The developmental stage is a critical parameter for accurate assessment of the drug-induced liver injury (DILI) potentials of drugs with the zebrafish larval liver model

Abstract

Prediction of drug-induced liver injury (DILI) potential of drugs is one of the most challenging issues of drug development. Zebrafish larvae provide an in vivo and robust test platform. Due to the ease of handling developing larvae between 2 - 5 days post fertilization (dpf) has been extensively used as a DILI test model. However, the liver is not fully functional at this stage. Here, the importance of larval liver maturation was tested by applying selected known DILI-rank drugs to liver reporter zebrafish between 2-5 dpf and 5-7 dpf. Acetaminophen (most-DILI) treatment caused a significant dose-dependent reduction in liver size only at the early stage. Isoniazid (most-DILI) administration after liver maturation induced hepatomegaly, while it induced liver size reduction between 2-5 dpf. Chlorambucil (less-DILI) treatment induced opposing effects on liver size, in the two stages tested. A non-DILI agent chloramphenicol did not induce any liver size change in either larval stage. Clinical observations were better reproduced when isoniazid and chlorambucil were administered after liver maturation. Our findings show that often-overlooked liver maturity status is a critical parameter for the evaluation of DILI.

Keywords

• Acetaminophen
• Isoniazid
• Chloramphenicol
• Chlorambucil
• Hepatic injury

References

1. Akdogan, Y., Sozer, S. C., Akyol, C., Basol, M., Karakoyun, C., & Cakan-Akdogan, G. (2022). Synthesis of albumin nanoparticles in a water-miscible ionic liquid system, and their applications for chlorambucil delivery to cancer cells. Journal of Molecular Liquids, 367, 120575. https://doi.org/10.1016/-j.molliq.2022.120575
2. Ali, S., Champagne, D. L., & Richardson, M. K. (2012). Behavioral profiling of zebrafish embryos exposed to a panel of 60 water-soluble compounds. Behavioural Brain Research, 228(2), 272–283. https://doi.org//0.1016//j.bbr.2011.11.020
3. Almazroo, O. A., Miah, M. K., & Venkataramanan, R. (2017). Drug metabolism in the liver. Clinics in liver disease, 21(1), 1-20. https://doi.org/10.1016/j.cld.2016.08.001
4. Andrade, R. J., Chalasani, N., Björnsson, E. S., Suzuki, A., Kullak-Ublick, G. A., Watkins, P. B., Devarbhavi, H., Merz, M., Lucena, M. I., & Kaplowitz, N. (2019). Drug-induced liver injury. Nature Reviews Disease Primers, 5(1), 58. https://doi.org/10.1038/s41572-019-0105-0
5. Andrade, R. J., & Robles‐Díaz, M. (2020). Diagnostic and prognostic assessment of suspected drug‐induced liver injury in clinical practice. Liver International, 40(1), 6–17. https://doi.org/10.1111/liv.14271
6. Cassar, S., Adatto, I., Freeman, J. L., Gamse, J. T., Iturria, I., Lawrence, C., Muriana, A., Peterson, R. T., Van Cruchten, S., & Zon, L. I. (2019). Use of zebrafish in drug discovery toxicology. Chemical Research in Toxicology, 33(1), 95–118. https://doi.org/10.1021/acs.chemrestox.9b00335
7. Chan, R., & Benet, L. Z. (2017). Evaluation of DILI predictive hypotheses in early drug development. Chemical Research in Toxicology, 30(4), 1017–1029. https://doi.org/10.1021/acs.chemrestox.7b00025
8. Chen, M., Vijay, V., Shi, Q., Liu, Z., Fang, H., & Tong, W. (2011). FDA-approved drug labeling for the study of drug-induced liver injury. Drug Discovery Today, 16(15–16), 697–703. https://doi.org/10.1016/j.drudis.2011.05.007

9. Choi, T., Ninov, N., Stainier, D. Y. R., & Shin, D. (2014). Extensive conversion of hepatic biliary epithelial cells to hepatocytes after near total loss of hepatocytes in zebrafish. Gastroenterology, 146(3), 776–788. https://doi.org/10.1053/j.gastro.2013.10.019
10. Chu, J., & Sadler, K. C. (2009). New school in liver development: lessons from zebrafish. Hepatology, 50(5), 1656–1663. https://doi.org/10.1002/hep.23157
11. Cornet, C., Calzolari, S., Miñana-Prieto, R., Dyballa, S., Van Doornmalen, E., Rutjes, H., Savy, T., D’Amico, D., & Terriente, J. (2017). ZeGlobalTox: an innovative approach to address organ drug toxicity using zebrafish. International Journal of Molecular Sciences, 18(4), 864. https://doi.org/10.3390/ijms18040864
12. Feder Jr, H. M. (1986). Chloramphenicol: what we have learned in the last decade. Southern Medical Journal, 79(9), 1129–1134.
13. Garcia-Cortes, M., Robles-Diaz, M., Stephens, C., Ortega-Alonso, A., Lucena, M. I., & Andrade, R. J. (2020). Drug induced liver injury: an update. Archives of Toxicology, 94, 3381–3407. https://doi.org/10.1007/s00204-020-02885-1
14. Goldstone, J. V, McArthur, A. G., Kubota, A., Zanette, J., Parente, T., Jönsson, M. E., Nelson, D. R., & Stegeman, J. J. (2010). Identification and developmental expression of the full complement of Cytochrome P450 genes in Zebrafish. BMC Genomics, 11(1), 1–21. https://doi.org/10.1186/1471-2164-11-643
15. Guo, Q., Yang, W., Xiao, B., Zhang, H., Lei, X., Ou, H., Qin, R., & Jin, R. (2015). Study on early biomarkers of zebrafish liver injury induced by acetaminophen. Toxin Reviews, 34(1), 28–36. https://doi.org/10.3109/15569543.2014.986282
16. Higuchi, A., Wakai, E., Tada, T., Koiwa, J., Adachi, Y., Shiromizu, T., Goto, H., Tanaka, T., & Nishimura, Y. (2021). Generation of a Transgenic Zebrafish Line for In Vivo Assessment of Hepatic Apoptosis. Pharmaceuticals, 14(11), 1117. https://doi.org/10.3390/ph14111117
17. Iruzubieta, P., Arias-Loste, M. T., Barbier-Torres, L., Martinez-Chantar, M. L., & Crespo, J. (2015). The need for biomarkers in diagnosis and prognosis of drug-induced liver disease: does metabolomics have any role? BioMed Research International, 2015. https://doi.org/10.1155/2015/386186
18. Jagtap, U., Basu, S., Lokhande, L., Bharti, N., & Sachidanandan, C. (2022). BML-257, a Small Molecule that Protects against Drug-Induced Liver Injury in Zebrafish. Chemical Research in Toxicology, 35(8), 1393–1399. https://doi.org/10.1021/acs.chemrestox.2c00100
19. Katoch, S., & Patial, V. (2021). Zebrafish: An emerging model system to study liver diseases and related drug discovery. Journal of Applied Toxicology, 41(1), 33–51. https://doi.org/10.1002/jat.4031
20. Klotz, U. (2012). Paracetamol (acetaminophen)–a popular and widely used nonopioid analgesic. Arzneimittelforschung, 62(08), 355–359. https://doi.org/10.1055/s-0032-1321785
21. Kyle, R. A., Greipp, P. R., Gertz, M. A., Witzig, T. E., Lust, J. A., Lacy, M. Q., & Therneau, T. M. (2000). Waldenström’s macroglobulinaemia: a prospective study comparing daily with intermittent oral chlorambucil. British Journal of Haematology, 108(4), 737–742. https://doi.org/10.1046/j.1365-2141.2000.01918.x
22. Lecaudey, V., Cakan-Akdogan, G., Norton, W. H. J., & Gilmour, D. (2008). Dynamic Fgf signaling couples morphogenesis and migration in the zebrafish lateral line primordium. https://doi.org/10.1242/dev.025981
23. Lin, H.-S., Huang, Y.-L., Wang, Y.-R. S., Hsiao, E., Hsu, T.-A., Shiao, H.-Y., Jiaang, W.-T., Sampurna, B. P., Lin, K.-H., & Wu, M.-S. (2019). Identification of novel anti-liver cancer small molecules with better therapeutic index than sorafenib via zebrafish drug screening platform. Cancers, 11(6). https://doi.org/739. 10.3390/cancers11060739
24. Metushi, I., Uetrecht, J., & Phillips, E. (2016). Mechanism of isoniazid‐induced hepatotoxicity: then and now. British Journal of Clinical Pharmacology, 81(6), 1030–1036. https://doi.org/10.1111/bcp.12885
25. Nakayama, J., Tan, L., Li, Y., Goh, B. C., Wang, S., Makinoshima, H., & Gong, Z. (2021). A zebrafish embryo screen utilizing gastrulation identifies the HTR2C inhibitor pizotifen as a suppressor of EMT-mediated metastasis. Elife, 10, e70151. https://doi.org/10.7554/eLife.70151
26. Nanton, S. E., Bu-Ghanim, M., Ponnambalam, A., Nathan, R., & Fisher, S. E. (2004). Isoniazid induced fulminant hepatic failure in a teenager: 971. Official Journal of the American College of Gastroenterology| ACG, 99, S319. Nawaji, T., Yamashita, N., Umeda, H., Zhang, S., Mizoguchi, N., Seki, M., Kitazawa, T., & Teraoka, H. (2020). Cytochrome P450 expression and chemical metabolic activity before full liver development in zebrafish. Pharmaceuticals, 13(12), 456. https://doi.org/10.3390/ph13120456
27. Nguyen, X.-B., Kislyuk, S., Pham, D.-H., Kecskés, A., Maes, J., Cabooter, D., Annaert, P., De Witte, P., & Ny, A. (2017). Cell imaging counting as a novel ex vivo approach for investigating drug-induced hepatotoxicity in zebrafish larvae. International Journal of Molecular Sciences, 18(2), 356. https://doi.org/10.3390/ijms18020356
28. North, T. E., Babu, I. R., Vedder, L. M., Lord, A. M., Wishnok, J. S., Tannenbaum, S. R., Zon, L. I., & Goessling, W. (2010). PGE2-regulated wnt signaling and N-acetylcysteine are synergistically hepatoprotective in zebrafish acetaminophen injury. Proceedings of the National Academy of Sciences, 107(40), 17315–17320. https://doi.org/10.1073/pnas.1008209107
29. Park, Y. M., Dahlem, C., Meyer, M. R., Kiemer, A. K., Müller, R., & Herrmann, J. (2022). Induction of Liver Size Reduction in Zebrafish Larvae by the Emerging Synthetic Cannabinoid 4F-MDMB-BINACA and Its Impact on Drug Metabolism. Molecules, 27(4), 1290. https://doi.org/10.3390/molecules27041290
30. Patel, S. P. (2000). First report of liver failure: case report. Reactions, 818, 9. https://doi.org/10.2165/00128415-200008180-00016
31. Patel, S. P., Nast, C. C., & Adler, S. G. (2000). Chlorambucil-induced acute hepatic failure in a patient with membranous nephropathy. American Journal of Kidney Diseases, 36(2), 401–404. https://doi.org/10.1053/ajkd.2000.8995
32. Robles-Díaz, M., Medina-Caliz, I., Stephens, C., Andrade, R. J., & Lucena, M. I. (2016). Biomarkers in DILI: one more step forward. Frontiers in Pharmacology, 7, 267. https://doi.org/10.3389/fphar.2016.00267
33. Sato, Y., Dong, W., Nakamura, T., Mizoguchi, N., Nawaji, T., Nishikawa, M., Onaga, T., Ikushiro, S., Kobayashi, M., & Teraoka, H. (2023). Transgenic Zebrafish Expressing Rat Cytochrome P450 2E1 (CYP2E1): Augmentation of Acetaminophen-Induced Toxicity in the Liver and Retina. International Journal of Molecular Sciences, 24(4), 4013. https://doi.org/10.3390/ijms24044013
34. Shah, R., Ankale, P., Sinha, K., Iyer, A., & Jayalakshmi, T. K. (2016). Isoniazid induced lupus presenting as oral mucosal ulcers with pancytopenia. Journal of Clinical and Diagnostic Research: JCDR, 10(10), OD03. https://doi.org/10.7860/JCDR/2016/22543.8629
35. Sienkiewicz, P., Bielawski, K., Bielawska, A., & Pałka, J. (2005). Inhibition of collagen and DNA biosynthesis by a novel amidine analogue of chlorambucil is accompanied by deregulation of β1-integrin and IGF-I receptor signaling in MDA-MB 231 cells. Environmental Toxicology and Pharmacology, 20(1), 118–124. https://doi.org/10.1016/j.etap.2004.11.001
36. Singhal, K. K., Mukim, M. D., Dubey, C. K., & Nagar, J. C. (2020). An Updated Review on Pharmacology and Toxicities Related to Chloramphenicol. Asian Journal of Pharmaceutical Research and Development, 8(4), 104–109. https://doi.org/10.22270/ajprd.v8i4.671
37. Skoutelis, A. (2000). Chlorambucil/methylprednisolone. Reactions, 811, 22. https://doi.org/10.2165/00128415-200008110-00011
38. Tierbach, A., Groh, K. J., Schönenberger, R., Schirmer, K., & Suter, M. J.-F. (2018). Glutathione S-transferase protein expression in different life stages of zebrafish (Danio rerio). Toxicological Sciences, 162(2), 702–712. https://doi.org/10.1093/toxsci/kfx293
39. Vliegenthart, A. D. B., Tucker, C. S., Del Pozo, J., & Dear, J. W. (2014). Zebrafish as model organisms for studying drug‐induced liver injury. British Journal of Clinical Pharmacology, 78(6), 1217–1227. https://doi.org/10.1111/bcp.12408
40. Wolf, A. D., & Lavine, J. E. (2000). Hepatomegaly in neonates and children. Pediatrics in Review, 21(9), 303–310.https://doi.org/10.1542/pir.21-9-303
41. Zhang, X., Li, C., & Gong, Z. (2014). Development of a convenient in vivo hepatotoxin assay using a transgenic zebrafish line with liver-specific DsRed expression. PloS One, 9(3), e91874. https://doi.org/10.1371/journal.pone.0091874
42. Zhang, Y., Cen, J., Jia, Z., Hsiao, C.-D., Xia, Q., Wang, X., Chen, X., Wang, R., Jiang, Z., & Zhang, L. (2019). Hepatotoxicity induced by isoniazid-lipopolysaccharide through endoplasmic reticulum stress, autophagy, and apoptosis pathways in zebrafish. Antimicrobial Agents and Chemotherapy, 63(5), e01639-18. https://doi.org/10.1128/aac.01639-18