Whole Zebrafish Cytochrome P450 Assay for Assessing Drug Metabolism and Safety - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 104

quantifying levels of CYP3A65 (CYP3A4) activity in zebrafish treated with azamu-
lin, a known mammalian CYP3A4 inhibitor, and rifampicin, a known mammalian
CYP3A4 inducer. Zebrafish treated with 50 M dexamethasone for 24 h were used

as a positive control. Zebrafish were also treated with no-effect compound
a

m

-naphthoflavone as a negative control (Cali et al., 2006), and vehicle control was
0.1%DMSO. At the end of treatment, CYP3A4 activitywas quantitated in control and
drug-treated zebrafish, as described in Section 10.3. Treatment with 0.1% DMSO for
24 h had no effect on CYP3A4 functional activity (data not shown), indicating that
vehicle solvent did not affect CYP3A4 function. 0.01, 0.1, 1, 10, and 100

M
concentrations of azamulin inhibited CYPA4 by 0%, 13%, 20%, 30%, and 31%,
respectively (Fig. 9.1a). 0.01, 0.1, 1, 10, and 100

m

M concentrations of rifampicin
induced CYPA4 by 0%, 17%, 37%, 65%, and 99%, respectively (Fig. 9.1b). Spec-
ificity of the assay was further supported by insensitivity to

m

a -naphthoflavone, a no-

effect negative control compound (data not shown).

9.4.2 Determination of Optimum Assay Conditions

We also assessed effects of zebrafish developmental stage and found that CYP3A4
level was higher at 3dpf than at 2dpf. However, since rifampicin treatment caused a
statistically significant effect ( P 0.05)

(Fig. 9.2), we used 2dpf zebrafish as the optimum stage for performing the CYP3A4
assay.
To develop a reproducible assay, we also assessed the relationship between
number of zebrafish per microwell and luminescence intensity. Three dpf zebrafish
(one, two, and three zebrafish per well) were deposited into 96-well plates and
incubated in substrate for 30min. Untreated zebrafish without substrate were used as
background controls. After subtracting nonspecific background, a linear relationship
between chemiluminescent signal and number of zebrafish per well was observed
(data not shown). These data further confirmed specificity and sensitivity of the
microplate-based whole zebrafish CYP3A4 assay.

< 0.001) in 2 but not in 3dpf zebrafish ( p >

9.4.3 Validation of Whole Zebrafish CYP
Microplate Assay

To further validate use of the microplate-based whole zebrafish CYP3A4 functional
assay for assessing drug metabolism and safety, using the optimum zebrafish stage
and assay procedures, we assessed five additional human CYP3A4 inhibitors, dis-
opyramide, erythromycin, fluvoxamine, omeprazole, and cimetidine, and six addi-
tional human CYP3A4 inducers, carbamazepine, hydrocortisone, prednisone, preg-
nenolone-16 -carbonitrile, lovastatin, and phenytoin (Gomez-Lechon et al., 2003;

Daly, 2004; Luo et al., 2004; Cali et al., 2006). We selected these drugs because CYP
effects have been evaluated either in mammalian models or in human clinical trials.
Drug treatment and the zebrafish CYP3A4 assay were performed as described
above. Zebrafish treated with 50

a

m M concentration of dexamethasone for 24 h were