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Institut de Radioprotection et de Sûreté Nucléaire,

Inhibition of DNA double strand break repair by uranium and links with effects on reproduction of the zebrafish. C. Adam-Guillermin , S. Pereira, S. Bourrachot, S. Barillet, I. Cavalié, V. Camilleri, M. Floriani, J. Garnier-Laplace. Institut de Radioprotection et de Sûreté Nucléaire,

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Institut de Radioprotection et de Sûreté Nucléaire,

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  1. Inhibition of DNA double strand break repair by uranium and links with effects on reproduction of the zebrafish C. Adam-Guillermin, S. Pereira, S. Bourrachot, S. Barillet, I. Cavalié, V. Camilleri, M. Floriani, J. Garnier-Laplace Institut de Radioprotection et de Sûreté Nucléaire, Laboratoire d’ECOtoxicologie des radionucléides (LECO), Cadarache, France

  2. nuclear fuel cycle, military, industrial & agricultural applications Uranium Contamination of aquatic ecosystems Background [U] surface water : ~ ng/L to 6 µg/L Vicinity of former uranium mines : ~ tens – 100 µg/L U drinking water guideline : 15 µg/L WHO Environmental Quality Standards range from 1 µg/L to 100 µg/L C. Adam, IRPA13, Glasgow, May 16th, 2012 2/14

  3. Biological endpoints Ecological relevance Ecosystem • Genotoxicity • DNA is the 1st cellular target for radionuclides • DNA damages in : • somatic cells : carcinogenesis • germ cells : teratogenesis and reprotoxicity Population Organism Tissue • Objective • Assess genotoxicity markers as predictive biomarkers for U effects on individuals or populations Cell Molecule Mechanisms 3/14 2

  4. 32¢ Zebrafish • Easy breeding (mature in 3 month) • Fully sequenced genome v v • Model for developmental studies C. Adam, IRPA13, Glasgow, May 16th, 2012 4/14

  5. 50 12 10 40 8 30 Mean DNA tail (%) 6 20 4 10 2 0 control 250 µg/L 20 µg/L 0 male female control 20 µg/L 250 µg/L DNA damages and reproduction Zebrafish exposure to uranium (20 days) and reproduction Comet assay (gonads after 20 d exposure) Bourrachot, 2009 Spawning events per female for 16 days Significant increase of DNA damage in males and females gonads Decrease of fecundity & of egg and larvae viability C. Adam, IRPA13, Glasgow, May 16th, 2012 5/14

  6. Reversible DNA damages ? Comet assay in male gonad cells Exposure of zebrafish to 20 µg/L of uranium during 27 days U depuration during 28 days U accumulation U depuration ** ** ** * Mean DNA tail (%) Time (d) * p<0.05 ** p<0.01 control exposed C.Adam, 2012 • DNA damages are still observed after 28 days of depuration  occurence of DNA Double Strand breaks (DSBs) ? correlation with the decrease of fertility ? C. Adam, IRPA13, Glasgow, May 16th, 2012 6/14

  7. Does U induce DNA DSBs in zebrafish? DNApK P P P DSB detection DSB repair Dose-response experiments Time-response Experiments ATM P P Cytogenetic abnormalities MRE11 dependant recombination Pathway MRE11 Micronuclei number Cells in G0/G1 DNA Double Strand Breaks H2AX DNA repair kinetics Number of foci at 10min, 1h,2h, 4h, 24h Ku70 Ku80 DNApK NHEJ repair Pathway C. Adam, IRPA13, Glasgow, May 16th, 2012 7/14

  8. ZF4 cells (embryonic fibroblasts) Uranium induces DSB in ZF4 cells 5 µm Pereira et al, Aquatic Toxicology, 2011 • No cytotoxicity up to 500 µM • Increase of the nb of -H2AX foci per cell up to 100 µM C. Adam, IRPA13, Glasgow, May 16th, 2012 8/14

  9. Occurrence of SSBs concomitant to DBSs formation Alkaline comet assay Pereira et al, Aquatic Toxicology, 2011 C. Adam, IRPA13, Glasgow, May 16th, 2012 9/14

  10. Genotoxicity of uranium on ZF4 cells Increasing of U precipitates with the decrease of DSBs U U Pereira et al, AquaticToxicology, 2011 C. Adam, IRPA13, Glasgow, May 16th, 2012 10/14

  11. Genotoxicity of uranium on ZF4 cells Urchins-like thin needle-shaped uranium structures, mainly concentrated in lyzosome-like vesicles M. Floriani Pereira et al, Aquatic Toxicology, 2011 C. Adam, IRPA13, Glasgow, May 16th, 2012 11/14

  12. Unrepaired DBSs at low U concentrations Pereira et al, Aquatic Toxicology, 2011 NHEJ repair was disrupted in uranium contaminated ZF4 cells: no DNApK foci was observed after 24 h of 10 µM uranium exposure C. Adam, IRPA13, Glasgow, May 16th, 2012 12/14

  13. These damages are probably linked to U reprotoxicity (similar internal [U] concentrations for in vivo & in vitro studies) UO2 100 µg/L ZF4 cells ♂ gonad cells H2AX Conclusions • Uranium induces DNA SSBs, DSBs and micronuclei • Uranium detoxication in lyzosome-like structures may explain the low cytotoxicity (< 10 %) • Inhibition of NHEJ (DNA PK) complemented by another non specific DNA repair mechanism ? C. Adam, IRPA13, Glasgow, May 16th, 2012 13/14

  14. Thank you for your attention ANR HEMI-BREAKS Envirhom project christelle.adam-guillermin@irsn.fr 14 C. Adam, IRPA13, Glasgow, May 16th, 2012 14/14

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