Gen. Pharmac. Vol. 22, No. 4, pp. 677-684, 1991
0306-3623/91 $3.00 + 0.00 Copyright © 1991 Pergamon Press plc
Printed in Great Britain. All rights reserved
EXPRESSION OF A R Y L H Y D R O C A R B O N HYDROXYLASE, EPOXIDE HYDROLASES, GLUTATHIONE S-TRANSFERASE A N D U D P - G L U C U R O N O S Y L T R A N S F E R A S E S IN H5-6 HEPATOMA CELLS MARINA ROQUES,* DENYSE BAGREL,JACQUESMAGDALOUt and GERARDSIEST Centre du M6dicament, U.R.A. CNRS No. 597, Facult6 des Sciences Pharmaceutiques et Biologiques, 30 rue Lionnois, 54000 Nancy, France [Tel. 83-32-29-23; Fax 83-32-13-22] (Received 16 October 1990)
Ahstract--l. The presence of arylhydrocarbon hydroxylase (cytochrome P-450 IAI dependent), glutathione S-transferase, two distinct forms of epoxide hydrolases and UDP-glucuronosyltransferases was detected in H 5 ~ hepatoma cell homogenates using model substrates, selective inhibitors and specific antibodies. 2. The activity of arylhydrocarbon hydroxylase decreased strongly at the first days after plating and remained at a minimal value (1.5 pmol/min per mg) after 5 days of culture. 3. The hydratation of trans-stilbene oxide catalyzed by the soluble form of epoxide hydrolase was very low (ll.0pmol/min per mg), whereas the hepatoma cells contained appreciable amounts of the membrane-bound epoxide hydrolase and glutathione S-transferase measured with cis-stilbene oxide as substrate (maximal specific activity: 1.46 and 2.73 nmol/min per mg, respectively). 4. These cells also glucuronidated l-naphthol efficiently (6 nmol/min per mg) and, at a lower extent, bilirubin (12 pmol/min per mg). 5. Addition of fenofibrate (70 ~ M) into the culture medium for 1 3 days failed to significantly stimulate the activity of cytosolic epoxide hydrolase. Only bilirubin glucuronidation increased 2-fold after 2 days of presence of the drug.
INTRODUCTION
Compared to laboratory animals, hepatoma cells provide an attractive and alternate tool to investigate various aspects of cellular biology and especially drug metabolism. Hepatoma cells are generally easy to handle and present a better stability of their enzymatic functions, when compared to hepatocytes. The characterization of proteins involved in the transformation of drugs and other potentially hazardous substances can contribute whether to choose this model to investigate the metabolic pathways, which account for the elimination of these substances from the cell, and for their possible toxic effects. Moreover the response of drug metabolizing enzymes towards inducers helps in the understanding of the regulation mechanism of these proteins at a molecular level. H5-6 hepatoma cell line, issued from H5, a cell line which derives from the rat Reuber hepatoma H35, has been recently established (Sperling et al., 1980). Until now, the characterization of drug metabolizing enzymes has not been extensively performed. Wiebel et al. (1984) reported that H5 expressed a cytochrome P-450 inducible by 3-methylcholanthrene but not the *Present address: Unit6 INSERM No. 61, 3, avenue Moli~re, 67200 Strasbourg, France. tTo whom all correspondence should be addressed.
phenobarbital inducible forms. In a preliminary experiment by Western blotting with several specific cytochromes P-450 antibodies, we were only able to detect in H5-6 a protein corresponding to the isoform IA1 (non-published observations). In this work the activity of arylhydrocarbon hydroxylase (AHH, EC 1.14.14.2), supported by cytochrome IAI, was therefore measured. In order to determine if H 5 ~ cells have the capacity to metabolize drugs, the presence of membrane bound and cytosolic epoxide hydrolases (mEH, cEH, EC 1.11.1.6), glutathione S-transferase (GST, EC 2.5.1.18) and UDP-glucuronosyltransferases (UDPGT, EC 2.4.1.17) was investigated. These enzyme systems play a crucial role in the destruction of reactant epoxides and elimination of drugs as hydrophilic glucuronides (Glatt and Oesch, 1977; Siest et al., 1987; Wixtrom and Hammock, 1985). Their activity is known to be enhanced by inducers such as phenobarbital, 3-methylcholanthrene and clofibrate (Owens, 1977; Fournel et al., 1987; Ding and Pickett, 1985). The presence of several isoenzymes was determined with model substrates and selective inhibitors of the corresponding hepatic enzymes, as well as from their immunoreactivity with antibodies raised against the corresponding purified liver enzymes. Finally the inducing effect of the hypolipidemic drug fenofibrate (2-[4-(4-chlorobenzoyl)phenoxy]-2-methylpropanoic acid 1-methylethyl ester) was investigated. 677
GP 22/~-H
MARINA ROQUESet al.
678 MATERIALS AND METHODS
Chemicals Tritiated trans-stibene oxide (TSO), tritiated cis-stilbene oxide (CSO) and 4-fluorochalcone oxide were kindly supplied by Professor Hammock (University of California, Davis, CA). Cyclohexane 1,2-oxide and l,l,l-trichloropropane 2,3-oxide were purchased from Aldrich Chemic (Steinheim, Germany). l-Chloro-2,4-dinitrobenzene and benzo(a)pyrene was provided by Sigma (St Louis, MO). Fenofibrate was kindly supplied by Laboratoires Fournier (Daix, France). l-[l-~4C]naphthol (58 mCi/mmol, 2.15 GBq/ retool) was purchased from Amersham (Les Ulis, France). UDP-glucuronic acid (sodium salt) was provided by Boehringer (Mannheim, Germany), and bilirubin by Merck (Darmstadt, Germany). All the other reagents were of the best purity commercially available. Ce.II euhures
Hepatoma H5 6 cells have been cloned from the H4IIEC3 cell line (Pitot et al., 1964), which has been established from Reuber H35 rat hepatoma (Reuber, 1961). Cells were grown at 3TC in a humidified atmosphere of 95% air and 5% CO_,, in Dulbecco's modified Eagle medium (Gibco, Cergy-Pontoise, France), supplemented with 10% v/v Nu-Serum (Collaborative Research, Bedford, MA) in the presence of 1% v/v antibiotic and antimycotic mixture (penicillin I00 UI/ml, streptomycin 100/~g/ml and fungizone 0.25 #g/ml, Gibco). Treatment of cells
Cells were seeded in 100 mm-diameter plastic dishes, at a density of 1 × 106 per 10 ml medium. These conditions allow to perform the experiments during the exponential phase of growth. The medium was replaced with fresh medium 48 hr after seeding. For induction experiments, the fresh medium contained increased concentrations of fenofibrate in the range 15 280pM and dissolved in a same amount of DMSO. The final solvent concentration was 1% v/v. Two control experiments were simultaneously run in which the cells were grown either in presence of DMSO or without any solvent. After 24, 48 and 72 hr of treatment, the culture medium was collected, centrifuged at 170g for 5 min and frozen at - 2 0 C . The cells were washed twice with ice-cold phosphate-buffered saline, harvested and suspended in this solution. They were frozen at - 8 0 C after centrifugation again at 170 g for 5 rain. Evaluation o/" cytotoxicity
The viability of the cells was estimated with the Trypan blue exclusion test. Release of lactate dehydrogenase (LDH) was measured in the medium according to the method of Mathieu et al. (1982). Biochemical assays
Before measurement of epoxide metabolizing enzyme activity, cells were suspended in the buffer used to assay the corresponding enzymes and homogenized in a glass-glass homogenizer in ice, in order to make the cells completely broken, as revealed by observation under light microscopy. For UDPGT, thawing was enough to remove the enzyme latency. Additional homogenization procedure resulted in complete loss of activity. Immunoreactivity and biochemical properties of the drug metabolizing enzymes were determined on cells cultured for 96 hr. The concentration of cellular proteins was determined by the method of Lowry et al. (1951) with bovine serum albumin as standard. Hydroxylation of benzo(a)pyrene was followed according to the fluorimetric assay of Nebert and Gelboin (1968) modified by Wiebel et al. (1977). The activity of mEH, cEH and GST measured with CSO or TSO was determined at 37'C with the radiometric partition assays described by Wixtrom and Hammock (1985). Measurement
of cEH activity was performed in presence of 1.25 mM 1-chloro-2,4-dinitrobenzene to deplete glutathione, according to Schladt et al. (1986). For inhibition experiments, 4-fluorochalcone oxide (10 _3 M) in 1 ~1 DMSO, cyclohexane 1,2-oxide (10-5_10 3 M) or I,l,l-trichloropropane 2,3-oxide (10 3 M) in 1 #l ethanol or 1-chloro-2,4-dinitrobenzene (10 ~M) in 1/~1 ethanol were added 2 rain before starting the enzyme assay of the EHs and the GST. Glucuronidation of Inaphthol was followed according to the method of Bock et al. (1974). A modification of the procedure described by Heirwegh et al. (1972) was used for the measurement of bilirubin glucuronidation. The incubation mixture (300 #1) consisted in 0.5rag of cellular proteins and 0.17mM bilirubin. After 30 min of incubation at 37C, the reaction was stopped with addition of 0.5 ml of 2 M glycine-HC1 buffer (pH 2.7). The diazo reagent (0.5ml) was added and the diazo by-products formed were extracted with 2-pentanone and quantified by spectrophotometry at 530 nm. bnmunochemical detection
After solubilization in 2% w/v sodium dodecylsulfate, 5 mM dithiothreitol and 50 mM iodoacetamide, the cell homogenates were fractionated by gel electrophoresis (Laemmli, 1970), using 12.5% (w/v) polyacrylamide in the separating gel and 5% (w/v) in the stacking gel. Proteins were transferred with a Trans-Blot system (Bio-Rad, Richmond, CA) at room temperature overnight at 50V in 20mM Tris-HCl buffer (pH 8.3) containing 20% v/v methanol and 150mM glycine (Burnette, 1981). The proteins on the nitrocellulose sheets immunoreacted with specific rabbit antibodies raised against purified rat liver mEH, UDPGT isozymes active towards phenols and bilirubin (Pham et al., 1989; Thomassin et al., 1986; Zhiri et al., 1987), and mouse liver cEH (Dietze et al., 1990). The antisera were diluted 750 2000-fold. Revealation of the bands was carried out with peroxidase-conjugated sheep anti rabbit IgG (Institut Pasteur, Paris, France) and 3,Ydiaminobenzidine as detector agent. Statistical analysis
The data were analyzed using the Student's t-test for small samples and non-paired series. A difference between groups of P < 0.05 was considered significant. RESULTS Constitutive expression o f A H H , epoxide metabolizing enzymes and U D P G T
Figure 1 represents the evolution p a t t e r n of A H H activity in function of time of culture. Hydroxylation of benzo(a)pyrene was at a maximal level at the beginning of culture. The reaction rate decreased dramatically thereafter to reach a residual a n d stable value after 5 days (1.5 p m o l / m i n per mg). The optimal conditions for the m e a s u r e m e n t of epoxide metabolizing enzymes were determined. The reactions proceeded linearly in function of time up to 20 m i n for c E H and up to 6 rain for m E H and GST. The reaction rates were linear in function of the protein content up to 1.2, 0.6 a n d 0.2 mg for cEH, m E H a n d GST, respectively (data not shown). In Table 1 are indicated the specific activities m e a s u r e d for cEH, m E H a n d G S T in function of time a n d various treatments of the cells with D M S O and fenofibrate. In control cultures (without D M S O ) , the specific activity o f c E H decreased by 6 5 % after 4 days of culture. By contrast, the expression of m E H and G S T was r a t h e r stable for the time scale considered.
Drug metabolism in H5-6 hepatoma
The presence o f m E H was also revealed by Western blot [Fig. 2(A)]. The antibodies i m m u n o r e a c t e d with a 50 k D a - b a n d , which c o r r e s p o n d s to the molecular weight o f the rat liver purified enzyme. W h e n antibodies raised against the mouse liver cEH were used, they cross-reacted with the rat liver enzyme [59 kDa, Fig. 2(B), lane e]. They also revealed a faint band o f similar molecular weight in cell h o m o g e n a t e s [Fig. 2(B), lane d]. N o a p p a r e n t increase in the immunoreaction staining, upon treatment with fenofibrate (lane c), could be seen. The activity o f 1-naphthol and bilirubin glucuronidation, which is catalyzed by two separate UDP-glucuronosyltransferases is reported in Table 3. G l u c u r o n i d a t i o n o f 1-naphthot increased slightly, especially after 5 days, whereas bilirubin glucuronidation remained stable through the time course o f the experiment. The use o f antibodies immunoreacting with the enzyme forms which glucuronidate phenols and bilirubin revealed a main band o f 5 4 k D a [Fig. 2(A)].
! 30 A
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0306-3623/91 $3.00 + 0.00 Copyright © 1991 Pergamon Press plc
Printed in Great Britain. All rights reserved
EXPRESSION OF A R Y L H Y D R O C A R B O N HYDROXYLASE, EPOXIDE HYDROLASES, GLUTATHIONE S-TRANSFERASE A N D U D P - G L U C U R O N O S Y L T R A N S F E R A S E S IN H5-6 HEPATOMA CELLS MARINA ROQUES,* DENYSE BAGREL,JACQUESMAGDALOUt and GERARDSIEST Centre du M6dicament, U.R.A. CNRS No. 597, Facult6 des Sciences Pharmaceutiques et Biologiques, 30 rue Lionnois, 54000 Nancy, France [Tel. 83-32-29-23; Fax 83-32-13-22] (Received 16 October 1990)
Ahstract--l. The presence of arylhydrocarbon hydroxylase (cytochrome P-450 IAI dependent), glutathione S-transferase, two distinct forms of epoxide hydrolases and UDP-glucuronosyltransferases was detected in H 5 ~ hepatoma cell homogenates using model substrates, selective inhibitors and specific antibodies. 2. The activity of arylhydrocarbon hydroxylase decreased strongly at the first days after plating and remained at a minimal value (1.5 pmol/min per mg) after 5 days of culture. 3. The hydratation of trans-stilbene oxide catalyzed by the soluble form of epoxide hydrolase was very low (ll.0pmol/min per mg), whereas the hepatoma cells contained appreciable amounts of the membrane-bound epoxide hydrolase and glutathione S-transferase measured with cis-stilbene oxide as substrate (maximal specific activity: 1.46 and 2.73 nmol/min per mg, respectively). 4. These cells also glucuronidated l-naphthol efficiently (6 nmol/min per mg) and, at a lower extent, bilirubin (12 pmol/min per mg). 5. Addition of fenofibrate (70 ~ M) into the culture medium for 1 3 days failed to significantly stimulate the activity of cytosolic epoxide hydrolase. Only bilirubin glucuronidation increased 2-fold after 2 days of presence of the drug.
INTRODUCTION
Compared to laboratory animals, hepatoma cells provide an attractive and alternate tool to investigate various aspects of cellular biology and especially drug metabolism. Hepatoma cells are generally easy to handle and present a better stability of their enzymatic functions, when compared to hepatocytes. The characterization of proteins involved in the transformation of drugs and other potentially hazardous substances can contribute whether to choose this model to investigate the metabolic pathways, which account for the elimination of these substances from the cell, and for their possible toxic effects. Moreover the response of drug metabolizing enzymes towards inducers helps in the understanding of the regulation mechanism of these proteins at a molecular level. H5-6 hepatoma cell line, issued from H5, a cell line which derives from the rat Reuber hepatoma H35, has been recently established (Sperling et al., 1980). Until now, the characterization of drug metabolizing enzymes has not been extensively performed. Wiebel et al. (1984) reported that H5 expressed a cytochrome P-450 inducible by 3-methylcholanthrene but not the *Present address: Unit6 INSERM No. 61, 3, avenue Moli~re, 67200 Strasbourg, France. tTo whom all correspondence should be addressed.
phenobarbital inducible forms. In a preliminary experiment by Western blotting with several specific cytochromes P-450 antibodies, we were only able to detect in H5-6 a protein corresponding to the isoform IA1 (non-published observations). In this work the activity of arylhydrocarbon hydroxylase (AHH, EC 1.14.14.2), supported by cytochrome IAI, was therefore measured. In order to determine if H 5 ~ cells have the capacity to metabolize drugs, the presence of membrane bound and cytosolic epoxide hydrolases (mEH, cEH, EC 1.11.1.6), glutathione S-transferase (GST, EC 2.5.1.18) and UDP-glucuronosyltransferases (UDPGT, EC 2.4.1.17) was investigated. These enzyme systems play a crucial role in the destruction of reactant epoxides and elimination of drugs as hydrophilic glucuronides (Glatt and Oesch, 1977; Siest et al., 1987; Wixtrom and Hammock, 1985). Their activity is known to be enhanced by inducers such as phenobarbital, 3-methylcholanthrene and clofibrate (Owens, 1977; Fournel et al., 1987; Ding and Pickett, 1985). The presence of several isoenzymes was determined with model substrates and selective inhibitors of the corresponding hepatic enzymes, as well as from their immunoreactivity with antibodies raised against the corresponding purified liver enzymes. Finally the inducing effect of the hypolipidemic drug fenofibrate (2-[4-(4-chlorobenzoyl)phenoxy]-2-methylpropanoic acid 1-methylethyl ester) was investigated. 677
GP 22/~-H
MARINA ROQUESet al.
678 MATERIALS AND METHODS
Chemicals Tritiated trans-stibene oxide (TSO), tritiated cis-stilbene oxide (CSO) and 4-fluorochalcone oxide were kindly supplied by Professor Hammock (University of California, Davis, CA). Cyclohexane 1,2-oxide and l,l,l-trichloropropane 2,3-oxide were purchased from Aldrich Chemic (Steinheim, Germany). l-Chloro-2,4-dinitrobenzene and benzo(a)pyrene was provided by Sigma (St Louis, MO). Fenofibrate was kindly supplied by Laboratoires Fournier (Daix, France). l-[l-~4C]naphthol (58 mCi/mmol, 2.15 GBq/ retool) was purchased from Amersham (Les Ulis, France). UDP-glucuronic acid (sodium salt) was provided by Boehringer (Mannheim, Germany), and bilirubin by Merck (Darmstadt, Germany). All the other reagents were of the best purity commercially available. Ce.II euhures
Hepatoma H5 6 cells have been cloned from the H4IIEC3 cell line (Pitot et al., 1964), which has been established from Reuber H35 rat hepatoma (Reuber, 1961). Cells were grown at 3TC in a humidified atmosphere of 95% air and 5% CO_,, in Dulbecco's modified Eagle medium (Gibco, Cergy-Pontoise, France), supplemented with 10% v/v Nu-Serum (Collaborative Research, Bedford, MA) in the presence of 1% v/v antibiotic and antimycotic mixture (penicillin I00 UI/ml, streptomycin 100/~g/ml and fungizone 0.25 #g/ml, Gibco). Treatment of cells
Cells were seeded in 100 mm-diameter plastic dishes, at a density of 1 × 106 per 10 ml medium. These conditions allow to perform the experiments during the exponential phase of growth. The medium was replaced with fresh medium 48 hr after seeding. For induction experiments, the fresh medium contained increased concentrations of fenofibrate in the range 15 280pM and dissolved in a same amount of DMSO. The final solvent concentration was 1% v/v. Two control experiments were simultaneously run in which the cells were grown either in presence of DMSO or without any solvent. After 24, 48 and 72 hr of treatment, the culture medium was collected, centrifuged at 170g for 5 min and frozen at - 2 0 C . The cells were washed twice with ice-cold phosphate-buffered saline, harvested and suspended in this solution. They were frozen at - 8 0 C after centrifugation again at 170 g for 5 rain. Evaluation o/" cytotoxicity
The viability of the cells was estimated with the Trypan blue exclusion test. Release of lactate dehydrogenase (LDH) was measured in the medium according to the method of Mathieu et al. (1982). Biochemical assays
Before measurement of epoxide metabolizing enzyme activity, cells were suspended in the buffer used to assay the corresponding enzymes and homogenized in a glass-glass homogenizer in ice, in order to make the cells completely broken, as revealed by observation under light microscopy. For UDPGT, thawing was enough to remove the enzyme latency. Additional homogenization procedure resulted in complete loss of activity. Immunoreactivity and biochemical properties of the drug metabolizing enzymes were determined on cells cultured for 96 hr. The concentration of cellular proteins was determined by the method of Lowry et al. (1951) with bovine serum albumin as standard. Hydroxylation of benzo(a)pyrene was followed according to the fluorimetric assay of Nebert and Gelboin (1968) modified by Wiebel et al. (1977). The activity of mEH, cEH and GST measured with CSO or TSO was determined at 37'C with the radiometric partition assays described by Wixtrom and Hammock (1985). Measurement
of cEH activity was performed in presence of 1.25 mM 1-chloro-2,4-dinitrobenzene to deplete glutathione, according to Schladt et al. (1986). For inhibition experiments, 4-fluorochalcone oxide (10 _3 M) in 1 ~1 DMSO, cyclohexane 1,2-oxide (10-5_10 3 M) or I,l,l-trichloropropane 2,3-oxide (10 3 M) in 1 #l ethanol or 1-chloro-2,4-dinitrobenzene (10 ~M) in 1/~1 ethanol were added 2 rain before starting the enzyme assay of the EHs and the GST. Glucuronidation of Inaphthol was followed according to the method of Bock et al. (1974). A modification of the procedure described by Heirwegh et al. (1972) was used for the measurement of bilirubin glucuronidation. The incubation mixture (300 #1) consisted in 0.5rag of cellular proteins and 0.17mM bilirubin. After 30 min of incubation at 37C, the reaction was stopped with addition of 0.5 ml of 2 M glycine-HC1 buffer (pH 2.7). The diazo reagent (0.5ml) was added and the diazo by-products formed were extracted with 2-pentanone and quantified by spectrophotometry at 530 nm. bnmunochemical detection
After solubilization in 2% w/v sodium dodecylsulfate, 5 mM dithiothreitol and 50 mM iodoacetamide, the cell homogenates were fractionated by gel electrophoresis (Laemmli, 1970), using 12.5% (w/v) polyacrylamide in the separating gel and 5% (w/v) in the stacking gel. Proteins were transferred with a Trans-Blot system (Bio-Rad, Richmond, CA) at room temperature overnight at 50V in 20mM Tris-HCl buffer (pH 8.3) containing 20% v/v methanol and 150mM glycine (Burnette, 1981). The proteins on the nitrocellulose sheets immunoreacted with specific rabbit antibodies raised against purified rat liver mEH, UDPGT isozymes active towards phenols and bilirubin (Pham et al., 1989; Thomassin et al., 1986; Zhiri et al., 1987), and mouse liver cEH (Dietze et al., 1990). The antisera were diluted 750 2000-fold. Revealation of the bands was carried out with peroxidase-conjugated sheep anti rabbit IgG (Institut Pasteur, Paris, France) and 3,Ydiaminobenzidine as detector agent. Statistical analysis
The data were analyzed using the Student's t-test for small samples and non-paired series. A difference between groups of P < 0.05 was considered significant. RESULTS Constitutive expression o f A H H , epoxide metabolizing enzymes and U D P G T
Figure 1 represents the evolution p a t t e r n of A H H activity in function of time of culture. Hydroxylation of benzo(a)pyrene was at a maximal level at the beginning of culture. The reaction rate decreased dramatically thereafter to reach a residual a n d stable value after 5 days (1.5 p m o l / m i n per mg). The optimal conditions for the m e a s u r e m e n t of epoxide metabolizing enzymes were determined. The reactions proceeded linearly in function of time up to 20 m i n for c E H and up to 6 rain for m E H and GST. The reaction rates were linear in function of the protein content up to 1.2, 0.6 a n d 0.2 mg for cEH, m E H a n d GST, respectively (data not shown). In Table 1 are indicated the specific activities m e a s u r e d for cEH, m E H a n d G S T in function of time a n d various treatments of the cells with D M S O and fenofibrate. In control cultures (without D M S O ) , the specific activity o f c E H decreased by 6 5 % after 4 days of culture. By contrast, the expression of m E H and G S T was r a t h e r stable for the time scale considered.
Drug metabolism in H5-6 hepatoma
The presence o f m E H was also revealed by Western blot [Fig. 2(A)]. The antibodies i m m u n o r e a c t e d with a 50 k D a - b a n d , which c o r r e s p o n d s to the molecular weight o f the rat liver purified enzyme. W h e n antibodies raised against the mouse liver cEH were used, they cross-reacted with the rat liver enzyme [59 kDa, Fig. 2(B), lane e]. They also revealed a faint band o f similar molecular weight in cell h o m o g e n a t e s [Fig. 2(B), lane d]. N o a p p a r e n t increase in the immunoreaction staining, upon treatment with fenofibrate (lane c), could be seen. The activity o f 1-naphthol and bilirubin glucuronidation, which is catalyzed by two separate UDP-glucuronosyltransferases is reported in Table 3. G l u c u r o n i d a t i o n o f 1-naphthot increased slightly, especially after 5 days, whereas bilirubin glucuronidation remained stable through the time course o f the experiment. The use o f antibodies immunoreacting with the enzyme forms which glucuronidate phenols and bilirubin revealed a main band o f 5 4 k D a [Fig. 2(A)].
! 30 A
E .c
E 20 CL
._> [3 nT
