Page 363 - The Toxicology of Fishes

Liver Toxicity 343

2d14h Ov
S2
L
E GB

Y
La
(A) 50 µm (B) 200 µm

FIGURE 7.12 (See color insert following page 492.) (A) In ovo imaging of medaka liver anlage (La) emerging from the
ventral endoderm 62 hours after fertilization. In medaka, this occurs below the first to third somite (S2). Distinct at this
stage of development are hepatic tubule formations, elucidated here with the cytochrome P450-3A substrate 7-benzylox-
yresorufin (7-BR). Embryonic medaka exposed to aqueous concentrations of 7-BR exhibit CYP3A activity, indicated by
the red fluorescence of resorufin (the metabolic byproduct of CYP3A, via dealkylation of 7-BR) in the tubule lumens of
the developing liver (red punctuate features). (B) By 5 days after fertilization, the liver (L) of medaka is found in a left
lateral orientation, with the gallbladder (GB) at the liver caudal margin. Red fluorescence in the liver and gallbladder is the
fluorescent CYP3A byproduct resorufin. Here, the fluorophore is seen in transit through the intrahepatic biliary passageways
of the liver, with concentration in the gallbladder. E, eye; Ov, otic vesicle; Y, yolk (sac).

Selected Landmarks of Fish Liver Development

The development of fishes is considered a simple model for understanding this process in other verte-
brates. Simplicity is derived from the fact that the chorion is transparent in certain fishes and provides
detail for imaging key events and relationships of development in vivo. One species that has been used
extensively as a model for studying vertebrate ontogeny is the zebrafish (Danio rerio), particularly for
the study of liver development (Field et al., 2003; Ober et al., 2003). Another model teleost species,
medaka, is an excellent experimental animal (Yamamoto, 1975). The see-through medaka is genetically
deficient in pigments (Wakamatsu et al., 2001) and is being studied as a model for morphological and
molecular investigations on organogenesis in the later stages of fish development and throughout the
life span. Recent published and unpublished observations of the see-through medaka, made in the Hinton
laboratory (Hinton et al., 2004), are used to illustrate the following: liver appearance in the embryo,
anatomical position of the embryonic and larval liver, and assumptions regarding the adult vascular
supply and drainage, as well as biliary function.
The liver of fishes arises from the ventral foregut. In the medaka (Figure 7.12), a bulge is seen at
somites one to three on the left side of the embryo during the second day after fertilization (Iwamatsu,
2004; Iwamatsu et al., 2003) (Table 7.4). This bulge continues to grow until the left lateral longitudinal
liver leaflet (L5) is formed (Figure 7.12 B). As described below, L5 is the liver of the embryo and early
larval stages. The caudal-most extent of this leaflet is closely associated with a prominent, round
gallbladder (Figure 7.12) the fluid of which becomes pigmented (Hinton et al., 2004), suggesting that
metabolism of yolk is linked to formation of bile in the embryonic medaka. L5 is the liver during mid-
and late stage embryos and extends into the first week following hatching (Hinton et al., 2004).
Unpublished electron microscopic observations from this laboratory reveal that bile canaliculi are present
on lateral plasma membranes of hepatocytes. In addition, in vivo microscopic analysis clearly shows
that the L5 liver contains hepatic tubules. As is shown in Figure 7.13, transverse sections of embryonic
tubules are comprised of six to eight hepatocytes whose apical plasma membranes are in contact with
the tubule lumen while their basal plasma membranes face the microvasculature; therefore, even during
early development, hepatocytes are polarized. Bile of the gallbladder and intestinal lumen fluoresces
under ultraviolet light and may be detected through the body wall and chorion of the embryo using
brightfield and widefield fluorescence microscopy (Figure 7.14). In addition, when embryos are exposed
to 7-benzyloxyresorufin, a cytochrome P450 substrate, the phase I enzyme cleaves the alkyl group from