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Submitted on 16 May 2020
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Genomic cytosine methylation level: a molecular marker of stress in the species Gammarus fossarum?
Pauline Cribiu, Arnaud Chaumot, Geffard Olivier, J.L. Ravanat, T. Bastide, Sylvie Bony, Alain Devaux
To cite this version:
Pauline Cribiu, Arnaud Chaumot, Geffard Olivier, J.L. Ravanat, T. Bastide, et al.. Genomic cytosine methylation level: a molecular marker of stress in the species Gammarus fossarum?. SETAC Europe 28th annual meeting, May 2018, Rome, Italy. pp.1, 2018. �hal-02608378�
Materials and methods
Genomic cytosine methylation level: a molecular marker of stress in
the species Gammarus fossarum ?
Pauline Cribiu
1, Arnaud Chaumot
2, Olivier Geffard
2, Jean-Luc Ravanat
3, Thérèse Bastide
1, Alain
Devaux
1, Sylvie Bony
11ENTPE, INRA, CNRS UMR 5023 LEHNA, rue Maurice Audin 69518 Vaulx-en-Velin CEDEX, France
2IRSTEA- Groupement de Lyon, unité de recherche RiverLy, Laboratoire d’écotoxicologie, 5 rue de la Doua 69626 Villeurbanne CEDEX, France 3CEA , LAN, 17 rue des martyrs 38054 Grenoble CEDEX 9, France
Introduction and objectives
In the context of global change, understanding species response to environmental stressors is a major issue.
In the last decades, epigenetic modifications of genomic DNA have been highlighted as an environment-sensitive pathway,
reversible and potentially transmitted between generations.
The aim of this study is to explore natural and environment-induced modulations of the global genomic cytosine methylation levels in the ecologically relevant freshwater species Gammarus fossarum following 3 steps:
① Optimization of a DNA extraction protocol & assessment of the natural variability of the genomic cytosine methylation level ② Measurement of effect of two environmental stressors i.e. temperature and starvation
③ Measurement of effect of a chemical stressor i.e cadmium
Results and discussion
Conclusions
① Natural variability of the total genomic cytosine
methylation level in Gammarus fossarum
Total genomic cytosine methylation level is in average 0.49% (±0.08)
Lower than in vertebrates (5-10%) In the same range as in most
other invertebrates (0.1-2%).
No influence of sex
Adult gammarids > juveniles.
② Effect of temperature and starvation
Temperature
Gammarids from Cress population exposed in the laboratory
1 month starvation results in a higher DNA methylation level.
No effect for a shorter duration (14d).
In gammarids from various reference sites, DNA methylation level varies from 0.59% to 1.38%.
Total genomic DNA methylation is sensitive to natural and chemical stresses but taking into account the kinetics of response to environmental stress seems to be
important to interpret methylation level as a marker of stress.
Further field experiments will be carried out in order to investigate the possible application of genomic cytosine methylation level as a stress marker.
In complement, transgenerational experiments after a parental exposure are currently in progress for studying the potential transfer of this molecular mark of stress
through the offspring.
1 mm
Acknowledgments to Sylvain Caillat2, Nicolas Delorme3, Adeline François3
DNA extraction
QIAGEN Dneasy® Blood & Tissue Kit
One gammarid per sample Mechanical disruption Chemical lysis (proteinase K) at 56°C during 3h DNA adsorption onto silica Silica membrane gammarid lysate Elution column Piston Pellet Eppendorf® Grinder Washing & DNA elution
DNA methylation analysis
DNA extract
Enzymatic
digestion DNA strands Free nucleosides HPLC/MS-MS
% DNA methylation :
5-methylcytosines / total cytosines
Ionizer Analyzer Detector Processing electrical signals Triple quadrupole
analyzer spectrumMass
Gammarids exposed to thermic stress (18°C) for 1 month exhibit higher DNA methylation level than those exposed to temperatures encountered in the river.
No effect for a shorter duration (14d).
③ Effect of a chemical stress: cadmium
Starvation Basal level in gammarids collected in an unpolluted site
(Cress population)
High variability in DNA methylation level among reference sites ranging between 0.59%
to 1.38% Inter-population variability
Gammarids collected at 4 unpolluted sites showing different physico-chemical characteristics
Optimal water temperatures Optimal water
temperatures
After 14 days of Cd exposure, gammarids show a lower DNA methylation level than in control.
After 1 month of Cd exposure a non significant
hypermethylation trend occurs.
Similar modulations were observed in rat liver (Takiguchi et al., 2003), human cells (Huang et al., 2008; Jiang et al., 2008) and gastropod hepatopancreas (Nica et al., 2017).
Mechanistic hypothesis (Takiguchi et al., 2003)
DNA methyltransferase inhibition due to Cd early hypomethylation
Then, compensatory effect after a long Cd exposure time late
hypermethylation
References :
Huang D, Zhang Y, Qi Y, Chen C, Ji W. 2008. Global DNA hypomethylation, rather than reactive oxygen species (ROS), a potential facilitator of cadmium-stimulated K562 cell proliferation. Toxicology letters 179 : 43 – 47. Jiang G, Xu L, Song S, Zhu C, Wu Q, Zhang L, Wu L. Effects of long-term low-dose cadmium exposure on genomic DNA methylation in human embryo lung fibroblast cells. Toxicology 244: 49-55.
Nica DV, Popescu C, Draghici GA, Andrica FM, Privistirescu LA, Gergen II, Stöger R. 2017. High-level dietary cadium exposure is associated with global DNA hypermethylation in the gastropod hepatopancreas. PLoS ONE 12(9) : e0184221.
Takiguchi M, Achanzar WE, QU W, Li G, Waalkes MP. 2003. Effects of cadmium on DNA-(Cytosine-5) methyltransferase activity and DNA methylation status during cadmium-induced cellular transformation. Experimental Cell Research 286 : 355 – 365.
