radiosensitivity and transgenerational effects in non ... adam-guillermin presentation.pdf ·...

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

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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


▌ 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) ?


• 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


• 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



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 :


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)



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).


▌ …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


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


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


0

0.05

0.1

0.15

0.2

0.01 0.1 1 10 100

% M

eth

ylat

ed c

yto

sin

es


0

0.05

0.1

0.15

0.2

0.01 0.1 1 10 100

% M

eth

ylat

ed c

yto

sin

es


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

, %)


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



16/19

Specific methylation of genome (bisulfite sequencing)

▌ Decrease of F0 daphnid fecundity (exposed to IR)

Trijau et al., in prep



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



▌ 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:


19/19

Thank you !

Any question?


20/22

Key species

Radiosensitvity factors

Highlighting (or not) long term

effects

Sensitive and early

biomarkers

Fingerprints/signatures

« OMICS »

Application for the environmental protection


21/22

Arena et al 2014 What explains radiosensitivity

in plants ?


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)


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)


**

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

)



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



Arabidopsis thaliana - Phenotypical characterization

▌Number of leaves on the rosette, rosette diameter, inflorescence frequency, inflorescence height, amount of seed pods, seed weight, germination capacity.


▌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


25/22


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

,%)


**

*

Global hypermethylation of DNA in S1 and S2

What about field studies?


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