Protein expression patterns and activities of two metabolic enzymes (CS and LDH) highlight a disturbance in the metabolic pathways of tree frogs living in the Chernobyl Exclusion Zone
Résumé
While wildlife is chronically exposed to various sources and levels of ionizing radiation in the environment, studies regarding the long-term impact of long-lived radio-contaminant released in case of accident (137Cs, 90Sr, Pu), are still scarce, patchy, and sometimes contradictory1. Among the species relevant to understand and quantify the effects of radiological exposure on wildlife, amphibians such as the tree frog are sentinel organisms of choice2; they (i) have a highly permeable skin to pollutants, (ii) are exposed to pollutants via diversified habitats (aquatic and terrestrial), (iii) have a limited migration capacity, (iv) are among the reference animals listed by the International Commission on Radiological Protection (ICRP) to assess the toxic risk of ionizing radiation3.
In radiocontaminated areas immediately after the Chernobyl (nuclear power plant) and Kychtym (Maïak, reprocessing plant) accidents, the effects described on brown frogs (Rana arvalis) were a reduction in male fertility, egg size and egg laying3. Beyond this acute short term results, in more recent IRSN studies on the tree frog Hyla orientalis living in the Chornobyl Exclusion Zone (CEZ) for ~15 generations, analyses of genetic diversity and transcriptomics studies showed a high rate of mitochondrial mutations, a modulation of genes relative to energy metabolism, and, a small population size with a higher affiliation rate for those living in the most radio-contaminated areas4,5. This raises questions on the ability of populations to maintain themselves and on their health status.
In that way, to go further, proteomic analyses of frog muscles of the same individuals were performed after protein extraction and digestion, peptide separation and analysis via nanoHPLC (Ultimate 3000RSLC) and mass spectrometry (Orbitrap Fusion Lumos Tribrid - Thermo Fisher Scientific). In addition, the activity of two enzymes involved in the energetic metabolism, i.e. citrate synthase6 (aerobic metabolism) and lactate dehydrogenase7 (anaerobic metabolism), was measured. All results were analyzed in relation to individual dose rates.
Regarding proteomics, the full detected protein set was around 3600 proteins, whereas for data treatment the data sets used were either of 2370 proteins (i.e. 70% proteins with quantitative values for each site), or of 1700 proteins (i.e. 100% proteins with quantitative values, no missing data). Differential analysis between extrema conditions (lower vs higher dose rates) highlighted ~150 proteins oppositely modulated, that could correspond to a protein signature of radio-contamination present in the CEZ. highlighted differential signature and abundancy depending on the area of sampling (highly contaminated in the Chernobyl Exclusion Zone (CEZ) vs control area). Study of underlying biological pathways is in process and will be presented. Parallelly, dose-response modelling of proteomic data has been proceeded using DRomics8 on the full quantitative data set. Results showed 364 significant proteins successfully modelled over the dose gradient. GO enrichment highlighted four significant enriched pathways (FDR 5%) i.e. ‘Carboxylic acid metabolic process’, ‘Cellular lipid catabolic process’, ‘Fatty acid beta-oxidation’ and ‘Small molecule catabolic process’.
Regarding enzyme activities, preliminary results showed a tendency to modify the ratio between anaerobic and aerobic metabolisms.
All these results seem to confirm by two further approaches a disturbance in the metabolic pathways, notably the ones linked to fatty acids (β-oxidation, catabolism). Investigations will be carried on deciphering the biological pathways involved in long term effects of radiocontamination and conclude on CEZ tree frog health status.
Acknowledgements to G. Orizaola (Oviedo University, Spain), P. Burraco (Doñana Biological Station, Spain) for sampling campaign help; S. Gashack and Y. Gulyaichenko (Chornobyl Center for Nuclear Safety, Radioactive Waste and Radioecology, Ukraine) for sampling campaign help and sample radioactive activity measurements.
1 Beresford, N. A., N. Horemans, et al. (2020). Journal of Environmental Radioactivity 211.
2 Helbing, C. C. (2012). Frontiers in Genetics 3(MAR).
3 ICRP (2008). ICRP Publication 108. : 206.
4 Car, C., A. Gilles, et al. (2022). Evolutionary Applications 15(2): 203-219.
5 Car, C., A. Gilles, et al. (2023). BMC Biology 21(1).
6 Alp, P., Newsholme, E. et al. (1976) Biochem. J. 154, 689-700
7 Crabtree, B. and Newsholme, E. (1972) Biochem. J. 126, 49-58
8 Larras, F., E. Billoir, et al. (2018). Environmental Science and Technology 52(24): 14461-14468.
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