Functional units in rainbow trout Salmo gairdneri Richardson liverII. The biliary systemкод для вставкиСкачать
THE ANATOMICAL RECORD 221:619-634 (1988) Functional Units in Rainbow Trout (Salmo gairdneri, Richardson) Liver: II. The Biliary System JAMES A. HAMPTON, R. CLARK LANTZ, PETER J. GOLDBLATT, DARREL J. LAUREN, AND DAVID E. HINTON 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.) ABSTRACT 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; et 1984; 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. 620 J.A. HAMPTON ET AL. 621 TROUT INTRAHEPATIC BILIARY SYSTEM 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. MATERIALS AND METHODS 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). Fixation 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% in 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- 622 J.A. HAMPTON ET AL. TROUT INTRAHEPATIC BKIARY SYSTEM 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. RESULTS Parenchymal Arrangement 623 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. 626 J.A. HAMPTON ET AL. 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. TROUT INTRAHEPATIC BILIARY SYSTEM 627 628 J.A. HAMPTON ET AL. 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. TROUT INTRAHEPATIC BILIARY SYSTEM 629 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. TROUT INTRAHEPATIC BILIARY SYSTEM 631 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, 632 J.A. HAMPTON ET AL. 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). DISCUSSION 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., 1962). 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. TROUT INTRAHEPATIC BILIARY SYSTEM 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 tubules. 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. ACKNOWLEDGMENTS 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. LITERATURE CITED Andrew, W. H. H. (1979) Liver. Studies in Biology No. 105. University Park Press, Baltimore, p. 1. Aoki, K., and H. Matsudaira (1977) Introduction of hepatic tumors in a teleost (Oryzias latipes) after treatment with methylazoxymethano1 acetate. J. Natl. Cancer Inst., 59:1,747-1,749. Ashley, L.M. (1973) Animal model: Liver cell carcinoma in rainbow trout. Am. J. Pathol., 72345-348. Ayers, J.L., D.J. Leed, J.H. Wales, and R.O. Sinnhuber (1971)Atlatoxin structure and hepatocarcinogenicity in rainbow trout (Salmo gairdneri). J. Natl. Cancer Inst., 46.561-564. Bockman, D.E. (1976) Anastomosing tubular arrangement of the exocrine pancreas. Am. J. Anat., 147:113-118. Bockman, D.E. (1981) Cells of origin of pancreatic cancer: Experimental animal tumors related to human pancreas. Cancer, 47tl5281534. Cohen, D.M. (1970) How many recent fishes are there? Roc. Calif. Acad. Sci., 38:341-345. Cormack, D.H. (1987)Ham's Histology Ninth Edition. J.B. Lippincott, Philadelphia, pp. 518-520. 633 Cornelius, C.E. (1985)Hepatic ontogenesis. Hepatology, 51213-1221. Couch, J.A., and J.C. Harshbarger (1985)Effects of carcinogenic agents on aquatic animals. An environmental and experimental overview. Env. Care. Rev., 3:63-105. Damjanov, I. (1987) Biology of disease: Lectin cytochemistry and histochemistry. Lab. Invest., 57:5-20. David, H. (1961) Zur submikroskopischen Morphologie intrazellularer Gallenkapillaren. Acta Anat., 47:216-224. Egami, N., Y. Kyono-Hamaguchi, H. Mitani, and A. Shima (1981) Characteristics of hepatoma produced by treatment with diethylnitrosamine in the fish, Oryzias latipes. In: Phyletic Approaches to Cancer. C.J. Dawe, J.C. Harshbarger, S. Kondo, T. Sugimura, and S. Takayama, eds. Japan Sci. Soc. Press, Tokyo, pp. 205-216. Franklin, R.B., C.R. Elcombe, M.J. Vodicnik, and J.J. Lech (1980) Comparative aspects of the disposition and metabolism of xenobiotics in fish and mammals. Fed. Proc., 39:3144-3149. Frasca, J.M., and V.R. Parks (1965) A routine technique for doublestaining ultrathin sections using uranyl and lead salts. J. Cell Biol., 25:157-161. Gingerich, W.H. (1982) Hepatic toxicology of fishes. In: Aquatic Toxicology, Vol. 1.L.J. Weber, ed. Raven Press, New York, pp. 55-105. Grisham, J.W. (1980)Cell types in long-term propagable cultures of rat liver. Ann. N.Y. Acad. Sci., 349:128-137. Hacking, M.A., J. Budd, and K. Hodson (1977) The ultrastructure of the liver of the rainbow trout: normal structure and modifications after chronic administration of a polychlorinated biphenyl Aroclor 1254. Can. J. Zool., 56:477-491. Hampton, J.R., P.A. McCuskey, R.S. McCuskey, and D.E. Hinton (1985) Functional units in rainbow trout (Salrno gairdneri, Richardson) liver. I. Histochemical properties and arrangement of hepatocytes. Anat. Rec., 213:166-175. Hand, A.R. (1976) Salivary glands. In: Orban's Oral histology and embryology. S.N. Bhaskar, ed. C.V. Mosby Co., St. Louis, pp. 328360. Hendricks, J.D. (1982) Chemical carcinogenesis in fish. In: Aquatic Toxicology, Vol. 1. L.J. Weber, ed. Raven Press, New York, pp. 149211. Hendricks, J.D., T.R. Myers, and D.W. Shelton (1984) Histological progression of hepatic neoplasia in rainbow trout (Salmo gairdneri). Natl. Cancer Inst. Monogr., 65:321-336. Hinton, D.E., and C.R. Pool (1976) Ultrastructure of the liver in channel catfish Ietalurus punctatus (Rafinesque). J. Fish Biol., 8:209219. Hinton, D.E., J.A. Hampton, and P.A. McCuskey (1985) The Japanese medaka (Oryzias latipes) liver tumor model: Review of literature and new findings. In: Water Chlorination Chemistry, Environmental Impact and Health Effects, Vol. 5. R.L. Jolley, R.J. Bull, W.P. Davis, S. Katz, M.H. Roberts,. Jr., and V.A. Jacobs, eds. Lewis Publishers, Inc., Chelsea, MI, pp. 439-450. Hinton, D.E., R.C. Lantz, and J.A. Hampton (1984a) Effect of age and exposure to a carcinogen on the structure of the medaka liver: A morphometric study. Natl. Cancer Inst. Monogr., 65239-249. Hinton, D.E., E.R. Walker, C.A. Pinkstaff, and E.M. Zuchelkowski (1984b) Morphological survey of teleost organs important in carcinogenesis with attention to fixation. Natl. Cancer Inst. Monogr., 65291-320. Hinton, D.E., R.C. Lantz, J.A. Hampton, P.R. McCuskey, and R.S. McCuskey (1987) Normal versus altered anatomy: Considerations in morphologic responses of teleosts to pollutants. Environmental Health Perspectives, 71:139-146. Ishikawa, T., T. Shimamine, and S. Takayama (1975) Histologic and electron microscopy observations on dimethylnitrosamine-induced hepatomas in small aquarium fish (Orpias latipes). J. Natl. Cancer Inst., 55:909-916. Ito, S., and M.J. Karnovsky (1968) Formaldehyde-glutaraldehydefixatives containing trinitro compounds. J. Cell Biol., 39:168a-l69a (Abstract). Ito, T., A. Watanabe, and Y. Takahashi (1962) Histologiche und cytologische Untersuchungen der Leber bei Fischen und Cyclostomata, nebst bemerkungen uber die Fettspecicherzellen.Arch. Histol. Jap., 22429-463. Jungermann, K. (1986) Functional heterogeneity of periportal and perivenous hepatocytes. Enzyme, 35: 161-180. Kiernan, I?. (1833) The anatomy and physiology of the liver. Phil. Trans. Roy. Soc. (London), 123:711-770. Lech, J.J., and M.J. Vodicnik (1984)Biotransformation of chemicals by fish An overview. Natl. Cancer Inst. Monogr., 65:355-358. McCuskey, P.A., R.S. McCuskev, and D.E. Hinton (1986) Electron microscopy of the hepatic sinusoids in rainbow trout (Salmo gairdnerz). In: Cells of the Hepatic Sinusoid. A. Kirn, D.L. Knook, and E. Wisse, eds. Kupffer Cell Foundation, Leiden, pp 489-494. 634 J.A. HAMPTON ET AL. Matsushima, T., and T. Sugimura (1967) Experimental carcinogenesis in small aquarium fishes. Prog. Exp. Tumor Res., 20:367-379. Matsumoto, T., R. Kornori, T. Magara, T. Ki, M. Kawakasui, T. Tokuda, S. Takasaki, H. Hayashi, K. Jo, H. Hano, H. Fujino, and H. Tanaka (1979)A study on the normal structure of human liver, with special reference to its angioarchitecture. Jikeikai Med. J., 26:1-40. Mugnaini, E., and S.B. Harboe (1967) The liver of Myxine gbtinosa: A true tubular gland. Zeit. Zellforsch., 78r341-369. Peek, W.D., E.W. Sidon, J.H. Youson, and M.M. Fisher (1979) Fine structure of the liver in the larval lamprey, Petromyzon marinus L.; hepatocytes and sinusoids. Am. J . Anat., 156:231-250. Pliss, G.B., and V.V. Khudoley (1975)Tumor induction by carcinogenic agents in aquarium fish. J. Natl. Cancer Inst., 55:129-136. Popper, H., and F. Schaffner (1957) Liver: Structure and Function. McGraw-Hill, New York, pp. 103-106. Rappaport, A.M. (1976) The microcirculatory acinar concept of normal and pathological hepatic structure. Beit. Path. Bd., 157:215-243. Roberts, R.J. (1975) Melanincontaining cells of teleost fish and their relation to disease. In: The Pathology of Fishes. Rebelin, W. and G. Migake, eds. University of Wisconsin Press, Madison, pp. 339-428. Scarpelli, D.G. (1967) Ultrastructural and biochemical observations of trout hepatoma. In: Trout Hepatoma Research Conference Papers. J.E. Halver and I.A. Mitchell, eds. U.S. Fish and Wildlife Service, Washington, D.C., pp. 60-71. Schmidt, D.C., and L.J. Weber (1973)Metabolism and biliary excretion of sulfobromouhthalein by rainbow trout. J. Fish. Res. Bd. Can., 30:1301-1308: Shore, T.W., and H.L. Jones (1889) On the structure of the vertebrate liver. J. Physiol. (London), 10:408-428. Simon, R.C., A.M. Dollar, and E.A. Smuckler (1967) Descriptive classification on normal and altered histology of trout livers. In: Trout Hepatoma Research Conference Papers. J.E. Halver and LA. Mitchell, eds. Res. Rept. #70, U.S. Fish and Wildlife Service, Washington, D.C., pp. 18-28. Sinnhuber, R.O., J.D. Hendricks, J.H. Wales, and G.B. Putnam (1977) Neoplasms in rainbow trout, a sensitive animal model for environmental carcinogenesis. Ann. N.Y. Acad. Sci., 298:389408. Stanton, M.F. (1965)Diethylnitrosamine induced hepatic degeneration and neoplasia in the aquarium fish, Branchydanio rerio. J. Natl. Cancer Inst., 34:117-130. Stegeman, J.J., B.R. Woodin, and R.L. Binder (1984) Patterns of benzdakyrene metabolism by varied species, organs and developmental stages of fish. Natl. Cancer Inst. Monogr., 65:371-377. Steiner, J.W., and J.S. Carruthers (1961) Studies on the fine structure of the terminal branches of the biliary tree. Am. J. Pathol., 38:639661. Sternlieb, I., and N. Quintana (1985) Biliary proteins and ductular ultrastructure. HeDatoloev. 5:139-143. Tanuma, Y. (1980) Electron &roscope observations on the intrahepatocytic bile canalicules and sequent bile ductules in the crucian, Carassius carassius. Arch. Histol. Jpn., 43r1-21. Trump, B.F., E.A. Smuckler, and E.P. Benditt (1961) A method for staining epoxy sections for light microscopy. J. Ultrastruct. Res., 5:343-348. Tsao, M-S., and J.W. Grisham (1987)Hepatocarcinomas, cholangiocarcinomas and hepatoblastomas produced by chemically transformed cultured rat liver epithelial cells. A light and electron microscopic analysis. Am. J. Pathol., 127:168-181. Tsao, M-S., J.D. Smith, K.G. Nelson, and J.W. Grisham (1984)A diploid epithelial cell line from normal adult rat liver with phenotypic properties of “oval” cells. Exp. Cell Res., 154~38-51. Venable, J.H., and R. Coggeshall(1965) A simplified lead citrate stain for use in electron microscopy. J . Cell Biol., 25:407408. Wachstein, M., and E. Meisel (1957) Histochemistry of hepatic phosphatases at a physiologic pH. Am. J. Clin. Pathol., 27:13-23. Wake, K. (1980) Perisinusoidal stellate cells (fat-storing cells, interstitial cells, lipocytes), their related structure in and around the liver sinusoids, and vitamin A-storing cells in extrahepatic organs. Int. Rev. Cytol., 66:303-353. Wales, J.H., R.O. Sinnhuber, J.D. Hendricks, J.E. Nixon, and T.A. Eisele (1978)Aflatoxin B1induction of hepatocellular carcinoma in the embryos of rainbow trout (Salmo gairdneri). J. Natl. Cancer Inst., 60:?133-1137. Wolf, K. (1963)Physiological salines for fresh-water teleosts. Prog. Fish Cult., 25:135-140. Yamamoto, T. (1965) Some observations of the fine structure of the intrahepatic biliary passages in goldfish (Carasszus auratus). Z. Zellforsch., 65:319-330. Youson, J.H. (1981)The liver. In: The Biology of Lampreys, Vol3. M.W. Hardisty, and I.C. Potter, eds. Academic Press, London, pp. 263332. Zimmerman, H.J. (1978) Hepatotoxicity. The adverse effects of drugs and other chemicals on the liver. Appleton-Century-Crofts, New York, pp. 47-90.