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Laboratory of Toxicokinetics and Metabolism, Department of Industrial Hygiene and Toxicology, Finnish Institute of Occupational Health, Helsinki, Finland (E.E., J.M., L.L.); Viikki Drug Discovery Technology Center, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland (J.T.); UMR 7561 Centre National de la Research Scientifique-Université Henri Poincaré Nancy, Vandoeuvre-lès-Nancy, France (L.A., S.F.-G., J.M.); and Department of Molecular and Cellular Pathology, Ninewells Hospital and Medical School, University of Dundee, Dundee United Kingdom (B.B.)
(Received June 18, 2004; accepted September 13, 2004)
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Abstract |
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; ACGIH, 2001). Catechol itself is classified as a rodent carcinogen (IARC Working Group, 1999). It is thus noteworthy that biotransformation of common industrial carcinogens, such as benzene and polycyclic aromatic hydrocarbons (PAHs), involves pathways generating catechols (Lovern et al., 2001) and various catecholic PAH derivatives, respectively (Penning et al., 1999). Catechols are also present in dietetic products (Carmella et al., 1982). Natural catechols from plant extracts, such as catechins and polyphenols from green tea, show anticancer activity (Colic and Pavelic, 2000).
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Dopamine and other neurotransmitters are functionally important endogenous catechols. Entacapone and tolcapone are selective and reversible catechol-O-methyltransferase (COMT) inhibitors (Fig. 1), which prevent the peripheral O-methylation of levodopa and therefore enhance the bioavailability of this dopamine precursor in the brain. These nitrocatechols, developed as adjuncts to antiparkinsonian medication, prolong the duration and improve the efficacy of dopatherapy in patients with fluctuating Parkinson's disease (Kurth and Adler, 1998; Männistö and Kaakkola, 1999; Poewe, 2004). Tolcapone has been withdrawn or its use restricted following findings of acute hepatic dysfunction in several patients and, in three cases, fatal fulminant hepatitis (Olanow, 2000; Spahr et al., 2000). In the light of the experience increasing worldwide (over 300,000 patient years), entacapone is potentially not a hepatotoxin (Brooks, 2004). In the rat model, high doses of tolcapone (500 mg/kg/day), but not of entacapone, cause hepatotoxicity and uncoupling of mitochondrial oxidative phosphorylation (Haasio et al., 2002). Entacapone and tolcapone are metabolized and eliminated in bile and urine mainly as glucuronides (Wikberg et al., 1993; Jorga et al., 1999; Keski-Hynnilä et al., 2002).
Catechols undergo two main metabolic pathways, oxidation and conjugation. Oxidation of catechols to electrophilic and redox active semiquinones and o-quinones generates quinoid and reactive oxygen species, which cause damage by covalent binding to and oxidation of DNA and other cellular macromolecules. The formation of catechol o-quinones is proposed to imply a unifying mechanism in the development of cancer by catechols, catechol PAH, and catechol estrogens (Bolton, 2002; Penning et al., 1999), and also in the initiation of Parkinson's disease and other neurodegenerative disorders by dopamine (Cavalieri et al., 2002).
Characterization of catechol metabolism by sulfation, methylation, and glucuronidation has been evaluated with a set of 53 compounds to clarify common structural features that govern their conjugation pathways. Investigation of the UDP-glucuronosyltransferase (UGT; EC 2.4.1.17
[EC]
) isoforms that catalyze the glucuronidation of catechols has been performed using five human (h) and two rat (r) cloned and expressed isoenzymes. Thirty-eight catechols were glucuronidated at least by one of the forms hUGT1A1, hUGT1A6, hUGT1A9, hUGT2B7, hUGT2B15, rUGT1A6, and/or rUGT2B1. When these catechols were classified as substrates of recombinant COMT and sulfotransferase isoenzymes, 45 were methylated and 48 sulfated, respectively (Lautala et al., 2001; Antonio et al., 2002; Taskinen et al., 2003).
The aim of this study was to investigate the inductive effect of five model catechols on the glucuronidation of catechols themselves and noncatecholic UGT probe substrates (1-naphthol, 1-hydroxypyrene) in rats. The well known inducer, PCB, was used as a positive control. Hepatotoxicity of treatment doses was estimated by blood plasma transaminase tests [alanine and aspartate aminotransferases (ALT and AST)]. Since UGT1A9 and 1A6 isoforms have been previously shown by us to be greatly involved in catechol glucuronidation (Lautala et al., 2000; Antonio et al., 2002, 2003), the catechol drug entacapone and the PAH phenols, 1-naphthol and 1-hydroxypyrene (1-OHP), were chosen as substrates.
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Materials and Methods |
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). Uridine 5'-diphosphate-glucuronic acid (UDPGA, sodium salt) and UDP-[U-14C]glucuronic acid (350 mCi/mmol) were obtained from Roche Diagnostics (Mannheim, Germany) and Amersham Biosciences Inc. (Saclay, France), respectively. 1-Hydroxypyrene was obtained from Acros Organics (Geel, Belgium). Animals and Study Design. Sixty male Wistar rats (271 ± 19 g, from the Helsinki University Breeding Centre) were housed in plastic cages bedded with aspen wood shavings in an air-conditioned room (23-25°C) with artificial lighting from 7:00 AM to 7:00 PM. They had free access to the pellet chow (Rat Chow, Altromin 1320; Altromin International, Lage, Denmark) and tap water. Five rats per group were treated with different catechols for 3 days by oral gavage (see Table 1). 4-Methylcatechol (100 or 150 mg/kg body weight/day) and 4-nitrocatechol (100 or 200 mg/kg/day) were administered in 5% sucrose syrup (2.5 ml/kg). Entacapone (200 mg/kg/day), tolcapone (200 mg/kg/day), and DHN (300 mg/kg/day) were administered in olive oil (3 ml/kg). Catechol- and vehicle-treated animals were killed 24 h after the third dose. PCB-treated rats and their controls were killed 5 days after a single 0.55 g/kg dose administered intraperitoneally in olive oil (2.5 ml/kg). Food was withdrawn 3 h before each treatment and before sacrifice. Blood (6-8 ml) was drawn under CO2 anesthesia by cardiac puncture with heparin-rinsed plastic syringes and transferred in plastic tubes (in an ice bath). The livers were perfused in situ with phosphate-buffered saline, removed, weighed, cooled in ice, and stored frozen (-70°C). The experiments were approved by the local Ethical Committee for Animal Studies (the guidelines of the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes were followed).
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Preparation of Microsomes from Rat Liver. The thawed livers were homogenized in 2 volumes (w/v) of cold 0.250 M sucrose/1 mM Tris-HCl (pH 7.4) buffer with a Potter-Elvehjem glass homogenizer (Thomas tubes with Teflon pestle). Enzyme fractions were prepared at 5°C by differential ultra-centrifugation. The supernatant of the first centrifugation (10,000g, 15 min) was run at 106,000g for 60 min to obtain as a pellet the microsomal fraction. The pellet was homogenized in 10 ml of buffer and centrifuged again at 106,000g for 60 min. The microsomes were resuspended in the buffer to give a protein content of 10 to 20 mg/ml, divided in small aliquots, and stored frozen in Eppendorf tubes (-70°C) until use. The protein concentrations were determined (Lowry et al., 1951) using commercial protein standards (Precise Eiweiss/Protein; Roche Diagnostics).
Blood Plasma Enzyme Analyses. Blood specimens were centrifuged at 3000g for 20 min (at 20°C) to obtain the plasma samples stored at -70°C until use. The activity of ALT and AST in plasma was kinetically determined by recommended methods (ECCLS, 1988) using enzyme reagent kits from Medix Biochemica (Kauniainen, Finland).
Glucuronidation Activity Analyses. Entacapone glucuronidation. The activity was determined by an HPLC method. The incubation mixture (final volume 250 µl, in Eppendorf tubes) contained 50 mM potassium/sodium phosphate buffer (pH 7.4), 8 mM MgCl2, 250 µM entacapone (added in assay buffer), and 40 to 80 µg of microsomal protein. The reaction was started with 50 µl of 25 mM UDPGA (5 mM), incubated for 15 min at 37°C, and stopped with 25 µl of cold 4 M perchloric acid. The tubes were placed in an ice bath for 10 min (protein precipitation) before being mixed and centrifuged (13,000 rpm, 5 min). The resulting supernatants were injected into a Shimadzu HPLC/CLASS VP 5.021 chromatography system (Shimadzu, Kyoto, Japan) equipped with a Nova-Pak C18 column (4 µm, 150 x 3.9 mm; Waters, Milford, MA), an SPD-10AV UV-visible detector (at 305 nm), and an SIL-10A autoinjector. Acetonitrile/0.5% acetic acid in Millipore H2O (25:75, v/v) was used as the mobile phase for isocratic runs at a flow rate of 1 ml/min. Entacapone 3-O-ß-D-glucuronide was used for product identification (RT = 3.1 min; see Fig. 2) and quantitation by peak area calculation. Calibration was based on a seven-point (0-4 µM) concentration curve (r 
0.99). The enzyme reaction was linear with incubation time (
60 min) and protein concentration (25-150 µg/250 µl).
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1-OHP glucuronidation assay. The reaction mixture contained 25 µl of rat liver microsomes (0.3-3 µg of protein) in 1% bovine serum albumin, 120 µl of 50 mM potassium/sodium phosphate buffer/0.15 M KCl (pH 7.4) with 10 mM MgCl2, 100 µl of 50 mM potassium/sodium phosphate buffer/0.15 M KCl (pH 7.4), with 11 mM UDPGA (samples) or without UDPGA (controls). The reaction was started (after a 5- to 7-min preincubation at 37°C) with 5 µl of 1-OHP in dimethyl sulfoxide, incubated for 10 min, stopped with 50 µl of cold 15% ZnSO4 solution, and cooled for 15 min in an ice bath (protein precipitation) before addition of 500 µl of acetonitrile. The tubes were mixed by vortex (10 s), sonicated (2 min), and centrifuged (13,000g, 10 min, at 20°C). The supernatants were analyzed as described previously (Luukkanen et al., 2001) in a Shimadzu HPLC/CLASS VP 5.021 chromatography system (Shimadzu) using an RF-10Axl fluorescence detector (
ex 242 nm, em 382 nm) and 60% acetonitrile in a 0.5% aqueous acetic acid for isocratic reverse-phase C18 runs. 1-Pyrenyl ß-D-glucuronide was used for product identification (RT = 2.5 min) and quantitation by six-point (0-56 nM) linear calibration curves (r 0.99) and peak area calculation. The enzyme reaction was linear with incubation time (30 min) and protein concentration (0.3-4 µg/250 µl).
1-Naphthol glucuronidation. This activity was determined using 70 to 100 µg of rat liver microsomal protein per 250-µl reaction mixture and a 500 µM 1-naphthol substrate concentration; but, otherwise, the reaction conditions were identical to those of the 1-OHP UGT incubation assay described above. The supernatants were diluted (1:31) in 30% acetonitrile/0.5% aqueous acetic acid (eluent) for isocratic analysis (5-µl injection, flow rate 0.9 ml/min) with a reverse-phase C18 guard cartridge (Spherisorb S5 ODS2, 30 x 4.6 mm) and an analytical column (S3 ODS2, 150 x 4.6 mm, Spherisorb; Waters, Wexford, Ireland) using an RF-10Axl fluorescence detector (ex 290 nm, em 332 nm) and an SIL-10ADvp autoinjector in a Shimadzu HPLC/CLASS VP 6.12 SP2 chromatography system. 1-Naphthol ß-D-glucuronide was used for product identification (RT = 4.1 min) and quantitation by six-point linear calibration curves (0, 0.1, 0.3, 0.6, 0.8, 1.0, and 1.5 µM; r = 1.00) and peak area calculation. The enzyme reaction was linear with incubation time (20 min; protein 150 µg/250 µl) and protein concentration (37-300 µg/250 µl; 10-min incubation).
Glucuronidation of catechol, 4-methylcatechol, 4-nitrocatechol, DHN, entacapone, and tolcapone. A thin-layer chromatographic method was used for this purpose (Antonio et al., 2002). Briefly, microsomes (100 µg of protein) were incubated in 100 mM Tris-HCl buffer (pH 7.4), 10 mM MgCl2 in the presence of radiolabeled UDP-[U-14C]glucuronic acid (2.5 mM, 0.1 µCi) and 0.5 mM catechol substrate dissolved in dimethyl sulfoxide for 1 h at 37°C. The reaction was stopped by addition of 40 µl of ethanol in ice. The precipitated proteins were removed by centrifugation for 10 min at 4000g and the supernatant was loaded onto LK6DF silica gel plates (250-µm thickness; Whatman, Clifton, NJ). The plates were developed with a mobile phase composed of n-butanol, acetone, acetic acid, 25% aqueous ammonia, and water (70:50:18: 1.5:60 v/v), dried, and sprayed with 1% (v/v) 2-(4-t-butylphenyl)-5-(4-biphenyl)-1,3,4-oxadiazole in toluene. The radioactivity associated with the glucuronide was visualized by autoradiography with BioMax MR-2 Kodak films (Sigma-Aldrich) after 3 days of exposure at -20°C. The silica gel was scrapped and the radioactivity was measured by liquid scintillation counting with a Tri-Carb 2100 TR spectrometer, with Fluoran Safe Ultima Gold (PerkinElmer Life and Analytical Sciences, Rungis, France) as scintillation cocktail.
Enzyme Kinetics. Apparent kinetic constants, Km and Vmax, toward entacapone and 1-OHP were determined by fitting the initial glucuronidation velocities (V) and substrate (S) concentrations to the Michaelis-Menten equation by a nonlinear least-squares method (Leonora v1.0; Cambridge University Press, Oxford, UK). The constants were determined also by the Eadie-Hofstee method, by plotting the observations (V versus V/S).
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Results |
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Glucuronidation of Catechols, 1-Naphthol, and 1-OHP in Liver Microsomes of Rats Pretreated with Model Catechols or PCB. Induction of catechol glucuronidation upon exposure to five different model catechols (Fig. 1) was assessed by using the inducing agents themselves as probe substrates in vitro. The specific glucuronidation activity toward these catechols and the catechol itself was determined at a 0.5 mM aglycone and a 2.5 mM glucuronic acid substrate level. The results are shown in Fig. 3. The catechol glucuronidation rates, when calculated from the means of the four vehicle-treated subgroups, were in control rat liver as follows (mean ± S.D., n = 4): 0.12 ± 0.04, 0.54 ± 0.16, 5.7 ± 1.1, 6.8 ± 1.2, 7.0 ± 0.5, and 22.0 ± 1.1 nmol/min/mg microsomal protein for entacapone, tolcapone, 4-methylcatechol, catechol, DHN, and 4-nitrocatechol, respectively. A multifold difference was consistently observed between the conjugation rates of the COMT inhibitors and the small catechols. The difference displayed between entacapone and 4-nitrocatechol was greater in vehicle-treated (183-fold) and catechol-treated rat groups (77- to 245-fold) than in PCB rats (14-fold), because the PCB treatment stimulated the metabolism of entacapone more than that of 4-nitrocatechol (15-fold versus 1.3-fold). The inducing effect of catechols was weak. No autoinduction was observed.
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The ability of catechols to induce glucuronidation metabolism in rat liver was determined by the substrates entacapone, 1-naphthol, and 1-OHP as shown in Table 2. Aroclor 1254 treatment enhanced the glucuronidation of 1-naphthol, entacapone, and 1-OHP by 5-, 8-, and 16-fold, respectively, whereas the catechols exhibited only a weak or no effect at all. Some slight increases were observed in rats treated with 4-methylcatechol (low dose), DHN, entacapone, or tolcapone in the activities toward 1-naphthol (24-30%), entacapone (25-50%), and 1-OHP (11-39%). For the UGT enzyme induction studies shown in Table 2, the substrate concentrations selected for screening of UGT by 1-OHP and entacapone are based on the enzyme kinetics shown in Figs. 4 and 5. The 1-OHP UGT assay is extremely sensitive, allowing activity measurement at low substrate concentrations. When the glucuronidation activity was determined at a concentration as low as 1 µM, the turnover was 16 times faster in PCB microsomes than in control microsomes. At the higher probe concentrations, 15, 100, and 150 µM, the glucuronidation rates were increased 10-fold, 4-fold, and 5-fold, respectively (Table 2).
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Kinetics of Entacapone and 1-OHP Glucuronidation in Rat Liver Microsomes. The initial velocities of the glucuronidation of 1-OHP (Fig. 4) or entacapone (Fig. 5) were measured in liver microsomes (pooled from five rats per treatment) under conditions with a substrate turnover of 10% at tested aglycone concentrations. The apparent Km and Vmax values were determined by the Michaelis-Menten equation for hyperbolic enzyme kinetics. These plots and the corresponding Eadie-Hofstee plots are presented in Figs. 4 and 5. Characterization of 1-OHP glucuronidation in microsomes from control (olive oil) or DHN-, entacapone-, or tolcapone-treated rats gave results consistent with monophasic Michaelis-Menten kinetics. The apparent Km and Vmax values were similar after these treatments, ranging from 6.0 to 6.4 µM and from 8.3 to 10.1 nmol/min/mg, respectively. Variance in intrinsic clearance was small (Vmax/Km, 1.3-1.8 ml/min/mg). For PCB microsomes, the Km was 7.6 µM and the Vmax 22.1 nmol/min/mg (Vmax/Km = 2.9 ml/min/mg). The monophasic or multiphasic nature of the Eadie-Hofstee plots (yielding one or three straight lines) suggested that 1-OHP glucuronidation was catalyzed in catechol compound-treated rats predominantly by one isoenzyme but in PCB microsomes by several forms. Up to three components could be resolved for 1-OHP glucuronidation data: Km1, Km2, Km3 = 0.8, 9.7, and 63 µM, and Vmax1, Vmax2, Vmax3 = 11, 24, and 55 nmol/min/mg, respectively. The corresponding Vmax/Km values were 13.8, 2.5, and 0.9 ml/min/mg, respectively (Fig. 4.).
Entacapone glucuronidation kinetics were analyzed in control and PCB microsomes, giving the results: Km = 237 µM, Vmax = 0.37 nmol/min/mg (Vmax/Km = 1.6 x 10-3 ml/min/mg) and Km = 130 µM, Vmax = 1.62 nmol/min/mg (Vmax/Km = 12.5 x 10-3 ml/min/mg), respectively. The changes observed in kinetic constants suggested UGT enzyme induction. Despite the present findings implicating an increase in catalytic affinity toward entacapone, only one component was resolved by the Eadie-Hofstee method (Fig. 5.)
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; Burchell et al., 1995, 1998; Mackenzie et al., 1997; Tukey and Strassburg, 2000). The reaction leads to the formation of hydrophilic glucuronides, which can be excreted into bile and urine (Wikberg et al., 1993; Jorga et al., 1999; Radominska-Pandya et al., 1999; Antonio et al., 2002, 2003; Keski-Hynnilä et al., 2002). In this work, we determined, using dedicated thin-layer chromatography or HPLC methods, the glucuronidation activity of different catecholic substrates, and of the antiparkinsonian drugs tolcapone and entacapone. The average rates at which these catechols were glucuronidated in liver microsomes of control rats differed up to 183-fold, entacapone being glucuronidated at the lowest rate, whereas 4-nitrocatechol was actively glucuronidated. These data revealed the importance of the structure of the chemical group on the catechol ring on the glucuronidation reaction.
Compared with the effects by the highly potent inducer, PCB, the glucuronidation rate of catechols showed only marginal alterations in response to the present catechol treatments. Under the experimental conditions used here, catechols appeared to present no inducing properties. The data on the inductive effect of PCB on catechol glucuronidation could be favorably compared with those of Antonio et al. (2002), who determined the induction potency of 3-methylcholanthrene, phenobarbital, or the peroxisome proliferator, clofibrate, on the glucuronidation of 41 catechols. The results indicated that only 3-methylcholanthrene could enhance catechol glucuronidation, thus indicating that UGT isoforms (UGT1 family) could be involved in catechol glucuronidation. The PAH type of induction, e.g., by 3-methylcholanthrene, ß-naphthoflavone, 2,3,7,8-tetrachlorodibenzo-p-dioxin, PAH, or PCB, is regulated by aryl hydrocarbon receptor, which functions as a ligand-activated transcription factor in over-expression of the forms rUGT1A6, rUGT1A7, hUGT1A6, and hUGT1A9 (Bock et al., 1998, 1999). Other typical coeffects achieved by using Aroclor 1254 as the inducing PCB agent (Safe, 1994) are a phenobarbital type of induction and hepatomegaly, which accounted for a 55% increase in the liver weights (Table 1).
PCB induction enhanced the glucuronidation of all six catechols significantly, as shown in Fig. 3, and that of 1-naphthol and 1-OHP as well (Table 2). Although the activity increased relatively more with entacapone (8-15 times) than with 4-nitrocatechol (1.3-fold), 4-nitrocatechol was still conjugated 14 times faster than entacapone. In control microsomes, entacapone was conjugated 4 times slower than tolcapone, but upon UGT induction by Aroclor 1254, the rates became similar. Creosote is also a PAH-type inducer that enhances entacapone glucuronidation more than that of tolcapone (Lautala et al., 1997). The finding that entacapone glucuronidation was enhanced relatively more than the tolcapone glucuronidation suggests differential kinetics of PCB-inducible UGTs toward these COMT-inhibitor drugs. Moreover, we have found that entacapone glucuronidation was enhanced in rat liver over 6-fold upon exposure to ß-naphthoflavone, benzo(a)pyrene, or 3-methylcholanthrene, whereas exposure to 
-naphthoflavone or chrysene had an intermediary effect (a 2-fold increase). Finally the PAHs, naphthalene, phenanthrene, and pyrene, caused no effect (E. Elovaara and L. Luukkanen, unpublished results).
Entacapone was glucuronidated in rat liver microsomes by an as yet uncharacterized rUGT. When we compare the apparent Km and Vmax values shown in Fig. 5 with the kinetic parameters reported in human liver microsomes (Km = 47 µM, Vmax = 6.8 nmol/min/mg) by Lautala et al. (2000), it appears that entacapone is conjugated by rUGTs less efficiently than by forms expressed in human liver (UGT1A9). Moreover, a manifold induction of entacapone glucuronidation was consistently detected in PCB-treated animals (Table 2; Fig. 3). Enzyme kinetic data (Fig. 5) showed that the apparent Km value for entacapone-metabolizing activity was lower in PCB rats (130 µM) than that in control rats (237 µM).
1-Naphthol was used as a phenol UGT marker substrate for rat UGT1A6. As shown by Jackson et al. (1988), 1-naphthol is one of the most rapidly glucuronidated substrates by this enzyme, which exhibits a restricted specificity toward planar phenols. Also, with this substrate, a significant UGT induction was detected in PCB-treated rats (Table 2). This finding is in agreement with that reported for Aroclor 1254 by Oesch et al. (1992). We have also found that 1-naphthol glucuronidation is enhanced in rat liver 3- to 4-fold upon exposure to PAH-type inducers such as ß-naphthoflavone, benzo(a)pyrene, or 3-methylcholanthrene, whereas exposure to PAHs like naphthalene, phenanthrene, or pyrene has no effect (E. Elovaara and J. Mikkola, unpublished results).
1-OHP is a sensitive probe for detecting UGT induction by chemical inducers of the PAH type. We have shown that 1-OHP (at a 3 µM concentration) is glucuronidated 5 and 8 times faster in liver microsomes of rats treated by 3-methylcholanthrene and PCB, respectively, than by control microsomes (Luukkanen et al., 1997). 1-OHP is also a UGT-selective substrate as inferred from enzyme kinetic data obtained in rats treated with different model inducers: control < pyrazole < acetone < phenobarbital < 4,4'-methylenebis-(2-chloroaniline) < 3-methylcholanthrene < creosote < PCB (Vmax/Km, 0.11, 0.19, 0.22, 0.43, 0.46, 2.05, 2.41, and 3.01 ml/min/mg, respectively). Application of 1-OHP as a selective marker of the PAH-type inducible phenol UGT forms (low Km, high Vmax/Km) requires testing at a low substrate concentration. Despite the high sensitivity of the 1-OHP UGT assay, only slight inductive effects could be observed in catechol-pretreated rats, whereas in the PCB-treated rats, the glucuronidation rate rose drastically (Table 2).
The glucuronidation kinetic constants estimated from the Michaelis-Menten and Eadie-Hofstee plots gave very similar values for Km and Vmax. An exception was the 1-OHP kinetic data analyzed in PCB-induced microsomes (Figs. 4 and 5). The enzyme kinetic data of both 1-OHP and entacapone glucuronidation agree with the above findings (Table 2; Fig. 3), which indicate that catechols are poor inducers of hepatic UGT activities in the rat. In the light of the kinetic data, we can conclude that PCB induced several UGT activities and, in particular, high-affinity enzyme forms, probably efficient catalysts of phenolic compounds, including catecholic compounds.
Entacapone (hUGT1A9), 1-naphthol (hUGT1A6), and 1-OHP (hUGT1A6/1A9) are preferentially conjugated by these two human isozymes, which are, in most cases, recognized as the major catalysts of catechol glucuronidation. Large differences are, however, observed in the glucuronidation rates among individual catechols as found here and in previous studies (Antonio et al., 2002; Taskinen et al., 2003). For this reason, it is important to study in each case, also, the glucuronidation by the catechol of interest. Although it is unclear by which UGTs entacapone, 1-naphthol, and 1-OHP are catalyzed in rat liver, they may be used as sensitive tools for screening rUGT activity that may also catalyze the glucuronidation of many catechols. Compared with the assay methods based on entacapone and 1-naphthol, the great advantage provided by the 1-OHP glucuronidation assay is its extraordinarily high sensitivity. By using 1-OHP as a surrogate substrate, it is possible to quantify UGT activity (that is not readily detected by catechol glucuronidation methods) in micro-size samples such as human liver microsomes (E. Elovaara, J. Mikkola, and O. Pelkonen, unpublished results) or very-low-activity samples such as human blood lymphocytes (E. Elovaara and J. Mikkola, unpublished results). Our findings suggest that induction of UGTs by catecholic compounds is very low in comparison with an effect displayed by potent inducers such as the Aroclor 1254-PCB mixture. Based on this and previous work, it appears that PAH-type enzyme inducers are the most powerful agents, which may greatly enhance the activity of catachol-metabolizing UGTs in the liver.
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ABBREVIATIONS: DHN, 2,3-dihydroxynaphthalene; COMT, catechol-O-methyltransferase; HPLC, high performance liquid chromatography; 1-OHP, 1-hydroxypyrene; PAH, polycyclic aromatic hydrocarbon; PCB, polychlorinated biphenyl; UDPGA, UDP-glucuronic acid; UGT, UDP-glucuronosyltransferase; h, human; r, rat; ALT, alanine aminotransferase; AST, aspartate aminotransferase; RT, retention time.
1 Present address: Division of Pharmaceutical Chemistry, Department of Pharmacy, FIN-00014 University of Helsinki, Finland. 
Address correspondence to: Dr. Eivor Elovaara, Department of Industrial Hygiene and Toxicology, Finnish Institute of Occupational Health, FIN-00250 Helsinki, Finland. E-mail: Eivor.Elovaara{at}occuphealth.fi
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, p < 0.05 (Student's t test), differs significantly from the corresponding control group.
