Page 361 - The Toxicology of Fishes

Liver Toxicity 341

following vascular perfusion fixation (McCuskey et al., 1986) were used to study fenestrae. In goldfish
(Cyprinus carpio), 15 to 20 fenestrae were grouped as sieve plates, like their mammalian counterparts
(Wisse, 1972). Trout fenestrae were not as tightly grouped as those of carp. When their diameters were
estimated, they ranged from 75 to 150 nm (McCuskey et al., 1986).
Sinusoidal endothelium has proved to be particularly reactive for alkaline phosphatase in enzyme
histochemical preparations. In addition, the antibodies directed against the cytochrome P450 isozymes
LM2, LM4, and CYP1A label endothelial cells (Lorenzana et al., 1989). Location immediately adjacent
to blood cells and the presence of attenuated cytoplasm helps to differentiate endothelial cells from
neighboring cells; for example, the strong response with alkaline phosphatase assists in differentiating
endothelial cells from hepatic stellate cells (Hinton, 1993b). Little attention has been given in fish to
differentiation of venous from arterial endothelium of liver.

Hepatic Stellate Cells
The morphology and functional properties of Stellate cells in various vertebrates have been reviewed
(Hinton, 1993b; Wake 1980). The connective tissue cells of the teleost liver include hepatic stellate cells
(Ito and Nemoto, 1952), fibroblasts associated with the hilus of the liver, and cells incorporated as a
sheath around major blood vessels and bile ducts and located immediately beneath the liver capsule and
the blood cells. These cells (see Figure 7.10) and the fibroblasts provide a supportive framework for the
liver. Hepatic stellate cells contain an elongated and frequently indented (by lipid droplets, the solvent
of vitamin A) nucleus with peripheral heterochromatin. Well-developed granular endoplasmic reticulum
with dilated cisternae also characterizes these cells (Rocha et al., 1997). In one significant review, livers
from 48 species of fishes were investigated, and fat-storing cells were reported in all but three species
(Ito et al., 1962). Since then, two studies (Hinton et al., 1984b; Lauren et al., 1990) have demonstrated
the presence of fat-storing cells in medaka (Oryzias latipes), one of the three species originally reported
to lack them. Another variant of the hepatic stellate cell is devoid of lipid droplets (Nopanitaya et al.,
1979a). These cells are fibroblast-like cells with well-developed granular endoplasmic reticulum (Ito
and Shibasaki, 1968). First described in human adults and embryos, the cells have been described in
various fishes and reptiles. In teleosts, injection of vitamin A causes the number of fibroblast-like cells
to decrease while the number of lipid-laden cells increases (Fujita et al., 1980; Takahashi et al., 1978).
Perhaps the heaviest accumulation of lipids is found in the Antarctic fish Dissostichus mawsoni (Eastman
and deVries, 1981). Numerous desmosomal junctions between hepatic stellate cells and other liver cells
suggest a mechanical, supporting role for the cells. Numerous cytofilaments are also distinguishing
features of these cells. If the cytofilaments are contractile, as has been suggested, the orientation of the
hepatic stellate cells may have a role in the regulation of blood flow within the sinusoid. The contribution
of these cells to the volume of the parenchyma was estimated for trout (Hampton et al., 1989); they
occupied 1.37% of the parenchymal space of females, and the male hepatic stellate cell compartment
was about half that of females. Actual volume may be underestimated, as cellular processes are elongated
and the terminal portions may be difficult to differentiate from portions of other cell types.
Vitamin A shows apple-green autofluorescence, which fades upon continued excitation. Alcian blue
pH 2.5 is positive for acidic glycosaminoglycans of fat-storing cells. When present, lipid vacuoles stain
positively with any of a variety of lipid stains. Semi-thin sections of Epon-embedded material stained
with toluidine blue reveal the lipids of fat-storing cells as aquamarine droplets. To differentiate hepatic
stellate cells from others, their location, shape, and contents (or lack thereof) have been found to be
useful. Differentiation of hepatic stellate cells from endothelial cells appears to be the greatest challenge.
Their common origin (mesoderm) and the presence of cytoplasm, vesicles, and cytofilaments illustrate
the degree of shared features of the cell types. Alkaline phosphatase, positive in endothelial but absent
in fat-storing cells, may be the strongest differentiator at present for fish.

Interhepatocytic, Perisinusoidal Macrophages
Interhepatocytic, perisinusoidal macrophages appear as single cells and are often devoid of pigment. They
have been reported in rainbow trout (Oncorhynchus mykiss) (Hampton et al., 1985; McCuskey et al.,
1986) and European brown trout (Salmo trutta fario) (Rocha et al., 1997). Interestingly, when quantitative