Introduction

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The UDP-glucuronosyltransferases (UGTs)¹ are a large family of enzymes that play a key role in the inactivation and elimination of many endogenous and exogenous compounds, such as bile acids, steroids, drugs, environmental pollutants, and dietary constituents. Two families of UGT proteins have been defined and termed UGT1 and UGT2 based on sequence similarities (Burchell et al., 1997; Mackenzie et al., 1997). The UGT1 family members are encoded by a unique gene complex, UGT1, with identical carboxy-terminal domain, and differing amino-terminal domains that confer aglycone specificity. UGT2 family members, in contrast, seem to be coded by independent genes (Ritter et al., 1992; Meech and Mackenzie, 1997). Each UGT isoenzyme exhibits unique, but overlapping profile of substrate and tissue specificity and regulation by exposure to endogenous and xenobiotic compounds. The UGT1 family includes enzymes that are capable of conjugating bilirubin, quinones, and phenols. Some are inducible with phenobarbital, or with polycyclic aromatic hydrocarbons, respectively, and numerous enzymes are constitutively expressed. It is very difficult to determine or predict the complete substrate profile of any transferase because multiple transferase isoforms overlap and metabolize many different substrates (Sutherland et al., 1993; Guengerich et al., 1997). Phenol derivatives such as p-nitrophenol (pNP), 1-naphthol, and bilirubin have been widely used to estimate conjugation capacity of UGT1 isoenzymes (UGT1A6 and UGT1A1, respectively); however, neither of them can be considered as specific substrate of any UGT (Ciotti et al., 1997). These isoforms are inducible and the regulation of their activity may, therefore, be an important determinant of drug detoxification and elimination (Prough et al., 1996).

Conjugation with glucuronic acid is one of the main metabolic pathways of the thyroid hormones, thyroxine (T4), and 3,3',5-triiodothyronine (T3) in the rat. Some chemicals, like 3-methylcholanthrene (MC) and clofibrate (Cl), which induce the activity of some UGTs, have been shown to enhance the metabolic clearance of T4; others, like pregnenolone-16-carbonitrile and polychlorinated biphenyls, have been shown to increase the clearance of both T3 and T4, and result in a decrease in serum T3 and/or T4 concentration (Curran and DeGroot, 1991; Visser et al., 1991; Barter and Klaassen, 1992; Brucker-Davis, 1998). The low plasma level increases the secretion of thyroid-stimulating hormone, and chronic exposure of rats to UGT inducers may cause morphological changes in the thyroid (Saito et al., 1991). Induction studies on T4 and T3 metabolism have described that T4 and T3 are glucuronidated by different UGTs, also suggesting that both of them are substrates for more than one UGT isoenzymes (Beetstra et al., 1991; Brucker-Davis, 1998). T3 is ranged among UGT2B3 substrates in case of rat, and T4 among UGT1*1 and UGT1*02 substrates in case of humans (Burchell et al., 1997). T4 was shown to be conjugated by rat UGT2B1-expressing V79 cells, whereas T3 was not (Pritchard et al., 1994).

The purpose of the present work was to study the effect of MC, dexamethasone (DEX), Cl, and MC combined with DEX and Cl, respectively on T4 and pNP metabolism, to gain more information concerning the enzymes involved in T4 metabolism in the rat.

	Materials and Methods

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Chemicals. T4, UDP-glucuronic acid, MC, clofibric acid, DEX, D-saccharic acid 1,4-lactone, Williams Medium E, and collagenase were purchased from Sigma Chemical Co. (St. Louis, MO), Triton X-100 and pNP were obtained from Reanal (Budapest, Hungary). SDS and acetonitrile were purchased from Merck (Darmstadt, Germany). Other chemicals were of analytical grade of the highest purity available.

Primary Hepatocyte Cell Culture. Hepatocytes were prepared from male Wistar rats (200 g) by in situ liver collagenase perfusion (Seglen, 1976). Cell viability (>85%) was determined by trypan blue exclusion. Cells were seeded on collagen-coated dishes (60 × 60 or 100 × 100 mm) at a density of 4.5 × 10⁶ and 11.25 × 10⁶ cells per dish, respectively, in Williams Medium E containing 10% fetal calf serum, 100 nM DEX, 100 nM insulin, 2.5 µg/ml amphotericin B, 0.1 mg/ml gentamicin, and 30 nM Na₂SeO₃. Calf serum and amphotericin B were present for the first 24 h, then omitted. Cells were maintained at 37°C in a humidified atmosphere of 95% air/5% CO₂. Four hours after plating and every day thereafter the medium was changed to Williams Medium E supplemented with 3.7 µM MC, 10 µM DEX, 100 µM Cl, 3.7 µM MC combined with 10 µM DEX, and 3.7 µM MC combined with 100 µM Cl, respectively. Control medium contained 0.1% dimethyl sulfoxide as it was the solvent of the inducers. The induction period lasted for 72 h. Cells washed twice with PBS were either incubated with T4 or with pNP for 2 h or for 20 and 40 min, respectively. For preparing microsomes, cells were scraped and sonicated on ice followed by centrifugation at 10,000g for 30 min, and the microsomes were sedimented at 100,000g centrifugation for 60 min. Protein concentration was determined by the method of Lowry, using BSA as protein standard (Lowry et al., 1951). Microsomes were stored at 70°C, and were used for the determination of UGT activity and for Western immunoblot analysis.

Enzyme Assays. UGT activity was determined in intact cell culture using pNP and T4 as substrates. Cells were treated with Williams Medium E containing pNP (0.1 mM) and T4 (0.25 mM). pNP UGT activity was measured by HPLC detection of pNP-glucuronide peak, using pNP-glucuronide for a standard curve. Twenty microliters of the medium taken at 20 and 40 min of incubation were injected directly into the HPLC. The chromatographic equipment consisted of two Isco model 2350 pumps, an Isco V⁴ type UV detector (Isco, Lincoln, Nebraska), a Hypersil ODS 150 × 4 mm reversed phase column with particle size of 3 µm (BST, Budapest, Hungary). The mobile phase for determination of p-NP and its glucuronide consisted of 15% acetonitrile, 85% 0.02 M potassium phosphate buffer pH 1.8, and 2.5 mM SDS. The flow rate was 1 ml/min, and the wavelength was 305 nm. In medium containing 0.1 mM pNP the rate of conjugation was linear up to 60 min. The detection limit for pNP-glucuronide was 1 µM.

Western Immunoblot Analysis. The microsomes were prepared for immunoblot analysis by suspension in sample loading buffer, heated at 100°C for 5 min, and separated by SDS-polyacrylamide gel electrophoresis (7.5%). After separation, the proteins were transferred to nitrocellulose membranes, treated with PBS-Tween 20 (0.1%) containing 3% BSA overnight at 4°C (Towbin et al., 1979). The membranes were washed in PBS containing 0.1% Tween 20 before the addition of primary antibodies. Rat anti-liver antibody (RAL) (1:3000 dilution of 46.5 mg of protein/ml) originated from the laboratory of Professor Brian Burchell (University of Dundee, Dundee, UK). RAL was a gift from László Braun, Semmelweis Medical University, Budapest, Hungary. After incubation with primary antibodies, the membranes were washed and incubated with secondary antibody, horseradish peroxidase-conjugated anti-sheep IgG (Calbiochem, San Diego, CA; dilution as recommended by the supplier). Immunoreactive proteins were visualized by the chemiluminescence method (Amersham International, Arlington Heights, IL).

	Results

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We have developed a sensitive method for the measurement of pNP and T4 UGT activities in cultured hepatocytes analyzing the medium by HPLC. Each experiment for studying the induction of UGT activity of hepatocytes was performed in monolayers prepared from a single rat, cells treated with 0.1% dimethyl sulfoxide served as control. Six independent experiments were conducted for both substrates with three parallels for each inducer or combination of inducers and for control as well. UGT activities of control hepatocytes for pNP and T4 were 0.592 ± 0.061 nmol/min/10⁶ cells and 8.17 ± 3.83 pmol/min/10⁶ cells, respectively. The values represent mean ± S.D.

For 96 h of incubating T4 UGT activity remained relatively constant, and no change in cell viability was observed. Addition of Cl and DEX to the medium produced a 3- and 2-fold increase in T4 UGT activity, respectively, by 96 h. MC treatment increased the T4 UGT activity, reaching a value 7.5 times higher than that of the control cultures by 72 h, but then declining during the next 24 h, still remaining 5 times higher than that of the control (Fig. 1.).

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Time courses for induction of T4 UGT activity by MC, Cl, or DEX in cultured rat hepatocytes. Enzyme activity was measured in the supernatant fraction of cultured hepatocytes treated with either vehicle or with 3.7 µM MC, 100 µM Cl, or 10 µM DEX for various times as described in Materials and Methods. Activities are expressed as mean ± S.D. for three culture dishes.

The concentration dependence of T4 UGT activity by Cl is shown in Fig. 2. Incubation was carried out for 48 h. Fifty micromolar Cl enhanced T4 UGT activity to 165%, and an additional increase was observed in the presence of 100 µM Cl (215%). Addition of 200 µM Cl to the medium resulted in a dramatic decrease in T4 glucuronide formation: T4 UGT activity dropped to 30% of the value observed in control cultures, although the cell viability did not appear to change. For additional induction studies 100 µM Cl concentration and 72 h of induction were chosen.

View larger version (11K): [in this window] [in a new window]   Fig. 2.   Concentration-dependent induction of T4 UGT activity by Cl. Enzyme activity was measured in the supernatant fraction of cultured hepatocytes treated either with vehicle or with various concentrations of Cl as described in Materials and Methods. Activities are expressed as mean ± S.D. for three culture dishes.

As shown in Fig. 3A, MC, Cl, and DEX significantly increased the activity of T4 UGT. The combination of MC and Cl showed additive effect, the T4 UGT activity was enhanced significantly compared with either MC or CL treatment alone (617, 441, and 217% of the control, respectively). The combination of MC and DEX did not result in higher T4 UGT activity than MC treatment alone. Figure 3B shows the effect of inducers on pNP UGT activity. Cl treatment did not affect the rate of pNP conjugation, either alone or in combination with MC. Both MC and DEX treatment enhanced the level of pNP UGT activity significantly (182 and 162% of the control, respectively), and the combination of MC with DEX produced an additional enhance of activity (228% of the control).

View larger version (35K): [in this window] [in a new window]   Fig. 3.   Induction of T4 UGT (A) and pNP UGT (B) activities by MC, Cl, DEX, and MC combined with Cl or DEX. Enzyme activity was measured in the supernatant fraction of cultured hepatocytes treated either with vehicle or with 3.7 µM MC, 100 µM Cl, 10 µM DEX, or 3.7 µM MC combined with 100 µM Cl or 10 µM DEX, as described in Materials and Methods. The data shown are expressed as mean ± S.D. of six separate experiments conducted with three replicate dishes at each point. *, statistically different (P < .01) from control; **, statistically different from the activity of cells treated with MC alone.

To visualize any change in enzyme concentrations, the microsomes prepared from cells after treatment with the inducers were analyzed by Western blot. RAL antigen reacts with UGT1A1, UGT1A6, and two enzymes of the UGT2 family (not identified). According to the representative Western blot shown, the amount of UGT1A6 was increased by MC either alone or in combination with Cl or DEX (Fig. 4). The Western blot also suggests that the protein concentration of the UGT1A6 fraction was not affected by Cl and DEX. The intensity of the UGT1A1 band was enhanced by both Cl and DEX. The effect was more pronounced in the case of Cl treatment. MC had no effect on the UGT1A1 protein level.

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Western blot analysis of microsomes prepared from rat hepatocytes after induction by MC, Cl, DEX, and MC combined with Cl or DEX using an anti-rat UGT antibody. Microsomes were prepared and Western blotting was carried out as described in Materials and Methods; 10 µg of protein were loaded in each line.

	Discussion

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Glucuronidation plays a central role in the metabolism of endobiotic and exobiotic compounds. Many human and rat UGT enzymes have already been identified by gene sequencing and cDNA cloning (Visser et al., 1993a; Coffman et al., 1995; Emi et al., 1995). The substrate specificity and tissue distribution of the individual UGTs have also been investigated, but little is known about the in vivo role of these enzymes in the metabolism of different substrates (McGurk et al., 1998). The formation of inactive T4-glucuronide is the main metabolic pathway of T4 metabolism in rat, and it is well documented that administration of inducers like -naphthoflavone, MC, Cl, and polychlorinated biphenyls increased specific hepatic microsomal UGT activity toward T4 (Visser et al., 1993b). In this manuscript we demonstrated that primary hepatocyte culture is a reliable assay system for studying this induction process. Combination of MC and Cl treatment resulted in an additive inductive effect on T4 glucuronidation, which was detected at the level of enzyme activity. The UGT protein pattern revealed that the two inducers MC and Cl stimulate the synthesis of different UGTs, UGT1A6 and UGT1A1, respectively. On the other hand, it is well documented that phenobarbital, a good inducer of UGT1A1, has no effect on T4 glucuronidation (Burchell et al., 1997). These data suggest that although, like phenobarbital, Cl is an inducer of UGT1A1, another Cl-inducible enzyme is apparently involved in T4 metabolism. This hypothesis is supported by the results of DEX, which enhanced the amount of UGT1A1 and had only a slight effect on T4 glucuronidation.

Previous studies demonstrated the potentiation of polycyclic aromatic hydrocarbon induction of cytochrome P-4501A1 and UGT1A6 by DEX (Prough et al., 1996). In our experiments both MC and DEX doubled the rate of pNP-glucuronide formation, and their joint effect was additive. On the contrary, although MC induced T4 conjugation more than pNP glucuronidation, DEX was ineffective on T4 conjugation, suggesting that UGT1A6 is not the only MC-inducible UGT enzyme responsible for T4 conjugation. This hypothesis is in agreement with the results of Xiao et al. (1995), which have provided evidence that UGT1A6 induction by benzanthracene was potentiated by DEX at the level of enzyme activity, protein, and mRNA.

The decline of T4 UGT activity observed at 200 µM Cl resembles the findings of Boie et al. (1993), which have described that the responsiveness of peroxisome proliferator-activated receptor to the peroxisome proliferator peaks and declines at higher concentrations for all proliferators tested. The reason for the decrease in T4 UGT activity is not clear, but it does not appear to be the result of a change in cell viability as judged by trypan blue exclusion.

In conclusion, our results suggest that there are enzymes other than UGT1A1 and UGT 1A6 that can be induced by Cl and/or MC and are responsible for T4 conjugation.