Transgenerational effects of parental exposure
Yuri E Dubrova
Department of Genetics University of Leicester, UK Radiation-induced genomic instability
0.20
0.15
0.10 γ-irradiated
Aberrations/cell F0 0.05 control
0.00 5 10 15 20 25 30 35 40 Population doublings
Are they unstable? F1 Let’s go transgenerational…
0.5 Gy of fission neutrons Expanded simple tandem repeat (ESTR) loci F0 = microsatellite loci
Paternal
mutations
Mother
F1 Father
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 Is transgenerational instability strain-specific? Fission neutrons, 0.4 Gy: CBA/H; C57BL/6 Acute X-rays, 2 Gy: CBA/H CBA/H Acute X-rays, 1 Gy: BALB/c
F0 BALB/c ♂ ♀
F F C57BL/6J 1 1
F2 F2
F3 F3
From: Barber et al., 2002, PNAS 99, 6877-82 Transgenerational instability in three inbred mouse strains
Control 0.4 F1 F 2 ESTR mutation rates are elevated in 0.3 both generations of all inbred strains
0.2
ESTR mutation rate, 95% ESTRCI rate, mutation 0.1
0.0 C57BL CBA/H BALB/c 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
0.6
20 A genome-wide destabilisation
, 95% CI 95% , -6
15 0.5
10 hprt is X-linked 0.4gene
5
Frequency of chrmosome aberrations chrmosome Frequencyof 0.3
Hprtmutation10 x frequency XY XX 0 ♂ ♀ 24 30 33 36 39 42 48 CBA/Ca BALB/c Hours after partial hepatectomy
From: Barber et al., 2006, Oncogene 25, 7336-42 From: Vorobtsova, 2000, Mutagenesis 15, 33-38
♂ ♀ ♂ ♀ XY XY 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? Alkylating agent ethynitoesurea, ENU mostly base damage results in base substitutions Anticancer drug mitomycin C, MMC ~ no ENU-induced DSBs alkylated monoadducts & crosslinks 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 Initiation of an epigenetic ♂♂ instability signal in the directly exposed male germ cells F0
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 1.0
9 Control 0.9 Control 0.8
8 F1 CI 95% foci, F1 0.7 7 1.9-fold 1.7-fold 2-fold γ-H2AX 0.6 6 0.5 2.3-fold 5 Mean Comet tail, 95% CI 95% tail, Comet Mean 0.4
4 number Mean 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 40
Ex vivo exposure of bone The efficiency of DNA repair in marrow to X-rays, 10 Gy the offspring30 is not compromised Alkaline Comet
20 Tail DNATail
10
0
0 20 40 60 0 20 40 60
From: Barber et al., 2006, Oncogene 25, 7336-42 Time post-treatment, min Oxidative DNA damage in the F1 offspring (FPG Comet) Oxidative stress 2.4 Control 2.2 DNA damage: F1 modified bases 2.0 single-strand breaks double-strand breaks 1.8
Hallmark: 1.6
Accumulation of CI 95% tail, Comet Mean oxidatively damaged 1.4 nucleotides in DNA 1.2 CBA/Ca BALB/c
From: Barber et al., 2006, Oncogene 25, 7336-42 The efficiency of DNA in the F1 offspring is OK No sign of oxidative stress in the F1 offspring What else? The effects of paternal irradiation on F1 gene expression
14
Dbp 56 transcripts @ FDR 0.05 12 Dbp Per2
10 10 10 Arntl Npas2 8 Arntl Npas2 Per3 Tef Mtf1 Arntl 6 Ppard
Nr1d2 Probability, -log Probability, Probability, -log Probability, 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 0.4 P = 1.52 0.8x 10 1.2 GO:0006355 Regulation of transcription, P = 1.62 x 10-6 Mean fold-change, log2 From: Gomes et al., Mutat Res 787, 33-37 DNA-dependent, 11 genes 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 what is transmitted? What can we expect to find?
From: Taudt et al., 2016, Nat Rev Genet 17, 319-332 Spermatogenesis in mice & humans
Transcription Diploid spermatogonia
Meiotic primary spermatocytes
Meiotic secondary spermatocytes
Post-meiotic spermatids
SpermX cells 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 1 week
Ratio to control, s.e. control, to Ratio 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 Potential transgenerational mechanisms involving sperm RNAs
Extacellular vesicles, Evs may be regarded as the transgenerational messenger
From: Chen et al., 2016, Nat Rev Genet 17, 733-743 And so what? Transgenerational effects in the children of irradiated parents
Childhood cancer survivors Chernobyl clean-up workers
survivors control families partners irradiated families children
Stable? Unstable?
Stable?
From: Tawn et al., 2005, Mutat Res 523, 198-206; Aghajanyan & Suskov, 2009, Mutat Res 523, 52-7 From mice to humans....
Experiment one: Male mice exposed to 10 – 100 cGy acute γ-rays ♂ ♀ or 100 cGy chronic γ-rays
Experiment two: Male mice exposed to clinically-relevant doses of 3 anticancer drugs: Cyclophosphamide Mitomycin C Sperm, brain, bone marrow Procarbazine Paternal exposure to acute & chronic γ-rays Paternal exposure to anticancer drugs
4.0 2.5 Instability may not exist among the 3.5 sperm children of irradiated cancer patients 3.0 brain bone 2.5 2.0 The children of cancermarrow patients sperm treated by these drugs may be unstable 2.0
1.5
Ratio to control, s.e. control, to Ratio 1.5 Ratio to control, s.e. control, to Ratio 1.0 150 mg/kg 5 mg/kg 50 mg/kg 0.5 acute chronic 1.0 10 25 50 100 100 Paternal dose, cGy Cyclophosphamide Mitomycin C Procacbazine
From: Mughal et al., 2012, PLoS ONE 7, e41300 From: Glen & Dubrova, 2012, PNAS 109, 2984 Doses per single chemotherapy procedure Dose per single radiotherapy procedure 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 Hamdy Ali Abouzeid Dominic Kelly Robert Hardwick Mariel Voutounou Carole Yauk Tim Hatch Peter Hickenbotham Morag Shanks Carles Vilarino-Guell Bruno Gutierrez Isabelle Roux Karen Burr Karen Monger Julia Brown Natalya Topchiy Peter Black Demetria Pavlou 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