MiR-378a-3p and miR-491-5p as markers of xenon abuse in doping control

Xenon stimulates the synthesis of the hormone erythropoietin, which leads to improved oxygen supply to tissues, increased endurance and can be used by athletes to gain an undue advantage in competitions. The World Anti-Doping Agency (WADA) banned its use. The determination of xenon in biological fluids, in particular, in blood plasma samples, is difficult due to the narrow detection window. Its indirect detection is possible by changing in some blood parameters during a clinical analysis (RET%, HGB, HCT, etc.), however, this analysis is nonspecific and the use of other erythropoiesis-stimulating agents can lead to similar changes.

Aims: The aim of the study was to search for long-term microRNA markers, the expression of which is specific and markedly altered by inhaled xenon.

Methods: Quantitative real-time PCR was performed on CFX96 Bio-Rad analyser using miRCURY® LNA® miRNA SYBR® Green PCR Kit and panels for studying the expression profiles of mature microRNAs of the hypoxia signaling pathway miRCURY LNA™ miRNA Focus Panel.

Results: Based on statistical data analysis, it was found that the expression of hsa-miR-378a-3p and hsa-miR-491-5p in blood plasma increases significantly (more than 70 times) when xenon inhalations are used as an erythropoiesis stimulator. Measurement of hematological parameters before and after inhalation showed no significant changes that could affect endurance or give competitive advantages.

Conclusion: The evaluated difference in microRNA expression levels before and after administration of the xenon mixture (Xe/O2) makes hsa-miR-378a-3p and hsa-miR-491-5p potential candidates for the role of long-term markers of xenon abuse.

1. Hou B., Li F., Ou S., Yang L., Zhou S. Comparison of recovery parameters for xenon versus other inhalation anesthetics: systematic review and meta-analysis. J. Clin. Anesth. 2016:29:65–74. https://doi.org/10.1016/j.jclinane.2015.10.018

2. Risberg J. Regional cerebral blood flow measurements by 133Xe-inhalation: methodology and applications in neuropsychology and psychiatry. Brain Lang. 1980;9(1):9–34. https://doi.org/10.1016/0093-934x(80)90069-3

3. Tzigankov B.D., Shamov S.A., Rykhletskiy P.Z., Davletov L.A. The possibilities of xenon application in complex therapy of psychopathologic disorders in patients of narcologic profile. Russian Medicine. 2013;(4):11–13 (In Russ.).

4. Brücken A., Coburn M., Rex S., Rossaint R., Fries M. Current developments in xenon research. Importance for anesthesia and intensive care medicine. Anaesthesist. 2010;59(10):883–895. https://doi.org/10.1007/s00101-010-1787-6

5. Tassel C., Le Dare B., Morel I., Gicquel T. Xenon: from rare gas to doping product. Presse Med. 2016;45(4):422–430. https://doi.org/10.1016/j.lpm.2016.01.025

6. Bukhtiyarov I.V., Kalmanov A.S., Kislyakov U.U., Nikiforov D.A., Christov S.D., Shvetskiy F.M., Bubeyev U.A. Studying of xenon adaptability within training process for functional state correction of sportsmen. Phys. Ther. Sports Med. 2010;78:22–29

7. Maze M., Laitio T. Neuroprotective Properties of Xenon. Mol. Neurobiol. 2020;57(1):118–124. https://doi.org/10.1007/s12035-019-01761-z

8. Goetzenich A., Hatam N., Preuss S., Moza A., Bleilevens C., Roehl A. B., Autschbach R., Bernhagen J., Stoppe C. The role of hypoxia-inducible factor-1α and vascular endothelial growth factor in late-phase preconditioning with xenon, isoflurane and levosimendan in rat cardiomyocytes. Interact. Cardiovasc. Thorac. Surg. 2014;18(3):321–328. https://doi.org/10.1093/icvts/ivt450

9. Stoppe C., Coburn M., Fahlenkamp A., Ney J., Kraemer S., Rossaint R., Goetzenich A. Elevated serum concentrations of erythropoietin after xenon anaesthesia in cardiac surgery: secondary analysis of a randomized controlled trial. Br. J. Anaesth. 2015;114:701–703. https://doi.org/10.1093/bja/aev060

10. Dias K.A., Lawley J.S., Gatterer H., Howden E.J., Sarma S., Cornwell 3rd W.K., et al. Effect of acute and chronic xenon inhalation on erythropoietin, hematological parameters, and athletic performance. J. Appl. Physiol. 2019;127(6):1503–1510. https://doi.org/110.1152/japplphysiol.00289.2019

11. Bezuglov E., Morgans R., Khalikov R., Bertholz V., Emanov A., Talibov O., Astakhov E., Lazarev A., Shoshorina M. Effect of xenon and argon inhalation on erythropoiesis and steroidogenesis: A systematic review. Heliyon. 2023;9(5):e15837. https://doi.org/10.1016/j.heliyon.2023.e15837

12. The 2023 List of prohibited substances and methods [internet]. Available at: https://www.wada-ama.org/en/resources/world-anti-doping-program/prohibited-list (accessed 19 July 2023).

13. Thevis M., Piper T., Geyer H., Thomas A., Schaefer M.S., Kienbaum P., Schänzer W. Measuring xenon in human plasma and blood by gas chromatography/mass spectrometry. Rapid Commun. Mass Spectrom. 2014;28(13):1501–1506. https://doi.org/10.1002/rcm.6926

14. Thevis M., Piper T., Geyer H., Schaefer M.S., Schneemann J., Kienbaum P., Schänzer W. Urine analysis concerning xenon for doping control purposes. Rapid Commun. Mass Spectrom. 2015;29(1):61–66. https://doi.org/10.1002/rcm.7080

15. Frampas C., Ney J., Coburn M., Augsburger M., Varlet V. Xenon detection in human blood: Analytical validation by accuracy profile and identification of critical storage parameters. J. Forensic Leg. Med. 2018;58:14–19. https://doi.org/10.1016/j.jflm.2018.04.005

16. Kwok W.H., Choi T.L.S., So P.-K., Yao Z.-P., Wan T.S.M. Simultaneous detection of xenon and krypton in equine plasma by gas chromatography-tandem mass spectrometry for doping control. Drug Test. Anal. 2017;9(2):317–322. https://doi.org/10.1002/dta.1971

17. Postnikov P.V., Ishutenko G.V., Polosin A.V., Potapov S.V., Zhovnerchuk E.V., Mochalova E.S. Study of a Possibility of the Direct Determination of Xenon in the Equilibrium Vapor Phase of Whole Blood and Plasma Samples by GC-MS/MS after Inhalation by Healthy Volunteers. Journal of Analytical Chemistry. 2022;77:1737–1743. https://doi.org/10.1134/s1061934822140064

18. Athlete biological passport (ABP) operating guidelines [internet]. Available at: https://www.wada-ama.org/en/resources/world-anti-doping-program/athlete-biological-passport-abp-operating-guidelines (accessed 19 July 2023).

19. Postnikov P.V., Efimova Yu.A., Pronina I.V. Circulating MicroRNAs as a New Class of Biomarkers of Physiological Reactions of the Organism to the Intake of Dietary Supplements and Drugs. Microrna. 2022;11(1):25–35. https://doi.org/10.2174/2211536611666220422123437

20. Postnikov P.V., Pronina I.V. Preanalytical features of the determination of circulating microRNAs as new specific biomarkers of the body’s response to physical activity. Sports medicine: research and practice. 2021;11(4):90–103 (In Russ.). https://doi.org/10.47529/2223-2524.2021.4.1

21. Weber J.A., Baxter D.H., Zhang S., Huang D.Y., Huang K.H., Lee M.J., Galas D.J., Wang K. The microRNA spectrum in 12 body fluids. Clin. Chem. 2010;56(11):1733–1741. https://doi.org/10.1373/clinchem.2010.147405

22. Declaration of Helsinki of the World medical association [internet]. Available at: http://acto-russia.org/index.php?option=com_content&task=view&id=21 (accessed 19 July 2023).

23. International Standard for Laboratories [internet]. Available at: https://www.wada-ama.org/en/resources/world-anti-doping-program/international-standard-laboratories-isl (accessed 21 July 2023).

24. Postnikov P.V., Radus F.V., Efimova Yu.A., Pronina I.V. Determination of possible microRNA-markers of cobalt abuse by real-time qPCR using hypoxia signaling pathway panels. Fine Chem. Tech. 2023;18(1):65–74. https://doi.org/10.32362/2410-6593-2023-18-1-65-74

25. Ma D., Lim T., Xu J., Tang H., Wan Y., Zhao H., Hossain M., Maxwell P.H., Maze M. Xenon preconditioning protects against renal ischemic-reperfusion injury via HIF-1alpha activation. J. Am. Soc. Nephrol. 2009;20(4):713–720 https://doi.org/10.1681/ASN.2008070712

26. Sessa F., Salerno M., Bertozzi G., Cipolloni L., Messina G., Aromatario M., Polo L., Turillazzi E., Pomara C. miRNAs as Novel Biomarkers of Chronic Kidney Injury in Anabolic-Androgenic Steroid Users: An Experimental Study. Front. Pharmacol. 2020;11:563756. https://doi.org/10.3389/fphar.2020.563756

27. Patten I.S., Arany Z. PGC-1 coactivators in the cardiovascular system. Trends Endocrinol. Metab. 2012;23(2):90–97. https://doi.org/10.1016/j.tem.2011.09.007

28. Krist B., Florczyk U., Pietraszek-Gremplewicz K., Józkowicz A., Dulak J. The Role of miR-378a in Metabolism, Angiogenesis, and Muscle Biology. Int. J. Endocrinol. 2015;2015:281756. https://doi.org/10.1155/2015/281756

29. Eichner L.J., Perry M.-C., Dufour C.R., Bertos N., Park M., St-Pierre J., Giguère V. miR-378(*) mediates metabolic shift in breast cancer cells via the PGC-1β/ERRγ transcriptional pathway. Cell Metab. 2010;12(4):352–361. https://doi.org/10.1016/j.cmet.2010.09.002

30. Gagan J., Dey B.K., Layer R., Yan Z., Dutta A. MicroR-NA-378 targets the myogenic repressor MyoR during myoblast differentiation. J. Biol. Chem. 2011;286(22):19431–19438. https://doi.org/10.1074/jbc.M111.219006

31. Davidsen P.K., Gallagher I.J., Hartman J.W., Tarnopolsky M.A., Dela F., Helge J.W., Timmons J.A., Phillips S.M. High responders to resistance exercise training demonstrate differential regulation of skeletal muscle microRNA expression. J. Appl. Physiol. (1985). 2011;110(2):309–317. https://doi.org/10.1152/japplphysiol.00901.2010

32. Kim S.W., Kim H.W., Huang W., Okada M., Welge J.A., Wang Y., Ashraf M. Cardiac stem cells with electrical stimulation improve ischaemic heart function through regulation of connective tissue growth factor and miR-378. Cardiovasc. Res. 2013;100(2):241–251. https://doi.org/10.1093/cvr/cvt192

33. Nagalingam R.S., Sundaresan N.R., Gupta M.P., Geenen D.L., Solaro R.J., Gupta M. A cardiac-enriched microRNA, miR-378, blocks cardiac hypertrophy by targeting Ras signaling. J. Biol. Chem. 2013;288(16):11216-11232. https://doi.org/10.1074/jbc.M112.442384

34. Jeon T.-I., Park J.W., Ahn J., Hwa Jung C., Ha T.Y. Fisetin protects against hepatosteatosis in mice by inhibiting miR-378. Mol. Nutr. Food Res. 2013;57(11):1931–1937. https://doi.org/10.1002/mnfr.201300071

35. Chen L.-T., Xu S.-D., Xu H., Zhang J.-F., Ning J.-F., Wang S.-F. MicroRNA-378 is associated with non-small cell lung cancer brain metastasis by promoting cell migration, invasion and tumor angiogenesis. Med. Oncol. 2012;29(3):1673–1680. https://doi.org/10.1007/s12032-011-0083-x

36. Lee D.Y., Deng Z., Wang C.-H., Yang B.B. MicroR-NA-378 promotes cell survival, tumor growth, and angiogenesis by targeting SuFu and Fus-1 expression. Proc. Natl. Acad. Sci. U. S. A. 2007;104(51):20350–20355. https://doi.org/10.1073/pnas.0706901104

37. Loboda A., Jozkowicz A., Dulak J. HIF-1 versus HIF-2 is one more important than the other? Vascul. Pharmacol. 2012;56(5–6):245–251. https://doi.org/10.1016/j.vph.2012.02.006

38. Hua Z., Lv Q., Ye W., Amy Wong C.-K., Cai G., Gu D., et al. MiRNA-directed regulation of VEGF and other angiogenic factors under hypoxia. PLoS One. 2006;1(1):e116. https://doi.org/10.1371/journal.pone.0000116

39. Jia X., Wang F., Han Y., Geng X., Li M., Shi Y., Lu L., Chen Y. miR-137 and miR-491 Negatively Regulate Dopamine Transporter Expression and Function in Neural Cells. Neurosci Bull. 2016;32(6):512–522. https://doi.org/10.1007/s12264-016-0061-6

40. Pramod A.B., Foster J., Carvelli L., Henry L.K. SLC6 transporters: structure, function, regulation, disease association and therapeutics. Mol. Aspects Med. 2013;34(2-3):197–219. https://doi.org/10.1016/j.mam.2012.07.002

41. Nikolaus S., Antke C., Hautzel H., Mueller H.W. Pharmacological treatment with L-DOPA may reduce striatal dopamine transporter binding in in vivo imaging studies. Nuklearmedizin. 2016;55(1):21–28. https://doi.org/10.3413/Nukmed-0764-15-08

42. Cheng M.H., Bahar I. Molecular mechanism of dopamine transport by human dopamine transporter. Structure. 2015;23(11):2171–2181 https://doi.org/10.1016/j.str.2015.09.001

43. Shen G., Li X., JIA Y.-F., Piazza G. A., Xi Y. Hypoxia-regulated microRNAs in human cancer. Acta Pharmacologica Sinica. 2013;34(3):336–341. https://doi.org/10.1038/aps.2012.195

44. Choudhry H., Mole D.R. Hypoxic regulation of the non-coding genome and NEAT1. Briefings in Functional Genomics. 2016;15(3):174–185. https://doi.org/10.1093/bfgp/elv050

45. Nallamshetty S., Chan S.Y., Loscalzo J. Hypoxia: A master regulator of microRNA biogenesis and activity. Free Radic. Biol. Med. 2013;64:20–30. https://doi.org/10.1016/j.freeradbiomed.2013.05.022

46. Tang W., Guo Z.-D., Chai W.-N., Du D.-L., Yang X.-M., Cao L., et al. Downregulation of miR-491-5p promotes neovascularization after traumatic brain injury. Neural Regen. Res. 2022;17(3):577–586. https://doi.org/10.4103/1673-5374.314326

47. Yang Y., Wang H., Yang S. Meta-Analysis of miR-NA Expression and Diagnostic Value in Human Glioma. Journal of Biological Regulators and Homeostatic Agents. 2023;37(4):1783–1790. https://doi.org/10.23812/j.biol.regul.homeost.agents.20233704.177