Biomedical Engineering Reference
In-Depth Information
mating; on average, 50-100 zebrafish per mating were generated. Zebrafish were
maintained in fish water (5 g of Instant Ocean Salt with 3 g of CaSO
4
in 25 L of
distilled water) at 28
C for 24 h before sorting for viability. Because the zebrafish
embryo receives nourishment from an attached yolk sac, no additional maintenance
was required.
9.3.2 Reagents and Drugs
CYP3A4 chemiluminogenic luciferin-BE substrate and luciferin detection reagent
were purchased from Promega (Madison, WI). CYP inhibitors and inducers and
controls (azamulin, dexamethasone, rifampicin,
-naphthoflavone, disopyramide,
erythromycin, fluvoxamine, omeprazole, and cimetidine, carbamazepine, hydrocor-
tisone, prednisone, pregnenolone-16
a
-carbonitrile, lovastatin, and phenytoin, and
DMSO) were obtained from Sigma (St. Louis, MO).
a
9.3.3 Drug Treatment
Stock solutions of chemicals/drugs were diluted directly in fish water. Two dpf
zebrafish were treated with five drug concentrations (0.01, 0.1, 1, 10, and 100
M) for
24 h. Ten zebrafish per concentration were exposed in a total volume of 1 mL (ratio of
100
m
L/zebrafish) using 24-well plates. Zebrafish were maintained in test medium
throughout the experiments. Controls with and without vehicle solvent were included
in all experiments. To protect compounds from light-induced decomposition, experi-
ments were carried out at a constant temperature (28
C) in the dark.
m
9.3.4 Zebrafish CYP3A4 Functional Activity Assay
After drug treatment, zebrafish were transferred to untreated, white, opaque, flat-
bottom96-well microplates, one zebrafish per well. Excess fishwater was removed by
pipetting and then 25
m
L Millipore filtered water and 25
m
L of 400mM KH
2
PO4-
100
M luciferin-BE substrate were added to each well. Microplates were incubated at
37
C for 30min and 50
m
L of reconstituted luciferin detection reagent was added. Final
KPO
4
concentration was 200mM and final luciferin-BE substrate concentration was
50
m
M. Sampleswith no substratewere used as background control. After mixing on an
orbital shaker for 10 s or gently tapping the plate, luminescence was recorded with a
microplate reader. Net chemiluminescence was calculated by subtracting background
luminescence signal from the no substrate negative control wells. Experiments were
performed at least three times and final results were expressed as mean
m
SD.
9.3.5 IC
50
and EC
50
Estimation
Best-fit concentration-response curves for CYP3A4 inhibition or induction were
generated using the linear regression function of JMP statistical software (SAS, Cary,
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