Metabolomics in drugs research on zebrafish-based cardiotoxicity models: endothelial and mitochondrial dysfunction, oxidative stress

The aim. To investigate the metabolic profile of zebrafish embryos when exposed to drugs with known risks of cardiotoxicity, such as acetaminophen, carbamazepine, salbutamol, ketorolac, bisoprolol, and metoprolol. The analysis is aimed at detecting changes in the level of amino acids (including branched chain BCAAs), products of carnitine metabolism (acylcarnitines) and related metabolic indices reflecting mitochondrial dysfunction, oxidative stress and disorders of the nitric oxide signaling pathway.

Materials and methods. Zebrafish embryos were incubated with the test substances in a concentration gradient (0.5–10×NOEC). A quantitative targeted metabolomics analysis was performed using high-performance liquid chromatography with tandem mass spectrometry (HPLC–MS/MS) with a panel of 98 metabolites (amino acids, nitric oxide metabolism products, vitamins, nucleosides and acylcarnitines). The obtained concentrations of metabolites were compared with the control (0.1% DMSO). Statistically significant deviations were expressed as the ratio of concentration to control on a base 2 logarithmic scale (log2FC).

Results. Changes in concentrations of metabolites under the influence of cardiotoxic drugs were revealed. There was an accumulation of BCAAs (the sum of leucine, isoleucine, valine; log2FC≈0.5–2.2; p <0.05) compared with the control, as well as an increase in the level of acylcarnitines, indicating mitochondrial dysfunction: for example, metoprolol and bisoprolol caused an increase in the ratio of the sum of acylcarnitines to free carnitine by more than 4–6 times (log2FC=+3.8 for bisoprolol and -1.27 for metoprolol; p <0.01), as well as accumulation of long-chain acylcarnitines. Pronounced changes in indicators related to oxidative stress were noted: in the samples after exposure to beta-1 blockers (bisoprolol, metoprolol) and ketorolac, the concentration of methionine sulfoxide (by 80–130%, p < 0.01), the product of methionine oxidation, and the ratio of methionine sulfoxide/methionine increased, whereas when exposed to salbutamol, on the contrary, the level of methionine sulfoxide decreased (-120%, p <0.01), indicating a multidirectional effect on the oxidative status. Violations of the nitric oxide signaling pathway were reflected in an increase in the level of asymmetric dimethylarginine.

Conclusion. Each of the analyzed compounds produced a specific metabolic “imprint” in Zebrafish samples, reflecting the mechanisms of their cardiotoxicity. An increase in BCAA levels and related indicators indicates a violation of myocardial energy metabolism, the accumulation of long-chain acylcarnitines indicates incomplete beta-oxidation of fatty acids. An increase in the concentration of ADMA is associated with endothelial dysfunction, and an increase in methionine sulfoxide is associated with increased oxidative stress.

1. Onakpoya IJ, Heneghan CJ, Aronson JK. Post-marketing withdrawal of 462 medicinal products because of adverse drug reactions: a systematic review of the world literature. BMC Medicine. 2016;14:10. DOI: 10.1186/s12916-016-0553-2

2. Ferdinandy P, Baczkó I, Bencsik P, Giricz Z, Görbe A, Pacher P, Varga ZV, Varró A, Schulz R. Definition of hidden drug cardiotoxicity: paradigm change in cardiac safety testing and its clinical implications. Eur Heart J. 2018;40(22):1771–7. DOI: 10.1093/eurheartj/ehy365

3. Ussher JR, Elmariah S, Gerszten RE, Dyck JRB. The Emerging Role of Metabolomics in the Diagnosis and Prognosis of Cardiovascular Disease. J Am College Cardiolog. 2016;68(25):2850–70. DOI: 10.1016/j.jacc.2016.09.972

4. Singh A, Bakhtyar M, Jun SR, Boerma M, Lan RS, Su LJ, Makhoul S, Hsu PC. A narrative review of metabolomics approaches in identifying biomarkers of doxorubicin-induced cardiotoxicity. Metabolomics. 2025;21:68. DOI: 10.1007/s11306-025-02258-8

5. Rhee EP, Gerszten RE. Metabolomics and Cardiovascular Biomarker Discovery. Clin Chem. 2011;58(1):139–147. DOI: 10.1373/clinchem.2011.169573

6. Holeček M. Branched-chain amino acids in health and disease: metabolism, alterations in blood plasma, and as supplements. Nutrition & Metabolism. 2018;15:33. DOI: 10.1186/s12986-018-0271-1

7. Gao C, Hou L. Branched chain amino acids metabolism in heart failure. Front Nutrition. 2023;10:1279066. DOI: 10.3389/fnut.2023.1279066

8. Kozhevnikova MV, Belenkov YN, Shestakova KM, Ageev AA, Markin PA, Kakotkina AV, Korobkova EO, Moskaleva NE, Kuznetsov IV, Khabarova NV, Kukharenko AV, Appolonova SA. Metabolomic profiling in heart failure as a new tool for diagnosis and phenotyping. Sci Rep. 2025;15(1):11849. DOI: 10.1038/s41598-025-95553-2

9. Kozhevnikova MV, Kakotkina AV, Korobkova EO, Kuznetsov IV, Shestakova KM, Moskaleva NE, Appolonova SA, Belenkov YN. Metabolomic Panel for the Diagnosis of Heart Failure with Preserved Ejection Fraction. Int J Mol Sci. 2025;26:2102. DOI: 10.3390/ijms26052102

10. Aitken-Buck HM, Krause J, Zeller T, Jones PP, Lamberts RR. Long-Chain Acylcarnitines and Cardiac Excitation-Contraction Coupling: Links to Arrhythmias. Front Physiology. 2020;11:577856. DOI: 10.3389/fphys.2020.577856

11. Kukharenko A, Brito A, Kozhevnikova MV, Moskaleva N, Markin PA, Bochkareva N, Korobkova EO, Belenkov YN, Privalova EV, Larcova EV, Ariani A, La Frano MR, Appolonova SA. Relationship between the plasma acylcarnitine profile and cardiometabolic risk factors in adults diagnosed with cardiovascular diseases. Clin Chim Acta. 2020;507:250–256. DOI: 10.1016/j.cca.2020.04.035

12. Sibal L, Agarwal SC, Home PD, Boger RH. The Role of Asymmetric Dimethylarginine (ADMA) in Endothelial Dysfunction and Cardiovascular Disease. Cur Cardiology Rev. 2010;6(2):82–90. DOI: 10.2174/157340310791162659

13. Markin SS, Ponomarenko EA, Romashova YA, Pleshakova TO, Ivanov SV, Bedretdinov FN, Konstantinov SL, Nizov AA, Koledinskii AG, Girivenko AI, Shestakova KM, Markin PA, Moskaleva NE, Kozhevnikova MV, Chefranova ZhYu, Appolonova SA. A novel preliminary metabolomic panel for IHD diagnostics and pathogenesis. Scientific Reports. 2024;14:2651. DOI: 10.1038/s41598-024-53215-9

14. Brito-Martins M, Harding SE, Ali NN. beta(1)- and beta(2)-adrenoceptor responses in cardiomyocytes derived from human embryonic stem cells: comparison with failing and non-failing adult human heart. Br J Pharmacol. 2008;153(4):751–9. DOI: 10.1038/sj.bjp.0707619

15. Asnani A, Moslehi JJ, Adhikari BB, Baik AH, Beyer AM, de Boer RA, Ghigo A, Grumbach IM, Jain S, Zhu H; American Heart Association Council on Basic Cardiovascular Sciences; Cardio-Oncology Science Subcommittee of Council on Genomic and Precision Medicine and Council on Clinical Cardiology; Council on Peripheral Vascular Disease; and Council on Arteriosclerosis, Thrombosis and Vascular Biology. Preclinical Models of Cancer Therapy-Associated Cardiovascular Toxicity: A Scientific Statement From the American Heart Association. Circ Res. 2021;129(1):e21–e34. DOI: 10.1161/RES.0000000000000473

16. Wang W, Gao X, Liu L, Guo S, Duan JA, Xiao P. Zebrafish as a Vertebrate Model for High-Throughput Drug Toxicity Screening: Mechanisms, Novel Techniques, and Future Perspectives. J Pharm Analysis. 2025:101195. DOI: 10.1016/j.jpha.2025.101195

17. Markin PA, Brito A, Moskaleva NE, Tagliaro F, La Frano MR, Savitskii MV, Appolonova SA. Short- and long-term exposures of the synthetic cannabinoid 5F-APINAC induce metabolomic alterations associated with neurotransmitter systems and embryotoxicity confirmed by teratogenicity in zebrafish. Comp Biochem Physiol C Toxicol Pharmacol. 2021;243:109000. DOI: 10.1016/j.cbpc.2021.109000

18. Markin PA, Brito A, Moskaleva NE, Tagliaro F, Tarasov VV, La Frano MR, Savitskii MV, Appolonova SA. Short- and medium-term exposures of diazepam induce metabolomic alterations associated with the serotonergic, dopaminergic, adrenergic and aspartic acid neurotransmitter systems in zebrafish (Danio rerio) embryos/larvae. Comp Biochem Physiol Part D Genomics Proteomics. 2021;38:100816. DOI: 10.1016/j.cbd.2021.100816

19. Dyballa S, Miñana R, Rubio-Brotons M, Cornet C, Pederzani T, Escaramis G, Garcia-Serna R, Mestres J, Terriente J. Comparison of Zebrafish Larvae and hiPSC Cardiomyocytes for Predicting Drug-Induced Cardiotoxicity in Humans. Toxicol Sci. 2019;171(2):283–295. DOI: 10.1093/toxsci/kfz165

20. Zakaria ZZ, Benslimane FM, Nasrallah GK, Shurbaji S, Younes NN, Mraiche F, Da'as SI, Yalcin HC. Using Zebrafish for Investigating the Molecular Mechanisms of Drug-Induced Cardiotoxicity. Biomed Res Int. 2018;2018:1642684. DOI: 10.1155/2018/1642684

21. Tusher VG, Tibshirani R, Chu G. Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A. 2001;98(9):5116–21. DOI: 10.1073/pnas.091062498

22. Morris CR, Hamilton-Reeves J, Martindale RG, Sarav M, Ochoa Gautier JB. Acquired Amino Acid Deficiencies: A Focus on Arginine and Glutamine. Nutr Clin Pract. 2017;32(1_suppl):30S–47S. DOI: 10.1177/0884533617691250

23. Rees CA, Rostad CA, Mantus G, Anderson EJ, Chahroudi A, Jaggi P, Wrammert J, Ochoa JB, Ochoa A, Basu RK, Heilman S, Harris F, Lapp SA, Hussaini L, Vos MB, Brown LA, Morris CR. Altered amino acid profile in patients with SARS-CoV-2 infection. Proc Natl Acad Sci U S A. 2021;118(25):e2101708118. DOI: 10.1073/pnas.2101708118

24. Masoodi M, Gastaldelli A, Hyötyläinen T, Arretxe E, Alonso C, Gaggini M, Brosnan J, Anstee QM, Millet O, Ortiz P, Mato JM, Dufour JF, Orešič M. Metabolomics and lipidomics in NAFLD: biomarkers and non-invasive diagnostic tests. Nat Rev Gastroenterol Hepatol. 2021;18(12):835–856. DOI: 10.1038/s41575-021-00502-9

25. Leonetti S, Herzog RI, Caprio S, Santoro N, Tricò D. Glutamate-Serine-Glycine Index: A Novel Potential Biomarker in Pediatric Non-Alcoholic Fatty Liver Disease. Children. 2020;7(12):270. DOI: 10.3390/children7120270

26. Da Silva KM, Iturrospe E, Bars C, Knapen D, Van Cruchten S, Covaci A, Van Nuijs ALN. Mass spectrometry-based zebrafish toxicometabolomics: a review of analytical and data quality challenges. Metabolites. 2021;11(9):635. DOI: 10.3390/metabo11090635

27. Moskaleva NE, Shestakova KM, Kukharenko AV, Markin PA, Kozhevnikova MV, Korobkova EO, Brito A, Baskhanova SN, Mesonzhnik NV, Belenkov YN, Pyatigorskaya NV, Tobolkina E, Rudaz S, Appolonova SA. Target Metabolome Profiling-Based Machine Learning as a Diagnostic Approach for Cardiovascular Diseases in Adults. Metabolites. 2022;12(12):1185. DOI: 10.3390/metabo12121185

28. Drygała S, Radzikowski M, Maciejczyk M. β-blockers and metabolic modulation: unraveling the complex interplay with glucose metabolism, inflammation and oxidative stress. Front Pharmacol. 2024 Dec 20;15:1489657. DOI: 10.3389/fphar.2024.1489657

29. Förstermann U, Sessa WC. Nitric oxide synthases: regulation and function. Eur Heart J. 2012;33(7):829–837, 837a–837d. DOI: 10.1093/eurheartj/ehr30

30. Adding LC, Agvald P, Artlich A, Persson MG, Gustafsson LE. Beta-adrenoceptor agonist stimulation of pulmonary nitric oxide production in the rabbit. Br J Pharmacol. 1999;126(3):833–9. DOI: 10.1038/sj.bjp.0702369

31. Cheng ML, Wang CH, Shiao MS, Liu MH, Huang YY, Huang CY, Mao CT, Lin JF, Ho HY, Yang NI. Metabolic disturbances identified in plasma are associated with outcomes in patients with heart failure: diagnostic and prognostic value of metabolomics. J Am Coll Cardiol. 2015;65(15):1509–20. doi: 10.1016/j.jacc.2015.02.018

32. Sabbatino F, Conti V, Liguori L, Polcaro G, Corbi G, Manzo V, Tortora V, Carlomagno C, Vecchione C, Filippelli A, Pepe S. Molecules and Mechanisms to Overcome Oxidative Stress Inducing Cardiovascular Disease in Cancer Patients. Life (Basel). 2021;11(2):105. DOI: 10.3390/life11020105

33. Ramachandran A, Duan L, Akakpo JY, Jaeschke H. Mitochondrial dysfunction as a mechanism of drug-induced hepatotoxicity: current understanding and future perspectives. J Clin Translat Res. 2018;4(1):75–100. DOI: 10.18053/jctres.04.201801.005

34. Bashir S, Morgan WA. Inhibition of mitochondrial function: An alternative explanation for the antipyretic and hypothermic actions of acetaminophen. Life Sci. 2023;312:121194. DOI: 10.1016/j.lfs.2022.121194

35. Hosohata K. Role of Oxidative Stress in Drug-Induced Kidney Injury. Int J Mol Sci. 2016;17(11):1826. DOI: 10.3390/ijms17111826

36. Fine KS, Wilkins JT, Sawicki KT. Circulating branched chain amino acids and cardiometabolic disease. J Am Heart Association. 2024;13:e031617. DOI: 10.1161/JAHA.123.031617

37. Truby LK, Regan JA, Giamberardino SN, Ilkayeva O, Bain J, Newgard CB, O'Connor CM, Felker GM, Kraus WE, McGarrah RW, Shah SH. Circulating long chain acylcarnitines and outcomes in diabetic heart failure: an HF-ACTION clinical trial substudy. Cardiovasc Diabetol. 2021;20(1):161. DOI: 10.1186/s12933-021-01353-z