Whole Zebrafish Cytochrome P450 Assay for Assessing Drug Metabolism and Safety - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 101

synthetic steroid, upregulated zebrafish CYP3A gene expression, whereas nifedipine
did not. In addition, the antibiotic clotrimazole, a human CYP3A inducer, increased
zebrafish CYP3A expression (Bresolin et al., 2005). These findings suggest that
zebrafish CYP3A65 exhibits the same functions as human CYP3A4.
9.2.3 CYP Experimental Models
During the past decade, a number of CYP assays for assessing drug metabolism have
been developed (Luo et al., 2004). Most of these approaches rely on use of subcellular
liver fractions or human hepatocytes. However, results using these in vitro assays
have been shown to differ from results in vivo (Gomez-Lechon et al., 2003; Vermeir
et al., 2005). CYP assays in rat and mouse models, including LPTA CYP3A4-luc
transgenic mice, have recently been investigated; however, reliance on complex
technologies and low throughput make them largely unsuitable for drug screening
(Zhang et al., 2003, 2004; Gonzalez and Yu, 2006).
Although LC/UVis generally considered to be the gold standard for assessing drug
metabolism, this method is relatively slow, expensive, and low throughput. A second
widely used method relies on synthetic fluorogenic CYP substrates. Although adapted
for high-throughput screening, these substrates are not pharmaceutically relevant, and,
given the complexity of CYP active sites, in order to fully explore their potential for
CYP inhibition or induction, test compounds may require use of five separate
fluorogenic substrates. Furthermore, results using fluorogenic substrates to assess a
wide range of CYP3A4 inhibitors were less predictive than results using LC/UV
(Gomez-Lechon et al., 2003; Vermeir et al., 2005; Yueh et al., 2005; Cali et al., 2006).
For recently developed luminogenic assays, CYP enzyme activity is coupled to
the light generating reaction of firefly luciferase (Cali et al., 2006). These in vitro
assays were initially designed to measure CYP activity for recombinant CYPs, liver
microsomes, or hepatocytes. Compared to HPLC and radiochemical-based methods,
luminogenic CYP assays are rapid and safe. Compared to fluorogenic methods, this
approach offers high sensitivity and low interference between optical properties of
test compound and substrate. These homogenous assays are robust tools for high-
throughput screening for early drug discovery. Development of new rapid assay
methods and animal models for assessing drug metabolism and predicting potential
drug interactions are needed. A rapid and robust whole zebrafish CYP assay,
amenable to automation in a microplate format (Serbedzija et al., 2003), will speed
up drug metabolism and safety profiling and reduce the possibility of market
withdrawal or termination in late stages of drug development.
9.3 MATERIALS AND METHODS
9.3.1 Zebrafish Handling
Phylonix AB zebrafish were generated by natural pairwise mating in our aquaculture
facility, as described by Westerfield (1993). Four to five pairs were set up for each