Introduction

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APAP¹ (Tylenol) is a widely used over-the-counter analgesic/antipyretic drug and model hepatotoxin. Although it is considered safe at normal doses, at higher doses it is associated with a predictable, dose-dependent centrilobular hepatotoxicity (Black, 1984) by a mechanism involving its metabolism to a toxic quinone imine (Cohen and Khairallah, 1997; Bessems and Vermeulen, 2001). APAP undergoes detoxification by competing phase 2 conjugation reactions, glucuronidation and sulfation, which convert APAP to nontoxic conjugates for elimination in bile or urine. The main type of APAP conjugation in most species, including rat and human, is glucuronidation. It has been proposed that individuals who metabolize APAP at slower rates are at higher risk of developing hepatotoxicity, based partly on the known greater susceptibility of animals that are deficient in the glucuronidation of phenols, such as cats (Feloidae) (Court and Greenblatt, 2000; Bessems and Vermeulen, 2001) and the Gunn rat (de Morais and Wells, 1988, 1989).

A common assumption has been that the increased susceptibility of cats and Gunn rats to APAP toxicity is due to inactive UGT1A6, a major liver-expressed phenol UGT. The wild-type 1A6² enzyme from rat and human is effective in APAP catalysis (Bock et al., 1993), and in rats, 1A6 is induced by 3-methylcholanthrene (Munzel et al., 1994; Bock et al., 1999), which results in increased APAP glucuronidation (Gregus et al., 1990; Bock et al., 1993). In cats, the 1A6 gene is not expressed and possesses characteristics of a pseudogene (Court and Greenblatt, 2000). In Gunn rats, the UGT1A6 gene is expressed but is nonfunctional due to a frame shift mutation that inactivates the entire set of 1A enzymes (Iyanagi et al., 1989). However, evidence supports the involvement of other UGT isozymes in APAP glucuronidation. A second human UGT1A family member, the 1A9 form, was reported to have high activity toward APAP, although it exhibited significantly lower affinity (K_m of ~50 versus 2 mM for 1A6) (Bock et al., 1993). In addition, the observation that APAP glucuronidating capacity is not totally absent in Gunn rats (de Morais and Wells, 1989) suggests that a UGT2 isoform also contributes to APAP glucuronidation.

Although the rat is the best characterized animal model for drug metabolism studies, the UGT system from this species is not well understood. Rat liver expresses an array of UGT1A forms similar to that of human with some notable differences. The rat, like human, expresses 1A1 and 1A6 at relatively high levels in liver. However, 1A9 is a pseudogene in rats (Emi et al., 1995), and in this species, its function may be replaced by 1A7, which exhibits a similar preference for "bulky" phenol-type substrates. In humans, 1A7 is not significantly expressed in liver and is considered an extrahepatic UGT (Strassburg et al., 1997). 1A6 and 1A7 are both markedly induced in rat liver by exposure to inducing agents in the polycyclic aromatic hydrocarbon and dithiole thione families (Grove et al., 1997). Based on the reported characteristics of 1A9, we considered the possibility that rat 1A7 is also significantly active toward APAP. The main goal of the current study was to directly assess the APAP UGT activities of 1A7 and the other major UGT1A isoforms expressed in rat liver.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

APAP hydrochloride and APAP glucuronide were obtained from Sigma Chemical (St. Louis, MO). BNF and phenobarbital were purchased from Aldrich Chemical (Milwaukee, WI) and Sigma Chemical, respectively. Oltipraz was from Aventis (Strasbourg, France). The lambda ZAP cDNA library synthesis kit and cloned Pfu DNA polymerase were from Stratagene (La Jolla, CA). [-³²P]dCTP (3000 Ci/mM) was from ICN Pharmaceuticals (Costa Mesa, CA). The human embryonic kidney-derived HEK 293 cell line and G418 sulfate were purchased from American Type Culture Collection (Reston, VA) and Mediatech (Herndon, VA), respectively. Human UGT1A common region anti-peptide antibody was purchased from GENTEST (Woburn, MA).

Animals and Drug Treatment. All animal experiments were conducted according to the National Institutes of Health guidelines for the care and use of animals using protocols approved by the Virginia Commonwealth University Institutional Animal Care and Use Committee. Male rats (6-8 weeks old, 160-200 g) were obtained from Harlan Sprague-Dawley (Frederick, MD). After arrival, the rats were housed in steel-wire cages and acclimated for at least 1 week before the experiment during which they were given free access to food and water. In the inducer study, Sprague-Dawley rats were exposed for 3 days to oltipraz, BNF, or phenobarbital. Oltipraz was administered by oral gavage as a micronized suspension in 30% polyethylene glycol-8000 (75 mg/kg/day for 3 days). BNF was administered by intraperitoneal injection in corn oil (12 mg/ml, 80 mg/kg/day for 3 days). Phenobarbital was given as a single intraperitoneal injection (80 mg/kg) in water, followed by exposure for the next 3 days via drinking water (0.1%). In an experiment with Gunn rats, BNF (80 mg/kg/day) was delivered as a suspension in 30% polyethylene glycol by gavage for 3 days. Twenty-four hours after the final dose, livers were removed for the preparation of liver microsomes as previously described (Kessler and Ritter, 1997). The final microsome pellets were resuspended in 100 mM K₂HPO₄, pH 7.4, 1 mM EDTA, and 20% glycerol by homogenization (three passes in a Potter Elvehjem tube) and sonication (five 1-s pulses delivered at medium power on ice). The protein concentration was determined using the BCA Protein Assay kit (Pierce Chemical, Rockford, IL). Microsomes were stored frozen at 80°C until analysis.

Preparation of Recombinant Rat UGT-Expressing HEK Cell Membranes. Complementary DNAs representing the major UGTs expressed in livers of oltipraz-treated rats were isolated by preparing and screening a lambda ZAP cDNA library with poly A+ RNA from an adult male Sprague-Dawley rat pretreated with oltipraz (300 mg/kg/day for 3 days). All probes used for screening corresponded to the 5'-end region of the respective cDNAs and were labeled by random priming in the presence of [-³²P]dCTP or dATP. Fragments representing UGT1A1 (bases 8-300 of GenBank accession number D38065) and UGT1A5 (bases 11-317 of GenBank accession number D38069) were obtained from rat liver cDNA by using polymerase chain reaction as described (Emi et al., 1995). Fragments representing UGT1A6 (bases 29-862) and UGT1A7 (bases 8-689) were derived from clones pR1A6 (Kessler and Ritter, 1997) and pLC14 (Grove et al., 1997). After plating and hybridization, filters were washed at low stringency (final wash in 2× standard saline citrate-0.05% SDS) and exposed to film. Under these conditions, it was established that the 1A5 and 1A7 probes would cross-react with other forms in the 1A2 to 1A5 and 1A7 to 1A9 subfamilies, respectively. For each probe, a total of 10 to 20 clones was plaque purified and analyzed by restriction endonuclease site mapping and partial nucleotide sequencing. This resulted in the successful isolation of full-coding clones for 1A1, 1A5, 1A6, and 1A7.³ The 1A8 cDNA was obtained by reverse transcriptase-polymerase chain reaction by using rat liver cDNA as template (D. Auyeung and J. Ritter, manuscript in preparation).

APAP UGT Activity Determination. APAP UGT activities were determined at 37°C in the presence of 50 mM Tris-Cl, pH 7.6, 10 mM MgCl₂, 2.5 mM APAP, and microsomal/crude membrane protein (0.5 mg/ml). After preincubation for 5 min at 37°C, UDP-glucuronic acid was added (3 mM final concentration) to start the reaction. After 1 h, reactions were stopped by the addition of perchloric acid (5.7% final concentration). Precipitated protein was removed by centrifuging at 14,000 rpm for 2 min, and the supernatants were transferred to a fresh tube and stored at 80°C until the analysis.

Kinetic Studies. In the experiments to determine the apparent Michaelis-Menten constants for APAP glucuronidation, the UDP-glucuronic acid concentration was 3 mM and the APAP concentration was varied between 0.33 and 40 mM. The apparent K_m and V_max were calculated using Tallarida Pharm-PCS software (version 4.0), which estimates K_m and V_max based on standard linear regression analysis.

Western Immunoblot Analysis. Relative levels of UGTs in rat liver microsomes and the UGT-expressing HEK cell membranes were determined using an enhanced chemiluminescence-based immunoblotting procedure essentially as described (Ritter et al., 1999; Guillemette et al., 2000). The phenol UGTs were analyzed using antisera raised in mice immunized with 6Xhis-tagged fusion proteins representing amino acids 14 to 132 of human 1A6 or 21 to 149 of rat 1A7. The human 1A6 antiserum displays moderate cross-reactivity toward rat 1A6 at a low dilution (1:500). Total UGT1A protein was analyzed using an anti-peptide antibody directed against the UGT1A common region (GENTEST). The absolute levels of expressed enzymes in crude cell membranes were established using a bacterially expressed 6Xhis-tagged fusion protein developed for this study. The fusion protein contains C-terminal residues 21 to 531 of r1A7 at its C terminus. The fusion protein was purified by standard Ni-NTA Sepharose affinity chromatography (QIAGEN, Valencia, CA). The concentration of the 58-kDa standard in the preparation was estimated by silver staining of SDS-polyacrylamide gel electrophoresis gels containing known amounts of human serum albumin standard (66 kDa).

Primary Rat Hepatocyte Cultures. Hepatocytes from male Sprague-Dawley or Gunn j/j rats were obtained from the Hepatocyte Isolation Core Facility of the Liver Center at Virginia Commonwealth University. Cells (800,000/35-mm gelatin-coated dish) were plated in Williams' E medium containing 10% fetal bovine serum, 100 U/ml penicillin, 100 µg of streptomycin, 0.125 U/ml insulin, 1 µM T4, and 0.1 µM dexamethasone. After the cells had attached (~4 h after plating), the medium was changed to serum-free medium. The following day, vehicle (0.1% v/v dimethyl sulfoxide) or vehicle containing oltipraz was added (50 µM final concentration) to induce 1A6 and 1A7 expression. Twenty-four hours later, medium with APAP (4 mM) was added, and aliquots were removed at the indicated times for analysis of APAP glucuronide.

	Results

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Effect of 1A6- and 1A7-Inducing Agents on Rat Liver Microsomal APAP UGT Activity. An initial study was conducted to determine the effects of prototypical microsomal enzyme-inducing agents on relative liver microsomal 1A6 and 1A7 levels and APAP UGT activities. Antibodies raised against the N-terminal sequences of human 1A6 and rat 1A7 were tested for reactivity toward various UGT1A family isoforms expressed in HEK cells (to be described further below). The human 1A6 antisera was strongly reactive with r1A6 and had no apparent reactivity toward either r1A1, r1A5, r1A7, or r1A8 (Fig. 1A). In contrast, the rat 1A7 antisera also exhibited high selectivity, reacting preferentially with r1A7 and, to a lesser extent, r1A8.

View larger version (34K): [in this window] [in a new window]   Fig. 1.   Effect of UGT1A6/1A7 and UGT2B1-inducing agents on liver microsomal APAP UGT activities and phenol UGT expression in normal rats. A, selectivity of the human 1A6 and rat 1A7 antisera toward different rat UGT1A isoforms. Membranes from different clonal UGT-expressing cell lines were prepared and analyzed by Western immunoblotting, as described under Materials and Methods. An immunoblot probed with human UGT1A common antibody is included for comparison. B, effects of inducers on UGT1A6, UGT1A7, or total UGT1A protein determined by immunoblotting with the indicated antibodies. Sprague-Dawley rats were treated subacutely with vehicle (VH), oltipraz (OTP), BNF, or phenobarbital (PB). Liver microsomes from two control or inducer-treated animals were prepared and analyzed as described under Materials and Methods. The average magnitude of induction determined by densitometric analysis (n = 2) is indicated. C, APAP UGT activities determined by HPLC assay. The data represent the mean ± standard error of the mean (n = 3 rats/group).

View larger version (52K): [in this window] [in a new window]   Fig. 2.   APAP glucuronidation activities of liver microsomes from UGT1A1-normal and UGT1A1-defective rats. APAP UGT activities of liver microsomes from homozygous UGT1A1-normal (+/+), homozygous UGT1A-defective (j/j), or heterozygous UGT1A-defective (j/+) rats were treated with BNF and analyzed as described under Materials and Methods. Columns represent the activities determined for two different rats.

Cloning, Sequence Characterization, and Expression of Rat UGT1A Family cDNAs in HEK Cells. To characterize the activities of the major UGT1A isoforms in rat liver, cDNA clones representing each of these forms were isolated from a lambda ZAP Sprague-Dawley rat liver cDNA library. Screening of the library with 5'-end directed probes indicated high abundance of 1A6 and 1A7-like positive clones followed in relative order by 1A1- and 1A5-like clones. Restriction analysis of purified clones suggested that 1A1, 1A5, 1A6, and 1A7 were the only UGT1A forms significantly expressed in adult male Sprague-Dawley rat liver.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Quantitative immunoblotting approach to determine the content of expressed 1A7 in clonal line r1A7-3. Known amounts of a purified 6Xhis-tagged rat UGT1A7 fusion protein (r1A7FL) were electrophoresed in parallel with serially diluted samples of a clonal HEK cell line expressing r1A7 (r1A7-3) and analyzed by Western blotting with the r1A721-149 antisera. Densitometric analyses enabled estimation of the absolute level of expression of r1A7 protein in the r1A7-3 membranes.

Kinetic Analysis of APAP UGT Activities in Expressing HEK Cell Protein and Liver Microsomes. To compare the apparent K_m and V_max of the two most active forms, the effect of varying the substrate concentration on initial reaction velocities was assessed. In these assays, UDP-glucuronic acid concentration was held constant at 3 0.0 mM. Recombinant 1A6 and 1A7 exhibited similar apparent affinities of 3.47 ± 0.4 mM versus 4.56 ± 1.5 mM, respectively (Fig. 4A). The V_max values were also comparable (0.60 ± 0.04 and 1.41± 0.10 nmol/min/mg protein, respectively). Expressed in units of specific activity, 1A7 had a 2.9-fold higher specific V_max (7.83 versus 2.69 nmol/min/nmol).

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Kinetic analysis of APAP glucuronidation catalyzed by rat 1A6 and 1A7 and comparison with rat liver microsomes with normal or defective UGT1A isozymes. A, membranes from r1A6-1 and r1A7-3 were assayed for APAP UGT activity by using different concentrations of APAP and 3 mM UDP-glucuronic acid. The reciprocals of substrate concentration and reaction velocity were graphed to yield the Lineweaver-Burk plots shown. B, corresponding analyses were performed using liver microsomes from either a homozygous normal (+/+) or homozygous defective (j/j) Gunn rats.

Induction of APAP Glucuronidation in Cultured Primary Rat Hepatocytes by Oltipraz. An experiment was performed to study the glucuronidation of APAP by cultured primary hepatocytes from normal and UGT1A-deficient rats and the influence of pre-exposing the hepatocytes to oltipraz. Western analysis of lysate from control cells and cells exposed to oltipraz for 24 h before addition of APAP to the cell culture medium suggested that 1A6 and 1A7 UGT levels were elevated by oltipraz at the time of APAP addition (Fig. 5A). The glucuronidation of APAP was stimulated >2-fold in the oltipraz-exposed cells, whereas the sulfation pathway was not visibly altered (Fig. 5B). In contrast, hepatocytes from UGT1A defective Gunn rats (j/j) formed APAP-glucuronide at reduced rates compared with Sprague-Dawley hepatocytes, and there was no apparent stimulation of glucuronidation or sulfation by oltipraz (Fig. 5C).

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Influence of oltipraz pretreatment on the formation of APAP glucuronide in cultured primary hepatocytes from normal and UGT1A-defective rats. Freshly isolated rat hepatocytes (800,000 cells/35-mm well) were exposed to either dimethyl sulfoxide (0.1%) (open symbols) or 50 µM oltipraz added to the culture medium. After 24 h, the medium was changed, and APAP was added (4 mM final). At the times indicated, the culture medium was sampled for analysis of APAP glucuronide (squares) and sulfate (circles). A, immunoblot analysis for 1A6, 1A7, and UGT1A total protein in Sprague-Dawley hepatocytes. B, oltipraz stimulates APAP glucuronidation in Sprague-Dawley rat hepatocytes. C, oltipraz has no effect on APAP glucuronidation in homozygous defective (j/j) Gunn rat hepatocytes.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

APAP is commonly viewed as a specific "probe" substrate for the 1A6 isoform (Esteban and Perez-Mateo, 1999; Fisher et al., 2000). However, results in this study show that glucuronidation of APAP is catalyzed by multiple rat UGT isoforms, and that, at least in rats, APAP is a somewhat nonspecific substrate. Our data confirm a previous report suggesting high APAP activity of rat 1A6. Using rat 1A6 expressed in V79 Chinese hamster lung fibroblasts, Bock et al. (1993) reported an apparent K_m of 2.7 mM, in good agreement with the value determined for HEK cell-expressed enzyme in the current study (3.9 mM). The activity of 1A7 toward APAP has not been previously described. We found that 1A7 has comparable APAP binding affinity and superior catalytic efficiency. These data suggest considerable potential for the 1A7 isoform to compete with 1A6 and contribute significantly to the capacity of rat liver to glucuronidate APAP. The percentage contributions of 1A6 and 1A7 to APAP glucuronidation will depend on their relative levels in the liver endoplasmic reticulum. Although these are not known precisely at present, both appear to be expressed at significant levels in rat liver, especially after treatment with inducing agents (Grove et al., 1997).

Our study also confirms earlier work indicating that 1A1, the major bilirubin-glucuronidating UGT, is not involved in APAP catalysis. Although it was observed that 1A1 did not significantly metabolize APAP, we found that the preparation was highly active in bilirubin glucuronidation (data not shown). This is of interest in view of reports linking Gilbert's syndrome with reduced APAP glucuronidation and a possible increase in susceptibility to APAP hepatotoxicity (Ullrich et al., 1987; de Morais et al., 1992; Esteban and Perez-Mateo, 1999). The primary cause of Gilbert's in the Caucasian population is reduced UGT1A1 expression related to the UGT1A1*28 allele (TA repeat) in the 1A1 gene promoter. The suggestion by Esteban and Perez-Mateo (1999) that a significant percentage of Gilbert's patients (with the TA genetic change) may also possess a linked mutation in the phenol transferase gene appears to be possible based on a report by Lampe et al. (1999) that 8% of individuals homozygous for the *28 allele were also homozygous for the 1A6 variant allele (UGT1A6*2) associated with reduced enzymatic activity (Ciotti et al., 1997).

The model for rat APAP glucuronidation catalyzed by UGT1A isoforms described in this study resembles that of humans with some apparent differences. A major similarity is that 1A6 in both species is expressed at high hepatic levels and exhibit similar apparent APAP binding affinity (2.0 mM for human versus 2.7-3.9 mM for rat) (Bock et al., 1993). Both species also express a member of the "bulky phenol" subgroup of UGT1A forms (UGT1A7-UGT1A10 subfamily) that is significantly active in APAP glucuronidation. In the case of human, this form is 1A9 rather than 1A7. There are apparent differences, however, in the kinetic properties of rat 1A7 and human 1A9, especially compared with the respective 1A6 forms from these two species. In contrast to rat, where 1A7 has similar apparent affinity and marginally higher catalytic activity than 1A6, human 1A9 was reported to possess much lower affinity (K_m of ~50 mM) than its 1A6 counterpart (2 mM). In the human study, comparisons of intrinsic clearance (V_max/K_m) for 1A6 and 1A9 were not possible, because the levels of expressed enzyme protein in transfected cells could not be determined. Thus, the relative V_max of human 1A6 compared with 1A9 remains unclear at present.

Animal studies support the idea that the rate of APAP glucuronidation impacts the hepatotoxic potency of APAP (de Morais and Wells, 1988, 1989, 1992; Court and Greenblatt, 2000). APAP is an intermediate clearance drug whose rate of elimination depends in part on the APAP conjugate-forming capacity of the liver. Factors that modulate the levels of APAP UGTs, for example, exposure to oltipraz, are predicted to affect APAP clearance and organ toxicity. In this report, we were able to show using the cultured hepatocyte model that induction of UGTs 1A6 and 1A7 by oltipraz was associated with a stimulation in the rate of APAP glucuronidation. In vehicle-exposed hepatocytes, we found that ~26% of APAP was conjugated after 26 h, with the sulfoconjugate slightly predominating over the glucuronide (13.5 versus 12.3%, respectively). In hepatocytes exposed for 24 h to oltipraz, the total percentage conjugated increased to 37%, due almost entirely to an increase in the fraction glucuronidated. Oltipraz treatment shifted the ratio in favor of the glucuronide over the sulfate conjugate (22.8 to 14.0%, respectively). Despite our efforts, we were not able to demonstrate the association between UGT induction and protection against APAP toxicity in the cultured hepatocyte model. Others have reported that treatment of primary rat hepatocytes with APAP at concentrations in the range used in our study (4 mM) results in cytotoxic effects (Milam and Byard, 1985; Thibault et al., 1991). However, we were unable to observe any significant changes in cell morphology or biochemical indices (lactate dehydrogenase and aspartate transaminase) after a 24-h exposure to 4 mM APAP (data not shown). The lack of effect is likely related to the general insensitivity of rat as a model for APAP toxicity (Bessems and Vermeulen, 2001), due possibly to high glucuronidation or low bioactivation rates. It is noteworthy, however, that in hamsters, which are known to be highly sensitive to APAP, treatment with oltipraz has been shown to be protective against APAP-induced hepatotoxicity (Davies et al., 1991) by a mechanism involving induction of APAP glucuronidation (Davies and Schnell, 1991). These observations suggest that hamsters exhibit lower basal rates of APAP glucuronidation and that hamsters have corresponding oltipraz-inducible phenol UGTs.

Even under conditions when UGT1A isoforms are absent, it is clear that rat hepatocytes still possesses significant APAP glucuronide-forming activity. These data indicate a role for other UGTs in this process, presumably a UGT2B subfamily member. Although 2B1 and 2B12 are considered the major "xenobiotic-metabolizing" UGT2B family members, neither is active toward APAP (Pritchard et al., 1994; Green et al., 1995). Several UGTs in the rat 2B family, including 2B2, 2B3, and 2B6, each have been reported inactive toward simple phenolic substrates (Mackenzie, 1987). At least one remaining candidate to be investigated is the 2B5 form (GenBank accession number U27518). Data from the kinetic analysis of homozygous Gunn rat microsomes (Fig. 5) predict that this UGT has lower affinity for APAP (K_m of ~26.0) than either 1A6 or 1A7. Evidence has also been reported for a corresponding form in humans. Axelrod et al. (1957) described deficient, albeit not totally absent, APAP glucuronidation in a patient with severe hereditary unconjugated hyperbilirubinemia, who presumably had a UGT1A-inactivating mutation analogous to Gunn rats.

In summary, this study reveals evidence for the involvement of multiple UGT isoforms in the glucuronidation of APAP by rat liver. Two of these, 1A6 and 1A7, are members of the UGT1A family and are implicated in the increased sensitivity of homozygous Gunn j/j rats to APAP-induced hepatotoxicity. Both isozymes were found to possess similar kinetic properties in APAP catalysis. Their relative contributions to the total activity in vivo are predicted to depend on their levels of expression determined by dietary and/or environmental exposures. A third form that is active toward APAP, most likely from the UGT2 subfamily, remains to be identified.