Whole Zebrafish Cytochrome P450 Assay for Assessing Drug Metabolism and Safety - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 103

NC) and drug concentrations for 50% inhibition (IC 50 ) and 50% induction (EC 50 )
were estimated based on best-fit concentration-response curves.

9.3.6 Statistics

Since multiple concentrations were assessed, ANOVAwas used to determine if drug
effect was significant ( P 0.05). Dunnett's test, a multiple pairwise comparison test,

was then performed to identify concentrations that exhibited significant effects.

<

9.3.7 Vertebrate Animal Care and Safety

The Office of Laboratory Animal Welfare (OLAW), National Institutes of Health
(NIH), approved our Animal Welfare Assurance effective through February 2012. We
euthanize zebrafish of all ages by overexposure to tricaine methanesulfonate. This
procedure is consistent with theAmericanVeterinaryMedical Association's (AVMA)
Panel on Euthanasia.

9.4 RESULTS

Using a commercially available human CYP3A4-specific chemiluminescent sub-
strate, we developed a microplate-based zebrafish whole animal functional assay to
assess drug metabolism and drug safety. Specificity of the zebrafish CYPA65
(CYP3A4) assay was initially validated by assessing (a) mammalian CYP3A4
inhibitors, (b) mammalian CYP3A4 inducers, and (c) a no-effect compound. The
assay was further validated using five additional mammalian CYP3A4 inhibitors and
six mammalian CYP3A4 inducers. Overall prediction success rate was 87% (13/15):
100% for inhibition (6/6) and 75% for induction (6/8). We established that treating
2dpf zebrafish for 24 h was optimum. Our results demonstrate that zebrafish exhibit
comparable CYP drug metabolism profiles as mammals, supporting use of the whole
zebrafish microplate CYP3A4 functional activity assay for preliminary drug
screening.

9.4.1 Whole Zebrafish CYP Microplate Assay
Development

In an initial pilot investigation, we found that similar to CYP3A4 response in humans,
CYP3A4 functional activitywas upregulated in zebrafish treated with dexamethasone
at 10 and 50

M concentrations. Our results were consistent with a recent report that
showed that a low dose of dexamethasone (10

m

m M) enhanced zebrafish CYP3A65

transcription, whereas a high dose (100 M) did not (Tseng et al., 2005). We initially

established specificity of the in vivo microplate-based zebrafish functional assay by

m