XENOBIOTICA,1991, VOL.
21, NO. 4,461471
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Characterization and induction of xenobiotic metabolizing enzyme activities in a primary culture of rainbow trout hepatocytes M. PESONENTS and T. ANDERSSONS
t Department of Physiology, University of Kuopio, PL 6, 70221 Kuopio, Finland $ Department of Zoophysiology, University of Goteborg, Box 25059, 40031 Giiteborg, Sweden Received 9 M a y 1990; accepted 24 October 1990
1. A primary cell culture from rainbow trout (Oncorhynchus mykiss) liver was prepared and evaluated for biotransformation of xenobiotics. 2. T h e hepatocytes maintained cytochrome P-450 content, as well as their cytochrome P-450-dependent activities, stable for 5-6 days in serum-free medium. Protein and glutathione levels, as well as other enzyme activities important for biotransformation, were close to their fresh cell levels throughout the culture period. 3. T h e cells were also responsive to cytochrome P-450 inducers. Both ( T C D D ) caused an 8-naphthoflavone (BNF) and 2,3,7,8-tetrachlorodibenzo-p-dioxin increase in ethoxyresorufin 0-deethylase (EROD) activity, which was dose-dependent over the concentration ranges of 3.6-360 nM and 2.5-100 PM, respectively. T h e induced activities in BNF-exposed cells returned to basal lzvels within 48 h after replacing the medium with a BNF-free medium. Exposure of cells to T C D D (100pM) for 48 h induced E R O D activity which, in contrast to response of BNF-exposed cells, continued to increase after the medium had been replaced with TCDD-free medium. 4. T h e results show that trout hepatocytes in primary culture afford a reliable in vitro method for studying the regulation and functions of xenobiotic biotransformation enzymes, and for defining toxic effects of aquatic pollutants in cells.
Introduction T h e biotransformation of many lipophilic, organic xenobiotics to polar, excretable products generally comprises a sequence of reactions, which can be divided into two phases; in phase I the cytochrome P-450-dependent monooxygenases introduce functional groups into the molecule, and in phase I1 these molecules then serve as substrates for conjugating enzymes such as glucuronosyltransferase. In fish, as in mammals, the liver is the main organ involved in the biotransformation of xenobiotics. An important characteristic of these enzymes is their inducibility by various xenobiotics. Fish liver cytochrome P-450 enzymes are induced by polycyclic aromatic hydrocarbons (PAH) such as p-naphthoflavone, 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin(BNF, MC, T C D D ) , whereas fish, in contrast to rat, seem to be refractive to phenobarbital-type inducers (Vodicnik et al. 1981). This induction of cytochrome P-450 in organisms by exposure to certain xenobiotics is a process used for adapting to changes in the environment, and can affect the biotransformation of foreign compounds both quantitatively and qualitatively. T h e high susceptibility to PAH-type inducers has made the fish liver cytochrome P-450 system a sensitive tool for use in monitoring certain types of organic pollutants in aquatic environments (Stegeman 1987, Payne et al. 1987, Anderson et al. 1988). 0049-8254/91 $3.00 0 1991 Taylor & Francis Ltd.
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46 2
M . Pesonen and T.Anderson
Primary hepatocyte cultures from mammalian species are used as an in vitro model for investigating the regulation and functions of various xenobiotic metabolizing enzymes (Suolinna 1982, Guillouzo 1986). T h e advantage in using hepatocyte cultures is that various types of biotransformation reactions can be studied under strictly controlled conditions. Furthermore, in intact hepatocytes a complete intracellular organization is maintained; thus xenobiotic metabolism reactions can be studied in relation to other cellular events. There is, however, a disadvantage associated with the primary culture of mammalian hepatocytes, i.e. the decline in cytochrome P-450 monooxygenase activities that occurs during the culture period. Hormone mixtures, trace elements and haem precursors have been used in the culture medium to stabilize these enzymes (Guillouzo 1986, Dickins and Peterson 1979, Grant et al. 1985). Because of the instability of some cytochrome P-450 forms during culture the results of studies on regulatory mechanisms and toxic properties of chemicals can be uncertain. In this study hepatocytes were isolated from rainbow trout and maintained in primary culture on plastic Petri dishes for 5-6 days using synthetic culture medium. Cellular levels of cytochrome P-450, cytochrome P-450-mediated 7-ethoxyresorufin-0-deethylase (EROD) activity and aryl hydrocarbon hydroxylase (AHH) activity remained stable throughout the culture period. Furthermore, the cytochrome P-450 monooxygenase system was highly responsive to PAH-type inducers.
Materials and methods Animals Cultured immature rainbow trout (Oncorhynchus mykzss), ranging in weight between 150 and 250g, were obtained from local hatcheries in Sweden and Finland. T h e fish were fed with commercial food and kept in laboratory basins with aerated, filtered, circulating fresh water at n temperature of 10°C. A 12 h light-I2 h dark photoperiod was used. T h e fish were acclimatized to laboratory conditions for a minimum of 2 weeks before use in the experiments. Chemicals Chemicals were purchased from the following sources: antibiotic-antimycotic solution and I.-glutamine from Gibco Ltd (Scotland), collagenase from Roehringer Mannheim (Germany), 7-eth~txyresorufinand resorufin from Pierce Eurochemie BV (Rotterdam, Holland), Medium 199, 8-naphthoflavone, 3,4-benzo(o)pyrene and glutathione from Sigma (St Louis, MO), p-nitrophenol and l-chloro-2,4-dinitrobenzenefrom Fluka A G (Buchs, Switzerland), 2,3,7,8-tetrachlorodibenzo-p-dioxin (‘I‘CDD) was a kind gift from Dr S. Karenlampi, Department of Biochemistry, University of Kuopio, Finland. All other chemicals were of analytical grade. Isolation of liver cells All liquids and glassware were sterilized prior to use by filtration and autoclaving, respectively. IIepatocytes were isolated by a two-step perfusion method as described by Berry and Friend (1969), and modified for fish hy Anderson and Forlin (1985). Fish were stunned by a blow to the head and incised from thc urogenital pore to the gills. T h e portal vein was cannulated, and thc perfusion was started in sifu with a Ca2+-frer salmon buffer (Lockwood 1961) containing lOmM ethyleneglycolbis-(8-amino cthyl ether) N,N-tetra acetic acid (EGTA) using a flow rate of 5 ml/g liver per min. T h e liver was carefully removed from the fish and placed in an organ chamber. After 5-7 min the perfusion was continued with salmon buffer containing collagenase (0.5 mg/ml) for 30 min. T h e liver was then dispersed gently by using a ’I’eflon rod and the cell suspension was subsequently shaken for 5 min in the collagenase buffer. After filtration through cotton gauze the cells were centrifuged for 45 s at 60g and washed twice with culture medium (medium 199 supplemented with lOml/l of antibiotic-antimycotic solution, 10 ml/l of 1.glutamine, 0.9 g/1 of HEPES, 0 3 5 g/l of NaHCO,, 1.6 g/l of Na,HPO,, 0.10 g/l of CaC1,). T h e osmolarity of the culture medium was adjusted to 300mOsm. T h e cells were counted using a Biirker chamber, and the cell viability was routinely determined using the Trypan Blue exclusion test. Cell culture T h e hepatocytes (lo’) were seeded on plastic Petri dishes (100mm) and incubated at 10°C. Liver cell morphology was observed daily and photographed using phase contrast inverted microscopy with an
Xenobiotic metabolism in trout liver cells
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Olympus CK2 microscope. T h e medium was changed 24 h after seeding and every second day thereafter. The hepatocytes were harvested by flushing the Petri dishes with culture medium. T h e cells were pelleted by centrifugation at 60g for 2 min and resuspended in 0 1 M phosphate buffer, p H 7.4, containing 20% glycerol to a final concentration of lo7cells/ml. Cell suspensions were frozen in liquid nitrogen and stored at -80°C no longer than 1 week. BNF and T C D D were dissolved in dimethyl sulphoxide (DMSO) and added to the culture medium after 24 h of preculture. Control cultures received DMSO only. T h e final concentration of DMSO in the medium never exceeded 0.1%. T h e hepatocytes in both control and treated groups were always obtained from the same fish.
Assays T h e whole cell homogenates, prepared by sonicating cell suspensions with ultrasound for 5 S , were used for biochemical analyses. Protein content was determined according to Lowry et al. (1951) using bovine serum albumin as a standard. Total glutathione (GSH) content was measured according to the method described by Adams et al. (1983). The concentration of cytochrome P-450 was measured spectrophotometrically as described by Matsubara et al. (1974). NADPH-cytochrome c reductase activity was determined by the method of Dallner et al. (1966). The assay solution (1 ml) contained 0 1 M potassium phosphate buffer, 0 0 4 mM cytochrome c, 0.08 M NADPH and 0.05 mg of cell homogenate protein. The reduction of cytochrome c was followed at 550 nm and at 18°C. AHH-activity, using 3,4-benzo(a)pyrene as a substrate, was determined according to Nebert and Gelboin (1968). The incubation was carried out in a 0 5 ml mixture containing 85 p~ 3,4-benzopyrene, a NADI’H-generating system; 0.45 mM NADP, 5 mM isocitric acid, 75 mM Tris-HC1, 22 mM KC1, 5 mM MgCI,, 5 p~ MnCl,, 0.24U isocitric acid dehydrogenase and 0.154.2mg of cell homogenate protein. The reaction was started by adding cell homogenate and performed for 30 min at 18°C. 7-Ethoxyresorufin-0-deethylase (EROD) was measured as described by Burke and Mayer (1974). T h e assay, prepared in the fluorometer cuvette, was carried out in 2 ml of solution consisting of 0.1 M TrisHCI buffer (pH 7.8), 0.5 PM ethoxyresorufin and 0.34.4 mg cell homogenate protein. T h e reaction was initiated by adding lop1 NADPH (10 mM). The progressive increase in fluorescence (excitation wavelength 530 nm, emission wavelength 585 nm) was measured at 18°C for 1 min. UDP-glucuronosyltransferase activity towards p-nitrophenol was determined as described by Anderson et al. (1985). Cells were treated with Triton X-100 before making measurements (final concentration of detergent in the incubation mixture was 0.01%). T h e incubation was carried out in a 0125 ml mixture containing 0 5 M potassium phosphate buffer (pH 7.0) and 7 mM U D P glucuronic acid. T h e incubation was started by adding cell homogenate (0.2254.3mg) and lasted 40min at 18°C. Clutathione-S-transferase activity was measured according to Habig et al. (1974) using l-chloro-2,4dinitrohenzene as substrate. The assay, prepared in the cuvette, was carried out in a 2.5 ml mixtureof 0.1 M phosphate buffer (pH 7.4) and 60 m~ 1-chloro-2,4-dinitrobenzene. T h e reaction was initiated by adding 25 pI cell homogenate (003-0.05 protein). The increase in absorbance at 344 nm was recorded at 18°C for 1 min. Aldehyde dehydrogenase (ALDH) activity was assayed according to Torronen et a l . (1989) by monitoring the reduction of NAD at 340nm and 18°C. T h e assay mixture (final volume 1 ml) contained 70mM sodium pyrophosphate buffer (pH 8), 1 mM pyrazole, to inhibit alcohol dehydrogenase activity, 1 mM NAD as co-factor, and 5 mM propionaldehyde as substrate. Blanks, containing vehicle without aldehyde, were run for each sample. Lactate dehydrogenase (LDH) activity was assessed in cell homogenate and in culture medium according to Bergemayer and Bernt (1974). The assay was carried out in a 3 m2 mixture containing 0.1 M sodium phosphate buffer (pH 7.4), 0.81 mM NADH and 4mM pyruvate. T h e reaction was initiated by adding 5Opl of sample (0.074.1 mg cell homogenate protein) and was carried out at 18°C. T h e linear change in absorbance at 340 nm was recorded for 30 s. Statistics Statistical analysis was performed using a Mann-Whitney U-test. T h e significance levels were set at p < 0.05.
Results and discussion The present study describes a method for culturing liver cells from rainbow trout in a serum-free medium in plastic Petri dishes. With this method several cell properties important for the functioning of the systems involved in xenobiotic metabolism, were maintained throughout the 5-day culture period. Freshly isolated cells were round and morphologically intact (figure 1). Each isolation gave 100-200 x lo6cells/g liver weight. In addition to hepatocytes the isolated cell material also contained small non-parenchymal cells which were not
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464
M . Pesonen and T . Anderson
Figure 1.
T r o u t hepatocytes after
2 h in culture.
Cells are attached weakly to plastic culture dishes. Small nan-parenchymal cells can be seen between hepatocytes ( x 75).
counted. T h e hepatocytes become firmly attached to plastic culture dishes within 24 h after plating and formed a monolayer, which is necessary for prolonged survival of cells in culture. Thus it was possible to change medium without incurring an obvious loss of cells. Mammalian liver cells will not attach to the dish unless divalent cations (Ca”, M g Z f )and serum attachment factors, such as fibronectin are present (Rlaauboer and Paine 1979, Cjessing and Seglen 1980). Although we did not use serum we did find that hepatocytes attached more firmly to the substratum when the CaCl, level in the medium was elevated from 0.14 to 0.24 g/l. T h u s we did not have a problem with poor attachment of the trout liver cells to plastic culture dishes as reported earlier by Klaunig (Klaunig 1984, Klaunig et al. 1985) and Lipsky et al. (1986). Cell integrity was measured by the Trypan Blue exclusion test before plating of thc cells and by 1,DH leakage to the medium at 24-h intervals for the culture period. T h e Trypan I3luc exclusion test indicated that over 90% of the freshly isolated cells were viable. During the culture period leakage of LDH into the medium accounted for 15% of the total activity in the cells during the first 24 h, but decreased thereafter and was only 5% at 1 2 0 h . T h e total content of protein in the cell culture did not change during the culture period. Table 1 presents the contents of cellular components and enzyme activities in primary liver cell culture, measured at 24-h intervals for 5 days. T h e NADPHcytochrome c rcductase, which transfers reducing equivalents to the monooxygenase reaction, and the total content of cytochrome P-450 remained stable throughout the culture period. T w o cytochrome P-450-dependent activities, EROD and AHH activity, were measured. Both activities were maintained at their fresh cell levels during the 5-day culture period. These results indicate that the monooxygenase
8.7 f 0.2 87 2 22 8.8 f1.2 13.6 k 0.3 23.8 f 5.7 7.0 f 0.6 91 _+5 223 +32
24 10.8f2.0 96+6 9.4+ 1.1 20.9 k 1.1 38.8 k 1 7 3 6.3 f0.1 91 + 1 226 f 29
48
+
16.9 5.6 74f21 6.6 f1.7 17.4 f0.1 36.8 k9.5 6 4 f 2.2 80+6 228 f 25
72
17.2 f 2.6* 85213 9.6 1.7 1 6 6 F 2.6 3 4 2 k8.6 4.9fO.1 84f8 217+12
96
+
300k2.1 5.6 f1.7 85f8 269 f 20
ND
19.3 f0.7f 91 +20 7.0 1.0
120
Each point represents means & SD of data obtained, with the number of Petri dishes in square brackets. Statistical analysis was not performed. *P