Zebrafish: A New In Vivo Model for Identifying P-Glycoprotein Efflux Modulators - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 171

caffeine, did not cause significant inhibitory effects. These results were similar to results
in mammalian models and 100% correct prediction is considered “excellent” by the
Interagency Coordinating Committee on the Validation of Alternative Methods
(ICCVAM). A compelling advantage of using zebrafish for this application is that
animals are transparent throughout development and level of fluorescence, which
correlates with drug efflux in the brain region, can be quantitated in vivo .
14.1.1 Brain ABC Transporters Play a Role
in Drug Resistance
ABC efflux transporters, present in several tissue compartments, including the
gastrointestinal tract and brain microcapillary endothelial cells, have been shown
to inhibit drug absorption and retention. Seven different brain ABC transporters,
including ABCB1, ABCC1, ABCC2, ABCC3, ABCC4, ABCC5, and ABCG2 (Dean
et al., 1997), are known to play a role in drug resistance; ABCB1/MDR1/Pgp,
ABCC1/MRP1, and ABCG2 are the most well characterized. Several ABC trans-
porters have been identified at the BBB and the blood cerebrospinal fluid barrier
(BCSFB) (Loscher and Potschka, 2008), where they are found in the plasma
membranes on both the luminal and abluminal sides of microcapillary endothelial
cells (de Vries et al., 2001). ABCB1/MDR1, also known as Pgp, is the most widely
studied efflux transporter; it is an important component of active efflux transport
(AET) and serves as a gatekeeper to actively efflux small molecules from the brain to
the blood (Tsuji et al., 2006; Stewart et al., 2001; Schinkel, 1999). Three factors make
Pgp a key factor regulating drug entry into the brain: location, potency, and broad
specificity (Begley, 2004). Localization of Pgp on brain microvessels is consistent
with its role as an efflux modulator. Potent ATP-driven pumping prevents drug
accumulation in the brain. Broad Pgp specificity ensures that it can pump out a large
number of drugs fromdifferent classes (Bauer et al., 2005) and Pgp inhibition has been
shown to lead to drug retention. Underscoring the important role Pgp plays in drug
efflux, a Pgp knockout mouse exhibited a hundred-fold increase in drug absorption
and a significant decrease in drug elimination from the CNS (Schinkel et al., 2002;
van Asperen et al., 1998).
14.1.2 Zebrafish: A Transparent Model for
Investigating Drug Effects on ABC Transporters
The BBB, present in all vertebrates, has been confirmed in zebrafish (Cserr and
Bundgaard, 1984; Jeong et al., 2001). In previous studies (Fig. 10.6), using ZO-1
antibody staining and Evans blue dye injection into the circulation, at 3dpf, presence
of tight junctions was confirmed and blockage of dye diffusion from the vasculature to
brain tissue was observed (McGrath et al., 2004). Using a monoclonal antibody, Pgp
expression was detected in zebrafish liver at 5dpf (Sadler et al., 2010). In addition,
a phylogenetic study identified two genes encoding Pgp, ABCB1a and ABCB1b
(Annilo et al., 2006), and Pgp expression has also been detected in adult zebrafish