Kar Lai POON1,*, Xingang WANG1,*, Ashley S NG1, Wei Huang GOH1, Claudia MCGINNIS2, Stephen FOWLER2, Tom J CARNEY1,3,C, Haishan WANG1,+, Phillip W INGHAM1,3+
1 Institute of Molecular and Cell Biology (IMCB), A*STAR (Agency for Science, Technology and Research), 61 Biopolis Drive, 138673, Singapore
2 Roche Pharmaceutical Research & Early Development (pRED), Pharmaceutical Sciences, Roche Innovation Center Basel, Basel, Switzerland
3 Lee Kong Chian School of Medicine, Nanyang Technological University, 50 Nanyang Avenue, 639798, Singapore
* Joint first authors
+ Joint senior authors
C Corresponding author
Published in Archives of Toxicology in March 2017.
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 (ADME) 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.
Humanized zebrafish expressing CYP3A4 shows increased sensitivity to APAP toxicity and enhanced MDZ metabolism. a. Schematic representation of construct used for generation of humanized CYP3A4 zebrafish showing the zebrafish lfabp liver specific promoter (green) driving the expression of mCherry (red) tagged human CYP3A4 (blue). The construct is flanked by tol2 elements (purple triangles). b. Lateral micrograph of a transgenic larva immunostained for hCYP3A4 (green) and mCherry (red) confirms that tagged hCYP3A4 is expressed in the liver. c. The mCherry fluorescence intensities of dissected adult transgenic livers vary. The value below the picture indicates the measured fluorescence level determined from grey value intensities in Image J. d. Liver GSH content after 24h of treatment with APAP in both wildtype and humanized transgenics.: n=2 for all groups, ANOVA, P<0.001 for WT and TG (DMSO vs APAP); p<0.05 APAP (WT vs TG); P>0.05 DMSO (WT vs TG). e. The ratio of metabolites (OHMDZ to MDZ, %) in wildtype zebrafish adult livers compared to humanized transgenics livers after 6hpe. Two-tailed t-test were performed for WT (n=6) vs TG (n=15); p = 0.0002. f. The mCherry fluorescence intensities of humanized transgenics livers (n=15) correlate with hCYP3A4 metabolic activity as measured by MDZ biotransformation. Pearson correlation coefficients, P<0.0001; linear regression, r2 = 0.6991. g. The ratio and types of various metabolites to MDZ (%) measured from freeze dried medium that contained adult wildtype zebrafish or humanized transgenics collected at 6hpe (n=2)
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