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Research Report 2: The role of Pgp in zebrafish cardiac glycosides chronotoxicity

Starokozhko, V. (2013) Research Report 2: The role of Pgp in zebrafish cardiac glycosides chronotoxicity. Master's Thesis / Essay, Biology.

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Introduction: Circadian rhythms influence susceptibility and responses of organisms to xenobiotic exposure. ATP binding cassette transporters have a major influence on bio-availability, metabolism and excretion of drugs. Recently it has been shown that the gene expression of P-glycoprotein (Pgp), is under circadian transcriptional regulation. It has been hypothesized that Pgp expression might contribute to the observed diurnal variation in cardiac glycosides toxicity in human. Accordingly, the aims of this study were to establish a cardiac glycosides chronotoxicity model using zebrafish and to investigate the role of Pgp expression in observed diurnal changes in toxicity. Materials and Methods: Three or eight days post fertilization (dpf) zebrafish were incubated for 3 hours with digitoxin (DT) at two different time points: at the beginning (Zeitgeber, ZT 1-4) and at the end (ZT 10-13) of the light cycle. In the case of Pgp inhibitory studies, 3dpf or 8 dpf fish at two different circadian time points were pre-soaked for 1 hour with verapamil (VP) or ketoconazole (KT) before 3h incubation with DT. Toxicity was assessed using toxicity scoring systems. Furthermore, qRT-PCR was performed to measure gene expression levels of the gene for Pgp MDR1. Results: Diurnal variation in DT toxicity was demonstrated on 3 and 8 dpf zebrafish: significantly higher toxicity occurred at ZT 10-13 versus ZT 1-4. Additionally, a trend toward reduced expression levels of MDR1 in 3 and 8dpf fish ZT 13 in comparison with ZT 4 was observed. Increase in toxicity was observed when zebrafish were pre-soaked with KT before incubation with DT at ZT 1-4. VP in the tested concentration did not cause an increase in DT toxicity. Discussion: The present study highlights the suitability of zebrafish as a model to perform cardiac glycosides chronotoxicity studies, since a significant diurnal difference in DT toxicity has been demonstrated on 3 and 8 dpf zebrafish. The observed augmented toxicity of DT with KT could be explained by inhibition of both Pgp and CYP3A, which cannot be differentiated in the present study. Although the diurnal difference in MDR1 expression between ZT 4 and ZT 13 was not found to be significant, the general fluctuation trend in MDR1 expression levels corresponded well with variation in toxicity manifestation.

Item Type: Thesis (Master's Thesis / Essay)
Degree programme: Biology
Thesis type: Master's Thesis / Essay
Language: English
Date Deposited: 15 Feb 2018 07:52
Last Modified: 15 Feb 2018 07:52

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