HAL Id: hal-02606964
https://hal.inrae.fr/hal-02606964
Submitted on 16 May 2020
HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-entific research documents, whether they are pub-lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.
L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.
Effects of biotic and abiotic factors on the DNA methylation status in Gammarus fossarum
Pauline Cribiu, J.L. Ravanat, Arnaud Chaumot, Geffard Olivier, Sylvie Bony, Alain Devaux
To cite this version:
Pauline Cribiu, J.L. Ravanat, Arnaud Chaumot, Geffard Olivier, Sylvie Bony, et al.. Effects of biotic and abiotic factors on the DNA methylation status in Gammarus fossarum. 18th International Symposium on Toxicity Assessment (ISTA), Jul 2017, Limeira, Brazil. 2017. �hal-02606964�
Effects of biotic and abiotic factors on the DNA methylation status
in Gammarus fossarum
Pauline Cribiu
1, Jean-Luc Ravanat
3, Arnaud Chaumot
2, Olivier Geffard
2, Sylvie Bony
1, Alain Devaux
1*
1ENTPE, INRA, CNRS UMR 5023 LEHNA, rue Maurice Audin 69518 Vaulx-en-Velin CEDEX, France
2IRSTEA- Groupement de Lyon, unité de recherche MAEP, 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
The interest of epigenetic regulation is becoming increasingly important in ecotoxicology. Among epigenetic marks, DNA
methylation could be a biomarker of interest.
The aim of this study is to investigate the measurement of genomic DNA methylation level as a possible stress
biomarker in the ecologically relevant freshwater species Gammarus fossarum.
For this, 3 steps were followed : ① Optimization of a DNA extraction protocol and evaluation of a basal level of genomic
methylation in Gammarus fossarum.
② Measurement of the effect of natural factors on the genomic DNA methylation level.
③ Study of this epigenetic biomarker response to a chemical stress.
Materials and methods
Results and discussion
Conclusion
① Basal level of DNA methylation
in Gammarus fossarum
Genomic DNA methylation in
Gammarus fossarum ranges
between 0.38% and 0.61%
Lower than in vertebrates (5-10%)
but in the same range as in other
invertebrates (0.1-2%).
No influence of sex :
♀
=♂
. Adult gammarids > juveniles.
Thanks to the high sensitivity of
HPLC/MS-MS method, subtle
variations in DNA methylation can be measured.
② Effects of natural factors on Genomic DNA methylation level
Water temperature
Gammarids sampled at reference unpolluted site exposed in laboratory
Gammarids exposed to 18°C for 1
month exhibit higher DNA
methylation than those exposed to lower temperatures.
No influence of temperature for
shorter exposure duration.
Food starvation
1 month starvation results in a higher DNA methylation level than in fed gammarids.
No effect of starvation for
shorter duration.
Origin of populations
Gammarids from the same genetic line collected at various reference sites showing different chemical and physical characteristics
High variability in DNA methylation level among reference sites
0.59% to 1.24%.
③ Response to various chemical stress
Gammarids sampled at a reference unpolluted station were caged in sites impacted by various human activitiesSite T°C
Conductivity
(μS.cm-1) pH Land use
Mandorne
(reference) 8.4°C 367 8.5 Natural forest St Jean
d’Ardières 7.7°C 238 8.1 Wineyard
Bourbre 9.2°C 589 8.2 Crop growing & urban Turdine 7.9°C 420 8.6 Urban & industrial
After 7 days and 1 month of caging, no significant effect of chemical stress on DNA methylation was shown.
In gammarids from various reference sites, DNA methylation varies largely from 0.38% to 1.24%.
The use of the genomic DNA methylation level as a contamination biomarker in Gammarus fossarum requires further investigations since :
DNA methylation was shown to be highly sensitive to natural factors such as temperature, starvation and life history.
Chemical stress had no effect on DNA methylation level after a one-month exposure in contaminated sites.
1 mm
Optimum
physiological T°C
Acknowledgments to Sylvain Caillat2, Nicolas Delorme3, Thérèse Bastide1, Adeline François3
DNA extraction
Kit QIAGEN Dneasy® Blood & Tissue
One Gammarid per sample
Mechanical lyse Chemical
lyse (proteinase k) at 56°C during 3h Centrifugations DNA adsorption onto silica Silica membrane Gammarid extract Elution column Piston Pellet Eppendorf® Grinder Wash steps DNA elution
DNA methylation analyze
DNA extract
Enzymatic
digestion DNA strands Free nucleotides Mass spectrometry
HPLC/MS-MS
% DNA methylation = The amount of methylated
Cytosines/The total amount of Cytosines (methylated and unmethylated)
Ionizer Analyzer Detector Processing electrical signals
Triple quadrupole
analyzer spectrum Mass
