Development and characterization of a rainbow trout liver cell line ...

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DEVELOPMENT AND CHARACTERIZATION OF A RAINBOW TROUT LIVER CELL LINE EXPRESSING CYTOCHROME P450-DEPENDENT MONOOXYGENASE ACTIVITY LUCILA E.J. LEE 1, JANINE H. CLEMONS 2, DANIEL G. B E C H T E L 1, SARAH J. C A L D W E L L 1, KYU-BO HAN 1, MARIA PASITSCHNIAK-ARTS I, DICK D. MOSSER 2, and NIELS C. BOLS 2

1 Department of Veterinary Anatomy, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada 2 Department of Biology, University of Waterloo, Waterloo, Ontario, Canada A ceil line, RTL-W1, has been developed from the normal liver of an adult rainbow trout by proteolytic dissociation of liver fragments. R TL-W1 can be grown routinely in the basal medium, L-15, supplemented with 5% fetal bovine serum. In this medium, the cells have been passaged approximately 100 times over an 8-year period. The cells do not form colonies or grow in soft agar. The cultures are heteroploid. The cell shape was predominantly polygonal or epithelial-like, but as cultures became confluent, bipolar or fibroblast-like cells appeared. Among the prominent ultrastructural features of RTL-W1 were distended endoplasmic reticulum and desmosomes. Benzo[a]pyrene was cytotoxic to RTL-W1. Activity for the enzyme, 7-ethoxyresorufin O-deethylase (EROD), which is a measure of the cytochrome P4501A1 protein, increased dramatically in RTL-W1 upon their exposure to increasing concentrations of either ~-naphthoftavone (BNF) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). With these properties, RTL-W1 should be useful for studying the expression of the cytochrome P450 enzymes and as a tool for assessing the toxic potency of environmental contaminants. INTRODUCTION The CYP1A1 induction response in fish can be a valuable indicator for monitoring environmental pollution (Goks0yr and F6rlin, 1992). The CYPIA1 gene is one of many cytochrome P450 genes (CYP) but the only member of CYP1A subfamily in rainbow trout (Heilmann et al., 1988). In 1. Address all correspondence to: Dr. L.E.J. Lee, Department of Veterinary Anatomy, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Sask. S7N 0W0 Canada. 2. Key Words: cytochrome P450; 7-ethoxyresorufinO-deethylase; trout liver cell line. 3. Abbreviations: AHH, aryl hydrocarbon hydroxylase; B[a]P, benzo[a]pyrene; BNF, [3-naphthoflavone; CHSE-214, Chinook salmon embryo cells; CYP, cytochrome P450; DMSO, dimethyl sulfoxide; ECOD, 7-ethoxycoumarin O-deethylase; EDTA, ethylenediaminetetraacetic acid; EROD, 7-ethoxyresorufin Odeethylase; FBS, fetal bovine serum; HEPES, N-[co-hydroxyethyl]piperazine-N'-[2-ethanesulfonicacid]; H4IIE, rat hepatoma cells; PCB, polychlorinatedbiphenyls; PHH, planar halogenated hydrocarbons; TCDD, 2,3,7,8-tetrachlorodibenzo:p-dioxin; TEM, transmission electron microscopy; TEFs, toxic equivalent factors; TS, 0.05 mM Tris 0.2 M sucrose; RLE, rat liver epithelial cells; RTL-Wl, rainbow trout liverWaterloo 1 cells. Cell Biology and Toxicology, Vol. 9, No. 3, pp. 279-294 Copyright 9 1993 Kluwer Academic Publishers ISSN: 0742-2091

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fish exposed to environmental pollutants, expression of CYPIA1 increases in the liver (Goksr and F6rlin, 1992). This can be detected by measuring the level of either CYP1A1 mRNA with a DNA probe, the P4501A1 protein product with antibodies, or catalytic activity with assays such as arylhydrocarbon hydroxylase (AHH), 7-ethoxycoumarin O-deethylase (ECOD), and, most commonly, 7-ethoxyresorufin O-deethylase (EROD). For studying this induction in vitro, fish primary hepatocyte culture systems are being developed (Devaux et al., 1992; Pesonen et al., 1992), but suitable fish liver cell lines have yet to be identified. Few cell lines have been developed from the liver of fish (reviewed in Bols and Lee, 1991), and the cytochrome P450 system has been examined in only two: RTH-149, which was derived from a rainbow trout hepatoma (Fryer et al., 1981), and PLHC-1, which was initiated from hepatic tumors in the topminnow (Hightower and Renfro, 1988). In RTH-149, AHH activity was induced weakly by benz[a]anthracene and more strongly by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and, for the first time in fish, the Ah (aromatic hydrocarbon) receptor, which mediates AHH and EROD induction, was detected and characterized (Lorenzen and Okey, 1990). On the other hand, RTH-149 cells did not have sufficient intrinsic P450 enzyme activity to metabolize benzo[a]pyrene (B[a]P) to cytotoxic intermediates (Babich et al., 1989; Babich and Borenfreund, 1991), but in contrast PLHC-1 cells did have adequate activity (Babich et al., 1991). As well, PLHC-1 cells showed an increase in ECOD activity after exposure to Aroclor 1254 (Babich et al., 1991). However, because the proportions of different cytochrome P450 enzymes are altered in tumor-bearing livers of rats (Buchmann et al., 1985) and rainbow trout (Lorenzana et al., 1989), hepatoma-derived cell lines might not be completely representative of some events, such as the regulation of cytochrome P450 enzyme induction, in the normal liver. Liver epithelial cell lines have been initiated on numerous occasions from the normal rat liver by treating minced liver with proteolytic enzymes, but the in vivo cellular origin of these cells is uncertain (Williams et al., 1971; Tsao et al., 1984; Marceau et al., 1986). They have been suggested to have a blastic multipotent nature and to originate from a stem cell compartment that is located in bile ductular structures and has the capacity to differeniiate into both bile duct epithelial cell and hepatocyte lineages (Grisham, 1980; Bisgaard and Thorgeirsson, 1991). The differentiated functions of hepatocytes are expressed partially and variably by these cell lines (Tsao et al., 1984; Fausto et al., 1987). AHH activity has been detected but has shown considerable variability between lines (Diamond et al., 1973; Borenfreund et al., 1975; Idoine et al., 1976). In at least one line, benz[a]anthracene induced AHH activity (Diamond et al., 1973). On the other hand, EROD activity has been found recently to be undetectable and uninducible with benz[a]anthracene in a number of different rat liver epithelial cell lines and in bile duct cells (Schrenk et al., 1991). In this report, Dispase treatment of rainbow trout liver fragments has been used to initiate the development of a liver epithelial line, RTL-Wl. In contrast to another fish cell line, CHSE-214, RTL-Wl expresses cytochrome P450 enzymes: the cells are sensitive to B [a]P and induce EROD activity in response to either BNF or TCDD. This should make RTL-Wl useful for studying

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cytochrome P450 enzyme expression and as a tool for assessing toxic potency of environmental contaminants. METHODS

Routine Cell Culturing Two cell lines were used: rainbow trout liver, RTL-W1, which was derived as described in the following section, and the Chinook salmon embryo, CHSE-214, which was obtained from the American Type Culture Collection (Rockville, MD, USA). For routine growth, the basal medium, Leibovitz's L-15, was purchased from ICN/Flow (St. Laurent, PQ, Canada), as were the medium supplements: penicillin-streptomycin, glutamine, and fetal bovine serum (FBS). L-15 is designed for use in an atmosphere of 100% air (Leibovitz, 1963) and has been effective in maintaining differentiated functions of adult rat hepatocytes in culture (Sawada et al., 1987). After supplementation, the L-15 contained penicillin at 100 IU/ml, streptomycin at 100 gg/ml, and glutamine at 2raM. The FBS supplement was used at 5% (v/v) for routinely growing RTL-Wl and at 9% (v/v) for CHSE-214. The cells were grown at room temperature in flasks in free gas exchange with the air. The flasks were manufactured by Nunc (Roskilde, Denmark) and were of two different growth areas, 24cm 2 and 83 cm 2. On some occasions, Primaria (25-cm 2 style) flasks, which are manufactured by Falcon (Lincoln Park, NJ, USA) and are more positively charged than conventional tissue culture labware, were used. Cells were removed from growth surfaces with 0.1% (w/v) bovine trypsin (Sigma, St. Louis, MO, USA).

Origin of RTL-W1 The primary culture that eventually led to the RTL-W1 cell line was prepared from the liver of a 4-year-old male rainbow trout (Oncorhynchus mykiss). The liver appeared normal upon gross inspection. Large pieces from the interior of the liver were cut into very small pieces. These pieces were incubated at room temperature with Dispase I (0.6 U/ml) (Boehringer Mannheim, Laval, PQ, Canada) in Hanks phosphate-buffered saline with 10% FBS and without Ca2+ and Mg 2+. After several hours of incubation, the result was a mixture of single cells and clumps of tissue. These were collected by centrifugation, resuspended in L-15 with 10% FBS, and again pelleted by centrifugation. The pellet was resuspended in L-15 with 10% FBS and plated into Nunc flasks (24cm2). After several weeks, the medium with unattached cells and tissue fragments was removed and replaced with fresh L-15 with 10% FBS. This left only a few attached single cells and a few tissue fragments from which some cells had migrated out over the growth surface. With time one area of one flask became covered with a sheet of polygonal cells. After 5 months, the cells were removed from this flask with trypsin, collected by centrifugation, and plated into a single Nunc flask (83 cm2). This flask became confluent 4 months later. Since this time, spring 1984, the cells have been grown continuously in L-15 with 5% FBS by repeatedly allowing them to proliferate to cover the growth surface of a flask, removing them from a confluent flask, and putting them into two new flasks. This passaging has been done approximately 100 times over 8 years with conventional trypsin treatment. The cells were very

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difficult to remove from the growth surface during the first few passages, but this became easier with progressive passaging. After a 5-min trypsinization, the plating efficiency was as high as 95%. The time for flasks to become confluent declined from several months for the early passages to become relatively stable at between 7 and 10 days for later passages. About 14 months after the initial treatment of liver pieces with Dispase, there were sufficient cells to test some for the presence of mycoplasma by the method of Chen (1977) and to attempt storing others by putting them into cryovials with 10% (v/v) dimethyl sulfoxide (DMSO) in growth medium and placing the vials into liquid nitrogen. The cells were free of mycoplasma and were successfully thawed after being frozen in liquid nitrogen. Since then, cells have been frozen at different passages each year. The experiments reported in this paper were done with cells between passages 30 and 50.

Cell Proliferation The ability of RTL-W1 to proliferate in L-15 with different FBS concentrations was tested. Cells in 5% FBS were seeded at approximately 105 cells/well into Nunc 6-well plates that have a growth area of 9.6 cm2 per well. Twenty-four hours after seeding, cells were removed with trypsin from three wells and counted with a hemocytometer. For the remaining wells, the medium was changed to L-15 with either 0, 1, 5 or 10% FBS, and cell number per well was determined every 3 days for 12 days. For each FBS concentration and time point, cell number was determined in three wells. Two other growth properties were examined. The ability of RTL-W1 to form colonies was determined by plating approximately i x 103 cells in L-15 with 5% FBS into Nunc petri dishes, each of which had a growth area of 20.8 cm 2. The dishes were examined periodically with an inverted phase-contrast microscope for the presence of colonies. Also, the ability of RTL-Wl to grow in soft agarose in L-15 with 5% FBS was determined. Agarose type VII (Sigma) was used because the low gelling point is more suitable than other agars and agaroses for mixing in salmonid cells, which are sensitive to temperatures above 26~ (Bols et al., 1992). Cells were mixed with agarose to give a final agarose concentration of 0.6% (w/v), which at room temperature produces a loose, semisolid mixture. This was plated into petri dishes with or without a base of 0.6% (w/v) agar, which is solid at room temperature. The growth of RTL-Wl in dishes without agar indicated that being mixed in warm agarose was not lethal to the cells.

Transmission Electron Microscopy (TEM) For TEM, cells were grown in 60-mm Falcon petri dishes. The dishes were rinsed twice in L-15 without serum and gently flooded with 3% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2), fixing the cells in situ. Cells were rinsed with 0.22M sucrose in 0.1 M sodium cacodylate buffer, postfixed with 0.8% OSO4, and stained with aqueous 1% uranyl acetate containing 0.22 M sucrose. After gradual dehydration in ethanol, the cells were embedded in Epon and allowed to polymerize for 24 hr at 55~ The embedded cultures were allowed to cool slightly and were separated from the plate while the plastic was still warm. The optical quality of the polymerized Epon permitted the recognition and marking of the cells using phase-contrast microscopy. Those parts containing the marked cells were cut out. Ultrathin sections were cut parallel to the plane

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of the flask on an LKB3 ultramicrotome, poststained with Reynold's lead citrate and 2% uranyl acetate, and examined in a Phillips 410LS electron microscope.

Chromosome Preparation Cells in the logarithmic phase of growth were treated with colcemid (0.1 gg/ml in L-15) for 2 to 2.5 hr at room temperature, removed from the growth surface with trypsin-EDTA, treated with 0.075 M KC1, fixed in ice-cold methanol-glacial acid (3 : 1) at 4~ overnight, mounted on glass slides, and air dried for Giemsa (Harleco, Gibbstown, NY, USA) staining. Chromosome spreads were observed under oil immersion. Exposure to BNF and TCDD The exposure of cells to either [3-naphthoflavone (BNF) (Sigma) or 2,3,7,8-tetrachlorodibenzop-dioxin (TCDD) (Cambridge Isotope Laboratories, Woburn, MA, USA) was done at room temperature in Nunc petri dishes. Each dish had a growth area of 20.8 cm 2 and was seeded with approximately 6 x 103 cells in 5 ml of growth medium. When these dishes had become confluent, which was 3-5 days after seeding, the medium was replaced and then either BNF or TCDD was added at different concentrations (see Figure 5). Each concentration was applied to three cultures. Inasmuch as stock solutions of BNF or TCDD were prepared in DMSO, control cultures received 0.5 % (v/v) DMSO. The usual exposure period was 72 hr, although in two experiments with TCDD the exposure period was only 12 hr. EROD Assay After the incubation period, the cells were removed from the growth surface and sonicated. Removal was by rinsing the monolayer once with phosphate-buffered saline and then scraping off cells with a cell lifter (Costar, Cambridge, MA, USA) into 4 ml of 0.05 mM Tris 0.2 M sucrose (TS buffer). The cells were pelleted by centrifugation at 5~ for 11min at 2800rpm in a Damon/IEC-PR6000 with a 276 rotor. The cell pellet was resuspended into 500 gl of TS buffer and sonicated at 20 watts and 20% duty cycle in a Branson Sonicator Cell Disrupter 200 (Danbury, CT). Aliquots of 5 gl and 150 gl were removed to measure, rest~ectively , protein by the Bradford assay (BioRad, Richmond, CA, USA) and 7-ethoxyresorufin O-deethylase (EROD) activity by the following method. EROD activity was assayed by measuring fluorometrically the formation of resorufin from 7-ethoxyresorufin (Pohl and Fouts, 1980). The complete reaction mixture contained 0.1 M HEPES (pH 7.8), 0.27mM NADPH, 1.75mg/ml bovine serum albumin (Fraction V, Sigma), 5.3 mM MgSO4, 1.24 gM 7-ethoxyresorufin (Sigma), and 150 gl of cell extract in a final volume of 1.24 ml. The reaction was routinely carried out at 25"C in 16 • 100 mm glass test tubes for 10 min. The reactor was linear with time for at least 15 min and with protein concentrations of up to at least 0.7 mg/ml. The addition of 2.0 ml cold methanol terminated the reaction and precipitated protein, which was pelleted by centrifugation at room temperature for 30 min at 2800 rpm in a Damon/IEC-PR6000 with a 276 rotor. Resorufin was measured fluorometrically in the supernatant with a Turner 430 Spectrofluorometer at an excitation wavelength of 550 nm with a slit width

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of 15 nm and an emission wavelength of 585 nm with a slit width of 60 nm. The activity was expressed in picomoles of resorufin per milligram protein per minute (pmol/mgP/min). In some assays a-naphthoflavone (Sigma) in DMSO was added to reaction mixtures to final concentrations of either 1, 10 or 100 gM. In these cases, control reaction mixtures received equivalent volumes of DMSO. a-Naphthoflavone is an inhibitor of cytochrome P450-dependent activities in hepatic microsomes of rainbow trout (Elcombe and Lech, 1979).

Cytotoxicity Assay The measure of cytotoxicity was a decline in [3H]thymidine incorporation into acid-insoluble material as the concentration of benzo[a]pyrene (B[a]P) was increased. Previously, macromolecular precursor incorporation has been used successfully as a measure of polychlorinated biphenyl (PCB) cytotoxicity to fish cells in culture (Denizeau and Marion, 1984). Cells in L-15/FBS were plated into 6-well plates at 105 cells/well. After 24 hr, the medium was changed to L-15/FBS with varying concentrations of B[a]P (Sigma). Within an experiment, three wells were exposed to each concentration. The stock solution was made up in DMSO. As a result, control wells received DMSO at concentrations never exceeding 0.1% (v/v). Twenty-four hours after the addition of B[a]P, [3H]thymidine (2 ~tCi/ml) (ICN) was added to each well. As outlined previously (Lee and Bols, 1989), incorporation into acid-insoluble material was measured 24 hr after the addition of the radioactive DNA precursor. The results were expressed as mean dpm/105 ceils for statist i.cal analysis and as a percentage of the incorporation into the control wells for graphic presentation. For each experiment, a single-factor analysis of variance (Zar, 1974) was used to test whether B[a]P had an effect on [3H]thymidine incorporation~ Two-tailed hypotheses (null hypothesis: treatment had no effect) were tested and the maximum probability of a type I error was set at 0.05. If a difference was detected, Dunnet's test (Zar, 1974) was used to compare mean incorporation by B[a]P-treated cultures to mean incorporation by control cultures. RESULTS

Cell Growth RTL-W1 grew in L-15 supplemented with FBS but not in L-15 alone (Figure 1). In 5% and 10% FBS, cultures became confluent, but confluency was not reached in 1% FBS. Cell number increased most rapidly in 10% FBS. The cells did not form colonies or grow in soft agarose. Morphology The shape of RTL-W1 cells grown in 5% FBS was predominantly polygonal (Figure 2), but as cultures became confuent, bipolar cells appeared. With the continued maintenance of a confluent culture, these bipolar or fibroblast-like cells piled up into ridges or mounds between which the polygonal or epithelial-like cells remained as a monolayer (Figure 2). This cycle of change in cell shape occurred in Primaria flasks as well as in conventional tissue culture labware and was observed from early in the development of RTL-W1 through approximately 100 passages for

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over 8 years. The proportion of fibroblast-like cells in cultures appeared to be higher in 10% FBS and lower in 1% FBS.

Ultrastructure The ultrastructure of RTL-W1 was examined in confluent cultures by TEM. Nuclei were round to oval with one or more nucleoli. Polysomes and free ribosomes were abundant in the cytoplasm. Many cells had swollen or dilated profiles of rough endoplasmic reticulum (Figure 3). Usually the mitochondria were highly elongated. The cytoplasm also contained lysosomes and Golgi complexes. Vesicles and coated vesicles were seen adjacent to the cell membrane. Some cells were linked by desmosomes from which arose clumps of intermediate filaments (Figure 3). Small microvillus-like projections were found occasionally on the cell surface.

Chromosome Analysis For chromosome analysis, metaphase chromosome spreads were counted from two independent preparations of RTL-W1 at between 38 and 44 passages. Chromosome number ranged between 30 and 180. A plot of the distribution revealed two peaks (Figure 4). One ranged from 48 to 62 and had a modal number of 54-56. This peak and the modal number of this peak represented 67.5% and 32.5%, respectively, of all spreads. The second peak ranged from 100 to 112 and accounted for 21.9% of all spreads. The modal number of this peak was 108, which represented 6.9% of the total spread.

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FIGURE 2. Morphology of RTL-W1 monolayers. (A) Three-day-old subconfluent monolayer of RTL-W1 cells. A polygonal shape is adopted by the majority of the cells, which arrange in an irregular cobblestone pattern. Mitotic figures (arrows) are prominent. (B) Nine-day-old overconfluent monolayer of RTL-Wl. Ridges of bipolar cells (arrows) appear in cultures that have been allowed to grow past confluency. Ceils were visualized under a Nikon inverted phase-contrast microscope at xl00 (reproduced at 50%). EROD Induction EROD activity increased in RTL-W1 upon their exposure to increasing concentrations of either BNF or TCDD (Figure 5). A 72 hr exposure to TCDD elicited activity at a lower concentration and induced a higher activity than BNF. The lowest concentration to induce EROD activity was 4--6 pM for TCDD and 9-18 nM for BNE The maximum activities were achieved after exposure to TCDD concentrations of 49 pM and greater, and at between 92 and 183 nM BNE Without TCDD or BNF exposure, the EROD assay of cell extracts was at the limit of its detection and activity was not found. With TCDD, activity could be detected as early as 12hr after exposure (data not shown). The induced EROD activity was completely inhibited by the addition of 1, 10 or 100 ~tM ct-naphthoflavone to the reaction mixture.

As a comparison, the response of another salmonid cell line, CHSE-214, to TCDD was also

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FIGURE 3. Ultrastructure of RTL-W1. The upper photograph (A) (x2250) illustrates nucleus (nuc), polysomes (pol), free ribosomes (rib), rough endoplasmic reticulum (rer), cistemae (cis) of dilated rough endoplasmic reticulum, mitochondria (mit) and vesicles (v). The bottom photograph (13) (• shows desmosomes (des), intermediate filaments (ill), mitochondria (mit), smooth endoplasmic reticulum (ser) and coated vesicle (cv). studied. These cells had no detectable EROD activity even after a 3-day exposure to TCDD over the range of 1.7-97.5 pM.

Cytotoxicity of B[a]P B[a]P significantly reduced [3H]thymidine incorporation into acid-soluble material by RTL-W1 but not by CHSE-214 (Figure 6). Incorporation by RTL-Wl cultures exposed to B[a]P concentrations of 0.1 gg/ml and higher was significantly less than incorporation by control cultures (p < 0.05). At 0.5 ~tg/ml incorporation was inhibited by 50%. By contrast, in CHSE-214 cultures, incorporation was reduced only slightly and not statistically significantly by B[a]P concentrations as high as 5.0 gg/ml. Nine-day exposure to B[a]P (5 gg/ml) had little effect on CHSE-214 cultures but caused overt signs of toxicity in RTL-W1 cultures. Many RTL-Wl cells detached from the growth surface and these cells stained with trypan blue. The remaining attached cells showed

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FIGURE4. RTL-W1 chromosome distribution. RTL-Wl cells at passage 44 were prepared for karyotyping as described in Methods. In this preparation, a total of 136 metaphase spreads were counted.

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ultrastructural changes: increased cytoplasmic vacuolization, degenerating mitochondria, swollen nuclear envelope, and clumping of chromatin. DISCUSSION This study demonstrates that an epithelial cell line expressing inducible EROD activity can be derived from the liver of an adult rainbow trout and suggests that the development of fish liver cell lines useful for toxicological studies should be generally possible. At this time RTL-Wl can be regarded as an immortal or continuous cell line but not a neoplastic one, although the criteria for these categories are poorly developed for piscine cells (Bols and Lee, 1991). RTL-Wl is judged immortal because cultures have been grown continuously for approximately 100 passages over an 8-year period. Such spontaneous immortalization has been observed in cell cultures from a variety of fish organs (Nicholson, 1989; Bols and Lee, 1991), which now includes the adult liver. For rainbow trout cells this was a slow process, taking approximately 14 months. The inability of RTL-Wl to grow as colonies or in soft agarose suggests that they are not neoplastically transformed. Also, rat liver epithelial cell lines generally become neoplastic only after treatment with chemical carcinogens (Fausto et al., 1987), although some have been observed to undergo spontaneous neoplastic transformation upon continued passaging (Diamond et al., 1973; San et al., 1979; Narita, 1990). RTL-W1 shares some properties with rat liver epithelial cell lines (RLEs) but differs from them

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in other ways. Cells of both have similar polygonal morphologies, and the shape change of RTL-W1 at confluency has been observed in at least one rat liver liver epithelial cell line, WIRL-3C (Diamond et al., 1973). RTL-W1 showed both ultrastructural similarities with and differences from RLEs, which have been examined by Tsao etal. (1984). They both contained dilated rough endoplasmic reticulum, desmosomes, lysosomes, and microvillus-like projections. Ultrastructural differences were the greater abundance of cytoplasmic filaments, free ribosomes, and smooth and rough endoplasmic reticulum and the larger size of mitochondria in RTL-W1 than in RLEs. EROD activity was inducible in RTL-W1 but not in RLEs (Schrenk et al., 1991). RLEs are usually described as being diploid or near-diploid (Tsao etal., 1984; Bisgaard and Thorgeirsson, 1991), whereas the RTL-W1 cultures were heteroploid, although over 60% of the cell spreads had a near-diploid number. The origin of RTL-W1 from among the many cell types in the rainbow trout liver is not known, but one potential source, which is suggested by work with RLEs (Grisham, 1980; Bisgaard and Thorgeirsson, 1991), is a compartment of stem cells located in bile ductular structures. Biliary epithelial cells are abundant in the rainbow trout liver (Hinton et al., 1987), and in the scup they showed inducible P4501A1 after the fish had been injected with planar halogenated hydrocarbons (PHHs) (Smolowitz et al., 1991). Another possible origin is the spontaneous immortalization of hepatocytes, but if the behavior of mammalian hepatocytes can serve as a guide, this is unlikely. Continuous mammalian hepatocyte cell lines arise not spontaneously but through directed immortalization with agents such as SV40. Even then, defined medium must be used during immortalization in order to obtain cell lines expressing differentiated functions, including cytochrome P450 and associated mixed function oxidases (Bayad et al., 1991). In addition, the original primary culture likely had few hepatocytes because subsequent work has shown that rainbow trout hepatocytes attach poorly to plastic tissue culture dishes (Klaunig et al., 1985; Lipsky et al., 1986; Blair et al., 1990).

Other possible origins that are unlikely but cannot be ruled out are connective tissue, which has been the source of a mouse liver cell line (Borojevic et al., 1985), and endothelial cells, which from the human liver have been immortalized by polyoma virus (Hering et al., 1991). These origins are less likely because, relative to hepatocytes and biliary epithelial cells, these ceils are much less abundant in the rainbow trout liver (Blair et al., 1990). In the scup, P4501A1 was induced in most cell types of the liver, including endothelial cells, but not in fibrous connective tissue (Smolowitz et al., 1991). This result would favor an endothelial over a connective tissue origin for RTL-W1. However, if the liver endothelial cells of trout are similar to those of rat and attach poorly to plastic tissue culture dishes (Pertofl and Smedsrod, 1987), few would have been present in the original primary culture. Finally, inasmuch as RTL-Wl have been difficult to clone, which is characteristic of many fish cell lines (Bols and Lee, 1991), the possibility exists that RTL-W1 contains multiple cell lineages. An answer to the question of in vivo origin will have to await two developments: one is the development of markers for different rainbow trout cell

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lineages; the second is improvements in the growth medium that will allow cloning from this population. Regardless of their origin, RTL-W1 respond to classic inducers of the CYP1A1 response, BNF and TCDD, in a manner similar to primary rainbow trout hepatocyte cultures. As in primary cultures (Pesonen and Andersson, 1991; Pesonen et al., 1992), TCDD was a better inducer of EROD activity in RTL-Wl than BNE The lowest concentration of TCDD that brought about a significant increase in EROD activity was similar and was at between 4 and 6pM. The maximal increase in EROD activity was achieved with 80 pM TCDD for primary cultures (Pesonen and Andersson, 1991) and 49 pM for RTL-W1. Like primary cultures, an increase in EROD activity was observed as early as 12 hr after the exposure of RTL-Wl to TCDD. The response of RTL-W1 to TCDD has been very stable over a 2-year period: similar levels of EROD activity have been induced in cultures at different passages, These properties should make RTL-Wl useful for studying the regulation of the CYP1A1 response.

With respect to the expression of P450 enzymes in other fish cell lines, RTL-W1 appears similar to some lines but different from rainbow trout hepatoma line, RTH-149. As judged by a decline in [3H]thymidine incorporation, B[a]P was cytotoxic to RTL-W1. With neutral red uptake as the cytotoxic endpoint, five different piscine cell lines had sufficient P450-dependent activity to metabolize B[a]P to cytotoxic intermediates, and only RTH-149 lacked adequate activity (Babich et al., 1991; Babich and Borenfreund, 1991). Induction has been demonstrated in three piscine cell lines. AHH activity was induced in RTH-149 by TCDD (Lorenzen and Okey, 1990), and ECOD activity in PLHC-1 by the PCB mixture, Aroclor-1254 (Babich et al., 1991). Recently, the induction of P4501A1 by TCDD was detected immunologically in a zebrafish embryo cell line (Collodi et al., 1992).

In contrast to the above results, in the Chinook salmon embryo cell line, CHSE-214, EROD activity was undetectable and uninducible by TCDD. Also, B[a]P failed to elicit a cytotoxic response from these cells. These results suggest that the presence of P450-dependent monooxygenase activity is not a property of all fish cell lines.

The inducibility of EROD activity in the rat hepatoma cell line, H4IIE, has been used to compare the toxic potency of different environmental contaminants relative to TCDD (Tillitt and Giesy, 1991). As RTL-W1 are like H4IIE in responding to TCDD by inducing EROD activity, they too could be used to evaluate toxic potencies. Currently, toxic equivalent factors (TEFs) for PHHs are being derived with both RTL-Wl and H4IIE in order to evaluate the cell line most suitable for assessing the impact of environmental contaminants on fish (Clemons et al., 1992). In addition, the potential to neoplastically transform RTL-Wl with aflatoxins is being studied (Bechtel and Lee, 1993).

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