C. Adam-Guillermin (IRSN/PSE-ENV/SRTE/LECO)
N. Horemans (SCK•CEN)
On behalf of the related ALLIANCE topical Working Group
Radiosensitivity and transgenerational
effects in non-human species
Working group on effects of ionising radiation on wildlife
2/19
15 groups involved from 11 countries
Christelle Adam-Guillermin / IRSN – ERPW –October12th 2017 © IRSN
Prefered pet organism
Group Species of interest
HZ-IRE Bacteria, C. elegans ,
Danio rerio, plant cell cultures
UIAR Plants: A thaliana, Pinus
CEH-MEEG Earthworms, C. elegans
CEH-CG Large mammals
IRSN C.elegans, Danio rerio,
Daphnia magna, autochtonous
fish sp.
UoP Fish: stickle backs+
autochtonius
UGent Daphnia
SCK•CEN Plants: Ath, Oryza sativa, L
minor
RIRA Plants: scots Pine
NMBU Danio rerio, C.elegans
SU Interacting species (small
ecosystems/foodwebs)
CEA bacteria 3/19
4/19Christelle Adam-Guillermin / IRSN – ERPW –October12th 2017 © IRSN
▌ Actions▪ Inter-comparison on the analysis of biomarkers of
effects▪ Project submission to international calls (COMET
European project (2013-2017); BIOMARKER(CONCERT))
▪ Roadmap▪ Position paper on “DNA methylation as a potential
biomarker for environmental radiologicalcontamination”, Horemans et al. (to besubmitted)
▪ Paper for the proceedings of this congress“Radiosensitivity and transgenerational effects innon-human species”, Adam-Guillermin et al.
Working group on effects of ionising radiation on wildlife
5/19
Daly MJ, (2012) DNA Repair
(Rev)
and ref. therein
Blanchard and de Groot, 2017
Adineta vaga
Radioresistant organisms found in the three orders
Deinococcus: highest radiation resistance to acute exposure
Phylum
Deinococcus / Thermus
Directed evolution
Bacteria :Bacillus pumibus, Salmonella typhimurium,
E. coli => Highly radiation resistant E. coli
mutants (3000 Gy)
Cancer cells :radioresistance developed during
fractionated exposures to g rays
D10(10% survival)
1200 Gy
6000 Gy
4000 Gy
12000 Gy
4000 Gy
Byrne et al., (2014) eLiFE
Evolution (and directed evolution) of radioresistanceTopic 1 : radiosensitivity
Christelle Adam-Guillermin / IRSN – ERPW –October 12th 2017 © IRSN
0
10
20
30
40
50
60
70
80
90
100
1 10 100 1000 10000 100000 1000000
Percentage of Affected Fraction (%)
Capra
hircus
Sus c
rofa
Rattus n
orv
egic
us
Mus m
usculu
s
Ophry
otr
ocha d
iadem
a
Ple
uro
necte
s p
late
ssa
Poecili
a r
eticula
ta
Neanth
es a
renaceodenta
ta
Sola
num
tubero
sum
Canis
fam
iliaris
Vitis
vin
ifera
Pin
us r
igid
aP
orc
elli
o s
caber
Abie
s
bals
am
ea
Ory
zias l
atipes
Oncorh
ynchus t
shaw
yts
cha
Pis
um
sp.
Dahlb
om
inu s
p.
Eis
enia
fetida
Laru
s r
idib
undus
Physa h
ete
rotr
opha
Gallu
s d
om
esticus.
Triticum
sp.
Pin
us b
anksia
na
Merc
enaria m
erc
enaria
.D
aphnia
magna
Fagopyru
m e
scule
ntu
a.
Hord
eum
sp.
Calli
necte
s s
apid
us.
Daphnia
pule
x.
Vertebrates
Plants
Invertebrates
Dose rate (µGy/h)
Radiosensitivity between species ranges over 6 orders of
magnitude under chronic g controlled exposure (Effect
Dose Rate10)
Topic 1 : radiosensitivity
6/19Christelle Adam-Guillermin / IRSN – ERPW –October 12th 2017 © IRSN
Garnier-Laplace et al., 2010
7/19Christelle Adam-Guillermin / IRSN – ERPW –October12th 2017 © IRSN
▌ Mechanistic understanding of ≠ in radiation sensitivity across species
▌ Identification of sensitive species that may require special attention in
monitoring and radiation protection
▌ How do differences in sensitivity between species lie behind overall
effects at higher levels (community, ecosystem) ?
Topic 1 : radiosensitivity
• Genome size ; but highly condensed nucleoid may prevent the dispersion of the
DNA fragments generated by irradiation thus facilitating DNA repair (e.g.
Deinococcus or spz)Species Haploid genome
size (Mbp)
D10 survival
(Gy) (10%
survival)
Number of
survivable
DSBs per
haploid
genomea
DSB/Gy/Mbp
(approximate
linear density
of DSBs in vivo)
E. coli (MG1655) 4.6 700 6 0.002
D. radiodurans 3.3 12,000 118 0.003
Topic 1 : radiosensitivity
• Efficiency of checkpoint control mechanisms and DNA repair (HR vs NHEJ,
UNSCEAR 2000 ; “extended synthesis-dependent strand annealing” (ESDSA)
Zahradka et al., 2006)
• Reactive oxygen species scavenging :
• Higher [Mn]/[Fe] ratios in Deinococcus sp. (de Groot et al.; 2009)
• Melanized pigments in radioresistant fungi in Chernobyl (Dadachova and
Casadevall, 2009)
• Carotenoid pigments in bacteria (e.g. Rubrobacter radiotolerans) and
birds (Møller et al., 2005)
• Pheomelanin-based colorations in birds and mammals (Galvan et al., 2011
; Boratynski et al., 2014)• Induction of cell death ; tissue regeneration ; life stage
• Metabolic rate (high metabolic rate=sensitive species, Baas and Koijman, 2015)
2015) 8/19Christelle Adam-Guillermin / IRSN – ERPW –October 12th 2017 © IRSN
Organisms in their natural environmental are 8 times more sensitive than in
laboratory controlled conditions (Garnier-Laplace et al., 2013)
• Combination of factors such as gene diversity, competition, predation,
and abiotic factors.
• Evolutionary adaptation or increased sensitivity in different environ-
mental extremes, for long-term exposure.9/19
Topic 1 : radiosensitivity
Christelle Adam-Guillermin / IRSN – ERPW –October12th 2017 © IRSN
0
10
20
30
40
50
60
70
80
90
100
1 10 100 1000 10000 100000 1000000
Percentage of Affected Fraction (%)
Capra
hircus
Sus c
rofa
Rattus n
orv
egic
us
Mus m
usculu
s
Ophry
otr
ocha d
iadem
a
Ple
uro
necte
s p
late
ssa
Poecili
a r
eticula
ta
Neanth
es a
renaceodenta
ta
Sola
num
tubero
sum
Canis
fam
iliaris
Vitis
vin
ifera
Pin
us r
igid
aP
orc
elli
o s
caber
Abie
s
bals
am
ea
Ory
zias l
atipes
Oncorh
ynchus t
shaw
yts
cha
Pis
um
sp.
Dahlb
om
inu s
p.
Eis
enia
fetida
Laru
s r
idib
undus
Physa h
ete
rotr
opha
Gallu
s d
om
esticus.
Triticum
sp.
Pin
us b
anksia
na
Merc
enaria m
erc
enaria
.D
aphnia
magna
Fagopyru
m e
scule
ntu
a.
Hord
eum
sp.
Calli
necte
s s
apid
us.
Daphnia
pule
x.
Vertebrates
Plants
Invertebrates
Dose rate (µGy/h)
Chernobyl Exclusion Zone
10/19
Chronic exposure of microcrustaceans to Am-241 (15 mGy/h max).
Altered survival and fecundity in F1 and F2 but not in F0 (Alonzo et al.,
2008). Effects higher in F2 than in F1 (5-fold)
Topic 2 : long term and transgenerational effects
Field studies: observation of morphologic abnormalities in
butterflies exposed at Fukushima at 3 µSv/h. Worse
morphologic abnormalities observed in the following
generations obtained in laboratory controlled conditions (Hiyama
et al., 2012, 2015)
▌ Increase of sensitivity :
Christelle Adam-Guillermin / IRSN – ERPW –October12th 2017 © IRSN
11/19
▌ Decrease of sensitivity :
Chronic exposure of nematodes to uranium. Drastic decrease of fertility
until F3, then, from F3 to F21, selection of individuals being the more
fertile and having the fastest growth (Dutilleul et al., 2014)
0
2
4
6
8
10
12
14
16
1
gH2
AX
fo
ci n
um
be
r
ZF4 irradiées 24h à 100mGy/j
ZF4 adaptation 100 mGy/j
ZF4 irradiées 24h à 750mGy/j
ZF4 adaptation 750 mGy/j
4 mGy/h 31 mGy/h
24 h continuous
irradiation
4 h prior-irradiation+20 h
continuous irradiation
Radioadaptation effect
observed in ZF4 cells
(zebrafish fibroblasts),
exposed to a prior
irradiation dose (Pereira et
al., 2014)
Topic 2 : long term and transgenerational effects
Christelle Adam-Guillermin / IRSN – ERPW –October12th 2017 © IRSN
12/19
▌ Role of genetic mechanisms (mutation rates and types ; genetic
diversity)…
Genetic…Decrease of fertility and increase of mutation rates,
DNA damages or to chromosomes in sparrows (Ellegren et al., 1997 ;
Bonisoli-Alquati et al., 2010) and mice in Chernobyl (Pomerantseva et al.,
1997) or in daphnids in the lab (>7 µGy/h) (Parisot et al., 2015).
Topic 2 : long term and transgenerational effects
▌ …but not only
Adaptation of pines to high doses in Chernobyl despite deformities
and DNA damages (Kovalchuck et al., 2003). These adaptive
mechanism can not be only genetic (10-5-10-6 mutation per germ
cell)…increase of DNA methylation : epigenetic mechanisms
Christelle Adam-Guillermin / IRSN – ERPW –October12th 2017 © IRSN
What are epigenetic mechanisms
13/19
▌“The causal interactions between genes and their products, which bring the phenotype into being” Waddington 1942
▌“Epigenetics, in a broad sense, is a bridge between genotype and phenotype— a phenomenon that changes the final outcome of a locus or chromosome without changing the underlying DNA sequence” Goldberg et al 2007
Goldberg et al. 2007 reprinted from
Waddington 1957
• Three interacting players:
– DNA methylation
– Histone modifications
– Non-codingRNA
Christelle Adam-Guillermin / IRSN – ERPW –October12th 2017 © IRSN
DNA methylation : potential biomarker of IR
DNA methylation
DNA damages
FukushimaCEZ
European Project COMET
Gombeau et al. in prep
14/19
DNA methylation
pattern A. caliginosa
DNA methylation
pattern O. lacteum
0
0.05
0.1
0.15
0.2
0.01 0.1 1 10 100
% M
eth
ylat
ed c
yto
sin
es
Total dose rate (µGy h-1)
0
0.05
0.1
0.15
0.2
0.01 0.1 1 10 100
% M
eth
ylat
ed c
yto
sin
es
Total dose rate (µGy h-1)
0
0.05
0.1
0.15
0.2
0.01 0.1 1 10 100
% M
eth
ylat
ed c
yto
sin
es
Total dose rate (µGy h-1)
0
0.05
0.1
0.15
0.2
0.01 0.1 1 10 100
% M
eth
ylat
ed c
yto
sin
es
Total dose rate (µGy h-1)
CEZ
DNA methylation
in C. bursa pastoris of
FEZ
0
2
4
6
8
10
12
0 10 20 30 40 50
Met
hyl
ated
Cyt
osi
nes
(5-m
dC
/dC
, %)
Total dose rate (µGy h-1)
DNA methylation
in Pinus sp. of CEZ
DNA
methylation
Micro-
RNA
Histone
modification
Epigenetic
modifications
Transcription Translation
DNA mRNA PROTEIN
***
***
*
***
***
***
**
Control Recovery Exposed
P-value : *** < 0,001 ; **<0,01 ; *<0,05 ; 0,05<.<0,1
Wild type N2Strain DAMT-1 : DNA 6mA
methyltransferase depleted
Strain SET-30 : H3K4me
methytransferase depleted
Cum
ula
ted
num
ber
of
larv
ae
per
org
anis
m
****
*****
*.
*
Involvement of mutated pathways in the sensitivity and heritability of
irradiation effects
C. elegans: Methylations of histone H3K4 and adenines jointly control the epigenetic
inheritance of phenotypes
Topic 2 : long term and transgenerational effects
15/19Christelle Adam-Guillermin / IRSN – ERPW –October12th 2017 © IRSN
16/19
Specific methylation of genome (bisulfite sequencing)
▌ Decrease of F0 daphnid fecundity (exposed to IR)
Trijau et al., in prep
Topic 2 : long term and transgenerational effects
Christelle Adam-Guillermin / IRSN – ERPW –October12th 2017 © IRSN
F0 F1 F2 F3
mGy/h 0.007 40 0.007 40 0.007 40 0.007 40
Survival Ø Ø Ø Ø Ø Ø Ø Ø
Growth Ø Ø Ø Ø Ø Ø Ø Ø
Reproduction Ø + Ø Ø Ø Ø Ø Ø
17/19
▌ Hypomethylation of DNA in generations F2 and F3 coming from mother
exposed to IR and heritability of this methylation pattern between F2
and F3 generations : b-mpp (biogenesis of mitochondrial proteins), Hsp
70 (protein repair, IR), rpl28 (protein synthesis, IR)
▌ → Epigenetic biomarkers for IR exposure and effect
Topic 2 : long term and transgenerational effects
Christelle Adam-Guillermin / IRSN – ERPW –October12th 2017 © IRSN
▌ No significant effect on average global methylation levels was
observed in generations F0, F2 and F3, at 0.007 and 40 mGy/h
47
535
60
6
56
83
11
35
49
9
60
101
11
50
123
21
87
91
13
76
104
24
105
90
9
67
77
20
71
87
6
61
87
16
73
F0 F2 F3
0.0
06
5 m
G/h
41
.3 m
Gy
/h
hyper hypo hyper hypo hyper hypo
0
50
100
150
200
0
50
100
150
200
Nu
mb
er
of
DM
Cs
Exon
Intron
Intergenic
▌ Differentially (hypo- or
hyper-) methylated cytosines
(DMC) were detected in
various genomic locations in
generations F0, F2 and F3
after F0 exposure to 0.0065
and 41.3 mGy/h
Synthesis and perspectives
D. magna C. elegans
Multigenerational effects
on reproduction
✓ 35 mGy/h in F0
✓ 70 µGy/h in F2
✓ 26 mGy/h in F0
✓ 7 mGy/h in F2
Transgenerational effects
on reproduction
F1, F2, F3 (41mGy/h) ✓ F1, F2, F3 (50 mGy/h)
Epigenetic changes DMCs: F0, F2, F3 Increase in radioresistance
of mutants depleted on 6mA
and H3K4me2
(Parisot et al., 2015 ; Buisset-Goussen et al., 2014 ; Lecomte-Pradines et al., 2017 ; Trijau et al., in prep.)
Chronic effects has to be considered in a multigenerational context
Epigenetic mechanisms play a major role in these effects
Need to understand the link between molecular process and phenotype
changes (Adverse Outcome Pathway)
High throughput analyses to identify finderprints of ionising radiation effects
and early and sensitive biomarkers
Most realistic conditions of exposure (lower dose rates, more generations,
use of complex systems)
Perspectives:
18/19Christelle Adam-Guillermin / IRSN – ERPW –October12th 2017 © IRSN
19/19
Thank you !
Any question?
Christelle Adam-Guillermin / IRSN – ERPW –October12th 2017 © IRSN
20/22
Key species
Radiosensitvity factors
Highlighting (or not) long term
effects
Sensitive and early
biomarkers
Fingerprints/signatures
« OMICS »
Application for the environmental protection
Christelle Adam-Guillermin / IRSN – ERPW –October12th 2017 © IRSN
21/22
Arena et al 2014 What explains radiosensitivity
in plants ?
Topic 1 : radiosensitivity
Increasing Sensitivity Decreasing Sensitivity
Large nucleus Small nucleus
Large chromosomes Small chromosomes
Acrocentric chromosomes
Low chromosome number
Diploid or haploid
Sexual reproduction
Long intermitotic time
Long dormant period
Metacentric chromosomes
High chromosome number
High polyploid
Asexual reproduction
Short intermitotic time
Short or no dormant period
Genetic and reproductive factors
influencing the sensitivity of plants to
radiation (Sparrow and Miksche, 1961)
Christelle Adam-Guillermin / IRSN – ERPW –October12th 2017 © IRSN
a Aab
B
ab
B
bB
0
20
40
60
80
100
120
P0 S1
Enzy
me
act
ivit
y G
PO
D (U
g-1
FW)
22 mGy/h 38 mGy/h 86 mGy/h 457 mGy/h
**
Co
ntr
ol (
a,A
)Enzyme capacities
22/22
Higher activity of ROS detoxifying enzymes in S1
a Aab
B
ab
B
bB
0
20
40
60
80
100
120
P0 S1
Enzy
me
act
ivit
y G
PO
D (U
g-1
FW)
22 mGy/h 38 mGy/h 86 mGy/h 457 mGy/h
**
Co
ntro
l (a,
A)
a Aa
A
a
A
a
B
0
0,05
0,1
0,15
0,2
0,25
0,3
0,35
0,4
P0 S1
En
zym
e a
ctiv
ity
CA
T (
U g
-1FW
)
*
Co
ntr
ol (
a,A
)
Topic 1 : radiosensitivity
Christelle Adam-Guillermin / IRSN – ERPW –October12th 2017 © IRSN
Enzyme capacities
23/22
ab
B
ab
Bb
B
c
B
0,00
0,20
0,40
0,60
0,80
1,00
1,20
1,40
1,60
P0 S1
En
zym
e a
ctiv
ity
SPX
( U
g-1
FW)
0,00
0,50
1,00
1,50
2,00
P0 S1
Chart Title
22mGy/h 38 mGy/h 86 mGy/h 457 mGy/h
SPX + GPX ↑ in S1
Syringaldazine = Lignin
precursor
+
GPX involved in
cell wall metabolism
Cell wall strengthening as
defense
Topic 1 : radiosensitivity
Christelle Adam-Guillermin / IRSN – ERPW –October12th 2017 © IRSN
Arabidopsis thaliana - Phenotypical characterization
▌Number of leaves on the rosette, rosette diameter, inflorescence frequency, inflorescence height, amount of seed pods, seed weight, germination capacity.
Topic 2 : long term and transgenerational effects
▌Delay of inflorescence day in irradiated plants and of germination in irradiated plants maintained in control conditions
0 1 2 3 4
Control
22 mGy/h
38 mGy/h
86 mGy/h
457 mGy/h
Time (days)
0 1 2 3 4
G0
G1
G2
G3
G4
Phase 0 Phase 1 Phase 2 Phase 3 Phase 4 Phase 5 Phase 6
24/22Christelle Adam-Guillermin / IRSN – ERPW –October12th 2017 © IRSN
25/22
Topic 2 : long term and transgenerational effects
ab A abaAB ab
b
Bab
ab
Bb
0
1
2
3
4
5
6
7
8
P0 S1 S2
Leve
l of
me
thyl
ate
d c
yto
sin
es
(5-m
dC/
dC
,%)
22 mGy/h 38 mGy/h 86 mGy/h 457 mGy/h
**
*
Global hypermethylation of DNA in S1 and S2
What about field studies?
Christelle Adam-Guillermin / IRSN – ERPW –October12th 2017 © IRSN