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Functional units in rainbow trout Salmo gairdneri Richardson liverII. The biliary system

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THE ANATOMICAL RECORD 221:619-634 (1988)
Functional Units in Rainbow Trout (Salmo gairdneri,
Richardson) Liver: II. The Biliary System
Department of Anatomy, School of Medicine, West Virginia Uniuersity, Morgantown, West
Virginia 26506 (J.A.H., R.C.L., D.E.H.); Department of Pathology, Medical College of Ohio at
Toledo, Toledo, Ohio 43699 (J.A.H.,P J . G.); Department of Medicine, School of Veterinary
Medicine, University of California, Dauis, California 95616 @.J.L,, D.E.H.)
The intrahepatic biliary system was studied in the rainbow trout
(Sulmo guirdneri), a teleost known to form liver neoplasms after exposure to various
carcinogens. Normal adults (N = 25) were examined using light microscopic, enzyme histochemical, and transmission and scanning electron microscopic methods.
In light micrographs, longitudinal arrays of hepatocytes appeared as double rows
incompletely divided by elongated darkly stained cells. Electron micrographs showed
tubules of five to nine pyramidally shaped hepatocytes with their apices directed
toward a central biliary passageway and their bases directed toward sinusoids.
Sequentially, beginning with hepatocytes, biliary passageways included canaliculi,
preductules, ductules, and ducts. Canaliculi were short and joined transitional
passageways (preductules) formed by junctional complexes between plasma membranes of hepatocytes and small, electron-dense cells with a high nuclear to cytoplasmic ratio. Ductules, completely lined by biliary epithelial cells, occupied central
regions of hepatic tubules. Relatively elongated, ductular cells were intimately
associated with surrounding hepatocytes, separated from them by only a thin extracellular space devoid of a basal lamina. Epithelium of bile ducts included cuboidal
through mucus-laden columnar cells, surrounded by basal lamina and, in larger
ducts, by fibroblasts, smooth muscle cells, and a capillary plexus. Bile ducts and
hepatic arterioles, but not venules, were distributed together. The ultrastructure of
biliary epithelium, periductular, and periductal cells is presented.
Teleost fishes possess hepatic mixed-function oxidase
systems comparable in many respects to those found in
mammals (Franklin et al., 1980) and that are active in
bioaccumulation and biotransformation of xenobiotics
nech and Vodicnik, 1984; Stegeman et al., 1984).Discoveries of tumor epizootics among feral teleost populations
indicate the need for further development of these as
models for studies in environmental carcinogenesis
(Couch and Harshbarger, 1985). In laboratory exposures
to known carcinogens, the rainbow trout (Salmo gairdneri), Japanese medaka (Oryzius Zutipes), and guppy
(Lebistes reticulutus) have shown sensitive tumorigenic
responses (Ayers et al., 1971; Pliss and Khudoley, 1975;
Aoki and Matsudaira, 1977; Sinnhuber et al., 1977;
Wales et al., 1978; Egami et al., 1981). The liver has
been a major site for tumor development (Matsushima
and Sugimura, 1976; Hendricks, 1982; Hendricks et al.,
1984) and neoplastic lesions frequently are composed of
ductule cells as well as hepatocytes (Stanton, 1965; Scarpelli, 1967; Ishikawa et al., 1975; Aoki and Matsudaira,
i977; ~ ~ ~ n h u et
b e r 1977;
Hinton et al., 1984a, b, 1985).
In an effort to define the normal StrUCtWe Of the liver
in one representative teleost, our studies have focused
0 1988 ALAN R. LISS, INC.
on arrangement and histochemical properties of hepatocytes (Hampton et al., 1985) and on cells of the sinusoidal wall (McCuskey et al., 1986) in the rainbow trout
(Salmo guirdneri). Enzyme histochemical localization of
magnesium-dependent adenosine triphosphatase was
compared in livers of trout and rat (Hampton et al.,
1985). The patterns of reaction product differed markedly between the two species and indicated structural
differences of the intrahepatic biliary system.
Since the bony fishes number approximately 20,000
species (Cohen, 19701, it is somewhat surprising that
morphologic descriptions of the liver in these vertebrates are so limited. Our literature review revealed a
partial description of the biliary system in two species
of the family Cyprinidue (David, 1961; Yamamoto, 1965;
Tanuma, 19801, and one each of the families Siluridae
(Hinton and Pool, 1976) and Salmonidue (Hacking et al.,
1977). The objectives of the present investigation were:
1) to examine thoroughly the intrahepatic biliary sysReceived June 18,1987; accepted November 19,1987.
Address reprint requests to David E. Hinton, Ph.D., Professor of
Fish Pathology, Department of Medicine, School of Veterinary Medicine, University of California, Davis, CA 95616.
Fig. 3. One-pm epoxy section illustrating circular (two) and ellipsoidal (one) profiles of hepatic
tubules. By tracing the trabecular profile of a hepatic tubule in a counterclockwise fashion
(from 1 to 2 to 3), the curvilinear extent of this structure can be appreciated. Note darkly
stained cells in central regions of tubules (arrows). Darkly stained particulate material in
apical hepatocyte cytoplasm (L)is presumptive lysosomes. Toluidine blue. x 918.
tem in a carcinogen-sensitive teleost species, 2) to correlate light microscopic and enzyme histochemical findings
with the ultrastructure Of Component cells, and 3) to
describe cellular relationships thereby facilitating determination Of rOle(S) Of Specific Cell types in neoplastic
alterations of liver.
Animal Care and Maintenance
Adult, 5-year-old male (N = 9) and female (N = l6)
rainbow trout (Salmo gairdneri) were obtained from the
National Fish Health Center, Kearneysville, wv. Conditions of maintenance were identical to those described
previously (Hampton et al., 1985).
Except where stated otherwise, fixative was administered by vascular perfusion. n o u t were anesthetized
with MS-222 (tricaine methane sulfonate, 50 mg/l;
Sigma) and the subintestinal tributary of the hepatic
portal vein was catheterized (PE 50). Blood was cleared
with chilled (15°C) oxygenated Cortland saline (pH 7.7)
(Wolf, 1963) at 5 mlkglmin (Schmidt and Weber, 1973).
The livers were then perfused with chilled (15°C) halfstrength Karnovsky’s fluid (It0 and Karnovsky, 1968;
light microscopy and transmission electron microscopy)
or buffered glutaraldehyde (2.5% in 0.05 M Na cacodylate, p~ 7.7; scanningelectron microscopy),for 15-30
minutes. Livers were then removed, sliced in chilled
fixative, and stored overnight at 0-4°C.
Tissue Processing
Fig. 1. Section of paraffin-embedded trout liver showing linear arrays of longitudinally oriented hepatic tubules separated by sinusoids
(S). Arrows point to h e a r arrays of darkly stained cells between
individual rows of hepatocytes. V-venule. H + E stained paraffin section (5 pm). ~ 2 5 0 .
Fig. 2. GMA-embedded trout liver after portal venous perfusion
fixation reveals ellipsoidal (common) and circular profiles (less common; see brackets) of hepatic tubules Cr). Arrows point to darkly stained
cells occupying central portions of cross and longitudinally sectioned
hepatic tubules. Methylene bluehasic fuchsin. X250.
Tissue slices (5 x 5 x 3 mm) from different regions of
each liver were sectioned (4-6 pm, paraffin; 1-2 pm,
GMA) and stained with hematoxylin and eosin (H+E)
or methylene blue and basic fuchsin. Additional tissue
slices from different regions of each liver were minced
(1 mm3), pooled, and postfixed for 1 hour in 1%
0 .1 M cacodylatebuffer p~ 7.2. At least 15 tissuepieces
from each liver were
and embedded in Epon
812 (E.F. Fullam, Inc., Schenectady, NY). Semithin (0.5-
1.0 pm) sections from 3 randomly selected blocks of each
liver were cut and stained with toluidine blue (Trump
et al., 1961). Thin sections were cut from 3 randomly
selected blocks of each liver, mounted on copper grids,
and stained with uranyl acetate (Frasca and Parks, 1965)
and lead citrate (Venable and Coggeshall, 1965).Stained
sections were examined under a Philips 410 electron
microscope at a n accelerating voltage of 80 or 100 kv.
For scanning electron microscopy, tissue slices were
quenched in isopentane at liquid Nz temperature and
fractured with precooled metal forceps. These pieces
were dehydrated in a graded ethanol series, critical point
dried, sputter coated, and fractured surfaces were observed with a Phillips 501 scanning electron microscope
at 15 kv.
Mg ++-DependentAdenosine Triphosphatase (ATPase)
To label luminal surfaces of plasma membranes within
the biliary system, the lead method of Wachstein and
Meisel (1957) was used. Following brief aldehyde fixation, liver tissue was frozen and 12-pm cryostat sections
were made. Following air drying, coverslips with sections attached were incubated at room temperature for
40 minutes a t pH 7.2 using adenosine 5’-triphosphate,
disodium salt (A-5394, Sigma) as substrate. Deposits of
lead sulfide (brown) marked sites of enzyme activity.
Controls included similarly processed rat liver sections
and incubation of sections (trout and rat) in the absence
of substrate.
Parenchymal Arrangement
Analysis of semithin sections stained by toluidine blue
(Fig. 3) confirmed and extended the above observations.
Circular and ellipsoidal profiles of hepatic tubules were
seen. In addition, bending and anastomosis of tubules
were suggested by the curvilinear path of longitudinally
oriented profiles, tapering of tubular profiles, and confluence of tubules (Fig. 3). When seen in transverse
section, 6 to 9 pyramidally shaped hepatocytes were
arranged in circular configuration with their bases directed toward sinusoids and their apices directed toward
a bile canaliculus (not resolved in Fig. 3) or toward
darkly stained cells (Fig. 3). Frequently, small, rounded,
darkly stained bodies (presumptive lysosomes) were seen
in apical hepatocyte cytoplasm. Regions of hepatocyte
cytoplasm, which stained with low to intermediate intensity with toluidine blue (Fig. 3),corresponded to large
glycogen deposits seen by electron microscopy or following periodic acid-Schiff s reagent staining (Hampton et
al., 1985).
A representative field from a fixed-frozen section of
trout liver reacted for M$ +-dependent ATPase is shown
(Fig. 4). Sinusoidal lumina did not stain, and these white
spaces contrasted with tubules of hepatocytes, the cytoplasm of which was a light gray color. Reaction product
was distributed as thin black lines midway across the
width of individual tubules or as black dots at the center
of circular or elliptical tubular profiles (Fig. 4). The
finest diameter lines were interpreted as canaliculi, and
two or more of these joined larger diameter lines corresponding to bile preductules. Remaining within the parenchyma, still larger, less numerous accumulations of
reaction product were considered to represent bile ductules (Fig. 4). The largest accumulations of reaction
product were distributed as circular profiles surrounding obvious lumina, which, in stained GMA preparations (Fig. 51, corresponded to intrahepatic bile ducts.
The transition between biliary ductule and bile duct
occurred with the appearance of a connective tissue
sheath surrounding a duct, a n hepatic arteriole, and
usually one or more pigment-laden melanomacrophages
(Roberts, 1975). These biliary-arteriolar tracts (BAT)
(Fig. 5) contained no venous profiles and demonstrated
the independent distribution of portal venous and biliary systems in trout liver. This pattern of distribution
differs from that of mammalian liver in which “portal
tracts” contain portal venule, bile ductule, a n hepatic
arteriole, and a lymphatic vessel (Popper and Schaffner,
1957; Steiner and Carruthers, 1961).
Following portal venous perfusion fixation, hepatic
sinusoids were cleared of blood cells and definition of
hepatic parenchyma was enhanced. A section from paraffin-embedded liver stained with H&E reveals features
common to trout hepatic parenchyma (Fig. 1).Longitudinal arrays of individual hepatic tubules were separated by sinusoids. Branching and anastomosis of
hepatic tubules were apparent. Within individual tubules, thin, darkly stained cells observed in longitudinal
sections were often encountered, and these appeared to
lie between individual rows of hepatocytes (Fig. 1).
The tubular arrangement of trout hepatic parenchyma
was even more apparent in sections (1-2 pm thick) of
GMA-embedded liver. Both circular and, more commonly, ellipsoidal profiles were seen (Fig. 2). Individual
tubules appeared to curve and anastomoses were occasionally encountered. Darkly stained cells commonly
Electron Microscopic Observations
occupied centrotubular sites (Fig. 2 arrowheads).
Bile canaliculi
Canaliculi were formed solely by junctional complexes
between plasma membranes of hepatocytes (Fig. 6). Lumina of canaliculi were nearly completely filled by hepatocyte microvillar processes. Filaments within indiFig. 4. Magnesium-dependent adenosine triphosphatase reaction
product in fixed-frozen section (8-10 pm) of trout liver. Reaction prod- vidual microvilli continued into the apical cytoplasm of
uct is localized over canaliculi (tips of arrows, the finest diameter hepatocytes and contributed to a nearly complete terlines), bile preductules (BPI, biliary ductules (BD), and bile ducts (D). minal web (not shown in figures). Circular vesicles
Note intratubular location of canaliculi, preductules, and ductules. within the terminal web fused with the plasma memV = venule. ~ 9 6 .
brane, and although not confirmed by this study, appear
Fig. 5. Biliary-arteriolar tracts (BAT) are frequent within trout liver. to provide a n apical transport system between hepatoContained within a common connective tissue sheath, a BAT is com- cyte cytoplasm and canalicular lumen. Parallel arrays
posed of a small branch of the hepatic artery (A) and one or more bile of granular endoplasmic reticulum (GER) extended apiducts (BD). M = melanomacrophages. Note darkly stained cells (arrows) in central regions of hepatic tubules (T). Methylene bluehasic cally to contact the terminal web. Here, vesicles associated with peripheral GER appear to give rise to apical
fuchsin stained GMA section (1-2 pm). ~ 5 0 0 .
Fig. 6. Transmission electron micrograph of trout liver illustrating a bile canaliculus (BC)
formed by 3 hepatocytes whose plasma membranes are joined at junctions (TI. Apical vesicles
(AV) are within the hepatocyte terminal web and fusing with plasma membrane (arrow). Golgi
(G)apparatus; N = hepatocyte nucleus; SER = smooth endoplasmic reticulum; Ly = lysosome;
M = mitochondria. Uranyl acetate and lead citrate. X19,880.
Fig. 7. Joined to hepatocytes by junctions (TIand desmosomes (D), bile preductular cells (BP)
are small, contain scant cytoplasm, and possess processes that extend between adjacent hepatocytes (HI. Processes of bile preductular cells are joined to other bile preductular cells by
junctions (arrows). A basal lamina is absent. A Golgi apparatus (G) and apical vesicles (AV) are
observed in hepatocytes. M = bile preductular cell mitochondria. Uranyl acetate and lead
citrate. ~22,600.
Fig. 8. High magnification view of cytoplasm from a bile preductular cell. Cytoplasm contains
tonofilaments associated with desmosomes (D), and endocytotic vesicles (arrow), smooth surfaced vesicles (V), sparse granular endoplasmic reticulum (GER), and polyribosomes (P).
H = hepatocyte, N = preductular cell nucleus, C = canaliculus filled with hepatocyte microvilli. Uranyl acetate and lead citrate. ~ 3 1 , 2 0 0 .
vesicles. Golgi apparatus and associated vesicles (Fig. 6;
see also Fig. 8>,mitochondria and electron-dense bodies
were prominent. Electron-dense bodies were identical in
shape and appearance to hepatocyte lysosomes of channel catfish (Ictalurus punctatus) demonstrated positive
for aryl sulfatase and acid phosphatase (Hinton and
Pool, 1976).Although glycogen-richareas extended close
to lateral cell membranes of hepatocytes (Fig. 61,glyco‘The terms “bile preductule” and “bile preductular cell” were used
in preference to “terminal bile ductule” in a n effort to present elements of the biliary tree in the sequence with which bile is formed
and transported. Based on location with respect to hepatocytes, these
units are centrotubular comprising a transition from canaliculus to
ductule, the initial bile passageway completely surrounded by biliary
epithelial cells. Steiner and Carruthers (1961) considered the term
“canaliculus” or “canal” to refer to a passage that possesses no
specialized lining cell, whereas the term “ductule” or “duct” implies
a channel provided with its own lining cells. Furthermore, these
workers used the terms “bile preductule” and “Canal of Hering”
synonymously to designate connecting channels between bile canaliculi in lobules and bile ductules in portal tracts. Due to the further
extent of biliary epithelial cells into the tubules of hepatocytes and
due to the absence (Gingerich, 1982) or less apparent nature (Simon
et al., 1967) of lobules within trout liver, it may be impossible to
construct a precise analogy of trout and mammalian Canals of Hering. Based on the position of a connective tissue sheath, we propose
the connection between trout bile ductule and the initial portion of
the bile duct a s analogous to the mammalian Canal of Hering.
gen was excluded from apical regions. The apical-most
extent of glycogen areas tapered to a point and contained relatively abundant vesicular profiles of granular
and smooth endoplasmic reticulum (Fig. 6).
Bile preductules’
When orientation of hepatic tubules was longitudinal,
fields frequently contained two or more modified canaliculi (Fig. 7). Whereas the contents of opposed apical
hepatocyte cytoplasm were the same as above, an additional cell type was seen (Fig. 7). This elongated cell
contained a single, elongated nucleus (Fig. 7) and cytoplasm containing polyribosomes, oval-shaped mitochondria, intermediate (10 nm diameter) filaments, microtubules, smooth membranes, and vesicles (Figs. 7, 8).
Fig. 9. TEM of biliary ductule (BD) formed by 2 or more epithelial
cells. No basal lamina between ductule cells and hepatocytes is seen.
Desmosomes (D) are frequent. Canaliculus (C) lies within a single
hepatocyte. Biliary ductular cells contain mitochondria, dense bodies,
tonofilaments associated with desmosomes, intermediate filaments (10
nm), polyribosomes, smooth membranes, vesicles, and a well-developed
terminal web P W ) . Arrows indicate the intimate contact between
ductular epithelium and hepatocytes. Uranyl acetate and lead citrate.
x 16,500.
Fig, 10. Scanning electron micrograph of fractured surface of trout liver. Hepatocytes are
arranged as tubules (T) with their apices directed toward bile canaliculi, bile preductules
(arrows), or ductule (D). Sinusoids ( S )separate adjacent tubules and are related to basal portions
of hepatocytes. X5,OOO
Based on its position between canaliculi and subsequent
ductules, this cell has been named bile preductular epithelial cell (BPE) (Steiner and Carruthers, 1961).
In trout liver, BPE shared apical junctional complexes
with hepatocytes (Fig. 7) forming a portion of the wall of
bile preductules. In addition, desmosomes were abundant between the two cell types. Between hepatocytes
and BPE, a thin uniformly shaped intercellular space
showed no evidence of a basal lamina or other intervening structure (Figs. 7,8). Microvilli of preductules occupied apparently enlarged luminal spaces. Bases of
microvilli were connected only to hepatocytes (Figs. 7,
8).Whereas apical cytopIasm of hepatocytes contained a
terminal web with an apical caveolar complex (Fig. 71,
only a slight specialization was apparent in the apical
cytoplasm of bile preduetular cells (Fig. 8).
lamina, providing morphologic evidence of the retained
intimacy of the two cell types in trout liver. Electron
density of biliary epithelial cells varied but was always
greater than that of surrounding hepatocytes. Analysis
of the cytoplasm of ductular epithelial cells showed occasional dense bodies, relatively few but elongated mitochondria, tonofilaments associated with desmosomes,
intermediate filaments, smooth membranes, and vesicles (Fig. 9). Near the lumen a well-defined terminal
web was observed (Fig. 9). Our ATPase histochemistry
suggests that bile ductules receive preductules and canaliculi directly (Fig. 5).
Fig. 11. Low magnification view of anastomosing biliary channels at
the transition between a biliary ductule and bile duct (BD). The chanBile ductules
nels are formed by cuboidal epithelial cells joined apically by tight
As diameters of bile preductules enlarged, additional junctions and basally by desmosomes. Short sparse microvilli project
biliary epithelial cells contributed to the channel wall. into the channel. A wispy basal lamina is present (arrows). SurroundThe transition from bile preductule to ductule occurred ing the epithelial cells are periductal fibroblasts and stellate fat-storwhen the biliary lumen was completely surrounded by ing cells (FSC). S = sinusoid. Uranyl acetate and lead citrate. ~8,100.
biliary epithelial cells, usually two or three, joined by
Fig. 12. High magnification of epithelium illustrated in Figure 11.
junctional complexes (Fig. 9). Ductular lumina were usu- Cytoplasm is electron dense and contains mitochondria (MI, granular
reticulum (GER), polyribosomes (PI, dense bodies, smooth
ally patent and contained few to no microvilli. Where endoplasmic
vesicles (V), tonofilaments associated with desmosomes, and
adjacent biliary epithelial cells overlapped, desmosomes surfaced
other intermediate filaments. Apically, a well-developed terminal web
were seen (Fig. 9). Analyses of space between hepato- (TW) with transport vesicles is observed. BM = indistinct basal lamcytes and epithelial cells of ductules showed no basal ina, FSC = fat-storing cell. Uranyl acetate and lead citrate. X20.160.
Fig. 13. Electron micrograph of a large bile duct illustrating columnar epithelium with welldeveloped luminal microvilli (MV) and apical mucous granules (Mu). Apically, adjacent cells
are joined by a junctional complex and more basal demosomes are seen. Macrophages (M)are
present basally between epithelial cells. A well-developed basal lamina (BL) separates epithelium from peribiliary smooth muscle cells (SMC) and fibroblasts (F). C = capillary with nucleated red blood cell. Uranyl acetate and lead citrate. X3,800.
Fig. 14. Perisinusoidal, interhepatocytic macrophage of trout liver CM). BD = ductuIe, H =
hepatocytes, S = sinusoidal lumen. Uranyl acetate and lead citrate. X6,OOO.
Scanning electron microscopy of fractured surfaces in
trout liver supported the tubular, glandular arrangement of hepatocytes (Fig. 10).In addition, biliary epithelial cells were clearly demonstrated in a centrotubular
location. An apparently small ductule (Fig. 10)appeared
to increase in size as contributions were made from bile
preductular cells (Fig. 10, arrows), which separated apical portions of adjacent hepatocytes. Sinusoids were related to basal aspects of hepatocytes.
Bile ducts
Cuboidal epithelial cells formed the wall of larger
structures identified as bile ducts (Fig. 5). The transition
between ductules and ducts was characterized by the
presence of a basal lamina separating epithelial cells
from surrounding periductal cells (Figs. 11, 12). Periductal cells either contained lipid and appeared identical to
stellate, fat-storing cells of the space of Disse (Wake,
1980; McCuskey et al., 19861, or, particularly around
larger ducts, took on characteristics of fibroblasts (Fig.
11). Apically, lateral surfaces of adjacent small duct cells
were attached by junctional complexes (Fig. 111, whereas
desmosomes were observed more basally (Figs. 11, 12).
Cells displayed a sparse number of short but broad luminal microvilli. As small ducts enlarged, interdigitations of lateral plasma membranes became more
apparent. Basally located, nuclei of small duct cells were
somewhat flattened with a n irregular ovoid contour.
The cytoplasm, like that of other biliary cells, was electron dense and characterized by intermediate filaments,
dense bodies, mitochondria, smooth membranes, and
vesicles (Fig. 12). A well-developed terminal web in apical cytoplasm showed occasional vesicles (Fig. 12).
Epithelial cells of larger bile ducts were columnar and
a distinct basal lamina separated epithelium from peribiliary connective tissue (Fig. 13). A peribiliary capillary
plexus surrounded large ducts. Collagen and nerve fiber
bundles were intermingled with fibroblasts and smooth
muscle cells (Fig. 13). We concur with Tanuma (1980)
that smooth muscle cells associated with bile ducts may
provide for peristaltic movement of bile. Extensive interdigitations of the lateral walls of adjacent epithelial
cells were seen in some but not all ducts. Apically, lateral surfaces of adjacent duct cells were attached by
terminal bars, whereas basally desmosomes were observed (Fig. 13). Duct cells displayed well-developed luminal microvilli, irregularly shaped nuclei, and mucous
granules (Fig. 13). A well-developed apical terminal
web was seen. Basal cytoplasm of duct cells displayed a
well-developed Golgi apparatus, elongate mitochondria,
smooth membranes and vesicles, and relatively sparse
GER (data not shown). Basally located cells of low electron density (Fig. 13)showed macrophagelike characteristics. In addition to positions within the space of Disse,
macrophages were observed to extend to biliary ductular elements and separate the latter from surrounding
hepatocytes (Fig. 14).
The observations of this study are consistent with
a n anastomosing tubular arrangement of hepatocytes,
which is identical to that described in cartilaginous and
bony fishes by anatomists of the past century (see Shore
and Jones, 1889). In this arrangement, transverse sections reveal as few as 4 and as many as 9 hepatocytes
clustered about a biliary passageway, with their apices
directed toward the biliary lumen and their bases associated with sinusoids across the perisinusoidal space of
Disse. Similar patterns have been described for the hagfish, Myxine glutinosa (Mugnaini and Harboe, 1967), the
larval lamprey, Petromyzon marinus (Peek e t al., 1979;
Youson, 19811, and the eel Anguilla japonica (It0 et al.,
Elements of the intrahepatic biliary system of trout
bear similarity to certain mammalian glands. Although
intercalated duct cells of salivary glands do not extend
into the acini, their position, intermediate between serous acinus and striated duct (Hand, 19761, is similar to
that of cuboidal cells lining small bile ducts of trout
liver. The centrotubular position of bile preductular and
ductular epithelial cells resembles that of the centroacinar cell of the exocrine pancreas (Cormack, 1987).
Furthermore, analysis of serial sections of the rat exo-
crine pancreas and of the wax reconstructions made
from them showed the arrangement of zymogen-containing cells of that organ to be a continuous branching
mass of tubules that anastomosed with each other (Bockman, 1976). This arrangement provided a basis for understanding the apparent proliferation of ducts and the
concomitant decrease in “acini” observed during induction of pancreatic tumors (Bockman, 1981). Interestingly, Andrew (1979) postulated that the liver probably
originated as a digestive gland but added new functional
dimensions involving excretion of specific compounds,
metabolism and storage of nutrients, and homeostasis
of the organism’s internal environment. Hepatic and
pancreatic similarities between invertebrate digestive
or midgut gland(s) and the vertebrate exocrine pancreas
have been recently reviewed (Cornelius, 1985).
Bile ducts of trout liver accompanied arterioles and
were ensheathed in connective tissue. However, venule
profiles were not included. This condition, first described
by Shore and Jones (1889) in flounder and eel, has been
confirmed by two more recent reviews (Ashley, 1973;
Gingerich, 1982) of hepatic histology including findings
in additional teleost species? From a practical standpoint, with conventional histologic preparations, that
feature that provides the investigator with a means of
differentiating portal venules from their hepatic venule
counterparts in the mammalian liver is absent in teleost
liver. This difference makes it difficult to determine
functional units, lobules (Kiernan, 1833),acini (Rappaport, 19761, or modified lobules (Matsumoto et al., 1979)
in the tubular liver of teleosts. We are presently using
microvascular casting and in vivo microscopy (Hinton et
al., 1987) in trout exposed to reference hepatotoxicants
of known zonal toxicity for mammals (Zimmerman, 1978)
to identify teleost analogues to periportal versus perivenous (Jungermann, 1986) regions.
The basic arrangement of bile canaliculi drained by a
hierarchy of ducts is identical in livers of fishes and
mammals; however, the extensive length of the bile
preductular and ductular cells within the hepatic tubule
is a significant difference. In the mammal, the major
intralobular biliary structure is the canaliculus (Popper
and Schaffner, 1957; Steiner and Carruthers, 1961). Intralobular ductules or bile preductules (Steiner and Carruthers, 1961) a t the lobule periphery connect canaliculi
with ductules inside portal tracts. According to Popper
and SchafTner (19571, the eponym, Canal of Hering, is
used by some to describe only the junction between
canaliculi and ductule, and by others the entire ductule.
Based on the above, the portion of the trout intrahepatic
biliary system more closely analogous to the Canal of
Hering is the ductule, and the smallest diameter duct
into which it drains.
Although once regarded as a n inert conduit (Popper
and Schaffner, 19571, biliary epithelial cells have recently been proposed a s a route whereby plasma-derived
proteins may reach the bile (Sternlieb and Quintana,
1985).The anatomical intimacy of peribiliary capillaries
2Despite a lack of histological evidence in conventionally processed
tissues, it is common in histopathologic reports of responses of fish
liver to toxicants to find usage of such terms as centrolobular, periportal, and zonal. Until a means to unequivocally identify afferent and
efferent venules in teleost hepatic parenchyma is forthcoming, use of
these mammalian terms should be avoided.
with bile ducts of trout could permit selective exchanges
of contents between the two compartments. Indirect evidence for this hypothesis is based on the prompt recovery, within time intervals that seem too brief to allow
for passage via both the parenchymal cells and the void
volume of the biliary tree, of radiolabeled macromolecules in bile (Sternlieb and Quintana, 1985). Trout biliary ductules may be accessible by isolation from liver
parenchyma or by micropuncture in superficial hepatic
The final objective of this study was to provide information that might be used in determining role(s) of
specific cell types in liver neoplasia. Although hepatocellular and cholangiocellular tumors are often found in
the same liver following exposure of trout to various
hepatocarcinogens (Hendricks et al., 19821, the contribution of the various segments of the intrahepatic biliary system to such lesions is not known. By electron
microscopy correlated with immunohisto- and cytochemical methods, selective labeling of specific cell types may
be achieved (Damjanov, 1987). Presence or absence of
intermediate filaments, mucous granules, basal lamina,
pericytes, connective tissue, and arterioles, sharing of
junctional complexes with hepatocytes, and type and
relative extent of plasma membrane specializations are
some of the potential identifying characteristics emanating from the present study.
Bile ductular epithelial cells have been shown to be
the cell of origin for oval cells and long-term cultures of
hepatic epithelial cells in rats (Grisham, 1980; Tsao et
al., 1984). When the latter were exposed in vitro to the
direct-acting carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine, and subsequently injected at subcutaneous
and intraperitoneal sites within 1-day-old isogeneic
Fischer-344 rats, hepato- and cholangiocarcinomas resulted (Tsao and Grisham, 1987). The stem cell characteristics of these biliary epithelial cells were further
suggested by the growth of mixed epithelial-mesenchyma1 tumors and sarcomata (Tsao and Grisham, 1987).
Similar studies in selected fish species are proceeding.
This project was supported by grant number PCM8316002 from the National Science Foundation, grant
number CR-811017 from the United States Environmental Protection Agency, and by NCI grant CA-45131from
the United States Public Health Service.
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