Transgenerational effects of parental exposure. Yuri E Dubrova

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1 Transgenerational effects of parental exposure Yuri E Dubrova yed2@le.ac.uk Department of Genetics University of Leicester, UK

2 Radiation-induced genomic instability 0.20 Aberrations/cell γ-irradiated control F Population doublings Are they unstable? F 1

3 Father Mother Let s go transgenerational 0.5 Gy of fission neutrons F 0 Expanded simple tandem repeat (ESTR) loci = microsatellite loci Paternal mutations F 1 F 2 Maternal mutation ESTR mutation rates were evaluated in the germline of: irradiated F 0 males = mutation induction non-exposed F 1 offspring = transgenerational instability From: Dubrova et al., 2000, Nature 405, 37

4 Transgenerational germline instability in the F 1 offspring of CBA/H male mice exposed to 0.5 Gy of fission neutrons 5.6-fold The non-exposed offspring of irradiated parents are unstable 4.5-fold

5 Is transgenerational instability strain-specific?

6 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 F 0 C57BL/6J F 1 F 1 F 2 F 2 F 3 F 3 From: Barber et al., 2002, PNAS 99,

7 Transgenerational instability in three inbred mouse strains Control F 1 F 2 ESTR mutation rates are elevated in both generations of all inbred strains 0.2 From: Barber et al., 2002, PNAS 99, ESTR mutation ra C57BL CBA/H BALB/c

8 Is transgenerational instability tissue-specific?

9 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, ; 2009, Mutat Res 664, 6-12

10 Is transgenerational instability specific for tandem repeat loci?

11 Transgenerational instability at the mouse hprt locus Chromosome aberrations in the F 1 offspring of irradiated rats 20, 95% CI A genome-wide destabilisation hprt is X-linked gene 0.4 Hprt mutation frequency x XY CBA/Ca BALB/c Frequency of chrmosome aberrations Hours after partial hepatectomy From: Barber et al., 2006, Oncogene 25, From: Vorobtsova, 2000, Mutagenesis 15, XY XY XX

12 Is transgenerational instability sex-specific?

13 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

14 Can paternal exposure to chemical mutagens destabilise the F 1 genomes?

15 Alkylating agent ethynitoesurea, ENU mostly base damage results in base substitutions ~ no ENU-induced DSBs Anticancer drug mitomycin C, MMC alkylated monoadducts & crosslinks base substitutions crosslinks can result in DSBs Anticancer drug cyclophosphamide, CPP alkylated monoadducts & crosslinks results in base substitutions crosslinks can result in DSBs after replication/repair Anticancer drug procarbazine, PCH alkylated monoadducts free radical species base substitutions & SSBs

16 ESTR instability in the F 1 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, Dubrova et al., 2008, Environ Mol Mutagen 49, Glen, Dubrova 2012, PNAS 109, 2984

17 Mechanisms

18 Initiation of an epigenetic instability signal in the directly exposed male germ cells F 0 F 1 Transmission of an epigenetic instability signal to the offspring & its manifestation

19 Endogenous DNA damage in controls & the F 1 offspring of irradiated males Single-strand DNA breaks Comet assay, bone marrow Double-strand DNA breaks γ-h2ax assay, spleen Control 0.9 Control Mean Comet tail, 95% CI 8 F fold CBA/Ca From: Barber et al., 2006, Oncogene 25, BALB/c 2-fold foci, 95% γ-h2ax CI Mean number F fold CBA/Ca 1.7-fold BALB/c

20 DNA repair in the F 1 offspring of irradiated males 40 CBA/Ca BALB/c The efficiency of DNA repair in the offspring 30 is not compromised Ex vivo exposure of bone marrow to X-rays, 10 Gy Alkaline Comet Tail DNA From: Barber et al., 2006, Oncogene 25, Time post-treatment, min

21 Oxidative DNA damage in the F 1 offspring (FPG Comet) Oxidative stress DNA damage: modified bases single-strand breaks double-strand breaks Control F 1 Hallmark: Accumulation of oxidatively damaged nucleotides in DNA CBA/Ca The efficiency of DNA in the F 1 offspring is OK No sign of oxidative stress in the F 1 offspring What else? Mean Comet tail, 95% CI BALB/c From: Barber et al., 2006, Oncogene 25,

22 The effects of paternal irradiation on F 1 gene expression 14 From: Gomes et al., Mutat Res 787, Probability, -log Dbp Dbp FDR < FDR 0.05 Per2 Per3 Nr1d2 Tef Mtf1 Ppard Lhx2 Nfil3 Arntl Arntl Arntl Npas2 Npas2 GO categories: GO: Rhythmic process, 6 genes P = 1.25 x 10-9 GO: Circadian rhythm, 5 genes P = 1.52 x GO: Regulation of transcription, P = 1.62 x 10 Mean fold-change, log -6 2 DNA-dependent, 11 genes

23 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

24 And what is transmitted?

25 From: Taudt et al., 2016, Nat Rev Genet 17, What can we expect to find?

26 Spermatogenesis in mice & humans Diploid spermatogonia Transcription Meiotic primary spermatocytes Meiotic secondary spermatocytes Post-meiotic spermatids X Sperm cells

27 Adult <1 week Sperm Instability?

28 Adult 3 weeks Spermatids Instability?

29 Adult 6 weeks Spermatogonia Instability?

30 in utero Primordial stem cells Instability?

31 5 CBA/H germline BALB/c germline BALB/c bone marrow 4 3 weeks 6 weeks Transgenerational effects manifest in the offspring regardless the stage of paternal irradiation 3 in utero 6 weeks 2 Ratio to control, s.e. From: Barber et al., 2002, PNAS 99, ; 2006, Oncogene 25, ; 2009, Mutat Res 664, 6-12; Hatch et al., 2007, Oncogene, 26, week 6 weeks -tids -gonia sperm -gonia in utero sperm -gonia in utero Stage of paternal irradiation 1 week in utero

32 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,

33 And so what?

34 Transgenerational effects in the children of irradiated parents Childhood cancer survivors Chernobyl clean-up workers survivors partners children control families irradiated families Stable? Unstable? Stable? From: Tawn et al., 2005, Mutat Res 523, ; Aghajanyan & Suskov, 2009, Mutat Res 523, 52-7

35 From mice to humans... Experiment one: Male mice exposed to 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 Procarbazine Sperm, brain, bone marrow

36 Paternal exposure to acute & chronic γ-rays Paternal exposure to anticancer drugs Instability may not exist among the children of irradiated cancer patients sperm brain sperm bone marrow The children of cancer patients treated by these drugs may be unstable Ratio to control, s.e acute chronic Paternal dose, cgy Ratio to control, s.e mg/kg 5 mg/kg 50 mg/kg Cyclophosphamide Mitomycin C Procacbazine From: Mughal et al., 2012, PLoS ONE 7, e41300 Dose per single radiotherapy procedure From: Glen & Dubrova, 2012, PNAS 109, 2984 Doses per single chemotherapy procedure

37 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!

38 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