytokines (IL-6, IL-4, IL-10, TNF-α), Igs, CRP,
ROS and erythrocyte sedimentation rate were measured. All showed significant but weak association
with radiation dose (except IL-4) and age. Multivariate analysis of selected cytokines/factors revealed
that different combinations of factors, including or excluding ROS, could explain a large proportion ofradiation and age dependency, suggesting the existence of different pathways associated with radiationand age-associated inflammation. The effects of aging on inflammation appeared more pronounced thanthe effects of radiation. Radiation exposure may potentiate age-dependent inflammation.
295. In order to evaluate other age-related changes of the innate immune system, Kajimura et al. [K8]analysed the abundance and response to stimulation of circulating dendritic cells in 229 atomic bombing
survivors. These individuals were categorized by the authors into non-exposed (median dose 0.001 Gy,
range 0–0.002 Gy), and exposed to low (median dose 0.13 Gy, range 0.12–0.49 Gy) and high (mediandose 0.98 Gy, range 0.50–3.80 Gy) radiation dose groups. The number of total dendritic cells,
plasmacytoid and classical dendritic cells was not modulated by radiation exposure in men, while thenumber of plasmacytoid dendritic cells decreased with increased radiation dose in women. Of note, theANNEX C: BIOLOGICAL MECHANISMS RELEVANT FOR THE INFERENCE OF CANCER RISKS ...

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total number of dendritic cells, plasmacytoid and classical dendritic cells decreased with age also only inwomen. Ex vivo dendritic cells activation, evaluated by RT-qPCR and secretion of cytokines was
influenced by the age of donors, but not by radiation dose or sex.
296. To determine the effects of radiation exposure early in life on the functionality of the immune
system, the efficiency of vaccination against influenza virus was measured in a group of 292 aged atomic
bombing survivors (non-exposed, low-moderate exposure, high exposure) who donated blood before andafter subcutaneous vaccination with commercial trivalent inactivated influenza vaccine during the 2011or 2012 flu season [H16]. The response to vaccination was evaluated by quantification ofhemagglutination inhibition antibody titres in serum, and the production of cytokines by peripheral bloodmononuclear cells stimulated in vitro with the relevant influenza vaccine. Although the response tovaccination was modest, as expected in this aged populations, it was similar in control and exposedgroups, indicating that radiation exposure early in life does not prevent nor impair vaccine response
decades later. Thus, the functional decline of the immune system does not seem to be aggravated by past
radiation exposure.

(c) Environmental, occupational, medical and accidental exposures
297. Attar et al. [A25] compared immune parameters in residents of a HBRA in Ramsar, Islamic
Republic of Iran, and of a control area. They reported modulation of lymphocyte numbers, cytokine andantioxidant levels in the serum, and neutrophil activity, suggestive of a shift of T-lymphocyte populations
from Th1 to Th2, and activation of innate immunity, including antioxidant defences in HBRA residents.
In a different study Borzoueisileh et al. [B52] addressed the influence of the duration of residence in thesame region on immune parameters in a group of 50 healthy adults who lived in this area for 10 to 35 years(mean exposure duration 22±8.7 years). They observed a significant increase in the frequency of blood
CD4+ T-cell and a decrease of blood CD107a+ cells (cytotoxic CD8+ T-cells and NK cells) with time ofresidence, i.e. cumulative exposure. This last result was partially confirmed in an independent study
comparing the abundance of CD107a+ cells in the blood of a group of 30 HBRA residents and a group of
30 residents from a control region [B52]. HBRA group included individuals living in this area for at least
10 years, and the mean annual effective radiation dose was 9.77 mSv (4.5–57 mSv). For the control
group, the mean annual effective radiation dose was 1.2 mSv (0.57–3.0 mSv). The frequency and numberof white blood cells and lymphocytes was similar in both groups, but the concentration (cells/µL) of
CD107a+ cells was lower in donors from the HBRA group.
298. Li et al. [L22] studied immune parameters in two comparable groups of 100 healthy women living
in China, one living in the Yangjiang district HBRA (mean age 65±10 years) and the control one in the
Enping district (median age 61±8.1 years). Cumulative dose exposure in HBRA subjects was160±38 mSv (range 59–249 mSv) and 44±7.3 mSv (range 31–66 mSv) in the control group. There wasno statistical difference in the frequency of total or CD4+ blood T-lymphocyte. However, the frequency
of CD8+ T-cells increased with cumulative dose. Furthermore, the number of CD8+ T-lymphocytes wasincreased in function of body mass index after adjustment for age and dose. The analysis of serum
cytokine levels by antibody array identified a group of 10 factors elevated by 1.5 to 2.6 times in HBRA
residents. The levels of 6, including sIL-6R, MCP-1, IFN-γ, EGFR and CRP were confirmed by ELISA
to increase with increasing cumulative dose, in a linear relation for CRP, MCP-1 and sIL-6R.
299. In a different experimental approach, Jain and Das [J10] performed an in-depth transcriptional
profiling (Affymetrix Gene Chip) of peripheral blood mononuclear cells isolated from residents of theKerala coast region in India. They analysed cells from four groups of nine healthy donors exposed to<1.5 mGy/y (group I), 1.51–5.0 mGy/y (group II), 5.01–15 mGy/y (group III), and >15 mGy/y (groupIV). The number of differentially expressed genes (up- or down-regulated with fold changes of 1.3, 1.5

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UNSCEAR 2020/2021 REPORTand 2.0) increased with exposure in groups II, III and IV. In each group, the number of genes regulated
decreases as the fold change increases. Gene ontology analysis showed that immune response pathways
are among the over-represented biological processes defined by differentially expressed genes in groups
III and IV, with 24 and 28 pathways, respectively. In group IV, some pathways related to T-cell receptor
signalling, B-cell receptor signalling, and cytokine-cytokine receptor signalling are up-regulated, whereassome other related to T-cell receptor signalling, Toll-like receptor signalling, and cytokine-cytokine
receptor signalling are down-regulated. Expression of certain genes including NFKB2 (up-regulated) andCXCR1, TNFS10, CCR2 (down-regulated) is dependent of the radiation dose in groups II–IV. The
regulation of expression of 30 genes was validated by RT-qPCR. This study illustrates the complexity of
the regulation of the immune system/immune processes in response to chronic low-dose exposure.
300. To evaluate the effects of chronic exposure/internal contamination on the immune system, Sajjadieh
et al. [S7] measured the levels of IL-4 and INF-γ together with the CD4+ T-cell population in Ukrainianchildren living in a post-Chernobyl contaminated area, with or without irritable bowel disease without
inflammation. Compared to children from the same area without irritable bowel disease, children withirritable bowel disease had less CD4+ T-cells, a higher level of IL-4 and a lower level of IFN-γ, suggestinga stronger polarization toward a Th2 phenotype. There was no difference with age, suggesting that there
was no radiation-dose effect.
301. Apoptosis of lymphocytes and mutations in the T-cell receptor genes in circulating lymphocytes
were analysed in Techa river basin residents chronically exposed for years to a combination of external
and internal radiation [B41]. The study group consisted of 161 people (mean age 69 years, range 58–83,
69% women) with mean accumulated red bone marrow dose of 0.96 Gy (range 0.01–4.5 Gy), and amaximum annual dose to red bone marrow of 0.26 Gy and included 38 people who survived chronic
radiation syndrome. Chronic radiation syndrome is a clinical syndrome which develops in man after
whole body annual radiation exposures exceeding 0.7–1.0 Gy and cumulative doses >2–3 Gy over 2–
3 years, characterized by inhibition of haemopoiesis and immune reactions, structural and functional
disorders of the central nervous, cardiovascular and other organ systems [I2]. The control group consisted
in 72 unexposed individuals with similar characteristics. The frequency of apoptotic cells was foundincreased in the study group (0.35%) compared with the control group (0.25%). The frequency of T-cellswith mutated T-cell receptor genes was increased three times (0.33%) in the study group and even reached0.41% in the chronic radiation syndrome survivors. The induction of apoptosis may limit theaccumulation of mutated T-lymphocytes at higher accumulated doses.
302. A comprehensive survey of immune parameters was conducted in a different group of residents of
the Techa river basin in relation to their level of mutation in T-cell receptor genes [A8]. The study group(N=66) was divided into a main group (N=29), consisting of individuals with higher levels of T-cell
receptor gene mutations, and a comparison group (N=37). The mean age, sex ratio and socio-economic
status of both groups were similar. The absorbed dose to red bone marrow was 0.89±0.09 Gy (range
0.09–2.0 Gy) and 1.03±0.07 Gy (range 0.30–2.3 Gy) for the main and comparison groups, respectively.
The absorbed dose to soft tissue was 0.07±0.01 Gy (range 0.01–0.33 Gy) and 0.07±0.02 Gy (range 0.01–
0.49 Gy) for the main and comparison groups, respectively. Adaptive (number of B- and T-lymphocyte
subsets, serum Ig concentration) and innate (number of NK and NKT cells, myeloid cells, theirphagocytic and lysosomal activities, cytokine levels) immune parameters were quantified in blood. The
lysosomal activity of neutrophils, the number and frequency of NKT cells and the amount of IL-1α inserum were found to be increased in the main group together with a decrease in IL-2 and GM-CSF levels.
lysosomal activity of neutrophils increase was correlated to the dose rate and absorbed dose to red bonemarrow, whereas the amount of NKT cells was associated to the soft tissue dose rate. The authors view
these changes as the reaction of the immune system to the increased frequency of mutated T-cells in
exposed individuals.

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303. Analysis of immunological changes was also performed in workers from the Mayak production
association workers cohort [R23] occupationally exposed to cumulative doses of external gamma rays
ranging from 0.5 to 3.0 Gy (N=14, mean dose 1.8±0.76 Gy) or to a combination of gamma rays (N=77,
range 0.7–5.1 Gy, mean dose 1.8±0.76 Gy) and internal alpha radiation from plutonium incorporation(mean body burden 1.8±0.23 kBq, range 0.3–16.4 kBq). The control group included 43 matched subjects
with no history of exposure. The amount of serum IgM, IgG, IgA, IgE, MMP-9, TNF-α, IFN-γ, IL-10
and TGF-β1 and the proportion and number of selected circulating T-lymphocyte and NK cell
populations were measured. The level of IgM, IgA, MMP-9, and the frequency of NK cells wereincreased while the NK cell frequency of T-lymphocytes was decreased in both groups of exposed
workers compared to the control group. The level of IgG and the frequency of total and T-helper
lymphocytes were reduced while the amount of TGF-β1, IFN-γ, the frequency of NK and NKT cells andthe number of NKT cells were increased in externally exposed workers. Finally, the frequency of helper
T-cells was significantly lower and the amount of serum TNF-α and TGF-β1 significantly higher inworkers with mixed exposure compared to workers exposed to external radiation only. In the externalradiation group, the proportion of CD4+ helper T-cells was inversely correlated with the level of gammaradiation exposure. Thus, chronic occupational exposure leads to long-term modulation of immune
parameters, different according to the exposure scenario.
304. Howell et al. [H37] described the response of rodents captured in the Chernobyl exclusion zone tochemotherapeutic agents and lipopolysaccharide stimulation. After intra-peritoneal injection of
lipopolysaccharide (0.5 mg per kg body weight), the induction of the glutathione peroxidase 1 andsuperoxide dismutase 2 genes in the liver was significantly lower in animals from contaminated areas
than in control animals. The results indicate a modulation of the response to lipopolysaccharide in animals
living in contaminated environments.
305. Occupational exposure of medical staff is an important consideration. Karimi et al. [K15] comparedhaematological parameters, lymphocyte response to mitogenic stimulation and cytokine levels in a groupof 30 radiology staff who received less than 50 mSv (“the maximum permissible annual dose”) and 20
unexposed control laboratory staff. Detailed exposure information is not available. It was found thatlymphocytes from radiology workers proliferate more intensely in response to phytohaemagglutinin thanthose of unexposed workers. Radiology workers had higher levels of IFN-γ and lower levels of IL-4 thanunexposed donors, suggesting a shift toward a Th1 status.
306. Zakeri et al. [Z2] analysed chromosomal damage and selected markers of cellular and humoral
immune responses in serum of interventional cardiologists compared to unexposed clinical physicians.
The group of 37 cardiologists was exposed to an annual mean dose of 8.1±7.8 mSv, with a total dose of
30.5±24.3 mSv over the last five years. The frequency of aberrant cells, chromosomal breaks anddicentrics plus centric rings increased 2, 4 and 5 times, respectively in cardiologists when compared to acontrol group of clinical physicians. Interventional cardiologists had a more than 10-fold higher serum
IgG level, but without any correlation with duration of employment and exposure. The levels of IL-2 andIL-10 were respectively significantly up- and down-regulated in cardiologists. No variation was foundfor IL-4, IL-8, IFN-γ and IL-6. There was no variation in the number and frequency of the main
lymphocyte subsets in cardiologists at the steady state level. However, upon phytohaemagglutinin
stimulation, CD4+CD69+ T-lymphocytes proliferate more.
307. Gyuleva et al. [G21, G23] analysed several immune parameters in a cohort of 438 workers (421 men
and 17 women) of the Kozloduy nuclear power plant in Bulgaria and a control group of 65 persons (49men and 16 women) without any work-related exposure. Nuclear power plant workers were grouped
according to employment duration (mean dose of 9.5±5.8 mSv in group 1 to 350±120 mSv in group 4).
The frequency of blood CD4+ T-cells was decreased in group 1 (dose range of 0.1–20 mSv) and group 3(dose range of 100–200 mSv) when compared to the control group; the frequency of CD8+

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T-lymphocytes was decreased in group 1, and the frequency of NKT cells was found to be decreased ingroups 1 and 3, and that of T-lymphocytes with NK activity in all groups. However, all these fluctuations
were found to be within the range of normal reference values. No changes were observed in thepercentages of B-lymphocytes, total T-lymphocytes and NK cells in the different groups of workers. Ina follow-up study, the frequency and absolute number of CD4+CD62L+ memory T-lymphocytes in groups
1 and 2, and of CD4+CD25+ Treg cells in group 1 were found decreased compared to control group. The
frequency of CD8+CD28+ cytotoxic T-lymphocytes was decreased only in group 4 (>200 mSv). Hereagain, all these parameters were within the normal reference values.
308. These analyses were repeated and extended in a second cohort of Kozloduy nuclear power plant
workers (105 workers stratified in four groups with mean doses of 11.8±9, 53.2±21, 130±30 and
324.6±89 mSv, respectively). The frequency and absolute number of B-lymphocytes, total
T-lymphocytes, CD4+ and CD8+ T-lymphocytes, cytotoxic CD8+CD28+ T-lymphocytes, NK cells and
NKT cells were found to be unaffected by radiation exposure. Within the CD4+ T-lymphocyte population,naïve and terminal effector T-cells were found to be unchanged, but the absolute number of central
memory CD4+ T-lymphocytes was found to be decreased in group 1 and increased in group 4 whencompared to a control group of 32 unexposed workers. No changes were observed in the levels of
circulating Igs (IgG, IgA and IgM) or cytokines (IL-2, IL-4 and IFN-γ) in exposed workers [G22].
Collectively, these studies indicate that work-related exposure with cumulative doses up to 350 mSv does
not induce any pathological changes in the frequency and absolute number of the main blood lymphocyte
and NK-cell populations in these occupationally-exposed workers.
309. Chi et al. [C22] reported on the follow-up of three subjects accidentally irradiated while sterilizing
food products. Subjects A, B and C received an estimated physical dose of 11.2, 1.82 and 1.0 Gy of 60Co,
respectively in about 20 minutes after entering a defective radiation-sterilization room. Subjects A and B
received bone marrow transplantation on days 14 and 21 after the accident, while subject C received GMCSF 48 hours after exposure. Three years later, they all showed a reduced percentage of CD4+
T-lymphocytes, and a reduced CD4+/CD8+ ratio. B-lymphocytes and NK cells were still decreased onlyin subject A. Analysis of gene expression in leukocytes showed persistent modulation of the numerous
genes involved in inflammation, including PTGS2, coding for the COX-2 enzyme, and oxidative stressmanagement, showing that radiation exposure, at least at high doses, has long-lasting molecular andcellular consequences.
310. Li et al. [L21] report on the 10-year follow-up of the accidental “persistent” exposure of a group of
54 people (29 males and 25 females, median age 31.5, range 20–42 years) exposed to 192Ir between May
9 and July19, 2002. Neither the exact exposure scenario nor the method used for dosimetry are provided.
Subjects are said to have been exposed to doses estimated to be between 0.05 and 0.65 Gy by biologicaldosimetry (lymphocyte chromosome aberration). Ninety per cent of the subjects exhibited physicalsymptoms one month post-exposure. About half of them had abnormal white blood cell counts, and most(90%) exhibited abnormal morphology of bone marrow granulocytes, erythrocytes and megakaryocytes
at that time. These abnormalities decreased with time (analysis at 1, 3, 5 and 10 years), but two of thesubjects still had abnormal white blood cell counts, and abnormal bone marrow cell morphology 10 years
after the accident, respectively. Chromosomal aberrations (dicentrics and rings assessed using
conventional staining) sharply decrease with time post-accident. The number of CD4+ and of CD8+
T-lymphocyte and the number of NK cells in blood were decreased for up to 1 year post-accident, but
returned to normal levels thereafter. Despite the lack of information on the scenario and dosimetry of
exposure, this study provides valuable information about long-term follow-up of the consequences ofaccidental exposure.

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99(d) Low-dose exposure for treatment of cancer, inflammatory, infectious andchronic diseases
311. Kojima et al. [K34] reported on the use of various regimens of radiation exposure as therapy forcancer and non-cancer patients. Patient A had surgery for prostate cancer, but his prostate-specific antigen
level rose again and remained high. Exposure to 30-weekly dose fractions of 150 mGy from X-rays (totalor half body irradiation) resulted in normalization of his prostate-specific antigen level. Patient B wasaffected by an inoperable prostate cancer with bone metastasis. A combination of X-rays (10 fractions of
150 mGy, three fractions each week) and radon exposure (use of a 44 × 93 cm radon sheet, surface
activity 37 µGy/h), six hours each day for ten months resulted in the disappearance of the bone metastasis
and normalization of his prostate-specific antigen, which stayed stable for at least six months after thetreatment. Finally, a patient afflicted with ulcerative colitis for 16 years was treated with a combinationof daily intakes of radon-containing water (200 mL of radon-containing water (330 Bq/L) with everymeal), exposure to radon in a room mimicking the conditions found in radon spas (9.8 kBq/m3) twice aweek for 40 minutes and daily exposure to radon at bedtime. The clinical symptoms disappeared, and thepatient’s bowel movements were restored to normal levels. This patient has now been healthy for morethan eight years.
312. Exposure to low doses of radon (effective dose estimated to be around 0.3 mSv) in radon spa
therapies is reported to relieve pain in patients suffering from musculoskeletal disorders associated withchronic inflammation. A lasting reduction in the serum concentration of collagen degradation products,
observed in patients from 12 weeks after the beginning of the treatment, suggests an amelioration of bone
resorption, which might result, at least in part, from a reduction in the concentration of the adipokine
vifsatin, which follows the same kinetics [C31]. This study also reported a decrease in Treg cells as
another sign of attenuated inflammation. Rühle et al. [R21] performed a detailed longitudinal monitoringof immune cell populations in the blood of a cohort of 100 patients undergoing radon spa therapy.
Immune cells were compared before the beginning and up to 30 weeks post-therapy. T-lymphocytes andmonocytes were slightly increased from 6 to 30 weeks. Within T-lymphocytes, the frequency of Treg
cells, effector and effector memory cytotoxic T-cells were up-regulated, whereas cytotoxic T-cells, naïve
T-cells and central memory cytotoxic T-cells were down-regulated at 6, 12 and/or 30 weeks. Importantly,
no change was observed in the frequency of CD4+, Th1 and Th2 cells. Myeloid and plasmacytoid
dendritic cells were only transiently increased at week 6, whereas neutrophils and eosinophils wereslightly decreased at that time. No changes were observed for B-cells. The frequency of total NK cells
remained constant during the 30 weeks, but different NK cell subsets were up- (NK1) and down-regulated(NK2 and NK3) after six weeks after therapy. The activation level of T-, B- and NK cells, as defined by
CD69 expression, is lastingly reduced after therapy, whereas a small subset of T-cells shows increased
expression of HLA-DR, indicating that they are activated. Thus, exposure to a very low dose of radon
can induce discrete but long-lasting changes in immune cell homeostasis and/or activation in patients,
which appears to impact on inflammatory status and the course of musculoskeletal diseases.
313. Kojima et al. [K35] describe a case report where low-dose radon exposure therapy using conditions
encountered in radon-spa therapy (40 minutes each day, 5 days per week in a room with radon activity
200 kBq/m3 plus inhalation of vapours from radon-containing water) resulted in a long-term reduction ofrheumatoid arthritis physical symptoms (swelling, pain, mobility) and biological markers (reduction tonormal levels of the circulatory concentrations of C-reactive protein and MMP9).
314. Low- to moderate-dose low-LET radiotherapy is also used to treat a variety of human chronic
inflammatory disorders, but the underlying mechanisms are still elusive. Arenas et al. [A15, A16]
analysed the effects of low-dose radiotherapy on the interactions between leukocytes and endothelial cells
in mice, i.e. the early stages of inflammation. Intravital microscopy showed that a single abdominal

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exposure to low and moderate doses of 0.1–0.6 Gy inhibits lipopolysaccharide-induced adhesion ofleukocytes to endothelial cells in intestinal venules. Leukocyte rolling is inhibited by doses of 0.1 and
0.3, but not by a dose of 0.6 Gy. A dose of 0.3 Gy was found to be the most effective, with effects visible
as early as five hours post-exposure and lasting for two (adhesion) or three (rolling) days. The inhibitionof leukocyte adhesion/rolling was accompanied by a concomitant increase in plasmatic TGF-β1. The
administration of anti-TGF-β1 neutralizing antibody allowed to partially rescue leukocyte adhesion, but
not rolling inhibition. The increase in circulatory TGF-β1 is mirrored by a local increased production in
intestinal tissues.
315. The mechanistic basis of the anti-inflammatory effects of radiotherapy was also investigated in vitro
by analysing the response to a range of X-ray doses (0.1–3 Gy) of EA.hy.926 endothelial cells [R11,
R12]. Several aspects of the response were found to be discontinuous or biphasic. For example, theactivation of the transcription factor AP-1 is biphasic with maxima at doses of 0.3 and 3 Gy. Adhesionof blood polymorphonuclear cells to irradiated EA.hy.926 cells were found to be more decreased andpolymorphonuclear cells produced less CCL20 chemokine after a dose of 0.5 Gy than after doses of 0.3,
1 or 3 Gy. This effect is partially controlled by TGF-β1, and totally abrogated by inhibition of theexpression of the X-linked inhibitor of apoptosis protein (XIAP), which is maximally induced after adose of 0.5 Gy. Inhibition of XIAP expression also resulted in decreased NF-κB activation and increased
apoptosis in 0.5 Gy exposed EA.hy.926 cells [R13].
316. A discontinuous response was also observed when the secretion of IL-1β by irradiated (doses of
0.01–1 Gy) murine activated peritoneal macrophages was investigated. A decrease in the production of
IL-1β after exposure of macrophages to doses of 0.5 and 0.7 Gy occurred only when the macrophages
were purified from the radiosensitive BALB/c mouse strain, but not from the radioresistant C57BL/6
strain, even if they express a human TNF-α transgene. Thus, it appears that the anti-inflammatory effects
of moderate-dose radiotherapy might be linked to the genetic background rather than to the inflammatory
status [F20].
317. Joo et al. [J24, J25] and other authors [C1] addressed the effects of low-dose gamma irradiation on
IgE-mediated allergic response. Acute doses (0.01 to 0.1 Gy) and protracted doses (0.01, 0.05 Gy)
inhibited the release of inflammatory mediators by IgE crosslinking in a rat mast cell line after in vitro
sensitization. This inhibition is also reflected at the level of inflammatory cytokine production andintracellular calcium mobilization. Low-dose exposure had no effects on the activation of mast cells by
lipopolysaccharide or a calcium ionophore. This inhibition results from a radiation-dependent decreased
activation of FcεR1-mediated intracellular signalling pathways. These results were confirmed in humanmast cells lines, although not with the same dose-effect relation. Low-dose radiation exposure was also
shown to affect the development of immediate-type allergic reactions and late-phase reaction in mice,with a concomitant in situ decrease of FcεR1 expression in tissues.
318. Chronic or long-term inflammation is one of the mechanisms contributing to carcinogenesis.
Inflammation is often associated with chronic disease. In the following paragraphs, this annex presents
studies addressing the effects of low-dose/low-dose-rate radiation on inflammation, in several models ofchronic disease, as it may influence the risk of cancer.
319. The effects of exposure to 0.05 Gy of X-rays (delivered at 0.5 Gy/min) and 0.05 Gy of 12C ion(90 MeV, LET 28.3 keV/µm) on human peripheral blood lymphocytes in vitro were compared 24 hours
post-irradiation. The proportion of the CD4+ and CD8+ T-lymphocytes, and of NK and NKT cells were
not affected by exposure. The expression of the genes coding for IL-2, TNF-α and IFN-γ was increased
and the secretion of these cytokines was accordingly up-regulated. The cytotoxic activity of peripheral
blood lymphocytes was also up-regulated after 12C ion and X-ray exposure. For all these parameters, theeffects of 12C ions appeared always stronger than those elicited by X-rays [C17]. A similar study was

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performed on peripheral blood lymphocytes obtained from patients suffering from alimentary tract cancer(oesophagus, colon, gastric cancer). Here, the proportion of total T-lymphocytes as well as of CD4+ =and
CD8+ T-lymphocytes was found to increase after exposure to 0.05 Gy of X-rays or 12C ions (90 MeV,LET 28.3 keV/µm). The frequency of the different NK cell populations did not change. The expressionof IL-2 and IFN-γ was again found to be increased both at the mRNA and protein level, but the expressionof TNF-α was unchanged. Thus, the outcome of the radiation response may be influenced by the healthstatus at the time of exposure [C18].
320. The effects of repeated low-dose radiation exposure (25 mGy X-rays at 12.5 mGy/min every otherday for 2, 4, 8, 12 or 16 weeks) on the development and pathophysiological consequences of typeI diabetes were investigated in a murine model of experimentally induced disease [Z8, Z9]. Induction oftype 1 diabetes in C57BL/6 mice results in systemic and local (kidney, heart) inflammation (elevatedserum levels of TNF-α, MCP-1, IL-18, ICAM-1), and kidney and heart damage (oxidative stress,
fibrosis). Exposure of diabetic mice to 25 mGy every other day for 2 to 16 weeks reduces both systemic
and local inflammation and diabetes indicators, including oxidative kidney damage as early as 2 to
4 weeks after diabetes induction [Z8]. Repeated low-dose exposures also reduced cardiac inflammationand heart damage [Z9]. Thus, repeated low-dose exposure can significantly suppress several aspects ofdiabetes-induced inflammation and associated renal and heart dysfunction. It must, however, be notedthat repeated low-dose exposure also induces a low level of systemic and local (kidney, heart)
inflammation in healthy control C57BL/6 mice.
321. Beneficial effects of multiple low-dose exposures to X-rays were also found in a mouse model ofdiabetes after exposure of a different regimen of multiple low-dose radiation exposures. Diabetic
nephropathy develops in kidneys in diabetic mice and this was prevented by low-dose-rate radiation
exposures in two studies from the same group [C20, C21]. Diabetes was induced in 10-week-old
C57BL/6J mice by repeated exposure to streptozotocin, and then animals were exposed to 12.5, 25 or
50 mGy every other day for 4 or 8 weeks. Mice exposed to all of these doses of radiation showed lessfibrosis and some reduction in creatinine and proteins increased in diabetic animals such as connective
tissue growth factor.
322. Moderate-dose exposure protection in diabetic animals was found in rats which otherwise sufferfrom diabetic testicular atrophy [Z17]. Type 2 diabetes was induced in rats over two months by high fat
diet coupled with exposure to streptozotocin. Development of this disease triggered testicular atrophy,
unless exposure to moderate-dose radiation was carried out. Exposure of rats to 25 mGy of X-rays every
second day for four weeks until the accumulated dose reached 350 mGy protected animals from testicularatrophy. These rats had close to normal expression of anti-oxidative proteins NQO1, SOD, and catalase
as well nearly normal expression of glucose metabolism relevant proteins Akt and GSK-3b.
323. The effects of repeated low-dose radiation exposure on the development of experimentally induced
type 2 diabetes was also analysed. Mice were exposed to 25, 50 or 75 mGy of X-rays at 12.5 mGy/min
every other day for 4 or 8 weeks after disease induction [S27]. Radiation exposure was found to attenuate
dyslipidaemia, renal inflammation and kidney dysfunction in diabetic mice. Repeated exposures of
25 mGy had no impact on hyperglycemia but repeated exposures of 50 and 75 mGy transiently reduce
insulin resistance at 4 weeks. Differences in the response to different radiation doses were observed: theeffects of repeated 50 and 75 mGy exposures are often transiently visible at 4 but not 8 weeks, while theeffects of repeated 25 mGy exposures are often visible at 8 weeks only (for example for levels of urinary
albumin, plasma HDL, renal tri-glycerides, TNF-α, PAI-1, malondialdehyde, Nrf2 expression).
Expression of Nrf2 target genes (SOD1, HO-1 and NQO-1) was increased at 4 and 8 weeks for allradiation doses. These results were partially confirmed in a follow-up study comparing the effects ofrepeated low-dose exposures (50 mGy every other day for 4 weeks) and fibroblast growth factor 21 (FGF21) alone or in combination on the development of type 2 diabetes induced nephropathy [S28]. FGF-21

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is known to regulate carbohydrate and lipid metabolism and have beneficial effects in diabetes. The
attenuation of diabetes consequences/indicators was better after the combination than any treatment
alone. Of note, repeated low-dose exposures and FGF-21 have opposing effects on the expression of Nrf2
and its target genes, but the combination resulted in a normalization of their expression. Low dose rateand FGF-21 act independently to protect against type 2 diabetes pathophysiological consequences.
324. In a study using diabetic mice, Tsuruga et al. [T21] exposed 11 weeks old female db/db mice
(BSK.Cg-Leprdb +/+ Leprddbb/Jcl) and heterozygous littermates, db/m mice (BKS.Cg-m +/+Leprdb/Jcl) to 940 µGy/h of gamma rays for 24 days. Under these conditions, diabetic animals had
improved tolerance for glucose while no changes were found in healthy heterozygous littermates. Thisfinding was ascribed to improved MnSOD production in irradiated db/db animals.
325. Alpha-radiation exposure was also found to protect mice from renal damage induced by CCl4
injection, which induces a strong oxidative stress in kidney. Hence, 24 hours post-injection, mice exhibit
an increase in blood creatinine, and kidney lipid peroxidation, and a decrease in renal SOD and catalase
activity and morphological changes such as dilatation of Bowman’s space and glomerular atrophy.
Inhalation of radon (1,000 or 2,000 Bq/m3 for 24 hours) before CCl4 administration reduces creatinine
and lipid peroxidation increase and limits or reverses the effects of CCl4 on SOD and catalase activity,
respectively, but does not prevent tissue damage [K18]. Some of these effects are similar to those inducedby α-tocopherol, suggesting that radon inhalation can augment anti-oxidative defences.
326. The effects of radiation exposure on the development of thyroid autoimmune diseases wereanalysed in two mouse models [N2]. The exposure to a single dose of 0.5 Gy gamma rays 1 week before
beginning the supplementation of drinking water with NaI for 8 weeks exacerbates the development of
autoimmune thyroid disease (Hashimoto’s thyroiditis) in susceptible NOD-H2h4 mice, revealed by anincrease in the level of circulating anti-thyroglobulin. Total-body irradiation before (0.05 or 3 Gy) or after(0.05, 0.5 or 3 Gy) the beginning of NaI supplementation or exposure of the thyroid only (0.05 Gy) did
not increase the severity of the disease. The pre-exposure of animals to 0.05 Gy either 1 or 2 weeks before
exposure to 0.5 Gy does not significantly modulate thyroiditis scores. No effects of radiation exposure(single or repeated 0.05 Gy) were observed in a different experimental model of thyroid autoimmunity(induction of Grave’s hyperthyroidism in Balb/c mice). Thus, radiation may, under a specific exposure
regimen, exacerbate the development of certain autoimmune thyroid disease in susceptible animals witha discontinuous dose response pattern.
327. The effects of chronic low-dose-rate exposure on the development of asthma were investigated ina murine model of sensitization to ovalbumin (OVA) [K25]. Female C57BL/6 mice received on days 0
and 14 two intraperitoeal injections of OVA emulsified in aluminium hydroxide and were challenged atdays 21, 22 and 23 by inhalation of nebulized OVA (1% weight/volume). Mice were chronically exposed
to gamma radiation (137Cs) at 0.554 mGy/h or 1.818 mGy/h from the first injection to the last challenge,
i.e. 24 days for total doses of 0.3 and 1 Gy, respectively. They were sacrificed 48 hours later for
histopathology analysis of lungs, and analysis of inflammatory markers in bronchoalveolar lavage andserum. Radiation doses of 0.3 and/or 1 Gy were found to significantly attenuate histopathological signs(inflammatory cell invasion of lungs) and systemic markers (IL-4, IL-5, anti-OVA IgE) of asthma. Thesechanges were associated with a marked reduction in Mucin-5, p-ERK and p-JNK in lungs. The exposure
to intermediate doses delivered at low dose rate can therefore attenuate airway and systemic inflammation
during asthma [K25].
328. Decrease of arthritis in TNF-α transgenic mice was achieved by a single partial-body exposure to
500 mGy of X-rays [D15]. Endpoints such as mineralization properties of osteoblasts and neutrophil
infiltration were explored in vivo. The authors ascribed this success to the notion that 500 mGy exposure
induces apoptosis of fibroblast-like synoviocytes that reduced expression of the inflammatory phenotype.

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329. Behçet’s disease is a multi-systemic disorder of unclear aetiology characterized by blood vessel
inflammation throughout the body and, among others, mouth, skin, eyes sores, painful and swollen joints
that may result from a dysfunction of Th1 CD4+ T-lymphocytes. Kang et al. [K12] used a virally induced
murine model of Behçet’s disease to investigate whether low-dose radiation exposure could modulate theproduction of cytokines. Induced mice were exposed (whole-body or half-body only) to 0.1, 2 or 10 Gyof X-rays. The level of intracellular and serum IFN-γ (Th1) and IL-4 and IL-10 (Th2) were measured byfluorescence-activated cell sorting and ELISA, respectively. Whole-body exposure to 0.1 Gy results inincreased levels of all cytokines in cells and serum four to six days post-radiation. The IL-4/IFN-γ ratio
was clearly in favour of IL-4, suggesting an increased Th2 immunity. Half-body exposure to 0.1 Gy
results in an increased serum concentration of these cytokines compared to whole-body irradiation. Bothexposures to 0.1 Gy lead to a reduction of the severity score two weeks post-exposure.
330. The effects of low and high doses of 137Cs gamma ray (1 mGy–2 Gy at 1 mGy/min) exposure on
Escherishia coli-induced sepsis was investigated in C57BL/6 mice [S58]. It was found that 0.1 Gy totalbody irradiation 48 hours prior to infection significantly rescued mice from death (75% survival at
72 hours compared with 25% in non-irradiated animals). One day post-infection, the bacterial burden
was significantly decreased in the blood, spleen and kidney of pre-exposed mice, and the cytokine storm(large increase in serum IL-1β, IL-6 and IL-10) was prevented. The number of peritoneal macrophages
was lower in pre-irradiated infected mice than in non-irradiated mice. The frequency of CD8+ T-cells andmacrophages was increased in the spleen of pre-exposed mice, but exposure to radiation did not modulate
the expression of activation markers on splenocytes. In contrast, radiation exposure reduced the inductionof iNOS and increased the expression of Nfr2 and HO-1 in splenocytes. In vitro, pre-exposure of murine
RAW264.7 macrophage cells before Escherishia coli infection mostly recapitulates the same effects.
Thus, part of the effects observed in vivo could result from the stimulatory effects of low-dose exposure
on anti-microbial macrophage activity.
331. With the similar aim to evaluate the effects of radiation exposure on immune response in vivo,
Misra et al. [M35] evaluated the effects of internal contamination at a young age on the ability of adultmice to respond to influenza A infection. Fourteen-days old pups received an intra-peritoneal injectionof 50 µCi/185 kBq of 137Cs, resulting in a mean accumulated dose of 2.5–2.7 Gy over the next 50 days.
They were infected 26 weeks later with 100 HAU (haemagglutinating units) of influenza A/HKx31(H3N2) virus. Contaminated mice recovered over a 2-week period and had serum antibody titres andlevels of MCP-1 and club cell secretory protein mRNA in the lung similar to control mice. Thus, 137Cs
contamination early in life resulting in an accumulated dose of 2.5–2.7 Gy in the following several weeks
does not impair efficient anti-viral responses at later times.

(e) Low-dose/low-dose-rate exposure effects in healthy animals
332. The analysis of C57BL/6 mice exposed to 137Cs gamma radiation at low dose rate (0.7 mGy/h and3.95 mGy/h for 12 and 21 days for total dose of 0.2 and 2 Gy, respectively) seven days post-exposure did
not show any changes in body weight, in haematological parameters nor in the distribution of B- and Tlymphocytes in the spleen. The analysis of the level of circulating cytokines in the serum (analysed by
antibody arrays) showed, however that a number of cytokines were significantly up- or down-regulated,with changes suggestive of the recruitment, differentiation and activation of myeloid cells and a shift
toward Th2 type immunity [S39]. Thus, even if no overt changes in the homeostasis of immune cells
were visible, a modulation of inflammatory mediators involved in intercellular signalling occurs at thesystemic level and may shift the balance between Th1 and Th2 immunity.
333. However, a different study addressed the response of C57BL/6 mice to 0.01, 0.05, 0.1, 0.5 and 2 Gyof gamma radiation, from 4 hours to 7 days post-exposure. Apoptosis was already detected as early as

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4 hours post-radiation in CD4+ T-cells after 0.01 and 0.05 Gy. In NK cells and dendritic cells a dose of
0.05 and 0.1 Gy has no effects, whereas higher doses induce apoptosis in all populations analysed,
including CD8+, Treg and B-cells. The number of CD4+ T- and Treg-cells was transiently increased atday 1 after 0.01 Gy. All populations decreased, more or less dose dependently by day 3, and recovery
was already evident by day 7. These changes may be related to changes in the expression profile ofcytokines. Several of the cytokines analysed show a dual profile, with a reduction or no change at 0.01
and 0.05 Gy and an increased expression after 2 Gy. The response to 0.1 Gy seems intermediate between
these two patterns. It is not possible to discern a Th1 or Th2 trend. The expression of cytokines withhaematopoietic activity (GM-CSF and IL-5) is dose dependently increased from 4 hours to day 3. These
data illustrate the different response in time of splenic immune cells to low- and moderate-dose radiationin vivo [B46].
334. The effects of acute or fractionated low-dose radiation on immune cells was analysed in the spleenof C57BL/6 mice exposed to 0.001, 0.01 or 0.1 Gy of 137Cs gamma rays delivered either acutely or
fractionated in three equivalent doses delivered on three consecutive days. Spleen cell number is stable,
except for a sporadic increase at day 7 in the acutely exposed mice exposed to 0.001 Gy. In acutely
exposed mice, the proportion of splenic CD4+ T-cells increases transiently at day 2, while dendritic cells
and macrophages decrease. These factors are normalized in later days, but the frequency of CD8+ T-cells
decreases for higher doses at day 7 and 14. The expression of activation markers (CD69, CD28, CD86)
is unchanged or decreased for all doses at all timepoints. Fractionation only slightly changes this pattern.
However, the pattern of cytokine production by splenocytes differs after acute and fractionated dosedelivery. Some of these changes can be explained by changes in the expression of protein involved insignal transduction (Nrf2, IκBα), differently regulated according to dose and fractionation. These results
suggest that acute exposure elicits a mixed response that evolves to a Th1 type profile, whereas
fractionated exposure evokes first a Th1 response that evolves towards a Th2 phenotype [S57].
335. The effects of acute low- or intermediate-dose exposure and aging on the murine T-cell receptor(TCR) repertoire expressed by peripheral blood T-lymphocytes were compared in CBA/Ca mice after asingle irradiation of 0.1 or 1 Gy 250 kV X-rays with 1.2 mm HVL Cu [C3]. High throughput profiling ofrearranged TCR-β chain genes in the blood of irradiated mice 1, 3 and 6 months post-exposure showedthat radiation affected the homeostasis of the T-cell pool. The effects of 0.1 Gy were more pronounced
and lasted longer than those elicited by 1 Gy, which were partially reversed over time. These effects canbe assimilated to accelerated aging. They result, at least in part, from effects of radiation on
haematopoietic stem cells.
336. To investigate how extracellular vesicles participate in systemic intercellular signalling, including
bystander cell-to-cell communication, extracellular vesicles purified from C57BL/6 mice exposed to 0.1
or 2 Gy of X-rays were injected into non-irradiated syngeneic mice (bystander mice). Changes in thelevel of cytokines in the plasma were compared between bystander mice and directly irradiated mice 24 hafter injection or irradiation. M-CSF and pentraxin-3 levels were up-regulated in both low- and high-dose
irradiated mice and bystander mice injected with extracellular vesicles purified from 0.1 and 2.0 Gy
irradiated mice. In addition, CXCL16 and Lipocalin-2 were increased in mice exposed to 2 Gy and in
bystander mice injected with extracellular vesicles from mice exposed to 2 Gy, and C-C motif chemokine
ligand (CCL) 5 and CCL11 were similarly down-regulated in low and high-dose bystander mice.
Extracellular vesicles purified from the blood of 0.1 and 2 Gy exposed mice were found to containmiRNAs targeting similar pathways (acute myeloid leukaemia, T-cell receptor signalling, MAPK, TGFβ), but no common miRNA was identified. Thus, extracellular vesicles can recapitulate some of theeffects of direct radiation exposure in recipient mice, and some of these effects may result from themiRNA cargo of these extracellular vesicles [S75].

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337. The potential protective effects of nicaraven, a hydroxyl-radical scavenger radioprotectant, on
haematopoietic stem cells was tested in mice exposed for 30 consecutive days to 50 mGy of gamma
radiation (137Cs). Nicaraven was injected 30 minutes before each irradiation. There were two sets ofcontrols: One were mice that were irradiated with the same protocol and received no nicaraven (nontreated irradiated mice) and the second were mice that were not irradiated (non-irradiated controls). Mice
were sacrificed 24 hours after the last irradiation. Cytopenia was clearly reduced in nicaraven treatedmice, and there was no loss of circulating c-kit+ haematopoietic stem cells compared with the nonirradiated control mice, in contrast with non-treated irradiated mice. The number of c-kit+ bone marrowcells was also higher in irradiated mice which received nicaraven compared with non-treated irradiatedmice but was only about half of non-irradiated controls. Notwithstanding, the number of colony-forming
unit in the blood and bone marrow of nicaraven-treated mice was similar to non-irradiated controls, while
it was clearly reduced in non-treated irradiated mice. These effects could not be attributed to changes inintracellular ROS levels. Analysis of gene expression on dedicated RT-PCR arrays (370 genes
representing 13 pathways) showed that the majority of the genes representing the inflammatory/immunity
pathway (pro- and anti-inflammatory cytokines, co-activation molecules) were significantly downregulated. Thus, modulation of immune responses but not of the redox status participates in the protectionof haematopoietic stem cell by nicaraven in vivo after repeated exposure to low-dose radiation [A12].
338. The effects of exposure to radiation with different LET on the homeostasis and function of immune
cells in vivo and ex vivo was also addressed, including eventual induction of an adaptive response by lowdose and low-dose-rate X-irradiation prior to subsequent exposure to high-LET radiation. In mice exposed
to 0.01, 0.05 or 0.1 Gy of protons (210 MeV) delivered over several days at an average dose rate of 1 mGy/hfor 12 hours per day, the number of white blood cells, lymphocytes, monocytes/macrophages andgranulocytes in the blood and spleen were unchanged in the three weeks following exposure. However, amore detailed analysis revealed a modulation of the splenic T-lymphocytes in mice exposed to 0.01 Gy, due
to a decrease in CD4+ T-lymphocytes at the end of the exposure period (day 0) and at day 21, but not at
day 4. Splenic CD8+ T-lymphocytes were not affected. B-lymphocytes were reduced at day 21. The
expression of genes related to T-cell subsets differentiation was analysed by RT-qPCR using a dedicated
PCR array in splenic CD4+ T-cells purified from control mice and mice exposed to 0.01 and 0.1 Gy at day 0.
Five and nine genes were up-regulated in CD4+ T-cells exposed to 0.01 and 0.1 Gy, respectively, while
three genes were commonly up-regulated (CD40, CEBPB and TNFSF4). Only three genes were downregulated after 0.01 Gy, and none after 0.1 Gy. The down-regulated genes code for the p105 subunit of NFκB, JNK1 kinase and IL-4 receptor. Thus, protracted low-dose proton exposure induces early dosedependent modulation of signalling in CD4+ splenic T-cells after whole-body irradiation [G12].
339. In a different series of experiments, low-dose and low-dose-rate (0.01 Gy delivered at 0.18 mGy/h)
exposure to gamma radiation was found to result in a transient reduction in B- and T-lymphocytes (CD4+T-lymphocytes but not CD4+CD25+ and CD4+CD25+Foxp3+ Tregs) in the spleen of irradiated animals,
visible at day 4 but no longer at day 21. This low-dose and low-dose-rate exposure does not apparently
modulate the response of mice and CD4+ T-cells to a subsequent exposure to proton radiation representing
a simulated solar particle event (sSPE, 1.7 Gy, 30 to 210 MeV) [G13]. However, it was later found that
low-dose and low-dose-rate exposure induces changes in the level of several key proteins (NF-κB p65,
JNK1, p38 MAPK, Lck, ZAP70, up- or down-regulated at 4 and 21 days post-exposure) involved insignal transduction in CD4+ T-lymphocytes. CD4+ T-lymphocytes purified from mice at day 21 andactivated in vitro secreted three times more IL-4 and slightly less TGF-β1 than control mice, suggesting
a possible polarization towards a Th2 type response [R9]. Pre-exposure to low-dose and low-dose-rate
exposure could prevent or reduce most of the changes induced in CD4+ T cells by a 1.7 Gy sSPE
exposure. Thus, low-dose and low-dose-rate exposure prior to a sSPE results in radio-adaptation and canmodify intracellular signalling elicited by high-dose proton exposure [R9]. Despite these findings,
protracted low-dose and low-dose-rate exposure was found to have only a marginal influence on the

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effects of proton exposures (2 or 3 Gy, 250 MeV, 0.4 KeV/µm) on the dynamics of leukocytes
populations, platelets and erythrocytes in mice 4 and 17 days post-proton exposure [L51].
340. To determine whether protracted low-dose and low-dose-rate exposure affected only a subsequent
high-LET radiation response, a direct comparison was performed. Mice exposed or not to 0.01 Gy gamma
radiation delivered at 0.3 mGy/h were subsequently exposed to 2 Gy gamma radiation (0.9 Gy/min) or
2 Gy protons (250 MeV, LET 0.4 KeV/µm, 1 Gy/min). The different leukocyte populations as well as Tcell populations, their secretion of cytokines and their content in active signalling protein kinase wereanalysed 21 and 56 days later. Low-dose and low-dose-rate exposure alone had limited impact on cellhomeostasis and cytokine secretion, indicating long-lasting changes suggestive of a shift towards Th1
type immunity. Prior low-dose and low-dose-rate exposure did not overtly modulate the response of miceand CD4+ T-cells to 2 Gy gamma or proton radiation. Only a limited number of subtle changes could beobserved on Tregs homeostasis or secretion of VEGF. Thus, the radio-adaptive ability of low-dose andlow-dose-rate exposure to a challenge dose of high-dose gamma or protons radiation is quite limited[G14]. The liver of mice exposed to similar radiation exposure regimen (proton energy was only
230 MeV) showed no overt signs of inflammation at day 56 and only a small increase in oxidative burst
capacity by stimulation with S. cerevisiae Zymosan was observed after low dose and low dose rate +proton exposures. The expression of apoptosis-related genes, analysed by dedicated RT-qPCR array(84 genes), did not change in the liver of mice exposed to low-dose and low-dose-rate exposure only, but
pre-exposure to low-dose and low-dose-rate exposure modulates the gene expression in mice exposed to
2 Gy gamma rays or to 2 Gy of protons. Thus, only two genes are down-regulated in 2 Gy gammairradiated mice, but 11 are modulated (up- and down-regulated) in mice previously exposed to low doseand low dose rate. Similarly, 6 and 12 genes are modulated (up or down) in proton compared with lowdose and low dose rate + protons exposed mice. Thus, low-dose and low-dose-rate exposure can modify
the apoptotic response of liver cells to high-dose gamma or high-dose proton radiation long after
exposure. However, this study does not allow a conclusion to be drawn as to whether this radio-adaptation
is beneficial (increased apoptosis of exposed cells) or potentially detrimental (increased survival of
possibly exposed damaged cells) [G14].

(f) Effects of low-dose radiation on natural killer cells, dendritic cells andmacrophages
341. Yang et al. [Y3] analysed the effects of a range of low (25–75 mGy) and moderate (150 – 500 mGy)dose X-ray exposure in vitro on human NK cells generated from peripheral blood mononuclear cells after
culture with IL-2 and OK432 (anti-neoplastic compound). A dose of 75 mGy was found to induce theexpansion of in vitro generated NK cells and stimulate their production of TNF-α and IFN-γ and their
specific cytotoxicity. These effects were abrogated by p38-MAPK inhibitors, which also inhibited themRNA expression of FasL and perforin, two molecules involved in NK cytotoxic activities.
342. Human NK cells are however extremely radiosensitive. They were found to be the most radiosensitiveof the human peripheral blood mononuclear cells after in vitro exposure of freshly purified cells. The
fraction of viable NK cells is reduced 48 hours after exposure to 50 mGy X-rays, and clear signs of apoptosis
and secondary necrosis are detected 48 hours after exposure to 0.1 Gy [F6]. Furthermore, a transcriptomic
analysis of human whole blood exposed in vitro showed that numerous genes related to NK cell activity are
dose-dependently down-regulated 48 hours after exposure to 0.5–8 Gy of gamma radiation. Importantly,
the magnitude of this decrease is much more important than the loss of NK cells in culture, implying thatthese genes are specifically down-regulated in response to radiation [P7].

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107343. In contrast, Sonn et al. [S59] reported that in vitro exposure to a moderate radiation dose (0.2 Gy
gamma rays) of purified murine NK cells increased their cytotoxicity towards tumour target cells. Luzhna
and Kovalchuk [L53] reported that low-dose exposure (0.1 Gy X-rays) induced up-regulation ofnumerous genes belonging to the Kyoto Encyclopedia of Genes and Genomes NK cell mediatedcytotoxicity pathway in the mammary gland of rats, four days post-exposure.
344. However, chronic exposure of SJL mice for eight months to very low dose rate (annual dose of
100 mGy) induced only a transient increase in splenic NK cell frequency at weeks 28 and 32, without
increasing their cytotoxic activity. The only other change observed among immune cell populations wasa transient increase in CD8+ T-lymphocytes in lymph nodes at week 32 [L1]. At the interface of innate
and adaptive immunity, dendritic cells play a key role in the development of immune responses. The
effects of low-dose gamma irradiation on dendritic cells were analysed from a functional point of view,
both in human and mice. Different results were obtained in each experimental system.
345. Human dendritic cells were produced in vitro from purified CD14+ monocytes from peripheral
blood mononuclear cells. Sorted cells were allowed to differentiate into dendritic cells for four days in
the presence of IL-4 and GM-CSF, with or without fractionated (one dose per day) X-ray exposure(0.162 Gy/min), to a total of 0.05, 0.5, 1.0 or 2 Gy delivered in 4 fractions of 0.0125, 0.125, 0.25 or
0.5 Gy, respectively. Radiation did not affect dendritic cell differentiation, phenotype and activation,
maturation or cytokine secretion following lipopolysaccharide stimulation. Thus, exposure did not affectthe ability of dendritic cells to stimulate the proliferation and activation of allogeneic T-cells in co-culture.
Though in vitro, the study showed that radiation exposure has no effects on the differentiation, maturationand functionality of human dendritic cells generated from purified monocytes [J8].
346. The effects of low- to moderate-dose radiation exposure in vivo were analysed in C57BL/6 mice
exposed to 0.1, 0.25 and 2 Gy X-rays. Changes in the phenotype of splenic dendritic cells were foundonly in 2 Gy irradiated mice. Exposure to 0.1 Gy does not stimulate antigen uptake and, by contrast,
decreases antigen presentation to T-cells by irradiated dendritic cells. Cytokine secretion is only mildly
affected by low-dose exposure (small increase in IL-10, no changes in IL-1α, IL-1β, IL-6, IL-12, TNFα), whereas the secretion of all these factors is increased by 0.25 and 2 Gy. Thus, in vivo low-doseradiation exposure does not activate or even reduces murine dendritic cell functions, in contrast tomoderate- and high-dose radiation exposure [P14]. However, when mature CD11c+ dendritic cells
purified from the spleen of BALB/c mice were exposed in vitro to 0.02, 0.05, 0.1, 0.5 and 1.0 Gy before
being used to stimulate allogeneic purified T-cells in a mixed lymphocyte reaction, dendritic cells isolated
from 0.05 Gy exposed mice could stimulate T-lymphocyte proliferation. Although their phenotype
remains unchanged (no induction of activation markers), these cells produce more IL-12, IL-2 and IFNγ, but similar amounts of IL-10 when compared with control cells or dendritic cells from 1 Gy exposed
animals. Hence, ex vivo low-dose exposure can specifically activate dendritic cells to promote Th1 CD4+
T-lymphocyte differentiation [S31]. The differences observed in these two murine studies could result
from in vivo compared with ex vivo exposure, the different mouse strains or differences in dendritic cell
identification and sorting purity.
347. Inter-cellular communication is paramount in immune responses. Wunderlich et al. analysed if andhow radiation exposure interferes with these events using an in vitro system [W16]. Murine peritoneal
macrophages were activated or not activated in vitro with lipopolysaccharide and then exposed to a single
dose of X-rays ranging from 0.01 to 2 Gy. Irradiation (>0.1 Gy) of lipopolysaccharide-activatedmacrophages reduces their expression of MHC II molecules. The ability of these macrophages to induce
allogenic CD4+ T-cell proliferation is reduced only after a dose of 2 Gy. Conditioned medium collectedfrom irradiated activated macrophages cultures did not modulate the maturation of bone-marrow derived
dendritic cells, nor their capacity to induce T-cell proliferation. Thus, radiation exposure appears todifferentially modulate paracrine signalling from activated macrophages (see also section III.F.3).

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UNSCEAR 2020/2021 REPORT348. Radiation exposure can also elicit paracrine signalling from non-immune cells. The effects of low
and moderate dose radiation on the expression of inflammatory factors by skin cells for example, in
EpiDermFT skin explants exposed to 0.03, 0.1 or 2 Gy X-rays, were analysed ex vivo one and two days
post-irradiation [V5]. The modulation of the secretion of 23 cytokines was monitored. A small but
significant induction (1.4-fold) and reduction (0.9-fold) of the secretion of IFN-γ and CCL3, respectively,
was observed 48 hours after an exposure of 0.03 Gy. Exposure to 0.1 Gy results only in an induction (3.3
times) of IL-2 two days after irradiation, while 2 Gy exposure induces the up-regulation of several
cytokines (IL-2, IL-10, IL-13, IFN-γ, CCL3, TNF-α) one and/or two days post-exposure. The secretionof a limited number of cytokines (IL-1α, IL-6, IL-8, IL-10, TGF-β) was also compared in EpiDermFTskin explants exposed to intermediate and high doses of X-rays or 12C ion [S46]. X-ray exposure (2 Gy)
increased moderately the production of IL-6 and IL-8 1 and 2 days after irradiation, but exposure to 0.5
or 10 Gy had no effects. 12C ions irradiation induced a small increase in IL-10 and IL-8 secretion 2 days
after 0.5 and 2 Gy, respectively. Thus, low- and moderate-dose exposure induces only subtle changes in
inflammatory cytokine secretion in reconstituted skin explants. The profile of secretion evolves with time,dose and the quality of radiation.

(g) Summary and conclusions on immune system
349. Radiation was found to affect several aspects of the immune system. It is, however, difficult to draw
any general conclusion regarding the effects of low-dose/low-dose-rate exposure, as different
experimental systems were used, and different endpoints were analysed among the various reported
studies. The eventual interrelationship between low-dose radiation and the immune system in cancer
development is illustrated by the observed association of variant alleles of immune genes with breast
cancer in a cohort of radiological technologists.
350. For human exposure studies, some of the late effects of radiation exposure in atomic bombing
survivors suggest that radiation can accelerate discrete aspects of aging of the immune system sometimes
in a dose-dependent manner. However, immune functions (e.g. vaccination response) appear normal inaging survivors. Other human exposure studies (occupational, environmental, accidental) failed to
produce any consistent results, probably because of differences in exposure scenarios.
351. Studies analysing the effects of low-dose/low-dose-rate exposure in vivo and on purified cells
illustrate the diversity of responses (increase, decrease, no response) obtained in different experimental
systems. The effects of low-dose radiation exposure are different according to the initial inflammatory
status. One study reported that internal contamination in early life did not impair the response to influenza
24 weeks later.
352. In conclusion, low-dose/low-dose-rate radiation exposure may affect the homeostasis and/or
modulate the activation of the immune cells, but it remains unclear whether these effects are activating
or inhibiting the immune system; additionally, the precise mechanisms responsible and the short- andlong-term consequences remain elusive, especially in relation to cancer development. The resilience of
the immune system may, however, prevail in the long-term.

2.

In vivo effects on gene inversion and other genetic/cellular endpoints

353. In order to evaluate the potential of low-dose radiation to cause DNA modifications, this area of
research often employs transgenic animals in which mutation induction can be studied in a simplified
way. For example, when a bacterial gene is incorporated into mouse genome – mutations in this foreignANNEX C: BIOLOGICAL MECHANISMS RELEVANT FOR THE INFERENCE OF CANCER RISKS ...

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DNA are irrelevant for animal health while they permit easy means for monitoring mutations. The
Escherishia coli gene lacZ has been used for this purpose in rodents. In pKZ1 mice lacZ is incorporated
in inverse orientation adjacent to the chicken beta actin promoter; provided that an inversion occurs lacZ
expression leads to production of blue beta galactosidase in the presence of X-gal substrate. Work with
pKZ1 mice in Japan has been extensive, not the least because this strain was originally developed in Japan[M24]. This type of transgenic mouse model was used, among others, for studies at the Institute forEnvironmental Sciences [B53].
354. A colony of pKZ1Tg/+ heterozygotes maintained by backcrosses with C57BL/6J mice was used inextensive work published by the Sykes laboratory [D6, D7, D8, D9, O17, S71, S72, S73, Z5];
interestingly, different tissues were found to have similar degrees of chromosomal inversions. In prostatesof mice exposed to a range of doses (0.001, 0.005, 0.01, 0.02, 1, 10 and 1,000 mGy; delivered at dose
rates 0.0014, 0.0017, 0.34, 13.9 or 180 mGy/min) inversion frequencies scored in tissues obtained threedays post-exposure increased (at 0.005 and 0.01 mGy), decreased (at 1 and 10 mGy) and then increased
again (at 1,000 mGy). These differences were found to be statistically significant [Z5]. The same maleanimals were also explored in a different study by the same group [H35]. Dose-associated changes infrequencies of lacZ inversions in spleen documented in that study were similar (once again “bimodal”)
with findings documented in prostate. Authors of this work have proposed the following explanation forthe bimodal data. They propose that a low fidelity, error-prone DNA repair preventing apoptosis functionsin the ultra-low-dose region below 0.01 mGy (the NEOTRANS model); on the other hand, maximal
protection from mutation fixation was provided by p53-independent, high fidelity DNA repair coupledwith apoptosis (proposed “PAM” or p53-independent protective apoptosis-mediated process) in the lowdose region around 1–10 mGy.
355. The same group of researchers again used pKZ1Tg/+ heterozygotes to investigate lacZ inversions
three days post-exposure in prostates of mice exposed to an adapting dose of 0.001, 0.01 mGy (dose rates
0.0014 mGy/min), 1 mGy (dose rate 0.34 mGy/min) or 10 mGy (dose rate 13.9 mGy/min) followed fourhours later by a 1,000 mGy (dose rate 180 mGy/min) challenge dose of X-rays. All low-dose exposuresprotected equally well from 1 Gy challenge dose suggesting that there is no threshold in the range ofpriming doses used in this study, for protective X-ray exposure [D6]. When challenge doses were altered
to include 4,000 mGy, no increase in lacZ inversions was found unless two 4,000 mGy doses were given
in sequence. At the same time, two 1,000 mGy doses in sequence led to fewer inversions than a single
1,000 mGy dose [D8]. Additional studies with very low doses of radiation and their potential protective
effects were also extended to exploration of whether low doses can be effective if delivered after
challenge doses. When 0.01 or 1 mGy doses were delivered four hours after 1,000 mGy dose, total
mutations levels were lower than in controls, while 1 mGy dose on its own (or followed by 0.01 mGydose) decreased numbers of inversions compared to controls [D8]. It is important to note, however, that
post-irradiation period variation modulated registration of radiation-induced inversions [O17]; inversion
frequencies differences were the most pronounced when analyses are carried out at one or three days but
not seven days after exposure.
356. Among recent reviews that compared different strains and genotypes of animals, especially valuable
is work by Shimura and Kojima [S34]. The focus of this review is to pinpoint “lowest doses” withrecorded effects. This review paper considering research over past 20 years covering exposure of humans
and animals, focusing on functional endpoints (e.g. fetal abnormalities) as well as cellular events, frommicronuclei formation to nucleoplasmic bridges and, using chromosomal inversions in pKZ1 mice. No
universal conclusion could be drawn because different studies used different strains of animals andendpoints; again, animals with genetic changes were the ones to show the most intense effects (e.g.
chromosomal aberrations in SCID mice after 50 mGy). Otherwise, the lowest doses with recorded effects
in wild-type animals were exposures to 100 mGy of gamma rays or 9 mGy of fast neutrons from a 241AmBe neutron source. Another well researched review focused on animal research was published by Tang

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UNSCEAR 2020/2021 REPORTet al. [T9]. In this work the authors emphasize both beneficial and harmful effects found in numerous invivo studies.
357. Eight-week-old male and female pKZ1Tg/+ heterozygote mice were given either regular or tritiumcontaining water at activity levels of 10 kBq/L, 1 MBq/L or 20 MBq/L HTO over a period of 28 or
224 days; these three exposures correspond to total doses in the range 0.01–180 mGy [B12]. Externalbeam radiation with dose rates matching two highest HTO exposures was also used in this study. Nochanges in either spleen weight, cell numbers or recombination of beta galactosidase were found in anyof the treatment groups.
358. Wild-type mice with differences in radiosensitivity were used in a study focused on bone marrowcells [Z24]. C57BL/6 and CBA/Ca mice, susceptible or resistant, respectively, to myeloid leukaemia andradiation-induced chromosomal instability were exposed to X-rays at 8 to 12 weeks of age. A dose rateof 1 mGy/s was used to deliver total doses up to 100 mGy, for higher total doses (500, 1,000 or
3,000 mGy), a dose rate of 7.5 mGy/s was used. Bone marrow cells were inspected for expression of p21,
p53, Casp-3 or TUNEL assay staining. Positive p53 and p21 cells were noted at doses of 50 or 100 mGy
in C57BL/6 or CBA/Ca mice, respectively, while caspase and TUNEL became detectable only at doses
500 mGy or greater, regardless of genetic background.
359. Use of cells isolated from living organisms for temporary maintenance in cell culture is frequent inradiation research. Quite often these cells are peripheral blood mononuclear cells and leukocytes. Whilemost often such studies irradiate cells after they have been isolated, other approaches are sometimes used
too. An interesting example of such work is the study by Nafee et al. [N1] who isolated peripheral bloodmononuclear cells and leukocytes from three-month-old female Wistar rats after they have been exposed
to 9 mGy of fast neutrons (241Am-Be neutron source, dose rate 0.2 mGy/h). Comet assays were used toevaluate DNA damage and this study contends that nearly complete DNA repair occurred within threehours post-irradiation.
360. Less frequently used for preparation of single-cell isolates are muscle cells. Nevertheless, anexample relevant for low-dose work comes from Masuda et al. [M21]. In this work, 12 week-old
C57BL/6 male mice were gamma irradiated with dose rates of 0, 2, 10, 50 and 250 mGy/d over a periodof 30 days, resulting in cumulative doses of 60, 300, 1,500 and 7,500 mGy. Animals were sacrificed
immediately or three months after being irradiated, and satellite cells (muscle stem cells) were isolated
from muscles, stained by anti-Pax7 antibody and 4′,6-diamidino-2-phenylindole and counted. Exposures
to 60 and 300 mGy did not reduce cell numbers immediately after radiation, but there was a small thoughstatistically significant decrease in cell numbers for all doses used when animals were sacrificed at threemonths after irradiation.
361. Spleen cells isolated from irradiated animals were used in a study published by Koturbash et al.
[K40] designed to explore DNA damage caused by fractionated low doses of radiation [K40]. Wild-type
C57BL/6 male mice were whole body irradiated on day 60 of age with 100 mGy of X-rays at dose rateof 50 mGy/s. At 6 or 24 hours post-exposure the first group of mice was sacrificed and spleens extracted.
For additional groups of mice, irradiation was repeated each 24 hours, as well as the pattern of animal
harvest until experimental day 6. At the end of experiment, spleens of mice exposed to 100, 200, 300,
400 or 500 mGy and sacrificed 6 or 24 hours post-exposure were collected. DNA strand breaks weregenerally greater at 6 compared to 24 hours, and total DNA damage was increased in 400 and 500 mGy
exposed mice compared to the lower dose counterparts, except in 100 mGy exposed mice at 6 hours.
Similarly, apoptosis was greatest in 400 and 500 mGy exposed animals, both at 6 and 24 hours; for otherdoses, 24-hour group animals had more apoptotic cells. A dose-dependent increase in p38 protein was
found, while spleens from 200 mGy exposed mice had the most BCL2+ cells. Expression of Ki67 and
PCNA followed BCL2 as well. Phospho-ATM was increased only in 100 mGy exposed mice (bothANNEX C: BIOLOGICAL MECHANISMS RELEVANT FOR THE INFERENCE OF CANCER RISKS ...

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timepoints) while p53 was decreased compared to control. Authors concluded that the repair occurringin these animals was p53- and ATM-independent, which is in keeping with the concept of “PAM” (p53independent protective apoptosis-mediated process) for the low-dose region, postulated by the Sykes
laboratory, albeit for pKZ1 mice and doses around 1–10 mGy.
362. Healthy wild-type mice were also used in a study that looked at the fate of bone marrow cells
ex vivo after moderate-dose radiation exposure of live mice [L30]. Four-month-old female mice of thestrain BALB/cBYJ were used for this study. X-ray irradiations were carried out with doses of 170, 500
or 1,000 mGy at a dose rate of 180 mGy/min. Animals were sacrificed at days 3 or 21 post-exposure.
Bone microarchitecture was affected by exposure to radiation differently when different endpoints wereconsidered. The fate of isolated bone marrow cells was explored, and the following conclusions were
made: differentiation into adipocytes and growth of adipocyte colonies was increased only by exposure
to 1,000 mGy in cells isolated after 21 days. On the other hand, osteoblastogenesis was increased on
day 3 in cells from mice exposed to 170 mGy, and in cells on day 7 after exposure to 1,000 mGy.
363. Partial body irradiation studies were focus of several interesting recent studies. For example,
Yamada et al. did thoracic irradiations of female Wistar rats irradiated at 1, 5 or 15 weeks of age. Dose
rates about 500 mGy/min were used and total doses were 1, 3 or 5 Gy [Y1]. No life shortening was foundfor any of the doses when irradiated animals were 1-week old. In addition, total thoracic dose of 1 Gy did
not cause life shortening in any of the rats. However, cancer types found at necropsy were different in
irradiated mice compared with controls. Pituitary cancer was the most frequent cancer type in nonexposed rats, but not in exposed rats. The frequency of lung cancer was at least doubled with statisticalsignificance in all irradiated animal groups compared to controls, while mammary cancers increased onlyin 3 and 5 Gy groups. While it is difficult to compare moderate-dose partial body exposures with lowdose exposures, this type of research (i.e. in vivo studies with animals allowed to live out their lifespanand exposed to radiation at different ages) informs us that it may be necessary to broaden the spectrumof questions that need to be considered when discussing radiation-induced cancer.
364. Exposure to radioactive particles by inhalation can be considered as special case of partial body
irradiation. Extensive work of this type was carried out with beagle dogs in the United States between
1950s and 1990s; in many cases, these studies were concluded decades after final exposures. An example
of such a study is work by Park et al. [P4]. In this study, groups of 8 to 24 dogs inhaled 0.14, 0.63, 3.2,
13, 44 or 210 kBq of weapons-grade 239PuO2 aerosol (particle sizes of 2.3 µm) and were kept andmonitored throughout their lifespan. Among the dogs (20 per dose, 10 per sex) exposed to the two lowest
doses (0.012 or 0.059 kBq/kg), median life expectancy was not reduced compared to controls while lungcancer incidence was lower than in controls. At the same time, only incidences of bone cancer were foundin animals exposed to 0.012 or 0.059 kBq/kg and the same two groups of animals included some thatdied of bacterial pneumonia. Nevertheless, it should also be mentioned that in depth consideration ofthese and some additional data led another group of researchers to conclude that genetic inbreeding is an
important factor in proper analyses of dog data [W11]. Work by Wilson et al. suggested that use of
Wright’s coefficient of inbreeding as an explanatory variable led to improved model fitting with lungfibrosis data in beagle dogs that had inhaled 238Pu and 239Pu.

3.

Effects on gene and protein expression in experimental animals

365. A recent study investigating proteomics changes in liver compared acutely exposed C57BL/6J malemice given 4 Gy of X-rays at 10 weeks of age and animals exposed to 4 or 8 Gy at 24 weeks of age, withchronically exposed mice [N6]. Chronic exposures were carried out with C57BL/6J male mice exposed todose rate of 1 mGy/d, total dose of 400 mGy, or a dose rate of 20 mGy per day, total dose of 8,000 mGy.

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These mice were sacrificed immediately or three months after chronic exposure, while acutely exposed mice
(8 Gy) were sacrificed six days or three months later. Protein expression changes in irradiated mice weregenerally sustained over three months post-exposure. Laminin-2, DRAK1, BID, Bim, Bmf had persistently
altered expression in chronically exposed mice, while MyD88 and Bcl-xL had altered expression
immediately after acute exposure to 8 Gy and three months after acute exposure to 4 Gy.
366. In a similar study, liver proteins were measured in C57BL/6J chronically exposed to low-doseradiation [N5]. Twenty-two hours a day irradiation for 485 days was carried out with 137Cs gamma
irradiator at dose rates of 0.032, 0.65 and 13 µGy/min. This led to total accumulated doses of 21,420 and
8,000 mGy. After 2D-electrophoresis and protein identification, protein rhodanese expressed in
mitochondria was found to be significantly overexpressed in 420 mGy animals.
367. Liver mRNA expression in seven weeks old brown rats (Ratus norvegicus) was also investigatedafter exposure to low-dose radiation [N14]. Irradiations with gamma rays were carried out with
140 mGy/d for two weeks and a total dose of 2,000 mGy. Unfortunately, the time between irradiation
and animal sacrifice was not specified in this work. Up-regulated genes confirmed by RT-PCR included
Btg2, Gadd45, Jun, Plk2, Agpat3 and 9, and Mir-27b.
368. The liver proteome has been a primary focus of the Ding laboratory [Y7, Y8, Y9]. In order to evaluate
different potential environmental exposures, a “local” source of gamma rays was used in the form of“uranium tailings”. The material was accumulated in quantities needed to deliver dose rates of 30, 100 and
250 µGy/h for 22 hours each day for 500 days [Y9]. In this study, glycine N-methyltransferase andnucleophosmin were both up-regulated at 250 μGy/h, while glutathione S-transferase was down-regulated
in all irradiated groups. A shorter-term experiment was carried out with, once again, male six to sevenweek-old C57BL/6J mice. Gamma rays were delivered by a 137Cs source at dose rates of <50, 50–500 and
500–1,000 µGy/h for 22 hours a day for 90 days and then sacrificed. Liver tissue was used for 2Delectrophoresis followed by mass spectrometry and Western blots for several selected proteins, as well as
real-time PCR. Genes responding to radiation in this work include catalase (mRNA increase beginning with
<50 µGy/h group), glycine N-methyltransferase (increased from 50–500 µGy/h group) and glutathione Stransferase P1 (increased in 500–1,000 µGy/h group). The same group focused especially on calreticulin[Y8] in a study using six-week-old C57BL/6J male mice. Radiation doses were doubled, but dose rates kept
the same as in previous experiment; the most interesting proteins from this screen were catalase, glutathione
S-transferase P1 and calreticulin. Other than catalase and glutathione S-transferase P1 and a few other notemphasized proteins such as, e.g. serum albumin, relatively few proteins overlapped between livers of mice
irradiated over 90 or 180 days.
369. Recently, Bakshi et al. [B9] investigated effects of in utero irradiation on heart proteome in mice.
Inbred C57Bl/6J animals, mated during a two-hour time period to ensure synchronous embryonicdevelopment, were whole body irradiated while pregnant at embryonic day 11. Total doses of 0.2, 0.5, 1
or 10 Gy were delivered as X-rays at a dose rate of 350 mGy/min. Male offspring were sacrificed at six
months and female offspring at two years of age; two dose groups shared 13 or 14 proteins in common
as deregulated (this included both up- and down-regulated proteins). Only a few proteins were
deregulated in common when six-month-old male and two-year-old female animals were compared; e.g.
Peroxiredoxin 5 and MACRO domain-containing 1 ribose deacetylase are such examples. It would beinteresting to explore the causes for such low overlap, e.g. age or sex of animals. In previous work, maleoutbred NMRI mice were exposed to radiation on postnatal day 10 using 20, 100, 500 or 1,000 mGy
doses of gamma rays produced by a 137Cs source (dose rate 0.023 mGy/min). Heart proteome changes
were analysed seven months after irradiation [B7]. Nine proteins were affected by all used doses ofradiation; several of them were commonly regulated by PPAR alpha. Amounts of phospho-PPAR alphawere decreased in all irradiated samples. Subsequent exploration of liver proteome by the same groupgave similar results [B8]. Again – 10-day-old neonate male NMRI mice were exposed to 20, 50, 100 or

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1,000 mGy and proteomic liver analyses carried out with samples collected at day one or seven months
after exposure. About 40 proteins were found deregulated by 20 or 50 mGy exposures after either one
day or seven months. In comparison, 60 or 70 proteins at day one or seven months were deregulated inmice exposed to 500 or 1,000 mGy. Four or five proteins were common to all exposures at different
timepoints. Most importantly, PPAR alpha could again be found as regulator of commonly affected
proteins. In liver, activity of this protein dropped at 100 mGy exposures.
370. Partial body (heart “only”) irradiation of C57BL/6 mice with 200 mGy did not lead to significant
changes in mitochondrial proteome at 40 weeks post-exposure [B19]. Only five proteins responsible forcytoskeleton desmin, actin, and three forms of myosin were found to be up-regulated.
371. Brain proteome and miRNA changes were also studied by the same group [K21]. Mice at post-natal
day 10 were exposed to 100 or 500 mGy and their brains harvested 24 hours later, separated into cortex
and hippocampus and analysed for expression of mitochondrial proteins. Findings include twofold
changes for mitochondrial complex I proteins which were decreased at both doses in both parts of thebrain, and transcription factor phospho-CREB (binding to cyclic AMP response elements) increased incortex at both doses. Additional proteins relevant for synapses were modulated as well as several different
miRNAs. In most cases, miRNAs were similarly regulated in response to radiation both in cortex andhippocampus. Most miRNA changes were found in cortex after exposure to 100 mGy.
372. In addition, the same groups collaborated to produce brain samples from female C57Bl/6 ApoE−/−
mice [K22]. Eight-week-old mice were chronically irradiated with 1 or 20 mGy/d with 137Cs gamma rays
over a period of 300 days. Irradiations were carried out for 22 hours a day and cumulative doses were
300 or 6,000 mGy. Hippocampi from each hemisphere were processed separately as well as the two brain
hemispheres. Almost none of the proteins had altered expression but their phosphorylation patterns werevastly different. The phosphorylation profile for neurofilament and eukaryotic translation initiation factor(Eif4b) showed up-regulation, but down-regulation in phosphorylation of G-protein coupled receptor 158
at both doses. Results of pathway analysis for these data were detailed in the study; for example, in micechronically irradiated with 300 mGy pathways related to synaptic plasticity and altered in comparison tocontrol were glutamate receptor signalling, Wnt/β catenin signalling, and tight junction signalling. Basedon immunocytochemistry, no cell number changes were seen either for Ki67 or GFAP positive cells(proliferating and neural stem cells, or for mature neurons – NeuN+ cells, and Casp-3 pattern ofexpression was the same as in controls). Only immunocytochemical change detected was in increased
density of post-synaptic density protein 95 and the microtubule-associated protein 2 (MAP2), but notphospho-MAP2, in dentate Gyrus. Finally, at 300 but not 6,000 mGy there was a decrease in Iba1+
microglia, which is a marker of neuroinflammation, and a reduction in TNF-α and lipid peroxidation
quantified as total protein content that was modified with malondialdehyde.
373. Using the data and materials from study by Uehara et al. [U1] (dose rates of 20.0, 1.00 or
0.050 mGy/d for 401 days accumulating to total doses of 8, 0.4 and 0.02 Gy 137Cs gamma ray,respectively), the same group of researchers explored the possibility to use complex data obtained fromlow-dose irradiation of mice to look for possible transcription factors [V4]. Sequences from 68 genes thatwere shown to be modulated by low-dose exposures and 93 genes that were not affected by the low-dose
exposures were used for this study. The sequence span of 3 kb upstream and 1 kb downstream from thecoding sequence were chosen and searched for transcription factor binding sites using TFSEARCH
database. Potential binding sites were found for 154 transcription factors and their frequencies were
calculated both in genes modulated or not affected by low-dose exposures. Binding sequences forglucocorticoid receptor, fork head domain factor HFH-1, AP-1, sterol regulatory element-binding protein
1 and olfactory neuron-specific factor, several early growth response genes, NF-κB p65, heat shock factor
1, POU factor Brn-2, ectopic viral integration site 1 encoded factor, among others, were found to be

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associated with low-dose responding genes in liver of low-dose irradiated mice. Of these, only AP-1 and
NF-κB were known to be affected by low-dose radiation prior to this study.
374. Neuronal effects of moderate to high irradiation were the focus of work by Wei et al. [W8]. Oneyear-old Kunming mice (weighing 30–35 g) received 300 or 3,000 mGy of X-rays and were sacrificed
for mRNA analysis 7 days later or immunohistochemistry 14 days later. Exposure to 300 mGy led tohigher numbers of Wnt1, Wnt3a, Wnt5a, and β catenin expressing cells by immunohistochemistry as well
as expression measured by Western blot and RT-PCR. The ratio of nestin/β catenin and BrdU/β catenindouble positive cells was also the highest in 300 mGy exposed mice. These data support increased neuralstem cell proliferation and neurogenesis at 300 mGy compared to controls, while opposite is true for
3,000 mGy exposures: Neurogenesis drops compared to control mice; also, these mice perform less wellin behavioural studies.
375. Mice of NMRI (Naval Medical Research Institute) were exposed to whole body radiation from a
Co irradiator 10 days after birth. Doses included 350 or 500 mGy delivered at a dose rate of 20 mGy/min[B59]. The animals were sacrificed a day or six months after irradiation and changes in protein expression
noted, especially in 500 mGy exposed animals. For example, tau protein was increased in the cerebral
cortex and decreased in hippocampus one day after exposure, and while it increased to usual level inhippocampus by six months of age, tau expression remained increased in central cortex of six-month-old
mice. Proteins CaMKII, GAP-43 and synaptophysin showed milder changes.
60

4.

Modulation of different genetic effects by low-dose exposures

376. Mice heterozygous for APC1638N/+ and wild-type female C57BL/6J mice were used in a study by
Suman et al. [S68]. Animals irradiated with 250 mGy X-rays were either monitored for the developmentof intestinal cancer themselves or mated two days after exposure with other exposed or unexposed
animals. Heterozygous mice from both-parents- and male-parent-irradiated groups had significantly moretumours than mice from a no-parents-irradiated cross. With regard to sex, male F1 heterozygotes had
significantly higher tumour frequency in all experimental groups including animals that were irradiated.
377. Female mice of three different genotypes: SCID (C.B-17/Icr-SCID), C.B-17/Icr wild-type and F1
heterozygotes (Prkdc+/−) were used in the experiment conducted by Ishii-Ohba et al. [I11]. Irradiations
were carried out with gamma rays produced by 137Cs; 1, 2, 3, 5 or 6.5 Gy doses were delivered at dose
rate of 500 mGy/min; doses of 50, 100, 250 or 500 mGy were delivered at dose rate of 80 mGy/min.
Finally, in order to induce thymic lymphoma in wild-type mice a dose of 1.6 Gy was delivered once aweek for four weeks. In SCID mice the background incidence of lymphoma was about 38% and thisnumber did not change with a 50 mGy exposure; for 100 mGy exposure, however, the incidence
increased to 53% and the percentage continued to increase until it plateaued at a dose of 2 Gy. In wildtype mice or heterozygotes, the incidence of lymphoma did not increase compared to the incidence in
non-irradiated mice except at 5 or 6.5 Gy doses. Solid tumours were far more frequent in these mice, but
their frequency was not modulated dependent on the total dose received. In SCID mice non-lymphoma
tumour frequencies also did not change with a change in dose. While lymphoma-free survival in SCID
mice decreased for all doses above 50 mGy, it increased in wild-type mice exposed to 250 mGy.
378. Low and moderate doses of radiation were found to be protective on their own but in some cases,
they also have strongly synergistic harmful effects in combination with chemical mutagens in an animalmodel of induced carcinogenesis. Bruce et al. used a mouse model for lung cancer induction – A/J strain
in which lung tumours are chemically induced by benzo[a]pyrene and progression from hyperplastic focito adenoma and carcinoma is easily quantified [B57]. Ten-week-old females were exposed to six fractions

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of whole-body irradiation (gamma rays, 137Cs irradiator) at dose rates of 1.07 or 1.33 mGy/s over three
weeks for total doses of 60 and 600 mGy, respectively. Benzo[a]pyrene injection was carried out fourweeks before first irradiation and all animals were sacrificed for lung evaluation at 46 weeks after
chemical exposure. Low-dose irradiation alone protected these mice from the development ofhyperplastic foci (about 33% incidence compared to 65% incidence in controls), however, while about
92% of mice exposed to benzo[a]pyrene had hyperplastic foci, combination of chemical exposure and
60 mGy exposure led to development of foci in all mice. Moreover, this dose of radiation andbenzo[a]pyrene was associated with increased adenoma and carcinoma. Exposure to 600 mGy showedintermediate effects.
379. Exploration of experimental schemes where low-dose exposure is followed by high-dose has been
mentioned already several times, sometimes low-dose pre-exposure to adapting doses was found to beprotective. However, that is not the case when central nervous system is considered [A5]. Low-dose
exposures of 100 mGy followed 24 hours later by high dose of 2,000 mGy (both delivered as gamma
rays from a 137Cs irradiator) were found to have synergistic effect on central nervous system of twomonths old C57BL/6 wild-type mice with respect to formation and maturation of neurons. Transgenic
male mice with knockdown of CCR2 were not affected in the same way.
380. Mota et al. [M42] used X-ray fluorescence microscopy to study elementalomics of peripheral bloodand cardiac formalin fixed paraffin embedded tissue in low- and high-dose irradiated animals. Male
Wistar rats were exposed to 0.1 or 2.5 Gy total body irradiation with X-rays (150 kV, 10mA and
17 mGy/s), and samples collected at 24 hours after exposure. Elemental decreases for K and Ca(indicating cell death but not very reliable with formalin fixed paraffin embedded) and S, Fe and Zn werenoted at both doses.

5. Effects on vascularization, cell migration/invasion and epithelialmesenchymal transformation
381. Several examples support the notion that moderate doses and possibly low doses of radiation
support growth of new vasculature. That is one way in which low or moderate doses may contribute tocarcinogenesis, facilitating the vascularization and therefore growth of tumours.
382. A study focused on angiogenesis as a factor critical for support of tumour growth used X-rays todeliver a dose of 300 mGy to NOD-SCID mice [S53]. Two in vivo endpoints were investigated withrodents – outgrowth of vasculature into Matrigel plugs implanted into animals 24 hours after exposure(each mouse serving as its own control, which was possible because of partial body irradiation), and thesuccess rate for implantation and volume of tumour cells (MOLT-4 cells) injected into
immunocompromised mice, again 24 hours post-exposure. Both the growth of new blood vessels on theirown and tumour burden were increased in mice that were exposed to 300 mGy.
383. Diabetes was induced in male Wistar rats by a single intraperitoneal injection of streptozotocin and
60 days; after induction of diabetes, a skin wound was created in diabetic and control animals [G20]. Halfof the animals were exposed to X-rays in fractions of 75 mGy five days a week for 1, 2 or 3 weeks,
accumulating to 375, 600 or 825 mGy. Wound healing was most rapid in control animals, but in moderate
dose exposed diabetic animals wound healing progression was better than in unexposed animals induced
into a diabetic state by the injection of streptozotocin, with more PCNA (cell proliferation) and vascularendothelial growth factor receptor (VEGFR) (blood vessel growth stimulation) found byimmunohistochemistry, more tissue remodelling enzyme activity (e.g. MMP-2 and MMP-9). Finally,

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more CD34+ cells and blood vessels and more CD31+CD34+ cells were found in circulation of moderate
dose exposed diabetic rats compared to non-exposed diabetic animals.
384. Protection from reperfusion ischaemia injury by moderate doses of radiation was the primary
question explored in work by Ministro et al. [M32]. In this study, 22-week-old female C57BL/6 mice
were used as recipients of cells isolated from nine-week-old female C57BL/6-Tg(CAG-EGFP)10sb/J
mice. Critical limb ischaemia was induced in mice by surgery and 12 hours later the mice were exposed
to 300 mGy of X-rays each day for one to seven consecutive days – total doses of 300, 600, 900, 1,200 or
2,100 mGy. No benefits were found for doses below or above 1,200 mGy; however, this dose lead tosignificantly improved capillary density (CD31+) especially between 15 and 45 days post-injury. This
work also demonstrated that the radiation regimen increased numbers of circulating endothelial
progenitor cells and subsequent collateral vessel development. RT-PCR for these cells showed increased
expression of Vegfr2, Vegfr1, Fgf2, Angpt2, Pdgfc, Tgfb2, Hgf, and Met in mice exposed to radiation.
When the study included bone marrow transplantation from GFP-expressing animals prior to injury andirradiation GFP+CD31+ cells were found to be increased (while only CD31+ cells were increased in wildtype mice).
385. Male Sprague–Dawley rats were used to test effects of moderate to high-dose radiation on bonehealing [C15]. After a bone fracture was introduced, animals were irradiated with 500 or 5,000 mGy Xrays or left as controls. Cell viability and proliferation were greatest in rats exposed to 500 mGy;
moreover, higher gene expression associated with osteoblast differentiation was also documented in thisgroup at later timepoints. Regenerative capacity may also rely on better growth of new vasculature.
386. Recent work by Marques et al. [M18] used human endothelial cells (HUVEC) in the in vitro woundhealing assay and blood vessel growth assay in chicken embryo in the presence of conditioned media
from irradiated and control pre-adipocyte or adipocyte cell cultures. Conditioned media from mouse 3T3L1 pre-adipocytes exposed to 0.1 or 0.3 Gy X-rays (Clinac, 300 MU/min dose rate) produced the mosteffects in support of faster wound healing or more extensive vasculature growth. This effect was lesspronounced when irradiated mature adipocytes were used. Thus, this work may provide mechanistic
explanation that connects low and moderate dose-induced obesity in mice (e.g. [B53, T4]) with improved
neoangiogenesis described in this section. Production of VEGF-A and MMP1 and 2 by preadipocytes
could at the same time aid wound healing and yet lead to cancer progression.
387. The later stages of tumour progression are characterized by features such as increased cell migrationand invasiveness, and transformation of epithelial cell morphology to a more fibroblastic form, known as
the EMT. Each of these features relate to the increased metastatic potential of solid cancers. Unlike thestudies described above, much work on invasion, migration and EMT utilizes in vitro cell models. This
work is included in this chapter as it relates to cancer progression in vivo. Relevant literature issummarized in table 6.
388. Relatively few studies consider the effects of low or moderate doses of low-LET radiations. Zhang
et al. [Z15] report that X-irradiation at moderate-high doses promotes EMT, cell migration and cell
invasiveness. Ghosh et al. [G5] found that 100–200 mGy 60Co gamma irradiation enhanced the migrationand invasiveness of four cancer cell lines but not primary cells, 50 mGy exposures are reported to haveno effect. The previously cited work of Sofia Vala et al. [S53] indicated that moderate dose (0.5–0.8 Gy)
photon irradiation enhanced wound healing. By contrast, 100 mGy or 10 fractions of 10 mGy 137Cs
irradiation are reported to reduce migration and invasion of human breast cancer cell lines [K19]. One
study suggests that 0.5 Gy X-irradiation increases cell migration and invasion [O9], interpreted to suggestthat conventional radiotherapy may enhance metastatic spread of remaining tumour cells. The impact of
low-dose irradiation on these endpoints relating to metastatic spread are therefore inconclusive at present.

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389. Several studies relating to the use of protons and heavy ions for radiotherapy have reported effectsof particle irradiation on cell migration and invasion. The effects of the irradiation are generally reported
to reduce cell migration and invasion [F24, H3, O9, O10]. In these studies, the effects are interpreted toindicate that proton and heavy ion therapies provide a therapeutic advantage by not only direct killing of
tumour cells but also through reducing metastatic spread. The pro-migratory effects of high dose lowLET radiations may be mediated by the release of exosomes with cargos rich in pro-migratory factors[A20, M47]. By contrast, the effects of heavy ion and proton irradiation on EMT are reported to bepositive [W4, W5]. That is the irradiation causes or enhances EMT, which is a feature that is considered
to enhance metastatic spread of solid cancers.
.

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Species/cell type

Irradiation conditions

Effect reported at low doses/dose-rate

Other reported effects

Reference

Pancreatic carcinoma cell line,
PANC-1

6 MeV photons
Dose: 2–6 Gy
Dose rate: 4.18 Gy/min

2 and 4 Gy reduced migration and invasion,
especially in presence of plant extract[H3]

Human fibrosarcoma cell line
HT1080, mouse osteosarcoma cellline LM8190 MeV/nucleon protons
Dose: 4–8 Gy

All exposures increased adhesion to extracellularmatrix proteins; migration and invasion decreased
by protons and C ions, increased by X-rays; C ions
reduced number of metastases in transplant model[O9]

Protons reported to reduce invasion and migration[O10]

C ions reduce migration and invasion in all but one
cell line[F24]

290 MeV/nucleon C ions
Dose: 4–8 Gy
4 MeV LINAC (X-radiation)
Dose: 0.5 Gy
Dose rate: 1.8 Gy/min

Human lung adenoma cell line
A549

190 MeV/nucleon protons
Dose: 4–8 Gy
290 MeV/nucleon C ions
Dose: 4–8 Gy
4 MeV LINAC (X-radiation)
Dose: 0.5 Gy
Dose rate: 1.8 Gy/min

Human pancreatic cancer cell lines

290 MeV/nucleon C ions, 80 keV/mm
Dose: 0.5–4 Gy

Aleutian mink lung cell line, Mv/Lu;
hTERT immortalized oesophagealcells

170 MeV/nucleon Si ions, 99 keV/mm;
600 MeV/nucleon Fe ions, 180 keV/mm.
Dose: 0.1–2 Gy
Dose rate: 0.25–1 Gy/min

Si and Fe ions cause mild EMT,
even at 0.1 Gy[W4]Aleutian mink lung cell line, Mv/Lu;
hTERT immortalized oesophageal
cells

100 MeV protons, mean energy 5 MeV and
10 MeV/mm.
Dose: 0.1–4 Gy
Dose rate: 0.25–1 Gy/min

0.1 Gy and higher doses enhance TGFβ
induced EMT; mild induction of EMT in
absence of TGFβ

[W5]Human tumour cell lines U87,
BMG-1, A549, HeLa

Gamma radiation (60Co)
Dose: 50–200 mGy
Dose rate: 1.034 Gy/min

100–200 mGy increased connexin 43
expression and increased glioma cellline migration. No effect of 50 mGy. No
observed effects in primary cancer celllines

Connexin expression effect reported to be mediatedby sustained activation of p38 and up-regulation of
p21

[G5]

UNSCEAR 2020/2021 REPORT

Table 6. Endpoints relating to cell migration, invasiveness and epithelial-to-mesenchymal transition

Species/cell type

Irradiation conditions

Effect reported at low doses/dose-rate

Other reported effects

Reference

X-radiation
Dose rate: 0.4 Gy/min or 20 Gy/day(13.9 mGy/min)

EMT-like changes and increased migration andinvasion[Z15]

Human lung endothelial cells (nontransformed)

LINAC (Varian Clinac 2100 CD) photons
Dose rate : 300 MU/min

0.5–0.8 Gy dose increased wound healing – asurrogate for migration[S53]

Human breast cancer cell lines
MCF-7, MDA-MB231

Gamma radiation (137Cs)
Dose: 10 fractions of 0.01 Gy
Dose rate: 0.1 Gy/min

Reported effect mediated by inhibition of
JAK1/STAT3 signalling[K19]

Human glioblastoma cell lines andprimary astrocytes

X-radiation
Dose: 2–8 GyDose rate: 2.3 Gy/min

Increased release of exosomes carrying cell migration
related factors that enhance migration in recipient
cells

[A20]

Human head and neck squamouscell carcinoma cell lines

Gamma radiation (137Cs)
Dose: 3–9 Gy
Dose rate: 0.45 Gy/min

Exosomes derived from irradiated head and necksquamous cell carcinoma cells conferred a
migratory phenotype to recipient cancer cells,
linked to radiation-regulated exosomal proteinsthat increase AKT-signalling[M47]

100 mGy or 10×0.01 mGy reduced
migration and invasion, no evidence of
EMT

ANNEX C: BIOLOGICAL MECHANISMS RELEVANT FOR THE INFERENCE OF CANCER RISKS ...

Human breast carcinoma cell line
MCF-7

119

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UNSCEAR 2020/2021 REPORT

6.

Carcinogenesis

390. An extensive study using Sprague–Dawley rats included irradiations of 4,016 animals of both sexes
in order to evaluate the health of their offspring. Irradiations were both acute and fractionated (10 doses
once every 4 weeks) at three dose levels (0.1, 1 and 3 Gy). Pregnant dams irradiated with 0.1, 0.5 and1 Gy at day 12 of pregnancy produced 2,799 offspring, while male breeders exposed acutely to 0.1, 1 and3 Gy before mating produced 2,557 offspring. Finally, dams fed on day 12 of pregnancy with standard
feed irradiated by 1,000 or 4,000 Gy produced 1,139 offspring that were fed the same feed until
spontaneous death. Animals exposed to external beam were irradiated at six weeks of age with 60Co
gamma rays to total doses of 100, 1,000 or 3,000 mGy. While both lethal tumour and any tumour
incidence increased in rats exposed to 1,000 or 3,000 mGy, no significant increase was found in the group
exposed to 100 mGy [S52]. Life shortening occurred in both high dose groups and was the greatest infemales exposed to 3,000 mGy. The only biological endpoint in this study modulated by 100 mGy
exposure was development of atypical hyperplasia in mammary glands.
391. An interesting approach to study carcinogenesis came from work by Sasaki and Fukuda [S10]. In
this work, B3C3F1 mice (cross between C57BL/6JNrs and C3H/HeNrs) were exposed to 100, 480 or
950 mGy of 137Cs gamma rays at seven days of age. In addition to usual comparisons such as life
shortening (excess relative risk increased by 0.102, 0.697 and 1.206 for the three doses), other evaluations
were carried out: numbers of tumours per mouse were counted as well as numbers of tumours in groupsof mice dying within a certain age window. This evaluation provided new insights into health status ofanimals exposed to low doses of radiation. For example, even though no significant change in life
expectancy was seen in mice exposed to 100 mGy compared to controls, their causes of death weredifferent. In animals that reached age of 950 days, mice in the control group were dying from non-tumour
causes, while tumours accounted for most of the deaths of younger animals. On the other hand, 950 days
old mice that received 100 mGy continued to die predominantly of cancer (similar to younger animals).
Moreover, four distinct tumour types could be found in this group of mice but not in the controls. In termsof specific tumour types, ovarian and lung cancers had greater frequency in mice exposed to 100 mGycompared to controls (probably at the expense of non-tumour diseases).
392. An extensive review of years of work carried out at the low-dose-rate irradiation facility in Japan’s
Institute for Environmental Sciences was published recently [B53]. This review paper succinctly informs
about studies in mice of several different genotypes (B6C3F1, C3H, C57BL/6 and transgenic (gptdelta×SWR) F1), irradiated 22 hours a day by a 137Cs source over a range of dose rates from 0.04–
0.06 mGy/d, 0.8–1.1 mGy/d and 16–21 mGy/d. Total irradiation doses after exposure periods of 400 to
485 days were either 20–21, 400–420 or 8,000–8,050 mGy. Ten different endpoints – lifespan, neoplasm
incidence, body weight, tumour cell transplantability, changes in chromosome structure, gene mutations,changes in mRNA and protein levels, and transgenerational effects on lifespan and neoplasm incidence
were all studied in these mice, in both sexes. Statistical analyses considered P<0.05 as significant for
non-cancer endpoints and P<0.01 for neoplasia. While most findings from this review are covered in
specific references throughout this annex, this review is very valuable as a resource with the most succinct
information provided by the researchers themselves. It should also be noted that a not yet published study
is mentioned, where work was carried out with C57BL/6J mice (groups of 215–278 mice per sex perdose). Importantly, only male mice exposed to 20 mGy/d with total dose of 8,000 mGy demonstrated life
shortening. Males exposed to the total accumulated dose of 20 or 400 mGy did not show life shortening.
By comparison (based on [T3]), in B6C3F1 strain animals both sexes had shorter lifespans in groups
exposed to 20 mGy/d with total dose of 8,000 mGy, while females showed life shortening at the dose rateof 1 mGy/d with total dose of 400 mGy.
393. Yamauchi et al. [Y2] used 60 animals per group of 6-week-old male B6C3F1/Jcl mice, put onto acalorie-restriction diet and exposed to chronic 137Cs gamma ray radiation for 400 days at 20 mGy/d,ANNEX C: BIOLOGICAL MECHANISMS RELEVANT FOR THE INFERENCE OF CANCER RISKS ...

121

resulting in a total dose of 8 Gy. Life expectancy of low calorie animals was greatest, followed by low
calorie irradiated mice which survived longer than non-irradiated, standard calorie diet mice. Life
shortening by radiation in regular diet mice was not significant.
394. Lawrence et al. [L7] investigated effects of radiation in a mouse model prone to development of
prostate cancer – transgenic adenocarcinoma of the mouse prostate (TRAMP). Six weeks old TRAMPFVB F1 mice were exposed to a single high dose of 2 Gy (1 Gy/min X-irradiation), a single low dose of
50 mGy (13.9 mGy/min X-rays) or five weekly exposures of 50 mGy X-rays. There was no significant
difference in the time to palpable tumour between sham-irradiated animals and mice exposed to 2 Gyradiation. With regard to low to moderate doses of radiation (either 50 or 5×50 mGy) no changes withrespect to control were found in either apoptosis frequency or any other endpoint examined in this study(e.g. Ki67 staining, histopathological status of the prostate).
395. Development of lymphoma in mice with thymocyte-specific Bax overexpression was focus of thestudy published by Jacobus et al. [J6]. Three different genetically modified mice were used in this study:(a) C57BL/6 Lck-Bax transgenic mice have an insertion of additional copies of the mitochondriallocalized BAX gene under the control of the lymphocyte-specific protein tyrosine kinase promoter (Lck)and 50% of mice develop lymphomas in the absence of radiation by 40 weeks of age if male and by 70
weeks of age if female; (b) Lck-Bax38&1 mice additionally have increased levels of superoxide inthymocytes which still hastens development of lymphoma: 50% of non-irradiated mice develop it by 25–
30 weeks of age; in this genotype, no sex differences in development of lymphoma were noted; (c) LckBax38&1 mice were also crossed with SIRT3 knockout mice to produce double mutants for the purposeof this study – oxidative damage in these mice is still more pronounced and MnSOD activity decreased;
as a consequence mice that are a cross of Lck-Bax38&1 and SIRT3 knockout die even earlier than the
Lck-Bax38&1 mice and develop lymphoma at a rapid rate of increase. Irradiation with 100 or 1,000 mGy
gamma rays was provided by 137Cs irradiator at a dose rate of 350 mGy/min; in addition, some mice were
exposed to 100 or 1,000 mGy of Si- or Fe ions, administered over a period of approximately one minute.
In Lck-Bax1 mice, exposure to either dose significantly shortened latency of lymphoma in female mice,
while in male mice no significant differences between irradiated and sham-irradiated animals were found.
In Lck-Bax38&1 mice, no significant changes in latency were caused by exposure to either dose; withregard to sex, exposure to 100 mGy lead to a nearly statistically significant separation where females had
shorter latency than males. Finally, in Lck-Bax38&1 mice no significant change in latency were causedby exposure to either dose of Si ions and no sex differences were detected. Exposure to Fe ions, however,led to a dose-dependent shortening of disease latency; sexes were separated in this study with female
mice developing lymphoma sooner than males when exposed to 1,000 mGy of Fe ions. It should be notedthat in this study animal ages varied between four and nine weeks and this may be part of the reason forsome of the data variability; in other words, that data could perhaps have been more meaningful weremice more exactly age-matched.
396. In addition, a recent study using p53 heterozygotes found that a single X-ray exposure of 10 mGy
significantly delayed onset of cancer in irradiated compared to non-irradiated p53+/− animals [L18].
Moreover, multiple exposure to similar doses to those received from a CT scan (i.e. 10 exposures of
10 mGy, one per week) after a single high exposure (4 Gy) increased the overall lifespan of the 4 Gy
exposed mice, by approximately 8% (P=0.017) [L19].
397. Considering dependence of humans on other living organisms, and the fact that, for example,
intestinal flora plays a significant role in resilience to cancer (e.g. [K55]) it may be postulated that someof the complex effects of radiation of human surroundings may have indirect, yet real effects on cancer
induction. Several studies exploring high doses of radiation show this point. Goudarzi et al. [G8]investigated response of C57BL/6J 8-week-old male mice to high doses of radiation (5 and 12 Gy Xrays) through alterations of mouse metabolome dependent on intestinal microbiome. Collective gut

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UNSCEAR 2020/2021 REPORT

microbiome showed pronounced responses to radiation exposure, persisting in some cases for many days.
In a study focused on high-LET radiation exposures and gut microbiome [C6] C57BL/6J mice were fed
a consistent diet for five months, and then exposed at six months of age to 16O (600 MeV/n) as a heavy
ion. Delivery of doses of 100 and 250 mGy was found to alter the mouse gut microbiome extensively,
leading to functional metabolic changes (for example, benzaldehydes were increased in mice exposed to
100 and 250 mGy both at 10 and at 30 days post-exposure). Equivalent studies in animals exposed to low
doses of low-LET radiation are still not available. It is considered that any changes in microbiome would
also affect changes in animals’ cancer risk because of the metabolome changes that can be triggered.

(a) Low dose effects on transplanted tumours
398. A study focused on tumour cell transplantability [I13] used eight- to nine-week-old female BALB/cand C3H/HeN mice irradiated with 50, 100, 200, 500 or 1,500 mGy X-rays at a dose rate of 40 mGy/min.
Tumour cells used for transplantation were mammary sarcoma cell line EMT6 isogenic for BALB/c miceand SCCVII squamous cell carcinoma isogenic for C3H/HeN mice; both hind limbs were injected eitherwith 1,000 or 100 cells six hours after radiation exposure and tumours were allowed to grow for 50 days.
In both cases, only 1,500 mGy dose increased transplantability. In C3H mice, there was no difference forsham-irradiated mice and those exposed to doses of 500 mGy and below. In BALB/c mice animals
exposed to 200 mGy had the least successful implantations, however the differences were not significant.
399. A different approach to evaluation of tumour transplantability was tested when tumour cells usedcame from a mouse that developed cancer in response to exposure to high-dose-rate radiation – theovarian granulosa cell tumour cell line: OV3121, was derived from B6C3F1 mice and transplanted into
B6C3F1 and BALB/c mice [T2]. Recipient mice were exposed to chronic radiation at eight weeks of age
for 200 or 400 days, gamma rays were produced by a 137Cs source and total accumulated doses were
4,000 or 8,000 mGy. Cytologically, numbers of Th1 cells were increased in spleens of 8,000 mGy
exposed mice and proliferation induction by allo-Mixed Lymphocyte Reaction was decreased; all othercomparisons (for Th2, Th17, Treg etc.) failed to show significance. Tumour cells were injected into hindlimbs of recipient mice immediately after radiation was concluded and tumour growth was monitored for
60 days. Tumours grew in 8,000 mGy irradiated mice more than in controls. The difference was notstatistically significant when 4,000 mGy irradiated mice compared with controls, possibly because of thedifference in age of the mice.
400. Low-dose radiation hormesis was also studied with “radiation-emitting sheets” producing a rangeof low-dose alpha, beta and gamma emissions at dose rates between 35 or 130 µSv/h (the gamma raycontribution was about 10% in each case). When three-week-old female BALB/c mice were bred andmaintained on radiation emitting sheets with dose rate of 100 µSv/h, no changes in weight were observed[N9]. In addition, mice were maintained for seven weeks on radiation emitting sheets with dose rates of
40 or 100 µSv/h and then their hind limb was injected with syngeneic breast cancer tumour cell line
EMT6. In all cases (control, 40 µSv/h, 100 µSv/h, each 25–30 animals per group) tumours grew in threeout of four animals, but rates of tumour growth differed – the slowest rate of growth was recorded inanimals maintained on 40 µSv/h sheets.

(b) Low dose rate and liver, adrenal glands and ovaries
401. Many studies focused on animals continuously exposed to low-dose-rate gamma rays until
accumulation of medium to high total doses and explored cancer types and cancer incidence at the timeof sacrifice or at the end of lifespan. For example, eight-week-old female B6C3F1/Jcl mice were exposed
daily to 21 mGy/d of 137Cs gamma rays [T4] and the mice were sacrificed after 100, 200, 300 andANNEX C: BIOLOGICAL MECHANISMS RELEVANT FOR THE INFERENCE OF CANCER RISKS ...

123

400 days after beginning of irradiation (corresponding to total accumulated doses of 2,000, 4,000, 6,000
or 8,000 mGy). At that time exposures were stopped, and additional animals were sacrificed each hundreddays up to 400 days after conclusion of irradiation or allowed to live out the entire lifespan. Irradiatedanimal groups consisted of 60 to 90 mice, each group was accompanied with equal number of age
matched non-irradiated controls. This study found increased body weight in irradiated mice from sacrificedays 200 to 500 accompanied with many non-neoplastic lesions (adrenal subcapsular cell hyperplasia,
fatty degeneration of the liver, atrophy and tubulostromal hyperplasia of the ovaries). Cancers in all
animals included malignant lymphomas, which caused most of the life shortening in irradiated groups;
hepatocellular adenomas/carcinomas; bronchoalveolar adenomas and Harderian gland adenoma/
adenocarcinoma; adrenal subcapsular cell adenomas and ovarian neoplasms (tubulostromal adenoma,
granulosa cell tumours) especially had earlier incidence in irradiated mice. Finally, animals from sacrificedays 400 to 700 and in the lifespan group had increased incidence of multiple neoplasia [T4].
402. In an earlier study, Tanaka et al. [T3] exposed eight-week-old B6C3F1 mice (2,000 of each sex) at
dose rates of 21, 1.1 and 0.05 mGy/d 137Cs gamma radiation, again for 400 days total, leading toaccumulated total doses of 8,000, 400 and 20 mGy. In this study, animals were kept until natural death.
Among the most frequent cancers in all cases were myeloid leukaemias in males, soft tissue neoplasms
and malignant granulosa cell tumours in females, and haemangiosarcoma in both sexes. In mice receiving
the highest doses, the frequency of these diseases compared to controls were significantly increased, ageof onset for leukaemias was earlier, and frequency of multiple primary neoplasms per mouse wasincreased. While some life shortening was found in mice exposed to total doses of 400 and 20 mGy [T8]and more than 85% of all mice died of cancer, no specific fatal neoplasms could be associated with lowdose-rate, low-dose and medium-dose exposures. It should be noted that in this study too, increased bodyweight was noted in mice exposed to 21 mGy per day. Importantly, Nakamura et al. [N8] found that feed
consumption was not increased in irradiated female mice compared to non-irradiated littermates. Overall
increase in adiposity (body weight, adipose tissue weight, serum levels of leptin, and lipid contents of theliver and serum) must therefore be connected with metabolic alterations triggered by radiation exposures.
Considering that the same mice had highest cancer incidences as well, it is once again necessary to statethat the relationship between metabolic derangements and cancer needs further exploration. Importanceof metabolic profile as a modifier of long-term radiation responses has been found in humans, e.g. among
atomic bombing survivors [Y14].

(c) Low dose and lung cancer
403. Floyd et al. [F16] used the bi-transgenic CCSP-rtTA/Ki-ras mice (FVB/N background withdoxycycline inducible expression of the Ki-rasG12C gene in lungs) to develop a general model to study
the promotion of lung cancer, with multiple cancer foci in lungs developing after doxycycline exposure.
Munley et al. [M46] exposed these animals to low to moderate doses of X-radiation (80–160 mGy). Thisfurther increased the frequency of lung cancer incidence. There was a statistically significant sex
difference, with higher numbers of cancer foci in female than in male mice. The response to radiation
exposure of other organs in these animals remains to be investigated.
404. Puukila et al. [P26] examined data from several studies where beagles were exposed toaluminosilicate beads containing 90Sr, 144Ce, 91Y, 90Y with effective lung half-lives of 2.5 days to
600 days; the beagles were separated into three groups not on the basis of dose first but based on the life
shortening effect noted. Animals were grouped into those dying shortly after exposure (acute lung injury),those that die early (mostly lung cancer) and those whose death is non-significantly expediated.
Evaluation of total doses delivered to animals that died later in life due to lung cancer induction showedthat the total doses were not the same for all radionuclides used. The radionuclide with a long half-life,

124

90
90

UNSCEAR 2020/2021 REPORTSr (0.1–0.6 Gy/d), induced cancer with greater efficiency than the radionuclide with a short half-life,
Y (8–10 Gy/d), indicating the importance of cell death and stress-cell recovery in the radiation response.

7.

Role of sex in radiation responses

405. In Li et al. [L23], sex differences in ex vivo irradiated (moderate-high dose) human cells peripheral
blood treated with 0, 0.5, 1, 2, 3, 4, 6 and 8 Gy with 60Co gamma rays at a dose rate of 1 Gy/min were
explored; expression patterns of candidate genes using quantitative real-time polymerase chain reaction(qRT-PCR) at 6, 12, 24 and 48 hours post-irradiation were identified. Sex-dependent regression models
could explain 0.85 of variance at 24 hours and estimate absorbed dose up to 0.5–5 Gy.
406. Male and female C57BL/6 mice were exposed to X-radiation at eight weeks of age, either from
0.3 Gy to 3 Gy or 0.3 Gy followed by 3 Gy four days later. Ten males and ten females each weresacrificed 6 hours, 96 hours or 2 weeks after irradiation [W3] and miRNAs harvested and compared
between different treatments and animal of different sex. Sex differences were detected, confirming
previous findings by the same group [K38, K39]. Liver samples from female mice overexpressed miR29c only after 0.3 Gy irradiation but not after 3 Gy or 0.3 Gy followed by 3 Gy exposure. This finding is
additionally of interest because miR-29c expression is decreased in liver cancer mouse Hepa 1–6 (andhuman HepG2 and C3A) cell lines, but it is induced in normal mouse liver epithelial cell line NmuLi.
This finding can perhaps be combined with work of Tanaka et al. [T4] that finds signs of non-neoplasticchanges, e.g. fatty degeneration in livers of female B6C3F1 mice exposed to 21 mGy/d at 300 days after
beginning of irradiation while development of nodules occurs only at 200 days after the completion of
400 days of radiation.
407. Sex differences in responses to radiation exposure were noted in transgenic reporter miceexpressing luciferase from a promoter for secretory clusterin [K28]. This protein is an extracellularmolecular chaperone that clears cell debris, it is stress-induced and exposures to ionizing radiation atdoses greater than 20 mGy lead to its peak expression at 96 hours. Recombinant reporter gene hCLUpLuc was grafted onto an FVB/N genetic background. Mice of equal weight were used which is often apractice in experiments with large numbers of mice, however, in this case all mice were also used atexactly 10 weeks of age. It is important to emphasize experimental rigour of this approach because it
suggests that low-dose radiation research field could produce more accurate data if more attention toexperimental procedures was given. Irradiations with 100 mGy was carried out at dose rates of
3,880 mGy/min with a 137Cs source. At 24 hours after irradiation animals were injected with luciferin andfemale mice demonstrated greater reporter gene expression. This was explained by the fact that clusterin
is under testosterone repression. Isolated internal organs were also imaged ex vivo after 10 mGy
radiation; luciferase expression in bone marrow, spleen, colon, thymus and mammary glands wasincreased at 24 hours unless the animals were given 5 mg anti-TGF-β1 antibody per kg body weight. Thissuggests that expression of secretory clusterin in female mice exposed to 10 mGy depends on TGF-β1.
408. Takabatake et al. [T1] reported that B6C3F1 mice chronically exposed to a low dose rate 137Cs
gamma rays (21 mGy/d, total dose 8 Gy over 400 days) had shorter lifespans than controls. Irradiatedand non-irradiated females had shorter lifespans than males. Genetic evaluation of lymphomas (thepredominant cause of death in all cases, more frequent in females) showed that in irradiated mice there
was a gain of chromosomes 11 and 15 and partial gains of chromosomes 12, 13, 14, 16 and X werepredominant in lymphomas from non-irradiated mice. No sex differences were noted with respect tochromosomal changes.

ANNEX C: BIOLOGICAL MECHANISMS RELEVANT FOR THE INFERENCE OF CANCER RISKS ...

125409. Whoolery et al. [W10] demonstrated that 56Fe and 28Si ions interfered with hippocampal
neurogenesis. Neuronal proliferation decreased with dose when 28Si ions are used for whole body
irradiation of 9-week-old C57BL/6J and Nestin-GFP mice at doses of 0.2 Gy or 1 Gy (300 MeV/nucleon,
LET 67 keV/µm, dose rate 1 Gy/min). Sex differences were noted for controls with females having fewer
Ki67 positive cells. After radiation there were no significant differences for female mice but in long-term
male groups after radiation new neuron survival was reduced.

8.

Summary and conclusions on whole animal studies

410. Cancer induction in animals is one of the late effects of radiation, especially at low doses (seetable 7). Consequently, many research studies on mammals focus on mutated animals with increased
cancer frequency. In other animal systems, investigation is often focused on non-cancer events that would
be cancer initiating or promoting (or opposite) in mammalian model systems. In all cases, induction ofcancer by radiation varies with the tissue or cell type whether the exposure is external or internal, theLET of radiation, dose rate, the sex and age of the animal at the time of exposure etc. While some studies
suggest that exposures to low doses of radiation can reduce cancer incidence in experimental animals,
others show that similar low doses can increase cancer incidence. In all cases, variation in response andgroup size/statistical power can be limiting, thus reaching a consensus on the impact of low radiation
exposures on animals is not currently possible. It is clear that very careful control of experiments in termsof animal ages and weights is of great importance.

126

Type of animal, strain, age a

Genetic or
chemicalmodulation,
sex b

Irradiation conditions

Outcomes recorded,time post-exposure

Definitive findings

RNAs, proteins,metabolite, epigenetics c

Reference,comments

WILD-TYPE ANIMALS
Ra radiation source
Dose : 50, 100, 200,
400 mGy
Dose rate: 36 mGy/h d

226

Fruit fly
Canton-S

Lifespan, RNA (PCR)

X-radiation
Dose : 0.1, 1, 10, 50,
100, 200, 400 Gy dFruit fly
Canton-S,
24–48 hours

Alpha, beta and gamma
radiation via substrate

Silkworm
Eggs

Hormesis with respect to lifespan in
100 mGy males, 400 mGy females

Genes for circadianrhythm, DNA repair,Heat shock[Z18]

Hormesis with respect to lifespan for
100 mGy

Unique gene set for
low dose[A13]

Body weightNo adverse health effects at age
studiedDNA double-strand
break (TUNEL)

Linear increase in DNA double-strand
breaks at ≥100 mGy[B37, B38, B39]

DNA damage, healthstatusHatching time is shorter with ionizing
radiation; DNA damage andapoptosis at 570 mGy/d[G1][N9, S30]

Dose rate: 35–
130 µSv/h e
Zebrafish
Strain brass, 6 hours embryo,sibling groups

Menadione

Gamma radiation (137Cs)
Dose : 50–1 500 mGy
X-radiation
Dose : 1–50 mGy d

Zebrafish

Gamma radiation

Embryo, 3 hours postfertilization

Dose rate: 0.8,570 mGy/d for
96 hours e

Zebrafish

3.37 MeV protons
Dose : 10–
2 000 protons d

HealthTriphasic response: increased
apoptosis below 30 mGy and above
90 mGy, hormetic effect andreduced apoptosis between 30 and
60 mGy[C25]

X-radiation
Dose: 5–100 mGy
Different hardness d

Apoptosis

Triphasic response but shifted
depending on X-ray hardness (theharder the X-rays the lower the dosewith hormetic effect)[K36]2-cell embryo

Zebrafish

UNSCEAR 2020/2021 REPORT

Table 7. Summary of animal studies

Type of animal, strain, age a

Genetic or
chemicalmodulation,
sex b

Zebrafish
AB strain

Dog
BeagleBeagleMouse
B6C3F1, 8 week-old

Rat
Sprague–Dawley, 6 weeks
Rat

Bone injury

Sprague–Dawley

Rat

Streptozotocin –diabetes

Outcomes recorded,time post-exposure

Definitive findings

RNAs, proteins,metabolite, epigenetics c

Reference,comments

Few genes in F1 fromdifferent doses
overlapping, more
overlapping between
higher dose 1-month F1and lower dose 1-year
F1

[H42]

Gamma radiation (60Co)
Dose: 5.2, 31 Gy
Dose rate: 8.7,
53 mGy/h dMating, health etc.
month or year later,
health of F1

Gamma radiation
Dose rate: 3, 7.5,
18.8 mGy/d d

Myeloproliferative
diseases, life
shortening

No lymphoproliferative diseases
below 3 mGy/d[F14]
reviewGamma radiation(239PuO)
Activity inhaled: 0.14,
0.63, 3.2, 13, 44,
210 kBq f

Tumour incidence,life shortening

Lung cancer reduced in low dose
group

[P4]

Gamma radiation
Dose: 8 000, 400 and
20 mGyDose rate: 21, 1.1,
0.05 mGy/d for
400 days dTumour incidence

Body weight increase for 1.1 and21 mGy/d and higher tumour
incidence for 21 mGy/d (total dose
8 Gy)

[B53, T3]

Gamma radiation (60Co)
Dose: 0.1, 1, 3 Gy d

Tumour incidence

No tumour incidence increase at
100 mGy, only some mammary
hyperplasia

X-radiation
Dose : 0.5, 5 Gy d

Pathology, bonerecoveryBone healing with low doses; at
500 mGy high early osteoblast
viability and proliferation and highlate differentiation

X-radiation
Dose: 350 mGy
Dose rate: 25 mGy
every second day g

Diabetes testis
atrophy

Protection from atrophy[S52]

Proteins increased at
7 days in 500 mGy
COL1A, ALP,

[C15]

Osterix and OCN, PCNA,
etc.
[Z17]

ANNEX C: BIOLOGICAL MECHANISMS RELEVANT FOR THE INFERENCE OF CANCER RISKS ...

Dog

Irradiation conditions

127

128

Irradiation conditions

Outcomes recorded,time post-exposure

Definitive findings

RNAs, proteins,metabolite, epigenetics c

Reference,comments

PCNA and VEGFR
increased by IHC, MMP2 and MMP-9 increased[G20]

WistarStreptozotocin –diabetes

X-radiation
Dose: 375, 600,
825 mGy
Dose rate: 75 mGy
fractions, 5 days per
week for 1, 2, 3 weeks gDiabetes impairmentof skin woundhealing

Neovasculature growth (CD34+),
increased CD31+/CD34+ incirculation

Rat

FemaleFast neutrons(241Am-Be)
Dose: 9 mGy d

DNA repair

Repair nearly complete within 3 hours[N1]

FemaleThoracic X-radiation
Dose: 1, 3, 5 Gy d

Neoplasia, life
shortening

No life shortening for any age/dose,cancer types different than controls[Y1]

Gamma radiation (60Co)
Dose rate: 300 mGy/h,
6 weeks before
challenge d

Challenge dose
9 or 9.5 Gy survival

Better survival withpre-exposure

Streptozotocin
at 10 weeks –diabetes

X-radiation
Dose rate: 12.5, 25,
50 mGy fractions every
other day for 4–
8 weeks g

Diabetic nephropathy
evaluation

Less kidney fibrosis, protection by 12.5
or 25 mGy for 8 weeks best

Infection withscrapie strain
ME-7

Gamma radiation (60Co)
Dose: four fractions of
5–750 mGy per
fraction g

Prion disease
management

Four fractions at 500 mGy around day
50 post-infection effectively reduce
onset time and final decline[P20]

Gamma radiation
Dose rate: 1, 20 mGy/dfor 400 days dPathology

Tumour incidence and life shortening
significant in 8 000 mGy group[B53, T4]

Gamma radiation (137Cs)
Dose : 100, 480,
950 mGy d

Tumour incidence

Old mice from 100 mGy cohort die
more of tumours than from nontumour toxicities

[S10]

Rat

Wistar, 3 month-old
Rats
Wistar, 1, 5, 15 week-old
Mouse
DBA/2

Mouse
C57BL/6J

Mouse
C57BL/6, prion disease

Mouse
B6C3F1Mouse
B3C3F1 (C57BL/6JNrs ×
C3H/HeNrs),
7 day-old[E4]females had
higher LD50 in allgroupsLower creatinine andconnective tissue
growth factor[C20, C21]

UNSCEAR 2020/2021 REPORTType of animal, strain, age a

Genetic or
chemicalmodulation,
sex bType of animal, strain, age a

Genetic or
chemicalmodulation,
sex b

Mouse
C3H/HeN

Mouse

Male

C57BL/6, 12 week-old

C57BL/6, CBA/CaC57BL/6

Mouse

C57Bl/6
5 week-old

RNAs, proteins,metabolite, epigenetics c

Reference,comments

Chromosomal
abnormalities

Linear increase in number ofdicentrics in spleen cells over time
for both dose rates. Unstable
aberrations per cell show a spike at
1 Gy cumulative dose for 20 mGy/dgroup, data points not significant
however[B53, N5]

Gamma radiation
Dose: 60, 300, 1 500,
7 500 mGy
Dose rate: 2, 10, 50,
250 mGy/d for
30 days d

Muscle effects,
immediately after
ionizing radiation or
3 months afterSatellite cells isolated from muscles
had lower numbers when isolated
from animals after 3 months[M21]

X-radiation
Doses: up to 100 mGy
Dose rate: 1 mGy/s

Bone marrow
radiation-induced
chromosomal
instability

TUNEL assay not positive before
200 mGy

Total body X-irradiation
Dose: 200 mGy
Harvest 40 weeks
later gHeart mitochondrial
proteome

p53 and p21 cells werenoted at 50 or 100 mGy
in C57BL/6 or CBA/Ca
mice, respectively[Z24]

desmin, actin, and threeforms of myosin.

[B19]

None; Male16

O high-LET 600 MeV/n
Dose: 0.1, 0.25, 1.0 Gy
Dose rate: 0.21–
0.28 Gy/min f

Microbiome andmetabolome

Bacterial 16S rRNA amplicons;
Fecal metabolomics[C6]

FemaleX-radiation
Dose: 75 mGy fractions
before challenge
Dose rate: 1.2 mGy/hover 450 days g

Lymphoma incidence,
7.2 Gy (in 4 fractions)challenge

Chronic irradiation – better healthoverall, more CD4+, CD40+, more
efficient protection from lymphoma
but 75 mGy also protects[I7]

C57Bl/6 J

MouseDefinitive findings

Chronic total body
gamma irradiation
Dose: 200, 20 mGy
daily over 22 hours for
5–400 days from
8 weeks old dDoses: 500, 1 000,
3 000 mGy
Dose rate: 7.5 mGy/s d
Mouse

Outcomes recorded,time post-exposure

ANNEX C: BIOLOGICAL MECHANISMS RELEVANT FOR THE INFERENCE OF CANCER RISKS ...

Mouse

Irradiation conditions

129

130

Mouse

C57BL/6J,
7–8 week-old

Mouse

Gamma radiation
Dose: 0.3, 6.0 Gy
Dose rate: 1, 20 mGy/dfor 300 days dGene expression
analysis

FemaleGamma radiation (60Co)
Dose: 20, 100 or
500 mGy
Dose rate: 0.5 or
10 mGy/min d

Challenge dose 1 or
2 Gy, spleen
micronuclei

No protection from any of low doses(BALB/cJ always had more
micronuclei)[B13]

FemaleGamma radiation (60Co)
Dose: 20, 100 mGy d

Response to 2 Gy
challenge

Pre-exposure did not protect
splenocytes or thymocytes from
2 Gy

[B40]

Both or male fortritiated water

Gamma radiation (60Co)
Dose: 20, 100 mGy

Response of
splenocytes to 2 Gy
challenge, 24 hours
post-challenge

Pre-exposure by gamma rays
protected from apoptosis, tritiatedwater (in males) did not[F13]

Tritiated water
Dose: 10 kBq/L, or 1,
20 MBq/L for 1 month f

C57BL/6,
9–10 week-old[I12]

Differentially expressedgene lists[K22]

FemaleTritiated water
Dose: 10 kBq/L–
20 MBq/L f

Spleen cell damage,
micronuclei

No effects on splenocytes[B14]

FemaleGamma radiation
Dose : 50 mGy d

Health status after
2 or 3 Gy proton as
challenge, after
4 or 17 days

Organ weights, RBC and leucocyte
counts – none affected much but if
so – pre-exposure to gamma rays
modulated it[L51]

C57BL/6J, BALB/cJ,
2–5 month-old
Mouse

Reference,comments

FemaleC57BL/6J,
5–6 week-old
Mouse

50 mGy best protection at 6 hours and2 weeks before the challengeRNAs, proteins,metabolite, epigenetics c

Low dose protection
from LD50 5.9 Gy

C57BL/6J and BALB/cJ,
5-month-old

Mouse

Definitive findings

X-radiation
Dose : 50, 200,
400 mGy d

C57Bl/6

Mouse

Outcomes recorded,time post-exposure

Both

C57Bl/6,
4–8 week-old
Mouse

Irradiation conditions

UNSCEAR 2020/2021 REPORTType of animal, strain, age a

Genetic or
chemicalmodulation,
sex bType of animal, strain, age a

Genetic or
chemicalmodulation,
sex b

Irradiation conditions

Gamma radiation (137Cs)
Dose: 400 mGy
Dose rate: 1 mGy/d

Mouse
C57BL/6J, B6C3F1

Outcomes recorded,time post-exposureLife shortening,
neoplasias etc.

Dose: 8 000 mGy
Dose rate: 20 mGy/d e

Metabolomics can be carried out

None; Male

Gamma radiation (137Cs)
Dose : 50, 100,
1 000 mGy d

DNA damage(chromosomal
aberrations), 1 and 4
hours, 1 and 6
monthsNo long-term genomic instability
except 1 Gy, protection by 50 mGy[R6]

4 weeks before
ionizing
radiation

Gamma radiation (137Cs)
Dose: 60, 600 mGy
Dose rate: 1.07,
1.33 mGy/s over 3
weeks eLung cancer
development at
46 weeks

60 mGy alone is protective, increased
adenoma and carcinoma for
combined 60 mGy–benzo[a]pyrene,
600 mGy combined less cancer[B57]

Critical limb
ischaemia;
femaleX-radiation
Dose: 1–7 300 mGy
fractions, from
12 hours post-CLI g

Neovasculature;ischaemia recovery

CD31+ cells increased,
neoangiogenesis only improved by
4×300 mGy

Gamma radiation
Dose: 360, 3 600,
7 200 mGy
Dose rate: 20, 200,
400 mGy/d e

Pregnant mice
exposure form day 0
to day 18 ofgestation

Fetus size, fetal organ sizes, decreased
in all groups

BALB/cJ, C57BL/6J

C57BL/6, EGFR-expressing
C57BL, 22 week-old

Mouse
C57BL/6JJcl females matedwith C3H/HeNJcl males,
6 week-old

Differentially expressed
protein lists[B9]

Benzaldehydes and more

[C6]

ionizing radiation
increased Vegfr2,
Vegfr1, Fgf2, Angpt2,
Pdgfc, Tgfb2, Hgf, and
Met

[M32]

[G19]

ANNEX C: BIOLOGICAL MECHANISMS RELEVANT FOR THE INFERENCE OF CANCER RISKS ...

Metabolomics at 10
and 30 days

C57BL/6J, 6 month-old

Mouse[B53, T3]
2018 paper is areview – little
unpublished data

B6C3F1 females show life shortening
at 400 mGy and 8 000 mGy, males at
8 000 mGy; in C57BL/6J only males
live shorter at 8 000 mGy

600 MeV/n high-LET(16O)
Dose: 100, 250 mGy f

Mouse

A/J

Reference,comments

Proteomics

C57Bl/6J

Mouse

RNAs, proteins,metabolite, epigenetics c

X-radiation
Dose rate:
0.35 Gy/min d

Mouse

Mouse

Definitive findings

131

132

Mouse
BALB/c and SPRET/EiJ mice

Irradiation conditions

Outcomes recorded,time post-exposure

Total body X-irradiation
Dose: 100 mGy dGene expression

Definitive findings

RNAs, proteins,metabolite, epigenetics c

Reference,comments

Differentially expressedgene lists

[T10]

Mouse
Backcross of (BALB/c ×
SPRET/EiJ) × BALB/c

Syngeneic p53−/−
breasttumours,
femaleX-radiation
Dose : 100 mGy d

Pathology oforthotopic breast
cancerDecreased latency when recipient
mice receive 100 mGy

[Z12]

Mouse
BALB/c, C3H/HeN

Female,syngeneic
tumourstransplantation

X-radiation
Dose: 50, 100, 200, 500,
1 500 mGy 6 hours
before tumour
injection dProtection fromgrowth oftransplanted
tumours

Only a trend of protection in BALB/cmice animals exposed to 200 mGy

[I13]

Mouse
BALB/cA, 8 week-old

Syngeneic
tumourstransplantation

Gamma radiation (137Cs)
Dose: 4 000, 8 000 mGyover 200, 400 days e

Tumour cells injected
immediately after
ionizing radiation

Tumours grew faster in 8 000 mGy
recipients

[T2]

Mouse
BALB/cSyngeneic
tumourstransplantation

Alpha, beta and gamma
radiation via substrate
Dose rate: 40–
100 µSv/h e

Tumour cells injected
after 7 week-longexposure

The slowest rate of growth of hindlimb tumours was recorded inanimals maintained on 40 µSv/hsheets[N9]

X-radiation
Dose : 170, 500,
1 000 mGy dBone architecture 3 or
21 days later

Bone marrow cells growth ofadipocyte colonies only 1 Gy;
osteoblastogenesis increased on day
3 in 170 mGy[L30]

X-radiation
Dose: 75 mGy,
72 hours before DOX d

Oxidative stress inheart

Cardiomyocytes health increased, lessapoptosis

X-radiation
Dose : 200, 400 mGy d

Number and health ofthe embryos at days
19 or 18

No significant effects

Gamma radiation (137Cs)
Dose : 100, 1 000 mGy dReporter mice –luciferase increase
to ionizing radiation

Mouse
BALB/cBYJ,
4 month-old
Mouse
BALB/c, 4–6 week-old at
DOX administration
Mouse
CF1 and outbred ICR, 2-hourzygote in utero exposure
Mouse
hCLUp-Luc on FVB/N

Doxorubicin
7.5 mg/kg,
femaleReduced BAX, increased
Bcl2

[J18]

[J7]

TGF-β1 controls
expression fromclusterin promoter[K28]

UNSCEAR 2020/2021 REPORTType of animal, strain, age a

Genetic or
chemicalmodulation,
sex bType of animal, strain, age a

Genetic or
chemicalmodulation,
sex b

Mouse
Kunming, 12 month-old

Irradiation conditions

Outcomes recorded,time post-exposure

X-radiation
Dose : 300, 3 000 mGy d

mRNA, IHC one or two
weeks after

Definitive findings

Increased neural stem cellproliferation and neurogenesis at
300 mGyRNAs, proteins,metabolite, epigenetics c

Reference,comments

Increased number of
Wnt1, Wnt3a, Wnt 5a,and β-catenin[W8]

REPAIR DEFICIENT ANIMALS
Ku80 antisense

Zebrafish
Strain brass, same clutch 6
hour-old

DNA double-strand
break (TUNEL)

Linear increase in DNA double-strand
breaks at ≥10 mGy[B37]

X-radiation
Dose: 100 mGy single
or multiple fractions,or 6 Gy g

DNA damage
evaluated based onfoci, 0.5, 24, or
72 hours after
exposure

Cortical neurons, skin, intestine: ICC
for DNA break foci[S12]

X-radiation
Dose : 1–50 mGy d
SCID

SCID, ATM , ATM(Prkdc ), ATM

Mouse

SCID(Prkdc−/−),
ATM−/−

Lung gamma radiation
Dose: 100 mGy
Dose rate: 2 Gy/min, 5days a week for 2, 4, 6,
8, 10 weeks g

DNA damage in lungcells, 0.5, 24 or 48
hours after exposure

DNA damage decreased with time inwild-type mice (until 50 exposures),in SCID not so much, in ATM
knockout not at all[F15]

LIG4−/−, ATM−/−,
LIG4−/−, ATM−/−

X-radiation
Dose : 50–200 mGy d

DNA damage andapoptosis (DNAdouble-strand breakfoci and TUNEL)
1 hour after
exposureLess repair and more apoptosis inmutant embryos, spontaneously andafter ionizing radiation, spontaneous
damage decreases with age[B15]

SCID(Prkdc−/−),
human CD34+
cells from cord
blood grafted

High-LET 350 MeV/n(28Si)
Dose: 400 mGy

DNA damage incirculating humancells (1.2 ionstraverse each cell)

Chromosomal aberrations: 2x moredamage with
X-rays

−/−

+/−

−/−

SCID, ATM , ATM
−/−

+/−

Mouse
DNA ligase IV , ATM ,double mutants, E13.5
−/−

−/−

Mouse
NSG (NOD.Cg-Prkdc
Il2rgtm1Wjl/SzJ (NSG); 6–
8 week-old at grafting
scid

−/−

X-radiation
Dose: 1 Gy f

ANNEX C: BIOLOGICAL MECHANISMS RELEVANT FOR THE INFERENCE OF CANCER RISKS ...

Mouse

Gamma radiation
Dose : 50–1 500 mGy,

133

134

Mouse

SCID, C.B-17/Icr wild-type
and F1 heterozygotes for
Prkdc+/−
Mouse

Definitive findings

RNAs, proteins,metabolite, epigenetics c

Reference,comments

Gamma radiation (137Cs)
Dose : 50, 100,
1 000 mGy d

DNA damage (FISH) in
bone marrow cells,
1 and 4 hours,
1 and 6 months

No long-term genomic instability up
to 100 mGy based on chromosomal
aberrations[R8]

SCID(Prkdc−/−),
Prkdc+/−,

Gamma radiation (137Cs)
Dose: 50, 100, 250,
500 mGy d

Lymphoma incidence

Thymic lymphoma incidence
unchanged in SCID only at 50 mGy;
in wild type the latency extended at
250 mGy[I11]

SCID(Prkdc−/−)X-radiation
Dose : 300 mGy d

Success of MOLT-4
tumour growth,neovasculature

Tumour growth andneovascularization improved byionizing radiation[S53]

Ercc2+/−

Gamma radiation (60Co)
Dose: 63, 125,
500 mGy dBone marrowisolation at 4 and 24
hours, 2 or
18 months

No chromosomal or telomerechanges. Life expectancy increased
after 63 mGy, decreased at higher
doses

[D1]
study was geared
to study cataracts

FemaleSCID
Mouse

Outcomes recorded,time post-exposure

SCID(Prkdc−/−)

SCID, 10–12 week-old

Mouse

Irradiation conditions

F1 for ERCC2 mutants in CH3
crossed with C57BL/6J;
10 week-old

TUMOUR SUPPRESSOR MALFUNCTIONING ANIMALS
Mouse

p53+/−

X-radiation “CT scanquality”
Dose one or more
10 mGy fractions g

Tumour incidence,
apoptosis

Low dose protection from sarcomas
and lymphomas, extending lifespanin p53+/−; less apoptosis with more
fractions in wild type

[L18, L19, P17]

APC1638N/+ bothsexes

X-radiation
Dose: 250 mGy tosingle or both parents
or grandparents d

Transgenerational
tumour incidence

For males or both grandparents
irradiated, F2 mice had moretumours, in females bothgrandparents irradiated had moretumours[S68]
male F1
heterozygotes had
higher tumour
incidence thanfemaleheterozygotes p53 , 7–8
week-old
+/−

Mouse
APC
male or bothparents or grandparents
irradiated 2 days before
conceiving
1638N/+UNSCEAR 2020/2021 REPORTType of animal, strain, age a

Genetic or
chemicalmodulation,
sex bType of animal, strain, age a

Mouse

Genetic or
chemicalmodulation,
sex b
Ptch +/−

Ptch on background of
C57BL/6JCrl and C3H/He, 1
day-old

Irradiation conditions

Outcomes recorded,time post-exposure

Definitive findings

RNAs, proteins,metabolite, epigenetics c

X-radiation
Dose: 50, 100 mGy
Dose rate:
100 mGy/min

Life shortening, LOH,
microarrays, animals
exposed at day 1
after birth

Radiation induced LOH pattern
unique, at 50 mGy ratio of whatlooks like “spontaneous”: irradiated
LOH is 13:7, at 100 mGy 1:1[I10]

Complex events
related tomedulloblastoma

High dose worse on day E13.5, lowdose worse on day E16.5. Different
repair pathways predominate atdifferent embryo ages

[T12]

Reference,comments

Dose: 200–3 000 mGy
Dose rate:
650 mGy/min d
Mouse

X-radiation in utero
Dose: 250 mGy, 3 Gy at
E13.5 and E16.6 d

ONCOGENE OVER-EXPRESSING ANIMALS
Mouse
CCSP-rtTA/Ki-ras, 8 week-old

Mouse
AKR/J, 7 week-old

Mouse
AKR/J, 8 week-old

Rasoverexpression
in lung
induced by
DOX

X-radiation “CT scanquality”
Dose: 5, 15, 25 mGy
per fraction once aweek for 4 weeks gLung cancer burden

80–160 mGy correlates with increased
number of tumours per mouse but
not their size; no dose dependence
and no tumours if DOX was not used

Murineleukaemia viralgene, female

Gamma radiation (137Cs)
Dose: 2.1, 4.5 Gy
Dose rate: 0.7,
0.8 Gy/min,
respectively, over
130 days e

Progression of thymic
lymphoma

Earliest cases in low dose rate mice,but they still live longest of all

T- and B-cell activation
genes

[S38]

Murineleukaemia viralgene, female

Gamma radiation (137Cs)
Dose rate: 0.7 mGy/hfor 130 days total dose
2.1 Gy or acute e

Gene expression

Most apoptosis in low dose exposed
mice

Increased CDPdiacylglycerol
synthase 1 in low dose
rate mice; integrin-α4,integrin-β1 and Myc inboth groups[B49, B50, B51]

[M46]

ANNEX C: BIOLOGICAL MECHANISMS RELEVANT FOR THE INFERENCE OF CANCER RISKS ...

Ptc1 in CD1 backgroundcrossed with Rad54−/− andDNA-PKcs−/− mice on
C57BL/6 background; inutero exposure+/−

Ptc1+/−, Rad54−/−,
DNA-PKcs−/−

135

136

Mouse
C57B/6 Lck-Bax; Lck-Bax38 &1,
4–9 week-old

Thymocytespecific Bax

Irradiation conditions

Gamma radiation (137Cs)
Dose: 100, 1 000 mGy
Dose rate: 0.35Gy/min

Outcomes recorded,time post-exposure
Lymphoma onset

Definitive findings

RNAs, proteins,metabolite, epigenetics c

Lck-Bax1 shortened latency in females
only; Fe ion exposure shortens
latency

Reference,comments[J6]

Si or Fe ions
Dose : 100, 1 000 Gy
Dose rate: acuteradiation over 1
minute d, f
OTHER TRANSGENIC ANIMALS
Tritiated water
Dose rate: 10 kBq/L, or
1, 20 MBq/L f

LacZ inversions

No changes

Male

X-radiation
Dose : 0.001, 0.005,
0.01, 0.02, 1, 10,
1 000 mGy d

LacZ inversions inprostate 3 days after
ionizing radiation“Triphasic” response – inversions
decreased at 1 and 10 mGy but
increased at doses above and below
these[H35, Z5]

Male

X-radiation
Dose : 0.001, 0.01, 1,
10 mGy d

Challenge doses 4
hours later 1 or 4 Gy

Several dose combinations modulated
inversions[D6, D8]

ApoE−/−; female

Gamma radiation (60Co)
Dose: 25–2 000 mGy
Dose rate: 1,
150 mGy/min dHeart pathology andaorticatherosclerosis

Adaptive response at doses up to
0.5 Gy; increased capillary density

ApoE−/−; female

Gamma radiation (60Co)
Dose: 25–500 mGy
Dose rate: 1,
150 mGy/min d

Pathology of aortic
atherosclerosis

Disease slowed down by 25 or 50 mGy
delivered at 1 mGy/min[M37]

ApoE−/−; male

Gamma radiation (137Cs)
in vitro delivery
Dose: 4, 20, 100 kBq/L f

Pathology of aortic
atherosclerosis

Plaque stability increased,
inflammatory profile reduced

[L8]

Mouse
pKZ1

Tg/+

Mouse
pKZ1

Tg/+

Mouse
pKZ1Tg/+Mouse
ApoE−/− on C57BL/6J;
8 week-old
Mouse
ApoE on C57BL/6J; 2 or
8 month-old
−/−

Mouse
ApoE on C57BL/6J;
7–8 week-old
−/−

[B12]

Decreased inflammatory
vascular markers;changes of collagen IV
and Thy-1 tissue levels[M23]

UNSCEAR 2020/2021 REPORTType of animal, strain, age a

Genetic or
chemicalmodulation,
sex bType of animal, strain, age a

Mouse

Genetic or
chemicalmodulation,
sex b

X-radiation
Dose : 100 mGy d

Pathologies like
Alzheimer’s
monitored

No changes[W2]

FemaleGamma radiation (137Cs)
Dose rate: 0.35,
1.2 mGy/h dLife expectancy

Best improvements in life expectancy
1.2 mGy/h 5 weeks, some with
0.35 mGy/h[I6]

X-radiation
Dose : 500 mGy dControl of arthritis

Apoptosis of synoviocytes, reduced
expression of inflammatory
phenotype[D15]

At time of exposure or experiment initiating age.

b

When nothing is indicated both were used.

c

Indicated if recorded.
Low-dose radiation.
Low-dose-rate radiation.

fInternal emitters or high-LET.

g

Fractionated exposure.

[E1]

WT, or APP ortau; female

Transgenic TNF-αe

Cytokines (Il-4, Il-10, Il-13
and Il-18), catalase and
SODs up-regulated

Macrophages and gene expression
show adaptive response

Mouse

d

Reference,comments

Pathology ofatherosclerosis

MRL-lpr/lpr, 5 week-oldaRNAs, proteins,metabolite, epigenetics c

ANNEX C: BIOLOGICAL MECHANISMS RELEVANT FOR THE INFERENCE OF CANCER RISKS ...

MouseDefinitive findings

Gamma radiation (60Co)
Dose: 69, 161 mGy
Dose rate: 12,
28 µGy/h e

ApoE on C57BL/6J;
8 week-old

C57BL/6J, Tg2576, APP with
the Swedish mutation,
PS19 tau with the P301S
mutation, 10 week-old

Outcomes recorded,time post-exposure

ApoE−/−; male

−/−

Mouse

Irradiation conditions

137

138

UNSCEAR 2020/2021 REPORT

IV. INTEGRATION OF DATA AT DIFFERENT LEVELS OFORGANIZATION AND MODELLING OF CANCER MECHANISMS
411. This section presents mathematical models that were developed to describe different levels oforganization and to analyse mechanisms of carcinogenesis. Articles from 2006 and later were selectedthat the Committee considered informative regarding the effect of ionizing radiation on the process ofcarcinogenesis and its potential consequences. The framework of the models was required to be
sufficiently far developed with a robust scientific basis. The models should only depend on a limited
number of parameters so that the implications would not depend on too many assumptions. The models
should inform about cancer mechanisms that might be generalizable beyond the specific data that they
were applied to. Three areas with different description levels were identified. Firstly, biologically-based
mechanistic models of carcinogenesis were applied to epidemiological cohorts and the effect of exposure
on cellular processes and consequences for radiation risk were investigated (tables 8 and 9). Secondly,models of the bronchial airway were also developed to assess the impact of radon inhalation on cellular
and tissue effects in the lungs. Thirdly, models of intercellular signalling were applied to apoptosis andbystander mechanisms.
412. The UNSCEAR 2000 Report, annex G, “Biological effects of low radiation doses” [U5] included
a section on biologically-based modelling of radiation carcinogenesis. It was concluded that themodelling of epidemiological data tends to favour the view that, in general, cancer risk at low doses rises
as a simple function of dose. The report recognized that the further development and validation of
biologically-based models would have an important role in the future work of the Committee.

A. Biologically-based mechanistic models of carcinogenesis
413. Based on the idea that cancer develops from several successive cellular changes, Armitage and Doll
[A17] developed a mathematical model that was applied to human cancer rates. Using the observations
from Fisher and Hollomon [F12] and Nordling [N18] that cancer rates appeared to increase with about
the sixth power of age, Armitage and Doll proposed a multistage process that included seven stages.
Since initiated (or pre-malignant) cells, i.e. cells in an intermediate stage to cancer, may have a growthadvantage compared to normal cells, in a subsequent publication Armitage and Doll [A18] proposed a
two-stage model in which the cells in the first stage were allowed to grow exponentially and become
malignant after a second mutational event. On these grounds and using the hypothesis by Knudson thatcertain cancers such as retinoblastoma require two mutational events [K30], Moolgavkar and Venzon[M40] developed an entirely stochastic model of carcinogenesis with two stages in which both normal
cells and cells at an intermediate state were allowed to grow, differentiate and die. This model was named
the two-stage clonal expansion (TSCE) model and is an important representative of biologically-basedmodels of carcinogenesis.
414. These types of biologically-based models aim at describing the long-term process of carcinogenesis
by a series of molecular changes in cells towards malignancy that ultimately result in cancer. The complex
process of cancer development is reduced to a few basic steps. The parameters of the models can bederived from fits to epidemiological cohorts and biological input. With the assumption that the modeland its parameters capture underlying long-term carcinogenic processes, the model can then be used toestimate radiation risk for exposures different from the epidemiological study. Historically, the mostfrequently used model is the TSCE model that includes two basic steps from healthy stem cells towards
malignant cells. The first stage, the initiation, involves a transition, induced by genetic or epigenetic

ANNEX C: BIOLOGICAL MECHANISMS RELEVANT FOR THE INFERENCE OF CANCER RISKS ...

139

events, from healthy cells towards initiated cells. These initiated cells have a growth advantage, possibly
related to driver gene mutations, and can clonally expand. This process of clonal expansion is often
referred to as “promotion”. Further mutational events can induce malignant transitions from the initiatedcells towards cancer cells, referred to as “transformation”, that may manifest in a phenomenologicallydetectable cancer after a certain lag time. Extensions of this model include models with several stages, orthe inclusion of additional pathways, see Rühm et al. [R22] for a review on these models.
415. Such mechanistic models provide a general framework for the long-term development ofcarcinogenesis already in the absence of radiation. In such models, radiation can act on existing pathways,
either inducing transitions between different stages, in particular on the initiation or transformation step,or can accelerate clonal expansion. Furthermore, radiation-induced carcinogenesis might proceed along
different pathways compared to spontaneous cancer. By application of these models to radioepidemiological cohorts the type and form of radiation action can be assessed, and the shape of dose
response can be derived. While descriptive models of excess relative risk or models of excess absoluterisk are usually given as a function of total dose, in contrast, mechanistic models depend directly on dose
rate. The model parameters change during exposure and usually return to their spontaneous values after
exposure. The corresponding changes in the incidence/mortality rates and consequences for the dose–
response relationship can then be calculated.
416. Following the pioneering works, the mathematical structure of the models was studied in greater
depth and they were applied to various radio-epidemiological cohorts. Different endpoints after low-LET
radiation were studied in particular for the atomic bombing survivors of Hiroshima and Nagasaki (Life
Span Study cohort). Little [L33] studied 2-, 3- and 4-mutation models for solid cancers and leukaemia[L33]. Cancers of the stomach, colon and lung were analysed with the TSCE model by Kai et al. [K4]with particular focus on the dependence of the excess relative risk with age at exposure and time since
exposure. Different two-stage and multistage models were compared for all solid tumours and several
tumour sites by Heidenreich et al. [H19, H20]. For high-LET radiation, lung cancer after radon exposure
was studied in several cohorts. Luebeck et al. [L46] analysed lung cancer mortality in the Colorado
uranium miners with the TSCE model including smoking information, and found a highly significant
effect of radon on the clonal expansion of initiated cells. Subsequently, Little et al. [L32] extended theanalysis of the Colorado miners to models with three stages. Significant effects of radon on clonal
expansion were also found in analyses of lung cancer with the TSCE model in the Yunnan tin miners byHazelton et al. [H17] and of miners from the Czech Republic, France, China, and Colorado by
Heidenreich et al. [H22]. Furthermore, mechanistic models were used to study liver cancer in thorotrast
patients [H21, L16] and bone and head sinus cancers in radium dial painters [L15]. Additional
background information can be found in Little [L36] and Rühm et al. [R22].
417. The following discussion describes the efforts since 2006 to extend the range of applications of thebiologically-based models and to strengthen their link to biological processes. For low-LET radiation,
several studies were performed for the atomic bombing survivors of Hiroshima and Nagasaki. Further
investigations were carried out for occupationally, environmentally or medically exposed persons, oftenwith protracted low-dose-rate exposures. For high-LET radiation, a number of studies on lung cancer
after radon exposure among miners and on lung cancer after plutonium exposure in the Mayak workers
were performed. Tables 8 and 9 provide a summary of the findings. The mechanistic models were also
applied to non-radiation data, such as the Surveillance, Epidemiology and End Results registry, with theaim to gain more insight on the underlying processes and model structure, in particular on biologicaleffects such as genomic instability. This can improve the basis for implementation of radiation effects inthese models. Additional mechanistic studies also included analyses of animal data.

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1. Application of mechanistic models of carcinogenesis to low- linear
energy transfer radio-epidemiological cohorts
418. Incidence of cancer in the atomic bombing survivors has been analysed by Heidenreich et al. with
the TSCE model for stomach, colon, liver, lung and all solid tumours and for a collection of nine tumoursites with a follow-up period of 1958–1997 [H24]. Radiation was allowed to act either as initiating eventor on clonal expansion. The age-time patterns of risk suggested that both mechanisms play a role inradiation-induced carcinogenesis. Assuming that additional time-limiting steps were needed to obtain
intermediate cells with a growth advantage did not improve the quality of fit.
419. Jacob et al. [J3] analysed the Life Span Study mortality data for all solid, stomach and liver cancers
with a follow-up from 1950 to 2000. In addition to the effect of radiation on the mutation rates theinfluence of radiation-induced cell inactivation was studied. Cell survival data for liver cells, stomach
cells and a mixture of cells from different organs together with experimental data on low-dose
hypersensitivity were implemented in their TSCE model. A significant effect of radiation on the initiation
rate, i.e. the transition rate from a healthy cell towards a pre-malignant cell, was found for all endpoints.
Additional effects of radiation on the clonal expansion rate and the transformation rate towards malignant
cells were seen. Without consideration of radiation-induced cell inactivation the TSCE model would
predict a linear dose–response curve. However, if radiation-induced hyper-sensitivity influenced cell
inactivation then the initial slope at low doses of the resulting dose–response curve was steeper than athigh doses.
420. Shuryak et al. [S42, S43] have described cancer development involving a short- and long-term
component. The authors developed a model framework to describe the processes on both time scales
independently, and to integrate both formalisms. The short-term part of the model considered initiation,
inactivation, and repopulation of normal and pre-malignant stem cells during the radiation regimen anda recovery period of about a month. Niche takeover by pre-malignant cells was also considered a shortterm process. These short-term processes were assumed to be essentially completed by a few months
following exposure, before the long-term processes of spontaneous initiation in the absence of radiationand niche replication, which operate on the time scale of multiple years, start to have an appreciable
effect. Cancer incidence was assumed to be proportional to the total number of pre-malignant cells in all
niches, shifted by a lag time. These models were applied to the example of radiotherapy, to estimate risk
for nine solid cancer types using data on radiotherapy-induced second malignancies, on Japanese atomic
bombing survivors, and on background United States cancer incidence.
421. While excess relative risk for solid cancers in the Life Span Study decreased with increasing age at
exposure during childhood, Shuryak et al. [S44] observed that this pattern seemed to be different for
exposures during adult ages with constant or even increasing risk. Using data of excess relative risk fromthe atomic bombing survivors 1958–1998 of all solid, liver, colon, lung, breast, stomach, and bladdercancer, the authors applied the biologically-based model of radiation carcinogenesis derived in Shuryak
et al. [S43]. With the aim to assess whether such a potential age at exposure dependence would bebiologically plausible, radiation was allowed to act both on the initiation stage and on clonal expansion.
The authors concluded that the observed patterns of radiation induced cancer risks as a function of age at
exposure were not consistent with models of radiation carcinogenesis in which radiation solely initiatedpre-malignant cells but were consistent with models of radiation carcinogenesis that included bothradiation-induced initiation and clonal expansion.
422. Breast cancer incidence in the Japanese atomic bombing survivors was studied by Kaiser et al. [K5]with the TSCE model. The follow-up included the years 1958–1998 and 1,038 breast cancer cases.
Radiation was assumed to act linearly with dose on the initiation, clonal expansion and transformation
stages of the TSCE model. Three different models were found to describe the data similarly well. The

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first model included an effect of radiation on the initiation rate, the second a permanent increase of theclonal expansion rate, and the third a permanent simultaneous increase of the mutation rates for initiationand transformation. Permanent radiation enhancements of the mutation rates or the clonal expansion are
indications for long-lasting radiation effects, e.g. in the case of radiation-induced genomic instability.
Risk for women at age 70 years, exposed at age 30 years, was estimated to 10 (90% CI: 8.2, 13) cases
per 10,000 person-years at 1 Gy for the excess absolute risk, and 1.2 (90% CI: 0.72, 1.9) at 1 Gy for theexcess relative risk.
423. Colon cancer proceeds along two principal pathways. The majority of colon cancers show
chromosomal instability related to defects in the tumour suppressor gene APC. The second pathway isrelated to microsatellite instability, e.g. induced by defects in DNA mismatch repair genes such as MLH1.
Kaiser et al. [K6] developed a mechanistic model including the two pathways, and applied it to incidence
data 1958–1998 of the atomic bombing survivors of Hiroshima and Nagasaki with 1,508 colon cancer
cases. Biological information was used to estimate several model parameters, e.g. the number of stem
cells in the colon or cell division rates in adenoma and early carcinoma. In their preferred model, ionizing
radiation acted on the initiation rate among both sexes, in both pathways, while in addition it acted on theclonal expansion rate of chromosomal instability cells among males, in the chromosomal instability
pathway. Hence radiation acted by enhancing APC mutations, MLH1 methylation or suppression of cell
death. The latter effect might be caused by inflammation. The model allowed the prediction of the shareof microsatellite instability and chromosomal instability-type tumours for the Life Span Study coloncancers as a function of attained age. In the future, if molecular measurements from the Life Span Studycolon cancer cases were available these models predictions could be validated. On average, the model
predicted a share of microsatellite instability-type tumours of 25%, which is remarkably close to the shareof 15–20% reported for the clinically observed colorectal tumours in molecular studies of American coloncancer patients (from TCGA, the Cancer Genome Atlas).
424. Hazelton et al. studied lung cancer incidence among Canadian radiation workers with individual
annual dosimetry for protracted low-dose occupational gamma and tritium radiation exposure [H18].
Within the follow-up time 1969–1988, 322 male lung cancer cases were observed among 95,439 males.
The TSCE model and extensions of the model with up to 10 initiation steps were applied to the data.
Dose response to initiation, clonal expansion or malignant transformation in the TSCE model wasparametrized as a power law. In the TSCE model, dose response was significant for both clonal expansion
and malignant transformation, while it was only insignificant for initiation. The dose response of excess
relative risk had a downward curvature with an inverse dose-rate effect. For the multistage models, thebest fit was found for three initiation stages. However, clonal expansion was found to be highly significant
in all of these models.
425. All solid cancer incidence and mortality in the Techa River cohort was analysed with the TSCE
model by Eidemüller et al. [E11, E14]. In total, 1,889 incident solid cancers in the period 1956–2003
were observed among 17,069 persons, and 2,064 solid cancer deaths among 29,771 individuals for theperiod 1950–2003. For the incidence data, it turned out that when changes in the investigated rates were
only allowed during radiation exposure, initiation was the only process that was affected while theprocesses of clonal expansion and transformation were not affected. When a permanent effect of radiation
exposure on clonal expansion was added the fit to the incidence data was significantly improved. For the
mortality data, again a direct effect on initiation and a permanent effect on clonal expansion werepreferred, while a model with a smaller direct effect on initiation before a critical age of 24 years and astronger direct effect on initiation at older ages described the data only slightly worse. The analysis
showed a significant effect of ionizing radiation on the initiation stage, and good indication for an
additional effect on clonal expansion. However, no radiation effect on the transformation stage was seen.

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UNSCEAR 2020/2021 REPORT426. Breast cancer incidence from 17,200 female patients of the Swedish haemangioma cohort was
analysed by Eidemüller et al. [E13]. Exposure was at infancy mainly by 226Ra applicators. Within the
period 1958–2009, a total of 877 breast cancer cases was observed. Based on previous work [E10], theauthors had indications that radiation-induced cancer proceeded along a different pathway thanspontaneous cancer, and that this pathway hinted to long-lasting radiation-induced effects such as
genomic instability. Therefore, a structurally more advanced model was developed with a separate
pathway possibly related to genomic instability. The analysis showed that the two-pathway model was asignificant improvement compared to the TSCE model. Radiation was also found to induce the second
pathway with a linear dose–response relationship. Using biologically plausible assumptions on someparameters, the authors derived relationships between the spontaneous pathway and the one possibly
related to radiation-induced genomic instability. The speed of cancer development was increased in theradiation-induced genomic instability path and the model suggested that in particular the rate of mutations
from non-initiated cells towards initiated cells with a growth advantage was substantially elevated.
427. A number of studies provided evidence that overexpression of the CLIP2 gene on transcriptomic
and proteomic levels in tissue of PTC from patients undergoing surgery before 20 years of age served asa marker for the radiation-induced origin of the disease [H27, S23, S24]. To integrate the CLIP2 findings
into radio-epidemiological risk assessment, Kaiser et al. [K7] developed a model for two molecularpathways pertaining either to sporadic PTC development or to radiation-induced pathogenesis whichfeatures CLIP2 overexpression as a radiation marker. The model was applied to the Ukrainian-American(UkrAm) cohort of 13,183 subjects with 115 PTC who were exposed as children and adolescents toionizing radiation from ingested 131I after the Chernobyl accident. The authors showed that the dose
response for the probability of finding the CLIP2 marker in PTC tissue is directly related to the radiation
risk of the UkrAm cohort. Furthermore, the mechanistic model was applied to explore the impact ofradiation on molecular alterations such as mutations, genomic copy number alterations or altered mRNAand protein expression. The authors provided a comprehensive characterization of PTC carcinogenesis
for a number of clinical and molecular properties.
428. The effect of smoking and radiation on molecular pathways of lung adenocarcinoma was studied
by Castelletti et al. [C8]. To obtain a model structure reflecting the main pathways towards lungadenocarcinoma, the authors analysed molecular profiles from patients of the United States and China.
Grouping the clinical data by driver mutations revealed two main distinct molecular pathways to lungadenocarcinoma. The two-path model was applied to incidence data of Japanese atomic bombing
survivors that contain information on radiation and smoking but lack molecular genotyping of lung cancer
tissue. Consistent with the model structure, the best fit of the incidence data was achieved with a model
containing two pathways: one unique to transmembrane receptor-mutant patients that displayed publishedsignatures of radiation exposure and one shared between submembrane transducer-mutant patients andpatients with no evident driver mutation that carried the signature of smoking. For both agents, radiationand smoking, net clonal expansion was identified as the main biological target. The derived modelstrongly suggested that smoking and radiation in the development of lung adenocarcinoma act on
different pathways without noticeable synergy. These findings point to additive absolute risks of radiationand smoking with little biological interaction.

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Table 8. Mechanistic models applied to low-linear energy transfer studies
Cohort(low-LET)

Endpoint

Model

Analysis/Findings

StudyLife Span Study

Incidence 1958–1998 of
stomach, colon, liver, lungand all solid tumours andfor a collection of ninetumour sites

TSCE

Evidence for radiation action
both on initiation and clonal
expansion[H24]

Life Span Study

Mortality 1950–2000 for all
solid, stomach and livercancerTSCE

Modelling of cell inactivationin clonal expansion andconsequences for dose–
response relationship[J3]Life Span Study

Incidence 1958–1998 of
stomach, lung, colon,
rectal, pancreatic,
bladder, breast, central
nervous system andthyroid cancer

Short- and longterm model

Combining short-term
radiation effects with longterm cancer development[S42, S43]

Life Span Study

Incidence 1958–1998 of allsolid, liver, colon, lung,breast, stomach andbladder cancer

Biologicallybased
initiationpromotion
model

Risk assessment for exposureat middle ages

[S44]

Life Span Study

Incidence 1958–1998 ofbreast cancer

TSCE

Combination of models withdifferent radiation targets
using multi-model inference[K5]Life Span Study

Incidence 1958–1998 ofcolon cancer

Two-path model
adapted tomolecular
pathways

Separate models for two
molecular pathways ofchromosomal instability andmicrosatellite instability,
evidence for epigenetic
radiation action(hypermethylation)[K6]

Life Span Study

Incidence 1958–1999 oflung adenocarcinoma

Two-path model
adapted tomolecular
pathways

Distinct molecular pathways
for radiation and smoking.
Both radiation and smoking
affect clonal expansion[C8]

Canadianradiation
workers

Incidence 1969–1988 oflung cancer

TSCE andmultistage

Radiation effect on clonal
expansion and malignant
transformation[H18]

Techa River
population

Incidence and mortality
1956 (1950)–2003 of allsolid cancer

TSCE

Radiation effect on initiation,evidence of additional longterm effect on clonal
expansion[E11]

Swedishhaemangioma

Incidence 1958–2009 ofbreast cancer

Two-path model

Evidence for radiationinduced transition towards
genomic instability[E13]

UkrainianAmerican

Incidence 1998–2008 of
papillary thyroid cancer

Two-path model
includingbiomarker
statusIntegration of a radiation
biomarker into amechanistic model and risk
analysis

[K7]

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2. Application of mechanistic models of carcinogenesis to high-linear
energy transfer radio-epidemiological cohorts429. Jacob et al. [J2] used the TSCE model to analyse lung cancer mortality among the Mayak workers
from plutonium incorporation and external exposure [J2]. In the period 1948–2002 a total of 301 lungcancer deaths were observed among 6,293 male workers. In a previous analysis of this cohort thepreferred TSCE model allowed for a dependence of the clonal expansion rate both on smoking status andinternal plutonium dose, while external exposure modified the initiation step [J5]. Based on these results,
the authors studied the Mayak data with TSCE models that included smoking behaviour and a potential
radiation-induced bystander effect. The bystander effect was assumed to influence the initiation rate of
the model in a non-linear way. Under this assumption, the data were found to be incompatible with adetrimental bystander effect. Models with a protective bystander effect did not improve the quality of fit
compared to models without a bystander effect. Smoking was found to act on the clonal expansion rate.
The best models included a radiation action both on initiation and clonal expansion. While the radiation
action on initiation was linear, it acted on clonal expansion in a non-linear way with a levelling at large
plutonium exposure rate. The preferred TSCE model was sub-multiplicative in the risks due to smoking
and internal radiation, and more than additive. In a subsequent analysis with an updated data set for the
male Mayak workers, the authors applied a TSCE model that included a dose threshold for the bystander
effect [J4]. The preferred model suggested an effect of plutonium exposure with a dose threshold both on
initiation and clonal expansion.
430. Lung cancer mortality from radon exposure in the Wismut cohort was analysed with the two-mutation
carcinogenesis model by van Dillen et al. [V2]. The cohort comprised 35,084 workers among whom 461
died from lung cancer in the follow-up period 1955–1998. The two-mutation carcinogenesis model isstructurally similar to the TSCE model with two stages and a net clonal expansion of intermediate cells.
Assuming that radiation can only affect initiation or transformation, but not the net clonal expansion rate,
the authors found a significant cell killing contribution in the second mutation rate and a large value of thelag time of 13–14 years from a malignant cell to an observable tumour. To estimate the effect of missing
smoking information in this cohort, van Dillen et al. [V3] applied an imputation technique for smoking
based on Monte Carlo resampling. Smoking data from a case-control study were randomly projected onto
the corresponding cohort. A sensitivity analysis based on a set of 200 independent projections withsubsequent model analyses yielded narrow distributions of the free model parameters, indicating thatparameter values were relatively stable and independent of individual projections.
431. The TSCE model was used by Heidenreich et al. [H25] to analyse lung cancer mortality of European
miners from the Czech Republic, France and Germany from exposure to radon and its progeny. All threecohorts indicated a highly significant action of the alpha particles on clonal expansion. An initiating
action of the radiation only played a minor role and was not significant in the French cohort. A radiation
action on the transformation stage was tested but was not found to be significant. The pattern was notchanged when exposure uncertainties were included.
432. Lung cancer mortality from radon exposure was studied by Eidemüller et al. [E12] for the Eldorado
miners. The cohort included 16,236 male uranium miners and mill workers. In the follow-up period 1950–
1999 a total of 618 lung cancer deaths were observed. The model showed a strong action of the alpharadiation from radon progeny on the clonal expansion rate. The best fit of the TSCE model resulted in astrong increase of the clonal expansion rate until an exposure rate of about 20 working level month(WLM) per year, while this increase became smaller but still depended linearly on exposure rate, above
20 WLM per year. The authors speculated that this behaviour could be due to a bystander effect in whichat exposure rates lower than 20 WLM per year more cells than just those hit by alpha particles are affected

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by the radiation, while at higher exposure rates this effect saturates, and most of the observed effect isdue to directly hit cells.
433. The Mayak workers were investigated by Zöllner et al. to study lung cancer mortality fromplutonium exposure [Z21]. Three hundred eighty-eight lung cancer deaths among 8,604 males occurred
within the follow-up period 1948–2008. Only workers with full information on internal dose were
included. Plutonium remains in the lung and has a long physical half-life, and exposure conditions are
therefore different from radon. Information on smoking status and alcohol status was available for all
individuals in the cohort. The analysis was performed both with a standard mechanistic TSCE model,
and with more complex three-stage models with two phases of clonal expansion. For the TSCE model,
the alpha exposure from plutonium increased the rate of clonal expansion. The change of the clonal
expansion rate was linear at low dose rates and saturated at higher annual dose rates. On the other hand,no effect of radiation on initiation or malignant transformation was found. Similar to radiation, smoking
affected only the clonal expansion process. Among the possible three-stage models, three models were
found to provide a plausible fit to the data. In all of these models a highly significant effect of radiationon the clonal expansion phases was seen. Furthermore, in all models the clonal expansion rates showeda linear increase at lower dose rates with a levelling at higher dose rates. It was not possible to find amodel where radiation affected only the transition rates between the different stages. Depending on themodel, smoking could affect either clonal expansion or the mutation rates. Consequences of the models
for the shape of dose response were investigated. The preferred three-stage model predicted that at low
doses the dose response curve started linearly with an initial slope considerably flatter than predicted by
the TSCE model, then increased exponentially with dose and steeper than predicted by the TSCE model,
and finally levelled off. The dose response curves of the two other three-stage models were compatible
with the dose response of the preferred three-stage model. The findings give strong indications that the
influence of plutonium on the carcinogenic process is closely related to clonal expansion.
434. The TSCE model was used by Zaballa and Eidemuller [Z1] to study lung cancer mortality fromradon exposure among 58,695 male workers employed by the Wismut company in Germany for uranium
mining [Z1]. In the follow-up period 1946–2003, a total of 2,996 lung cancer deaths were observed.
Adjustment for exposure to silica dust was performed. The best model included a highly significant effectof exposure on the clonal expansion rate. The rate increased linearly with annual exposure at low dose
rates, and then saturated at higher exposure rates of about 100 WLM per year. No indication for additionalradiation effects on the initiation or malignant transformation rate was found.

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Table 9. Mechanistic models applied to high-linear energy transfer studiesaCohort(high-LET)

Endpoint

Model

Analysis/Findings

Study

Mayak workers

Lung cancer
mortality, 1948–
2002 (Plutonium)

TSCE

Modelling bystander effect on
mutation rates. Interaction ofsmoking and plutonium exposure[J2]

Mayak workers

Lung cancer
mortality, 1948–
2004 (Plutonium)

TSCE

Model with bystander effect and dosethreshold for plutonium exposure[J2]

Wismut miners

Lung cancer
mortality, 1955–
1998 (Radon)

Two-mutation
carcinogenesis

Radiation action on mutation rates
with cell killing contribution.
Radiation action on clonal
expansion not investigated[V2, V3]

Miners from theCzech Republic,
France andGermany

Lung cancer
mortality(Radon)TSCE

In all cohorts strongest effect of radon
exposure on clonal expansion withlinear-levelling. a Additional effecton initiation. Inclusion of
measurement errors[H25]

Eldorado miners

Lung cancer
mortality, 1950–
1999 (Radon)

TSCE

Radiation action on clonal expansion
with stronger increase for lower
exposure rates, and smaller increasefor higher exposure rates[E12]

Mayak workers

Lung cancer
mortality, 1948–
2008 (Plutonium)

TSCE and threestage model

Radiation action on clonal expansion
with linear-levelling a both in twostage and three-stage models[Z21]

Wismut miners

Lung cancer
mortality, 1946–
2003 (Radon)

TSCE

Radiation action on clonal expansion
with linear-levelling. a Correction forsilica exposure[Z1]

Linear-levelling: Clonal expansion rate increases linearly at lower exposure rate, and levels off at higher exposure rate.

3. Application of mechanistic models of carcinogenesis to
non-radiation epidemiological data
435. To gain more insight into the underlying model structure and implement biological effects, more
complex mechanistic models including destabilizing mutations were developed. Based on previous work
[L35], Little et al. set up a general model framework with a potentially arbitrary number of carcinogenic
mutations [L34, L35]. Each stage corresponds to increasingly carcinogenic cells with a higher number ofgenetic or epigenetic mutations. These pre-malignant cells can clonally expand. Finally, a pre-malignant
cell can acquire a malignant transformation towards a malignant cell that ultimately results in cancer. In
addition, healthy or pre-malignant cells can experience one or several destabilizing mutations towards
genomic instability. These models were applied to colon cancer incidence data for the period 1973–2002
from the Surveillance, Epidemiology and End Results registry [L34, L35]. Several models described thecolon cancer incidence as a function of age rather well. The rates of carcinogenic mutations in thepathways related to genomic instability were several orders of magnitude higher than in the pathway
without destabilizing events. It was commented that without further biological information, and only
based on age-incidence data, it is difficult to single out a specific model for genomic instability in coloncancer carcinogenesis.

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436. Multistage clonal expansion models with several carcinogenic mutations, but without separate
pathways for genomic instability, were applied to spontaneous incidence of four gastrointestinal
malignancies [L47]. The Surveillance, Epidemiology and End Results registries for the period 1975–
2008 were used to describe colorectal cancer, gastric cancer, pancreatic cancer, and oesophageal
adenocarcinomas. The analysis found a phase that increased exponentially with age up to an age of about
60 years, and a second phase that increased linearly at older ages. The model included an initiation step
on normal tissue stem cells involving a two-hit process and leading to pre-malignant progenitor cells.
These two hits may involve inactivation of tumour suppressor genes such as APC in the case of colorectal
cancer. The authors were able to derive different time scales of the carcinogenic process: The mean timerequired for a pre-malignant cell until occurrence of the first malignant cell (between 15.0 and
53.2 years), the mean time from a pre-malignant cell to appearance of the first malignant cell of asurviving malignant clone (between 37.9 and 58.6 years), and the mean time of persistent preclinicalcancers from the first malignant cell to time of cancer diagnosis (between 0.6 and 12.2 years).

4.

Further mechanistic studies and studies including animal data

437. While cancer risk at high and medium doses can be estimated from radio-epidemiological cohorts,
cancer risk estimates at low and very low doses rely on extrapolations. Brenner [B54] used biophysicalarguments to discuss the plausibility for a linear non-threshold extrapolation from low (about 10 mGy)
to very low doses (about or less than 1 mGy) [B54]. At doses of about 10 mGy each cell is traversed by
one or only a few radiation tracks. Reducing the dose, the number of traversed cells decreases linearlywith dose, however, the induced physical damage of each traversed cell would not change. This argument
suggests a biophysically-based rationale for a linear non-threshold extrapolation from low to very low
doses. Limitations to this biophysical argument include possible confounding effects of intercellularcommunication, potential differential effects of immunosurveillance when the number of (pre)malignant
cells is low, the possible impacts of stem cell competition, and the possibility of different types of
biological damage, and damage responses, dominating at very low doses, compared to low doses.
438. Heidenreich et al. [H23] determined doubling dose rates for lung cancer induction in male andfemale wild-type Janus mice for acute and fractionated gamma and fission neutron exposure. The model
considered initiation and promotion and included the notion that relative risk mounts gradually until
irradiations are completed for protracted exposures while it only drops off for acute exposures. Lungcancer doubling dose rate for males exposed to gamma rays was 13 mGy per day, and for neutrons
1.4 mGy per day; equivalent values for females were 7.1 mGy per day for gamma rays and 0.53 mGy/dfor neutrons.
439. Risk extrapolation based on animal data that directly compares exposures of interest can be used toevaluate the robustness of models used. For example, direct comparison of life expectancy for mice
exposed to acute or protracted external beam radiation with total doses below 1.5 Gy showed that suchcomparisons have to be made in order to evaluate how risk is modulated by dose fractionation.
Conversely, following the BEIR VII approach 1 and extrapolating risk from data only for acute exposureslead to overconfident estimates that became broader with inclusion of comparisons between experimental
data [H7].

1

Biological effects of Ionizing radiation, the BEIR VII report (see https://www.nap.edu/catalog/11340/health-risks-from-exposure-to-low-levels-of-ionizing-radiation

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UNSCEAR 2020/2021 REPORT440. To assess bone cancer mortality from 239Pu, Bijwaard and Dekkers [B35] combined studies of 239Pu
and 226Ra exposed animal data with radium dial painter data. They applied the same two-mutation
carcinogenesis model on the different data sets with the aim to find similarities in the risk transfer fromradium to plutonium. The animal data consisted of mouse, rat and beagle data sets with information on
exposure and the occurrence of fatal osteosarcoma. It was assumed that radiation could only affect initiation
or transformation, but not the net clonal expansion rate. While the parameter sets varied among the different
studies, the ratio of the strength of the radiation action between plutonium and radium was similar acrossthe studies with a factor of about eight. Using this factor, and applying the two-mutation carcinogenesis
model to the radium dial painters, the authors were able to suggest a model that could predict bone cancer
mortality from plutonium in humans that was in good agreement with published risk estimates based onother epidemiological and animal data. The authors argued that the higher toxicity of 239Pu compared to226
Ra might come from different metabolic behaviour of the radionuclides. Whereas radium diffuses in thebone volume, plutonium remains at the bone surface where the sensitive cells are located.
441. Dekkers et al. [D14] applied the two-mutation carcinogenesis model to radiation-induced acute
myeloid leukaemia mouse data [D14]. From experimental evidence that radiation-induced murine acute
myeloid leukaemia develops through two key steps that involve a deletion of one copy of the Sfpi1 gene,
followed by a mutation in the remaining copy, the authors adjusted the two-mutation carcinogenesis
model to several data sets of X-rays- and fission neutron-exposed CBA/H mice. All mice underwent
single acute exposure at young age. The model allowed for a radiation effect on the first or second
mutation rate, but not on net clonal expansion. Since the exposure was at young age where only a small
number of premalignant cells are present, no radiation effect on the second mutation could be determined.
The dose response at the first mutation rate was linear with a potential cell-killing effect at higher doses.
The obtained models provided good fits to the data. A comparison between the predictions for neutroninduced and X-ray-induced first mutation rates yielded a relative biological effectiveness for 1 MeV
fission neutrons of approximately three. The authors noted that the model could be considered as a firststep towards a model for human radiation-induced acute myeloid leukaemia.
442. Based on observations that more-rapidly renewing tissues could be at higher cancer risk because of
the larger number of cell divisions and consequently mutations [T17], Little and Hendry [L38] developed
generalizations of three recent cancer models that account for the joint maintenance and renewal of stemand transit cells. A particular focus of these mathematical models was the assessment of the probabilityof cancer associated with radiation or smoking. All three models demonstrated substantial variation incancer risks, spanning over 20 orders of magnitude. However, it was shown that if cancer occurred thechance that it was caused by a dominant mutagenic exposure was within an order of magnitude. In thesemodels few cancers arose from mutations occurring solely in stem cells rather than in a combination ofstem and transit cells. However, for cancers arising from 2–3 mutations, many had at least one mutation
derived from a mutated stem cell. The analysis suggested that the probability of a cancer being mutageninduced correlates significantly with the cumulative number of stem cell divisions.

5. Summary and conclusions
443. Mechanistic models of carcinogenesis provide a way to integrate biological information into cancer
risk assessment. While the structure of the models used so far is relatively simple compared to thecomplex process of cancer development, the models aim at describing essential rate-limiting steps. Thesemight be related to driver gene mutations. Most analyses were performed with the TSCE model including
two stages. However, several recent studies used more complex models, including three or more stages,
or additional pathways. The use of more complex models involves a trade-off between more realistic
descriptions of cancer development and parameter identifiability.

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444. The mechanisms of radiation-induced carcinogenesis are reflected in the age and dose dependenciesof the epidemiologically observed cancer incidence rates. For example, radiation action on initiation,
clonal expansion or transformation leads to different age and time-since-exposure dependencies ofradiation risk. The mechanistic models can therefore be used to generate and test hypotheses on the typeand form of radiation action. The predictive power of the models improves with increasing biologicalknowledge on the underlying model structure and model parameters. Nevertheless, such model studies
can only test whether the underlying assumptions on radiation carcinogenesis are compatible with theepidemiological data. They do not provide a proof that the assumed biological processes are correct[R22]. However, if the outcomes of a model study that has been carried out correctly are incompatible
with data, this does provide proof that the assumptions in the model are incorrect.
445. While the structures of mechanistic models are, in principle, informed by biological processes, thedirect identification of the model stages in experiments is a difficult task. Most data are derived fromcancer tissue which represents the final stage of the models. So far, little experimental data exist for the
pre-cancer stages from radiobiological cohorts. For example, for thyroid cancer it was possible to directly
include a radiation biomarker into radiation risk assessment [K7]. For characterization of the key eventsand estimation of the magnitude of the model parameters also studies of non-irradiated persons can beused. Further data from radiation effects on human, animal, and organoid culture systems might then be
used to refine the models and assess the impact of radiation.
446. Low-LET studies included the Life Span Study as well as several other cohorts for different
endpoints of solid cancer. Almost all studies found evidence for a radiation action on either initiation,
clonal expansion or both initiation and clonal expansion. A radiation effect on the transformation stage
seemed to be only of minor relevance. Recent studies extended the TSCE model towards models withdifferent pathways, and the effect of radiation on these pathways was analysed. Such pathways could be
related to genomic instability. A number of studies using biologically-based models, e.g. those used by
Kaiser et al. [K5, K6] and Castelletti et al. [C8], report reduced cell inactivation as the effective
mechanism to stimulate clonal growth of pre-neoplastic lesions. This mechanism has been detected
recently in the oesophagus of irradiated mice where p53-mutant cells could clonally expand due toresistance to radiation-induced oxidative stress [F10].
447. Lung cancer studies from high-LET radiation were mainly based on miners exposed to radon. In
addition, lung cancer from plutonium inhalation was analysed. Plutonium has further potential
chemotoxical and biological effects, however, such effects could not be taken into account. In almost all
studies, the radiation action showed a remarkable consistency and was different compared to the lowLET studies. It acted dominantly on the clonal expansion, both for radon and plutonium exposure.
Furthermore, the dose response of the clonal expansion rate was strongly non-linear. At low exposuresthe clonal expansion rate showed a steep linear increase with exposure rate, while for higher exposure
rates it either increased with a smaller slope or levelled off. Radiation-induced bystander effect andinflammation have been suggested as cause for such kind of dose response. In the mechanistic model
framework, the complex dose and age dependencies observed in miner studies, i.e. the inverse dose-rate
effect and the dependence of risk on time since exposure and attained age, are a consequence of this
distinctive form of radiation action. Since no residential cohorts were analysed with mechanistic models
it is not possible to compare the model structure and dose response directly to studies with low-dose highLET exposures.
448. In most studies a fixed lag time between the appearance of the first malignant cell and clinicalsymptomatic cancer was used. However, the progression from a potentially malignant cell towards cancer
is a complicated biological process. It includes the development of dormant tumours that may remain
dormant, become extinct, or continue with invasive growth after some delay time. The immune system
can play an important role in this process. Implementing such mechanisms, the modelling study by Fakir

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UNSCEAR 2020/2021 REPORTet al. [F5] found a substantially increased lag time with large variations. Currently it is unclear if suchmechanisms would lead to modifications of the conclusions drawn from the mechanistic models, sincethese effects can be partially compensated by higher initiation rates to account for the loss of malignant
cells. A detailed biological understanding is necessary to provide a more robust theoretical framework.
449. The mechanistic models are based on parameters that describe key steps in the carcinogenic process.
Advances in omics technologies provide new possibilities to link these parameters to molecularmeasurements. For example, using two whole-exome sequencing data sets for lung adenocarcinoma andcolorectal cancers, Tomasetti et al. [T16] analysed the number of driver gene mutations as rate-limiting
steps necessary for cancer development. It was inferred that only a relatively small number of driver genemutations appeared to be required for the development of advanced cancers of the lung and colon. In bothcases the most likely number of required mutations was three. The effect of ionizing radiation on key
pathways for the acquisition of tumour hallmark capabilities should be identified and quantified. Resultsof network analysis could support model design and development. While tissue microenvironment plays
an important role in carcinogenesis, this has not been well described by mechanistic models andconstitutes an important area of future research. An integration with other modelling approaches atvarious levels of disease development, and the integration with molecular data from radioepidemiological cohorts would represent an important step forward.
450. An added value of these models is that they provide a way to translate radiation-induced cancer
dynamics into risk assessment. If the assumed dynamics correlates with the real biological processes, themodels can suggest ways to extrapolate risk observed in an epidemiological cohort with specific exposure
conditions to different exposure situations, e.g. towards lower doses, for protracted exposures or fordifferent ages at exposure and attained age. The studies have demonstrated that potential non-targeted
effects such as radiation-induced genomic instability or bystander effect can affect the age and dose
dependencies of risk. Improved understanding of these non-targeted effects, combined with an integration
into mechanistic models, might add new insights into radiation-induced health consequences.
451. While the mechanistic models do not allow conclusive results on the shape of dose response, some
general observations can be drawn from the current evidence. For low-LET, the majority of studies founda radiation action on initiation or on clonal expansion consistent with a linear dependence on dose rate,though there are limitations in terms of the data available at dose rates of 1 mGy/h and above with uniform
irradiation. This is compatible with a linear dependence of risk at low total doses, however, different
forms of dose response cannot be excluded. Noteworthy, radiation action on initiation or clonal expansion
leads to different temporal risk dependencies. For initiation, radiation risk would increase only after arelatively long lag time but then remains elevated during lifetime. Radiation risk that originates from anincrease in clonal expansion would start earlier but become smaller with increasing time since exposure
or attained age. Little evidence was seen for radiation acting on the last transformation stage towards amalignant cell. The latter effect would manifest in a peak of radiation risk relatively short after exposure.
For high-LET, lung cancer risk of miners after radon exposure depends in a complex way on attained
age, age at exposure and time since exposure. Furthermore, the risk depends inversely on exposure rate.
Within the mechanistic models, these effects result from the specific non-linear dependence of the clonal
expansion rate on exposure rate. Nevertheless, for low exposure rates the clonal expansion rate isconsistent with a linear increase. An improved mechanistic understanding of the underlying processes
and robust parameter values among several studies and cohorts could provide a good basis for the transfer
of risk to other exposure scenarios.

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B. Bronchial airway deposition of inhaled radon progeny andcellular and tissue effects
452. Lung cancer from radon progeny poses an important radiogenic risk for the population and forworkers in places with elevated radon concentration. Because of the complex lung tissue structure andthe particular deposition patterns it can be expected that the carcinogenic mechanisms and, consequently,
the dose–response relationship and temporal dependencies of risk are different compared to other solidcancers after low-LET irradiation. The short-lived alpha particles emitted by radon progeny 218Po and214
Po are deposited in the bronchial airways of the lung. The alpha particles from the airway surfaces canhit the basal and secretory cells in the bronchial epithelium and induce or accelerate the bronchial
carcinogenic development.
453. Because of the biological variability of the lung structure, and the inhomogeneity of the local
radionuclide distributions within the airway bifurcations, local dose distributions are highly variable.
Cells located at carinal ridges receive highly elevated doses, also depending on particle size andmucociliary clearance [B10, H31]. Furthermore, because of the short range of alpha particles, theepithelial structure plays an important role when calculating the dose to sensitive target cells, and various
cell types in the bronchial epithelium are located at different depths in different airway generations [I4,M30]. Hofmann et al. [H32] modelled energy deposition and cellular radiation effects from radon
progeny for basal and secretory cell nuclei at different depths, and related these to inactivation, mutationand transformation frequencies using in vitro radiobiological data. Fakir et al. [F3, F4] performed
microdosimetric calculations in the bronchial airway bifurcations with a Monte Carlo code and related
these to transformation frequencies in sensitive target cells. More background information on lungmodels and radon progeny deposition patterns is provided in Hofmann el al. [H33]. In the following, themodelling efforts since 2006 are described to relate radon progeny deposition to potential mechanisms ofradiation-induced lung carcinogenesis.
454. Based on experimental evidence that the deposition pattern of radon progeny is highly non-uniform
within the airways of the lung, Szoke et al. applied a computational fluid and particle dynamics model toquantify local deposition patterns of inhaled radon progenies [S76]. For selected low-dose exposures,
microdosimetric quantities for all epithelial cells were calculated. A mathematical model of the cellular
structure of the bronchial epithelium was constructed with the aim to estimate the cell death and celltransformation probabilities. The deposition distributions of the attached radon progenies were highly
non-uniform both in mine and indoor exposure situations; however, the inhomogeneity was stronger inthe case of mines. For the unattached fraction, the molecular diffusion caused a more uniform deposition
distribution in both cases. The number of cellular hits and the hit probability were estimated for bothtypes of exposure for the same total amount of inhaled radon decay products. Exposure rates in mines
were significantly higher, and one week of exposure in homes was compared to 4.6 min of exposure inmines. The numbers of cells which receive multiple hits and the maxima of radiation burdens weresignificantly higher in mines.
455. Balashazy et al. [B11] simulated cellular hit probabilities of alpha particles emitted by inhaled radon
progenies in sensitive bronchial epithelial cell nuclei at low exposure levels characteristic of homes anduranium mines [B11]. Based on a bronchial airway deposition model, the results suggested that in thevicinity of the carinal regions of the central airways the probability of multiple cellular hits by alphaparticles can be quite high, even at low average doses, and increases non-linearly with radon
concentration. It was speculated that the high exposure burden in these hot spots might induce nonuniform dose effects or inflammation.

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UNSCEAR 2020/2021 REPORT456. With the aim to compute the spatio-temporal distributions of cell nucleus hits, cell killing and celltransformation events of inhaled radon and radon progeny, Farkas et al. [F8] simulated radioaerosol
deposition patterns in the central bronchial airway bifurcation with a human bronchial epithelium
model [F8]. The results indicated that the preferential radionuclide deposition at carinal ridges plays animportant role in the space and time evolution of the biological events. While multiple hits were generallyrare for low cumulative exposures, their probability might be quite high at the carinal ridges of the airway
bifurcations. Likewise, cell killing and transformation events also occurred with higher probability in thisarea. It was commented that the site-specificity of radionuclide deposition might impact not only direct
effects, but also non-targeted radiobiological effects due to intercellular communication. Regarding time
distributions, the probability of multiple hits in case of a uniform surface activity was low and the hits
were well separated in time, while they might be quite high for the cell nuclei located in the deposition
hot spot even at low doses.
457. A mathematical model of the bronchial epithelium was developed by Madas and Balashazy [M1]to study the biological effects of alpha particles emitted by inhaled radon progeny [M1]. Cell pillar types
consisted of basal, indeterminate, goblet, other secretory, ciliated and preciliated cells. Assuming that cell
survival probability decreased exponentially with the number of nuclear traversals, the number ofinactivated and surviving progenitor cells could be estimated. Assuming equilibrium between cell deathand cell division, cell division rates could be estimated at different exposure rates. The mean number of
induced mutations per progenitor cell per day was calculated applying a mathematical model for mutationinduction which considers both radiation-induced DNA double-strand breaks and cell divisions as events
which contribute to mutagenesis. The results indicated that the mutagenic effect of chronic exposure todensely ionizing radiation is dominated by the indirect increase in cell division rate of progenitor cells
due to cell inactivation in order to maintain homeostasis and not by DNA damage in surviving cells. Thissuggested that radiation burdens of non-progenitor cells play a significant role in mutagenesis in case of
protracted exposures to densely ionizing radiation, because of the acceleration of cell turnover due to cell
inactivation that increases mutation frequency. Mutation rates as a function of dose rate were found to
exhibit a convex shape below a threshold, indicating the exhaustion of the tissue regeneration capacity of
local progenitor cells. Further, it was suggested that progenitor cell hyperplasia occurs beyond thethreshold dose rate, giving a possible explanation of the inverse dose-rate effect observed in lung cancer
among uranium miners.
458. To study the effects of hyperplasia, bronchial epithelium models were developed by Madas et al.
with increased number of basal [M3] and goblet cells [M4] mimicking the hyperplastic epithelium in thedeposition hot spots. Basal and goblet cells were selected because histological and experimental studies
showed that these cell types can increase in number upon exposure to different drugs and irritants. It was
assumed that the hyperplastic epithelium is thicker than the normal one, and that the thickness of the
mucus layer covering the epithelium is proportional to the number of goblet cells. It was shown that the
average tissue dose resulting from radon exposure and the average hit number and dose of basal cells
decreased with the increase of both basal and goblet cell number. Exposure history of the tissue mightthus affect the microdosimetric consequences of present exposures. In this sense, hyperplasia induced byradon exposure could be considered as adaptive tissue response. Goblet cells can discharge mucus within
milliseconds upon exposure to ROS, and the increase in mucus thickness also significantly reduced theradiation burden of the tissue.
459. To study the possible effects of radon progeny on different stages in the TSCE model, Madas et al.
used a biophysical model for induction of mutations, i.e. the initiation and transformation steps, and of
clonal growth [M2]. Based on previous work for dose distributions and a mathematical model for the
bronchial epithelium [M1], the authors estimated the mutation and clonal expansion rates by the numberof non-repaired DNA double-strand breaks and by a geometric growth model, respectively. Cell survival
probability was supposed to decrease exponentially with cell nucleus hits. The number of cells in a clone

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increased quadratically with dose rate and was limited to the periphery of the clone, and its perimeterincreased linearly with dose rate and time. It was found that rates of initiation and transformation
increased monotonically with dose rate, whereas the effective clonal expansion rate decreased with time
but increased sublinearly with dose rate. The authors suggested that effects of radon exposure on both
mutational events and clonal growth are significant and should be considered in mechanistic models ofcarcinogenesis applied for analysing epidemiological data.
460. In an attempt to link cell-based processes to cancer development and epidemiological findings,
Drozsdik and Madas [D20] investigated whether progenitor cell hyperplasia can explain the levelling inclonal expansion rates above a given exposure rate observed by applying the TSCE model toepidemiological data of lung cancer among uranium miners. As hyperplasia reduces the local dose
consequences of radon exposure, and its measure is expected to increase with exposure rate, hyperplasia
should diminish the effects of exposure rate. A main assumption of the model was that the clonal
expansion rate obtained from epidemiological data is directly proportional to cell division rate quantified
by previous models to describe the bronchial epithelium, the alpha particle dosimetry and cell survival.
While the authors found a parameter set resulting in a cell division rate – exposure rate relationshipcorresponding to the plateau in clonal expansion rate, the simulation results were highly dependent on
the chosen model parameters and had a high uncertainty. The work provides an example how models at
different levels of description, cell and tissue-based radon models and mechanistic model ofcarcinogenesis, can be integrated to investigate consequences of underlying processes on radiation risk.

Summary and conclusions
461. In summary, the mechanisms of radon on lung carcinogenesis involve effects not only at cellularlevel, but also at higher levels. Alpha particles from radon and its progeny have a high cell killing
efficiency. Replacement of the killed cells increase the mutation rates in the descendants of theneighbouring cells. These additional cell divisions may be a significant source of mutations and may
exceed the mutations induced by DNA damage. Furthermore, already mutated cells with a growthadvantage might replace the missing cells faster. Cell killing may also change the tissue structure
especially in case of chronic exposures, e.g. by the induction of progenitor cell hyperplasia. Hyperplasia
reduces the local dose consequences of subsequent radon exposures and may in this sense be regarded as
an adaptive response. Such a mechanism might be a source of differences in risk regarding acutecompared with chronic effects, or regarding different dose rates. Radon progeny deposition in the
bronchial airways can be highly inhomogeneous. The consequences of this heterogeneous dose
distribution for cancer risk are not clear. Doses and dose rates at a macroscopic level might be very
different to the microscopic situation and might also be different for exposure situations in mines and at
home. Epidemiological studies of lung cancer risk from miners and residences find compatible risk perunit exposure, however, the dependence of risk on time since exposure, age at exposure or attained age
are still under discussion [U12]. High local radon progeny concentrations may also lead to immune
responses and result in inflammation.
462. These considerations imply that it will likely be very difficult to understand radon-induced
carcinogenesis at a purely cellular level. Different levels of biological organization, from cellular to tissuelevel, may be involved and interrelated in the carcinogenic process. First attempts tried to link the celland tissue-based bronchial airway models with mechanistic model of carcinogenesis to better understandthe particular patterns of radon-induced lung cancer risk. Future progress may be achieved by improved
integrated modelling at different levels of organization, together with experimental data that could guidesuch model development.

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C. Intercellular communication
463. A model for cell-to-cell communication including effects of ionizing radiation was developed by
Mariotti et al. [M14]. The study focused on cytokine signalling, and in particular the dynamics of IL-6cell releases. The model was applied to experimental data in which primary human AG01522 fibroblasts
were irradiated with 60Co gamma rays with a dose rate of 0.83 Gy/min. The seeding medium wascompletely replaced with fixed volumes of freshly made complete medium before collection started. In
the case of experiments with radiation, the medium was replaced with fresh complete medium before
irradiation. IL-6 concentrations in cell culture supernatants were determined with solid-phase sandwich
ELISA. The applied doses were 0.1, 0.25 and 1 Gy. In the experimental set-up, the net release rate of
IL-6 molecules in the control cells without irradiation was a truncated bell-shaped function of time after
medium change that reached its maximum release value at two hours and returned to near zero at 10
hours. Each cell emitted around one cytokine molecule per second at the time of maximum release. For
cells irradiated with a moderate dose of gamma rays at 0.25 Gy, the maximum release peaked at threehours. The radiation-induced response was around one third of the response induced by the mediumchanges with about 0.3 cytokines per cell and second and might contribute to radiation-induced bystander
effects. The study was extended including the role of ROS and the effect of alpha radiation [M15]. The
analysis indicated that ROS are constitutively important to produce IL-6, playing a crucial role for themodulation of the signal emission after irradiation. Alpha radiation from an 241Am source led to a similar
overall qualitative behaviour as for gamma irradiation, albeit with higher maximum IL-6 release.
464. A kinetic-based model of radiation-induced intercellular signalling was developed by McMahon etal. [M27] with the aim to study signal production and response kinetics in bystander experiments [M27].
In this model, irradiated cells generate a signal for an extended time period proportional to the delivereddose, which is spatially- and temporally-dependent. Exposure to this signal above a certain thresholdconcentration can lead to a damaging response in cells, both in hit and non-hit cells, with a probability
related to the time the cell is exposed to the signal above this threshold, rather than total signal exposure.
The model was applied to different experimental studies, including media transfer experiments andmodulated field exposures, and generally showed good agreement. The model suggested that bystander
effects play a significant role in determining cellular survival and that the inclusion of intercellularcommunication might be essential to produce robust models of radiobiological outcomes in clinically
relevant in vivo situations. Using a similar model, Kundrát and Friedland [K51] estimated that ionizing
radiation activated the emission of bystander signals from the hit cells for about a day, while the signal
itself decayed with a lifetime of about an hour, in medium transfer experiments with WTK1
lymphoblastoid cells [Z8].
465. When co-cultured with normal cells, oncogenic transformed cells are subject to the selective
removal by apoptosis induced via intercellular signalling [B23, B24]. Oncogenic transformed cells areused as an in vitro model system that mimics early-stage carcinogenesis. They do not obey contact
inhibition. Kundrát et al. [K50] presented a detailed analysis of the intercellular signalling involving
cytokines and ROS/RNS such as superoxide anions, nitric oxide and hydroxyl radicals [K50]; throughinducing membrane damage, the mitochondrial pathway of apoptosis was triggered. The selectivity ofthis process was given by the short range of superoxide anions released by the transformed cells.
Numerous in vitro studies demonstrated that this process is active for normal and transformed cells ofvarying types, indicating the generality of this phenomenon. Hence, this selective removal of transformed
cells upon signaling with their normal neighbours was suggested to represent a natural anti-carcinogenic
mechanism [B23, B24]. This suggestion was supported by mechanistic modelling, which predicts that
this phenomenon may stop the growth of a population of transformed cells and induce a kind of dormancy
in early-stage carcinogenesis. This may precede and/or enable the action of the immune system [K50].

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466. While radiation is known to increase cancer risk, it was shown to enhance the selective removal oftransformed cells in vitro, i.e. to act in an anti-carcinogenic way. In an effort to explain this discrepancy,
Kundrát and Friedland [K52] developed an extended signalling model to describe the selective removal
by apoptosis. Radiation-induced enhancements of the releases of superoxide, nitric oxide and/or
peroxidase from transformed or normal cells increased the induction of apoptosis under in vitro
conditions. However, the involved ROS/RNS possess much shorter lifetimes and the cell densities are
considerably higher in vivo than in vitro. The modelling study predicted that when a small population oftransformed cells is in proximity to a larger number of normal cells, as is the case in early-stage
carcinogenesis, ionizing radiation reduces rather than enhances the induction of apoptosis in transformed
cells. Thus, in addition to its well-known pro-transforming action, ionizing radiation loosens the growth
limit on the early-stage transformed clones. Small lesions that would be eradicated by apoptosis are
predicted to escape this control mechanism upon irradiation [K53]. The signalling behaviour was
predicted to be highly non-linear, arising due to spatial aspects of the underlying signalling scheme.

Summary and conclusions
467. In summary, several studies analysed in detail intercellular signalling and associated bystander
effects and apoptosis induction. Although the radiation-induced modulations of the intercellularsignalling are transient only, their sequelae might be of permanent nature [K52]. Importantly, theinvestigated signalling processes can be highly non-linear in radiation dose, time and space. It is unknown
how relevant these potential non-linear effects are for radiation-induced carcinogenesis. A big challenge
is the transfer of these mechanisms and results from in vitro to in vivo conditions. Further theoretical andexperimental research is needed to elucidate this issue. On the one hand, this includes experiments to test
model predictions for conditions close to those in vivo that still can be realized in vitro, for instancedensely seeded cells with increased levels of antioxidants in the growth medium. On the other hand, therespective models shall be further developed, in particular to study the effect of radiation exposure onthese processes. A promising way to infer on the consequences of radiation-modulated intercellularsignalling processes for cancer risk is the implementation of these processes and their models intomechanistic models of carcinogenesis.

V. IMPLICATIONS FOR CANCER RISK INFERENCE
468. In chapter IV recent findings relevant to mechanisms of radiation carcinogenesis at low radiation
doses have been considered. In this chapter the intention is to summarize the implications of thesefindings relevant to the inference of radiation cancer risks at low doses and low dose rates. These will betaken by section as presented earlier.

A. DNA damage
469. The basis of cancer risk inference at low doses and low dose rates currently draws upon theknowledge that DNA damage is induced in linear proportion to radiation exposure and any damage site
has a small but finite probability of leading to mutations of DNA sequence. Some such mutations may
modify the function of key genes controlling cell proliferation, alter transcription of key genes andexpression of encoded proteins, or other hallmark of cancer characteristic, thus increasing the risk of

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cancer presentation in the individual sustaining such a mutation. The forms of DNA damage consideredof most importance following ionizing radiation exposure are DSBs and sites of clustered damage that
may include any induced lesion type in close proximity to other induced lesions. An important role for
ROS and other reactive species is apparent at low dose exposures.
470. Substantial data are available from nuclear foci assays involving chromatin and core repair proteins(e.g. 53BP1, γH2AX) that are taken to reflect the frequency of induced DNA double-strand breaks. These
substantiate findings that DNA double-strand breaks are induced in linear proportion to dose down to
1 mGy at least. While double-strand breaks are of importance, other forms of DNA damage are likely tocontribute to the effects of radiation exposure, these include ROS-mediated damage, single-strand breaks
and base damages. Various assays (alkaline comet assay, specific ROS stains) point to linear inductionof these types of DNA damage.
471. The chromatin foci assays are not direct measures of DNA double-strand breaks but rather reflect
the early stages of DNA damage signalling (γH2AX) or repair (53BP1). Physical measurements of DNA
strand breakage are generally less sensitive than the foci assays but are direct measurements of damage.
Among the most sensitive of the physical assays for DSBs is the neutral comet assay, and the available
data are consistent with the conclusion that DSBs are formed in linear proportion to radiation dose.

B. DNA damage signalling, chromatin remodelling andepigenetics
472. The main molecular mechanism that drives DNA damage signalling, is post-translational
modification of specific proteins, including ATM, particularly by phosphorylation. Based on limited data
relating to phosphorylation of key proteins in the signalling pathway following acute exposures toionizing radiation (X- and γ-rays), DNA damage signalling appears to be linear with dose over a 10 mGy
to 1 Gy dose range.
473. The activation of the DNA damage response has been investigated using restriction enzyme
generated DSBs or breaks induced by other enzymatic systems as a model for radiation-induced breaks.
These enzyme-generated breaks are simpler in end structure than radiation-induced breaks. Nonethelesssingle and multiple DSBs generated by restriction enzymes do effectively trigger a DNA damage
response as assessed by the initiation of p53-dependent processes and the formation of repair complexes(e.g. [B6, W14, Z19]) and thus represent a valid model system for radiation-induced breakage. Whiledrawn from a model system, these findings are consistent with activation of the DNA damage response
by a single DSB per cell and the notion that the repair of such breaks utilizes similar pathways as does
the repair of radiation-induced DSBs, and tend to confirm the linear induction of phospho-ATM.
474. A general conclusion is that loss of functional chromatin remodelling and modification impairs therepair of DSBs and confers radiosensitivity to cells. The existence of radiation- and cancer-prone humandisorders associated with single DNA damage response gene mutations and the radiosensitivity andcancer proneness of mouse models carrying mutations in single DNA damage response genes, provide
evidence for crucial importance of functional DNA damage response genes for cancer risk. Most likely
this also holds for low-dose exposures based on outcome of model systems carrying a single DSB.
Evidently the cohort of genes that participate in the response to ionizing radiation-induced DNA damage,is substantially larger than previously anticipated and this may have implications for the proportion of
the population carrying heterozygous mutations in these genes and consequently is at increased risk ofcancer after exposure, and this may impact on the shape of cancer dose-response curves.

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475. Collectively, existing data suggest that radiation-induced effects on DNA methylation depend on
tissue/cell type, sex, and species. Moreover, radiation type or quality, dose, and time after exposure haveto be taken into consideration as well as the technologies to determine DNA methylation. The data alsosuggest that dose–response relationships are complex. The evidence from the scarce number of studies
suggests that low-dose radiation is capable of causing changes to DNA methylation and that the
methylation changes might differ between low and high dose. The methylation data are inconclusive
when related to transcriptional responses. To address the latter point, future work should focus on the useof high-resolution technologies to analyse simultaneously DNA methylation and transcription.
476. Epigenetic endpoints can be difficult to study because of the many different factors that influence
epigenetics to an equal extent as low doses of radiation and it can therefore be difficult to segregate
different influences from one another. A possible approach to deal with this complication was suggested
in the work by Tang et al. [T10]. This study, looking at lncRNAs compared responses to low doses ofradiation in two strains of mice after removing from the complete list those lncRNAs associated withphysiological states such as oestrus cycle.

C. Effects on signal transduction pathways
477. The effects of low-dose radiation exposure on intracellular signalling pathways have been addressed
mainly in immune cells, either purified from exposed animals or exposed in vitro after purification. Lowdose radiation exposure effects on signalling pathways can be seen in all cell types examined: humanpurified monocytes, human B- and mast cell lines, endothelial cells, murine and rat mast cells, splenic
CD4+ T-cells, splenocytes.
478. In these cells, radiation modulates the activity of several kinases and transcription factors belonging
to different signalling pathways. Distinct signalling pathways may be activated after exposure to lowand high-dose radiation in the same cell type. A given pathway can be activated or inhibited by low-doseradiation exposure in different cell types.
479. A radiation dose as low as 1 mGy can activate antioxidant and innate immune signalling pathways
in murine splenocytes. The kinetics of these effects are different according to the dose delivery (acute orin three fractions).
480. The downstream consequences of signalling pathway activation/suppression in relation tocarcinogenesis has received little attention. Therefore, conclusions on relevance of these observations tocancer risk inference cannot be drawn.

D. Gene and protein expression
481. There are many studies of gene expression responses to radiation exposure, many at high and moderate
doses and a few that include low doses. Much of the work has been carried out with a main purpose toimprove biological dosimetry and as such uses peripheral blood lymphocytes and their derivatives.
482. While some studies have observed a different set of genes or proteins responding to radiation, there
is no consensus on what defines a gene set that is uniquely responsive to high dose as opposed to an
exclusively low-dose responsive gene set. This may in part be due to inherent technical variability in the

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method predominantly used to date (i.e. gene expression analysis using microarrays), the variety ofmethods that have been applied and the selected time points which vary between studies. In the future
the application of more readily controlled methods including digital PCR may lead to improvements inconsistency and reproducibility, but it remains possible that the biological responses are in fact inherently
variable, depending on precise cells of origin, donor and probably other factors.
483. As has been stated, cancers can result from altered differentiation of cells leading to uncontrolled
proliferation and other hallmark characteristics, and patterns of gene expression are a substantial driverof differentiation state. Therefore, gene expression patterns are of relevance to carcinogenesis, andradiation has been shown to affect the expression of cancer-related genes, notably p53-responsive genes.
However, the majority of available data concern gene and protein expression alterations 24–48 hours
after radiation exposure, with relatively little information available on the persistence of such alterations.
484. The data available from gene and protein expression studies are insufficient to draw substantive
conclusions on the relevance of these studies for low-dose radiation cancer risk inference due to variable
findings between studies and the generally short time scale of reported studies.

E. DNA repair and effects on somatic cells
485. Several studies have shown that the damage revealed by chromatin/repair foci can be effectively
repaired by normal cells over the course of a few hours, the repair is generally observed to be completewith foci level returning to control levels. A potentially important exception to this finding concerns
repair after very low doses of one to a few mGy low-LET exposure in cultured human fibroblasts.
A number of studies report that foci levels in this situation are not found to be fully repaired, these studies
report that small residual level of excess foci remain, around one additional focus per 10 cells. Evidence
points to the lack of sufficient ROS after these very low doses to induce repair. Some evidence indicatingthat base damage repair can be induced by low radiation exposures is also available. In respect of theimplications of this incomplete/inducible repair for cancer risk, the interpretation is dependent on having
a clear understanding of the subsequent fate of the excess damage bearing cells. Some studies suggestthat the cells with excess residual damage enter apoptosis after one or a few cell cycles; this effectively
removes the cells from the pool of potential cancer cells. In contrast to the above-mentioned results, invivo studies on lymphocytes from individuals undergoing computed tomography examination are notconsistent with incomplete repair and so drawing general conclusions that apply to all cell types is not
currently possible. There is overwhelming evidence that DNA repair pathways NHEJ and HRR
contribute to repair of low dose induced DSBs, depending on cell cycle phase, except in mitosis. The
HRR pathway is less prone to errors and therefore plays a role in reducing cancer risk. It is notable thatsome evidence indicates that HRR is the predominant pathway on stem cell populations, likely protecting
the stem cell pool from the accumulation of induced mutations.
486. The repair assays based on chromatin/repair foci also revealed inter-individual variation in DSB
foci parameters in primary fibroblasts exposed to low doses of X-rays in a small survey of apparently
normal people suggesting that hypomorphic genetic variants in DNA damage response may contribute todifferential susceptibility to cancer after low-dose radiation.
487. The consequences of DNA damage in somatic cells can be observed in the form of DNA sequence
mutations and chromosomal damage. The induction of micronuclei, a sensitive endpoint that reflects
chromosomal breakage and whole chromosome loss events, has been observed to be linear with dose in
the low dose range down to 10 mGy both in vivo and in vitro. A plausible mechanism for the linear non-

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threshold induction of micronuclei by X-rays is the observation that DSB repair does not function in the
mitotic phase of the cell cycle, even not in repair proficient cells. It is conceivable that the mitotic period
without repair of DSB in wild-type (repair proficient) cells would allow the generation of micronucleus
in a frequency directly related to the initial frequency of DSB induction (which is linear). The formationand subsequent re-integration of micronuclei appears to be an important pathway for the generation ofchromothripsis, highly localized and extensive genome re-arrangement that has been observed in several
forms of cancer (though not specifically radiation-induced cancers). Whereas the induction studies
suggest that micronuclei are formed in linear proportion to dose, little is known on the dose relationshipfor the re-integration of micronuclei, although it has been observed that cells carrying micronuclei canundergo at least some cell divisions. Furthermore, low dose exposures have been reported to stimulate
the integration of DNA into chromosomal locations.
488. LOH is commonly observed in cancer genomes and can be generated, among other mechanisms,
through chromosomal translocations and deletions. Available data indicate that LOH is caused byradiation in linear proportion with dose down to 50 mGy low-LET radiation. The age-dependence of
induced chromosome aberration yields where some studies suggest that in the young yields are lower
than in adults, may have an impact on dose-response shapes in populations. However, such age-dependent
variation is not commonly observed in stem cells, and thus may not be a significant factor in determining
cancer risk at different ages.

F. Genomic instability, bystander effects, damage/effects on
non-nuclear cellular components, adaptive response andhyper-radiosensitivity
489. Thus far, the focus has been on direct damage to DNA and the consequences that directly follow.
However, radiation causes damage to all cellular components and observations indicate that there are
non-DNA targeted responses to radiation exposure.
490. Substantial attention has been given to effects of radiation exposure on mitochondria andmitochondrial function. Mitochondrial damage can mediate apoptosis independently of damage tonuclear DNA. Low-dose and fractionated exposures have been reported to lead to both mitochondrial
fusion and fission. Conflicting findings on the effects of radiation on ROS levels, mitochondrial massand cell proliferation have been reported. Nonetheless there appears to be a consensus that radiation
exposure even at low doses, e.g. 20–100 mGy, low-LET radiation can affect energy metabolism. Suchchanges may be of relevance to cancer risk inference given the hallmark characteristic of cancer of alteredcellular energetics, and generally a shift to glycolysis [Z22].
491. More recently available studies on the non-targeted effects of transmissible genomic instability andbystander phenomena do not affect or substantially challenge the Committee’s prior judgements as mostrecently set out in the UNSCEAR 2012 White Paper [U9].
492. Notwithstanding the above considerations, cancer risk modelling has demonstrated the potential
importance of transmissible genomic instability and bystander phenomena for cancer risk inference.
Thus, it remains important to generate more definitive data on the operation of these processes afterradiation exposure in vivo, particularly at low dose and low dose rate.
493. Adaptive response studies remain difficult to interpret and bring into any low-dose radiation risk
assessment framework. There remains no consensus on the adapting dose range or duration of the

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adapting signal, and this is further complicated by the lack of a consistent mechanistic basis for thephenomena. Additionally, some negative studies continue to be reported.
494. Given that all humans live in an environment with natural background radiation, it may be thateveryone is, to an extent, adapted. Also, if adaptive responses were a major modulator of cancer risks in
exposed populations, one might expect differences in the magnitude of cancer risks assessed in
occupational (chronic low/moderate exposures with changes in dose rate) studies and those in the atomic
bombing survivor studies (acute and frequently high-dose exposures), with the occupational studies
finding lower risk – this is not what is observed. Furthermore, given that radiation is not the sole, or
usually predominant cause of cancer, and there are multiple contributory risk factors, any modulation byradiation adaptive response is likely to be very small and therefore masked by the effects of the othercontributory risk factors such as diet, smoking and exposures to other agents. A broadly similar argument
can be made in relation to the small risks associated with low-dose and low-dose-rate exposures.
495. Hyper-radiosensitivity has been observed in cell culture systems and to a limited extent in vivo, andmost notably in stem cells in vitro and in vivo. The loss of cells carrying damage at low doses is expected
to serve to protect against cancer in that potentially induced mutation-carrying cells will be lost throughcell death and cannot therefore contribute to cancer development. However, the depletion of stem cellpools can cause an increase in proliferation of the stem cells to replenish the pool, this carries the risk ofaccumulation of replication-associated mutations. It is therefore difficult to interpret the impact of thehyper-radiosensitivity phenomenon on cancer risk.

G. Stem cells and target cell populations for radiation
carcinogenesis496. Stem and early progenitor cells are viewed as main target cell populations for radiation cancer due
to their long lifespan in the organism. Stem cell populations in several tissues have been shown to respondto low doses of radiation, and in terms of DNA damage response they appear to respond in a similar
fashion to other somatic cells, and this holds true for mutation frequencies that do not strictly correlatewith replication cycles traversed. The phenomenon of hyper-radiosensitivity has been described in stem
cell populations, as noted previously the impact of this in relation to cancer risk is not certain.
497. However, some stem cells, most notably embryonic stem cells, but also induced pluripotent stem
cells, depend more on HRR of DNA damage than NHEJ. The HRR pathway preference likely serves topreserve genome integrity and avoid mutation in the presence of DNA damage given the higher fidelityof recombinational repair compared to NHEJ. This is likely therefore to act to protect stem cells fromoncogenic mutations, much as the retention of parental DNA strands through DNA replication and cell
division that has been observed in some stem cell populations.
498. The basal and secretory cells are target cells for radon induced bronchogenic cancer. The alphaemitting radon decay products are located on the bronchial epithelium about 30 µm above the cells andthe high-LET alpha particles can produce direct DNA damage in the target cell populations.
499. Some stem cell populations enter apoptosis readily following radiation exposure, possibly due totheir hyper-radiosensitive phenotype, and this likely serves to protect from cancer. Loss of cells throughapoptosis though may lead to substantial proliferation of stem cell populations to restore tissue that brings
risk of replication-associated mutation, at low doses this is expected to be a minor consideration.

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500. A recent study has demonstrated how low dose (50 mGy) radiation exposures can lead to thepreferential expansion of cells and clones carrying mutations in the Trp53 gene [F10]. This supports arole of low doses in promoting the growth of pre-existing pre-neoplastic clones.

H. Effects at the whole organism level
1. Immune system and cytokine responses
501. In section III.H.1 recent work on the effects of low-dose/low-dose-rate radiation exposure on the
immune system was considered. This section includes studies considering in vivo human exposure,including atomic bombing survivors and environmental, occupational or accidental exposure, andexperimental exposure of cells and mice to low-dose, low-dose-rate and/or repeated low-dose exposure.
502. There remains no consensus from published studies whether low-dose radiation exposures serve tostimulate or reduce immune functions. This most probably stems from the nature of the immune system,
which is an integrated network of highly specialized/differentiated cells. Radiation exposure has different
effects on different cell types, and it is difficult to infer the effects on the protective functions of theimmune system at the organismal level. Low-dose radiation exposure effects may depend on the immune
status at time of exposure, with exposure on an inflammatory background reducing inflammation andexposure on a “neutral” background inducing inflammatory responses.
503. However, the association of certain alleles of immune genes with breast cancer development inradiological technologists suggests that dysregulation of immune functions may allow the developmentof cancers after protracted low-dose exposure. In addition, there is evidence that low-dose radiation
exposure can reduce the efficiency of transplantation of cancer cells in immuno-competent mice.
However, given the mixed nature of the findings, it is not possible to draw a conclusion on the effects of
low-dose/low-dose-rate exposure on cancer immunity, and especially cancer immunosurveillance/
immuno-editing.
504. Available studies on the immune status and immune functions of those living in areas with highnatural background radiation levels or professionally exposed individuals do not allow any consistentconclusions to be drawn at that time. Some of the late effects of radiation exposure in vivo appear toaccelerate the normal process of immunological aging. Premature immunosenescence may increase thesusceptibility to cancer and non-cancer diseases.
505. Clearly the immune system, and particularly cancer immunity, could be a very important factor inmodulating post-radiation cancer risk. However, the available data are not sufficiently consistent orrobust to draw conclusions relating to the use of such data in the inference of cancer risk.

2. Carcinogenesis506. Two themes that relate to low dose and carcinogenesis depend on the proliferation of stem and/or
lineage-specific cells. While moderate and high doses of radiation “exhaust” stem cell pools and lead toaccelerated aging, low doses of radiation seem to have the opposite effect. An extreme example of this is162

UNSCEAR 2020/2021 REPORTa transgenic model of accelerated aging homozygous mouse model, klotho knockout mice where
exposure to low doses significantly extended lifespan of animals.
507. Cells responsible for the development of new blood vessels proliferate following low/moderate
doses of radiation leading to increase of neovascularization in vivo. Several cancer and non-cancer studies
support this concept pointing to an alternative mechanism by which low/moderate exposures may
contribute to carcinogenesis.
508. Other endpoints that relate to later stages of cancer development such as cell migration, cell
invasiveness and EMT have not been studied in great depth following low-dose radiation exposure, andthe available evidence is mixed. There is the potential for low doses to affect these endpoints that relate
to tumour metastasis, and there are reports of low-dose exposure reduction of migration and invasion.
509. Both of these types of cell proliferation (i.e. stem cell proliferation and proliferation of vascular
cells and the growth of the vasculature) can be associated with carcinogenesis, both at the onset andduring cancer development.
510. Overall, the most serious obstacle in data interpretation from wild-type animal studies is the factthat the age of animals has great effects on all aspects of their health. This makes room for data variation.
Statistically significant findings are most often observed in studies where only animals of the same age
were used.
511. Not surprisingly, low-dose radiation studies of mutant animals where the mutation is relevant for
carcinogenesis, are far more affected by the presence of a mutation than by differences in animal age. For
that reason, studies on mutant homozygote or heterozygote animals exposed to low-dose radiation often
produce the most robust data. Some of these studies are focused on tumour suppressor genes where LOH
leads to cancer development.
512. Another group of studies explored animals with overexpression of different oncogenes. Variable
effects were observed ranging from increased to decreased to no effect on radiation induction of cancers.
While studies in mutant mice produce the most robust data – increase or decrease in cancer incidence
depends on what the exact mutation is and whether other mutagens are present in the environment at thesame time. Finally, studies focused primarily on DNA damage resolution – mutations introduced by lowdose radiation exposures in animals with intact repair are the most robust in those cases when animals ofsame age were used. While none of these studies could be directly related to carcinogenesis at this time,
there remain examples of animal studies where only apparently beneficial effects of low-dose exposures
were registered.
513. Because of the complexity of multicellular organisms and differences between cells and tissues itshould be appreciated that the same epigenetic modifications and mutations lead to different
consequences in different parts of the body. Thus, an animal may show no life shortening or even alifespan extension after exposure to a specific low dose, even if some of its non-proliferating cells
accumulate mutations that could under different circumstances lead to cancer development. With thedecrease of dose, ionization events became progressively less frequent and their burden on the organism
as a whole may cease to be stress to the organism.
514. As noted above, while many effects are context dependent, it is difficult to reach an overall
conclusion on the damaging compared with beneficial effects of radiation as reported in in vivo studies.
All animal experiments dealing with low doses and cancer induction suffer from the potential difficulties
of limited statistical power, and this could be contributing to the mixed evidence available.

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515. For most responses high-LET radiation is more damaging than the low-LET radiation neverthelessfor carcinogenesis the response is complex. It depends in many cases on whether the exposure is external
or internal, on the exact cancer, the tissue type of cancer, the sex and age of exposure of the animal, doseand dose rate and many other factors.

I. Integration of data at different levels of organization andmodelling of cancer mechanisms
516. In chapter IV, approaches to modelling and inferring cancer risk were considered. It is evident thatthe available models can incorporate information drawn from biological/mechanistic studies, although
the extent to which this has been successfully demonstrated varies with the study objective. The generalapproach of integrating biological data into cancer risk models clearly holds promise for the improvement
and refinement of cancer risk inference.
517. Broadly, the conclusions to date from modelling approaches suggest (a) low-LET radiations could
act to initiate or promote carcinogenesis; (b) high-LET radiations for lung cancer act largely throughpromotion/clonal expansion. This difference relating to the predominant stage at which radiation acts has
substantial implications on the dependence of radiation risk on age at exposure, attained age and timesince exposure. The inverse exposure-rate effect observed for lung cancer after radon exposure in minerstudies and the particular form of high-LET radiation action might be related to the lung-specific tissue
architecture and exposure situation.
518. Modelling of lung airway radiation dose deposition from radon inhalation suggests non-linear
effects due to the different levels of biological organization involved and the highly inhomogeneous radon
deposition in the bronchial airways. It is anticipated that cell repopulation and/or inflammation mightplay an important role in radon-induced lung carcinogenesis.
519. Models for intercellular signalling suggest processes that can be highly non-linear in radiation dose,time and space. It is currently unknown whether these effects might affect dose–response relationships
for cancers. A critical issue is the transfer of these mechanisms from in vitro to in vivo conditions.
520. To benefit most from the modelling approaches incorporating biological data, there are a numberof specific data requirements. These and other research needs are considered in chapter VI.

VI. DIRECTIONS FOR FUTURE RESEARCH
521. It is apparent that there remain many gaps in the evidence and knowledge on the biologicalmechanisms relevant for low-dose radiation cancer risk inference. Beside these gaps, there are areas
where findings need consolidation or replication to provide the robust evidence. It is noted particularlythat while many studies include consideration of effects of a 100 mGy low-LET exposure, relatively few
consider doses below that point. There remains a need for studies that explore the sub-100 mGy region
more thoroughly and include moderate doses for the purpose of comparison at different dose levels andextrapolation between dose levels. The following paragraphs summarize the key areas where theCommittee considers that further research is required. The order below reflects the order in which topics
were considered in the main body of the annex and does not reflect the Committee’s view on the relative
importance or urgency of the different areas of research.

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UNSCEAR 2020/2021 REPORT522. In the area of DNA damage, it is important to provide better quantitative data on the induction andfrequency of complex DNA damage sites, and to develop more sensitive approaches to provide physicalquantification of DNA double-strand breaks. Additionally, further information on the induction ofdamage to mitochondria by ROS, and the specific targets within the mitochondria could be useful.
523. Concerning DNA damage signalling, epigenetic effects and chromatin remodelling, the Committee
encourages research to:(a) understand the dose/dose-rate/quality dependence of epigenetic alterations caused by radiation
exposure and if dose–response relationships and/or persistence of induced changes vary substantiallyfrom those for the formation of mutations and chromosomal changes as this could have substantial
impact on judgements on the model used for low-dose risk inference;(b) understand the dynamics of post-translational modifications (especially phosphorylations/dephosphorylations) by cutting-edge proteomics, identification of the relevant kinases andphosphatases and integration with de novo transcriptional analysis. This understanding would
provide a basis to determine cell fate after radiation;(c) gather information on sites/genes that low-dose exposures methylate/demethylate, or otherwise
epigenetically alter and the impact of these alterations on transcription.
524. The Committee considers that research is needed to improve the reproducibility and intercomparability of results from gene/protein expression studies to determine what, if any, are thedifferences in response to radiation at differing dose levels, dose rates and with irradiations of different
types and qualities. As noted above, steady-state analyses should be complemented by analysis of denovo transcriptional response. Information on the duration of any alterations is required. Additionally, it
is important to follow any changes over longer time periods to help ascertain their relevance for diseases
or adverse outcome pathways of importance.
525. With respect to DNA repair and effects on somatic cells, the Committee encourages research to:(a) Provide a better understanding of the persistence of residual DNA damage and the fate of cells
carrying these after milligray-level exposures;(b) Understand if radiation exposures do increase the occurrence of chromothripsis and the relevant
exposure–response relationships, this will help elucidate the relative importance of the pathway for
carcinogenesis compared to conventional mutation/ chromosomal alteration/epi-mutation induction;(c) Provide greater insight into the generation and persistence of ROS and the consequent cellulareffects under physiologically realistic low O2 conditions;(d) Provide information on the frequency of integration of DNA fragments into the genomes ofirradiated cells to allow a better assessment of the contribution of chromothripsis to low-doseradiation carcinogenesis.
526. In relation to genomic instability, bystander effects, damage/effects on non-nuclear cellularcomponents, adaptive response and hyper-radiosensitivity, the Committee encourages research to:(a) Determine whether in vivo exposures lead to persistent elevation of mutation/chromosome
aberration/epi-mutation/chromothripsis frequencies that drive carcinogenesis in human or animalmodel systems, and to confirm if thresholds for the induction of genomic instabilities exist;

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165(b) Ascertain if bystander induction of cancer occurs in humans after ionizing radiation exposure
and whether bystander effects are generally cancer risk enhancing or risk reducing;(c) Assess the relative importance of adaptive response by comparison with the influences of othercontributory risk factors.
527. The immune system has potentially a substantial impact on cancer risk modulation. The Committee,
therefore, encourages research to determine how inflammation and immune functions are affected by
low-dose and low-dose-rate exposures in vivo (environmental, occupational and experimental) and how
any such effects modulate cancer risk.
528. In relation to carcinogenesis, the Committee considers that further data are required from humanand animal model radiation cancers, and organoid culture systems to build up a picture of the key events
that convert normal cells to cancer cells, and the dose- and dose-rate–effect relationships, particularly inthe low to moderate dose range for these events occurring prior to presentation of disease. Further workthat considers the impact of low-dose and low-dose-rate exposures on later stages of carcinogenesis isalso required, considering the preliminary information available on neovascularization, and endpoints
related to tumour metastasis. These data will be crucial for the further development, assessment andvalidation of mathematical modelling approaches to integrate biological with epidemiologicalinformation for cancer risk inference. Such work would be facilitated by the availability of validated
biomarkers of radiation disease/effects that could be integrated into epidemiological investigations.
Mathematical models provide a framework in which biological and epidemiological data can be
combined for risk inference. Important issues that could be addressed using models include: relative
importance of genomic instability/bystander effects/adaptive response, relative importance ofmutation/epi-mutation, etc. (when data of exposure dependence is available for each).
529. As stem and early progenitor cells are generally taken to be the target cells for radiation
carcinogenesis, the use of appropriate model systems is important as such cell populations may responddifferently to low-dose exposures. Notably, further research to provide a better understanding ofcompetition of stem cells in some tissues after milligray and sub-milligray exposure levels (i.e. sparsehits of incremental dose) is likely to be of importance for risk inference.
530. To obtain more robust evidence in the areas above, the Committee encourages the close
multidisciplinary working of radiobiology/epidemiology/mathematical modelling that has the potential
to generate the critical data required to develop predictive models for risk inference that make use of andcapitalize on all available robust and reliable knowledge of biological mechanisms and apply theknowledge to risk inference. It will be important that mechanisms defined using in vitro conditions aretranslated to in vivo conditions in humans; both experimental and theoretical approaches can be expected
to be informative.

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UNSCEAR 2020/2021 REPORTVII. CONCLUSIONS
531. Returning to the questions that were set out to guide this evaluation (see chapter I), below theCommittee summarizes the evidence and findings of this review relevant to each of the issues:
For which biological mechanisms and pathways is there evidence that indicates that they can affect thefrequency of cancers following exposure to ionizing radiation, including at low doses and dose rates?
532. There is very robust and reliable evidence that following the induction of DNA damage incomplete,failed or otherwise dysfunctional DNA damage responses contribute to induced mutation andchromosome damage and thereby affect the occurrence of cancers after exposures at all doses and doserates studied, these responses relate to (a) direct damage to DNA in the form of DSBs and complex
lesions; and (b) damage attributable to the generation of reactive oxygen and related species througheffects on mitochondria. DNA repair activities can serve to reduce yields of mutations andrearrangements, but they are not 100% effective, do not operate in mitosis and some repair pathways may
be induced or regulated by exposure. While not the main focus of this review, it is noted that thosecarrying certain variants of DNA damage response genes may be radiosensitive and at increased risk ofcancer spontaneously and after radiation exposures.
533. Additionally, there is evidence that variants of genes involved in chromatin remodelling also affectcancer risk, indicating that chromatin remodelling pathways are likely of relevance for radiation cancer
risk as well, though evidence relating to low dose/dose-rate exposures is not available; the same may
apply to epigenetic regulatory genes also. Modelling studies with some support from experimental work
indicate that low dose exposures can promote the growth of pre-existing pre-malignant cells and clones
in tissues. This represents a pathway in addition to the direct induction of mutations in cells by radiation
whereby low dose exposures are anticipated to increase cancer risk. Limited evidence from occupational
exposure of medical workers indicates that gene variants relating to immune functions can modulate
cancer risk, whether radiation exposure per se can stimulate or suppress such cancer immunity is unclear.
There is an emerging understanding that radiation exposure, a least at moderate dose levels may stimulate
tumour angiogenesis; while data at low dose levels are limited, if substantiated it may serve as a pathway
to promote carcinogenesis. Some experimental studies suggest that low doses can impact on endpoints
related to tumour metastasis but the available results are mixed and inconclusive at this stage.
What are the differences in utilization and/or activation of these pathways and mechanisms at low doses
compared with moderate doses?
534. DNA damage response operates at all dose levels, but with differences in utilization of specificpathways (for example at low doses responses related to reactive oxygen damage are likely to
predominate as that form of damage is considerably more frequent than DNA double-strand breaks). The
relative importance of complex/clustered damage sites and their repair is greater at higher dose levels(and after high-LET exposures) as such lesions are more common, nonetheless complex/clustereddamage does occur after low-dose exposure to low-LET radiation. Some pathways of DNA repair show
inducibility such that they are up-regulated after certain exposure levels, and this may apply to DSB repair
and base damage repair, it is not clear if this extends to complex/clustered damage sites. There are known
thresholds for the G2/M phase cell cycle checkpoint which is triggered only following doses in the range
200–500 mGy and above for low-LET radiation. Some studies of gene expression following radiation
exposure suggest differing responses at low as opposed to moderate/high doses. However, there is noclear consensus on the pathways specifically regulated at differing exposure levels and the alterations are
generally reported at only short times after irradiation, their relevance for carcinogenesis is therefore notclear. The dose range over which the potential promotional effects of radiation on pre-malignant cells

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operates is unclear. The dose range over which cancer immunosurveillance operates is not known, butevidence arises from low dose/chronically exposed occupational groups.
What evidence is available on the form of the dose–response relationships for these mechanisms?
535. The dose–response relationships for mutation (LOH) and micronuclei are linear in form in the lowdose range down to at least 50 and 10 mGy low-LET radiation, respectively. Similarly, the dose-response
for DNA damage response activation is linear down to 10 mGy low-LET radiation. The form of doseresponse for the cancer immunosurveillance is not known, but the process has been observed to protectfrom cancer in occupationally-exposed groups, and in mice, immune system activation signalling
operates at 1 mGy low-LET radiation and above. The potential promotional effects of radiation appear
to operate at 50 mGy low-LET radiation and above, but further data are required.
Considering the mechanisms identified above, can any conclusions be drawn as to their overall influenceon the dose–response relationship between cancers associated with radiation exposure at low doses
compared with moderate doses?
536. The knowledge of the mechanisms that affect cancer risk at low doses noted above suggests that an
overall threshold for cancer induction is unlikely, and there is evidence that the known mechanisms
operate at least down to 10 mGy. Furthermore, the mutational mechanism would imply a dose-risk
relationship without a threshold. At the lowest dose levels, where DNA double-strand breaks are inducedin say 1 in 10 or fewer cells (around 2 mGy low-LET exposure), ROS mediated effects are likely to
predominate, and these include the potential promotional action of radiation.
Are there ways to link information on the biological processes and mechanisms found to be relevant tohuman cancer and existing epidemiological data on incidence of disease in exposed populations?
537. The answer is positive, and the evidence considered in this review points to two ways in whichbiological information can be linked to existing epidemiological data. The first is through the applicationof radiation-related disease biomarkers in epidemiological investigations of risk. Such markers have thepotential to reduce the time taken to obtain epidemiological data through the use of robust surrogate
markers of disease when and where available and improve its accuracy by means of reducing the impact
of confounding by competing causes of death and co-morbidity. The second approach entails theintegration of qualitative and quantitative biological data into mechanistic modelling of cancer risk. The
accuracy of models can be enhanced by inclusion of data on the specific steps in carcinogenesis,
particularly where quantitative data are available. Furthermore, the modelling approaches can be used tohelp inform on judgements on the relevance of specific pathways for carcinogenesis. 2 Arguably themechanistic modelling of radiation risk as reviewed in this annex indicates that quantitative mechanistic
modelling of radiation risk is more advanced than that in the chemicals arena, though more work is
required to provide the qualitative and quantitative information for low dose and low dose-ratemechanistic modelling.

2 The Committee notes that other researchers and organizations recognize the value of integration of epidemiological and biological
data through mathematical models [Preston, 2015; Chauhan et al. 2019], notably the National Council on Radiation Protection andMeasurement that has published recently in its Report 186 “Approaches for integrating radiation biology and epidemiology forenhancing low dose risk assessment” [N11]. These publications advocate the application of adverse outcome pathway approaches
for low-dose radiation risk assessment as they are utilized in chemical risk assessment.

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UNSCEAR 2020/2021 REPORTIs there evidence for tissue-specific variation in the mechanisms of response to ionizing radiation that
relate to the differing sensitivity of tissues to radiogenic cancer?
538. There is long-standing evidence that the number of mutational steps required for leukaemia is lessthan in the case of solid cancers, and this impacts on the time to presentation of leukaemia by comparison
with solid cancers. Skin stem cell populations have been found to have different responses to radiation interms of apoptosis and this appears to relate to the higher risk of basal cell carcinoma. More generally
stem cell populations appear to have a greater dependence on HRR than NHEJ, this may serve to provide
a relative degree of protection of stem cell populations from induced mutation.
Are the mechanisms that operate and can be associated with disease development similar following lowand high-LET exposures?
539. Given the higher frequency induction of complex/clustered damage after high-LET exposure, theDNA damage response to these lesions will play a greater role than following low-LET exposures,independent of the high-LET radiation dose rate. The repair of complex/clustered damage is morechallenging for the cell and often results in the reorganization of lesions into repair centres. Evidence
indicates that high-LET exposures readily trigger the G2/M checkpoint independent of dose. Modellingstudies for lung cancer suggest that high-LET exposures may have a greater promotional effect on premalignant cells and clones; however, low doses (50 mGy) of low-LET radiation act to promote the growthof Trp53 mutation carrying clones and cells in oesophageal epithelium in one study. Modelling thatindicates an inhomogeneous distribution of radon (and progeny) in the lung leads to a protracted highdose-rate exposure of a small population of cells, and this protracted irradiation is likely to impact onlung carcinogenesis and radon-related risks. Further insight into the effectiveness of high-LET radiation
may be gained by a better mechanistic understanding of the resulting DNA damage at a microdosimetriclevel together with consideration of the impact of spatial aspects of dose distribution on DNA misrepair.
540. Some general conclusions may be drawn in addition to the above responses to the review questions
originally set out. In the paragraphs that follow, a brief summary of the findings of this report is provided.
In this annex, the Committee has undertaken a comprehensive evaluation of the biological mechanismsthat are considered to contribute to or modulate carcinogenesis following radiation exposure, particularly
at low exposure levels (100 mGy and below for low-LET radiations and at dose rates of 0.1 mGy/min
and below). A complete understanding of the mechanisms and modulators of carcinogenesis following
low-dose and low-dose-rate radiation exposures is not yet available. Additionally, an appendix
considering principles and criteria for ensuring the quality of the Committee’s reviews of experimental
studies of radiation exposure is provided.
541. Little in the way of robust data could be identified that would prompt the need to change the current
approach taken for low-dose radiation cancer risk inferenceas used for radiation protection purposes andfor the purpose of comparison with other risks. The potential contributions of phenomena such astransmissible genomic instability, bystander phenomena and adaptive response remain unclear. It isnotable that since the Committee’s last major evaluation of contributory mechanisms for radiation cancer[U7], there have been substantial new data on low-dose and low-dose-rate risk from epidemiologicalinvestigations, particularly of occupational and medical cohorts. These studies have added to theepidemiological evidence underpinning low-dose and low-dose-rate cancer risk estimation.
542. The Committee concluded that there remains good justification for the use of a non-threshold model
for risk inference given the robust knowledge on the role of mutation and chromosomal aberrations in
carcinogenesis. That said, there are ways that radiation could act that might lead to a re-evaluation of theuse of a linear dose-response model to infer radiation cancer risks. Some studies indicate that low-doseand low-dose-rate exposures can extend lifespan and possibly reduce tumour burdens in experimental

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animals. Generally, there is insufficient mechanistic understanding of these observations, however. Thissituation may be improved for example, if low-dose exposures were shown consistently andunequivocally to stimulate DNA damage response/repair, or immune responses preventing cancer
development, such a consistent evidence base has not been found in this review. In this case, someelements of risk reduction may have to be taken into consideration alongside the established DNA
damage–mutational, and potential promotional pathways. Another example where additional evidence
would help assessment of risk are the findings relating to stimulation of tumour vascularization by lowdose exposures, where there is greater consistency and coherence of the available data. Stimulation oftumour vascularization would serve to promote tumour development.
543. The implications of the studies on the induction of transmissible genomic instability, bystander
effects and adaptive responses are still not clear. Some studies suggested thresholds for the induction oftransmissible instability and bystander effects at around 100 mGy low-LET radiation; if true, this would
indicate the phenomena are not relevant for low-dose cancer risk inference. Further, adaptive response
studies remain without a confirmed mechanistic basis and are of mixed outcome; similarly studies ofsamples from persons inhabiting areas with high natural background radiation levels that are interpreted
by some as providing evidence for adaptive response are insufficiently coherent to adopt for risk
assessment purposes.
544. Looking to the future, the recommended approach to combine mechanistic understanding of lowdose radiation carcinogenesis with epidemiological studies is to use mathematical modelling integrating
data from experimental systems (e.g. dose–response data for induction of key mutations or epimutations).
There exist good multistage model frameworks for this purpose that have the flexibility to include dataon somatic events and germ line influences on risk. These approaches may be used to test hypotheses andprovide further insights for risk inference. Consideration should be given to the use of adverse outcome
pathway approaches to help define and formalize key mechanistic steps in carcinogenesis following lowdose exposures. Additionally, experimental investigations may identify cancer risk indicators, that whenvalidated could be integrated into epidemiological investigations to improve statistical power, or be used
for population screening.

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UNSCEAR 2020/2021 REPORTACKNOWLEDGEMENTS
The Committee acknowledges with gratitude the work of the members of the Expert Group involved in
preparing this annex, in particular, S. Bouffler (United Kingdom), who chaired the Expert Group. The
Committee would like also to thank A. Friedl and W. Rühm (Germany) and J. K. Lee (Republic of Korea)
for critically reviewing the manuscript. The views expressed in this annex remain those of the Committee
and do not necessarily represent the views of the United Nations or its Member States.
In drafting the appendix on principles and criteria for ensuring the quality of the Committee’s reviews ofexperimental studies of radiation exposure, the Expert Group have drawn extensively on the UNSCEAR
2017 Report, annex A, “Principles and criteria for ensuring the quality of the Committee’s reviews of
epidemiological studies of radiation exposure” [U11], which was an extremely valuable source for this work.

Members of the Expert Group
Chair: S. Bouffler (United Kingdom); Members: S. Candéias (France), M. Eidemüller (Germany),
L. Mullenders (Netherlands), M. P. Hande (Singapore) and G. Woloschak (United States).

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APPENDIX A. PRINCIPLES AND CRITERIA FOR ENSURING THE
QUALITY OF THE COMMITTEE’S REVIEWS OF EXPERIMENTALSTUDIES OF RADIATION EXPOSURE

I. INTRODUCTION
A1.
Evidence from experimental radiobiological studies forms an important part of the scientificevaluation of radiation effects conducted and reported by the United Nations Scientific Committee on the
Effects of Atomic Radiation (UNSCEAR). In many UNSCEAR evaluations, experimental studies arereviewed, and their results used to inform the assessment of health risks of radiation, particularly in doseand dose-rate ranges where direct epidemiological evidence is not available. In common with radiation
epidemiological studies, experimental studies have strengths and limitations, and each study requires
careful and systematic assessment to gauge its contribution to the topic being addressed. The Committee’s
UNSCEAR 2017 Report, annex A, “Principles and criteria for ensuring the quality of the Committee’s
reviews of epidemiological studies of radiation exposure” [U11] marked an important strengthening of
the robustness and transparency of its evaluations, and there was a clear need to develop similar principles
and criteria for experimental investigations, and there are many similarities in the consideration of qualityof epidemiological and experimental publications.
A2.
The Committee’s evaluations commonly need to cover a broad array of experimental studies
conducted at the level of molecules, cells and whole organisms. Such studies are frequently conductedwith widely differing motivations, objectives, designs and results. Clear and transparent criteria thatdefine the processes and decisions for the inclusion or exclusion of individual studies are essential toensure that its evaluations meet the key requirement of objectivity, i.e. to use sound evidence in an
undistorted manner, regardless of the composition of the group of experts conducting the evaluation for
the Committee´s scrutiny, with assessment unaffected by non-pertinent features of studies.
A3.
The principles of systematic review are clearly applicable and fundamental for the Committee’s
evaluations. Compiling, summarizing and comparing the results from all relevant and sound evidence isessential for achieving the goal of the Committee in producing high quality evaluations. In common with
the quality criteria developed for epidemiological studies, this requires that the question to be addressedis clearly defined, a search of all evidence is carried out, relevant studies fulfilling the inclusion criteria
are identified, their results compiled, and conclusions drawn from the evidence.
A4.
The UNSCEAR 2017 Report [U11] on quality criteria for epidemiological investigations notedthat systematic reviews are an important tool for providing guidelines in medicine, largely due to theevidence-based medicine movement since the 1990s [E2, S1]. Evidence-based medicine offers broad
principles for translating research into policies and practice. The need for it arose largely from conflicting
guidelines prepared by professional societies and other bodies, and also from difference in the quantityof scientific literature available. A key tenet of evidence-based medicine is the requirement that practiceof medicine should be based on the best available scientific evidence. The concept of science-based
medicine was not first introduced with evidence-based medicine, but it emphasizes that the evidence must
be directly applicable, i.e. based on real patients and outcomes of treatment. For health effects of radiation

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exposure, this would translate into underscoring the importance of human epidemiological studies forwhich the UNSCEAR 2017 Report is available [U11].
A5.
However, it is recognized that human data are not always available for specific exposure
situations, notably at low doses of exposure where epidemiological investigations may not have sufficient
statistical power to detect health effects. In these situations, supporting evidence from biologicalinvestigations of radiation effects is valuable for the Committee’s evaluations. Furthermore, theCommittee considers the role of specific biological mechanisms relevant for health risk evaluation.
Recent examples of the Committee’s evaluations of biological and mechanistic data include, annex C,“Non-targeted and delayed effects of exposure to ionizing radiation” and annex D, “Effects of ionizing
radiation on the immune system” of the UNSCEAR 2006 Report [U7].
A6.
Health risk assessment frameworks have also been developed, combining several lines ofevidence from toxicology, epidemiology and other disciplines for estimating the health impact of
exposure to potentially harmful substances. In general, health risk assessment is a process consisting of
hazard identification, exposure assessment, dose-response assessment and risk characterization [I9] andit provides the basis for risk management and risk communication. Health risk assessment has been
widely applied by expert committees mainly for chemical agents [I8]. Across the world, systematic
reviews are increasingly becoming the basis for these assessments [F9].
A7.
The Committee’s evaluations synthesize studies of effects of radiation exposure relevant tohuman health to guide decision-making, assist in preparing regulations and inform the scientificcommunity and the public. The reports comprise evidence summaries and aim to be comprehensive andsystematic to provide a definitive evaluation of the current scientific knowledge and provide evidencebased conclusions. The Committee has previously stated, in its UNSCEAR 2012 Report, that a balanced
evaluation involves avoiding unjustified causal associations (false positives) and also unjustified
dismissal of real health effects (false negatives) [U10]. In common with the quality criteria for
epidemiological studies, the approach to ensure the robustness of evaluation of biological investigations
requires several sequential steps:(a) Clearly define the topic and objectives of the specific evaluation;(b) Develop a search strategy and perform a search that allows identification of all studies withpotential to contribute to the evaluation and its defined objectives;(c) Apply a uniform approach to critically appraising the quality of the studies;(d) Synthesize the available evidence from the studies by summarizing their results from thosestudies meeting criteria for inclusion (providing relevant, high-quality evidence);(e) Develop overall conclusions drawn from the systematically retrieved, assessed and summarizedstudies.
A8.
This appendix extends the UNSCEAR 2017 Report, annex A, that provided guidelines to judge
the quality of epidemiological studies [U11]. It aims to recommend procedures for assessing experimental
radiobiological studies and criteria for conducting scientific reviews of biological studies for theCommittee’s evaluations. These criteria make use of the experience of development of the criteria for
epidemiological evaluations, which in turn drew on evidence-based medicine approaches. Furthermore,this annex also re-iterates the importance of adhering to the Governing Principles of the Committee 3,3 https://www.unscear.org/docs/GoverningPrinciples_ADOPTED_20150605.pdf

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notably to its process in the selection of experts according to defined terms of reference, qualifications
and competence, and the declarations of potential conflicts of interest are essential steps in securing
quality and scientific excellence of the Committee’s evaluations. All conflict of interest statements
collected in the framework of the Committee’s work are available at the Committee’s secretariat.

II. OVERVIEW OF EXPERIMENTAL RADIOBIOLOGICAL STUDY
TYPES
A9.
There is a wide range of biological and mechanistic study types that the Committee may consider
in its evaluations. Potentially relevant studies may be conducted at the molecular, cellular or wholeorganism level. All investigations examine the effect a given (and usually well defined) radiation
exposure has on a biological endpoint. There is a very wide range of endpoints that might be considered
in relevant studies, including but not restricted to reproductive outcomes, specific diseases such as cancers
or circulatory diseases, tissue damage, cell killing, damage to DNA/chromosomes, damage tomitochondria and other cellular components, enzymatic activities and molecular interactions.
A10.
Irrespective of organizational level or endpoint under consideration, there are some common
features required in all studies:(a) Definition and characterization of experimental model in which effects are to be investigated;(b) Definition and documentation of radiation exposure;(c) Definition and documentation of the endpoint under consideration;(d) Determining the magnitude and variability of the effect (if present), including power
calculations and robust statistical analysis where required;(e) Studying dose–response relationships;(f) Review the results generated and their relevance to human health.
A11.
A critical difference between experimental studies and epidemiological investigations is theability to control the conditions of an experiment. Inclusion of appropriate positive and negative controls
in experiments is essential for correct interpretation of results. Evaluation of experimental outcomes inblind fashion is an important contributor to the avoidance of bias. All investigations require the inclusionof exposed and non-exposed subjects and will often include investigation of dose-, dose-rate or radiation
quality-dependence of the effect under investigation.
A12.
Careful consideration of the experimental model selected is of great importance and impacts on
the extent to which findings and conclusions are generalizable. In the case of studies relating to humanhealth, a close relation to the human in evolutionary terms and a close relation to the disease in question
is important. More fundamental work might be investigating specific biological processes at the cellularor biochemical/molecular level and the relevance of these investigations to human health may only
emerge later.
A13.
Biochemical and molecular level investigations can provide information on the effects ofradiation exposure on fundamental biological processes. These may not necessarily relate directly to a

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radiogenic disease. As an example, a great deal of mechanistic detail of DNA repair pathways has been
gained through biochemical and molecular approaches, sometimes utilizing modelled DNA damage(e.g. restriction enzyme breaks as a model of radiation-induced DSBs).
A14.
Investigations at the cell level are perhaps the most common form of study, but these can include
a wide range of cell types of varying relevance to human health. It is intuitive that human cells are thebest model for human disease-related studies, and a similar consideration would apply if the research
related to a given non-human species. Primary cells at early passage are generally taken to be more similar
to cells in vivo. At later passages damage, mutational and epi-mutational changes may accumulate inculture. Specific adaptions to allow permanent culture such as viral transformation can present substantial
difficulties for cancer-related investigations and less weight should be given to studies with these celltype. The more recent advent of cell lifespan extension through TERT transformation is considered tohave fewer effects on normal cell function.
A15.
Cellular studies need to provide a cell culture environment close to the in vivo situation.
Differences between in vivo and in vitro oxygen levels can be substantial and impact on the results
observed. In general, 3D culture models are thought to more closely mimic the in vivo tissue environment
than classical 2D cell culture. Heterotypic cultures including more than one cell type should be a better
mimic of the in vivo situation by comparison with homotypic cultures. Both 3-D and heterotypic cell
culture methods remain at relatively early stages of development, and improvements can be anticipated.
A16.
Studies at the whole organism level, by definition, bear a closer physiological relationship toindividual humans but there are many constraints. In common with cellular studies, the use of organisms
with a close evolutionary relationship to the human is important. The range of available animal models
is in principle wide, but most utilize rodents, particularly mice, as a well characterized mammalian system
where both inbred and outbred strains are available. There is increasing use of genetically modified mousemodels where genes are inactivated or mutated in the laboratory. Such models can provide usefulmechanistic insights into the pathways of disease pathogenesis, but relatively rarely reflect genetic
variation within the human population. The use of models with closer relationship to the human, primatesfor example, is possible though relatively rare due to the housing requirements, lifespan, and ethicalconsiderations. Biologists have additionally developed lower eukaryote models such as Drosophila andthe nematode Caenorhabditis elegans, yeast and bacterial models have also been used among others.
A17.
Irrespective of the experimental model selected, a sufficient sample of observations is required
to ensure statistical significance of the investigation. Statistical power calculations can be performed to
inform on suitable sample sizes if there is sufficient prior information available on the endpoint/ outcome
measure (see chapter III below).
A18.
The outcome measure selected for study is another critical factor, with those most proximal toa disease or effect being preferred. The appropriate selection of the outcome measure is highly dependent
on the current state of knowledge on the disease/effect of interest. For example, there is a very large
knowledge on the mechanisms of carcinogenesis summarized in the “hallmarks of cancer” [H10], whileless is known of mechanisms contributing to cataract formation [H8]. The sensitivity of an endpoint andits variability due to technical variables or biological response variation is an important consideration.
A19.
All investigations require careful characterization and documentation of the radiation exposure
regime. The doses and dose rates used should closely parallel those of the natural/actual exposure underconsideration. But limitations are present due to the sensitivity of the endpoint under consideration. In
principle the use of relevant doses/dose rates and their adequate documentation is easy in a laboratory
situation, but not all papers clearly document the radiation source used, doses and dose rates employed.

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A20.
Both qualitative and quantitative investigations can be of use but in general quantitative
investigations are more informative. Quantitative studies require statistical evaluation of theirsignificance. The statistical robustness of a study can be affected by the sample size (see above), inherent
and technical variability of the endpoint. Technical variability can be assessed through the inclusion oftechnical replicates (i.e. utilization of the same sample with independent tests/assays). Assessment of
biological variability requires the use of fully independently generated biological samples.

III. MAIN FEATURES AFFECTING THE QUALITY OF
EXPERIMENTAL BIOLOGICAL STUDIES
A21.
Assessment of research evidence needs to consider the quality of studies available. This chapter
discusses the core features influencing the quality of experimental biological studies. All these issues are
relevant when reviewing study quality and should not be viewed separately. For studies of radiation
effects that can inform health risk assessment, the experimental model system selected and its relevanceto human and the experimental endpoint/outcome measure and its relevance to the disease/health effectunder consideration are of particular importance.

A. Experimental models system
A22.
As noted above this is of importance for studies that aim to improve human health risk
assessment. The model should have a close evolutionary relationship to human and as closely as possible
recapitulate the in vivo physiological situation. Thus, studies utilizing mammalian models would beviewed as more directly relevant than say studies using Drosophila. There are of course ethicalconsiderations that need to be taken into account as well as husbandry issues. Obtaining ethical approval
for experiments on primates, while closer to human in evolutionary terms, is more difficult. In addition,
the housing requirements and lifespan are likely to present constraints. Genetically modified models canbe of use to determine if a given pathway is relevant to a disease, direct translation of findings to humanneeds to be carried out with caution as rarely do similar genetic mutations exist in populations.
A23.
Cell models are ideally of human origin, but a wider range of mammalian cell lines have beenused to inform human disease mechanisms. 2D culture is not a realistic representation of tissue
architecture, and so 3D heterotypic cell models/organoids can be seen as more relevant. One factor thathas often been overlooked in cell culture experiments is the oxygen concentration. In vivo concentrationsof oxygen in tissue are lower than ambient concentrations. The use of low physiologically relevant
oxygen conditions can have a substantial impact on study outcomes and should be preferred.
A24.
When considering biochemical investigations, the use of proteins and other biochemicals ofhuman origin can be viewed as more directly relevant. Nevertheless, the above considerations potentiallyrelevant information can and has been derived from a wide range of experimental model systems at alllevels of organization and a wide range of species. Observations at one level or in one species can beextended to a more relevant model following initial observations and demonstration of principles.

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B. Radiation exposure
A25.
By contrast with epidemiological investigations, exposure to radiation can be carefully
controlled in experiments. The source used should be well characterized and exposure conditions well
documented. Dose and dose rate are of course important considerations and should be of relevance for
actual human exposures where possible. However, it is rarely possible to exactly mimic human exposurein terms of duration or dose. Based on current understanding, to be of relevance to the assessment of riskof low-dose exposures, doses should be of 100 mGy and less for low-LET exposures, and low-dose-rate
exposures should be 0.1 mGy/min or less.

C. Endpoint/outcome
A26.
Depending on the level of biological organization selected for an investigation, theendpoint/outcome may be a disease or a known or speculated intermediate step on a disease pathway.
Thus, many experimental rodent studies of radiation carcinogenesis are available. Here the disease ingeneral is clearly of relevance but some rodent cancers bear closer histopathological similarity to humandisease than others.
A27.
If an investigation concerns endpoints prior to disease presentation their relevance needs to beestablished. Many mechanistic investigations are undertaken to try to establish the relevance of an
endpoint or pathway to a particular disease while others are conducted simply to observe and describe
effects of radiation on the experimental system. Useful information for the Committee’s evaluations canbe found in both types of investigation.

D. Experimental conditions
A28.
The protocol adopted for any investigation requires to be documented and the fundamental
principles of experimental design must be respected. Controls, both positive and negative should be
included, independent biological and technical replicates should be included, sample sizes should be
sufficient to ensure suitable statistical power. Documentation of procedures must be sufficient to allow
for independent replication. Where an effect has been observed to occur using multiple analytic
approaches it can be viewed to be more firmly established.
A29.
Any experimental study of the effects of radiation is limited by the timeframe underconsideration. All biological processes are dynamic and so selection of a suitable timepoint(s) for
evaluation of outcome endpoints is important. Studies that take multiple timepoints are likely to be more
informative than those with one or a few. For example, studies of the effectiveness of DNA double-strand
break repair will depend critically on the timepoints for analysis selected, and caution should be taken in
analysis where any given process is not observed to come to completion.
A30.
A finding that has been replicated in an independent experiment in an independent laboratory ismore robust than a one-off reported finding. Exact replication of a given study may not always be
available but supporting findings help to provide a more robust evidence on which to base conclusions.

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E. Analysis
A31.
The analysis of results should be based on a study plan developed in advance. A clear andtransparent description of experimental results is necessary. To improve the transparency of researchfindings, making the original (non-patient/participant identifiable, if human samples are used) data
publicly available is encouraged, for example, by the open science framework and many journal editorial
policies. There are also well-established databases for genomic and transcriptomic data. Confidentiality
requirements have to be kept in mind when making study data available to the scientific community.

F. Variation
A32.
Some consideration of the variation in response between individuals or populations will be of
relevance to drawing conclusions on the general relevance of findings. Some variation in biologicalresponse is expected – usually taken to be around 10%, but important inter-individual or inter-groupdifferences may be apparent. For example, the induction by radiation of delayed transmissible genomic
instability has been reported in cells from some but not all individuals, and this may have a genetic basis(e.g. [K3]). As well as variation in response due to genetic factors, age may be of importance (e.g. [B4,G7, O8]). Similarly, sex may be a factor that affects individual response.

G. Reporting
A33.
While a study may have been conducted with great accuracy and care, the written report may
not necessarily be of the same quality due to using selective reports or omitting details of the study planand procedures. Reporting standards for a wide range of study types are available, e.g. qualitative studies[O1], the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments)
guidelines for quantitative PCR studies [B60].
A34.
It is sometimes the case that a research report is found to be erroneous and has to be withdrawn.
This may not always be apparent from literature searches, and so those reviewing studies should takesteps to ascertain if individual reports have been withdrawn.
A35.
The PRISMA 4 guidelines [M39] support transparent and comprehensive reporting of systematic
reviews and meta-analyses. While the focus of PRISMA is on healthcare interventions, the checklist
items can be applied with minor adaptation to systematic reviews of observational epidemiologicalstudies or experimental studies [M39].

4 Referred reporting items for systematic review and meta-analysis protocols.

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IV. PROCESS OF SYNTHESIS OF RESEARCH RESULTS
A36.
For radiation risk assessment, experimental/mechanistic studies can provide supporting
evidence to quantitative epidemiological assessments of risk. For traditional narrative reviews ofexperimental/mechanistic studies, no rigorous methodological standards have been established. Any such
review would aim to provide assessment of weight or strength of evidence, i.e. judgement of value orcontribution of studies commonly reporting a wide range of results, some contradictory. However, thedegree of credibility assigned to each study and formulation of summary measures does not tend to follow
formal procedures. Hence, the appraisal of the balance of evidence is heuristic and generally based on ad
hoc criteria. This methodological “softness” makes such reviews susceptible to judgemental bias,
however not intentionally.
A37.
For literature synthesis and evidence assessment, which are both essential components for theCommittee’s evaluation of experimental studies, appraising their quality of evidence is a key task. Twolevels of quality are to be considered. The first is the quality of any individual study used for the topic.
As noted in chapter III, many factors can affect the quality of experimental biological investigation.
A38.
The next step in the assessment of quality pertains to the totality of the evidence, i.e. all thestudies used for reporting on the topic of interest for the Committee’s evaluation. In the overall
assessment of the available evidence, low-quality studies and those using less relevant model systems or
endpoints should be assigned less weight than high-quality studies, or even excluded if the quality
limitations are critical.
A39.
For a scientific evaluation of a defined research topic according to established scientificprocedures and values (thus following Governing Principles of the Committee), a systematic approach isrecommended, including the following steps:−

Step A: Definition of the topic for which evidence is required;−

Step B: Transparent and systematic collection of information, based on a clearly defined scope,
objectives and protocol;−

Step C: Abstraction of relevant data from selected studies or other sources of information;−

Step D: Assessment of individual study quality following unambiguous and consistent
standards;−

Step E: Synthesis of information;−

Step F: Drawing of conclusions and consideration of future research needs.

A40.
Well-conducted and reported studies relevant to the topic in question utilizing relevant models
and endpoints should always form the main input into the Committee’s evaluations. Lower quality studies
can and should be identified and evaluated with a transparent decision either to exclude them or to include
them with a lower weight assigned to their results. Conclusions and summary statements should rest
primarily on the best quality studies. The lower quality studies can be assessed to attempt to determine
whether their results are generally consistent with or strongly deviate from the summary results.
A41.
The different steps in the process of research synthesis, including the definition of the study
question (topic), systematic evidence search, study identification, quality assessment, synthesis andderivation of conclusions have been described in the UNSCEAR 2017 Report, annex A [U11]. Verysimilar considerations apply to experimental studies.

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A. Definition of the topic for which evidence is required
A42.
A crucial issue at the planning stage of a scientific evaluation is a clear and precise definition of
the question or topic the evaluation seeks to cover. The scope of the planned work should be clearly
outlined in a document plan, ideally with precise and answerable study questions organized byexperimental model, exposure type, comparison, and outcome/endpoint. A protocol giving a clear
definition and description of exposure, including dose levels of interest and further exposure details,
should be produced. Likewise, the outcomes/endpoints of interest ideally need to be specified in advance,and eligible study designs and outcome measures outlined. This preparatory work should result in athorough and detailed work plan. Changes to the protocol and to the agreed scope/direction of theevaluation should be avoided. Any necessary changes should be clearly documented and endorsed by theCommittee or the scientific experts charged with an evaluation by the Committee.

B. Collection of information: searching the literature
A43.
Systematic reviews address a clearly defined objective, use transparent and reproducible
methodology and include a systematic literature search for all studies that meet predefined eligibility
criteria. The systematic literature search results in a set of research reports consequently used for theoverall evidence assessment and research synthesis. Systematic reviews should depend mainly on
primary peer-reviewed publications rather than reviews.
A44.
The substantial growth of new journals, particularly those with an open-access model, overrecent years has changed the scientific publishing landscape. There are many (online) journals with nontransparent quality control measures that nevertheless claim to be peer-reviewed and reputable. Thisdevelopment makes research synthesis more difficult and calls for even more emphasis on strict quality
assessment of publications that are to be included in evidence synthesis.
A45.
There are many ways to find relevant literature. A systematic search of one or more electronic
databases for specified key words should provide for completeness of information and extend theknowledge base, minimizing the possibility of selective inclusion of research reports. Nowadays, several
databases of scientific literature are used, such as Medline/PubMed, Scopus or EMBASE. These canprovide a good coverage of the published literature, but augmentation by expert databases and expert
knowledge of the respective literature is often warranted particularly for very specific topics that may not
be identified through keyword searches. A standard procedure is to also cover the reference lists ofincluded publications and of related research overviews. Through its revision and discussion process, theCommittee additionally ensures that further literature contained only in expert databases can be screened
for relevance and inclusion. Unpublished research (“grey literature”) is more difficult to find, and whileevidence-based medicine systematic reviews nowadays do include unpublished research, the Committee
usually does not use unpublished literature. However, reference lists of unpublished work may hint tofurther published research, and at times such work may be useful for hypothesis generation or for
highlighting previously unconsidered facts. Even if English is the predominant language in scientificpublishing, studies published in other languages can also contribute to the knowledge base and should,
as a rule, be identified and evaluated. Expert groups often comprise members from several different
countries and can thus potentially provide for expertise in different languages.
A46.
For searches in electronic databases, a translation of the review topic into the language of thedatabase (e.g. in terms of specific keywords) is required, and also a clear and concise use of search terms
for the model(s), endpoint(s) and exposure(s) of interest, for the comparisons (e.g. in relation to dose,

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dose rate or radiation quality) and the outcomes (cell level, molecular level or in vivo model system).
Searches may be limited to particular models/endpoints or publication types using suitable index terms.
Spelling variants and natural language variants of text terms should be considered [E18]. Overlap
between publications because of several publications deriving from the same material needs to be
identified and taken into account.

C. Abstraction of relevant data from selected studies or othersources of information
A47.
Documentation of the literature search and selection process is needed so that the search can be
repeated or updated at a later stage. Methods used for retrieval of information and data in the report should
be described, including evidence abstraction (e.g. was it based on titles and abstracts, review of full texts?),
criteria for inclusion, and quality assessment. The UNSCEAR 2017 Report, annex A, “Principles and criteria
for ensuring the quality of the Committee’s reviews of epidemiological studies of radiation exposure”
considers the presentation of systematic review protocols based on the PRISMA guidelines [U11].

D. Assessment of study quality
A48.
Once the studies to be included have been identified, core features are abstracted for further
assessment, as described in the UNSCEAR 2017 Report, annex A, “Principles and criteria for ensuring
the quality of the Committee’s reviews of epidemiological studies of radiation exposure” [U11]. In thecase of experimental studies, these features usually include study identifier (e.g. author, year), publication
type (e.g. peer-reviewed, review etc.), relevance of publication to review questions being addressed,radiation exposure characteristics – quality, dose rate, dose, and summary of main findings. Further
comments or details relevant to the respective study can be added. Study quality and relevance are also
assessed. Study quality assessment aims to provide a clear view of the robustness of the reported findings(statistical considerations, methodology, replication or verification by independent investigators). Study
quality assessment forms an integral part of evidence synthesis, and must be performed in a transparent
and reproducible fashion.
A49.
Risk of bias or quality assessments are focused on the methodological quality of the study, andnot on issues such as magnitude of effect or applicability of results. It should be noted that study size or
statistical power to identify effects (if existing) are not necessarily included in the quality assessment but
need to be considered in the overall assessment of the evidence. For the Committee’s work, the following
domains should be addressed: (a) exposure delivery; (b) methods for measuring endpoint/outcome;(c) sources of bias specific to the study design; (d) methods to control for confounding; (e) statisticalmethods; (f) study reporting; and (g) statement on conflict of interest of authors.
A50.
Based on the above assessments and information abstracted from publications a judgement canbe made on its relevance and importance for the review, and a decision made as to include or exclude
consideration of the publication in the Committee’s reports. A quality weighting approach can be used
but is not essential – such an approach would have to consider the relevance of the experimental model,
the endpoint/assay reported, relevance of the radiation exposure, quality of the experimental design,quality of the statistical analysis, study reproducibility.

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A51.
The result of the quality assessment/inclusion/exclusion for each study is documented, and thereview should then describe how the quality rating approach is used in the further steps of the review.
There is little use in assessing quality without utilizing its results. Studies with critical risk of bias or
methodological failings should generally be excluded. This also pertains to studies with seriously
inadequate dosimetry or endpoint assessment.
A52.
Applying the approach recommended by the Committee will ensure that research overviews will
be more informative as they identify and summarize the best and most relevant studies on a given topic
and put less or no weight on flawed or non-relevant investigations. Providing transparency in quality
assessment as outlined here will form a good scientific basis for the Committee’s discussions on the
merits of study-specific scientific evidence used for the Committee’s evaluations.

E. Synthesis of studies: meta-analysis and narrative
approaches
A53.
The subsequent step of the literature review process is the actual study synthesis andinterpretation. Narrative synthesis involves description and summary of the core findings from theincluded studies of varying structure and method. This involves grouping of the studies, for example
according to study design, population or outcome, and providing a verbal account of the body of evidence
relevant for the review question. On the basis of focused description of the individual studies, theevidence across the different studies is summarized and jointly assessed. When few studies are available
or are very diverse in terms of important characteristics, a narrative review may be more appropriate thanmeta-analysis of data that are not truly compatible. When large numbers of relevant studies are available
tabulation of the key features of each study can be very valuable. Such tabulations should document(a) the experimental model – species, cell or tissue type; (b) radiation exposures used, the radiation
quality, and both dose range and dose rate; (c) reported effect/endpoints; and (d) publication reference.
A54.
An important concern is the potential for publication bias, i.e. the selective reporting of studies,often those with statistically significant findings. Publication bias can seriously affect the synthesis ofevidence, and several methods to detect and evaluate publication bias have been developed for use inmeta-analyses [E9, J22].
A55.
Unlike the situation when synthesizing epidemiological evidence, rarely is it possible toundertake mathematical analyses of risk (or other relevant) values, thus meta-analysis and statisticalpooling is seldom employed. These analyses will need to be independently reviewed.

F. Reaching an overall conclusion from the evidence
synthesis
A56.
The final part of the evaluation of experimental studies should address and provide insight into
the systematic review questions posed at the outset. As noted, only very rarely is it possible to undertake
numerical analyses as one can in epidemiological studies of risk. Thus, reviews are generally of thenarrative type giving some indication of weight of evidence and confidence in the review findings.

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UNSCEAR 2020/2021 REPORTA57.
As described in the UNSCEAR 2017 Report, annex A [U11], a more articulated approach withwider assessment of various aspects of evidence has been developed within the Grading ofRecommendations Assessment, Development and Evaluation (GRADE) collaboration for the CochraneLibrary and has also been adopted by some international institutions involved in guideline development,
including the World Health Organization [W9]. The GRADE categorization [G9] is usually specific toan outcome, for example cancer site-specific incidence. Application of such criteria, or variants thereofin narrative reviews is possible but not essential.
A58.
Transparent organization and reporting of evidence is also an important step towards reducing
differences of understanding and subsequent interpretation between expert evidence synthesis, the way
the Committee uses this synthesis, and public understanding of the topic. To support transparency andclarity, standardized vocabulary and terminology with clear definitions are required. This would also help
to ensure that reports can be translated accurately into other languages.
A59.
The Committee’s draft evaluations, including syntheses of experimental evidence, have always
been subject to close scrutiny (independent critical review) and discussion at its annual sessions. This is
an important step in continuous quality control and encompasses all steps of scientific reportdevelopment, with critical revisions from experts with different scientific backgrounds. The Committee
also assesses the consistency and plausibility of experimental evidence synthesis with results fromepidemiology and indicates research needs arising from the overall assessment. The identification of
uncertainties regarding the respective topic and possible approaches to their reduction will usually be partof the Committee’s research recommendations.

V. CONCLUSIONS
A60.
Individual reported experimental studies on biological mechanisms relevant for the inference ofcancer risks from low-dose radiation provide the basic evidence on radiation effects, and this annex
provides guidance on assessing the quality of individual studies and of the synthesis of evidence fromseveral studies. Methods of evidence synthesis have evolved considerably during recent decades. The
current methodological standards define procedures for literature search, evaluation of quality, combining
research results and grading the overall strength of evidence. The current methods of evidence synthesis
are systematic reviews, which are regarded as the state-of-the-art scientific standards for pooling research
evidence and superior to traditional narrative reviews.
A61.
The Committee will benefit from adopting a framework that is informed by these developments.
However, the specific nature and scientific contents of experimental radiobiological studies makeapplication of generic quality criteria problematic. Therefore, this annex provides the Committee’s
approach to radiation experimental study quality assessment and to synthesis of findings across studies.
The approach provides for increased methodological rigour, which could enhance the degree of
coherence, transparency and objectivity in assessments.
A62.
The evidence synthesis approach outlined in this annex is focused on evidence fromexperimental radiobiological studies; previously the Committee has published similar quality criteria for
epidemiological studies [U11]. The Committee draws on these fields and others to reach conclusions on
the assessment of radiation health risk.
A63.
Overall, the Committee aims to continue working with a quality-oriented systematic review
approach for its evaluation wherever applicable, based on the concepts described in this appendix. WhileANNEX C: BIOLOGICAL MECHANISMS RELEVANT FOR THE INFERENCE OF CANCER RISKS ...

183

the focus on study quality and the explicit review of strengths and limitations of experimental
radiobiological studies is a long-standing feature of the Committee’s work, using well documented
quality criteria in a systematic way as outlined here has not been always applied to the Committee’s
evaluations. In summary, a high degree of transparency, quality and a systematic approach to collecting,analysing and interpreting information for the Committee’s evaluations and assessments will help tomaintain the high scientific standard necessary for its widely appraised reports.

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GLOSSARY
Note: The glossary is mainly based on the glossary included in the UNSCEAR 2012 Report[U10] and other relevant UNSCEAR reports to ensure consistency of terms and definitions.
Abscopal effect: A biological consequence induced by radiation that is observed at a distance from theirradiated tissue.
Absolute risk: Excess risk attributed to an agent and usually expressed as the numeric difference between
exposed and unexposed populations (e.g. five cancer deaths over a lifetime per 100 people, each irradiatedwith 1 Sv).
Absorbed dose, D: The fundamental dose quantity given by:

D=

dε
dm

where dε is the mean energy imparted to matter of mass dm by ionizing radiation. The SI unit for
absorbed dose is joule per kilogram (J/kg) and its special name is gray (Gy).
Activity: The rate at which spontaneous transformations occur in a given amount of radioactive material.
(Strictly, the expectation value of the number of nuclear transformations occurring in a given quantity ofmaterial per unit time.) It is measured in becquerels (Bq); 1 becquerel equals one transformation persecond.
Acute exposure: Exposure received within a short time period (see also “Protracted exposure” or“Chronic exposure”).
Adaptive response: Refers to the phenomenon by which cells irradiated with a sublethal dose of ionizing
radiation (an “adaptive” dose of a few centigrays) become less susceptible to subsequent exposure to highdoses of radiation (a “challenge” dose of several grays).
Adjustment: The process of statistically accounting for effects of differences between groups or
populations under comparison in order to control confounding. Adjustment is frequently performed whenestimating effect measures from epidemiological data, for example by stratification or multivariate
regression analysis.
Allele: One of several alternative forms of a gene (or DNA sequence) at a specific chromosome
allocation. At each autosomal locus, an individual possesses two alleles, one inherited from the father
and one from the mother.
Aneuploid: The chromosome number is not an exact multiple of the haploid number; an individual withan aneuploid chromosome number; usually refers to an absence (monosomy) or an extra copy (trisomy)
of a single chromosome.
Antioxidants: Substances preventing oxidation.
Apoptosis: Cell death caused by an intracellular pathway which can be activated by external stimuli.
Attained age: Age at observation or during the follow-up, usually refers to age as time-dependent
characteristic in a cohort study.

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Becquerel (Bq): The SI unit of activity, equal to one transformation per second. As the unit is so small,multiples are frequently used such as megabecquerels (MBq) which is 106 or a million becquerels (1 GBq
is 109 Bq; 1 TBq is 1012 Bq; and 1 PBq is 1015 Bq).
Bias: A statistical estimation procedure is “biased” if the expected value of the estimate of the quantityof interest is not equal to the true value of the quantity. The “bias” of the procedure is the difference
between the expected and true values.
Biodosimetry: The use of biological (or chemical, physiological) markers of exposure to reconstruct
acute or protracted radiation doses.
Bystander effect: A non-targeted cellular action of radiation exposure meaning that not only the cell that
was actually hit shows radiation consequences, but also neighbouring cells.
Candidate gene: Any gene by virtue of its property (function, expression pattern, chromosomal location,
structural motif, etc) is considered a possible locus for a given disease.
Carcinogen:An agent, chemical, physical, or biological, that can act on living tissue insuch a way as to cause a malignant neoplasm.
Solitary or complete: The agent does not need additional action of further
exogenous cancer risk factors to cause a neoplasm.
Indirect or pre-carcinogen: The agent has to be transformed to its active
molecular form (ultimate carcinogen) in the metabolism.

Case-control study: An epidemiological study type, where cases with the health outcome of interest are
selected from the population and compared in terms of exposure with control free of the outcome, who
represent the exposure distribution in the population. The effect of exposure on the outcome in a casecontrol study is expressed as an odds ratio.
Causation: The relationship between cause (here, radiation exposure) and outcome (radiogenic healtheffect or associated risk). The term can also mean the action of causing something.
Chromosomal aberration: A change in chromosome structure or number from the normal chromosome
complement of an organism.
Chromothripsis: A form of deleterious genome rearrangement where one chromosome is fragmented
and multiple parts are re-integrated into the genome of a somatic cell, observed in some cancers by wholegenome sequencing.
Chronic exposure: Exposure persisting in time. (See also: “Acute exposure” or “Protracted exposure”.)
Co-factor: A substance or agent that acts with another substance to bring about certain effects; e.g.
coenzyme, a low-molecular entity needed for enzymatic activity of the apoenzyme.
Coding sequence: Those parts of a gene from which the genetic code is translated into amino acidsequences of a protein.
Cohort study: An epidemiological study type, where people free of the health outcome of interest are
selected and subdivided into groups based on exposure of interest. The occurrence of the health outcome(typically incidence or mortality) is recorded for the entire study population and the effect of exposureon the risk of the outcome is expressed as a rate ratio, risk ratio or hazard ratio. Follow-up for development

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of disease cases since start of study period can be prospective or retrospective (collection of informationof the cases that have already occurred).
Confounding factor or confounder: A confounding factor is a variable that is correlated with both theexposure (e.g. radiation exposure or dose) and the outcome variable (e.g. risk of lung cancer) and, if notcontrolled for analytically, may distort the conclusions. For example, occupation may be a confounding
factor in a study of the relation between lung cancer incidence among non-smokers (dependent variable)
and medical radiation exposure (independent variable). For instance, air crew are exposed to higher levelsof radiation due to their employment (correlation with radiation exposure) while staff working in certain
recreation industries are often occupationally exposed to cigarette smoke (correlation with outcome lungcancer). This confounding might be controlled by introducing into the analysis an indicator for theoccupational group.
Cytokines: Small secreted proteins that mediate intercellular communication and signalling.
Deletion: Loss of a portion of a gene or chromosome; a type of mutation; a synonym for deficiency.
Diploid: A full set of genetic material, consisting of paired chromosomes – one chromosome from eachparental set; most animal cells except the gametes have a diploid set of chromosomes (compare haploid).
DNA methylation: The addition of methyl groups to the 5-position of cytosine by DNA
methyltransferases (Dnmts). High level methylation of genes or gene regulatory sequences usuallyindicates a transcriptionally inactive state. A form of epigenetic modification.
DNA sequence: The relative order of base pairs, whether in a fragment of DNA, a gene, a chromosome,or an entire genome.
Dose: A measure of the energy deposited by radiation in a target. Dose can be used as a shorthand forabsorbed dose and effective dose when the context is clear.
Dose rate: Dose relative to time during which the dose is received. In experimental studies dose rate isoften expressed per minute, while in epidemiological studies of long-term exposure dose per year isfrequently used.
Doubling dose (DD): An important concept used in genetic risk estimation. It is defined as the dose ofradiation required to produce as many mutations as those which occur spontaneously in a generation. It
is calculated as a ratio of the average spontaneous and induced rates of mutations in a defined set ofgenes.
EAR: See “Excess absolute rate/risk”.
Effective dose, E: The sum over all tissues and organs of the equivalent doses weighted by the tissue
weighting factor wT, which represents the contribution of that organ or tissue to the total detrimentresulting from uniform irradiation of the whole body. E has the unit joule per kilogram, which is given
the name sievert (Sv). The recommended values of wT are given in ICRP Publication 103 [I15].
Epigen, epigenetic: Changes in an organism brought about by alterations in the expression of genetic
information without any change in the genome itself; the genotype is unaffected by such a change but thephenotype is altered.
Epigenetic modification: The modification of DNA or associated chromatin proteins that leads to alteredexpression of genes. DNA methylation, histone acetylation and methylation are among the epigenetic
marks currently known.

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Equivalent dose: Fundamental dosimetric quantity for the purposes of radiological protection defined as
the product of the absorbed dose in the tissue or organ and the appropriate radiation weighting factor forthe type of radiation giving rise to the dose; measured in sieverts (Sv).
Equivalent dose, HT: The averaged absorbed dose in tissue or organ T, modified by the radiation
weighting factor, wR. The unit of equivalent dose is joule per kilogram, and it is given the special namesievert (Sv). The recommended values of wR are given in ICRP Publication 118 [I2].
ERR: See “Excess relative risk/rate.
Error: The difference between an observed or estimated value and the true (but unknown) value. Since
the true value is unknowable, error cannot be quantified. Error can be contrasted with uncertainty, which
can be estimated by repeated measurements or Bayesian inference.
Excess absolute risk/rate (EAR): The difference between the hazard rate in an exposed population andthe “baseline rate” in that population. This is often a function of dose, age (or some other measure oftime) and other factors (effect modifiers). Excess absolute rate is often called “excess absolute risk” or“excess rate”. Strictly risk would apply to a prospective estimate inferred from the data and reasoning,
while rate would be the direct statistic calculated from the data.
Excess relative risk/rate (ERR): The “relative risk/rate” minus one. The excess relative rate is strictlya statistic calculated from observed frequencies/rates, while the excess relative risk is a prospective
estimate inferred from the data and reasoning. The ERR is often considered as a function of dose andother factors.
Excess risk/rate: A measure of the statistical relationship between a given risk factor and a specificoutcome. Depending on the context it can refer to some characterization of the influence on rates such as
the “relative risk”, “excess relative risk”, or “excess (absolute) rate”, or, perhaps most appropriately, toestimates of the risk over some period of time (lifetime risk) associated with an exposure of interest. The
excess rate is strictly a statistic calculated from observed frequencies/rates, while the excess risk is a
prospective estimate inferred from the observations and reasoning.
Exon: A region of a gene containing a coding sequence. Most genes have several exons separated byintrons (non-coding) which are usually longer.
Exposure: Concentration, amount, or intensity of a particular physical or chemical or environmental
agent that reaches the target population, organism, organ, tissue, or cell, usually expressed in numericalterms of substance concentration, duration, and frequency (for chemical agents or microorganisms) or
intensity (for physical agents such as radiation). In the radiation field, exposure may also denote theelectrical charge of ions caused by X- or gamma rays per unit mass of air; however, the term is used inits more general sense as described here.
γH2AX foci: Nuclear foci or spots detected by immunofluorescence using antibodies specific for thephosphorylated form of histone γH2AX. An indicator of DNA double-strand breaks. A number of othernuclear proteins from foci after DNA damage.
Gene: The fundamental physical and functional unit of heredity. A gene is an ordered sequence of
nucleotides located in a particular position on a particular chromosome that encodes a functional product.
Genome: The complete DNA sequence of an organism.
Genomic instability: An all-embracing term to describe the increased rate of acquisition of alterationsin the genome. As compared with the direct actions of radiation, i.e. those consequence directly induced

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by energy deposition, radiation-induced instability is observed in cells at delayed times after irradiationand manifests in the progeny of exposed cells multiple generations after the initial insult.
Genotype: (a) the genetic constitution of an individual; (b) the types of alleles found at a locus in an
individual.
Gray (Gy): Unit of absorbed dose, equal to 1 joule per kilogram (J/kg).
Hazard: In epidemiology and statistics, a measure of occurrence obtained as time integral of risk andinterpreted as risk intensity. With respect to aspect of health and safety, the term “hazard” refers to any
source of potential damage, harm or adverse health effects.
Health effects (of radiation) (also radiogenic health effects):
Deterministic (health) effect: A health effect of radiation for which generally a threshold levelof dose exists, which varies with person and circumstance, above which the severity of the healtheffect is greater for a higher dose. The ICRP has introduced a term “tissue reactions” to describea group of health effects comprising deterministic effects and some health effects (such as
cataracts and fibrosis) that are not determined solely at the time of irradiation but can be
modified after radiation exposure [II15, I16].
Stochastic (health) effect: A radiation-related health effect, the probability of occurrence of
which depends on radiation dose and the severity of which (if it occurs) is independent of dose.
In radiation protection the so-called LNT-model is used (linear non-threshold) meaning that theassumption is made that one can linearly extrapolate from moderate/high doses to low and very
low doses without a threshold. Note that this should not be confused with the stochastic actions
or processes of radiation interaction at the molecular or cellular level.
Heterozygote: An individual with different alleles at some particular locus.
High dose: Doses above 1 Gy are high doses [U10], though the threshold value is necessarily arbitrary.
Homologous recombination repair: One of the pathways of DNA double-strand break repair that
requires an undamaged homologous stretch of DNA to provide a template for repair. Restricted to S andG2 phases of the cell cycle.
Homozygote: An individual with the same allele at the corresponding loci on the homologous
chromosomes.
Imputation: A statistical technique for generating a plausible value to replace a missing value.
In vitro measurement: A procedure used to determine the nature, activity, location or retention ofradionuclides in the body by analysis of material excreted or otherwise removed from the body.
Inference: The process of drawing conclusions from scientific observations, evidence and reasoning in
the presence of uncertainty. While this report is focussed on prospectively inferring risk, note that
estimating an assigned share (or probability of causation) is also inference, but retrospective.
Interaction: Refers to the situation in which the magnitude of the influence of one risk factor on disease
rates depends upon the magnitude of one or more other risk factors (see “Effect modification” or “Joint
effects”).

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Radiation: Dose from radioactive material deposited in the body.
Chemicals: (a) Amount of a chemical recently absorbed; measured, e.g. as
metal concentration in blood; (b) amount of chemical stored in one or several
body compartments or in the whole body (body burden); used mainly for
cumulative toxicants; (c) in the case of ideal biological monitoring, amount ofactive chemical species bound to the critical sites of action (target dose; e.g.
carbon monoxide binding to haemoglobin).

Inverse dose (or exposure) rate effect: A form of effect modification, where the risk per unit cumulative
dose is increased under circumstance of lower exposure intensity (dose rate).
Latency (period): The period between exposure and manifestation of a health effect. This is also theperiod after which statistically significant increases in frequency of occurrence of the health effect in a
population have been seen; theoretically, there might be an undetectable increased frequency ofoccurrence of the health effect in an exposed population during the presumed latency period, but thispossibility becomes vanishingly small in the period shortly after exposure because there is a finite timerequired for damaged cells to replicate in an uncontrolled manner and manifest as a cancerous growth.
LET (linear energy transfer): The average linear rate of energy loss of charged particle radiation in a
medium, i.e. the radiation energy lost per unit length of path through a material. That is, the quotient ofdE by dl where dE is the mean energy lost by a charged particle owing to collisions with electrons intraversing a distance dl in matter.

L=
The unit of L is J/m, often given in keV/µm.

dE
dl

Locus (plural: loci): A unique chromosomal location defining the position of an individual gene or DNA
sequence.
Low dose: Doses below 100 mGy are called low doses [U10], though the threshold value is necessarily
arbitrary.
Low dose rate: The Committee has defined “low dose rate” as 0.1 mGy per minute or less, averaged
over one hour, for radiations such as external X-rays and gamma rays.
Meta-analysis: In statistics, a meta-analysis combines the results of two or more studies that address a
set of related research hypotheses, for example by estimating a certain unknown parameter or parametricfunction common to two or more data sets, while controlling or adjusting for differences in otherparameters unrelated to those of immediate interest. The purpose is to obtain an estimate that is more
informative than any obtainable from a single study, while ensuring that other data do not corrupt or bias
the result. The term “meta-analysis” is also used for statistical pooling of results available in publishedform, in contrast with a pooled analysis of original data from two or more studies.
Micro RNA (miRNA): A class of small non-coding RNAs that are involved in the epigenetic regulationof gene expression.
Mitochondria: Sub-cellular organelles that are the main site of energy production. Mitochondria containa small circular DNA molecule that encodes some of their constituent proteins.
Mitochondrial DNA: DNA distinct from nuclear DNA in that it is mostly unique sequence DNA andcodes for proteins that reside in mitochondria.

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Model: An analytical or physical representation or quantification of a real system and the ways in which
phenomena occur within that system, used to predict or assess the behaviour of the real system under
specified (often hypothetical) conditions. This is in contrast to a relationship, which is simply the way
two or more factors are connected or related (i.e. relationships include causal relationships, those basedon models, and those that simply fit the observed data).
Moderate/medium dose: Doses between 100 mGy to 1 Gy are called moderate doses [U10], though thethreshold value is necessarily arbitrary.
Molecular epidemiology: This epidemiological approach uses the currently available biomarkers (notradiation-specific) combined with classical epidemiological methods. Central to this are the –omics(genomics, transcriptomics, proteomics, metabolomics and others) which allow the detection ofindividual characteristics on various molecular levels (genes, transcripts, proteins, metabolism andothers). The frequency of occurrence of these individual characteristics in exposed populations are thenstudied using classical epidemiological methods.
Mutagen: A substance that can induce heritable changes (mutations) of the genotype in a cell as aconsequence of alteration or loss of genes or chromosomes (or parts thereof).
Mutation: A hereditary change in genetic material. A mutation can be a change in a single base (point
mutation) or a single gene or it can involve larger chromosomal rearrangements such as deletions andtranslocations.
Non-homologous end joining: A DNA double-strand break repair process that is the predominant
pathway of repair in mammalian cells, predominately active in G1.
Nucleotide: A subunit of DNA or RNA consisting of a nitrogenous base (adenine (A), guanine (G),thymine (T) or cytosine (C) in DNA, adenine, guanine, uracil (U) or cytosine in RNA.
Occupationally exposed: Any person who is employed, whether full time, part time or temporarily, by
an employer, and who has recognized rights and duties in relation to occupational radiological protection.
The Japanese regulations use another similar term, radiation worker.
Oncogene: A gene, or more forms of which is associated with cancer; many oncogenes are involved,
directly or indirectly, in controlling the rate of cell growth.
Plausible: This relates to an argument or statement that seems reasonable or probable, without it
necessarily being so. For the purposes of this annex, the term is used to constrain the number ofpossibilities to those that currently appear to be supported by sound scientific reasoning, e.g. in the caseof health effects and inferred risks, those that can be reasonably supported by arguments based on
observed or known radiation action and response in biological systems. Other responses not supported
by mechanistic reasoning or other observations may be possible, but without such reasoning are notconsidered plausible.
Pooled analysis: A combined analysis of original data from two or more data sets bearing on a common
question of interest. The analysis may include parameters that distinguish between the different data sets.
(Contrast with “meta-analysis”).
Precursor: Substance from which another, usually more biologically active, substance is formed.
Prediction – well-founded and conditional: The conventional meaning relates to foretelling that a
specified outcome will occur in the future or will be a consequence of an action, event or set of conditions.
In this annex, a distinction is made between well-founded prediction and conditional prediction, both

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being included within the concept of science-based inference. A well-founded prediction is one that isbased on a hypothesis that is deemed proven by the application of scientific method; a conditional
prediction is one that is based on a hypothesis that is currently not deemed proven but is based on
scientific reasoning including the use of models and assumptions.
Progression: Increase in autonomous growth and malignancy; used in particular to describe the transition
from benign to malignant tumours and the progression of malignancy. There are probably numerous
stages of progression during neoplastic development. The process of progression features in the generalmodel of carcinogenesis as well as in the multi-stage model.
Promoter: Risk factors of cancer that are capable of triggering preferential multiplication of a cellchanged by initiation. Often, following initiation, a long-term action on the target tissue is necessary.
Promoters often cause enzyme induction, hyperplasia, and/or tissue damage. The essential primary effects
are considered to be reversible. As a rule, promoters do not bind covalently to cell components and do
not exert an immediate genotoxic action.
Proteome: The complete complement of proteins within an individual cell—investigated using thetechniques of proteomics.
Protracted exposure: Exposure received over a long time period (compare with “Acute exposure” and“Chronic exposure”).
Radiogenic health effects: See “Health effects (of radiation)”.
Radionuclide: A radioactive isotope of an element. Different isotopes of an element have the same
number of protons but different numbers of neutrons and hence different atomic masses. If there are too
many or too few neutrons, the nuclei of the isotope tend to be unstable and transform into the nuclei ofanother element and in the process emit radiation.
Radiosensitivity: Susceptibility of cells, tissues and organisms to effects induced by ionizing radiation,
especially, but not exclusively, in relation to radiation-associated carcinogenesis. The term“radiosensitivity” with regard to cancer induction refers to the rate of radiogenic tumour induction anddoes not refer to the degree of malignancy.
Radon levels: Radon concentration in air. The special unit for radon level is Becquerel per cubic metre(Bq/m3).
Rate: In general, the ratio of two quantities, where the denominator is usually a function of the period oftime at risk. Rates of interest in epidemiology (and radiation risk estimation) are of the form:e
PY

where e is the number of events over some time period of interest in a study population and PY (expressedas person-years) is the sum of time at risk during this time period for each person in the study population.
Rates for an event of interest can vary with such factors as age, sex, and dose. Characterization of how
rates depend on dose (or exposure) is central to radiation risk estimation. See also “Hazard function”, “Risk”,“Relative risk”, “Excess rate”, and “Relative risk”, and “Excess relative risk”.
Recessive: A trait that is expressed in individuals who are homozygous for a particular allele.
Recessive mutations: Those that produce a specific phenotype when present in the homozygous(autosomal recessive) or in hemizygous state (X-chromosomal recessive).

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Relationship: The way two or more factors are connected or related (i.e. relationships include causal
relationships, those based on models, and those that simply fit the observed data).
Relative risk (RR): Ratio between the cancer cases in the exposed population to the number of cases in
the unexposed population. A relative risk of 1.5 indicates a 50% increase in cancer due to the agent underconsideration. Excess relative risk (ERR) is RR – 1.
Shielding: The absorbing property of material between a radiation source and a receptor which results in
reduced exposure.
Sievert (Sv): Unit of equivalent dose and of effective dose, equal to 1 joule per kilogram (J/kg).
Signalling: Transmission of activatory or inhibitory signals in the form of association and/or dissociation
and/or modification of molecules that affect biological processes within a cell. Signalling can be intracellular or inter-cellular. Disruption of signalling can contribute to disease pathogenesis.
Single nucleotide polymorphisms: Variants of the genome at individual base pairs which can be usefulin genetic mapping of diseases or susceptibles.
Somatic cells: All cells in the body except gametes and their precursors.
Statistical power: The expected probability of being able to detect an effect of a specified magnitude,
estimated before the start of a study. It depends on study size, baseline risk and effect size. Statisticalpower reflects only random error in outcome occurrence, while ignoring measurement error and typicallyassumes no bias in the study.
Statistical significance: Statistical significance testing is a technique originally developed for decision
making, where the compatibility of the observations is contrasted with that expected in the absence ofany difference (null hypothesis). An alternative or study hypothesis needs to be specified positing a
difference, usually evaluating difference to any direction (called two-sided test), or to a specific direction(one-sided test). Conventionally, 0.05 has been used as the limit (critical value, alpha level or type I error)
for significance, corresponding to a frequency of 1/20 for occurrence of a difference equally large orlarger as observed in the absence of any true difference (under the null hypothesis). The choice of the
cut-off level is; however, arbitrary and statistical significance should not be seen as the sole criterion forinterpretation of the findings.
Target (biological): Any organism, organ, tissue, or cell that is subject to the action of a pollutant orother chemical, physical, or biological agent.
Telomere: The end of a chromosome. This specialized structure is involved in the replication andstability of linear DNA molecules.
Threshold dose: The minimum dose that will produce a biological effect. Dose below which no effects
occur (“true”, mechanistically derived threshold) or are measurable (apparent threshold). For a given
agent there can be multiple threshold doses, in essence one for each definable effect.
Tissue dose: Local dose in an organ or a functional or structural entity of an organ. See also Absorbed
dose and Internal dose-chemicals.
Toxicity: Capacity of an agent to cause injury to a living organism. Toxicity can only be defined inquantitative terms with reference to the quantity of substance administered or absorbed, the way in which
this quantity is administered (e.g. inhalation, ingestion, or injection) and distributed in time (e.g. single
or repeated doses), the type and severity of injury, and the time needed to produce the injury.

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UNSCEAR 2020/2021 REPORTTranscription: Process by which genetic information in DNA is converted into mRNA ahead oftranslation into protein.
Transmissible genomic instability: Persistent formation of genetic alterations (commonly mutations or
chromosomal aberrations) over many post-irradiation cell generations.
Working level (WL): Any combination of the short-lived decay products of radon in one litre of air thatwill result in the emission of 1.3×105 MeV of potential alpha energy (see also “Potential alpha energy
concentration”).
Working level month (WLM): The cumulative exposure from breathing an atmosphere at a concentration of
1 WL for a working month of 170 hours (1 WLM=3.54×10–3 J h/m3=6.38×105 h Bq/m3 EEC).

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LIST OF GENES AND PROTEINS
By convention gene symbols are italicized while proteins are not. Human genes/proteins are given incapital letters, those of other organisms are generally given with an initial capital letter only
53BP1

p53-binding protein 1

AEN

Apoptosis-enhancing nuclease

AGGF1

Angiogenic factor with G patch and FHA domains 1

AGPAT (AGPAT3,
AGPAT9, etc.)

1-acylglycerol-3-phosphate O-acyltransferase (3, 9, etc.)

AKT

AKT serine/threonine kinase, protein kinase B

ALC1

Amplified in liver cancer 1, also known as chromodomainhelicase-DNA binding protein 1-like (CHD1L)

ALP

Alkaline phosphatase

ANGPTL4

Angiopoietin-like 4

ANXA6

Annexin A6

AP-1

Activator protein 1 (transcription factor)

APC

Adenomatous polyposis coli

ATF6

Activating transcription factor 6

ATM

Ataxia-telangiectasia-mutated protein

ATR

Ataxia-telangiectasia and Rad3-related protein

ATRIP

ATR-interacting protein

B4GALT1

Beta-1,4-galactosyltransferase 1

BAALC

Brain and acute leukaemia, cytoplasmic

BACTIN

Beta-actin

BAX

BCL-2-associated X protein

BBC3

BCL2 binding component 3(also: p53 upregulated modulator of apoptosis)

BCL (e.g. BCL2, BCL-xL)

B-cell lymphoma (apoptosis regulators)

BID

BH3-interacting domain death agonist

Bim

BCL-2 interacting mediator of cell death(also: BCL-2-like protein 11)

Bmf

BCL-2 modifying factor

BMPR2

Bone morphogenetic protein receptor type II

BRCA1

Breast cancer type 1 susceptibility protein

BTG2

BTG anti-proliferation factor 2

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CAMKII, etc.)

Calcium/calmodulin-dependent protein kinase (ID, II, etc.)

CCL (CCL5, CCL20, etc.)

C-C motif chemokine ligand (5, 20, etc.)

CCNE1

Cyclin E1

CCR (CCR2, CCR4, etc.)

C-C chemokine receptor type (2, 4, etc.)

CD (CD4+, CD14, CD40, etc.)

Cluster of differentiation (4, 14, 40, etc.)

CDC (CDC6, etc.)

Cell division control protein (e.g. 6, etc.)

CDK2

Cyclin dependent kinase 2

CDKN1A (CDKN1A,
CDKN2A, etc.)

Cyclin dependent kinase inhibitor 1A (1A, 2A, etc.)

CDS1

CDP-diacylglycerol synthase 1

CEBP (CEBPA, CEBPB, etc.)

CCAAT/enhancer-binding protein (alpha, beta, etc.)

cGAS/STING

Cyclic GMP-AMP synthase/Stimulator of Interferon Genes

CHD

Chromodomain-helicase-DNA binding protein

Chk (Chk1, Chk2, etc.)

Checkpoint kinase (1, 2, etc.)

c-JUN

Jun proto-oncogene

CLIC-1Chloride intracellular channel protein 1

CLIP2

CAP-GLY domain containing linker protein 2

COL--A1 (COL1A1,
COL13A1, etc.)

Collagen, type -- (e.g. 1, 13, etc.), alpha 1

COX2

Cyclooxygenase-2

CRE

Creatinine

CREB

cAMP response element-binding protein

CRP

C-reactive protein

CtIP

CtBP (C-terminal binding protein) interacting protein

Cx (Cx26, Cx32, etc.)

Connexin (26, 32, etc.)

CXCL (CXCL1, CXCL16 etc.)

C-X-C motif chemokine ligand (1, 16, etc.)

CXCR1

C-X-C motif chemokine receptor 1

CYCSP27

Cytochrome C, somatic pseudogene 27

CYP4XI

Cytochrome P450, family 4, subfamily X, polypeptide 1

DBP

D site of albumin promoter binding protein

DCLRE (DCLRE1A,
DCLRE1C, etc.)

DNA cross-link repair (1A, 1C, etc.)

DDB2

DNA damage-binding protein 2

DLC1

Deleted in liver cancer 1

DNA-PKcs

DNA-dependent protein kinase, catalytic subunit

DRAK1

DAP kinase-related apoptosis-inducing protein kinase 1

DUSP (DUSP16, DUSP22, etc.)

Dual specificity protein phosphatase (16, 22, etc.)

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E2FTranscription factor E2F

EB13

Epstein-Barr virus induced gene 3

EFABP

Epidermal-fatty acid binding protein

EGF

Epidermal growth factor

EGFR

Epidermal growth factor receptor

EGR (EGR1, EGR4, etc.)

Early growth response (1, 4, etc.)

EIF (Eif2, Eif4b, etc.)

Eukaryotic translation initiation factor (2, 4, etc.)

Endo III

Endonuclease III

EPC2

Enhancer of polycomb homolog 2

ERCC (ERCC5, ERCC6, etc.)

Excision repair cross-complementing protein (5, 6, etc.)

ERK

Extracellular signal-regulated kinase

ESM1

Endothelial cell-specific molecule 1

FANCD (FANCD2, etc.)

Fanconi anemia group D protein (D2, etc.)

FasL

Fas ligand

FCGR (FCGR2B, FCGR3, etc.)

Fc fragment of IgG receptor (IIB, III, etc.)

FcεRI

Fc fragment of IgE receptor

FDXR

Ferredoxin reductase

FGF (FGF2, FGF21, etc.)

Fibroblast growth factor (2, 21, etc.)

FIS1

Mitochondrial fission 1 protein

FLJ35725

tRNA methyltransferase 44 homolog

FOLR1

Folate receptor 1

FTH1

Ferritin heavy chain 1

GADD45Growth arrest and DNA damage-inducible 45 gene

GAP-43Growth-associated protein 43

GFAP

Glial fibrillary acidic protein

GFP

Green fluorescent protein

GJC1

Gap junction gamma-1 protein

GM-CSF

Granulocyte-macrophage colony-stimulating factor

GNG11

Guanine nucleotide-binding protein subunit gamma-11

GPR124G-protein coupled receptor 124

GPX1

Glutathione peroxidase 1

GRIK3

Glutamate receptor, ionotropic kainate 3

GRP78

Glucose related protein 78

GSK-3b

Glycogen synthase kinase 3 beta

GSTP1

Glutathione S-transferase P

GTF2H2

General transcription factor IIH subunit 2

H (H3, H4, etc.)

Histone (3, 4, etc.)

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H2AX

H2A histone family member X

hCLUp-Luc

Human CLU promoter-luciferase

HDL

High-density lipoprotein

HERPUD1

Homocysteine-responsive endoplasmic reticulum-resident
ubiquitin-like domain member 1 protein

HFH-1

Hepatocyte nuclear factor-3 homologue 1

HGF

Hepatocyte growth factor

HIF1A

Hypoxia-inducible factor 1-alpha

HINT-1

Histidine triad nucleotide-binding protein 1

HIST3H2A

Histone H2A type 3

HLA (HLA-A2, HLA-DR, etc.)

Human leukocyte antigen (A2, DR, etc.)

HMGB (HMGB2, etc.)

High mobility group box (e.g. 2, etc.)

HNF4A

Hepatocyte nuclear factor 4 alphahNOP56

Human nucleolar protein 56

HO-1

Heme oxygenase 1

HSP (HSP27, HSPA1B, etc.)

Heat shock protein (27, 1B, etc.)

ICAM-1

Intercellular adhesion molecule 1

IFN

Interferon

IFNG

Interferon gamma

Ig (A, E, etc.)

Immunoglobulin (A, E, etc.)

IGFBP3Insulin-like growth factor-binding protein 3

IGH6

Immunoglobulin heavy chain 6

IL (IL17E, etc.)

Interleukin (17E, etc.)

INO80

INO80 Complex ATPase Subunit, a chromatin-remodelling
factoriNOSInducible nitric oxide synthase

IRAK1

Interleukin-1 receptor-associated kinase 1

ISG20LInterferon stimulated exonuclease gene 20

ISWI

Imitation Switch protein

ITG (ITGA4, ITGB1, etc.)

Integrin (alpha 4, beta 1, etc.)

JNK

c-Jun N-terminal kinaseJUN

Jun proto-oncogene

KRTAP2-1

Keratin associated protein 2-1

lacZ

Beta-galactosidase

Lck

Lymphocyte-specific protein tyrosine kinase

LCN (LCN2, LCN13, etc.)

Lipocalin (2, 13, etc.)

LDH-A

Lactate dehydrogenase A

LIG (LIG3, LIG4, etc.)

DNA ligase (3, 4, etc.)

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LILRB3

Leukocyte immunoglobulin-like receptor subfamily B member 3

LINE-1Long interspersed nuclear element 1

MAP2

Microtubule-associated protein 2

MAPK (MAPK10, etc.)

Mitogen-activated protein kinase (10, etc.)

MAT2A

Methionine adenosyltransferase 2A

MBD4

Methyl-CpG-binding domain protein 4

MBP2

Myrosinase-binding protein 2

MCM2

Minichromosome maintenance complex component 2

MCP-1

Monocyte chemoattractant protein 1

M-CSF

Macrophage colony-stimulating factor

MD2

Myeloid differentiation 2

MDC1

Mediator of DNA damage checkpoint protein 1

MDM2

Mouse double minute 2

MEK

Mitogen-activated protein kinase kinase, it is also known as
MapK2

MET

MET proto-oncogene, receptor tyrosine kinase

MHC II

Major histocompatibility complex II

MIR-27B

MicroRNA 27b

MLH1

MutL homolog 1

MMP (MMP1, MMP15, etc.)

Matrix metalloproteinase (1, 15, etc.)

MnSOD

Manganese superoxide dismutase

MOS

Oncogene MOS, Moloney murine sarcoma virus

MRE11

Double-strand break repair protein MRE11

MRN

Mre11-Rad50-Nbs1 complex

MSH (MSH1, MSH2, etc.)

MutS homolog (1, 2, etc.)

mTOR

Mechanistic (or mammalian) Target of Rapamycin

MUC5B

Mucin 5B, oligomeric mucus/gel-forming

MYC

MYC proto-oncogene

MyD88

Myeloid differentiation primary response 88

NBS1

NibrinncRNA PARTICLE

Promoter of MAT2A-antisense radiation-induced circulating
long non-coded RNA

NEIL3

Nei like DNA glycosylase 3

NeuN

Neuronal nuclei

NF-κB

Nuclear factor 'kappa-light-chain-enhancer' of activated B-cells

NIK

NF-κB-inducing kinase

NQO1

NAD(P)H quinone dehydrogenase 1

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UNSCEAR 2020/2021 REPORT

Nr4a2

Nuclear receptor subfamily 4, group A, member 2 (also known as
nuclear receptor related 1 protein)

Nrf2

Nuclear factor erythroid 2-related factor 2

OCLN

Occludin

OCN

Osteocalcin

OGG18-oxoguanine glycosylase

OK432

Picibanil

OVA

Ovalbuminp21

Alias for CDKN1A

p38MAPK

p38 mitogen-activated protein kinases

p53

Tumour protein 53

P70S6KP70 S6 kinase

PAGE1

Prostate-Associated Gene 1 Protein

PAI-1

Plasminogen activator inhibitor-1

PAM

p53-independent protective apoptosis-mediated process

PARP1

Poly [ADP-ribose] polymerase 1

Pax7

Paired box 7

PCNA

Proliferating cell nuclear antigen

PDI

Protein disulphide isomerase

PD-L1

Programmed death ligand-1

pERK

Phosphorylated extracellular signal-regulated kinase

PEX1

Peroxisome biogenesis factor 1

PF4

Platelet factor 4

PHPT1

Phosphohistidine phosphatase 1

PI3K/BTK

Phosphoinositide-3 kinase/Bruton’s tyrosine kinase

PIAS1

Protein inhibitor of activated STAT 1

Pim1

Proto-oncogene serine/threonine-protein kinase Pim-1

PKC

Protein kinase C

PLAU

Plasminogen activator, urokinase

PLC

Phospholipase C

PLK2

Polo like kinase 2 (also: serine/threonine-protein kinase PLK2)

POLB

DNA polymerase beta

POLD1

DNA polymerase delta 1

PP (PP2A, PPM1D, etc.)

Protein phosphatase (2A, 1D, etc.)

PPAR

Peroxisome proliferator-activated receptor

PPP2R5A

Serine/threonine-protein phosphatase 2A 56 kDa regulatory
subunit alpha isoform

PSA

Prostate-specific antigen

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Ptc1

Alias for PTCH

PTCH (PTCH1, etc.)

Protein patched homolog (1, etc.)

PTGS2

Prostaglandin-endoperoxide synthase 2PTIO

2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide

PYCARDPYD (pyrin domain) and CARD (caspase recruitment domain)
domain containing

RAD (RAD50, RAD51L1, etc.)

DNA repair protein RAD homolog

RAF1

RAF-1 proto-oncogene, serine/threonine kinase

RAPGEF4

Rap guanine nucleotide exchange factor (GEF) 4

RB1

Retinoblastoma 1

RET

Rearranged in transformation

REV3L

Protein reversionless 3-like (translesion DNA polymerase )RND1

Rho family GTPase 1

RNF (RNF8, RNF168, etc.)

RING finger protein, E3 ubiquitin-protein ligase (RNF8,
RNF168, etc.)

RPL10A

Ribosomal protein L10a

RRM (RRM1, RRM2, etc.)

Ribonucleotide reductase catalytic subunit (e.g. M1, M2, etc.)

S100A9S100 calcium binding protein A9

SAT1

Spermidine/spermine-N1-acetyltransferase 1

SERHL2

Serine hydrolase like 2

SERPINA4-PSI

Serpin family A member 4-presenilin 1

SESN (SESN1, SESN2, etc.)

Sestrin (1, 2, etc.)

SFRP1

Secreted frizzled-related protein 1

SGK3

Serine/threonine-protein kinase SGK3

SIRT (SIRT3, etc.)

Sirtuin (3, etc.)

SLC22A5

Solute carrier family 22 member 5

SMARCA (SMARCA2,SMARCA4, etc.)

SWI/SNF-related matrix-associated actin-dependent regulator ofchromatin subfamily A member (2, 4, etc.)

SMC

Structural maintenance of chromosomes

SOD1

Superoxide dismutase 1

Sox9

SRY-Box Transcription Factor 9

ST3GAL5

ST3 beta-galactoside alpha-2,3-sialyltransferase 5

STARD13

StAR-related lipid transfer domain protein 13

SUMO

Small Ubiquitin-like Modifier

Suv39h1

Histone-lysine N-methyltransferase SUV39H1

SWI/SNF

Switch/sucrose Non-Fermentable complex

TERF2

Telomeric repeat-binding factor 2

TERT

Telomerase reverse transcriptase

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UNSCEAR 2020/2021 REPORTTFRCTransferrin receptor

TGF (TGFB1, etc.)

Transforming growth factor (B1, etc.)

TIG3

Tazarotene-induced gene 3

Tip60

Histone acetyltransferase Tip60

TLR

Toll-like receptor

TMPO

Thymopoietin

TNF

Tumour necrosis factor

TNFSF (TNFSF10B)

Tumour necrosis factor superfamily

TP53

Alias for p53

TP53inP1

Tumour protein p53-inducible nuclear protein 1

TRAMP

Transgenic Adenocarcinoma of the Mouse Prostate

TSAP6Transmembrane protein tumour suppressor-activated pathway 6

UHRF2

E3 Ubiquitin-Protein Ligase UHRF2

UNG

Uracil-DNA glycosylase

USP2

Ubiquitin carboxyl-terminal hydrolase 2

VEGF

Vascular endothelial growth factor

VEGFR (VEGFR1,VEGFR2, etc.)

Vascular endothelial growth factor receptor (1, 2, etc.)

Wnt (Wnt1, Wnt3a, etc.)

Wingless-related integration site (1, 3a, etc.)

XIAP

X-linked inhibitor of apoptosis protein

XP (XPA, XPB, XPC etc.)

Xeroderma pigmentosum (complementation group A, B, C, etc.)

XRCC (XRCC1, XRCC4, etc.)

X-Ray Repair Cross Complementing (1, 4, etc.)

ZAP70

Zeta-chain-associated tyrosine protein kinase 70

ZBTB16

Zinc finger and BTB domain-containing protein 16

γH2AX

Phosphorylated H2A histone family member X

unscear.org

This publication contains:
VOLUME III
Annex C: Biological mechanisms relevant for the inference of cancer risks from low-doseand low-dose-rate radiation

E VA L U AT I N G R A D I AT I O N S C I E N C E F O R I N F O R M E D D E C I S I O N - M A K I N G
In 1955 the United Nations General Assembly established the Scientific Committee on the Effectsof Atomic Radiation (UNSCEAR) in response to concerns about the effects of ionizing radiation
on human health and the environment. At that time fallout from atmospheric nuclear weapons
tests was reaching people through air, water and food. UNSCEAR was to collect and evaluate
information on the levels and effects of ionizing radiation. Its first reports laid the scientific grounds
on which the Partial Test Ban Treaty prohibiting atmospheric nuclear weapons testing was
negotiated in 1963.
Over the decades, UNSCEAR has evolved to become the world authority on the global levels
and effects of exposure to ionizing radiation. UNSCEAR’s independent and objective evaluationsof the science are to provide for—but not address—informed policymaking and decision-making
related to radiation risks and protection.

V. 21-09153

ISBN 978-92-1-139208-1