Like father like son – transgenerational effects of parental exposure to mutagens
Yuri E Dubrova
Department of Genetics University of Leicester, UK The way we perceive the genetic risk
Random targeting DNA damage DNA repair Is it all or No ‘nasty’ there is Manifestation mutations something ‘Nasty’ induced else? mutation Radiation-induced genomic instability in somatic cells Yield of chromatid aberrations in MCF10A cells
Delayed mutations occur many cell divisions after exposure
γ-irradiated
control
From: Ullrich & Ponnaiya, 1998, Int J Radiat Biol 74, 747 What about the germline?
F0
F1
Are they stable or unstable? Let’s go transgenerational…
0.5 Gy of Expanded simple tandem fission neutrons repeat (ESTR) loci = microsatellite loci F0 Paternal mutations Mother
F1 FatherFa
Maternal mutation
F2
ESTR mutation rates were evaluated in the germline of:
‹ irradiated F0 males = mutation induction ‹ non-exposed F1 offspring = transgenerational instability
From: Dubrova et al., 2000, Nature 405, 37 Transgenerational germline instability in the F1 offspring of CBA/H male mice exposed to 0.5 Gy of fission neutrons
The non-exposed offspring of irradiated parents are unstable
5.6-fold
4.5-fold
From: Dubrova et al., 2000, Nature 405, 37 Is transgenerational instability strain-specific? CBA/H Fission neutrons, 0.4 Gy: CBA/H; C57BL/6 Acute X-rays, 2 Gy: CBA/H Acute X-rays, 1 Gy: BALB/c
BALB/c F0 ♂ ♀
C57BL/6J
F1 F1
F2 F2
F3 F3
From: Barber et al., 2002, PNAS 99, 6877-82 Transgenerational instability in three inbred mouse strains
Control F 1 ESTR mutation rates are elevated in F2 both generations of all inbred strains
From: Barber et al., 2002, PNAS 99, 6877-82 Is transgenerational instability tissue-specific? Transgenerational instability in the germline & somatic tissues
BALB/c CBA/Ca
ESTR mutation rates are equally elevated in the germline & somatic tissues
Early developmental onset of instability
From: Barber et al., 2006, Oncogene 25, 7336-42; 2009, Mutat Res 664, 6-12 Is transgenerational instability specific for tandem repeat loci? Transgenerational instability Chromosome aberrations in the
at the mouse hprt locus F1 offspring of irradiated rats
F1 of irradiated males Control 0.6 20 , 95% CI
-6 F1 A genome-wide destabilisation 15 0.5 3.3-fold 10 0.4
hprt is X-linked geneControl
5 of equency chrmosome aberrations mutation frequency x 10 mutation 3.7-fold Freq 0.3 Hprt mu Hprt 0 24 30 33 36 39 42 48 CBA/CaXY BALB/c XXHours after partial hepatectomy From: Barber et al., 2006, Oncogene 25, 7336-42 ♂ From: Vorobtsova,♀ 2000, Mutagenesis 15, 33-38
♂♀ ♂ ♀ XY XY For how long can an instability signal survive in the paternal germline? Adult <1 week ♂ ♀
Sperm
Instability? Adult 3 weeks ♂ ♀
Spermatids
Instability? Adult 6 weeks ♂ ♀
Spermatogonia
Instability? in utero
♂ ♀
Primordial stem cells
Instability? CBA/H BALB/c BALB/c 5 germline germline bone marrow
6 weeks Transgenerational effects manifest 4 in the offspring regardless 3 weeks the stage of paternal irradiation
3 6 weeks in utero to control, s.e. 1 week
Ratio to 1 week 2
6 weeks in utero 1
-tids -gonia sperm -gonia in utero sperm -gonia in utero Stage of paternal irradiation From: Barber et al., 2002, PNAS 99, 6877-82; 2006, Oncogene 25, 7336-42; 2009, Mutat Res 664, 6-12; Hatch et al., 2007, Oncogene, 26, 4720-4 Is transgenerational instability sex-specific? The offspring of irradiated females are stable Irradiated in utero Adult irradiation
From: Barber et al., 2009, Mutat Res 664, 6-12; Abouzeid Ali et al., 2012, Mutat Res 732, 21-5 Can paternal exposure to chemical mutagens destabilise
the F1 genomes? Radiation-induced damage to eukaryotic DNA (1 Gy of X-rays) ‹Base damage 2000 ‹ Single-strand breaks 1000 ‹ DNA-protein links 150 ‹ Double-strand breaks 40 ‹ Bulky lesions 40 ‹ Total 3300
From: Frankenberg-Schwager, 1990, Radiat Environ Biophys 29, 273-292
It has been suggested that radiation-induced double-strand breaks may represent one of the signals that can initiate the onset of the delayed genomic instability Alkylating agent ethynitoesurea, ENU ‹mostly base damage Anticancer drug mitomycin C, MMC ‹results in base substitutions ‹alkylated monoadducts & crosslinks ‹~ no ENU-induced DSBs ‹base substitutions ‹crosslinks can result in DSBs
Anticancer drug procarbazine, PCH Anticancer drug cyclophosphamide, CPP ‹alkylated monoadducts ‹alkylated monoadducts & crosslinks ‹free radical species ‹results in base substitutions ‹base substitutions & SSBs ‹crosslinks can result in DSBs after replication/repair ESTR instability in the F1 offspring of mutagen-treated male mice
sperm bone marrow
Instability signal is initiated by a generalised DNA damage
From: Barber et al., 2002, PNAS 99, 6877-82 Dubrova et al., 2008, Environ Mol Mutagen 49, 308-11 Glen, Dubrova 2012, PNAS 109, 2984 Mechanisms Some back of the envelope exercises…
‹~ 1,000 genes are involved in maintaining genome stability in mammals (DNA repair, apoptosis, cell cycle arrest etc)
‹ max spontaneous mutation rate 10-6 per locus
‹ exposure to 1 Gy of X-rays results in ~ a 3-fold increase in mutation rate
‹ if ANY radiation-induced mutation at ANY of 1,000 genes is DOMINANT and can substantially compromise the genome stability, then -6 1000 x 3 x 10 = 0.3% of the F1 offspring should be unstable
‹ according to our data ~100% of the F1 offspring of of irradiated males are unstable ♂ Initiation of an epigenetic instability signal in the directly exposed male germ cells
F1
Transmission of an epigenetic instability signal to the offspring & its manifestation Endogenous DNA damage in controls
& the F1 offspring of irradiated males Single-strand DNA breaks Double-strand DNA breaks Comet assay, bone marrow γ-H2AX assay, spleen
11 1.1
10 Control 1.0 Control 0.9 9 CI F 95% CI F 1 0.8 1
8 foci, 95 1.9-fold 0.7 7
-H2AX 1.7-fold 2-fold γ 0.6 6 0.5 5 2.3-fold Mean Comet tail, 95% C 0.4
4 Mean number 0.3
3 0.2 CBA/Ca BALB/c CBA/Ca BALB/c
From: Barber et al., 2006, Oncogene 25, 7336-42 DNA repair in the F1 offspring of irradiated males CBA/Ca BALB/c
Control F 1 ‹ Ex vivo exposure of bone marrow to X-rays, 10 Gy ‹ Alkaline Comet
The efficiency of DNA repair in the offspring is not compromised
From: Barber et al., 2006, Oncogene 25, 7336-42 Oxidative DNA damage
in the F1 offspring (FPG Comet) Oxidative stress
Control DNA damage: F ‹ modified bases 1 ‹ single-strand breaks ‹ double-strand breaks
Hallmark: Accumulation of oxidatively damaged nucleotides in DNA
‹ The efficiency of DNA in the F1 offspring is OK ‹ No sign of oxidative stress in the F1 offspring ‹ What else?
From: Barber et al., 2006, Oncogene 25, 7336-42 Transcriptome analysis of transgenerational effects 1 Gy of acute X-rays 1 Gy of acute X-rays
F0 F0 ♂ ♀ ♂ ♀
CBA/Ca BALB/c
F F ♂ 1 ♀ ♂ 1 ♀
Kidney NimbleGen 12x135K expression arrays: ‹135,000 probes per array; Liver 45-60mer long RNA ‹ Complete coverage of the mouse Spleen extraction transcriptome (42,576 transcripts) ‹ 3-4 probes per transcript Brain ‹ 12 arrays per slide
From: Gomes et al., submitted The effects of paternal irradiation on F1 gene expression
14
Dbp 12 Dbp 56 transcripts @ FDR 0.05 Per2 10
1010 Arntl Npas2 8 Arntl Npas2 Per3 Tef Mtf1 Arntl bility, bility, bility, -log-log 6 Ppard Nr1d2 ProbabilProbabil FDR < .05 Lhx2Nfil3 4
2 GO categories: -9 0 GO:0048511 Rhythmic process, 6 genes P = 1.25 x 10 -7 -1.2GO:0007623 -0.8 Circadian -0.4 rhythm, 0.05 genes 0.4P = 1.52 0.8x 10 1.2 GO:0006355 Regulation of transcription, P = 1.62 x 10-6 Mean fold-change, log DNA-dependent, 11 genes 2 From: Gomes et al., submitted Circadian trascriptome & circadian metabolism in mice
Circadian transcriptional regulators in liver Circadian transcripts in mouse liver
Among them those involved in: ‹ DNA replication, recombination & repair ‹ cell division & cell death
From: Koike et al., 2012, Science 338, 349; Akhtar et al., 2002, Curr Biol 12, 540; Maywood et al., 2007, Cold Spring Harb Symp Quant Biol 72, 85 And so what? Transgenerational effects in the children of irradiated parents
Childhood cancer survivors
survivors partners children Stable?
Chernobyl clean-up workers
control families Stable? irradiated families
Unstable?
From: Tawn et al., 2005, Mutat Res 523, 198-206; Aghajanyan & Suskov, 2009, Mutat Res 523, 52-7 From mice to humans and environment....
♂ ♀
Male mice exposed to 10 – 100 cGy acute γ-rays @ 50 cGy min-1 or 100 cGy chronic γ-rays @ 0.005 cGy min-1
Sperm, brain Paternal4.0 exposure to acute & chronic γ-rays
3.5 Instability signal is triggered by a stress-like response in irradiated cells 3.0 brain
2.5 Instability may not exist among the sperm children of irradiated cancer patients 2.0 Environmental exposure: 1.5 Ratioto control, s.e. ‹ mostly chronic 1.0 ‹ relatively low-dose 0.5 acute chronic Transgenerational instability may 10 25 50 100 100 Paternal dose, cGy not exist in many radioactively
From: Mughal et al., 2012, PLoS ONE 7, e41300 contaminated habitats?
However, the results of our study Dose per single Environmentaldescribe the effects of external radiotherapy exposurelow-LET paternal irradiation. procedure What about high-LET internal exposure from Pu etc.? The transgenerational effects of non-mutagenic substances
For example, diet The grandparental effects of poor food supply in mid-childhood on longevity of their grandchildren Paternal grandmothers→male probands Paternal grandmothers→female probands
good food availability poor food availability
Paternal grandfathers→female probands Paternal grandfathers→male probands
good food availability poor food availability
From: Kaati et al., 2007, Eur J Hum Genet 15, 784-90 Transgenerational reprogramming of gene expression in mice
Male mice fed control & low-protein diet (11%)
The offspring were fed control diet
Microarray analysis of RNA samples from liver
445 up- & down- regulated genes in the offspring
From: Carone et al., 2010, Cell 143, 1084-96 No transgenerational effects of methyl-deficient diet in mice
Treated F0 males Non-treated F1 offspring
From: Glen et al., 2015, Mol Nutr Food Res in press Conclusions ‹ High-dose acute paternal exposure to a number of mutagens can significantly destabilise the genomes of their offspring ‹ Transgenerational instability is a genome-wide phenomenon which affects the frequency of chromosome aberrations and gene mutations ‹ Transgenerational instability is triggered in the directly exposed germ cells by a stress-like response to a generalised DNA damage ‹ Transgenerational instability is attributed to the presence of a persistent subset of endogenous DNA lesions ‹ Transgenerational instability is attributed to the epigenetic changes affecting the expression of a subset of genes, involved in rhythmic process & regulation of transcription ‹ Transgenerational instability may represent an important component of the long-term genetic risk of human exposure to mutagens, but we need HUMAN data to prove it! Acknowledgements ‹ Dubrova’s lab Ruth Barber Colin Glen Safeer Mughal Andre Gomes Robert Hardwick Carole Yauk Mariel Voutounou Tim Hatch Dominic Kelly Peter Hickenbotham Morag Shanks Carles Vilarino-Guell Karen Monger Bruno Gutierrez Karen Burr Julia Brown Natalya Topchiy Isabelle Roux Peter Black Demetria Pavlou Hamdy Ali Abouzeid Adeolu Adewoye ‹ MRC Radiation and Genome Stability Unit, Harwell, UK Mark Plumb Emma Boulton Jan Fennelly Dudley Goodhead ‹ Department of Cancer Studies and Molecular Medicine, University of Leicester, UK George “Don” Jones Gabriela Almeida Comet Assay ‹ NI Vavilov Institute of General Genetics, Moscow, Russia Chronic irradiation Alexander Rubanovich Andrey Myazin ‹ Medical Radiological Research Centre, Obninsk, Russia Chronic irradiation Leonid Zhavoronkov Yuri Semin Albert Brovin Valentina Glushakova Valentina Posadskaya Olga Izmet’eva ‹ MRC Toxicology Unit, Leicester, UK Anticancer drugs Andy Smith ‹ Centre for Molecular Genetics and Toxicology, University of Wales, Swansea, UK George Johnson James Parry Hprt assay ‹ Catholic University of Nijmegen, The Netherlands Peter de Boer Alwin Derijck Godfried van der Heijden Sperm irradiation ‹ Gray Cancer Institute, Northwood, UK γH2AX assay Kai Rothkamm The EMF Biological Research Trust