Abstract
Understanding and predicting whether new drug candidates will be safe in the clinic is a critical hurdle in pharmaceutical development, that relies in part on absorption, distribution, metabolism, excretion and toxicology studies in vivo. Zebrafish is a relatively new model system for drug metabolism and toxicity studies, offering whole organism screening coupled with small size and potential for high-throughput screening. Through toxicity and absorption analyses of a number of drugs, we find that zebrafish is generally predictive of drug toxicity, although assay outcomes are influenced by drug lipophilicity which alters drug uptake. In addition, liver microsome assays reveal specific differences in metabolism of compounds between human and zebrafish livers, likely resulting from the divergence of the cytochrome P450 superfamily between species. To reflect human metabolism more accurately, we generated a transgenic “humanized” zebrafish line that expresses the major human phase I detoxifying enzyme, CYP3A4, in the liver. Here, we show that this humanized line shows an elevated metabolism of CYP3A4-specific substrates compared to wild-type zebrafish. The generation of this first described humanized zebrafish liver suggests such approaches can enhance the accuracy of the zebrafish model for toxicity prediction.
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References
Alderton W, Berghmans S, Butler P et al (2010) Accumulation and metabolism of drugs and CYP probe substrates in zebrafish larvae. Xenobiotica 40(8):547–557
Chang CT, Chung HY, Su HT, Tseng HP, Tzou WS, Hu CH (2013) Regulation of zebrafish CYP3A65 transcription by AHR2. Toxicol Appl Pharmacol 270(2):174–184
Cheung C, Gonzalez FJ (2008) Humanized mouse lines and their application for prediction of human drug metabolism and toxicological risk assessment. J Pharmacol Exp Ther 327(2):288–299
Cheung C, Ma X, Krausz KW et al (2005a) Differential metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in mice humanized for CYP1A1 and CYP1A2. Chem Res Toxicol 18(9):1471–1478
Cheung C, Yu AM, Ward JM et al (2005b) The cyp2e1-humanized transgenic mouse: role of cyp2e1 in acetaminophen hepatotoxicity. Drug Metab Dispos 33(3):449–457
Chng HT, Ho HK, Yap CW, Lam SH, Chan ECY (2012) An investigation of the bioactivation potential and metabolism profile of zebrafish versus human. J Biomol Screen 17(7):974–986
Corchero J, Granvil CP, Akiyama TE et al (2001) The CYP2D6 humanized mouse: effect of the human CYP2D6 transgene and HNF4alpha on the disposition of debrisoquine in the mouse. Mol Pharmacol 60(6):1260–1267
Dalvie D, Obach RS, Kang P et al (2009) Assessment of three human in vitro systems in the generation of major human excretory and circulating metabolites. Chem Res Toxicol 22(2):357–368
Dragin N, Uno S, Wang B, Dalton TP, Nebert DW (2007) Generation of ‘humanized’ hCYP1A1_1A2_Cyp1a1/1a2(−/−) mouse line. Biochem Biophys Res Commun 359(3):635–642
Goldstone JV, McArthur AG, Kubota A et al (2010) Identification and developmental expression of the full complement of Cytochrome P450 genes in zebrafish. BMC Genom 11(1):643
Granvil CP, Yu AM, Elizondo G et al (2003) Expression of the human CYP3A4 gene in the small intestine of transgenic mice: in vitro metabolism and pharmacokinetics of midazolam. Drug Metab Dispos 31(5):548–558
Guengerich FP (2011) Mechanisms of drug toxicity and relevance to pharmaceutical development. Drug Metab Pharmacokinet 26(1):3–14
Gustafson AL, Stedman DB, Ball J et al (2012) Inter-laboratory assessment of a harmonized zebrafish developmental toxicology assay—progress report on phase I. Reprod Toxicol 33(2):155–164
Hasegawa M, Kapelyukh Y, Tahara H et al (2011) Quantitative prediction of human pregnane X receptor and cytochrome P450 3A4 mediated drug-drug interaction in a novel multiple humanized mouse line. Mol Pharmacol 80(3):518–528
Her GM, Yeh YH, Wu JL (2003) 435-bp liver regulatory sequence in the liver fatty acid binding protein (L-FABP) gene is sufficient to modulate liver regional expression in transgenic zebrafish. Dev Dyn 227(3):347–356
Hesse RG, Kouklis GK, Ahituv N, Pomerantz JH (2015) The human ARF tumor suppressor senses blastema activity and suppresses epimorphic tissue regeneration. Elife 4:e07702
Jomaa B, Hermsen SA, Kessels MY et al (2014) Developmental toxicity of thyroid-active compounds in a zebrafish embryotoxicity test. ALTEX 31(3):303–317
Jones HS, Trollope HT, Hutchinson TH, Panter GH, Chipman JK (2012) Metabolism of ibuprofen in zebrafish larvae. Xenobiotica 42(11):1069–1075
Katoh M, Tateno C, Yoshizato K, Yokoi T (2008) Chimeric mice with humanized liver. Toxicology 246(1):9–17
Klieber S, Hugla S, Ngo R et al (2008) Contribution of the N-glucuronidation pathway to the overall in vitro metabolic clearance of midazolam in humans. Drug Metab Dispos 36(5):851–862
Kwan KM, Fujimoto E, Grabher C et al (2007) The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs. Dev Dyn 236(11):3088–3099
Li Y, Ross-Viola JS, Shay NF, Moore DD, Ricketts ML (2009) Human CYP3A4 and murine Cyp3A11 are regulated by equol and genistein via the pregnane X receptor in a species-specific manner. J Nutr 139(5):898–904
Lofgren S, Baldwin RM, Hiratsuka M et al (2008) Generation of mice transgenic for human CYP2C18 and CYP2C19: characterization of the sexually dimorphic gene and enzyme expression. Drug Metab Dispos 36(5):955–962
North TE, Babu IR, Vedder LM et al (2010) PGE2-regulated wnt signaling and N-acetylcysteine are synergistically hepatoprotective in zebrafish acetaminophen injury. Proc Natl Acad Sci USA 107(40):17315–17320
Ouellet D, Bramson C, Roman D et al (2007) Effects of three cytochrome P450 inhibitors, ketoconazole, fluconazole, and paroxetine, on the pharmacokinetics of lasofoxifene. Br J Clin Pharmacol 63(1):59–66
Pang SC, Wang HP, Li KY, Zhu ZY, Kang JX, Sun YH (2014) Double transgenesis of humanized fat1 and fat2 genes promotes omega-3 polyunsaturated fatty acids synthesis in a zebrafish model. Mar Biotechnol 16(5):580–593
Raldua D, Pina B (2014) In vivo zebrafish assays for analyzing drug toxicity. Expert Opin Drug Metab Toxicol 10(5):685–697
Scheer N, Roland Wolf C (2013) Xenobiotic receptor humanized mice and their utility. Drug Metab Rev 45(1):110–121
Scheer N, Wolf CR (2014) Genetically humanized mouse models of drug metabolizing enzymes and transporters and their applications. Xenobiotica 44(2):96–108
Schubert S, Keddig N, Hanel R, Kammann U (2014) Microinjection into zebrafish embryos (Danio rerio)—a useful tool in aquatic toxicity testing? Environ Sci Eur 26(1):1–8
Scornaienchi ML, Thornton C, Willett KL, Wilson JY (2010) Cytochrome P450-mediated 17β-estradiol metabolism in zebrafish (Danio rerio). J Endocrinol 206(3):317–325
Seo K-A, Bae SK, Choi Y-K, Choi CS, Liu K-H, Shin J-G (2010) Metabolism of 1′- and 4-hydroxymidazolam by glucuronide conjugation is largely mediated by UDP-glucuronosyltransferases 1A4, 2B4, and 2B7. Drug Metab Dispos 38(11):2007–2013
Shayeganpour A, El-Kadi AOS, Brocks DR (2006) Determination of the enzyme(s) involved in the metabolism of amiodarone in liver and intestine of rat: the contribution of cytochrome P450 3A isoforms. Drug Metab Dispos 34(1):43–50
Tseng HP, Hseu TH, Buhler DR, Wang WD, Hu CH (2005) Constitutive and xenobiotics-induced expression of a novel CYP3A gene from zebrafish larva. Toxicol Appl Pharmacol 205(3):247–258
van Herwaarden AE, Smit JW, Sparidans RW et al (2005) Midazolam and cyclosporin a metabolism in transgenic mice with liver-specific expression of human CYP3A4. Drug metabolism and disposition: the biological fate of chemicals 33(7):892–895
van Herwaarden AE, Wagenaar E, van der Kruijssen CM et al (2007) Knockout of cytochrome P450 3A yields new mouse models for understanding xenobiotic metabolism. J Clin Invest 117(11):3583–3592
Walsky RL, Obach RS (2004) Validated assays for human cytochrome P450 activities. Drug Metab Dispos 32(6):647–660
Williams PA, Cosme J, Vinkovic DM et al (2004) Crystal structures of human cytochrome P450 3A4 bound to metyrapone and progesterone. Science 305(5684):683–686
Xie W, Barwick JL, Downes M et al (2000) Humanized xenobiotic response in mice expressing nuclear receptor SXR. Nature 406(6794):435–439
Zanger UM, Schwab M (2013) Cytochrome P450 enzymes in drug metabolism: regulation of gene expression, enzyme activities, and impact of genetic variation. Pharmacol Ther 138(1):103–141
Zhang F, Qin W, Zhang JP, Hu CQ (2015) Antibiotic toxicity and absorption in zebrafish using liquid chromatography-tandem mass spectrometry. PLoS ONE 10(5):e0124805
Zhu B, Bush D, Doss GA, Vincent SH, Franklin RB, Xu S (2008) Characterization of 1′-hydroxymidazolam glucuronidation in human liver microsomes. Drug Metab Dispos 36(2):331–338
Acknowledgments
The authors would like to thank Stefan Hart and Diana Teo, ARC Roche Singapore, for helpful and effective administrative support; Thomas Weiser (Roche) and Steve Cohen (IMCB) for initiating the collaboration; Thomas Singer, Franz Schuler, Adrian B. Roth (Roche), for support of the project; Navrinder Kaur (IMCB) and Zhong Hua Zhao (IMCB) for technical assistance. The project was funded by Roche and IMCB, A-STAR, Singapore.
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Kar Lai Poon and Xingang Wang are joint first authors. Haishan Wang and Phillip W. Ingham are joint senior authors.
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Poon, K.L., Wang, X., Ng, A.S. et al. Humanizing the zebrafish liver shifts drug metabolic profiles and improves pharmacokinetics of CYP3A4 substrates. Arch Toxicol 91, 1187–1197 (2017). https://doi.org/10.1007/s00204-016-1789-5
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DOI: https://doi.org/10.1007/s00204-016-1789-5