EFFECTS OF IONIZING RADIATION
United Nations Scientific Committee on the
Effects of Atomic Radiation
UNSCEAR 2006
Report to the General Assemblywith Scientific Annexes

VOLUME II
Scientific Annexes C, D and E

UNITED NATIONSNew York, 2009

NOTE
The report of the Committee without its annexes appears as Official Records of the General
Assembly, Sixty-first Session, Supplement No. 46 and corrigendum (A/61/46 and Corr. 1). The
report reproduced here includes the corrections of the corrigendum.
The designation employed and the presentation of material in this publication do not imply theexpression of any opinion whatsoever on the part of the Secretariat of the United Nations concerning the legal status of any country, territory, city or area, or of its authorities, or concerning
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.09.IX.5
ISBN 978-92-1-142270-2

ANNEX C
Non-targeted and delayed effects of exposure to ionizing radiation
Contents
Page
INTRODUCTION. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3
I.

RADIATION-INDUCED GENOMIC INSTABILITY. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. Radiation-induced genomic instability in vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
B. Induced genomic instability after in vivo irradiation followed by in vitro analysis. . . . . . . . . . . . . . . . . . . . . .
C. Induced genomic instability after in vitro irradiation followed by in vivo analysis. . . . . . . . . . . . . . . . . . . . . .
D. Radiation-induced genomic instability in vivo. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
		 1. Mouse models for radiation-induced genomic instability in vivo. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
		 2. Human studies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
E. Genomic instability and radiation-induced leukaemia. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
		 1. Mouse models. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
		 2. Human studies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
F. Role of telomeres and telomerase in radiation-induced genomic instability. . . . . . . . . . . . . . . . . . . . . . . . . .
G. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5
5
12
12
13
17
18
21
21
21
21
22

II.

BYSTANDER EFFECTS AND RADIATION EXPOSURE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. Bystander effects in vitro. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
		 1. Bystander effects after cytoplasmic irradiation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
		 2. Bystander effects after low fluences of alpha particle irradiation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
		 3. Bystander effects after irradiation with a charged-particle microbeam. . . . . . . . . . . . . . . . . . . . . . . . . .
		 4. Bystander effects after transfer of medium from irradiated cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
B. Bystander effects in vivo. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

23
23
25
25
27
31
33

III. Relationship between Radiation-Induced Genomic Instability and Bystander Effects. . . . . . . . . .
A. Relationship between radiation hypersensitivity at low doses and bystander effects . . . . . . . . . . . . . . . . . .
B. Relationship between radiation adaptive response and bystander effects. . . . . . . . . . . . . . . . . . . . . . . . . . .
C. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

37
37
38
38

IV. ABSCOPAL EFFECTS OF RADIATION. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
B. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

39
39
40

V.

CLASTOGENIC FACTORS INDUCED BY IONIZING RADIATION. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
B. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

41
41
43

VI. IMPACT OF NON-TARGETED AND DELAYED EFFECTS OF RADIATION ON FUTURE GENERATIONS . . . . . . . . . . . .
A. Studies in non-mammalian species. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
B. Mouse studies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
		 1. Irradiation of the mouse zygote. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

45
45
46
46

1

		
		
		
		
C.
D.
E.
F.
G.
H.

Page
2. Pre-implantation embryo chimera assay. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
3. Mouse mutation assays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
4. Alterations in tandem repeat DNA sequences. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
5. Tumour induction in the offspring of irradiated parents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Malformation induction in the offspring of irradiated parents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Human studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Cancer incidence in the offspring of irradiated humans. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Pregnancy outcomes in the offspring of irradiated humans. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
Genetic damage and malformation induction in the offspring of irradiated parents. . . . . . . . . . . . . . . . . . . . 52
Impact of non-targeted and delayed effects of radiation on future generations. . . . . . . . . . . . . . . . . . . . . . . 52

VII. IMPLICATIONS OF NON-TARGETED AND DELAYED EFFECTS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

55

CONCLUDING REMARKS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

57

References

59. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2

INTRODUCTION
1. The goal of this annex is to summarize the evidence
for non-targeted and delayed effects of exposure to ionizing
radiation in vitro and in vivo. Currently, human health risk
estimates for effects associated with radiation exposures are
based primarily on the view that the detrimental effects ofirradiation occur only in irradiated cells. Over the years, anumber of non-targeted effects of radiation exposure havebeen described that challenge this concept. These non­targeted effects include genomic instability occurring in theprogeny of an irradiated cell, bystander effects, clastogenic
factors produced in plasma from irradiated individuals thatcan cause chromosome damage when cultured with nonirradiated cells, and heritable effects of parental irradiationthat can manifest across generations. This annex considers
whether these effects pose new challenges to evaluating risks
associated with radiation exposure, understanding radiationinduced carcinogenesis and interpreting epidemiological
data on radiation exposure.
2. A central tenet in the radiation sciences has been that theenergy from radiation must be deposited in the cell nucleus
to elicit a mutagenic and/or clastogenic effect and thus be relevant for its potential to cause damage (figure I). It is implicitin this tenet that the biological consequences of cellular irradiation affect only the irradiated cell and that non-irradiatedcells do not share the legacy of the radiation exposure.

Figure I. Prevailing paradigm for the biological effects of cellular exposure to ionizing radiation.
Ionizing radiation deposits energy in the nucleus of the cell. DNA damage is induced, and cellular responses to that damage can affect the fateof the irradiated cell. The damage can be removed and the genetic material restored by high-fidelity DNA repair. DNA repair systems may also
eliminate the damage, but error-prone processing can result in gene mutations and clastogenic effects leading to chromosomal rearrangements. Depending upon the cell type, various cellular processes may be initiated that result in carcinogenesis in somatic cells, heritable genetic
effects in germ line cells and developmental defects in foetal cells which may or may not be derived from mutational or clastogenic effects.
DNA damage might activate cell cycle checkpoint control and cause the damaged cell to go into a protracted senescent state. Alternatively,
if the damage is substantial, cell death may occur via a number of cellular pathways.

Radiation

High-fidelity
DNA repair

SenescenceDNA damage

Death- Apoptosis- Necrosis- Mitosis-linked

Mutations and/or
chromosome rearrangements

Somatic

Carcinogenesis

Germ line—

FoetalHeritable
genetic
effects—3

Developmental
effects

4

UNSCEAR 2006 REPORT: VOLUME II

3. When ionizing radiation is absorbed in biological material, excitations and ionizations occur that are non-randomly
distributed along localized tracks. The spatial distributionof these ionization/excitation events produced by different
particles varies considerably depending on the quality ofradiation. The term “linear energy transfer” (LET) is used
to classify radiation quality according to the average energy
transferred per unit length of the track. For the purposes ofthis annex, X- and gamma rays are considered to be lowLET radiation, protons and neutrons are considered to beintermediate LET radiation, and alpha particles and heavy
ions are considered to be high-LET radiation.
4. In contrast to the risks associated with exposures tolow doses of ionizing radiation (less than about 200 mSv,
UNSCEAR 2000 Report [U2]), the risks of cancer after highand moderate doses of radiation are relatively well understood. This understanding is based on data from detailed
epidemiological studies of the survivors of the atomic
bombings in Japan and other exposed groups, e.g. clinically
irradiated populations and those exposed as a result of theChernobyl accident (UNSCEAR 2000 Report, annex I).
However, risks at low doses are generally extrapolated fromthe high-dose data, applying dose and dose-rate effectiveness factors. Estimating risk is further complicated because
environmental exposures are predominantly protracted, lowdose, low-dose-rate exposures, or high-dose-rate exposuresdelivered in small fractions (see annex A, “Epidemiologicalstudies of radiation and cancer”). This contrasts with the
majority of laboratory studies and clinical exposure situations, where exposures are usually acute, high-dose, high-dose-rate exposures. In addition, inherent in many modelsof radiation risk is that only those cells or tissues actually
irradiated are burdened by the legacy of the radiation exposure. A number of non-targeted delayed effects of radiation
exposure have been described; the purpose of this annex isto summarize the evidence for these effects and indicate
present hypotheses on how they may affect the assessmentof health hazards associated with radiation exposure andradiation-induced carcinogenesis.
5. For the purposes of this annex, “non-targeted effects”
refers to radiation-induced effects manifesting in cells
whose nucleus was not subject to a direct hit by the radiation, i.e. no ionization events due to cellular irradiation
were deposited within the volume of that nucleus. In such
instances the radiation may have hit the cytoplasm, or neighbouring cells, tissues or organs, or even cells in anotherculture vessel, and a response is communicated from these
irradiated cells to non-irradiated cells to elicit an effect. It
must be stressed at this stage that the non-­targeted effectsof ionizing radiation described in this annex do not imply
that the well-documented targeted effects of radiation are
irrelevant or unimportant, or that the concept of “dose”
needs to be revised. That is not the case. Rather, the goalof this annex is to summarize the literature on non-targeted
effects associated with exposure to ionizing radiation and,
where possible, to evaluate how such effects may affectrisks associated with radiation exposure, the understandingof radiation-induced carcinogenesis, and the mechanistic
basis for interpreting epidemiological data on radiation
effects.

I.
A.

RADIATION-INDUCED GENOMIC INSTABILITY

Radiation-induced genomic instability in vitro

microsatellite (short tandem repeat) instabilities and/or
decreased plating efficiency (summarized in table 1), andhas been the subject of a number of reviews [K17, L14,
M11, M12, M14, M48, W12]. These observed delayed
effects can persist in unstable clones over time, and insome instances mimic those effects seen in tumour cells.
There are likely to be multiple pathways for initiating andperpetuating induced instability [K8, L11], and the relative contributions of the different pathways involved probably depend on the genetic background of the target cell ororganism [P3, W1] and on environmental factors (reviewed
in references [K4, M32, M33]).

6. Genomic instability is an all-embracing term todescribe the increased rate of acquisition of alterations in
the genome. As compared with the direct effects of radiation, i.e. those effects directly induced as a consequence ofenergy deposition, radiation-induced instability is observedin cells at delayed times after irradiation and manifests inthe progeny of exposed cells multiple generations after
the initial insult (figure II). Instability is measured as
chromo­somal alterations, changes in ploidy, micronucleus
formation, gene mutations and amplifications, mini- andFigure II. Radiation-induced genomic instability.
A single cell survives irradiation and is clonally expanded. During clonal expansion, a number of the progeny of that irradiated cell die (throughlethal mutations or delayed reproductive cell death), which results in a persistently reduced plating efficiency in this clone. Alternatively, or as aconsequence of the presence of these dead and dying cells, instability events can occur in the progeny of the irradiated cell. These may resultin chromosomal rearrangements, aberrations or gaps, micronuclei, mutations, gene amplifications and/or a failure of the cells to correctly
separate their chromosomes at mitosis, resulting in aneuploid cells.

Ionizing radiation

First
instability
event
occursCell death-Lethal mutation-Delayed reproductive
cell death

Secondinstability
event
occursAneuploidy

Multiple “subpopulations“ appear within a colonyclonally expanded from a single irradiated cell

5

6

UNSCEAR 2006 REPORT: VOLUME IITable 1

In vitro studies of radiation-induced genomic instabilityYear

End point

Radiation typea

Cell type

Comments

Reference

1991

Plating efficiency

Chinese hamster ovary cells

X-rays

[C13]

1992

Chromosomal aberrations

Murine haemopoietic stem cells

Alpha particles

[K3]

1992

Mutation frequency

Chinese hamster ovary cells

X-rays

[C12]

1992

Plating efficiency

Chinese hamster ovary cells

X-rays

[C12]

1993

Chromosomal instability

Human skin fibroblasts

Heavy ions: neon, argon

1993

Chromosomal aberrations

Human lymphocytes

X-rays[H7]

1993

Chromosomal aberrations

GM10115 human–hamster hybrid cells

X-rays[M2]

1993

Neoplastic transformation

HeLa × skin fibroblast human hybrid cells

Gamma radiation

[M31]

1993

Plating efficiency

HeLa cells

X-rays

1993

Plating efficiency

GM10115 human–hamster hybrid cells

X-rays[M2]

1994

Chromosomal aberrations

HeLa cells

X-rays

[B20]

1994

Chromosomal aberrations

Human haemopoietic stem cells

Alpha particles

[K1]

1994

Giant cell formation

HeLa cells

X-rays

[B20]

1994

Plating efficiency

HeLa cells

X-rays

[B20]

1994

Plating efficiency

Human keratinocyte cells

Gamma radiation;alpha particles

[O5]

1995

Chromosomal aberrations

Human lymphocytes

X-rays[H8]

1995

Chromosomal aberrations

Murine haemopoietic stem cells

Alpha particles; X-rays

[K2]

1995

Delayed TP53 mutation

Murine epithelial cells

Gamma rays

[S11]

1995

Plating efficiency

Murine haemopoietic stem cells

Alpha particles; X-rays

[K2]

1996

Apoptosis

V79 Chinese hamster ovary cells

X-rays(1–12 Gy)

Dose response up
to 3–4 Gy

[J5]

1996

Apoptosis

Human keratinocyte cells; CHOK hamster
cells

Gamma rays

Dose response

[L34]

1996

Chromosomal aberrations

Human epithelial cells

X-rays; alpha particles

[D18]

1996

Chromosomal aberrations

TK6 human lymphoblasts

X-rays[G8]

1996

Chromosomal aberrations

V79 Chinese hamster ovary cells

X-rays

Dose response

Dose response

Dose response up
to 3–4 Gy

[M30]

[F8]

[J5]

(1–12 Gy)
1996

Chromosomal aberrations

Human lymphocytes

Alpha particles

[K5]

1996

Micronucleus frequency

V79 Chinese hamster ovary cells

X-rays(1–12 Gy)

Dose response up
to 5 Gy

[J5]

1996

Morphological abnormalities

Human keratinocyte cells; CHOK hamster
cells

Gamma rays

Dose response

[L34]

1996

Mutation frequency

TK6 human lymphoblastoid cells

X-rays[G8]

1996

Plating efficiency

Human epithelial cells

X-rays; alpha particles

[D18]

1997

Chromosomal aberrations

GM10115 human–hamster hybrid cells

X-rays; gamma rays; 56Fe
ions; neutrons; gold ions

Dose response[L30, L31]

1997

Chromosomal aberrations

V79 Chinese hamster ovary cells

Alpha particles; X-rays

Dose response

[M28]

1997

Chromosomal aberrations

Murine haemopoietic stem cells

Gamma rays

[P9, P15]

ANNEX C: NON-TARGETED AND DELAYED EFFECTS OF EXPOSURE TO IONIZING RADIATION

Year

End point

7

Cell typeRadiation typea

Comments

Reference

Dose response

[M28]

1997

Micronucleus frequency

V79 Chinese hamster cells

Alpha particles; X-rays

1997

Mutation frequency

Murine haemopoietic stem cells

X-rays; alpha particles;
neutrons

[H12]

1997

Mutation frequency

Chinese hamster ovary cells

X-rays; alpha particles

[L29]

1997

Plating efficiency

V79 Chinese hamster cells

Alpha particles; X-rays

1998

Chromosomal aberrations

Human fibroblasts

X-rays; neutrons;
alpha particles

[K18]

1998

Chromosomal aberrations

Human epithelial cells

Gamma rays; neutrons

[U20]

1998

Chromosomal aberrations

GM10115 human–hamster hybrid cells

X-rays[D1]

1998

Chromosomal aberrations

Human lymphocytes

Gamma rays

[H9]

1998

Chromosomal aberrations

Murine haemopoietic stem cells

Alpha particles; X-rays

[K18]

1998

Chromosomal aberrations

GM10115 human–hamster hybrid cells

125

[K9]

1998

Chromosomal aberrations

Human lymphocytes

Gamma rays

[L32]

1998

Chromosomal aberrations

GM10115 human–hamster hybrid cells

X-rays; gamma rays; 56Fe
ions; neutrons; gold ions

1998

Chromosomal aberrations

Murine haemopoietic stem cells

Alpha particles

1998

Chromosomal aberrations

GM10115 human–hamster hybrid cells

X-rays; gamma rays; 56Fe
ions; neutrons; gold ions

1998

Chromosomal aberrations

V79 Chinese hamster ovary cells

X-rays[T3]

1998

Plating efficiency

GM10115 human–hamster hybrid cells

X-rays[D1]

1998

Plating efficiency

Murine haemopoietic stem cells

Alpha particles; X-rays

[K18]

1998

Plating efficiency

Murine haemopoietic stem cells

Alpha particles

[L24]

1999

Apoptosis; micronucleus
frequency

AGO l522B primary human fibroblasts

X-rays; alpha particles

Dose response

[B22]

1999

Chromosomal aberrations

GM10115 human–hamster hybrid cells

X-rays; gamma rays; 56Fe
ions; neutrons; gold ions

Dose response

[L6]

1999

Micronucleus frequency

Human SCL-II squamous carcinoma cells

X-rays

Dose response

[K20]

1999

Plating efficiency

AGO 1522B primary human fibroblasts

X-rays; alpha particles

Dose response

[B22]

1999

Plating efficiency

Human keratinocyte cells

Gamma rays;
alpha particles

1999

Plating efficiency

Human SCL-II squamous carcinoma cells

X-rays2000

Chromosomal aberrations

HPV-G and HaCaT human keratinocyte cells

Gamma rays;
alpha particles

2000

Chromosomal
rearrangements

HF19 human fibroblasts

2000

Chromosomal aberrations

Human lymphocytes

Alpha particles; X-rays

2000

Chromosomal aberrations

GM10115 human–hamster hybrid cells

X-rays; gamma rays; Fe
ions; neutrons; gold ions

2000

Plating efficiency

HPV-G and HaCaT human keratinocyte cells

Gamma radiation; alphaparticles

[M29]

2001

Apoptosis

WTKI human lymphoblastoid cells

56

Fe ions; 137Cs gamma rays

[S8]

2001

Chromosomal aberrations

TK6 human lymphoblasts

56

Fe ions; gamma rays

[E14]

2001

Chromosomal aberrations

Human lymphocytes

Gamma rays

[B11]

Dose response

I

Dose response

[M28]

[L9]
[L20]

Dose response

[P14]

[C14]
Dose response

[K20]
[M29]

No instability
observed

56

[G25]
[A6]

Dose response

[L12]

8

UNSCEAR 2006 REPORT: VOLUME II

Year

End point

Radiation typea

Cell type

Comments

Reference

2001

Chromosomal aberrations

Human lymphocytes

56

Fe ions

[G9]

2001

Chromosomal aberrations

Human lymphocytes

Alpha particles

[K19]

2001

Chromosomal aberrations

GM10115 human–hamster hybrid cells

X-rays; gamma rays; 56Fe
ions; neutrons; gold ions

2001

Chromosomal aberrations

WTK1 human lymphocytes

56

Fe ions; 137Cs gamma rays

[S8]

2001

Microsatellite instability;
gene amplifications

BEP2D human bronchial epithelial cells

Alpha particles; 56Fe ions

[P16]

2001

Minisatellite instability

4T1 murine mammary adenocarcinoma cells

Gamma rays

[L33]

2001

Mutation frequency

Saccharomyces cerevisiae

Gamma rays

2001

Mutation frequency

TK6 human lymphoblasts

56

Fe ions; gamma rays

[E14]

2001

Mutation frequency

Human lymphoid cells

Gamma rays
(12 Gy)

[G9]

2001

Mutation frequency

4T1 murine mammary adenocarcinoma cells

Gamma rays
(12 Gy)

[L33]

2001

Plating efficiency

Saccharomyces cerevisiae

Gamma rays

2001

Plating efficiency

Human lymphoid cells

56

2001

Recombination frequency

Saccharomyces cerevisiae

Gamma rays

2001

Telomere shortening

WTKI human lymphoblastoid cells

56

Fe ions; 137Cs gamma rays

[S8]

2002

Apoptosis

Mouse fibroblast clones; V79 Chinese
hamster ovary cells

X-rays

[C11]

2002

Chromosomal aberrations

Human fibroblasts

Gamma rays

[B21]

2002

Chromosomal aberrations

Murine haemopoietic stem cells

Gamma rays

[B23]

2002

Chromosomal aberrations

Mouse fibroblast clones; V79 Chinese
hamster ovary cells

X-rays

[C11]

2002

Chromosomal aberrations
and response to second
irradiation

TK6 human lymphoblasts

56

Fe ions; gamma rays

[E13]

2002

Mutation frequency

TK6 human lymphoblasts

56

Fe ions; gamma rays

[E13]

2003

Chromosomal aberrations

Normal diploid human fibroblasts

Low- and high-LET radiation

2003

Chromosomal aberrations

TK6 human lymphoblasts

Gamma rays

[E15]

2003

Global gene expression

Primary human lymphocytes

Gamma rays

[F16]

2003

Chromosomal instability andradiation-induced delayed
reproductive death

Haemopoietic stem cells(R-M26/2-1)

Gamma rays

2003

Delayed lethality and micronucleus formation

Human osteoblast cells

Depleted uranium

2003

Chromosomal aberrations

TK6 and NH32 human lymphoblasts

Gamma rays

Dose response up
to 5 Gy

[S9]

2003

Micronucleus frequency

Human peripheral blood lymphocytes

Gamma rays

Dose response

[J8]

2003

Chromosomal aberrations

Murine haemopoietic stem cells

Gamma rays

[M27]

2003

Delayed TP53 activation

HTI 080 human fibrosarcoma cells

X-rays; gamma rays

[S24]

2003

Mutation frequency

TK6 human lymphoblasts

56

Fe ions; gamma rays

[E15]

2003

Plating efficiency

Murine haemopoietic stem cells

Gamma rays

[M27]

Dose response

Dose response

Dose response

Fe ions

[L10]

[B19]

[B19]
[G9]

Dose response

No instability
observed

Independent of
TP53 status

[B19]

[D17]

[M27]

[M57]

ANNEX C: NON-TARGETED AND DELAYED EFFECTS OF EXPOSURE TO IONIZING RADIATION

Radiation typea

Cell type

Comments

9Year

End point

2004

DNA damage; comet assay

Chinese hamster ovary cells

X-rays

2004

GFP-based protein assay forhomologous recombination

Human RKO cells

X-rays

[H24]

2004

Cell viability; apoptosis;changes in MAP and ERK
signalling

Human lymphoblast cells

X-rays

[R13]

2004

Gene expression analysis

GM10115 human–hamster hybrid cells

X-rays

[S59]

2004

ROS production

Mouse m5S derived cl. 2011-14 cells

X-rays2004

Lethal sectoring;
division delay

HeLa S3-9IV cells

X-rays; alpha particles

[S47]

2004

Chromosomal aberrations

Scid mouse cells

X-rays

[U26]

2004

Mutagenic radicals

Human–hamster hybrid A (L) cells

X-rays2005

Chromosomal aberrations

Peripheral blood lymphocytes

Gamma irradiation

2005

HPRT frequency; apoptosis;
cell survival

Lymphoblastoid TK6 cells

2005

H2AX phosphorylation

Human fibroblasts

Si and Fe ions

High-LET

[D26]

2005

Global gene expression
profileHuman skin fibroblasts

X-rays2 cGy and 4 Gy

[D27]

2005

Chromatid and chromosomal
aberrations

Haemopoietic stem cells

Gamma rays

2005

Micronuclei; chromosomal
aberrations

Human peripheral blood lymphocytes

60

2005

Genomic patterns of­aberrations; radiation-induced
mouse lymphomas

Mouse genomic BACs

2005

Delayed apoptosis

CGL1 (HeLa × fibroblast) hybrid cells

X-rays

7 Gy

[M56]

2005

Chromosomal
rearrangements

TK6 cells and clones with differing TP53
statusGamma rays

2 Gy

[M54]

2005

Delayed apoptosis

GM10115 human–hamster hybrid cells

X-rays2005

Cell killing

V79 cells

X-rays2005

Microarray analysis of
isogenic clones to assay forgene expression

Human–hamster hybrid cells

X-rays2005

Gene expression influenced
by TP53 status

TK6; NH32; WTK1

Gamma rays

Low dose

Cell killing related
to time after
irradiation

High-LET

Reference
[G23]

[T6]

[W20]
[B33]
[C20]

Co gamma rays

[G22]
0–4 Gy

[J9]
[M55]

[N29]
Low dose

[S65]
[S61]

10 Gy at different
times

[T7]

a	It is not possible to include the range of doses used, as many studies used different doses, dose rates and radiation types within a single study. The reader is referred to theoriginal study for information on the range of doses used.

7. Of the multitude of end points associated with­radiation-induced instability, chromosomal changes are thebest described. While some investigators describe ­chromosome
gaps [S11, U20] or breaks [K3, L24] as the primary aberrations observed, it is unlikely that these contribute to longterm instability, as gaps have no known phenotype and breaks
are generally lethal [C2, C3]. Of more significance are grosschromosomal rearrangements, particularly chromosomal translocations, duplications and partial trisomies [G8, M2, S8],
which appear to involve amplification and recombination oflarge chromosomal regions by a currently unknown mechanism[D1, M16]. An example of chromosomal instability in human–
hamster hybrid GM10115 cells clonally expanded from a single
cell surviving X-irradiation is presented in figure III.

10

UNSCEAR 2006 REPORT: VOLUME II

Figure III. Metaphase spreads from human–hamster hybrid GM10115 cells clonally expanded from a single cell surviving
exposure to 5 Gy of X-rays.
A: Metaphase chromosomes from non-irradiated GM10115 cells. Using fluorescence in situ hybridization, the human chromosome 4 in thesehuman–hamster hybrid cells is painted green, and the hamster chromosomes are stained red. B–H: Representative metaphase cells showing
the chromosomal rearrangements observed in one colony of cells clonally expanded from a single cell. Radiation-induced instability stimulated
the dynamic rearrangement of the genetic material, resulting in multiple subpopulations of cytogenetically rearranged cells within the clonallyexpanded population. Such cytogenetic rearrangements in GM10115 cells have been used by Morgan and colleagues [K9, L6, L9, L30, L31,
M2] as a measure of genomic instability induced by both high- and low-LET radiation.

ANNEX C: NON-TARGETED AND DELAYED EFFECTS OF EXPOSURE TO IONIZING RADIATION

8. Instability is a frequent event in colonies of surviving
cells. Kadhim et al. [K3] reported karyotypic abnormalities
in 40–60% of murine stem cells exposed to doses of alphaparticles that would produce about one hit per cell. Sabatierand colleagues [S44] observed late passage non-random chromosomal instability in >50% of metaphase cells from humandermal fibroblasts irradiated with a wide range of high-LET
radiations (386 to 13,600 keV/µm). Likewise, Limoli et al.
[L6] observed that X-rays induced chromosomal instability in ~3% of surviving human–hamster hybrid GM10115
clones per gray of radiation. This increased to ~4% Gy–1
after high-LET iron ion exposure [L12, L13]. This observed
frequency of instability is grossly in excess of the reported
frequency for gene mutations at similar doses. Therefore it
is unlikely that mutation in a single gene or gene family is
responsible for the unstable phenotype in unstable clones.
Instead it is reasonable to suppose that factors contributing
to maintaining genomic instability over time include criticalpathways in DNA damage and repair [C10, H11, M48, Y3],
chromosomal replication [B32], cellular homeostasis [B3,B6, M24, M48] and alterations in gene expression [S59,S60, S61, S62].
9. The high frequency of induced instability observed indifferent systems raises the intriguing question as to what isreally being measured and of the significance of these observations. While the numerous in vitro studies summarized intable 1 have established the occurrence of radiation-induced
genomic instability, many of the cell lines used were not“normal” initially, and in many cases they involved tumourderived cell lines. Dugan and Bedford [D17] have pointed outthat instability is sometimes not observed in apparently normal cells after irradiation. However, there are many reports
of instability in the progeny of irradiated normal human andmurine bone marrow cells [K1, K2] and in cultured human
lymphocytes [B11, H7, H8, H9]. Some of this confusion
may relate to the role of a functional TP53 tumour suppressor gene. Both TP53-dependent [S9] and TP53-independent
pathways have been proposed [K5, L8], and Moore et al.
[M54] showed that genomic instability might differ bothquantitatively and qualitatively as a consequence of altered
TP53 expression. Furthermore, differences in cellular proliferation patterns and susceptibility to mutation between cells
and cell lines might also influence the reported results [C19].
Because the majority of reports indicate that irradiated normal
primary cells readily demonstrate the instability phenotype(table 1), this implicates ionizing radiation as the causative
agent. It also establishes induced instability as a phenotype
associated with radiation exposure. As always, however,­caution should be exercised when extrapolating from in vitro
cell culture systems to the human situation in vivo.
10. When cells or tissues are directly exposed to ionizing
radiation, biological effects are generally induced in a dosedependent manner. One perplexing feature of radiationinduced genomic instability, and of non-targeted effects ingeneral, is the lack of a well-defined dose response profile.
Most investigators report that non-targeted effects are independent of dose. However, some investigators have observed

11

a dose response at lower doses that tends to saturate at higher
doses, and a few investigators have observed consistent doserelated effects. These data are summarized in table 1 and arereviewed in references [L6, M32]. Furthermore, radiationinduced instability appears independent of dose rate, although
this has not been extensively investigated to date [L6]. In addition, there does not appear to be a significant LET effect forradiation-induced genomic instability, with both high- andlow-LET radiations being effective [H2, K34, L12].
11. Many of the genomic changes described under thetitle of induced instability are changes of the same type as
observed in human tumours. Radiation-induced cancers haveno known molecular signature, and continued investigation
aimed at understanding the processes and pathways by whichradiation induces genomic alterations in the progeny of irradiated cells should provide insights into the mechanisms ofradiation-induced transformation and carcinogenesis [M62].
Kennedy et al. [K10] demonstrated replication dependenceof radiation-induced transformation of C3H 10T½ cells.
A similar replication dependence was also reported for­radiation-induced mammary cancers in rats, in which expression of epigenetic initiation required replication of irradiatedmammary stem cells in the tissue microenvironment [K7].
12. While the multiple phenotypes associated with­radiation-induced genomic instability are relatively wellcharacterized, the molecular, biochemical and cellular events
that initiate and perpetuate instability remain unknown.
Directly induced DNA damage, e.g. induced DNA doublestrand breaks, is probably not causative [M13]. Instead,deficiencies in cellular responses to DNA damage [C10,Y3], changes in gene expression [B6] or perturbations incellular homeo­stasis [B3] are more likely to be involved,
and provide a rational explanation as to why the unstablephenotype can persist. In the GM10115 cell system, clones
of unstable cells continue to show the dynamic productionof novel chromosomal rearrangements for over four years
post-irradiation [N1]. Attempts to define the target for
induced instability indicate that, while the nucleus may be
the ultimate target [B6, K9], there is evidence for a persistent increase in reactive oxygen species (ROS) in cultures ofcells showing radiation-induced genomic instability. A rolefor enhanced oxidative stress in perpetuating the unstablephenotype was first described by Clutton et al. [C5], and waslater confirmed in studies by Limoli et al. [L7, L9, L10] andRedpath and Gutierrez [R3]. A persistent induction of ROS
has also been shown to cause delayed reinduction of TP53
in normal human fibro­blasts [R6]. A study by Roy et al. [R5]revealed that hypoxia (2% oxygen) significantly reduced
X-ray-induced delayed effects, specifically cell death, giant
cell formation and chromosomal aberrations, compared withcells cultured under their “normal” 20% oxygen conditions.
The role of ROS in radiation-induced genomic instability has
been reviewed in detail by Mikhailov and colleagues [B45,
M48]. It should be noted that oxygen tension in normal­tissue shows a typical ­Gaussian distribution of values witha median between 40 and 60 mm Hg, and no values below
10 mm Hg [A13]. Tumours, on the other hand, invariably

12

UNSCEAR 2006 REPORT: VOLUME II

show a distribution with much lower oxygen tension [B44].
As will be described later, a role for ROS in non-targetedradiation-induced bystander effects has also been described,
suggesting a potential commonality in processes involved inthese delayed effects of exposure to ionizing radiation.
13. It is well known that most mammalian cells do notdivide indefinitely in vitro or in vivo, owing to a processtermed replicative senescence. In human cells, replicative senescence can be caused by telomere shortening, butmurine cells senesce despite having long, stable telomeres.
Parrinello et al. [P19] showed that the phenotypes of senescent human fibroblasts and mouse embryonic fibroblasts(MEFs) differ under standard culture conditions that include
20% oxygen. The MEFs did not senesce in physiological(3%) oxygen levels, but underwent a spontaneous event thatallowed indefinite proliferation in 20% oxygen. The proliferation and cytogenetic profiles of DNA repair-deficient MEFs
suggested that DNA damage limits MEF proliferation in 20%
oxygen. Indeed, MEFs accumulated more DNA ­damage in
20% oxygen than in 3% oxygen, and more ­damage thanhuman fibroblasts in 20% oxygen. These results identify
oxygen sensitivity as a critical difference between mouseand human cells, explaining their proliferative differences inculture, and possibly their different rates of cancer induction
and ageing. Furthermore, they may contribute to explainingsome of the differences between mouse and human studies
described later in this annex.
14. There is emerging evidence implicating a role forextranuclear and even extracellular events in initiating andperpetuating radiation-induced chromosomal instability.
Kadhim et al. [K3] analysed chromosomal instability inmurine haemopoietic stem cells following alpha particle irradiation. Many of the surviving cells were those that were nottraversed by an alpha particle during irradiation. Expanding
these studies, Wright and colleagues used a protective metal
grid to shield regions of the cell culture flask and lethally
irradiated the non-shielded regions of the flask. They thencultured the non-irradiated, shielded cells and examined theclonal progeny for induced chromosomal instability. A highfrequency of instability was observed in the progeny of cells
that were not directly hit by radiation [L24]. Clearly, induced
instability has an extracellular component, and signals from
irradiated cells can stimulate chromosomal rearrangements
in non-targeted cells within the radiation environment(reviewed in reference [M10]). These observations have
implications for the fate of cells surviving radiation exposurein that some of these surviving cells may develop genomic
instability. These observations also indicate that even cells
outside the radiation field can manifest phenotypes similar
to those of irradiated cells.
B.

Induced genomic instability after in vivo
irradiation followed by in vitro analysis

15. Weissenborn and Streffer were the first to describe
induction of genomic instability after irradiation in vivo

followed by analysis in vitro. They reported structural andnumerical chromosomal anomalies as well as micronuclei
at the first, second and third mitosis after in vivo irradiationof one- or two-cell mouse embryos with X-rays or neutrons[W6, W7]. These observations were extended by Ullrich
and Davis [U18], who irradiated inbred BALB/c mice andat varying intervals after irradiation removed and cultured
the mammary glands in vitro. Cytogenetic analysis indicatedthat instability could develop and persist in situ in a mature,
fully differentiated tissue after in vivo irradiation. Furthermore, there was a dose-dependent increase in the frequency
of delayed aberrations at low doses (0.1–1 Gy) that reacheda plateau at higher doses [U18].
16. Cellular studies on radiation-induced murine mammary cancer demonstrated strain-dependent differencesin susceptibility, presumably resulting from differences insensitivity to neoplastic initiation [U17]. Similar strain susceptibility is apparent for in vivo irradiation followed byin vitro analysis of induced instability. Mammary cells from
BALB/c mice are more susceptible to radiation-induced
genomic instability than those from C57BL/6 or F1 hybrid
crosses of C57BL/6 and BALB/c mice [P9, U20]. Studies
of DNA repair in the radiosensitive BALB/c mouse revealed
inefficient end-joining of gamma-ray-induced double-strand
breaks in DNA. This is apparently due to reduced expressionof the DNA-PKcs protein and lowered DNA-PK activity inthese mice. This may impair the animals’ ability to appropriately respond to induced damage and may thus accountfor the increased instability [O4, Y3]. Most DNA repair pro­
cesses have evolved to prevent genomic instability induced
by endogenous lesions [L49] and induced DNA damage (fora comprehensive discussion, see the BEIR VII [C23] andFrench Academies [T8] reports).

C. Induced genomic instability after in vitro
irradiation followed by in vivo analysis
17. Conversely, instability induced in vitro can be transmitted in vivo following transplantation of irradiated cells
into recipient animals. Paquette and Little [P3] irradiated C3H 10T½ cells and cultured half in cell culture in
vitro; the other half was transplanted into syngeneic andnon-­immunosuppressed C3H mice. Interestingly, a higher­frequency of minisatellite instability was observed in those
irradiated cells injected into mice than those cultured invitro. Watson et al. [W3] reported the induction and longterm persistence of chromosomal instability after murinebone marrow cells were irradiated in vitro and then transplanted into female CBA/H mice that had received 10 Gy of
X-irradiation less than two hours before to eradicate the hostbone marrow. These studies were later extended to demonstrate that instability induced by X-ray or neutron irradiationin vitro can be transmitted in vivo [W4]. A recent analysis
of a series of radiation-induced sarcomas [G10] showed aprevalence (53%) of somatic TP53 mutations, which wassignificantly higher than that for sporadic sarcomas (16.8%).

ANNEX C: NON-TARGETED AND DELAYED EFFECTS OF EXPOSURE TO IONIZING RADIATION

The mutations were inactivating and associated with theloss of the other TP53 allele. This loss of heterozygosity
was due to the loss of a large fragment of the chromosome
or of the whole chromosome, probably indicating a moregeneral chromosomal instability similar to that previously
described [L1].
18. Watson et al. [W2] have provided convincing evidence
that the induction of genomic instability following in vitro
irradiation and in vivo expression can result from a nontargeted bystander-like effect. That is, rather than resulting
from the direct effect of radiation exposure being passed
on to the progeny of that irradiated cell, instability mightalso result from soluble signals being passed from irradiated cells to non-irradiated cells. When non-irradiated cells
were mixed with cells irradiated with 0.5 Gy of neutrons at
0.04 Gy/min and then transplanted into recipient CBA/H
mice, instability was observed in the non-irradiated cell
population [W2]. An elegantly conceived chromosomal
marker system allowed the investigators to distinguishbetween the irradiated and non-irradiated transplanted cells
and cells derived from the host mouse. Irradiated and nonirradiated cells were distinguished by using marrow from
CBA/H mice (40XY cells) and the congenic CBA/H strain(40XY6T6 cells) homozygous for the stable T6 reciprocal translocation between chromosomes 14 and 15. Using
this system, unambiguous evidence for non-clonal chromosomal aberrations was observed in clonal populations
derived in vitro from neutron-irradiated bone ­marrow cells.
Furthermore, after transplantation with neutron-­irradiatedcells, translocations and deletions were observed fora period of 3–13 months. Significantly, there was also ahigher frequency of unstable aberrations in the bone marrowof the recipient mouse. These results implicate an in vivo
bystander-like mechanism in the induction of chromosomal
instability, and suggest that the instability observed in the
non-irradiated cells is not an artefact of clonal selection.
This result was confirmed by Xue et al. [X2], who injectednude mice with a mixture of human colon LS174T adenocarcinoma cells and LS174T-cells prelabelled with lethal
doses of DNA-incorporated 5-[125I]iodo-2’-deoxyuridine(125IUdR). A distinct inhibitory effect on the growth of theunlabelled LS174T tumour cells was observed. Because
125
IUdR is incorporated into DNA, almost all the electrons
emitted during radioactive decay have a subcellular rangeof <0.5 µm. This led the authors to conclude that the inhibitory result was due to a bystander effect generated in vivo
by factors present within and/or released by the 125IUdRlabelled cells. However, it is also possible that debris andbreakdown products from the heavily irradiated cells might
affect bystander cells, and these non-labelled cells mighteven incorporate 125I released from dying cells.
19. Currently the mechanisms underlying the induction
and persistence of instability are not understood. The induction of chromosomal aberrations in vivo by a bystander-likemechanism might provide insights into the mechanisms aswell as link instability to bystander effects. Bystander effects
can be mediated by cell-to-cell gap junction communication

13

and secretion of soluble factors. These secreted factors[S39] might include extracellular cytokine-like factors [L2,N8] that are able to increase intracellular levels of ROS
in ­non-irradiated cells [L23, M10, M16]. Lorimore et al.
recently proposed a potential mechanism for these in vivo
radiation-induced bystander effects [L22]. They found persistent macro­phage activation combined with neutrophil
infiltration following 4 Gy whole-body irradiation of mice.
The inflammatory nature of the observed responses may
provide a mechanism for the long-term production ofgenetic damage by a bystander effect, ultimately contributing to radiation-induced instability and potentiallyleukaemo­genesis. This will be discussed in more detail inthe section on radiation-induced genomic instability andbystander effects.

D. Radiation-induced genomic instability in vivo
20. In reviewing the literature on in vivo non-targeted
effects of ionizing radiation, it becomes obvious when
considering the mouse studies that many of the observed
effects are highly dependent upon the mouse strain used
and the sex of the animal studied [M58]. Consequently,
there has been an effort throughout this annex to identify
the mouse strain used when comparing conflicting data. It
is also apparent that even the same mouse strain can varysignificantly when bred in different colonies in different laboratories. Differences due to sex might also exist,
but not all of the studies provide adequate details on thesex of the animals used. While much has been learned
from animal models [F7], caution should be exercisedwhen extrapolating from the animal studies to the humansituation.
21. The reports of radiation-induced genomic instability
in vivo are summarized in table 2, which also lists the end
point used to assay instability, the model system, the type ofradiation used and whether or not genomic instability wasobserved. In this section the methods of analysing instability in vivo will be highlighted along with potential areas ofconflict and associated caveats.
22. Nowell [N19] first proposed that genomic instability might be a driving force in tumorigenesis and a hallmark of many cancers [C7, L3]. There is accumulating
evidence suggesting that instability may represent a critical step in the genesis of certain radiation-induced cancers[L14, S3, U20]. Implicit in this annex is the hypothesis that­radiation-induced genomic instability provides relevant
underlying mechanistic contributions to some radiationinduced cancers. While the precise relationship between
radiation-induced genomic instability and radiation carcinogenesis remains to be determined, understanding themechanisms of induced instability might provide valuable
insights into health risks associated with radiation ­exposureand the carcino­genesis process in general.

14

UNSCEAR 2006 REPORT: VOLUME IITable 2

In vivo studies of radiation-induced genomic instability (RIGI) and transgenerational effects

Year

End point

Cellular system

Radiation type

RIGIa

Reference

1976

Graft versus host reactions; non-specificbystander activity(PVGc × Wistar) F1 hybrids; PVGc spleen
cells

X-rays

+

[J10]

1979

Chromosomal aberrations

Haemopoietic cells in atomic bombing
survivors

Neutrons; gamma rays

–

[K21]

1979

Tumour induction

Mouse localized exposures

X-rays; neutrons

+

[U19]

1980

Tumour induction

Mouse localized exposures

X-rays; neutrons

+

[U16]

1982

Foetal deaths, malformations in F1 mice

Mouse whole-body irradiation

X-rays

+

[K13]

1982

Tumour induction in F1

Mouse whole-body irradiation

X-rays

+

[N17]

1984

Foetal deaths, malformations in F1 mice

Mouse whole-body irradiation

X-rays

+

[K12]

1985

Tumour induction

Mouse whole-body irradiation

X-rays

+

[C6]

1988

Chromosomal aberrations

Skin fibroblasts (mouse zygotes irradiated)

X-rays; neutrons

+

[P1]

1988

Chromosomal aberrations

Mouse embryo

X-rays; neutrons

+

[W14]

1988

Congenital abnormalities

Skin fibroblasts (mouse zygotes irradiated)

X-rays; neutrons

+

[P1]

1988

Micronucleus frequency

Mouse embryo

X-rays

+

[W14]

1989

Cell proliferation in F1 and F2 generations

Mouse whole-body irradiation

Gamma rays

+

[O2]

1989

Chromosomal aberrations

Mouse zygotes

X-rays

+

[P17]

1989

Chromosomal aberrations

Mouse embryo

X-rays

+

[W6]

1989

Micronucleus frequency

Mouse zygotes

X-rays

+

[P17]

1990

Cancer prevalenceChildren of nuclear plant workers

X-rays; gamma rays

+

[G2]

1990

Foetal deaths, malformations

Mouse embryos

X-rays

+

[M26]

1991

Chromosomal aberrations

Blood lymphocytes of uranium miners after
whole-body irradiation

Alpha particles

–

[M34]

1991

Sister chromatid exchanges

Blood lymphocytes of uranium miners after
whole-body irradiation

Alpha particles

–

[M34]

1993

Chromosomal aberrations

Skin fibroblasts in atomic bombing survivors

Neutrons; gamma rays

–

[H13]

1993

Microsatellite instability in F0, F1 and F2
generations

Mice after whole-body irradiation

Neutrons; X-rays;
gamma rays

+

[D10]

1994

Cancer prevalenceChildren of nuclear plant workers

X-rays; gamma rays

–

[D6]

1994

Microsatellite instability in F0, F1 and F2
generations

Mice after whole-body irradiation

Neutrons; X-rays;
gamma rays

+

[S1]

1994

Minisatellite instability

C3H 10T½ murine cells (irradiated in vitro,then injected into mice)

X-rays

+

[P3]

1995

Cancer prevalence

Progeny of cancer patients who had undergone radiation therapy

X-rays

–

[H4]

1995

Microsatellite instability in F0, F1 and F2
generations

Mice after whole-body irradiation

Neutrons; X-rays;
gamma rays

+

[F2]

1995

Tumour induction in F1

Mouse whole-body irradiation

X-rays

–

[C4]

1996

Chromosomal aberrations

Murine bone marrow (in vitro irradiation)

Alpha particles

+

[W3]

1996

Minisatellite instability

Chernobyl survivors after whole-body
irradiation

Gamma rays

+

[D11]

1996

Minisatellite instability

Haemopoietic cells in atomic bombing
survivors

Neutrons; gamma rays

+/–

[S5]

ANNEX C: NON-TARGETED AND DELAYED EFFECTS OF EXPOSURE TO IONIZING RADIATION

15

RIGIa

Reference

X-rays+[U17]

Children of nuclear plant workers

X-rays; gamma rays

–

[D7]

Cell proliferation in F1 and F2 generations

Mouse whole-body irradiation

Gamma rays

+

[W10]

1997

Minisatellite instability

Chernobyl survivors after whole-body
irradiation

Gamma rays

+

[D12]

1997

Mutation frequencies in F1

Mice after whole-body irradiation

Gamma rays

+

[L25]

1998

Chromosomal aberrations

Human lymphocytes after whole-body
irradiation

Gamma rays

–

[S2]

1998

Chromosomal aberrations

Lymphocytes from uranium miners

Alpha particles (radon)

+/–

[S25]

1998

Chromosomal aberrations

Blood lymphocytes from plutonium workers

Gamma rays

–

[W9]

1998

Micronucleus frequency

Rat lung cells after partial-volume irradiation

Gamma rays

+

[K11]

1998

Micronucleus frequency

Lymphocytes from uranium miners

Alpha particles (radon)

+

[S25]

1998

Microsatellite instability in F0, F1 and F2
generations

Mice after whole-body irradiation

Neutrons; X-rays;
gamma rays

+

[D15]

1998

Tumour induction in F1

Mouse whole-body irradiation

Gamma rays

+

[L20]

1999

Chromosomal aberrations

Lymphocytes of Chernobyl survivors after
whole-body irradiation

Gamma rays

+

[G3]

1999

Chromosomal aberrations

Lymphocytes of Chernobyl recovery operations workers and workers from the nuclear
power plant

Gamma rays

+/–

[L36]

1999

Chromosomal aberrations

Haemopoietic cells in atomic bombing
survivors

Neutrons; gamma rays

–

[N6]

1999

Chromosomal aberrations

Mouse epithelial cells after whole-body
irradiation

X-rays

+

[U18]

1999

Chromosomal aberrations

Mouse bone marrow cells after whole-body
irradiation

X-rays

+

[X1]

1999

Prenatal mortality; developmental andskeletal defects in F2 mice

Mouse zygotes

X-rays

+

[P7]

1999

Sister chromatid exchanges

Lymphocytes of Chernobyl recovery operations workers and workers from the nuclear
power plant

Gamma rays

+

[L36]

1999

Minisatellite instabilityChildren of Chernobyl recovery operations
workers

Gamma rays

+/–

[L18]

2000

Chromosomal aberrations

Foetal haemopoietic cells of mice after
foetal irradiation

Gamma rays

+

[D3]

2000

Chromosomal aberrations

C57BL/6 mice after whole-body irradiation

Gamma rays

+

[S20]

2000

Chromosomal aberrations

Blood lymphocytes of patients after wholebody irradiation for radiotherapy

X-rays

–

[T2]

2000

Chromosomal aberrations

Mixture of irradiated and non-irradiatedmurine bone marrow cells

Neutrons

+

[W2]

2000

Micronucleus frequency

CBA mice after prenatal irradiation

Gamma rays

–

[A1]

2000

Microsatellite instability in F0, F1 and F2
generations

Mice after whole-body irradiation

Neutrons; X-rays;
gamma rays

+

[D16]

2000

Tumour induction in F1

Pregnant miceGamma rays

+

[U21]

2000

Minisatellite instability

Sperm from three seminoma patients

X-rays

–

[M5]

Year

End point

Cellular system

1996

Neoplastic transformation

Epithelial cells of whole-body-irradiated mice

1997

Cancer prevalence

1997

Radiation type16

UNSCEAR 2006 REPORT: VOLUME II

Radiation type

RIGIa

Reference

Mouse bone marrow cells after whole-body
irradiation

Alpha particles (224Ra);
X-rays–[B12]

Chromosomal aberrations

Mouse bone marrow cells after whole-body
irradiation

X-rays

+

[M1]

2001

Chromosomal aberrations

Blood lymphocytes of uranium miners after
whole-body irradiation

Alpha particles

–

[M35]

2001

Chromosomal aberrations

Blood lymphocytes of cancer patients after
whole-body irradiation

Gamma rays

–

[V1]

2001

Chromosomal aberrations

Murine bone marrow; irradiated cells orwhole-body irradiation

X-rays; alpha particles;
neutrons

+

[W4]

2001

Chromosomal aberrations

Blood lymphocytes from plutonium workers

Gamma rays

–

[W8]

2001

Ductal dysplasia

Epithelial cells of ATM+/- mice

X-rays

+

[W15]

2001

Minisatellite instabilityChildren of Chernobyl recovery operations
workers

Gamma rays

–

[L17]

2001

Minisatellite instability

Haemopoietic cells in atomic bombing
survivors

Neutrons; gamma rays

+/–

[N7]

2001

Minisatellite instabilityChildren of Chernobyl recovery operations
workers

Gamma rays

+

[W5]

2001

Mutation frequencies in F1

Mice after whole-body irradiation

Gamma rays

+

[N14]

2001

Signal kinase activity in F3

Mouse whole-body irradiation

Gamma rays

+

[B4]

2001

Sister chromatid exchanges

Blood lymphocytes of uranium miners after
whole-body irradiation

Alpha particles

–

[M35]

2002

APRT, HPRT mutation frequency

Mouse T-lymphocytes after whole-body
irradiation

X-rays

+

[L35]

2002

Cancer prevalence

Progeny of radiation workers

X-rays; gamma rays

+

[D20]

2002

Cell proliferation in F1 and F2

Mouse whole-body irradiation

Gamma rays

+

[B5]

2002

Chromosomal aberrations

Mouse T-lymphocytes after whole-body
irradiation

X-rays

+

[L35]

2002

Microsatellite instability in F0, F1 and F2
generations

Mice after whole-body irradiation

Neutrons; X-rays; gamma
rays

+

[B2]

2002

Microsatellite instability in F0, F1 and F2
generations

Mice after whole-body irradiation

Neutrons; X-rays; gamma
rays

+

[D9]

2002

Minisatellite instability

Chernobyl survivors after whole-body
irradiation

Gamma rays

+

[D19]

2002

Minisatellite instabilityChildren living in Semipalatinsk

131

+

[D8]

2002

Mutation frequencies in F1

Mice after whole-body irradiation

Gamma rays

+

[S26]

2002

Signal kinase activity in F3

Mouse whole-body irradiation

Gamma rays

+

[V2]

2003

ROS content; state of DNA structure

Bone marrow cells of male mice after
whole-body irradiation

X-rays (1.5 Gy)

+

[M58]

2003

Minisatellite instabilityChildren of Chernobyl recovery operations
workers

Gamma rays

–

[K37]

2004

DNA double-strand breaks; morula andgastrula formation

Oryzias latipes F3 embryos derived frommale founders

Gamma rays

+

[A18]

2004

Epigenetic global genomic DNA methylation
changes

Male and female mouse whole-body
irradiation

Low-dose X-rays

+

[K30]

2004

Latency of cancer risk

Cohorts of underground miners exposed to
radon

Radon

–

[L41]

Year

End point

2001

Chromosomal aberrations

2001

Cellular systemI, 90Sr and 137Cs

ANNEX C: NON-TARGETED AND DELAYED EFFECTS OF EXPOSURE TO IONIZING RADIATION

Year

End point

Cellular system

Radiation type

17

RIGIa

Reference

+

[H26]

2004

Chromosomal aberrations; transformation
frequency; cell killing; DNA damage

Mouse embryonic fibroblasts from mice
deficient in Hsp70.1 and Hsp70.3

2004

Epigenetic global genomic DNA methylation
changes

Male and female mouse whole-body
irradiation

High-dose X-rays(5 Gy)

+

[P21]

2004

Somatic mutation assay

F1 and F2 progeny of Oryzias latipes

Gamma rays

+

[S42]

2004

Destruction of haematopoietic progenitor andstem cells in F1

Mice after sublethal irradiation

+

[Z8]

2004

Chromosomal aberrations; tumour incidence;
blood counts

Pregnant mice; mice foetal liver and spleencells

1 Gy irradiation

+

[U22]

2004

Apoptosis; cell proliferation and differentiation

Mouse limb bud cells

0.3 and 5 Gy

+

[W21]

2004

Micronuclei; number of ovules; male fertility

Pea plant seedlings for two generations

Gamma rays

+

[Z8]

2004

Mini- and microsatellite instabilityChildren of Chernobyl recovery operations
workers

Gamma rays

–

[S55]

2005

Chromosomal aberrations

Pregnant Swiss albino miceGamma rays
(1–1.5 Gy)

+

[U23]

2005

Initiation of intestinal adenoma

Apc mice

X-irradiation

–

[E20]

2005

Locus-specific mutations

Oryzias latipes spermatogonial stem cells

Gamma rays
(0.03 cGy/min and
95 cGy/min)

+

[S43]

2005

Chromosomal instability

Congenic haematopoietic cells into irradiated mouse hostGamma rays

+

[L47]

2005

Chromatid aberrations; chromosomal
radiosensitivity

Prostrate patients with prostatic hyperplasia

Ionizing radiation

+

[H25]

2005

Clonogenic survival; apoptosis

Bladder explants from C57BL6 and CBA/Ca
mice after whole-body irradiation

Low-dose irradiation (0.5 Gy)

+

[H25]

2005

Chromosomal aberrations

Clonal T-cells from atomic bombing survivors

Gamma rays

–

[K29]

2005

Chromosomal instability

Haemopoietic stem cells

Gamma rays

+

[L47]

2005

Tumour formation

Female B6C3 F1 miceGamma rays (1.9 Gy)

+

[S66]

2005

Microsatellite instabilityChildren of Chernobyl recovery operations
workers

Gamma rays

–

[F15]

a +: genomic instability was observed; –: genomic instability was not detected.

1. Mouse models for radiation-induced
genomic instability in vivo
23. As described above, transmissible genomic instability has been observed after irradiation in vivo followed byculture in vitro and vice versa. The picture following irradiation in vivo and subsequent expression of instability invivo is less clear and more controversial. Following irradiation of bone marrow cells from 12 week-old CBA/H micewith 0.5 Gy of alpha particles, Watson et al. [W3] observed
a constant frequency (10–13.4%) of cells with stable chromosomal aberrations for up to 17½ months. This increased
to 49.8% at 24 months in pooled samples from three CBA/H
mice. They noted significant variation between individuals,
with a few animals exhibiting little or no induced instability
despite CBA/H being an inbred mouse strain. In contrast,

protracted whole-body gamma irradiation of prenatal CBACa mice at either 44, 99 or 265 mGy/day (to a total dose of
0.7, 1.6 or 4.2 Gy) did not induce damage in erythroid stem
cells that could be detected as persistent or delayed chromosomal aberrations as measured by micronucleated erythrocytes at 35 days after irradiation [A1]. Bouffler et al. [B12]also failed to find evidence of transmissible chromosomal
instability 50 or 100 days after in vivo exposure of CBA/H
mice either to alpha particles from the bone-seeking radio­
nuclide 224Ra or to X-rays. Likewise, chromosomal instability was not detected in peripheral blood lymphocytes from
C57BL/6 mice for up to 30 days after whole-body gamma
radiation [S21] or up to 21 months [S20]. These last resultsare ­consistent with in vitro studies indicating that induced
instability was not observed in C57BL/6 mice [P9, W1].
­Furthermore, no instability was reported in bone marrow

18

UNSCEAR 2006 REPORT: VOLUME II

cells from Swiss mice up to 100 days after exposure to
3 Gy of X-rays [X1]. These results were initially presumed
to indicate that the Swiss mouse strain, like the C57BL/6
mouse strain, is refractory to radiation-induced instability.
However, this does not appear to be the case. When Swiss
albino mice were exposed to 0.25–1.5 Gy of gamma radiation on day 14 or 17 of gestation, significant dose-dependent
increases in chromosomal aberrations, micronuclei and/or changes in ploidy were observed in the bone marrow at
12 months of age [D3]. The investigators concluded thatradiation-induced genomic instability in the foetal haemopoietic cells of the mouse persisted post-natally [D3]. These
data are summarized in table 2.
24. Although not designed to specifically investigate­radiation-induced genomic instability, a number of studies
have examined the persistence of cytogenetic rearrangements
in animals at delayed times after irradiation. Hande et al.
[H18, H19, H20, H21] used female Swiss mice to study the
induction and persistence of dicentrics and translocations in
splenocytes up to 112 days after exposure to 2 Gy of wholebody X irradiation. The frequencies of dicentrics decreased
exponentially with time, while the frequencies of translocations were constant in the period 0–7 days and then decreased
linearly or exponentially. No new chromosomal rearrangements were observed, suggesting that there was no delayed
cytogenetic instability in these animals. Similar studies using
other mouse models have reported similar results [T5].
25. In attempting to reconcile the apparently conflicting
results described above, Bouffler et al. [B12] have noted
the sensitivity of mouse bone marrow cells to perturbations
through transplantation and culture, and emphasized theneed for sound control experiments to be performed concurrently. For instance, some of the radiation-induced transmissible chromosomal instability reported by Watson et al.
[W3] could be attributed to the low background frequency ofaberrations observed in the control repopulating cells. This
is in contrast to the higher background described by Bouffler
et al. [B12]. It is also possible that the disparate literatureon radiation-induced genomic instability in vivo reflects theinherent variability between the inbred mouse strains usedand differences due to sex within the animal strains used.
26. To investigate the in vivo non-targeted effects of lowLET radiation, Lorimore et al. [L47] used the same congenic
sex-mismatch bone marrow transplantation protocol as usedby Watson et al. [W2] to repopulate the haemopoietic systemfrom a mixture of gamma-irradiated and non-irradiated haemopoietic stem cells such that host-, irradiated donor- andnon-irradiated donor-derived cells could be distinguished.
Chromosomal instability in the progeny of irradiated haemopoietic stem cells accompanied by a reduction in their contribution to the repopulated haemopoietic system was observed
and is consistent with a delayed genomic instability phenotype being expressed in vivo. However, chromosomal instability was also shown in the progeny of the non-­irradiatedhaemopoietic stem cells, implicating a bystander-likemechanism. Studies of the influence of irradiated recipient

stromal microenvironment and experiments replacing irradiated cells with irradiated cell-conditioned medium revealed
the source of the in vivo bystander effect to be the descendants of irradiated cells rather than the irradiated cells themselves. ­Lorimore et al. [L47] speculated that it is possiblethat a radiation-induced genomic instability phenotype invivo need not necessarily be a reflection of intrinsicallyunstable cells but the response to ongoing production of
inflammatory-type damaging signals [L22] as a long-term
unexpected consequence of the initial radiation.
27. While the literature is replete with apparently contradictory reports of radiation-induced instability in mouse model
systems, these results clearly indicate that genetic factors canplay a major role in the instability phenotype and that analysis of radiation-induced genomic instability in vivo is significantly more complicated than in vitro. Critical analysis ofradiation-induced genomic instability in vivo is not a trivial
undertaking. In any animal model there is likely to be someinherent genomic instability that complicates the selection of
appropriate control populations. Such experiments generally
involve inbred strains of mice, and even in radiation-­sensitive
populations only a small percentage, generally <50%, will
exhibit an instability phenotype. Further­more, extrapolatingsuch results to other mouse strains or outbred populations isdifficult at best. Until a careful study involving sound andrelevant controls as well as statistically relevant numbersof animals exposed to a homogeneous quality of radiation
is carried out, the induction of radiation-induced genomic
instability in vivo will remain controversial.
2.

Human studies

28. Radiation therapy has improved over recent years, andmany of the cancer patients treated with radiation are surviving longer than did those in the past. Second cancers occurring in the irradiated field have been reported in some of thesepatients, suggesting a direct role of the radiation exposure[B37, B38]. Data on second cancers occurring in children
irradiated for cancer indicate that some genetic predisposition to cancer may also predispose them to radiotherapy­related second cancers [D22, E19, F14]. Nevertheless,
it is still difficult to identify the radiation-induced lesions
initiating the second malignancy. At the time of diagnosis,
multiple genomic alterations are present in the tumours, andthe majority are likely to represent secondary events occurring during tumour evolution and subsequent selection.
This underscores that caution must be applied to analysis ofradiation-induced genomic instability and its role in humancarcinogenesis. The subsequent discussion in this section
highlights the controversies and contradictions inherent in
the human studies. To this end it is reasonable to expect thatanalysis of normal, healthy populations of individuals would
not provide evidence of instability regardless of the individuals’ radiation history. Indeed, the majority of studies investigating instability in radiation-exposed populations haveanalysed samples from normal, healthy individuals and did
not find evidence of instability [T2, T4]. It is also reasonable

ANNEX C: NON-TARGETED AND DELAYED EFFECTS OF EXPOSURE TO IONIZING RADIATION

to expect that analysis of instability in individuals manifesting phenotypic effects of radiation exposure, e.g. cancer or
leukaemia, might well show evidence of induced instability. Once again, limited studies indicate that this is the case[N6, N7]. Whether or not the observed instability is a director non-targeted effect of radiation exposure, or a secondary
selective effect of disease evolution, cannot be definitively
determined at present. Furthermore, this question is unlikely
to be resolved in the foreseeable future. This caveat should
be kept in mind in the following discussion.
29. As has been described utilizing the mouse as a model
system, both induction and lack of induction of transmissible radiation-induced genomic instability have been reported
in humans, and once again genetic factors appear to playa role in the observed instability [K4]. Induced chromosomal instability has been described in long-term culturesof human lymphocytes following irradiation and culture in
vitro [H9]. Using the same lymphocyte culture protocol,
chromosomal instability was reported in blood samples
from individuals exposed during the radiation accident inEstonia in 1994 [S2]. Radiation exposure was variable, protracted and not precisely determined. Furthermore, blood
samples were taken well after radiation exposure. No dose
response was apparent, and contrary to previous studies
from the Lambert laboratory, chromosomal instability was
also observed in long-term cultures from non-exposed controls [S2]. In contrast, cytogenetic analysis of 18 individuals who had received between 35 and 80 Gy of fractionatedradiation therapy for different cancers showed no increasein aberrant cell types as a function of time after completing therapy. Thus no cytogenetic evidence that fractionatedradiotherapy induced a persistent or late-manifesting stateof genomic instability was found [T2]. It should be stressedthat the majority of patients treated for different malignancies received localized, partial-body irradiation with emphasis on minimizing damage to normal tissue. Consequently,
different proportions of bone marrow stem cell populations
and peripheral blood lymphocytes would have been exposedto the radiation. It is likely that more cells than the number
actually analysed (<200 per patient), would have to be interrogated before evidence of persistent transmissible chromosomal instability would be observed in these individuals, if it
indeed existed [T2].
30. The availability of cultured lymphocyte preparations
from radiation workers with internal deposits of plutonium
has provided the opportunity to examine whether protracted
irradiation of bone marrow cells had induced a transmissible genomic instability in descendant cells in the peripheral
blood [W8]. Bone marrow dose calculations provided individual cumulative estimates at the time of sampling ranging
up to 1.8 Sv. Chromosome analysis revealed no significant
differences, either in comparisons between the total groupof plutonium workers and controls for comparable periods
or when the comparisons were restricted to a group of plutonium workers with initial bone marrow plutonium dosesof greater than 0.25 Sv. There was therefore no evidence
from this study for the induction of persistent transmissible

19

genomic instability in the bone marrow of radiation workers
with internal deposits of plutonium [W8]. Likewise, clonallyexpanded T-cell lymphocyte populations did not demonstrateincreased chromosomal instability using either G-band analysis or multicolour fluorescence in situ hybridization [K29].
31. The long-term effect of radiation exposure on uranium
miners employed by the Wismut uranium mining company
in the former German Democratic Republic was investigatedby scoring the frequency and percentage of micronuclei withand without a centromere. Kryscio et al. [K38] reported
that genomic instability had occurred in the lymphocytes of­miners, especially those with cancer.
32. A number of investigators have studied the alpha radiation risks in patients who received injections of Thorotrast,
an X-ray contrast medium used in Europe, Japan and theUnited States from the late 1920s to 1955. Thorotrast wascomposed of thorium dioxide and contained 232Th, a naturally occurring radionuclide. Because the physical half-lifeof 232Th is 14 billion years and Thorotrast is not appreciably eliminated from the body, the tissues in which it was
deposited are irradiated by alpha rays for the entire lifetimeof the subject. The major causes of death among the Thorotrast patients are liver cancer, liver cirrhosis, leukaemia andother cancers. Mutation analyses of the TP53 gene and lossof heterozygosity (LOH) studies at the 17p locus were performed by Ishikawa et al. [I2] to characterize the genetic
changes in Thorotrast-induced liver tumours. LOH was notfrequent; most mutations were transitions, suggesting thatgenetic changes in Thorotrast-induced cancers were mainly
delayed mutations and not the result of the direct effects ofradiation.
33. Likewise Iwamoto et al. [I3] analysed mutations in
TP53 from 20 Thorotrast recipients who developed cancer, mostly of hepatic bile duct and blood vessel origin. Of
the 20 cases, 19 had TP53 point mutations. Moreover, theaccompanying non-tumour tissues from these patients also
had TP53 mutations, albeit at lower frequency. The distribution pattern of the point mutations was significantly different between the non-tumour and tumour tissues, withmost mutations in malignant tissues located in the highly
conserved domains of the TP53 gene. These results support
the idea that TP53 mutations are important in the genesis of
Thorotrast-induced tumours but that these point mutations
are a secondary outcome of genomic instability induced by
the irradiation. A similar result was reported by Kamikawa
et al. [K24], who investigated mutations of the RAS and the
TP53 genes in archival sections of liver cancers induced byThorotrast. These investigators were unable to rule out thepossibility that genetic insults occurred indirectly in the proliferating cells adjacent to the necrosis rather than being a
direct effect of alpha particles.
34. Wada et al. [W17] also investigated genetic changes
in the TP53 gene in 19 autopsy cases of liver malignancies. LOH at the 17p13 locus and mutations in TP53 wereanalysed. A number of cases were informative: four cases

20

UNSCEAR 2006 REPORT: VOLUME II

showed LOH and eight contained mutations. The direct
action of alpha particles was thought to result in relatively
large deletions, such as those detected by LOH. Therefore
the low frequency of such changes (27%) compared withpoint mutations (47%) suggests that the genetic changes in
the TP53 gene in the liver tumours related to Thorotrast were
not caused mainly by direct actions of alpha particles but
rather by indirect effects that may have been due to cycles ofnecrosis and regeneration. This study was recently expanded
to compare Thorotrast-induced liver cancers to those notassociated with Thorotrast exposure. LOH at 37 loci wasinvestigated. Liu et al. [L46] found frequent LOH at micro­
satellite markers D4S1538, D16S2624 and D17S1303 to becommon to all the subtypes of liver cancer, independent of
the specific carcinogenic agent. In contrast, LOH at marker
D4S1652 was generally not observed in Thorotrast-induced
cancers. LOH analysis revealed that Thorotrast-induced cancers share some LOH features with cancers not induced byThorotrast, and Liu and colleagues concluded that induced
LOH is not simply due to direct insult to DNA by alpha particles, but can occur through complex mechanisms, including bystander effects [L46]. Such a conclusion is reasonablegiven the analysis of Goto et al. [G18], who used imaging
plate autoradiography to examine the microdistributionof alpha particles in pathological sections of tissues fromThorotrast patients. They found that the amount of thorium
deposited in tumour tissue was correlated with that in nontumour tissue, and that Thorotrast deposition was not associated with DNA damage determined by histochemistry.
Goto et al. [G18] concluded that radioactive thorium always
migrates in macrophages within the deposited organs, andthat the organs are evenly exposed to alpha particles.
35. In an evaluation of Thorotrast-induced genomic instability, Liu et al. [L45] analysed microsatellite instability inThorotrast-induced liver cancers. The frequency of microsatellite instability cases was 62.5% in Thorotrast-induced
cancers, whereas it was 22.7% in non-Thorotrast induced
cancers. Liu and colleagues suggested that microsatellite
instability induced by exposure to Thorotrast mainly reflects
clonal expansion of cancer cells and is partly due to inactivation of the DNA mismatch repair gene hMLH1 by hypermethylation. A recent finding also suggests that methylation
changes in DNA can be associated with radiation-induced
genomic instability [K35].
36. Littlefield et al. [L16] examined the cumulative genetic
damage in a 72-year-old man who was treated with a 32 mLbolus of Thorotrast during cerebral angiography performed
more than 40 years earlier. Peripheral T-lymphocytes were
cultured to quantify the frequencies and cellular distributions of asymmetrical and symmetrical types of chromosomal aberrations. Assays of glycophorin A (GPA) mutations in red blood cells were also performed. Their results
revealed that approximately 30% of the lymphocytes in thispatient contained one or more chromosomal aberrations,the majority of which were of the “stable” type. About one
third of the lymphocytes with chromosome damage carried
multiple aberrations, suggesting that significant numbers ofstem cells survived exposures to alpha particle radiation thatinduced complex genomic alterations. Increased frequencies of GPA mutations were observed, demonstrating thatgenomic damage was also induced in erythroid progenitors.
Despite the relatively severe burden of somatic cell damage
induced by 40 years of internal alpha particle irradiation, thepatient remained free of any serious illness. Furthermore,these results provided no in vivo evidence for the continued expression of genomic instability. A similar observation
was reported by Hande et al. [H3], using a fluorescence insitu hybridization technique that made possible the detection of intrachromosomal rearrangements and deletions.
They described the quantification of stable intrachromosomal aberrations in lymphocytes of healthy former nuclear
weapons workers who were exposed to plutonium. Even
many years after occupational exposure, more than half
the blood cells of the healthy plutonium workers containedlarge (>6 Mb) intrachromosomal rearrangements. The yieldof these aberrations was highly correlated with plutonium
dose to the bone marrow. It is significant that, despite therelatively high frequency of intrachromosomal aberrations,
there was no evidence of transmissible chromosomal instability and no obvious detrimental health consequences in the
populations sampled.
37. Nevertheless, a role for radiation-induced genomic
instability has been described for solid tumours developing after radiotherapy for bilateral retinoblastoma [L1].
Genome alterations of second tumours (five osteosarcomas,
one malignant peripheral sheath nerve tumour, one leiomyosarcoma) occurring in the field of irradiation of sevenpatients treated for bilateral retinoblastoma were studied.
Because of a germ line mutation in the retinoblastoma gene(RB1), these patients were predisposed to develop radiationinduced tumours. In all radiation-induced tumours analysed, the normal RB1 allele was lost, whereas the germ line
mutated allele was retained and the two TP53 alleles were
inactivated. A comparison of these tumours with the nonradiation-induced tumours led Lefevre et al. [L1] to conclude
that this loss was due to the radiation-induced chromosomal
instability rather than a direct effect of ionizing radiation. A
similar observation was reported by Ryabchenko and colleagues [R12] after analysis of chromosomal aberrations
in peripheral lymphocytes taken from Hodgkin’s disease
patients after prolonged (up to 31 years) remission periods.
The mean frequency and patterns of aberrations in remission
patients were significantly different from comparison groups(healthy donors and primary Hodgkin’s disease patients).
New cancer cases were diagnosed in a number of the remission patients, leading the investigators to suggest that thetumorigenic potential of radiochemotherapy is mediated via
induction of genomic instability in exposed cells. Long after
the therapy, the instability may become an initiating eventin the development of new malignancies in affected tissues,
whereas the instability induced in haemopoietic stem cells
may reveal itself in peripheral lymphocytes derived frompreviously exposed precursor cells. The caveat, of course,is that these individuals were cancer patients and may havebeen inherently predisposed to second cancers.

ANNEX C: NON-TARGETED AND DELAYED EFFECTS OF EXPOSURE TO IONIZING RADIATION

E.

Genomic instability and radiation-induced leukaemia
1.

Mouse models

38. Plumb et al. [P8] have reviewed the relationship between
radiation-induced genomic instability and radiation-induced
leukaemia. They presented evidence that genomic instability plays a role during radiation leukaemogenesis. However, with the exception of a high incidence of non-clonal
chromatid-type cytogenetic aberrations in neutron-induced
acute myeloid leukaemia in mice [B26], the genetic lesions
described (including non-clonal chromosomal aberrations,
LOH and minisatellite/microsatellite mutations) were similar to those detected in de novo leukaemias and cancers.
This damage was not transmissible and the authors interpreted these observations as evidence of apoptosis or othercell death. Nevertheless, this radiation-induced ­damage invivo was indistinguishable from de novo multistage leukaemogenesis. Thus it is not yet possible to define a type ofgenomic instability in radiation-induced leukaemias in micethat demonstrates a specific characteristic of immediate ordelayed effects of the initiating exposure to ionizing radiation.
Evidence for radiation-induced genomic instability in mouse
leukaemia and haemopoietic stem cells led M­ acDonald et al.
[M1] to conclude that the induced instability contributed significantly to the induced leukaemia. A similar conclusion wasreached by Ban et al. [B1], who suggested that loss of TP53
function triggers the tumorigenic process leading to stem cellleukaemia through the induction of chromosomal instability.
These authors also pointed out that the aetiology of stem cellleukaemia is likely to differ from that of myeloid leukaemia,
because different results were observed in acute myeloid
leukaemia (AML) [B1].
39. Interestingly, however, susceptibility to radiationinduced leukaemia is genetically separable from sensitivity
to radiation-induced genomic instability [B13]. A series ofmatings, backcrosses and intercrosses between CBA/H micesusceptible to radiation-induced acute myeloid leukaemia
and radiation-resistant C57BL/6 mice was carried out, andacute myeloid leukaemia and thymic lymphoma susceptibility was analysed. No simple genetic relationship between
susceptibility to radiation-induced leukaemia and the sensitivity of the haemopoietic stem cells to induced instability
was found.
2.

Human studies

40. Cytogenetic analysis of leukaemia patients among thesurvivors of the atomic bombings in Japan revealed thatpatients exposed to >2 Gy exhibited a higher incidence ofchromosomal aberrations and more complex chromosomal
rearrangements than did patients exposed to lower radiation
doses or unexposed patients [T1]. A more recent cytogenetic
analysis of the heavily exposed patients with acute myelocytic leukaemia or myelodysplastic syndrome in the cohort ofbombing survivors indicated persistent chromosomal instability [N6]. These cytogenetic observations are supported

21

by studies demonstrating high frequencies of micro­satellite
instability in those bombing survivors with acute myelocytic leukaemia and a history of high exposure [N7]. These
investigators concluded that this persistent instability mightstrongly influence the development of leukaemia in humans
exposed to ionizing radiation. This study stimulated two­letters to the editor of the journal in which it was published.
The first, by Little [L40], claimed that although there wasevidence that the microsatellite instability rate was higher
in the AML cases among the bombing survivors than in thecontrol group, the evidence that this higher rate was related to
the radiation dose these cases received was weak. Little went
on to show that the number of loci for which microsatellite
mutation data were not detectable was higher in the bombing
survivor cases than in the control group. The second letter,by Cox and Edwards [C15], raised the issue of the statisticalstrength of the dose-related association between expressionof genomic instability in AML and the probability of causation by radiation. In response to these letters, Plumb [P18]pointed out that, from a biological perspective, the striking
similarities in the leukaemias that arose in the bombing survivors and in therapy patients indicated that a significant
proportion of the AML cases among the bombing survivors
described by Nakanishi et al. [N6, N7] could indeed havebeen induced by the radiation. Reconciling mathematicalmodels [L40] and statistical analysis [C15] with the biological observations will always be difficult, however, particularly with the small number of AMLs observed in this uniquepopulation.
41. Mazurik and colleagues [M63] investigated molecular, biochemical and cytogenetic parameters in blood­samples from 17 radiation accident victims who between
1.7 and 43.8 years previously had suffered acute radiation
sickness ranging in severity from grade I to grade IV. All
patients showed ~25–30% reduction in oxidative status andincreased levels of both stable and unstable chromosomal
aberrations that correlated with the severity of the acuteradiation sickness. These data were interpreted as evidence
for delayed genomic instability in these radiation exposed
individuals.
F.

Role of telomeres and telomerase inradiation-induced genomic instability

42. Telomeres are specialized DNA–protein complexes atthe ends of linear chromosomes. They are composed of arepetitive DNA sequence and associated proteins. ­Telomere
alterations, caused by replication-mediated shortening,
direct damage or defective telomere-associated proteins, cangenerate chromosomal instability, which can be observedin senescence and during the immortalization process(reviewed in reference [M47]). Telomeres are essential for
proper maintenance of chromosomes and may play a role inageing and cancer [G13, G14]. Telomere length can be maintained by telomerase [B25], or by the alternative mechanismof telomere lengthening which is ­telomerase-independent[M7, S50]. Telomere length abnormalities observed in22

UNSCEAR 2006 REPORT: VOLUME II

radiosensitive cells suggest that, in some human cells, short
telomeres might correlate with radiation sensitivity [M49].
This may or may not be the case in mouse cells, as both
hypersensitivity [G19] and the absence of an effect havebeen described [M49]. To complicate the matter further,long but dysfunctional telomeres have been found to correlate with chromosomal radiosensitivity in a mouse AML cellline 7926 [F13].
43. Another role for telomerase appears to be the de novo
formation of telomeres, or chromosome healing to stabilize
broken chromosomes [M41, S35]. Telomere loss results insister chromatid fusion and prolonged breakage–fusion–bridge cycles leading to extensive DNA amplification andlarge deletions (reviewed in reference [M37]). Significantly,
the loss of a single telomere can result in the instability ofmultiple chromosomes [S46] and generate many of the typesof cytogenetic rearrangements commonly associated withhuman cancer. Telomere dysfunction can also trigger extensive DNA fragmentation and the evolution of complex chromosomal abnormalities in tumours in mice [A10] and humans[G11, M37]. This also appears to involve repeated cycles ofdicentric chromosome formation, anaphase bridging, subsequent breakage and refusion events [G12]. In addition, telo­mere dysfunction can result in increased mutation rates andgenomic instability (reviewed in references [F9, M37]).
44. It is noteworthy that exposure to ionizing radiation can
induce telomerase activity both in vitro [H23, N27] and invivo [H22]. This appears to be TP53-dependent [N26] andhas been suggested as a measure for monitoring the radiocurability of tumour cells [S49]. Radiation-induced telomerase activation depends on dose rate, is not related to cell
cycle redistribution or to the induction of cell death, and islikely to be the consequence of specific regulatory responses
to ionizing radiation [P20].
45. Telomere repeat-like sequences are also seen as discrete bands at distinct intrachromosomal sites in a numberof vertebrate species [M42]. These interstitial telomere sites
probably represent ancestral telomere fusion events or amplification of the repeat sequences in ancestral karyotypes aslatent telomeres [L39, M42]. There is compelling evidence
that some of these interstitial telomere sites may be hot spots
for both spontaneous [A11, H15] and radiation-induced
chromosome damage [A9, B27, S36, S57, S58]. While it isclear that interstitial telomere sequences can influence theradiation sensitivity of chromosomes, many of these studies
were performed in hamster cells where the interstitial telo­meres are located at or near pericentromeric heterochromatin. Analyses of radiation sensitivity in a naturally occurring
short interstitial telomere in a human chromosome (2q31,[A16]) and of a transfected 800bp telomeric repeat in humanchromosome 4q indicate that human interstitial telomere
sequences might not be prone to spontaneous [D24] orradiation-induced [D25] breakage.
46. The involvement of telomeric repeats in radiationinduced chromosomal instability was first described by

Sabatier et al. [S44] in high-LET-irradiated human fibroblasts. They demonstrated that instability acquired by humanchromosomes recurrently involved telomeric associations.
This was not due to drastic telomere shortening [S45]. Arole for recombination involving interstitial telomere-like
repeat sequences in inducing chromosomal instability waslater demonstrated by Day et al. [D1] in an in vitro ­Chinesehuman–hamster hybrid model system. In this instance it
was apparent that rearrangements involving unstable chromosomes occurred preferentially at the sites of interstitial
telomere sequences rather than at the true terminal telo­meres as observed by Sabatier [S45]. This difference islikely to be due to the telomeres on transformed ­hamster
chromosomes being very small relative to human telo­meres[S56]. Ojima et al. [O6] have reported that telomeres aredestabilized several generations after X-irradiation in normal human fibroblasts. Their data suggest X-irradiation
might not affect telomeres directly, but rather by inducing
a delayed instability.
47. Initially it was thought that an interstitial telomere-like
repeat sequence on chromosome 2 played an important rolein the deletions in somatic haemopoietic cells that characterized the earliest phases of radiation-induced AML in the
CBA/H mouse [B28]. Subsequent detailed molecular analysis of break points suggested that, while telomere sequenceswere located close to regions frequently involved, breakpoints were not exactly coincident with telomere sequences[F10]. The regions of frequent breakage appeared to haveproperties expected of matrix/scaffold attachment regions[F10]. At this stage one can only speculate on whether telomeric changes are a cause, an effect or a combination of bothin contributing to genomic instability independent of radiation exposure.

G.

Conclusions

48. Radiation-induced genomic instability is now wellestablished in a number of normal and transformed celllines in vitro. Instability can manifest as multiple different end points and can result from both targeted andnon-targeted events. The results of studies of induced
instability in vivo are more complex and contradictory. In
mice, observation of the instability phenotype appears tobe dependent upon the mouse strain used and the powerof the investigation in terms of the numbers of animals
investigated. The evidence for induced instability in
exposed human populations is controversial, with both
positive and negative effects being reported. It is possible that radiation-induced genomic instability provides
the impetus for those genomic alterations associated withradiation carcinogenesis. This view should be temperedby the high frequency with which instability is observed
both in vitro and in vivo, and the general lack of a dose–
response curve. Overall, a specific role for induced instability in the genesis of radiation-induced cancers has yet
to be definitively demonstrated.

II.

BYSTANDER EFFECTS AND RADIATION EXPOSURE[M50]. This underscores the critical role of adequate andappropriate controls. A historical perspective describing key
events in the study of radiation-induced bystander effects
has been presented by Mothersill and Seymour [M45].

49. There has been a resurgence of interest in radiationinduced bystander effects, largely because of the development of single-cell charged-particle irradiators. The term
“bystander effect” was adopted from the gene therapy literature, where it usually refers to the killing of several subpopulations of tumour cells by targeting only one “type” ofcell within a heterogeneous population [F6]. For the purposes of this annex, the definition proposed by Djordjevic
will be used [D5]. That is, “bystander effect” describes theability of cells affected by an agent to convey manifestationsof ­damage to other cells not directly targeted by the agent
or not necessarily susceptible to it per se. Thus radiationinduced bystander effects are effects manifesting in cells
that were non-irradiated neighbours of irradiated cells orthat received factors secreted or shed by irradiated cells. It is
implicit in this review that in vitro bystander effects are theresult of a signal generated by an irradiated cell interactingwith a non-irradiated cell [C22] and are not the result of radiation-induced changes in the culture medium [Z2], or due toexperimental variables such as the cell culture environment

Table 3Year

A.

Bystander effects in vitro

50. Radiation-induced bystander effects in vitro embrace anumber of different non-targeted experimental effects, someof which are likely to be detrimental to the cell, whereas
others are not. Different effects are observed in different cell types, and depend on the cell type producing the
bystander signal after irradiation and the cell type receiving
the bystander signal (summarized in table 3). Consequently,
no rigid rules can be applied to the multitude of responses
occurring in cells not targeted by radiation. For convenience,
bystander effects have been divided into four, not necessarily
mutually exclusive, subcategories.

In vitro studies of the bystander effectOrigin of bystander effectEnd point

Cell typeRadiation type

Reference

1992

Low-fluence alpha particle
irradiation

Sister chromatid exchanges

Human fibroblasts and epithelial
cells

Alpha particles

[N4]

1996

Low-fluence alpha particle
irradiation

Sister chromatid exchanges

Human lung fibroblasts

Alpha particles

[D2]

1997

Medium transfer

Sister chromatid exchanges

Human lung fibroblasts

Alpha particles

[L2]

1997

Medium transfer

Clonogenic survival

Human epithelial cells; humanfibroblasts

Gamma rays

[M18]

1997

Medium transfer

Clonogenic survival

Human keratinocytes

Gamma rays

[S12]

1997

Microbeam irradiation

Si- mutants induced

AL human–hamster hybrid cells

Alpha particles

[H14]

1998

Low-fluence alpha particle
irradiation

p53, p21, MDM2, CDC2, RAD5 1
protein levels

Human fibroblasts

Alpha particles

[A3]

1998

Low-fluence alpha particle
irradiation

Plating efficiency; chromosomal
aberrations

Murine bone marrow cells

Alpha particles

[L24]

1998

Medium transfer

Clonogenic survival

Human keratinocytes

Gamma rays

[M22]

1998

Microbeam irradiation

Micronucleus frequency; apoptosis

Human fibroblasts

Alpha particles

[P10]

1999

Cytoplasmic irradiation by
microbeam

Clonogenic survival; CD59 mutation
frequency

AL human–hamster hybrid cells

Alpha particles

[W13]

1999

Low-fluence alpha particle
irradiation

HPRT mutations

Chinese hamster ovary cells

Alpha particles

[N5]

23

24

UNSCEAR 2006 REPORT: VOLUME II

Year

Origin of bystander effectEnd point

Cell typeRadiation type

Reference

2000

Cytoplasmic irradiation by
microbeam

Clonogenic survival; CD59 mutation
frequency

AL human–hamster hybrid cells

Alpha particles

[Z1]

2000

Low-fluence alpha particle
irradiation

Gl checkpoint

Human fibroblasts

Alpha particles

[A8]

2000

Medium transfer

Clonogenic survival; AP-endonuclease;
TP53; ROS

Human lung fibroblasts (HFL-l)

Alpha particles

[I10]

2000

Medium transfer

Clonogenic survival; intracellular calcium levels; mitochondrial membranepotential; ROS levels

Human keratinocytes

Gamma rays

[L28]

2000

Medium transfer

Clonogenic survival

Human keratinocytes

Gamma rays

[S13]

2001

Co-culture

Clonogenic survival

V79 Chinese hamster cells (3-Dtissue culture model)

3

H beta particles

[B24]

2001

Co-culture

p53, HSP72 protein levels

A-172 human glioblastoma cells

X-rays[M3]

2001

Co-culture

Enhanced plating efficiency;
micronucleus frequency

Human salivary gland cells

X-rays or carbon
beam

[S30]

2001

Low-fluence alpha particle
irradiation

Changes in gene expression; inductionof DNA damage

Human fibroblasts and epithelial
cells

Alpha particles

[A4]

2001

Medium transfer

Clonogenic survival; apoptosis; transformation frequency

CGLI human HeLa × skin fibroblast
hybrid cells

X-rays[L5]

2001

Medium transfer

Clonogenic survival; intracellular calcium levels; mitochondrial membranepotential; ROS levels

Human keratinocytes

Gamma rays

[L38]

2001

Medium transfer

Clonogenic survival

Human urothelium cells

Gamma rays

[M39]

2001

Microbeam irradiation

Micronucleus frequency

Primary human fibroblasts

Alpha particles

[B9]

2001

Microbeam irradiation

Transformation frequency

C3H 10T½ murine fibroblasts

Alpha particles

[S6]

2001

Microbeam irradiation

Clonogenic survival

C3H l0T½ murine fibroblasts

Alpha particles

[A7]

2002

Co-culture

Enhanced plating efficiency andproliferation

Human salivary gland cells

Carbon beam

[S15]

2002

Co-culture on double Mylar dishes

Clonogenic survival; CD59 mutation
frequency

AL human–hamster hybrid cells

Alpha particles

[Z4]

2002

Low-fluence alpha particle
irradiation

Micronucleus frequency

Human fibroblasts

Alpha particles

[L43]

2002

Low-fluence alpha particle
irradiation

HPRT mutations; sister chromatid
exchanges

Human fibroblasts and epithelial
cells

Alpha particles

[L37]

2002

Low-fluence alpha particle
irradiation

Chromosomal aberrations

Chinese hamster ovary cells

Alpha particles

[N3]

2002

Low-fluence alpha particle
irradiation

HPRT mutations; sister chromatid
exchanges

Chinese hamster ovary cells

Alpha particles

[N21]

2002

Medium transfer

Clonogenic survival; AP-endonuclease;
TP53; ROS

Human lung fibroblasts (HFL-l)

Alpha particles[I1, I5]

2002

Medium transfer

Clonogenic survival; intracellular calcium levels; mitochondrial membranepotential; ROS levels

Human keratinocytes

Gamma rays

[L26, L27]

2002

Medium transfer

Clonogenic survival

Thirteen cell lines: human epithelial
carcinoma cells, SW48 humancolon carcinoma cells

Gamma rays

[M21,
M38]

2002

Microbeam irradiation

Micronucleus frequency; apoptosis

Sections of human and porcine
ureterAlpha particles[B7]

ANNEX C: NON-TARGETED AND DELAYED EFFECTS OF EXPOSURE TO IONIZING RADIATION

Year

Origin of bystander effectEnd point

Cell typeRadiation type25

Reference

2002

Microbeam irradiation

Clonogenic survival

V79 Chinese hamster cells

Alpha particles

[S27]

2003

Co-culture

Micronucleus frequency

Human fibroblasts

Carbon-ion beam:
beta particles

[S28]

2003

Co-culture

Enhanced plating efficiency andproliferation

Human salivary gland cells

Carbon beams

[S29]

2003

Low-fluence alpha particle
irradiation

HPRT mutations

Chinese hamster ovary cells

Alpha particles

[N20]

2003

Medium transfer

Clonogenic survival

Human keratinocytes

Gamma rays

[M27]

2003

Microbeam irradiation

Micronucleus frequency; apoptosis

Sections of human and porcine
ureterAlpha particles

[B8]

2003

Medium transfer

Apoptosis; micronuclei

Human–hamster hybrid cells(GM10115; Fe10-3; LS12)

X-rays[N1, N2]

2003

Carbon ion beam

Micronuclei; gap junctions; ROS

Human fibroblasts

Carbon ion beam

[S28]

2003

Helium ion microbeam

Micronuclei; nitric oxide

T98G cell nuclei from humanglioblastoma

Helium ion beam

[S16]

2003

Microbeam irradiation

AL cell mutagenic assay

AL cells

Charged particle
microbeam; alphaparticles; X-rays

[Z6]

2004

Medium transfer

RPA expression

Primary human fibroblasts

Gamma irradiation

[B42]

2004

Medium transfer

Micronucleus frequency

Chinese hamster ovary cells

Ultrasoft X-ray
microprobe

[K31]

2004

Medium transfer

Transformation frequency

HeLa × skin fibroblast hybrid cells

Low-dose X-rays

[K32]

2004

Co-culture

Cell proliferation using 3H-TdR
incorporation

Antigen presenting cells (J774A.1)
and T-lymphocytes(EL-4)

Low-dose irradiation

[L44]

2004

Chronic and acute irradiation

iNOS accumulation

WTP53 cells

Chronic gamma
rays; acute X-rays

[M59]

2004

Co-culture and medium transfer

Oncogenic transformation frequency

C3H 10T½ cells

High- and low-dose
X-rays

[M51]

2004

Cell–cell contact during irradiation

Oncogenic transformation frequency

C3H 10T½ cells

X-rays

[M43]

2004

Co-culture and medium transfer

Cloning efficiency; cell numbers

Repair-deficient human cell lines

Gamma rays

[M44]

2004

Co-culture

Chromosomal instability

Human fibroblast BJ1-htert

Alpha irradiation

[P22]

2004

Co-culture

Apoptosis; necrosis

LY (L5178Y) suspension cells;
human salivary gland cells

Carbon ions; X-rays

[S31]

2004

Co-culture

Micronuclei; nitric oxide

Glioma cells; primary humanfibroblasts

He ion particles

[S32]

2004

Co-culture

AL cell mutagenic assay

AL cells

Low-dose X-rays

[Z10]

2004

Microbeam irradiation

Micronuclei and cell cycle delays

Human fibroblasts

90 keV/µm alphaparticles

[P6]

2005

Co-culture

Transposition of chromosomal loci

Human lymphocytes

X-rays (10 cGy)

[T4]

2005

Medium transfer

Micronucleus frequency; apoptosis

X-rays

[K33]

2005

Medium transfer

Mutation and deletion in mitochondrialDNA

Human keratinocytes (HPV-G)

Gamma irradiation

[M60]

2005

Medium transfer

Micronucleus frequency; HPRT
mutation frequency

GM10115 human–hamster hybrid
cells

X-rays

[N30]

2005

Co-culture

Sister chromatid exchanges andchromosomal aberrations

Hamster cell lines (V3 and irs3)

Low-fluence alphaparticles

[N25]

26

UNSCEAR 2006 REPORT: VOLUME II

Year

Origin of bystander effectEnd point

Cell typeRadiation type

Reference

2005

Co-culture

Micronuclei; nitric oxide; ROS

Glioma cells (T98G) and fibroblasts(AG01522)

Helium particles

[S23]

2005

Medium transfer

Micronuclei; induction of p21waf1
protein; gamma H2AX foci; ROS;
clonogenic survival

Human fibroblasts

X-rays[Y5]

2005

Bystander assay

COX-2 signalling involving mitogenactivated protein kinases

Charged particle
beam

[Z5]

2005

Co-culture

Micronuclei; apoptosis

Mouse embryonic stem cells

Alpha particles

[Z7]

2006

Apoptosis from bystander effectIodine incorporation for apoptosis; gap
junctions; connexin-43 expression

Non-small-cell lung cancer

Ionizing radiation[Z9]

1. Bystander effects after cytoplasmic irradiation
51. The most convincing demonstration of the bystander
effect has come from studies using charged-particle micro­
beams [F3, F4, F5, R1]. The microbeam is capable of putting
an exact number of particles through a specific subcellularcompartment of a defined number of cells in a particularradiation environment.
52. Using the microbeam at the Radiological Research
Accelerator Facility of Columbia University in the UnitedStates [R1], Wu et al. [W13] targeted and irradiated thecytoplasm of human–hamster (AL) cells. They observed asignificant increase in mutations at the CD59 (S1) nuclear
gene locus while causing minimal cytotoxicity. Cytoplasmic
irradiation with a single alpha particle doubled the spontaneous mutation frequency, while a two- to threefold increase
was observed after four cytoplasmic traversals. The mutation spectrum was similar to the spontaneous, non-irradiatedmutation spectrum, but different from that observed aftertargeted nuclear irradiation. The addition of the free radical scavenger dimethyl sulphoxide or the intracellular glutathione inhibitor buthionine-S-R-sulfoximine indicated thatthe mutagenicity of cytoplasmic irradiation depended on thegeneration of ROS (figure IV). Shao et al. [S32] also useda charged-particle microbeam, at the Gray Cancer Institutein the United Kingdom, to target individual glioma cells
cultured alone or in co-culture with primary human fibro­
blasts. They found that even when only a single cell within
the glioma cell population was precisely traversed throughits cytoplasm with one helium ion, bystander responses were
induced in the neighbouring non-irradiated glioma cells or
fibroblasts. Significantly, the yield of bystander-induced
micronuclei was similar when the cytoplasm or nucleus ofa cell was targeted, indicating that direct DNA damage is
not required for switching on cell-signalling mechanisms
after low-dose irradiation. Two important conclusions canbe reached from these experiments. First, because bystander
effects are observed after cytoplasmic irradiation, the target
for genetic effects of radiation is larger than the nucleus.
Secondly, cytoplasmic traversal by alpha particles may be
more deleterious in the long term than nuclear traversal. Thisis because, as the number of nuclear traversals increases, theprobability of cell killing increases, whereas after cytoplasmic irradiation the increased mutagenicity occurs where
there is negligible killing of the irradiated cells [W13].
2.

Bystander effects after low fluencesof alpha particle irradiation

53. It is implicit in the evaluation of bystander effects that
bystander cells were not hit by the radiation but received
signals from an irradiated cell that generated a response in
the bystander cell. Broad-beam irradiation with low fluencesof alpha particles does not traverse every cell in the radiation environment, and data suggest that a sizeable portion of
the damage observed after exposure to low fluences of alphaparticles results from responses occurring in cells that were
not actually traversed by an alpha particle.
54. Nagasawa and Little [N4] observed small increases
in sister chromatid exchange frequency in ~30% of cells
analysed, even though <1% of the cell nuclei were actually traversed by an alpha particle. This observation waslater confirmed and extended by Lehnert and co-workers[D2, L2]. The induced sister chromatid exchanges could
be inhibited by superoxide dismutase, once again indicating a role for ROS [L2, N8, N9]. The alpha-particleinduced increase in ROS appears to be temporally linked
to enhanced production of tumour necrosis factor alphaand interleukin 1, which in turn operate in an autocrine
manner to up-regulate interleukin 8 [N9]. Low fluencesof alpha particles can also increase mutation yield [N5]and cause accumulation of the tumour suppressor protein
TP53 in a higher percentage of the exposed population thancalculated to receive a nuclear traversal by one or morealpha particles [H5]. Whether or not mutation induction or
induced gene expression is mediated by ROS is uncertain,
but whatever the mechanism, it involves gap-junctionmediated inter­cellular communication in the transmissionof damage signals from irradiated to non-irradiated cells(figure V[A]). By examining changes in gene expression
after low-fluence alpha particle irradiation, Azzam et al.
[A3, A4] demonstrated the involvement of connexin-43mediated intercellular communication in the transmission

ANNEX C: NON-TARGETED AND DELAYED EFFECTS OF EXPOSURE TO IONIZING RADIATION

27

correlated with the induction of micronuclei as a measureof DNA damage as well as with increased Ser-15 phosphorylation of TP53 (reviewed in reference [A5]).

of damage signals to non-irradiated cells. In gap-junctioncompetent cells, induction of p21Waf1 protein far exceeded
the fraction of cells whose nuclei had been traversed, andFigure IV. Induced S1 mutations in human–hamster AL cells per 105 survivors.
AL cells were irradiated with four alpha particles through the nucleus (1) or the cytoplasm (2). Dimethyl sulphoxide (8%) from 10 minutes
before until 10 minutes after cytoplasmic irradiation with four alpha particles significantly suppressed mutation yield (3). Treatment withd-dimethyl sulphoxide alone did not increase mutation frequency (4). In contrast, pretreatment of AL cells with a 10 μM dose of buthionineS-R-sulfoximine for 18 hours, which reduced the intracellular glutathione content to <5% of control levels, increased the mutagenicity ofcytoplasmic irradiation with four alpha particles by four- to fivefold (5). Treatment with buthionine-S-R-sulfoximine alone had no significant
effect on mutation frequency (6). These data strongly implicate ROS as being the mediator of the mutagenic response of cytoplasmic irradiation (adapted from reference [W13]). The insert represents a schematic of the irradiation protocol.
700

600

S1 MUTATIONS PER 105 SURVIVORS

ROS

500

∝-particle irradiation

400

300

200

100

0

1

2

3

4

5

6

TREATMENT

Figure V. Bystander effects are those effects occurring in cells that were not directly subjected to the deposition of energy
by radiation but were in contact with irradiated cells or received a signal from an irradiated cell.
A cell is irradiated through the nucleus with an alpha particle (vertical arrow). This irradiated cell then communicates a signal to a non­irradiated bystander cell by intercellular cell-to-cell gap junction communication (A) or the transmission of soluble factors from the irradiatedcell to the non-irradiated cell via the cell medium (B).

A.

B.
Gap junction
communication

Soluble factors

Alpha particle

UNSCEAR 2006 REPORT: VOLUME II

55. A bystander effect for chromosomal aberrations
induced by low fluences of alpha particles in wild-type
and repair-deficient (xrs-5) Chinese hamster ovary cells
has been described [N3]. Gross chromosomal aberrations
are generally associated with DNA double-strand breaks[M15], suggesting that the factors responsible for the
bystander effect are also ultimately capable of cleaving the
double helix. As an explanation of their data, Nagasawa
and Little [N3] proposed that the relatively small bystander
effect in wild-type cells was due to proficient double-strand
break repair as compared with the significantly enhanced
bystander effect, manifesting as dramatic increases in aberration yields in repair-deficient xrs-5 cells. This study was
expanded to examine potential bystander effects for sister
chromatid exchanges and chromosomal aberrations in hamster cell lines deficient in either DNA-PKcs (V3 cells, deficient in non-homologous DNA end joining) or RAD51C(irs3 cells, deficient in homologous recombination). Cells
were irradiated with very low fluences of alpha particles
such that <1% of the nuclei were traversed by an alphaparticle. Wild-type cells showed a prominent bystander
response for sister chromatid exchange induction; an evengreater effect was observed in V3 cells. On the other hand,no significant induction of sister chromatid exchanges wasobserved in the irs3 RAD51C-deficient bystander cells. In
contrast, a marked bystander effect for chromosomal aberrations occurred in V3 cells, and the induction of chromosomal aberrations in irs3 bystander cells was minimaland similar to that of wild-type cells. On the basis of thesefindings, Nagasawa et al. [N25] hypothesized that homologous recombination is essential for the induction of sister
chromatid exchange in bystander cells; but homologous
recombination is unable to repair the damage induced in
non-homologous end-joining-deficient bystander cells that
leads to either sister chromatid exchange or chromosomal
aberrations.

3. Bystander effects after irradiation witha charged-particle microbeam
56. Using human–hamster hybrid AL cells, Zhou et al.
[Z1] located all cells in the radiation environment andexposed 20% of these to 20 alpha particles using the Columbia University microbeam. This dose of radiation allowed
less than 1% of the irradiated cells to survive. They thenassayed surviving cells for mutations in the target humanchromosome and found a mutation frequency four times thatof background (figure VI). Since the irradiated cells were
exposed to lethal doses of radiation, these mutations musthave arisen in non-exposed bystander cells. Furthermore,
the mutation spectrum observed in bystander cells was significantly different from the spontaneous spectrum and fromthat observed after cytoplasmic irradiation, suggesting thatdifferent mutagenic mechanisms were involved in the twoprocesses [Z1].

Figure VI. CD59 mutants per 105 surviving AL cells.
(1) Non-irradiated cells. (2) Observed mutation yield when 20% of
AL cells were exposed to a lethal dose of 20 alpha particles per cell.
(All cells irradiated with 20 alpha particles should have been killed.)(3) Expected mutation yield when 20% of AL cells were exposed toa lethal dose of 20 alpha particles per cell. (4) Effect of two hours of
pretreatment and 72 hours of post-treatment with 40 μM Lindane
when 20% of AL cells were exposed to a lethal dose of 20 alphaparticles per cell. (5) Effect of 40 μM Lindane alone on AL cells(adapted from reference [Z1]).
250

200
MUTANTS/105 SURVIVING CELLS

28

150

100

50

0

1

2

3

4

5

TREATMENT

57. Unlike cytoplasmic irradiation [W13] or the bystander
effect reported by Lehnert et al. after low fluences of alphaparticle irradiation [L2, N8, N9], the bystander effectdescribed by Zhou et al. [Z1] was not modulated by additionof the free radical scavenger dimethyl sulphoxide. Instead,when AL cells were treated with the cell-to-cell gap junction
communication inhibitor Lindane, the bystander effect wassignificantly reduced but not eliminated (figure VI).
58. In a subsequent study, Zhou et al. [Z3] used the Columbia University microbeam to deliver exactly one alpha­particle through the nuclei of 5%, 10%, 20% or 100% of
the AL cells. Their results indicated that the frequencies of
induced mutations and chromosomal changes in populations
where known fractions of nuclei were hit were consistent
with non-hit cells contributing significantly to the response.
When 10% of a confluent mammalian cell population wasirradiated with a single alpha particle, the mutation yield
was similar to that observed when 100% of the cells were
irradiated. This effect was significantly reduced in cells pretreated with octanol, which inhibits gap-junction-mediatedinter­cellular communication, or in cells carrying a dominant negative connexin-43 vector. These results indicatethat a single alpha particle can induce genomic damage incells that were not irradiated. Since a cell cannot receive aANNEX C: NON-TARGETED AND DELAYED EFFECTS OF EXPOSURE TO IONIZING RADIATION

lower dose of radiation after exposure to alpha particles thana single traversal, these data suggest that at the very lowest radiation dose of alpha particles, i.e. a single particle,
the genotoxic risk for high-LET radiation may be underestimated. It should be noted that traversal of a single alphaparticle results in a dose of 0.074–0.17 Gy, depending upon
the nuclear cross-sections of a given cell type and assuming
an average LET of 90 keV/µm [R1].
59. The precision of intracellular irradiation and the highthroughput available at the microbeam facility at Columbia University have also enabled studies of oncogenictransformation to be carried out. Sawant et al. [S6] utilized
sparsely populated monolayers of mouse C3H 10T½ cells
and irradiated the nucleus of every cell, or every tenth cellat random, with either two or four alpha particles. The
yield of transformed foci was determined morphologically,
and the frequency of transformation was similar whether
100% or only 10% of the cells were irradiated (figure VII).

20

15transformation that cell-to-cell gap junction communication is the most important mediator of the bystander effectin C3H 10T½ cells after microbeam irradiation [M43].
60. Related studies using microbeam-generated alphaparticles have important implications for evaluating potential hazards associated with radiation exposure [M6]. One
alpha particle per nucleus, the lowest possible dose of highLET radiation, effectively elicits a bystander response indifferent cell types [B9, Z3]. By comparing the biological effectiveness of exposure to exactly one alpha particle
per cell with that expected from normal broad-beam irradiation similar to that used in the experiments describedabove (using a mean of one particle per cell), it is clearthat exactly one particle per cell is less biologically effective than a mean of one particle per cell. Miller et al. [M6]found that the transformation frequency after irradiation
with exactly one alpha particle per nucleus did not differ significantly from that observed in non-irradiated cells(figure VIII). This suggested that the increased transformation frequency observed after broad-beam alpha irradiation, where a mean of one alpha particle traversal per cell
results in a Poisson distribution of particles per cell, is aconsequence of the minority of cells subjected to multiple(≥2) alpha particle traversals.

Figure VIII. Transformation frequency in C3H 10T½ cells
in vitro.
(1) Non-irradiated controls for the microbeam experiment. (2) Cells
receiving exactly one alpha particle per nucleus from the Columbia
University microbeam. (3) Non-irradiated controls for the broadbeam experiment. (4) Cells receiving a mean of one alpha particle
per nucleus after broad-beam irradiation. Note that the transformation frequency in cells receiving exactly one alpha particle per
nucleus is only slightly different from that in the non-irradiatedcontrols (adapted from reference [M6]).

10

3

5

0

1

2

3

4

5

6

7

TREATMENT

Furthermore, Lindane (the cell-to-cell communication
gap junction inhibitor) reduced but did not eliminate thisbystander effect. In subsequent studies from the Columbia University group, a role for secreted factors or factors released from irradiated cells into the culture medium
has also been identified. However, it is clear from studies of bystander-induced cell killing and oncogenic

TRANSFORMATION FREQUENCY/104 SURVIVING CELLSTRANSFORMATION FREQUENCY/104 SURVIVING CELLS

Figure VII. Transformation frequency in C3H 10T½ cells
in vitro.
(1) Non-irradiated controls. (2) 10% of the cells received two alphaparticles. (3) 100% of the cells received two alpha particles. (4)10% of the cells received four alpha particles. (5) 100% of the cells
received four alpha particles. (6) 10% of the cells received eightalpha particles. (7) 100% of the cells received eight alpha particles(adapted from reference [S6]).

29

2.5

2

1.5

1

0.5

0

1

2

3

TREATMENT

430

UNSCEAR 2006 REPORT: VOLUME II

61. An important advantage of using low fluences of alphaparticles or a charged-particle irradiator is that it represents
environmentally relevant exposure conditions, where mostcells in a tissue would not be traversed by an alpha particle.
It should be stressed, however, that to irradiate cells in vitro
with a high-energy alpha particle, target cells must be grownas monolayers on a Mylar substrate. Under these culture
conditions, cells by necessity are more flattened and moreelongated that would reasonably be expected in a threedimensional in vivo situation. Consequently, the energy
deposited per traversal is much less biologically effective.
To address this issue, three-dimensional model tissue culture
systems are being developed. Any evaluation of potential
hazards associated with these types of high-LET radiationmust consider these unique cell culture conditions.

thus eliminating potential cell-to-cell gap junction communication [B9, P10]. This indicates that radiation-induced
soluble factors may be released into the culture medium andaffect non-irradiated cells (figure V[B]), and suggests that
there are at least two different types of bystander effect, thoseeffects mediated by cell-to-cell gap junction communication and those induced by secreted soluble factors (figure V).
Alternatively, there are at least two mechanisms for the same
biological effect. Intriguing data from Sawant et al. [S7] indicated that, as the number of alpha particles through a fixed(10%) number of cells increases, more bystander-induced
cell killing occurs (figure IX). These observations suggested
that increasing induced damage in 10% of the cells in the culture produced more cell-to-cell gap junction communication
and/or increased secretion of cytotoxic substances into theculture environment. A strategy combining medium transfer
experiments and gap junction inhibitor studies would enable
investigators to determine the relative contributions of these
bystander-mediated cytotoxic effects and ultimately thiswould aid in identification of the secreted factors.

62. There is also a microbeam at the Gray Cancer Institute,
where 3He ions with an LET of 100 keV/µm have been used
for targeted cellular irradiation [F5]. In addition, the Gray
Cancer Institute microbeam has recently been adapted so thatthe effects of ultrasoft X-rays can also be investigated [F3].
Using this microbeam, evidence for a bystander effect in primary human fibroblasts, as measured by micronucleus formation and cell killing, revealed significantly more damage than
expected on the basis of direct effects of radiation [B7, B8,
B9, P11, P12]. The bystander effects observed using the Gray
Cancer Institute microbeam can occur when cells are considerable distances apart from one another and not in contact,

63. Interestingly, some novel insights into the mechanismsof radiation-induced bystander effects in vitro have been
revealed using the single-cell microbeam at the Gray Cancer Institute. When human glioblastoma T98G cell nuclei
were individually irradiated with an exact number of helium
ions, it was found that, when only one cell in a populationof approximately 1,200 cells was targeted, cellular damage

Figure IX. Bystander effect for cell survival.
Ten per cent of the cells, selected randomly, were exposed to increasing numbers of alpha particles from 2 to 12, and the percentage of cell
survival was determined by reduction in plating efficiency. Open bars: experimentally determined survival. Filled bars: expected survival if only
irradiated cells were killed (adapted from reference [S7]).
120

100

% CELL SURVIVAL

80

60

40

20

0

0

1

2

4

6

8

NUMBER OF ALPHA PARTICLES IN 10% OF CELLS

1012ANNEX C: NON-TARGETED AND DELAYED EFFECTS OF EXPOSURE TO IONIZING RADIATION

measured as induced micronuclei was increased by 20%[S16]. When the percentage of cells individually targeted
was increased from 1% to 20%, the yield of micronuclei inthe population greatly exceeded that predicted on the basisof the yield when all of the cells were targeted assuming no
bystander effect. However, when 2-(4-carboxyphenyl)-4, 4, 5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO), a nitricoxide-specific scavenger, was present in the culture medium,the micronucleus yields decreased to the predicted values.
This indicates that nitric oxide contributed to the bystander
effect. Moreover, the medium harvested from cells targetedwith helium ions showed a cytotoxic effect by inducing
micronuclei in non-irradiated T98G cells, and this bystander
response was also inhibited by c-PTIO treatment. The induction of micronuclei in the population could also be decreasedby c-PTIO treatment when 100% of cells were individuallytargeted by one or two helium ions, indicating a complex
interaction of direct irradiation and bystander signals. A rolefor nitric oxide in the medium-mediated bystander effect has
now been described in a number of ­studies [S15, S16, S31,
S32]. Furthermore, the secretion of nitric oxide as a bystander
effect has been linked to the induction of radioresistance inrecipient cells [M3, M4]. However, it is not clear whether
nitric-oxide-mediated bystander effects occur in conjunction
with, or independent of, increased ­levels of ROS, and how
this might affect the characterization of subsequent radiation
sensitivity.
64. Microbeam experiments using high-Z elements(460 MeV 40Ar, 1,260 keV/µm and 260 MeV 20Ne, 380 keV/µm) have also been performed at the Japan Atomic Energy
Research Institute [S33]. Confluent normal human fibroblasts were targeted and the induced micronuclei evaluatedafter replating the cells. Even when only a single cell was
hit, a 1.4-fold increase in the frequency of micronuclei wasobserved, indicative of a bystander effect. The observed
increase in micronucleus frequency saturated when fourcells were targeted and could be suppressed when dimethyl
sulphoxide (a scavenger of ROS) or PMA (an inhibitor of
gap junction communication) was present at the time of irradiation. Thus a role for nitric oxide, ROS and cell-to-cell gap
junctions has been invoked in bystander responses.
65. It is likely, however, that there are multiple bystander
pathways. By using the Columbia University charged particle beam in conjunction with a strip dish design, Zhou et al.
[Z5] show that the cyclooxygenase-2 (COX-2, also known asprostaglandin endoperoxide synthase-2) signalling cascade
plays an essential role in the bystander process. Treatmentof bystander cells with NS-398, which suppresses COX-2
activity, significantly reduced the bystander effect. This provided evidence that the COX-2-related pathway, which isessential in mediating cellular inflammatory response, is a
critical signalling link for the bystander phenomenon in thisassay system. Furthermore, any signalling pathway likely to
be involved is complicated by the genotype of the exposedorganism. For example, Zhu et al. [Z7] examined the abilityof mouse embryonic stem cells differing in the status of the
DNA repair gene, Rad9, to express a bystander effect after

31

exposure to alpha particles. All populations, when confluent,demonstrated a dose-independent bystander effect with respect
to cell killing and apoptosis. Minimal alpha particle inductionof micronuclei in bystander cells was observed, except for theRad9-/- mutant, where a significant increase above backgroundwas detected. Therefore the Rad9 null mutation selectively
sensitizes mouse embryonic stem cells to spontaneous andhigh-LET-radiation-induced bystander apoptosis and micronucleus formation, but it has much less impact on cell killingby direct or bystander alpha particle exposure.
66. At present there is no detailed information on potential
bystander effects occurring after cellular exposure to lowLET radiation in non-irradiated cells subsequently cultured
in the same physical proximity. This is because the development of focused low-LET radiation sources has lagged wellbehind that of high-LET charged particle microbeams, owing
to practical and technical complications. Gamma rays interact with matter in a number of ways, including photoelectric
absorption, Compton scattering and pair production. Since
the electrons will undergo scattering as they exit the vacuum
system, it is not practical at present to irradiate single cells orsubcellular structures, as is possible with the charged particle microbeams described above. However, by using pulsed
electron beams, a low-LET microbeam can be constructedthat will mimic many important aspects of the interactionof gamma rays with cells. Such devices are currently available at the University of Maryland [S37] and Texas A&M
University [W16] in the United States. Consequently, it isanticipated that in the foreseeable future many of the studies
that have been performed with high-LET alpha particles orintermediate-LET ultrasoft X-rays can be replicated using
these new low-LET microbeams. In addition, the ability toexperimentally vary the LET distribution by changing theincident beam energy will allow investigation of the relative biological importance of various parts of the energetic
electrons’ track and hence evaluate how this might modulate
bystander responses [S38].
4.

Bystander effects after transferof medium from irradiated cells

67. Mothersill and Seymour [M18] demonstrated a significant reduction in plating efficiency in non-irradiatedcultures of human epithelial cells (but not fibroblasts) thatreceived culture medium from irradiated cultures. Theseobservations indicated that irradiated cells secreted a toxic
substance, a “bystander factor”, into the culture mediumthat can kill non-irradiated cells (figure X). Medium irradiated in the absence of cells had no effect on survival
when transferred to ­non-irradiated cells. Not all cells arecapable of producing the toxic bystander factor, nor are allcells capable of receiving and responding to the secreted
signal [M17, M18, M22, M25]. The effect was dependent on the cell number at the time of irradiation, could beobserved as early as 30 minutes post-irradiation, and wasstill effective when medium transfer occurred 60 hours
after irradiation.

32

UNSCEAR 2006 REPORT: VOLUME II

Figure X. Bystander effects in an immortalized human keratinocyte cell line as demonstrated by medium transfer
experiments.
Upper portion: schematic of the experimental protocol. A flask of cells is irradiated, and as a function of time post-irradiation, medium
is removed from the irradiated cells, filtered and transferred to non-irradiated cells. Clonogenic survival is then investigated in these non­irradiated cells. Bystander effects are indicated by a reduction in plating efficiency in the non-irradiated cells that were cultured in medium
from the irradiated cells. Lower portion: plating efficiency expressed as percentage of survival after transfer of medium from cells exposed toincreasing doses of ionizing radiation (adapted from reference [S13]).
FilterTransfer
mediumDetermine plating efficiency in
non-irradiated cells

Irradiated flask of cells
10090% CELL SURVIVAL

80
70
60
50
40
30
20
10
0

0

0.01

0.03

0.05

0.1

0.3

0.5

2.5

5

RADIATION DOSE (Gy)

68. Whether this is the same bystander phenomenon as
described above for bystander effects after low fluencesof alpha particles or irradiation with a charged particle
microbeam has yet to be determined. If it is not an identicalphenomenon, it is likely to be similar to those bystander
effects defined above that involve the secretion of soluble
factors [M43, S64, Z2]. However, one caveat in these studies should be mentioned. When AL cells were plated oneither one or both sides of double Mylar dishes before irradiation, and one side (with or without cells) was irradiatedwith alpha particles, different effects on different cellular
end points were observed for both survival and mutation[Z2]. When the side with cells was irradiated, the surviving fraction among the non-irradiated cells was significantly
lower than that of the controls after 48 hours co-culture.
However, such a change was not detected after 1 hour coculture or when medium alone was irradiated. Furthermore,
co-cultivation with irradiated cells had no significant effecton the spontaneous mutagenic yield of non-irradiated cells
collected from the other half of the double Mylar dishes.
These results suggested that the irradiated cells released
certain cytotoxic factors into the culture medium that killed

the non-irradiated cells. Importantly, such factors had­little effect on mutation induction, indicating that different bystander end points may involve different mechanisms
with different cell types. This is supported by the study by
Wang and Coderre [W18], who used a co-culture system to
examine bystander effects transmitted through the medium
from the directly targeted cells to tumour cells growing
on an insert well beyond the range of the alpha particles.
Alpha particle doses of 0.1–6.0 Gy to the targeted cells on
the Mylar membrane, followed by a 2 hour co-incubationof the cells on the insert in the irradiated medium above theirradiated cells, all caused an approximately 50% increasein micronucleus formation in the non-targeted co-cultured
cells. Addition of the radical scavenger dimethyl sulph­oxide to the medium during the irradiation and the 2 hourpost-irradiation incubation period completely blocked the
bystander effect, whereas addition of a nitric oxide scavenger had no effect.
69. Medium transfer experiments demonstrated that irradiation can lead to secretion of a factor or factors by irradiated cells that can reduce cloning efficiency, predominantly

ANNEX C: NON-TARGETED AND DELAYED EFFECTS OF EXPOSURE TO IONIZING RADIATION

by stimulating apoptosis [L26, L27, L28, S12], increasing
neoplastic transformation [L5] or inducing genomic instability [S12] in non-irradiated cells. The first detectable effecton recipient cells after transfer of medium containing the
bystander factor from irradiated cells was a rapid calcium
pulse (1–2 minutes) followed 30–120 minutes later by changes
in mitochondrial membrane permeability and the inductionof ROS [L28, L38, M24]. Cell-to-cell contact during irradiation was not required to induce killing of bystander cells, but
medium from cell cultures irradiated at high densities resulted
in the greatest amount of bystander-induced cell death [M22].
Furthermore, the use of apoptosis inhibitors or medium fromlactate dehydrogenase or glucose-6-phosphate dehydrogenasemutant cells reduced or prevented the bystander effect [M24].
Treatment with the antioxidants L-lactate and L-deprenyl­prevented bystander-factor-associated cell killing [M24], suggesting that energy/redox metabolism may be involved in themedium-mediated bystander response.
70. Mothersill and co-workers [M44] showed that repairdeficient human cell lines produced a moderate to severeamount of bystander-induced cell death after mediumtransfer to autologous cells or to a reporter cell line. Normal “repair-proficient” lines have much less severe, or no,
bystander-induced effects on cloning efficiency. These
results are in agreement with the observations of Little andcolleagues [L42, N3]. Mothersill et al. [M44] interpreted
these data as supporting the hypothesis that bystander effects
play a protective role in biological systems by terminating
division in cells containing DNA damage. Thus the repairdeficient cells, irrespective of the actual repair defect, may
respond to the occurrence of DNA damage in the population
by removing large numbers of cells from the proliferating
pool. It should be noted that repair-deficient cell lines tend
to show an increased frequency of induced genomic instability, again suggesting a commonality in the mechanism ofradiation-induced genomic instability and bystander effects.
71. The majority of medium transfer experiments reported
have utilized low-LET radiation to induce the bystander effectand have come primarily from a single laboratory (reviewed
in references [M19, M20]). A fascinating observation fromthese studies, but one that is difficult to reconcile with thoseof other studies, concerns the radiation doses required to elicit
a bystander response. In human keratino­cytes, low-LET 60Co
gamma ray doses of 0.01–0.5 Gy reduced clono­genic survival after transfer of irradiated medium. This was entirely
due to bystander effects. The magnitude of cell killing was
relatively constant and appeared to saturate at doses in therange 0.03–0.05 Gy [S13]. At doses of greater than 0.5 Gy,
cell killing was the result of the direct effects of radiation as
well as the dose-independent bystander effect [S13]. Theseobservations are difficult to explain in terms of the classicalcell survival curve, where there is very little if any cell killing observed at radiation doses of a few milligrays in directly
irradiated cells.
72. In contrast to the cell-killing effects reported by­Mothersill and Seymour after medium transfer from gamma-

33

irradiated cultures, Iyer and Lehnert [I4] have observed quite
different cellular responses in human fibroblast cells cultured in supernatants from alpha-irradiated fibroblasts. Theyobserved decreases in basal levels of TP53 and CDKN1AQ
in non-irradiated cells, rather than increases as described byothers (e.g. [A4, H5]). These decreases were accompanied
by increases in proliferating cell nuclear antigen and CDC2,
apparently mediated by TGF-beta 1 and the induction of
intracellular ROS [N8]. In contrast to the detrimental effects
on cell well-being characteristic of bystander effects so far
described, Iyer and Lehnert [I4] showed that their decreased
TP53/CDKN1A bystander effect correlated with enhanced
cell proliferation.
73. Attempting to reconcile these conflicting results raises
a number of questions. While the quality of radiation andthe cell types under investigation are different, these studies highlight the family of responses characterizing the
bystander effect. Mechanistically, factors transferred via
cell-to-cell gap junction communication or secreted into
the culture medium may interact with those non-irradiatedcells in an antagonistic manner, ultimately killing the nonirradiated cells (reviewed in reference [S39]), but the reasons
why cultured cells should secrete cytotoxic factors and why
no such dramatic reduction in plating efficiency is observed
when populations of cells are irradiated with low doses of
low-LET radiation are not immediately obvious. Likewise,
the pro-mitogenic response reported by Lehnert et al. [L2,
L48] after medium transfer is contradictory to those direct
effects observed in irradiated cells where irradiation caninhibit cell growth. Cell proliferation following irradiation
appears contrary to the long-term well-being of the cell,­tissue, organ or organism, whereas cell death would be likely
to protect against the possibility of detrimental mutations,chromosomal rearrangements, and so on. Clearly bystander
effects can modify cellular responses to radiation, and it
remains to be determined whether these effects characterized in non-irradiated cells in vitro have a major role in the
response of irradiated cells in vitro or in irradiated and nonirradiated cells in vivo.
B.

Bystander effects in vivo

74. Bystander effects have been observed predominantly
by using single-cell in vitro systems that do not have realistic multicellular morphology. Given that the bystander
pheno­menon must involve cell-to-cell interactions, the rele­vance of such single-cell in vitro studies is questionable.
However, Belyakov et al. [B36] have described bystander
responses in a three-dimensional, normal human tissue system. While not a true in vivo assay, this model skin systemdoes provide some semblance of multicellular interactions.
End points were induction of micronucleated and apoptotic
cells. Non-­irradiated cells up to 1 mm distant from irradiated cells showed a significant enhancement in the effectover background levels, with an average increase in effectof 1.7-fold for micronuclei and 2.8-fold for apoptosis. The
surprisingly long range of bystander signals in a human

34

UNSCEAR 2006 REPORT: VOLUME II

tissue model system suggests that bystander responses may
be important in extrapolating potential radiation effects fromepidemiologically relevant doses down to very low doses(<200 mGy), where non-hit bystander cells would likely
predominate [M36].
75. Compared with the number of in vitro studies on
bystander effects, there are relatively few studies on bystander
effects in vivo, and these are summarized in table 4. Manyof these studies appear to have been performed not to look

Table 4

specifically at non-targeted effects of radiation but for otherpurposes, so that critically evaluating bystander effects invivo is premature at present. It should be mentioned at thisstage that investigators are beginning to move from singlecell systems to multicellular systems using primary explanttechniques [B8, B36] as well as three-dimensional model
systems [P11]. Consequently, it is anticipated that as more
information from these model systems becomes available
and more focused studies in vivo are undertaken, it will bepossible to critically re-evaluate bystander effects in vivo.

In vivo studies of the bystander effectEnd point

Cellular system

Radiation type

Reference

Chinese hamster ovary and liver

239
PuO2 particles;
alpha particles

Mixture of irradiated and non-irradiated mice bonemarrow cells

Neutrons[W2]

Micronucleus frequency

Rat lung

Gamma rays

[K11, K22]

Calcium mobilization; alkaline phosphatase levels;
embryonic development

Rat incisor, thyroid and abdomen

X-rays[C8, H1]

Regenerative capacity

Earthworm

X-rays[M8]

Macrophage activation; respiratory burst;
NO activation; neutrophil infiltration

TP53-/- miceGamma rays

[L22]

Growth of tumour

Mixture of human colon LS174T adenocarcinoma cells
and 125I-labelled LS174T cells

125

[X2]

C57BL/6 miceGamma rays

[C1]

Chromosomal aberrations

76. Despite the caveats outlined above, there are studies indicating a bystander effect in vivo. Chinese hamsters
were injected with different sized particles of the internallydeposited alpha emitter plutonium. The radioactive particles
concentrate in the liver and produce chronic low-dose radiation exposure, with the dose and dose rate being highest to
cells located closest to the largest particles. However, analysis of induced chromosome damage in these livers revealed
increased cytogenetic damage that was not directly related
to the local dose distribution [B17]. These observations wereinterpreted as indicating that all the cells in the liver wereat the same risk of induced chromosome damage despite
only a small fraction of the total liver being exposed to theradiation. The cumulative incidence of liver cancer as afunction of time after plutonium injection and total dose wasalso determined. Neither the time of tumour onset nor thetumour incidence varied with particle size, indicating thatthe number of cells hit by alpha particles was not a factor intumour induction in irradiated livers [B16].
77. These two studies suggest that radiation-induced
genetic damage and ultimately tumour induction are related
to the total dose to the organ, i.e. the whole liver, rather thanthe dose to individual cells or the number of cells traversed

I beta particles[B16, B17, M40]

by an alpha particle [B16, B17]. Furthermore, these dataraise the intriguing possibility that the target for induced
bystander effects may actually be limited to the specific organ
irradiated and that adjacent non-irradiated organs are not targets for bystander effects. It is certainly not unexpected thatmulticellular organs function in response to genotoxic stressin a coordinated fashion [G24], and recently Barcellos-Hoffand Brooks have hypothesized that multicellular responses
through extracellular signalling are integral components
of predicting cancer risk after radiation exposure [B3]. To
this end, there is evidence from in vivo studies of radiationinduced genomic instability that delayed instability has asignificant bystander component [W2, X2]. Nevertheless, arecent report of the International Commission on Radiological Protection (ICRP) [I11] suggests that early initiating cellular and molecular events are the major determinants of risk
at low doses, rather than cell-, tissue- and host-modifying
factors.
78. The evidence for in vivo bystander effects has beenreviewed in detail [B43, K25] and shows that these effects
probably involve a genetic component [M61]. In addition
to damage directly induced by the deposition of energy
in the irradiated cell, consideration must now be given toANNEX C: NON-TARGETED AND DELAYED EFFECTS OF EXPOSURE TO IONIZING RADIATION

these indirect effects of radiation, and a model quantifying
these considerations has been proposed [B14]. An irradiated cell can send out a signal and induce a response in acell whose nucleus was not hit by radiation. Thus a detrimental bystander effect, e.g. chromosomal aberrations, inessence “modifies” the biological effectiveness of a given
radiation dose by increasing the number of cells that experience adverse effects over that directly exposed to theradiation. Significantly, these bystander effects appear to
be limited to the organ irradiated, i.e. are organ-specific35[B43]. Thus, at the present state of our knowledge, it isreasonable to assume that any bystander effect induced invivo is accounted for in models of organ risk evaluation.
As a result, it is unlikely that the resurgence of interest inthese non-targeted radiation effects will substantially alterrisk estimates as discussed in detail in the BEIR VII report[C23]. Nevertheless, it cannot be excluded that increasing
the knowledge basis for in vivo bystander effects at low
doses and low dose rates in specific organs may affect­current organ risk estimates.

III. Relationship between Radiation-Induced
Genomic Instability and Bystander Effects
79. The evidence for effects occurring in cells that themselves were not irradiated but are the progeny of irradiatedcells (radiation-induced genomic instability), and non­targeted cellular effects usually associated with direct exposure to ionizing radiation occurring in non-irradiated cells(bystander effects), has been reviewed in the previous paragraphs. Many of the end points associated with these two
phenomena are the same: induced chromosomal rearrangements, micronuclei, increased mutation, increased transformation and cell killing. So what is the relationship, if any,
between induced instability and bystander effects? Chromosomal instability in haemopoietic cells can be induced byan indirect, non-targeted bystander type of mechanism [K3,
L24]. Persistently increased levels of intracellular ROS havebeen reported in chromosomally unstable cells [C5, L7, L8,
L9, L10, R3, R6], and provide a plausible mechanism for
perpetuating instability over time (reviewed in references[M10, M16]). Considerable experimental support for thishypothesis comes from Wright and co-workers [L22, W2,
W4], and this has recently been reviewed in detail [L23]. The
resultant intercellular signalling cascades, cytokine production, nitric oxide production and persistent free radicals all
have the potential to mediate both instability and bystander
effects (reviewed in references [L23, M10]).

GM10115 cells. This indicates that GM10115 cells either donot secrete a cytotoxic bystander factor or are not susceptible
to a bystander factor, and that the death-inducing effect is
not the same as the bystander effect described by Mothersill
and co-workers. Likewise, chromosomal instability was notdetected in GM10115 cells after transferring medium from
irradiated cells as described by Mothersill and Seymour[M18, M21] for induced bystander effects.
81. It should be noted that Mothersill et al. [M44] havealso reported that they do not find a bystander response
in some Chinese hamster cell lines after medium transfer.
This implies that, like the human–hamster hybrid GM10115
cell line, CHOK1 hamster cells may be deficient in producing a bystander signal or in responding to that signal. It isinteresting that when medium from irradiated CHOK1 cells
was added to either non-irradiated CHOK1 cells or repairdeficient XR1 hamster cells, the plating efficiency actually
increased rather than decreased [M44]. However, significantly increased bystander effects after cellular exposure to
low fluences of alpha particles have been described in repairdeficient Chinese hamster cells compared with wild-type
hamster cells [N3, N20, N25].
82. Evidence increasingly suggests that induced instability and bystander effects are linked (reviewed in references[L23, M10, M16]), and it is likely, given the commonality ofthe end points observed, that both phenomena could be mani­
festations of the same non-targeted processes [M10, M16].
Furthermore, a significant contribution from bystander-likefactors could help explain the high frequency of radiationinduced instability reported, for example, in references [K3,L6, M2].

80. To critically evaluate the hypothesis that chromosomallyunstable GM10115 clones perpetuate instability by secretinga bystander-like factor into the culture medium, thus driving the delayed production of chromosomal rearrangements,
Nagar et al. took medium from a chromosomally unstableclone of GM10115 human–hamster hybrid cells, filtered it
and cultured non-irradiated GM10115 cells in this medium.
None of the non-irradiated GM10115 cells were able to survive and form colonies in medium from the unstable clone.
Nagar et al. called this novel effect, by which cells cultured in
medium from chromosomally unstable GM10115 cells die,the death-inducing effect [N1]. The unstable clones showing
the death-inducing effect also showed increased numbers of
apoptotic cells and elevated levels of intracellular ROS [N2],either or both of which might contribute factors to the culture medium responsible for the death-inducing effect. Furthermore, Nagar et al. [N1] have interpreted this observation
as indicating that unstable clones of cells do secrete factors
that, while generally not toxic to the unstable clone, most
likely contribute to the perpetuation of the unstable pheno­
type. It should be stressed that the death-inducing effectis separate from the bystander effect observed after transferring medium from irradiated cells. Nagar et al. did notobserve a reduction in plating efficiency when medium from
irradiated GM10115 cells was transferred to non-irradiated83. The Committee continues to hold the view that mechanistic information is important for its recommendations onradiation-induced health effects at doses of below ~200 mSv
and for risk assessment for individuals. The latter aspect isimportant because epidemiology always refers to populations, and genomic instability, for example, varies amongindividuals.
A. Relationship between radiation hypersensitivity
at low doses and bystander effects
84. To date, investigation of the relationship between
radiation hypersensitivity at low doses and bystander
effects has been limited to a single study. Joiner et al. [J4]described radiation hypersensitivity at radiation doses where
37

38

UNSCEAR 2006 REPORT: VOLUME II

the in vitro bystander effect would be expected to predominate. Interestingly, an investigation into the relationship between ­radiation-induced low-dose hypersensitivity
and the bystander effect indicated a weak inverse correlation between these two low-dose phenomena. Specifically,
those cells exhibiting a large bystander effect did not show
radiation hypersensitivity [M23]. Should these results beconfirmed, they would suggest that, at very low radiation
doses, bystander effects might dominate the overall cellular response. Furthermore, such bystander effects at dosesof a few milligrays could have some bearing on the apparent
elimination of damaged cells and the absence of repair ofradiation-induced DNA double-strand breaks in the very low
dose range (1.2 mGy) in X-irradiated normal human fibro­
blasts as reported by Rothkamm and Lobrich [R14].
B.

Relationship between radiation adaptive
response and bystander effects

85. The radiation adaptive response refers to the phenomenon by which cells irradiated with a sublethal dose ofionizing radiation (an “adaptive” dose of a few centigrays)
become less susceptible to subsequent exposure to high dosesof radiation (a “challenge” dose of several grays). There isa vast literature on “adaptive responses”, and this section is
not intended to be an exhaustive literature review. Instead,the goal is to provide examples of the main evidence for low
doses of radiation protecting against a subsequent high-doseradiation challenge. The adaptive response to radiation was
first described as a reduction in chromosomal aberration frequency in stimulated human lymphocytes [O7]. Subsequent
reported adaptive responses include reduction of cell killing[I6], micronucleus formation and sister chromatid exchange[I7, I8], mutation [K26, R11, U25] and transformation [A17,R8]. An adaptive response has also been described after
clinical [M53], environmental [G16] or occupational [B34]exposures to radiation. The mechanism for this radioadaptation is thought to be that low radiation doses enhance DNA
repair ability and antioxidant activity, resulting in more proficient cellular responses to the subsequent challenge [G21,I9, S48].
86. Reports of the adaptive response to radiation are conflicting, however, because radioadaptation is not consistently
a robust effect in all cell systems [A14, B35, B40, H16]. The
variation among different studies could be related to a numberof factors, including cell type [R7], cell culture conditions, cell

cycle effects, types of radiation used, doses and dose rates,
as well as time interval between irradiations [S54]. ­Current
uncertainties in interpreting experimental data from both
adaptive response and bystander investigations do not permit any firm conclusions regarding the relationships between
these two low-dose phenomena to be reached at present.
87. Nevertheless, radiation-induced bystander effects canbe considered a competing phenomenon with respect toan adaptive response [S7]. Using the Columbia University
charged particle microbeam and the AL cell mutagenic assay,
Zhou et al. [Z6] showed that pretreatment of cells with a low
dose of X-rays four hours before alpha particle irradiation
significantly decreased this bystander mutagenic response.
Furthermore, bystander cells showed an increase in sensitivity after a subsequent challenging dose of X-rays. Using thesame irradiation system, Mitchell et al. [M51] found that an
adaptive dose of 2 cGy of X-rays cancelled out the majority of the bystander effect produced by alpha particles. For
oncogenic transformation, but not cell survival, radioadaptation could occur in non-irradiated cells via a transmissible
signal.
C. Conclusions
88. Ionizing-radiation-induced bystander effects are thoseeffects occurring in cells that were not traversed by radiation
but were induced by signals from irradiated cells. Mechanistically, the signal is passed from cell to cell by gap junction communication or is secreted into the culture medium
where it can be transferred to non-irradiated cells. Bothpositive effects for the cell (e.g. increased cell proliferationor an induced radioprotective adaptive response) and negative effects (e.g. cytogenetic damage or cytotoxic bystander
responses) have been described. Bystander effects inducedby high-LET radiation have been described in a number ofdifferent cell types in studies using either low radiation fluences of alpha particles or charged particle microbeams. A
bystander effect induced by low-LET radiation is less wellestablished and to date has only been demonstrated after
medium transfer experiments. Experimental verification indifferent laboratories and the use of newly developed lowLET microbeams will extend these observations. To date,low-LET bystander effects appear to be a low-dose phenomenon, and reconciling low-dose bystander cytotoxicity withthe lack of directly induced cell killing at these same doses
is perplexing.

IV.
A.

ABSCOPAL EFFECTS OF RADIATION

Reviewand T241 tumour cells implanted into the midline dorsum
grew at a significantly slower rate when the leg of the animal
was exposed to five 10 Gy fractions of radiation comparedwith sham-irradiated animals. This suggests that the abscopal effect is not tumour-specific. When the radiation dose to
the leg was reduced (12 × 2 Gy), the inhibition of Lewis lungcarcinoma tumour growth was decreased, indicating a radiation dose dependency for the abscopal effect. In contrast,
when the legs of TP53 null animals or wild-type TP53 micetreated with pifithrin-alpha (a TP53 blocker) were irradiated(5 ×10 Gy), tumour growth was not delayed. These data
implicate TP53 as a key mediator of the radiation-induced
abscopal effect and suggest that pathways downstream of
TP53 are important in eliciting this response.

89. An abscopal effect may be defined as a significant­tissue response to irradiation in tissues definitively separate
from the region exposed to radiation. The response must
be measurable, and the distance separating the responding­tissues and the portal(s) of irradiation must be great enoughto rule out any possible effect of scattered radiation [N15].
Originally described by Mole [M52] in 1953, the word
abscopal comes from the Latin ab (position away from) andscopus (mark or target). The mechanism of the abscopal
effect is unknown, although a variety of underlying biological events can be hypothesized, including a possible role forthe immune system [M46, U24].
90. An example illustrating radiation-induced abscopal
effects is the study of early DNA damage induced in rat lungcells following single-dose, partial-volume (lung base andlung apex) irradiation [K11]. When the lungs were removed
at 16–18 hours after whole-lung irradiation, an average of~0.85 micronuclei per binucleate cell were observed in theirradiated animals, compared with 0.02 micronuclei per
binucleate cell in the lungs from control animals. When only
the lung base was irradiated, the frequency of micronuclei
in cells from the irradiated field was 0.85. However, nonirradiated cells from the out-of-field lung apex also showed asignificant increase in the frequency of micronuclei, 0.43 per
binucleate cell, significantly higher than the non-­irradiatedcontrol value. Cells from the lungs of rats injected withsuperoxide dismutase within one hour prior to irradiationof the lung base and processed 16–18 hours after irradiation showed a reduction in the number of micronuclei in theshielded lung apex, indicating the potential involvement ofoxygen radicals [K11].

92. Ionizing radiation can reduce tumour growth outside
the field of radiation [A15, E16, K27, R9, R10], but thisabscopal effect remains a rare and poorly understood event.
Ionizing radiation generates inflammatory signals and inprinciple could provide both tumour-specific antigens fromdying cells and maturation stimuli that are necessary for dendritic cells to activate tumour-specific T-cells. Demaria et al.
[D23] tested the hypothesis that the abscopal effect elicitedby radiation is immune-mediated. Mice bearing a syngeneic
mammary carcinoma, 67NR, in both flanks were treated withgrowth factor Flt3-Ligand daily for 10 days after local radiation therapy to only one of the two tumours at a single doseof 2 or 6 Gy. The second, non-irradiated tumour was used asindicator of the abscopal effect. Radiation alone led to growth
delay exclusively of the irradiated 67NR tumour. However,growth of the non-irradiated tumour was also impaired by
the combination of radiation and ligand. Importantly, theabscopal effect was shown to be tumour-specific, because
growth of a non-irradiated A20 lymphoma in the same micecontaining a treated 67NR tumour was not affected. More­over, no growth delay of non-irradiated 67NR tumours wasobserved when T-cell-deficient (nude) mice were treatedwith the combination of radiation and ligand. These results
demonstrate that in this cell system the abscopal effect isin part immune-mediated and that T-cells are required tomediate distant tumour inhibition induced by radiation.

91. Ohba et al. [O3] described the case of a 76-year-old
Japanese man with hepatocellular carcinoma that regressed
after radiotherapy for thoracic vertebral bone metastasis.
Serum levels of tumour necrosis factor alpha increased
after radiotherapy, and the investigators suggested thatsuch abscopal-related regression might be associated withhost immune response, involving cytokines such as tumour
necrosis factor alpha. To understand the potential mechanisms, Camphausen et al. [C1] examined whether the abscopal effect was mediated through TP53. Non-tumour-bearing
legs of C57BL/6 (wild-type TP53) and TP53 null B6.129S2Trp53(tm1Tyj) mice were irradiated to determine whether
an abscopal effect could be observed against Lewis lung carcinoma and T241 (fibrosarcoma) implanted at a distant site.
In the TP53 wild-type mice, both the Lewis lung carcinoma

93. Abscopal effects after partial-body irradiation have also
been described in earthworms [M8], White Leghorn cockerels [M9] and rats [C8, H1]. Abscopal reactions have also
been described in patients with chronic leukaemias, wherein
irradiation of an enlarged spleen or liver will induce a generalized remission with return of the bone marrow, whiteblood cell count and peripheral blood cells to normal ranges[N15]. In fact, there are a number of well-recognized effects
39

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UNSCEAR 2006 REPORT: VOLUME II

described by radiation therapists that occur beyond the radiation field. Goldberg and Lehnert [G7] have recently reviewed
these bystander-like effects but concluded that the clinicalliterature does not provide strong evidence for or against theexistence of radiation bystander effects, and by extension
abscopal effects, in vivo. They argue that many studies canbe interpreted as suggesting non-targeted effects in vivo, andrecommend that prospective clinical trials be carried out thatinvolve detailed field and dose information combined withwell-documented patient risk factors in order to investigate
potential bystander effects after radiation ­therapy [G7].

B.

Conclusions

94. The few studies discussed in this section that describe
the potential abscopal effects of ionizing radiation are generally descriptive in nature and provide little or no interpretation in terms of the mechanism underlying the response.
This together with the lack of confirmatory studies meansthat definitive conclusions on the impact of any potential abscopal effects are not possible. Additional focusedresearch involving well-designed prospective clinical trials
could clarify this issue.

V.

CLASTOGENIC FACTORS INDUCED BY IONIZING RADIATION
A.

Review

factors capable of inducing detrimental effects in unexposed cells. These “clastogenic factors” (or clastogenic
plasma factors) were first described by Parsons et al. [P5],
who observed bone marrow damage in the sternum of children with chronic granulocytic leukaemia whose spleens
had been irradiated. This report was corroborated by Souto[S19], who showed that rats injected with plasma from
irradiated animals developed mammary tumours at a significantly higher rate than rats exposed to plasma from nonirradiated animals. A number of reports eventually followedthat demonstrated that culturing normal human peripheral
blood lymphocytes in medium containing plasma obtained
from accidentally [G5] or therapeutically [H6, L15] irradiated individuals resulted in significantly more chromosomal aberrations than culturing lymphocytes in medium
with plasma from non-irradiated individuals. These observations led to the suggestion that, after in vivo irradiation,
exposed individuals can possess clastogenic factors in theirblood plasma that, when transferred to cell cultures from
unexposed individuals, can induce chromosome damage(figure XI).

95. Following whole-body irradiation, blood plasma fromsome animals and humans can contain factors that can cause
chromosome damage, hence the name clastogenic factors.
This indicates the persistence of soluble factors induced byradiation that are capable of causing effects in non-irradiatedcells. As such, clastogenic factors are not necessarily classical bystander effect factors but are included here to support
the concept of a role for secreted and released soluble factors in delayed effects associated with radiation exposure.
Strictly, under the definitions presented in this review, clastogenic factors, like abscopal effects, could well be considered bystander effects. However, for historical reasons, both
abscopal effects and clastogenic factors are considered separately. In reality, however, no mechanistic distinctions are
implied between these non-targeted effects of exposure toionizing radiation.
96. There is a large body of evidence demonstrating thatplasma from irradiated animals and humans can contain

Figure XI. Theoretical schematic for identifying clastogenic factors.
Plasma isolated from blood from an irradiated mouse is mixed with blood from a non-irradiated mouse and cultured for cytogenetic analysis.
Clastogenic factors in the plasma from the irradiated mouse can cause chromosomal aberrations in the peripheral blood lymphocytes of the
non-irradiated mouse. A: metaphase chromosomes from a peripheral blood sample from a non-irradiated mouse cultured in media containing
isolated plasma from a non-irradiated mouse. No chromosomal aberrations are observed. B and C: metaphase chromosomes from a peripheral
blood sample from a non-irradiated mouse cultured in isolated plasma from an irradiated mouse (B: chromatid deletions (arrows); C: chromatid
exchange and deletion (arrows)).
Ionizing radiation

Blood sample

Blood sample

Isolate plasma

Peripheral blood lymphocytes

Culture for cytogenetic analysis

A

B

C
41

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UNSCEAR 2006 REPORT: VOLUME II

97. Clastogenic factors have been described in plasma
from survivors of the atomic bombings in Japan [P2], personnel involved in salvage operations after the Chernobyl
accident [E8, E10] and children exposed as a consequence of
the Chernobyl accident [E11, G3]. In addition, clastogenic
factors have been reported in human [G1, L19, S10] and rat
blood plasma [F1] after in vitro irradiation. Clastogenic factors can be induced within 15 minutes of irradiation [F1]and appear to be very persistent or continuously regenerated: times of 10 weeks post-irradiation have been reported
for rats [F1], 7–10½ years for irradiated humans [E8, G5,
G6] and >30 years for the atomic bombing survivors [P2].
Clastogenic factors reflect neither radiation-induced depletion of protective factors nor radiation-induced changes innormal plasma components, but rather represent products
secreted or excreted by cellular elements as a result of irradiation [F1]. In vitro induction of clastogenic factors does notappear to be related to the dose [S10] or quality of radiation[G1]; however, this might not be the case in vivo [E8].
98. Emerit et al. [E8] investigated clastogenic factors in
the plasma of 32 civil workers from Armenia who had been
engaged as emergency workers around the Chernobyl atomic
power station in 1986. They also included 15 emergency
workers who had emigrated from the former Soviet Union
to Israel. Reference plasma samples were obtained from 41
blood donors from the Armenian Blood Center in Yerevan.
The samples were tested for their clastogenic activity inblood cultures from healthy donors. The samples from thefirst Armenian group, with the higher average radiation dose
(0.6 ± 0.6 Gy), were more clastogenic than those from thesecond group, which had been exposed to 0.2 ± 0.2 Gy. The
samples from the Israeli emergency workers also induced
significantly increased aberration rates (14.0 ± 3.9% aberrant cells). The clastogenic activity described above could be
inhibited by superoxide dismutase [E8], indicating that thechromosome-damaging effects of radiation-induced clasto­
genic factors are exerted via the intermediation of super­oxide radicals, as is known for clastogenic factors of ­different
origin [E18, F12].
99. It should be stressed that there is variability between
individuals in their ability to produce clastogenic factors[E11, G3], and not all irradiated individuals exhibit thiseffect [L4]. Indeed it is difficult to evaluate how common is
the induction of clastogenic factors in the human population.
On the one hand, blood samples from irradiated individuals
are a valuable commodity, usually studied for more conventional biomarkers of radiation exposure. On the other hand,
it is perhaps not surprising that there are very few reports
failing to detect clastogenic factors, as such negative results
are generally less likely to be published.
100. The precise nature of clastogenic factors is unknown,
but endogenous viruses and compounds that interfere with
DNA repair and/or increase the production of free radicals have all been implicated [E1, E2, E3]. On the basisof a number of inhibitor studies, the bulk of evidence suggests that the mechanism of action of clastogenic factors

is probably mediated by free radicals. Free radical scavengers such as superoxide dismutase, penicillamine, cysteineand various antioxidant plant extracts all reduce or eliminate clastogenic factor activity [E1, E2, E3]. The molecular mechanisms for this effect and the specific nature of thefactors capable of persisting, or of being regenerated overprotracted time intervals (>30 years in the case of someof the atomic bombing survivors [P2]), remain unknown.
Never­theless, it is tempting to speculate on the potential relationship between clasto­genic factors and ­factors
involved in the bystander effect. Both can be inducedby ionizing radiation and the factors produced can cause
genetic damage in non-irradiated cells. Given the current
interest in bystander effects resulting from secreted factors produced after cellular irradiation [S39], it may be an
appropriate time to revisit clastogenic factors and evaluate
the biological significance and nature of these radiationinduced secreted factors.
101. At present, the biological significance of clastogenic
factors remains unclear [H11]. Furthermore, it would be
misleading to imply that clastogenic factors are unique toradiation exposure. Transferable clastogenic effects havebeen described in blood plasma after whole-body stresses
as diverse as asbestos exposure [E7] and ischaemia reperfusion injury [E6], and occur spontaneously in patientswith HIV-1 [E1], hepatitis C [E12], Crohn’s disease [E5]and scleroderma [A2]. The reports of diffusible clastogenic
factors induced by irradiation also resemble the reports ofclastogenic activity in the plasma of patients with certain
inherited disorders, including Bloom’s syndrome [E4],ataxia-telangiectasia [S14] and Fanconi’s anaemia [E9].
Individuals with these chromosome breakage syndromes
show an increased incidence of cancer, which begs thequestion as to the role of clastogenic factors in creatinga cellular environment predisposed to increased genomic
instability and ultimately neoplastic transformation [H11,W11].
102. Emerit and colleagues have carried out many of thestudies investigating clastogenic factors. These investigators have described these factors in plasma from individuals exposed to different types of radiation under a variety
of exposure conditions. In addition, Emerit’s group has
described clastogenic factors after exposure to other DNAdamaging agents, as well as in individuals with a number of
medical conditions and various genetic diseases. It would be
misleading to imply that Emerit and colleagues are the only
ones to describe clastogenic factors in various disease states.
Reports from other laboratories lend support to these observations (e.g. [B10, G1, S14]), but the biological significanceand potential health hazards associated with clastogenicf­actors remain to be determined.
103. Nevertheless, the presence of clastogenic factors inperipheral blood samples from some irradiated individuals
raises intriguing questions concerning the role of chromosomal rearrangements as dosimeters of radiation exposure[S10]. Cytogenetic analysis of first division metaphase

ANNEX C: NON-TARGETED AND DELAYED EFFECTS OF EXPOSURE TO IONIZING RADIATION

cells from irradiated individuals reveals both asymmetrical (dicentric and polycentric chromosomes as well as ring
chromosomes) and symmetrical (reciprocal translocations)exchange-type aberrations, insertions, inversions and chromosomal breaks (reviewed in reference [C9]). The asymmetrical exchange-type aberrations generally lead to proliferative cell death in subsequent mitoses and decline over time.
Symmetrical translocations, on the other hand, are generally
stable over time and can persist, although they also appear to
decline, albeit at a much slower rate than asymmetrical aberrations [S20]. Since clastogenic factors from some irradiatedindividuals can induce chromosome damage, a role for thesefactors in the well-described persistence of chromosomal

43

rearrangements in blood samples from irradiated individuals
is possible but unlikely.
B.

Conclusions

104. It remains difficult to establish a clear descriptionof the relevance of clastogenic factors to overall cellularresponses to ionizing radiation, particularly at low doses.
In part, this is due to the paucity of data on the nature of thefactors and on their mechanism of action. Furthermore, howsuch clastogenic factors might influence the dose–response
curve at low doses is not possible to discern at this time.

VI.

IMPACT OF NON-TARGETED AND DELAYED EFFECTS
OF RADIATION ON FUTURE GENERATIONS

105. It is important to consider whether non-targeted effectsof radiation influence our consideration of the consequencesof irradiation of a parent on end points in offspring. The following sections summarize the data from non-human andhuman studies and consider them in the context of the Committee’s current position on heritable effects of irradiation.
Several diverse studies have provided a somewhat confusing
picture of the potential non-targeted or delayed effects of ionizing radiation in humans, mice and other organisms. Manyof the studies present technical difficulties and ambiguities in interpretation, for example, with respect to uncertain­radiation doses in human studies, potential strain dependencyof responses in mouse studies, and poorly defined criteria
used to define effects in some molecular studies. These complicating factors will be highlighted throughout this section.

109. There was also a significant dose-rate effect for thistype of mutation. Shimada et al. [S43] determined the frequency of “specific locus” mutations at five pigmentationloci in medaka spermatogonial stem cells after gamma
irradiation at 0.03 cGy/min and 95 cGy/min. At each totaldose, the mutation frequency was significantly lower in the
0.03 cGy/min group than in the 95 cGy/min group. The ratioof the induced mutation frequency at 0.03 cGy/min to that at
95 cGy/min was approximately 0.42 for doses of less than
1.9 cGy and approximately 0.33 for doses of 1.9–4.75 Gy[S43]. There was some specificity as to when such mutation
events can be induced during spermatogenesis. When sperm
and late spermatids were irradiated, the mutation frequency
within non-irradiated maternally derived alleles was approximately three times higher than in the control group. In the F2generation, however, no increase in mutation frequency wasobserved. Similarly, there was no significant increase in theF1 mutation frequency when stem cell spermatogonia were
irradiated. These data suggest that irradiation of sperm andlate spermatids can induce indirect mutations in F1 somatic
cells, supporting the idea that genomic instability arises during F1 embryonic development. Moreover, such instability
apparently arises most frequently when eggs are fertilized
just after the sperm are irradiated [S42]. It should be notedthat dose-rate effects and germ-cell-stage specificities formutational response were previously demonstrated for “specific locus” mutations in mice, and such findings have beenfactored into past decisions of the Committee regarding theestimation of the risk of hereditary effects. Although interesting, these new findings in fish provide no reason to modify
those estimates.

106. Many of the studies examine effects in the F1 offspring of irradiated parents, while other studies consider F2and later generations. It is only these studies of F2 and later
generations that can be unambiguously considered transgenerational. This is because the F1 studies may be revealing
mutations that occur during parental germ cell development.
Thus F1 studies address “heritable effects”, while F2 and later
generation studies address “transgenerational effects”.
A.

Studies in non-mammalian species

107. The first demonstrated transmission of genomic instability to subsequent generations was in Drosophila treatedwith ionizing radiation and mustard gas. However, the end
point used was lethality, which revealed little about thenature or the mechanisms of the processes involved [A12].

110. Microsatellite mutations have also been studied inplants grown in heavily contaminated areas near Chernobyl[K15, K16]. Kovalchuk et al. [K15] investigated the mutation rates of 13 microsatellite loci in wheat plants grownin a contaminated (900 Ci/km2) versus a control (<1 Ci/
km2) area, and found a 3.6-fold increase in germinal mutation rate in the contaminated versus the control plot. Ellegren et al. [E17] reported an increased frequency of partial
albinism, a morphological aberration associated with lossof fitness, among barn swallows, Hirundo rustica, breeding close to Chernobyl. Heritability estimates indicate that
mutations causing albinism were at least partly of germ line
origin. Furthermore, evidence for an increased germ line
mutation rate was obtained from segregation analysis at
two hypervariable microsatellite loci, indicating that mutation events in barn swallows from Chernobyl were two- totenfold higher than in birds from control areas in Ukraine
and Italy.

108. Shima and colleagues have developed a “specificlocus” test system using the Japanese medaka fish, Oryzias
latipes [S40]. The genetic end points available are dominantlethal mutations, total “specific locus” mutations and viable“specific locus” mutations. The medaka has a transparent egg
membrane and embryo body, and both visible mosaics andwhole-body mutations can be detected during development
at an early-expressed pigmentation locus [S40]. When wildtype +/+ males were gamma-irradiated and then mated withwl/wl females, the frequency of F1 embryos with both wildtype orange leucophores (wl/+) and mutant-type white leucophores (wl/wl*) (mosaic mutants) was ~5.7 × 10–3 Gy–1. The
frequency of embryos with only white leucophores (wholebody mutants) was ~1.3 × 10–3 Gy–1. These results suggest thatdelayed mutations arise frequently in medaka fish embryosthat have been fertilized with irradiated sperm [S41].
45

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UNSCEAR 2006 REPORT: VOLUME II

B.

Mouse studies

1. Irradiation of the mouse zygote
111. A long-held belief regarding radiation teratogenesis
was that developmental defects are only inducible when
the conceptus is irradiated during organogenesis. In contradiction to this, Streffer et al. [M26, P1, P7] demonstrated
that irradiation of a single cell, the zygote, can induce
developmental abnormalities, particularly gastroschisis, inthe resulting animal. Indeed, when irradiated during early
embryogenesis, induced teratogenesis is suppressed in a
TP53-dependent manner, where apoptotic cell death plays acritical role [K23, N23, N24].
112. Streffer’s studies used the Heiligenberger (now the
HLG/Zte) mouse, which has a spontaneous frequency ofgastroschisis of ~3%. One gray of X-rays to the zygote
increased this to ~11%, leading the investigators to conclude that ionizing radiation enhances latent damage alreadypresent in this predisposed mouse strain [H27, S34]. Similarfindings have been made by other investigators [G4, G20,
J7] after exposure to chemical mutagens [G15]. Streffer andcolleagues extended these studies to demonstrate congenital
malformations in the 19-day-old foetuses after paternal irradiation (2.8 Gy of 137Cs gamma rays) [M64]. This increased
lethality occurred after exposure of all stages of spermato­
genesis with the exception of early spermatogonia.

Figure XII. Pre-implantation embryo chimera assay.
F0 CD1 male mice were exposed to 1 Gy of 137Cs gamma rays andmated to non-irradiated CD1 females six weeks after F0 paternal
irradiation. F1 animals with a radiation history were mated with nonirradiated females to obtain F2 embryos with a paternal F0 radiation
history. These four-cell embryos were paired with non-irradiatedCD1 control four-cell embryos, and the resulting chimeras were
cultured to the blastocyst stage and transferred to a foster mother.
The pups resulting from the transfer were screened, and those withand without a paternal F0 radiation history were identified and bredwith normal females. Sex-matched pairs could then be evaluatedfor competitive cell proliferation disadvantage, protein kinase C,
MAP kinase and GST activities, as well as p21waf1 and TP53 protein
levels.
1 Gy 137Cs gamma rays

F0

Type B
Spermatogonia

F1113. One possible interpretation of the gastroschisis results
is that this mouse strain has a peculiarly high susceptibility
to this type of gross abnormality and that the radiation treatments induced many dominant lethal mutations in germ cells
or in the pronucleus that greatly exacerbated this extreme
inherent susceptibility. While it is important to be aware thatsuch situations exist, there does not seem to be any practical
way to apply data on a strain of mice with a strong predisposition to a serious anomaly to the estimation of hereditaryrisk in human populations.
114. The effect of in utero exposure to ionizing radiation
during the early phases of pregnancy has previously beenreviewed [J7, P1, S34]. The risk of congenital malformations, the role of genomic instability after irradiation during
the pre-implantation phase [S63], genetic susceptibility [J6]and health consequences [H17] have been reviewed in detail
and will not be further considered here.

Radiation history

F2

F3

Litter mates

2. Pre-implantation embryo chimera assay
ANALYSIS

115. Wiley et al. have used a pre-implantation embryo chimera assay to demonstrate adverse effects in embryos after
acute whole-body paternal irradiation (figure XII). They
measured the competitive cell proliferation disadvantage ofan embryo with a radiation history after challenge by directcell-to-cell contact with a normal embryo in an aggregation chimera [O1, O2, W10]. The F1 embryos conceived
6–7 weeks after paternal F0 irradiation were most likely todisplay the phenotype, indicating that the type B spermatogonia were the most sensitive [W10]. Recently, Baulch et al.
[B4, B5] have evaluated F3 mouse offspring from F0 paternal
mice exposed to 1 Gy of 137Cs gamma rays for gene productsthat can modulate cell proliferation rate, including receptor tyrosine kinase, protein kinase C and MAP kinases, andANNEX C: NON-TARGETED AND DELAYED EFFECTS OF EXPOSURE TO IONIZING RADIATION

protein levels of nuclear TP53 and p21waf1. All three-protein
kinase activities were altered, and nuclear levels of TP53
and p21waf1 protein were higher in F3 offspring with a paternal F0 radiation history than in non-irradiated litter-mates.
While there is clear evidence of effects, it is unclear how thisrather novel and artificial phenotype can be related to clinically important hereditary effects, and thus no attempt will
be made to apply these results to hereditary risk estimationin humans.
3. Mouse mutation assays
116. To investigate whether preconception paternal irradiation can lead to the heritable transmission of genomic
instability in mice, Luke et al. [L25] measured mutation frequency in a transgenic mouse model tagged with a lambda
shuttle vector. This assay system allowed mutations in thelacI gene from the shuttle vector to be analysed in vitro after
the animal had been irradiated in vivo. The results indicatedthat, as parental dose increased, there was a trend towards
higher mutation frequency in vectors recovered from DNA
from the bone marrow of F1 progeny. These data demonstrate
heritable transmission of factors leading to genomic instability in F1 progeny following paternal pre-conception irradiation, although the results with a lambda shuttle vector would
be complicated to apply in quantitative risk estimation forhumans because they do not involve mammalian genes.
117. An increase in micronucleus frequency in bone­marrow erythrocytes from the F1 progeny of male BALB/cmice exposed to chronic low-dose gamma irradiation wasobserved by Fomenko et al. [F11]. Mice were irradiatedwith 10, 25 or 50 cGy at dose rates of 1, 5 and 15 cGy/day,and were mated with non-irradiated females on day 15 after
irradiation. The obtained offspring had an elevated micronucleus frequency in bone marrow erythrocytes at the ageof two months. This suggests the transmission of genomic
instability from damaged germ line cells of irradiated maleparents to somatic cells of the progeny. It is unclear, however, if the effects on micronuclei represent chromosome
damage of consequence to clinical diseases beyond thosealready covered by the Committee’s current methods toestimate hereditary risk.
118. The pun mouse background allows visual detection of~70 kb DNA deletions in the pink-eyed unstable (pun) locus indeveloping mouse embryos. These are scored as black spots
on the light gray fur or black cells on the transparent retinal
epithelium of the offspring [R2]. In the fur spot assay, 10-dayold pups are observed for black spots on the light gray fur,and the number of animals with fur spots is counted. In theeye-spot assay, mice are sacrificed at ~20 days, the eyes are
removed and the number of black cells in whole mounts of
the unpigmented retinal pigment epithelium are determined[R2]. The C57BL/6Jpun/pun mouse strain contains a 70 kb
tandem duplication of the pink-eyed dilution (p) gene [B29],
the pun mutation. The pun mutation is autosomal recessive
and results in a dilute, light grey coat colour and pink eyes.

47

Intrachromosomal homologous recombination between the
70 kb repeats that delete one copy of a duplicated 70 kb DNA
fragment at the pun locus restores the p gene and produces
black pigment in the hair and retinal epithelium in wild-type
mice. On the C57BL/6Jpun/pun inbred background, 5–10% of
the mice spontaneously display fur spots and from four to six
eye-spots per unpigmented retinal pigment epithelium.
119. The pun fur spot assay was used to demonstrate that
exposure of the parental germ line to ionizing radiation
results in induction of delayed DNA deletions in mouse offspring [C16, S17]. Male pun assay/pun assay mice were irradiated with 1 Gy of X-rays and mated 28 days later with
non-irradiated pun assay/pun assay females. The offspring
showed a higher frequency of large fur spots. Since deletions
occurring early in embryogenesis should yield larger spots
than events occurring later, the large spots indicated deletion
events occurring early in embryo development and manycell divisions after irradiation [C16]. Shiraishi et al. [S17]irradiated male mice with 6 Gy and observed an increasein pun assay reversions resulting in eye-spots, in the irradiated paternal pun assay allele as well as in the non-irradiatedmaternal pun assay allele, indicating untargeted recombination in the offspring. The number of spots per retinal epithelium increased twofold when the male was irradiated at the
spermatozoa stage [S17], but pun assay instability was notobserved when radiation was delivered either to spermatogonial stem cells or to late spermatids. It is noteworthy thatradiation-induced instability of the pun assay allele has been
observed in F1 mice but not in the F2 generation (reported inreference [N22]).
120. Estimates by the Committee of hereditary risk for
exposures of males have been based on damage to spermatogonial stem cells because they are the only germ cells inmales that can accumulate appreciable doses under lowdose-rate exposure conditions. The finding that there is no
induced pun instability in spermatogonial stem cells suggeststhat there is no need to revise current risk estimates upward.
4.

Alterations in tandem repeat DNA sequences

121. Alterations in tandem repeat DNA sequences, suchas minisatellite DNA and expanded simple tandem repeats(ESTRs), in the genome have been used as markers ofgenetic change. Such loci have high spontaneous rates ofmutation, which facilitates the measurement of induced
mutation in relatively small numbers of samples. Alterations(mutations) are manifested as gains or losses in repeat units
and are detected either by pedigree screening or by amplifications [N22, Y4]. Mutations in both minisatellite sequencesand ESTRs appear to arise via indirect mechanisms rather
than by direct damage to the repeat locus itself [Y4]. The
significance of these DNA sequence changes is unknown. Ifthey are genetically neutral they will not affect risk; nevertheless, the fact that they happen indicates that exposure toionizing radiation can lead to genomic destabilization that
may occur by a non-targeted mechanism.

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UNSCEAR 2006 REPORT: VOLUME II

122. Minisatellites are tandem repeat loci, typically
0.5–30 kb long, with repeat units in the range 6–100 basepairs. Thousands of minisatellites exist in the genome andfrequently they show variability in repeat copy number andtherefore allele length [J1]. Some minisatellites are highly
unstable and undergo a frequent length change mutation
spontaneously in germ cells, sometimes as high as 10-1 per
gamete [J2]. ESTRs were formerly classified as minisatellites but are now recognized as a fundamentally different
entity. In contrast to minisatellites, ESTRs are composed oflong arrays, up to 16 kb in length, of short (<10 bp) repeat
units. Unstable ESTRs appear to be comprised almost exclusively of homogeneous arrays, with longer arrays exhibiting the highest rates of mutation [B30]. This is in contrast
to the complex variant repeat distribution that makes up
common minisatellite alleles. ESTRs exhibit high levels ofsomatic instability and also show different mechanisms ofmutation from that observed in the highly mutable GC-richmini­satellites [Y4]. Examples of alterations (mutations) inan ESTR in mice are presented in figure XIII.

Figure XIII. Mutation analysis at two mouse ESTR loci.
A: Ms6-hm loci. Lanes: 1: father; 2: normal offspring; 3: offspring with a maternal mutation (open arrow); 4 and 5: offspring with two different
paternal mutations (dark arrows); 6: mother.
B: Hm-2 loci. Lanes: 1: father; 2, 4, 6 and 7: normal offspring; 3 and 5: offspring with different paternal mutations (dark arrows); 8: mother.
[Figure kindly provided by Y. Dubrova, University of Leicester, United Kingdom.]

123. By analysing DNA fingerprints of the offspring of 60Co
gamma-irradiated mice, Dubrova et al. [D10] have shown thattandem repeat loci mutations appear to be induced in spermatogonia by low doses of ionizing radiation, with an estimateddoubling dose of 0.5 Gy. This estimated doubling dose wassubsequently revised downwards to 0.33 Gy [D14], which issimilar to that reported earlier in the “specific locus” test inmice [R4]. This is an assay that has been relied upon by theCommittee to a large extent in estimating hereditary risk.
124. Dubrova [D21] has since compared the spectra anddose response for mutations at ESTR loci in the germ line ofmale mice acutely exposed to low-LET X- or gamma rays at
pre-meiotic stages of spermatogenesis in five strains of laboratory mice. He found that most mutation events involved thegain or loss of a relatively small number of repeat units, andthe distributions of the observed length changes were indistinguishable between the exposed and the control males. Overalla significant bias toward gains of DNA repeats was detected,
with approximately 60% of mutants showing gains. The values for ESTR mutation induction produced doubling doses of
0.44–0.98 Gy. Doubling dose estimations were also made by
Niwa and his group, and values of 3.4, 0.893 and 4.0 Gy werereported for stem cells, spermatids and spermatozoa, respectively [F2, N14]. Inherent imprecision in the methodology
used is likely to be the cause of these discrepancies [N22].
125. To date, most laboratory studies demonstrating heritable effects of radiation exposure have involved paternal ­irradiation [B4, B5, D14, D15, N14, S1, W10], and an
approximately linear dose-response curve for paternal mutation induced at pre-meiotic stages was found [D14]. Germline mutation in mouse tandem repeat loci appears to be asensitive indicator of irradiation of pre-meiotic stage germ
cells [D10, F2, S1], and an elevated paternal mutation rate
was found after irradiation of mouse pre-meiotic spermatogonia. In contrast, post-meiotic irradiation of spermatids
gave a result similar to that in control litters [D14]. However, both pre- and post-meiotic exposures were reported toincrease mutation yield, suggesting that strains of mice may
differ in stage susceptibility [F2, N14, S1].
126. Dubrova et al. [D14] also analysed the maternal mutation rate in mice after paternal irradiation at different stagesof spermatogenesis, and found no difference in the frequency
of maternal mutation. In contrast, Niwa et al. have describedANNEX C: NON-TARGETED AND DELAYED EFFECTS OF EXPOSURE TO IONIZING RADIATION

a small but statistically significant increase in the maternallyderived Ms6hm allele, in addition to the paternally derived
allele, when only male parents were irradiated at the spermatozoa stage [F2, N14, S1]. Niwa and Kominami [N14]
demonstrated a statistically significant increase in maternal allelic mutation rate in F1 mice born to irradiated male­parents. The authors concluded that, as a consequence ofmale (sperm) irradiation, genomic instability is triggered in
the zygote, which then mutates the paternally derived allelein cis and the maternally derived allele in trans.
127. Mutation rates at two ESTR loci have been studied
in the germ line of first- and second-generation offspring ofinbred male CBA/H, C57BL/6 and BALB/c mice exposed toeither neutrons or X-rays. Paternal CBA/H exposure resulted
in increased mutation rates in the germ line of two subsequent generations. Comparable transgenerational effects wereobserved also in neutron-irradiated C57BL/6 and X-irradiated
BALB/c mice. The spontaneous mutation rates and radiationinduced transgenerational instability varied between strains(BALB/c>CBA/H>C57BL/6). Pre- and post-meiotic paternal
exposure resulted in similar increases in mutation rate in thegerm line of both generations of CBA/H mice, which suggeststhat radiation-induced expanded simple tandem repeat instability is manifested in diploid cells after fertilization [B2].
Although there are some difficulties in interpreting these data
[B41], they do suggest that excess transgenerational mutation at the unstable loci may be detected after irradiation ofdeveloping male germ cells.
128. Analysis of ESTR mutation induction in the mousegerm line suggests that mutations are likely to be due topausing of the replication fork and subsequent polymerase
slippage events [B31, D21]. The frequency of induced mutation appears to be significantly greater than the predicted frequency of radiation-induced DNA damage at a given tandem
repeat locus. This has led many investigators to conclude thatthe increased mutation rates observed at these repeats are
not necessarily the result of directly induced DNA ­damage
at the “specific locus”, but result from radiation-induced­damage elsewhere in the genome or cell [D13, D14, D15,
F2]. While alterations in tandem repeat DNA sequences may
provide useful biomarkers of induced germ line effects, thebiological significance of such mutations remains unknown.
The similarity of the calculated doubling doses to those published for “specific-locus” mutations in mice supports thevalidity of the tandem repeat DNA sequence data, although
the uncertainty in doubling doses in “specific-locus” experiments needs to be borne in mind [U1]. The comparative data
between germ cell stages for mutations that affect ­tandem
repeat DNA sequences remain of interest and potential
importance.
5.

Tumour induction in the offspring of irradiated parents

129. Nomura first reported a significant increase in lungtumours, mostly benign adenomas, in the F1 offspring of
X-irradiated ICR parental mice [N17, N18, N28]. This has

49

been confirmed in some studies [H10, L20, L21, V3] but notall. For example, Cattanach et al. [C4], using the optimal
experimental conditions defined by Nomura [N17], foundthat tumour incidence was no higher in the offspring of irradiated BALB/cJ mice than in the non-irradiated controls.
They did find that the proportion of fertile females and meanlitter size were affected by the radiation, showing a dosedependent, dominant lethal response. In attempting to reconcile these differences, Cattanach et al. [C4] proposed thatinconsistencies in the animal experiments may in part be due
to lack of an appropriate concurrent control, whose periodic
or cyclic variation in tumour incidence may have been outof phase with that in the treated animals. Alternatively, thereported differences could reflect strain differences in themice used.
130. Selby and Priest [S52] reported no induced leukaemias when male CBA/Ca mice were injected with 239Pu­citrate solutions at nominal activities of 6 and 60 Bq/g, togive absorbed doses of approximately 0.3 and 4.0 cGy, andwere mated to females of the same strain 54–68 days later.
Nomura [N16] found no increase in leukaemia in the offspring of ICR mice derived from spermatogonia after acuteirradiation. In contrast, when spermatogonia from the N5mouse strain were irradiated, Nomura found a 10-foldgreater incidence of acute lymphocytic leukaemia in the offspring than in the non-irradiated controls. Once again, thismay be explained by differences in genetic predisposition toleukaemia induction by radiation in these mouse strains.
131. Extending his original findings, Nomura hypothe­
sized that, if radiation-induced mutations in the germ line
led to heritable lung tumours in offspring, then all the cells in
the lung should be at increased tumorigenic risk. Following a
subsequent challenge with the carcinogen urethane, Nomura
described significantly increased clusters of tumour nodules
in the lung [N17, N18]. Vorobtsova and Kitaev [V3] reported
similar findings, but Cattanach et al. [C21] were unable toreplicate these results in C3H/HeH mice. Selby et al. [S53]suggested that the explanation for the surprisingly high rates
of induction of dominant mutations that cause tumours suggested by the experiments of Nomura and others might result
from the confounding effect of the radiation-induced dominant lethality that often occurs in such experiments. Other
possible non-mutational explanations for the high mutation
rates reported for dominant tumour mutations by Nomura
have been suggested by Selby [S51] and Cattanach et al.
[C4, C21].
132. Lord et al. have also investigated the heritable effects
of pre-conception paternal irradiation from injected plutonium (239Pu alpha particle irradiation), 137Cs gamma rays or
the Auger-electron-emitting radionuclide 55Fe. They demon­
strated perturbed haemopoiesis in offspring, as well as
enhanced sensitivity to methylnitrosourea (MNU, 50 mg/kg)
as a secondary carcinogenic insult [H10, L20, L21]. A majordifference from the tumour experiments discussed above
was that Lord et al. used much lower doses of radiation. As
a result, there would have been little or no induced dominant

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UNSCEAR 2006 REPORT: VOLUME II

lethality, and the possibility that this was a confounding
effect is therefore removed. The mutation rates calculated
from these results suggest that Nomura’s results actually
underestimated the extent of this phenomenon. Since thesestudies rely on a secondary treatment with a carcinogen,there seems to be no way to apply their results to revise theCommittee’s estimates of hereditary risk, which are madefor radiation alone.
C.

Malformation induction in the offspringof irradiated parents

133. There are several studies addressing the question of
malformation induction after irradiation of either female[K13, L50, M65, N17, N31, N32, N34, W22, W23] or male
[K12, K36, M64, N17, N31, N32, N34, R15] mice andlooking for malformations in the next or subsequent [L50,
L51, N33, P7] generations. In all of these studies, conditions were found that resulted in transmission of radiationinduced germ cell effects in the form of malformations to
the F1 or subsequent generations. The mechanism for thedevelopment of these malformations is apparently different under these conditions from that which is responsible
for the development of malformations induced by radiation
exposures during major organogenesis [S34]. The following
conclusions can be drawn from the experiments: comparatively high doses (>1 Gy) are required for transgenerationalmalformation effects to be detected. The effects seen after
female exposure are not due to indirect effects because ofradiation sickness of the mother, but must have, at least
partly, a genetic background [L50, W22, W23]. This genetic
background is quite obvious after radiation exposure of malemice. These transgenerational effects are not restricted to
low-LET radiation [K36]. It seems unlikely that there is adirect relationship between chromosomal translocations andcongenital anomalies [L50, N34], although some suspicion
in that direction has been expressed [R15]. The basis of themalformations observed seems to be heterogeneous: someare due to genetic changes of high penetrance that are rapidly eliminated; some are due to modification of genes oflow penetrance; and some are probably of non-genetic origin[L50]. There are strain-specific differences as to the extent of
the transgenerational effect [R15]. There are indications thata major proportion of mutations are eliminated in the first
generation and that only a minor proportion are transmitted
to later generations [L50].
134. At present there are too many uncertainties about thosedata suggesting that dominant mutations and/or genomic
instability cause tumours in progeny of irradiated mice to beable to apply such data in hereditary risk estimation.
D.

Human studies

135. To date, no radiation-induced genetic, i.e. hereditary, diseases have been demonstrated in human populations exposed to ionizing radiation. Neither the offspring ofindividuals treated with chemotherapy and/or radiotherapy
for cancer [B18] nor the offspring of women treated withradiation during infancy for haemangiomas [K6] demonstrated any significant effects attributable to parental exposure to chemicals or radiation [U1]. Furthermore, a numberof studies involving the children of survivors of the Hiroshima and Nagasaki atomic bombings have failed to detect
any transmitted genetic effects of radiation exposure [K14,N12, N13, S4, S5]. A cohort of 31,150 children born to parents who were within 2 kilometres of the hypocentre at thetime of the bombing was compared with a control cohort of
41,066 children. During the children’s early years, congenital defects, sexual development, physical development andsurvival were all investigated. Later, cytogenetic studies andthe electrophoretic properties of a series of serum proteins orerythrocytic enzymes were analysed, in addition to a complete medical evaluation. None of these indicators was significantly modified by parental radiation exposure (reviewed
in reference [N10]).
136. In addition, in a study that examined 50 families
exposed after the Hiroshima and Nagasaki atomic bombings
with 64 children, and 50 control families with 60 children[K14], minisatellite analyses revealed no genetic effects at
six human tandem repeat. This study was later expanded toinclude analysis of mutations at eight hypervariable minisatellite loci in the offspring (61 from exposed families,in 60 of which only one parent was exposed, and 58 from
unexposed parents) of atomic bombing survivors with meandoses of >1 Sv. They found 44 mutations in paternal ­alleles
and 8 mutations in maternal alleles, with no indicationthat the high doses of acutely applied radiation had caused
significant genetic effects [K28].
137. In contrast to these observations, Dubrova et al. [D11]described elevated mutation rates in DNA tandem repeat
sequences in humans living in rural areas of the Mogilev
district of Belarus, which was heavily contaminated withradionuclides from the Chernobyl reactor accident. The frequency of mutation was assayed both by DNA fingerprinting using one multilocus probe and by single-locus analysis
using four probes, and this revealed mutation rates approximately twofold higher in the offspring of exposed parents
when compared with an unexposed population from theUnited Kingdom. These initial observations were expanded
to include analysis of families from rural areas in the Kiev
and Zhytomir regions of Ukraine, which were heavily contaminated by radionuclides after the Chernobyl accident.
A statistically significant 1.6-fold increase in mutation rate
was found in the germ line of exposed fathers, whereas thematernal germ line mutation rate in the exposed families
was not elevated [D9].
138. The initial report by Dubrova et al. [D11] generated
commentary centred largely on the selection of a non-exposed
population from the United Kingdom for comparison with
exposed parents from Belarus [S4], the failure to exclude
other contaminants such as pollutants and viral infections[N11], and questions regarding the biological significance ofANNEX C: NON-TARGETED AND DELAYED EFFECTS OF EXPOSURE TO IONIZING RADIATION

increased mutations in hypervariable tandem repeat alleles[N10]. These criticisms initially appeared to detract from thesignificance of the findings of Dubrova et al. However, in a
subsequent report, Dubrova et al. [D12] recruited more families from the affected region and used five additional mini­satellite probes, and once again their data indicated a twofold
higher mutation rate in exposed families than in non-­irradiatedfamilies from the United Kingdom. They also used individual
radiation doses for external and internal chronic exposure to137
Cs as an indicator of long-term population exposure andfound a significant positive correlation between radiation doseand mutation rate over multiple loci with the exposed families.
Significantly, there were no obvious differences in the mutation spectrum observed between the exposed and the control families. In subsequent studies, children born to parents
who participated in recovery operations after the Chernobyl
accident also showed an elevated mutation rate at some loci
[L18, W5], as did children born to parents living around theS­emipalatinsk nuclear test site in Kazakhstan [D8].
139. It should be stressed that the analysis of tandem
repeat loci mutation rate demands sophisticated molecularbiology, and it is important that observed mutants be validated. ­Jeffreys and Dubrova [J3] suggested that technicalartefacts might explain the sevenfold increase in mutation
rate in child­ren of Chernobyl recovery operations workers described by Weinberg et al. [W5]. Questions of valid
paternity, sample mix-up, variation between DNA samples
and the demands of the required technology are all potential
sources of variability and could explain differences in results
between different investigators.
140. Nevertheless, other studies have failed to reproduce
these positive findings [F15, L17, S55]. A way of reconciling these apparently contradictory results was offered by­Livshits et al. [L17]. They measured the frequency of inherited mutant alleles at seven hypermutable minisatellite lociin 183 children born to Chernobyl recovery operations workers and in 163 children born to control families living in nonirradiated areas of Ukraine. No significant difference in thefrequency of inherited mutant alleles was found between the
exposed and the control groups. The exposed group was thendivided into two subgroups according to the time at which
the children were conceived in relation to the fathers’ workat the power plant. Eighty-eight children were conceived
either while their fathers were employed at the facility or up
to 2 months later (subgroup 1). The other 95 children wereconceived at least 4 months after their fathers had stopped
working at the Chernobyl site (subgroup 2). The frequencies
of mutant alleles were higher for the majority of loci in subgroup 1 than in subgroup 2, suggesting that the ­timing of irradiation during spermatogenesis had affected its ­mutagenic
potential.
141. Given the frequency of mutations in these hyper­variable
alleles and the lack of evidence for significant differences in
the mutation spectrum between control and exposed families[D12], it is unlikely that the minisatellite loci themselves are
the direct targets of the radiation. If the increased mutation

51

rate is not caused by DNA damage directly, it might well result
from non-targeted events associated with ­radiation-induced
genomic instability [D13, D16, F2, N14, S1].
142. The reasons for the discrepancy between the data­collected from the children of survivors of the Hiroshima
and Nagasaki bombings and the induction of human germline mutation in the majority of studies involving the offspring of parents living in radiation-contaminated environments are not easily reconciled. The types of radiation towhich individuals were exposed differed between the two
populations, and there are uncertainties associated with thedoses. The bombings resulted in a single acute exposure
to predominantly gamma radiation and a small amount of
neutron radiation, whereas contamination from Chernobyl
resulted in chronic exposures to internalized 131I. However,this is unlikely to be the only reason for the discrepancy,
because there are also negative results for the children ofrecovery operations workers exposed to low-dose-rate­external/internal exposures [F15, S55].
143. The controversy surrounding the induction of mutations in tandem repeat sequences is far from resolved. May
et al. [M5] examined the mutation frequency at hyper­variable
tandem repeats in sperm from three seminoma patients­following hemipelvic radiotherapy. Scattered radiation
doses to the testicles were monitored, and the mutation rates
in pre-treatment sperm DNA were compared with sperm
derived from irradiated meiotic and post-meiotic cells. No
evidence for radiation-induced germ line mutation at these
hyper­variable loci was observed even though the patientswere monitored for a period of 1 to 11 months.
144. Of all the reported studies, it is only those of the populations living in the Semipalatinsk region of Kazakhstan that
permit comment on the potential transgenerational effects(i.e. F2+ generations). In one such study, the F2 offspring of
those parents that received the highest external and internal
doses did not show any elevation of minisatellite mutation
frequency [D8].
145. These and related studies have been summarizedin table 2 and have recently been subject to critical review[B41]. To summarize the conclusions of Bouffler and theexpert review panel [B41]: only limited data are available
indicating that germ line mutation of minisatellites can bedetected in irradiated human populations. The data are inconsistent and show only limited evidence of dose dependence,and the panel found that additional work would be necessary
to establish the radiation responsiveness of these loci. Furthermore, the data on mutation of human tandemly repeated
DNA loci do not warrant a dramatic revision of germ line orcancer risk estimates for radiation at present [B41].
E.

Cancer incidence in the offspring of irradiated humans

146. The risk of cancer in the offspring of humans irradiated prior to conception is also controversial. No excess

52

UNSCEAR 2006 REPORT: VOLUME II

cancer incidence has been reported in children born to parents exposed to ionizing radiation by the atomic bombings inJapan [Y1, Y2] or in the offspring of cancer patients treatedwith radiotherapy [H4].
147. A major event early in this controversy was the conclusion by Gardner et al. [G2] from a case-control study thatthe increased incidence of leukaemia and non-Hodgkin’s
lymphoma among children living near the nuclear reprocessing plant in Sellafield in the United Kingdom was associatedwith paternal employment and the recorded external dose ofwhole-body radiation during work at the plant before conception. This conclusion was controversial [D6] and was notsupported by the excess of childhood leukaemia observed at
nearby Seascale [P4], or by an extensive study of radiation
workers and childhood cancers [D7]. Nevertheless, Dickinson and Parker [D4] published results of a cohort study that
supports the initial association with paternal radiation dose,
which suggests that it still remains a possible explanation[B15], although population mixing [G17] is regarded as apotentially important factor in this particular cluster.
148. The United Kingdom Committee on Medical
Aspects of Radiation in the Environment (COMARE), in
their seventh report, reviewed the evidence concerning
the incidence of childhood leukaemia and other cancers in
the offspring of parents occupationally exposed to radiation prior to conception [C17]. The COMARE agreed that,while a link between parental exposure and such effects
in the offspring was possible in principle, the epidemiological evidence from the offspring of radiation workers in
the United Kingdom, the United States and Germany failed
to support an increased rate of solid tumours in children.
Furthermore, COMARE concluded that the balance of evidence indicated that the likelihood of developing childhood
leukaemia or non-Hodgkin’s lymphoma was not related toradiation dose.
F.

Pregnancy outcomes in the offspringof irradiated humans

149. In their eighth report, COMARE reviewed pregnancy
outcomes following pre-conception exposure to ionizing
radiation in humans [C18]. They found that the available
epidemiological data were inadequate to allow definitive
statements about the effect of pre-conception radiation exposure on pregnancy outcomes. This was due to difficulties in
obtaining reliable figures for the end points of concern andto the possibility that the radiation exposures in most ­studies
may have been too low to produce a detectable effect. The
conclusion reached by COMARE was that, from all the epidemiological data examined, there was little evidence thatadverse reproductive outcomes in general are related toparental radiation exposure. Similarly, the limited data available did not link miscarriage or neonatal death with parental
irradiation. COMARE did point out, however, that almost
all of the published studies on pregnancy outcome following parental exposure to radiation in human populations

lack statistical power; this is probably due to the low doses
to which the populations were exposed and to the small­population sample size.
150. The Scientific Committee takes note of these conclusions by COMARE, which support its own view that the typesof genetic data discussed in this annex imply no modificationof its own estimates of hereditary risk. It has been the Scientific Committee’s position that the mutation rates in experimental organisms upon which it has based its estimates ofhereditary risk are sufficiently low as to make it unlikely thatanalyses of the radiation-exposed human populations available for study would show statistically ­significant increases
in hereditary diseases.
G.

Genetic damage and malformation inductionin the offspring of irradiated parents

151. The densely populated costal regions of Kerala state
in southwest India have deposits of radioactive monazitebearing sand and provide a unique opportunity to investigate
the effects of high levels of natural radiation on human populations [N35]. The background radiation levels range from
≤1.0 mGy to >35.0 mGy per year owing to naturally occurring thorium and its decay products. There is a comprehensive programme to assess the biological and health effectsof this radiation exposure in humans, focusing mainly on
constitutional chromosome abnormalities and the incidenceof congenital malformations in newborns. To date, the data
do not reveal any effect on cytogenetic aberrations in lymphocytes [C24] or the incidence of congenital malformations
in newborns [J11] that can be associated with exposure toionizing radiation.
H. Impact of non-targeted and delayed effectsof radiation on future generations
152. Heritable effects are observed in first-generation offspring and/or in later generations after one or both parents
have been irradiated prior to conception. Since it was established, the Committee has made estimates of the genetic
effects of radiation in humans in offspring of irradiated­parents based upon clear demonstrations that mutations canbe induced in experimental organisms, including experimental mammals. The UNSCEAR 2001 Report on the hereditaryeffects of radiation emphasized that no radiation-induced
genetic (i.e. hereditary) diseases have so far been demonstrated in human populations exposed to ionizing radiation [U1]. No demonstrable adverse reproductive outcomes
were described for the survivors of the atomic bombings inJapan, or for women irradiated during infancy for skin haemangiomas. No demonstrable hereditary effects of radiation
exposure resulting from the Chernobyl accident have been
described [U2]. Likewise, no increase in cytogenetic abnormalities [W19] or genetic effects [B39] has been reported insurvivors of childhood cancer exposed to ionizing radiation
before reproduction.

ANNEX C: NON-TARGETED AND DELAYED EFFECTS OF EXPOSURE TO IONIZING RADIATION

153. Ionizing radiation is considered a universal mutagen.
Experimental studies in plants and animals have demon­
strated that radiation can induce hereditary effects, andhumans are unlikely to be an exception in this regard. It wasfor this reason that the Committee estimated hereditary risk
in humans in the absence of direct evidence in humans. Thisannex presents a re-evaluation of some of the controversial
data, in view of newer findings, and also a review of some
types of damage not considered in earlier UNSCEAR reports.
Two assumptions are commonly made in the estimation ofgenetic risk: (1) that the seven “specific loci” in the mouse
constitute a suitable basis for extrapolation to genetic disease in humans; and (2) that heritable mutations are inducedby radiation damage (energy-loss events leading to doublestrand damage) occurring within the genome and are induced
linearly with dose, at least at low doses. The issues of main
importance in this section are whether the information on53

the types of mutation considered below might (1) be used to
improve the Committee’s estimates of hereditary risk, and(2) indicate some type of genetic instability that could leadto modification of risk through subsequent generations.
154. Overall it is clear that irradiation of the parent can leadto some changes in the offspring, but it is likely that most ofthese are due to the manifestation of direct damage caused byradiation in the original germ cell. The high incidence of offspring with “mutations” in ESTRs and the detection of mutations in maternal alleles after paternal irradiation ­suggest that
non-targeted instability may be induced in specific circumstances. There is only very limited evidence that instability
is transmitted across into the F2 generation, and human data
are negative. Therefore the Committee considers that thesedata are insufficient to justify modification of current risk
estimates for hereditary effects or cancer in humans.

VII.

IMPLICATIONS OF NON-TARGETED AND DELAYED EFFECTS

155. A wealth of information has been reviewed that deals
with possible radiation-induced non-targeted and delayed
genetic effects, as well as genetic end points that can occur
spontaneously. Most of the estimates of hereditary risk madeby the Committee in the past have been based on classicalmutation experiments. Such studies often require exceedingly
large samples of offspring. For the reasons described above,
it does not appear that the new findings necessitate changes in
the Committee’s estimates of hereditary risk. It is possible thatcertain types of genetic damage detected by some of the assays
have no relationship to clinically important phenotypes. It isalso possible that the methods of hereditary risk estimation
used by the Committee in the past adequately incorporate any
genetic risks of clinical rele­vance that might be associatedwith the damage detected by the various assays considered.
While it is clear that some of the assays permit demonstrationof effects of radiation using much smaller sample sizes thanmore classical methods, uncertainty remains as to whether
these effects correlate with the rare types of mutation thatcause clinically serious conditions. There is no convincing
evidence of transgenerational instability in humans causedby radiation that would lead to the propagation of clinicallyimportant effects over succeeding generations. Because theCommittee’s current risk estimates already assume transmission of many of the effects found in the first generation tolater generations, rare instances of such propagation would
have little impact on total risk estimates.

effects are essentially encompassed in current radiation risk
estimates for carcinogenesis.
157. Radiation-induced instability and the existence of
bystander effects are well established and incontrovertible. A
common observation of these responses is that they dominate
at low doses and saturate with increasing dose (reviewed inreferences [P13, S18]). In addition to damage directly inducedby the deposition of energy in the nucleus of the irradiatedcell, consideration must now be given to these indirect effectsof radiation. An irradiated cell can send out a signal and inducea response in a cell whose nucleus was not hit by radiation.
This might result in genetic damage, genomic instability or
lethality in non-irradiated cells. These non-targeted effects inessence “amplify” the biological effectiveness of a given radiation dose by increasing the number of cells that experience
effects over those directly exposed to the radiation.
158. Understanding of these non-targeted effects is still in its
infancy, and much of the data to date have been obtained fromin vitro studies. While the significance of these indirect effects
for human health remains to be elucidated, it would seem
prudent to consider the implications of non-targeted delayed
effects of radiation exposure when considering ­models ofradiation carcinogenesis, particularly at low doses.
159. In vivo non-targeted effects are not new and have previously been implicated in radiation-induced carcinogenesis[S22]. The recent revival of interest in these non-­targeted
effects and the subsequent influx of new data ­suggest that it istime to re-examine the concepts of radiation dose and ­target
size. Many of the indirect effects described indicate that thetissue volume in which detrimental effects of radiation may
be observed is larger than the precise ­volume irradiated. Thisissue may have important implications for human health. Life
exists in a radiation environment, the use of radiation has
become an integral part of modern life, and the applications ofradiation in medicine and industry bring tremendous bene­fits
to society. Over time, biological systems have demonstrated aremarkable ability to adapt to environments to which they are
gradually exposed, and low doses of radiation are no exception. Higher doses of radiation can cause neoplasia. This presumably occurs through a combination of direct damage andnon-targeted effects, and models of radiation-induced carcinogenesis should incorporate both direct and indirect effects
when evaluating radiation risks. Ultimately, understanding
the multitude of multicellular responses to radiation may provide a framework for evaluating health risks associated with­radiation exposure and a logical means of intervening in thedevelopment of suspected radiation-induced cancers.

156. The relevance of non-targeted and delayed effects
to the development of cancer and hereditary effects is not
yet clear. Carcinogenesis involves a progression of genetic
events that are associated with specific stages of the malignant ­process. It is tempting to speculate that induced
genomic instability can drive the progression of genetic
changes and thus provide the impetus for acquiring thosegenomic alterations associated with carcino­genesis. Yet
this must be tempered by the high frequency with which
instability is observed both in vitro and in vivo, and theobservation that instability generally tends to saturate at
low doses of radiation. Nevertheless, if radiation exposure
induces a transgenerational instability that could be passed
through the germ line and increase a child’s susceptibility
to ­cancer or genetic effects, this would have health ramifications. Bystander effects could also have significant
implications for human exposures, particularly to very
low fluences of high-LET radiation, e.g. to radon, where
only a small fraction of the cell population would be hit,i.e. subject to energy deposition events. However, bystander
effects appear to be limited to the irradiated organ, andsince risk estimates are to an organ and not a cell, bystander
55

CONCLUDING REMARKS
160. This annex has reviewed a multitude of studies on nontargeted and delayed effects of exposure to ionizing radiation. In addition to damage directly induced by the depositionof energy in the nucleus of an irradiated cell, consideration
should now be given to these indirect effects of radiation. An
irradiated cell can send out signals and induce a response in acell whose nucleus was not subject to energy deposition events
following irradiation. These non-targeted effects in essence“amplify” the biological effectiveness of a given radiationdose by increasing the number of cells that experience effects
over those directly exposed to the radiation.

determined directly. Such effects can provide mechanistic
information at doses of below ~200 mGy that could be pertinent to evaluating health effects at these low doses. However, in ascribing a mechanism to a particular biologicaleffect, the data in question should be independently replicated and show a strong coherence with the particular end
point considered. The UNSCEAR 2000 Report considered
the conventional view that the deposition of energy in the
nucleus and the subsequent cellular processing of induced
DNA damage were consistent with the observed cancer/
heritable effects induced by ionizing radiation.

161. In spite of the large body of new information available, considerable disagreement remains concerning any
definitive relationship between these non-targeted effects
and the observed health effects attributable to radiation.

164. In light of these considerations, the overall view of
the Committee is that the data currently available do not
require changes in radiation risk coefficients for cancer
and hereditary effects of radiation in humans. The Committee will maintain surveillance of developments in thearea of non-targeted and delayed effects, and recommends
that future research pay particular attention to study design
emphasizing replication, low-dose responses and associations with health effects particularly in the human population. Ultimately, understanding the range and multitude ofmulticellular responses to radiation will provide mechanistic insights into how radiation induces its observed healtheffects.

162. The Committee stresses that direct epidemiological observations and associated quantification of the healtheffects of radiation incorporate all mechanistic elements,
including the targeted (direct) effects of irradiation as well as
the non-targeted and delayed effects described in this report.
163. A specific role for non-targeted effects in the observed
health effects associated with radiation exposure cannot be

57

ANNEX C: NON-TARGETED AND DELAYED EFFECTS OF EXPOSURE TO IONIZING RADIATION

59

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