Kinetics of Acetaminophen Glucuronidation by UDP

Chem. Res. Toxicol. 2006, 19, 701-709

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Kinetics of Acetaminophen Glucuronidation by UDP-Glucuronosyltransferases 1A1, 1A6, 1A9 and 2B15. Potential Implications in Acetaminophen-Induced Hepatotoxicity Abdul E. Mutlib,*,† Theunis C. Goosen,† Jonathan N. Bauman,† J. Andrew Williams,† Shaila Kulkarni,‡ and Seva Kostrubsky‡ Departments of Pharmacokinetics, Dynamics and Metabolism, and Safety Sciences, Pfizer Inc., 2800 Plymouth Road, Ann Arbor, Michigan 48105 ReceiVed NoVember 14, 2005

The importance of uridine 5′-diphosphate-glucuronosyltranferases (UGT) 2B15 and other UGT enzymes (1A1, 1A6, and 1A9) in glucuronidating acetaminophen (APAP) is demonstrated. The kinetics and contributions of various UGTs in glucuronidating APAP are presented using clinically and toxicologically relevant concentrations of the substrate. UGT 1A9 and UGT 2B15 contribute significantly toward glucuronidating APAP when incubations were conducted in either phosphate or Tris-HCl buffers at 0.1 and 1.0 mM substrate concentrations. At 10 mM APAP, UGT 1A9 is a significant enzyme responsible for metabolizing APAP in either one of the buffers. UGT 1A1 is the next most important enzyme in glucuronidating APAP at this high substrate concentration. The contribution of UGT 1A6 at 10 mM APAP concentration became obscured by similar relative activities exhibited by UGTs 1A7, 1A8, and 2B7. These observations may reflect the differences in kinetic parameters for APAP glucuronidation by the individual UGTs. UGT 1A1 demonstrated Hill kinetics while UGT 1A9 displayed Michaelis-Menten kinetics. Substrate inhibition kinetics is observed with UGT 1A6, UGT 2B15, and human liver microsomes. The substrate inhibition is confirmed by employing stable isotope-labeled APAP as the substrate, while APAP glucuronide is used to test for inhibition of d4-APAP glucuronide. The in vitro hepatotoxicity caused by APAP in combination with phenobarbital or phenytoin is demonstrated in this study. The inhibition of APAP glucuronidation by phenobarbital leads to an increase in APAP-mediated toxicity in human hepatocytes. The toxicity to hepatocytes was further increased by coadministering APAP with phenytoin and phenobarbital. This synergistic increase in toxicity is postulated to be due to inhibition of UGTs (1A6, 1A9, and 2B15) responsible for detoxifying APAP through the glucuronidation pathway. Introduction It was recently shown that phenobarbital (PB) and phenytoin (PH) increased acetaminophen (APAP)-mediated hepatotoxicity in cultured human hepatocytes due to inhibition of uridine 5′diphosphate-glucuronosyltranferases (UGTs) (1). This was considered as a predictive example of hepatotoxicity resulting from an inhibitory effect on phase II metabolism (glucuronidation) of APAP. PB and PH were not glucuronidated during the inhibition process; hence, it was postulated that these inhibitors interacted directly with the UGTs responsible for APAP glucuronidation. During the course of that study, it was found that UGT 2B15 played a role in APAP glucuronidation. The results from recently conducted experiments in our laboratory described here suggest that UGT 2B15 is a significant enzyme in metabolizing APAP. Previously, the glucuronidation of APAP was attributed mainly to the UGT1A family (UGT 1A1, 1A6, and 1A9) (2-6). Apparently, the significance of UGT 2B15 in metabolizing APAP was overlooked or was not observed perhaps due to the limited availability of UGT 2B15 in earlier investigative studies. In this paper, we would like to provide additional data on the kinetic characterization and the importance of UGT 2B15 as well as other UGT 1A enzymes (1A1, 1A6, and 1A9) in metabolizing APAP. * To whom correspondence should be addressed. Tel: 734-622-2198. Fax: 734-622-1459. E-mail: [email protected]. † Departments of Pharmacokinetics, Dynamics and Metabolism. ‡ Safety Sciences.

Studies conducted in this laboratory showed that PB was able to inhibit recombinant UGT 2B15 (IC50 of 300 µM), which catalyzes the formation of APAP glucuronide (1). In comparison, it was found to be a less potent inhibitor of UGTs 1A6 and 1A9 with IC50 values of 1700 and 3700 µM, respectively (1). These differences in the IC50 values were considered sufficient enough to use PB as an inhibitor of UGT 2B15 in the studies conducted with human hepatocytes. It was postulated that at 1 mM concentration, PB would inhibit most of the UGT 2B15 activity while leaving the UGT 1A9 enzyme mostly unaffected. UGT 1A9 has been previously considered as one of the major enzymes responsible for glucuronidating APAP (6). Further studies conducted in this laboratory suggested another antiepileptic; PH was also able to inhibit UGTs 1A6 (IC50 ) 280 µM), 1A9 (IC50 ) 145 µM), and 2B15 (IC50 ) 600 µM) in forming APAP glucuronide (1). This greater potency in inhibiting the UGTs by PH was considered as one possible explanation for the greater degree of hepatotoxicity exhibited by APAP + PH than shown by APAP + PB in cultured human hepatocytes. Maximal toxicity, monitored by a decrease in protein synthesis (as a biomarker for hepatotoxicity), was caused by PH at 0.2 mM and PB at 2 mM when added with APAP (5 mM) to cultured human hepatocytes (1). At these concentrations of PB (2 mM) and PH (0.2 mM), the glucuronidation of APAP was reduced to less than 50% of the control values (1). The potent inhibition by PH in APAP glucuronide formation (and significant increase in cellular toxicity) implies a contribution of UGTs

10.1021/tx050317i CCC: $33.50 © 2006 American Chemical Society Published on Web 03/25/2006

702 Chem. Res. Toxicol., Vol. 19, No. 5, 2006

1A6, 1A9, and 2B15. The studies conducted with APAP coadministered with PB, which show greater selectivity toward inhibiting UGT 2B15, indicate that this enzyme also plays a significant role in glucuronidating APAP in cultured human hepatocytes. However, the combined inhibitory potential of PH and PB on APAP glucuronidation was not investigated. Neither was the toxicological outcome of using such a combination with APAP evaluated. The hypothesis was that by inhibiting UGTs 1A6, 1A9, and 2B15 activities by adding PH (at 0.1 mM, mostly inhibiting UGT 1A9) and PB (at 1 mM, mostly inhibiting UGT 2B15) together to human hepatocytes in the presence of 5 mM APAP, we should see greater inhibition of glucuronidation and subsequently a more pronounced extent of toxicity than observed with individual inhibitors. In this paper, in addition to describing the kinetics of APAP glucuronidation, we describe the attainment of this predicted synergistic toxicological effect (as measured by decrease in protein synthesis) mediated by simultaneous addition of PH and PB to human hepatocytes, confirming our hypothesis. It was postulated that a significant reduction in glucuronidation of APAP in the presence of these two UGT inhibitors would lead to a greater degree of bioactivation of APAP to the reactive intermediate, N-acetyl para-benzoquinoneimine (NAPQI). The higher levels of NAPQI are believed to be responsible for the increased toxicity to the hepatocytes. Furthermore, the significance of UGT 2B15 in glucuronidating APAP is demonstrated. It is postulated that UGT 2B15, present as a polymorphic enzyme in humans, plays a much more significant role in the metabolic disposition of APAP than has been previously realized.

Materials and Methods Chemicals and Supplies. HMM (modified Williams E) culture medium and supplements were from BioWhittaker (Walkersville, MD). Pooled human liver microsomes (HLMs) (lot HL-Mix-101, 20.4 mg/mL) and recombinant UGTs 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15, and 2B17 and insect control were purchased from BD Gentest (Woburn, MA). Two lots of UGT 2B15 were studied for the formation of APAP glucuronide, lots 7 (November 2003) and 23953 (June 2005). All recombinant UGTs were expressed using baculovirus-infected Sf-9 insect cells with a final protein concentration of 5 mg/mL. The levels of expressions were demonstrated to be comparable between the various UGT enzymes by the commercial supplier (BD Gentest) (data not shown). Control human microsomes were from Xenotech (Kansas City, KS). PH, PB, 4-acetamidophenol, acetaminophen glucuronide, uridine 5′-diphosphoglucuronic acid (UDPGA), NADPH, MgCl2, phosphate buffer (pH 7.4), and alamethicin were purchased from SigmaAldrich (St. Louis, MO). Ultrapure Tris-HCl buffer was purchased from In Vitrogen (Carlsbad, Ca). The isotopically labeled d4-APAP was obtained from CDN Isotopes (Pointe-Claire, Quebec, Canada). HPLC-grade water, methanol, and acetonitrile were purchased from Mallinckrodt Chemicals (Phillipsburg, NJ). All general solvents and reagents were of the highest grade commercially available. Formation of APAP Glucuronide by Various rUGTs in TrisHCl or Phosphate Buffers. The rUGTs included in this study consisted of 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15, and 2B17. Recombinant UGTs (0.25 mg/mL) were preincubated with assay buffer containing Tris-HCl or phosphate (50 mM, pH 7.5, at 37 °C), MgCl2 (5 mM), and alamethicin (80 µg/mg protein) on ice for 15 min. APAP (0.1, 1.0, or 10 mM) in methanol was added to a final incubation volume of 250 µL and preincubated at 37 °C for 3 min. Reactions were initiated by the addition of UDPGA (5 mM). Blank incubations were also performed without UDPGA. After 30 min, the reactions were stopped by transferring 100 µL of incubation mixture to 200 µL of acetonitrile containing 50 µg/L acetanilide as the internal standard.

Mutlib et al. In some cases, smaller aliquots of the incubation mixture were extracted, depending on the expected levels of the APAP glucuronide produced especially by rUGTs 1A1, 1A9, and 2B15. Quenched incubates were centrifuged at 12000 rpm for 10 min. Supernatants were transferred to another set of clean polypropylene tubes and dried under a stream of nitrogen at 30 °C. The dried extracts were reconstituted in the HPLC mobile phase (200 µL), and aliquots of 50 µL were analyzed by LC/MS (see below). To quantitate APAP glucuronide formed in the presence of various UGTs, a standard curve was constructed in the concentration range of 0.25-500 ng/mL. APAP glucuronide was added at various concentrations to an incubation mixture containing alamethicin (80 µg/mg protein), APAP (10 mM), MgCl2 (5 mM), UDPGA (5 mM), and insect control protein (0.25 mg/mL) with a final volume made up to 250 µL with either phosphate or Tris-HCl buffer on ice. The samples were precipitated with acetonitrile containing the internal standard and processed in the same manner as described above. Enzyme Kinetics for APAP Glucuronidation by rUGTs 1A1, 1A6, 1A9, and 2B15 and HLMs in Tris-HCl Buffer. To determine the enzyme kinetic parameters of various rUGTs (1A1, 1A6, 1A9, and 2B15) at 0.25 mg/mL, samples were preincubated with buffer containing 50 mM Tris-HCl (pH 7.5), MgCl2 (5 mM), alamethicin (80 µg/mg protein), and increasing concentrations of APAP (0.160 mM) on ice for 15 min. The incubation mixtures were subsequently transferred to a water bath maintained at 37 °C and equilibrated for 3 min prior to initiating the reaction with UDPGA (5 mM). Incubations were conducted in triplicate for each concentration of APAP. Aliquots of incubation mixture (10-200 µL) were removed after 30 min of incubation and quenched with 200 µL of acetonitrile containing the internal standard. Generally, smaller aliquots (10-20 µL) were removed from incubations containing high concentrations of APAP (>3 mM). A standard curve to quantitate APAP glucuronide was constructed in the range of 0.25500 and 25-10000 ng/mL for samples from rUGTs and HLMs, respectively. Quenched incubates were centrifuged at 12000 rpm for 10 min. Supernatants were transferred to another set of clean polypropylene tubes and dried under a stream of nitrogen at 30 °C. The dried extracts were reconstituted in the HPLC mobile phase (200-400 µL), and aliquots of 10-40 µL were analyzed by LC/ MS (see below). For enzyme kinetic data, substrate concentration [S] and velocity (V) data were fitted to the appropriate enzyme kinetic model by nonlinear least-squares regression analysis (Sigmaplot; SPSS, Chicago, IL) in order to derive the enzyme kinetic parameters Vmax (maximal velocity) and Km (substrate concentration at half-maximal velocity). The Michaelis-Menten model (eq 1), the uncompetitive substrate inhibition model (eq 2), the two-enzyme model (eq 3), and the substrate activation model (eq 4), which incorporates the Hill coefficient (n), were used as follows: V ) Vmax × S/(Km + S)

(1)

V ) Vmax × S/[Km + S × (1 + S/Ki)]

(2)

V ) Vmax1 × S/(Km1 + S) + Vmax2 × S/(Km2 + S)

(3)

V ) Vmax × Sn/(S50n + Sn)

(4)

where Vmax is the maximal velocity, Km or S50 is the substrate concentration at half-maximal velocity, n is an exponent indicative of the degree of curve sigmoidicity, and Ki is an inhibition constant (7). The best fit was based on a number of criteria, including visual inspection of the data plots (Michaelis-Menten and EadieHofstee), distribution of the residuals, size of the sum of the squared residuals, and the standard error of the estimates. Selection of models other than Michaelis-Menten was based on the F-test (P < 0.05) and the Akaike information criterion. Data are presented as the means ( standard errors of the mean (SEM). Studies with d4-APAP to Understand Substrate or Product Inhibition Kinetics. On the basis of enzyme kinetics exhibited by various rUGTs, a study was conducted to determine if the decrease

UGT2B15-Mediated Glucuronidation of APAP

Figure 1. Application of deuterated (d4)-APAP to study substrate inhibition kinetics exhibited by APAP in the presence of UGT 2B15.

in rate of APAP glucuronidation at higher substrate concentration was due to either substrate (APAP) or product (APAP glucuronide). To distinguish between the two possibilities, a deuterated analogue, whereby four deuteriums were incorporated on the aromatic ring of APAP (d4-APAP), was used as the substrate (see Figure 1). This permitted us to monitor the formation of the d4-APAP glucuronide as a product, while the commercially available APAP glucuronide was used a potential inhibitor. The phenolic hydroxyl group was unaltered, hence allowing rUGT-mediated glucuronidation to occur. Recombinant UGT 2B15 (0.25 mg/mL) was preincubated with assay buffer containing Tris-HCl (50 mM, pH 7.5 at 37 °C), MgCl2 (5 mM), and alamethicin (80 µg/mg protein) on ice for 15 min. d4-APAP (1.0 or 60 mM) was added to a final incubation volume of 250 µL and preincubated at 37 °C for 3 min. APAP glucuronide was added at various concentrations (0-300 µM) as a potential inhibitor to incubation mixtures. Reactions were initiated by the addition of UDPGA (5 mM). After 30 min, the reactions were stopped by transferring 100 µL of incubation mixture to 200 µL of acetonitrile containing 50 µg/L acetanilide as the internal standard. The peak area ratios of d4-APAP glucuronide to the internal standard were obtained. Unchanged peak area ratios in the presence of various concentrations of APAP glucuronide would suggest that the product was not inhibiting the formation of glucuronide. The samples were reanalyzed after adding a sufficient amount of APAP glucuronide to each reconstituted sample extract to give a final concentration equivalent to the 300 µM extract. Results before and after the addition of APAP glucuronide were compared. LC/MS Analysis of APAP Metabolites. Injections of reconstituted incubation extracts were made directly onto an HPLC column (Aqua C18, 150 mm × 2.0 mm, Phenomenex) coupled to an API 4000 mass spectrometer (Applied Biosystems/MDS SCIEX, Ontario, Canada), equipped with a TurboIonSpray source held at 275 °C. The electrospray needle was maintained at 4600 V with the declustering and exit potentials set at 46 and 10 V, respectively. Ultrapure nitrogen was used as the nebulizer and curtain gas. The mass spectrometer was operated in the multiple reaction monitoring (MRM) mode for quantitative analyses. MRM analysis was carried out using nitrogen as the collision gas. The collision energy was kept at 25 eV. Other parameter settings for the MRM analyses included arbitrary values of 6, 25, 40, and 40 for collision (CAD), curtain, nebulizer, and turbo gases, respectively. The mass transitions for the metabolites and the internal standard were as follows: 136 f 94 (acetanilide, internal standard), 232 f 152 (APAP sulfate), 271 f 140 (cysteine conjugate), 328 f 152 (APAP glucuronide), 328 f 182 (cysteinylglycine), 332 f 156 (d4-APAP glucuronide), and 457 f 328 (APAP glutathione). The peak areas from each of these transitions were obtained, and ratios of the analyte to the internal standard were obtained for each sample. The identities of APAP metabolites were based on previously reported data (8). The metabolites were separated on the HPLC column using a gradient solvent system consisting of acetonitrile and 0.1% formic acid with the flow rate set at 0.3 mL/min. The initial conditions

Chem. Res. Toxicol., Vol. 19, No. 5, 2006 703 consisted of a mixture of acetonitrile and 0.1% formic acid (4:96 v/v). For quantitation of the APAP glucuronide from the kinetic studies, the percentage of acetonitrile was increased linearly from 4 to 80% in 2 min. After an additional 1 min at 80% acetonitrile, the column was reequilibrated with the initial mobile phase for 5 min before the next injection. For studies requiring quantitation of all metabolites, a longer analysis was conducted for each sample. In such cases, the initial mobile phase conditions were maintained for 3 min after the sample was injected. The percentage of acetonitrile was increased linearly from 3 to 90% in the next 17 min. After an additional 5 min at 90% acetonitrile, the column was re-equilibrated with the initial mobile phase for 10 min before the next injection. The possibility of ion suppression, especially at high concentrations of substrate or in the presence of an inhibitor, was also assessed in cases of coeluting analytes. The chromatography ensured that the APAP metabolites were separated from the substrate. However, APAP glucuronide and d4-APAP glucuronide coeluted. Experiments were conducted to demonstrate the absence or presence of ion suppressions especially at higher concentrations in these studies. In Vitro Phase I Metabolism of APAP to NAPQI by HLMs in the Presence and Absence of UGT Inhibitors. APAP (0.1 and 1.0 mM) was incubated in the presence of HLMs (2 mg/mL), glutathione (GSH) (2 mM), and MgCl2 (3 mM) in 0.1 M phosphate buffer (pH 7.4) at 37 °C for 60 min. The effect of PB (0.1 and 1.0 mM) and PH (0.1 mM) on the P450 enzymes responsible for the formation of NAPQI was evaluated by adding these inhibitors prior to initiating the reactions with NADPH (2 mM final concentration). The relative levels of NAPQI formed were determined by monitoring the presence of trapped glutathione adducts by LC/MS. At the end of the incubation, the microsomal proteins were precipitated by adding 2 mL of ice-cold acetonitrile containing acetanilide as the internal standard, followed by centrifugation at 3000 rpm using an Allegra X-22R centrifuge equipped with a SX4250 rotor. The supernatant was removed, dried under a stream of nitrogen, and reconstituted in 400 µL of ACN:0.1% formic acid (4:96 v/v). An aliquot (20 µL) was injected onto LC/MS using the conditions described earlier. Mass spectral analyses (MRM mode) were conducted to monitor the GSH adduct formed in each sample. The peak area ratios of the GSH adduct to the internal standard (acetanilide) were obtained as described above. Another study was conducted to evaluate the effect of substrate concentrations on bioactivation of APAP to NAPQI. Incubations were conducted in the same manner as described above, except various concentrations of APAP (range 0.1-10 mM) were used. In addition to GSH adduct formation, the levels of cys-gly were also monitored by mass spectrometry. Hepatocyte Cultures and Treatment Protocol. Human hepatocytes were isolated from livers (n ) 2) not used for whole organ transplant. Hepatocytes were isolated by three-step collagenase perfusion as described previously (9). The viability of cells obtained, as measured by trypan blue exclusion test, ranged from 74 to 90%. Hepatocytes were plated in Williams E medium supplemented with 10-7 M dexamethasone, 10-7 M insulin, 100 units/mL penicillin G, 100 µg/mL streptomycin, and 10% bovine calf serum. Hepatocytes (2 × 106/well) were plated on six-well culture plates previously coated with type I (rat tail) collagen. Cells were allowed to attach for 4-6 h in 37 °C, at which time the medium was replaced with serum-free medium with the supplements listed above, and changed every 24 h thereafter. After 72 h in culture, cells were treated with either APAP (5 mM) alone or in combination with PB (1 mM) or PH (0.1 mM) for 24 h. A treatment consisting of APAP (5 mM), PB (1 mM), and PH (0.1 mM) was also conducted to study the net effect of this combination on glucuronidation and toxicity to cells. These culture conditions maintained sufficient levels of phase I and phase II drug metabolizing activities in hepatocytes after 96 h in culture as was demonstrated for several substrates (10, 11) and, at the same time, minimized the difference between activities in control cells prepared from different donors (12). Toxicity was determined by the measurement of total protein synthesis by pulse-labeling hepatocytes for 1 h with [14C]leucine,


Mutlib et al.

Figure 2. Formation of APAP glucuronide from APAP (0.1, 1.0, and 10 mM) in Tris-HCl or phosphate buffers in the presence of various rUGTs.

as described previously (13). All values were normalized per amount of cellular protein. Cells and culture media were also collected for determination of APAP metabolites. Analysis of Culture Media and Cell Lysates. The 24 h culture media and cell lysates were assayed for the presence of cysteine, cysteinylglycine, glutathione, sulfate, and glucuronide conjugates of APAP. Aliquots of media (100 µL) were diluted with water to a final volume of 2 mL, and internal standard (acetanilide) was added. An aliquot (10 µL) of each sample was injected onto LC/ MS without any further sample preparation. Quantitation of sulfate and glucuronide conjugates was conducted using the mass spectrometer operated in the MRM mode (see above). Standard curves for both metabolites were constructed in the range 100 ng/mL to 20 µg/mL. Peak area ratios for the GSH-related metabolites to the internal standard were obtained via MRM, and comparisons were made between various treatment groups. The cell pellets from the hepatocyte incubations were reconstituted in 0.1 M phosphate buffer (100 µL) and precipitated with 300 µL of acetonitrile containing the internal standard. After vortexing, aliquots of 200 µL were transferred to clean glass tubes and the solvent was removed under a stream of nitrogen. The dried extract was reconstituted with 2 mL of water and analyzed as described above for the culture media.

Results Formation of APAP Glucuronide by rUGTs in the Presence of Tris-HCl and Phosphate Buffers. APAP glucuronidation activities were measured at a clinically relevant (0.1 mM), sub-toxic (1.0 mM), and UGT enzyme saturating (10 mM)

substrate concentrations using commercially available recombinant UGTs. All UGTs were capable of glucuronidating APAP; however, UGTs 1A1, 1A6, 1A9, and 2B15 were most active on a pmol/mg total protein/min basis (Figure 2). At 0.1 and 1.0 mM APAP concentrations and in incubations conducted in TrisHCl buffer, four UGT enzymes were shown to be active in descending order of activity as follows: UGT 1A9, UGT 2B15, UGT 1A6, and UGT 1A1. At 10 mM APAP concentration, UGT 1A9 clearly showed the highest activity, more than 2-fold higher than UGT 1A1, the next major UGT contributing toward glucuronidating APAP at this substrate concentration. Most of the remaining UGTs showed very low although measurable activity. No activity could be detected with the vector control preparation. Incubations conducted in phosphate buffer showed a similar trend as the reactions conducted in Tris-HCl buffer, except for an apparent suppression of UGT 1A9 activity. Kinetics of UGTs 1A1, 1A6, 1A9, and 2B15 in Forming APAP Glucuronide in Tris-HCl Buffer. APAP glucuronidation kinetics (Table 1) differed markedly between UGT enzymes in that UGT 1A1 showed both substrate activation and inhibition (Hill kinetics) and UGT 1A9 displayed Michaelis-Menten kinetics, whereas UGT 1A6 and UGT 2B15 both showed substrate inhibition. UGT 1A1 (Figure 3) was a relatively highaffinity (S50 ) 5.5 ( 0.3 mM), high-capacity enzyme (Vmax ) 2620 ( 87 pmol/min/mg); UGT 1A6 (Figure 4) was a highaffinity (Km ) 4.0 ( 0.4 mM) and low-capacity enzyme (Vmax


Chem. Res. Toxicol., Vol. 19, No. 5, 2006 705

Table 1. Kinetic Parameters for APAP Glucuronidation with HLMs and Recombinant UGTs 1A1, 1A6, 1A9, and 2B15a matrix

Km or S50 (mM)b

Vmax (pmol/ min/mg)

Ki (mM)

HLM UGT 1A1 UGT 1A6 UGT 1A9 UGT 2B15

12 ( 1.7 5.5 ( 0.3 4.0 ( 0.4 9.2 ( 1.9 23 ( 14

16800 ( 1590 2620 ( 87 204 ( 10 2070 ( 157 3040 ( 1560

58 ( 12 23 ( 2.2

Hill coefficient (n) 3.2 ( 0.5

5.3 ( 3.2

Values are means ( SEM for three experiments. HLM and recombinant UGTs were pretreated with alamethacin (80 µg/mg protein) and incubated with increasing concentrations of APAP (0.01-60 mM) in Tris-HCl buffer to determine kinetic parameters as described in the Materials and Methods. b K or S depending upon Michaelis-Menten or Hill kinetics, respectively. m 50 a

Figure 5. Enzyme kinetics of APAP glucuronidation by UGT 1A9 in Tris-HCl buffer (pH 7.5) showing a typical Michaelis-Menten profile.

Figure 3. Enzyme kinetics of APAP glucuronidation by UGT 1A1 in Tris-HCl buffer (pH 7.5). The Eadie-Hofstee plot is shown as an insert to illustrate the substrate inhibition and activation kinetics.

Figure 4. Enzyme kinetics of APAP glucuronidation by UGT 1A6 in Tris-HCl buffer (pH 7.5) showing substrate inhibition. Eadie-Hofstee plot is shown as an insert to illustrate inhibition kinetics.

) 204 ( 10 pmol/min/mg); UGT 1A9 (Figure 5) was an intermediate affinity (Km ) 9.2 ( 1.9 mM), high-capacity enzyme (Vmax ) 2070 ( 157 pmol/min/mg), and UGT 2B15 (Figure 6) was a low-affinity (Km ) 23 ( 14 mM), high-capacity (Vmax ) 3040 ( 1560 pmol/min/mg) enzyme. Inhibition of UGTs 1A6 and 2B15 activities appears to occur at g10 mM APAP for both enzymes. The kinetic profile displayed by pooled HLMs is shown in Figure 7. The Km (12 ( 1.7 mM) and Vmax (16800 ( 1590 pmol/min/mg) reflect the contributions made by various UGTs in glucuronidating APAP. The substrate inhibition pattern exhibited by UGTs 1A6 and 2B15 was also observed with pooled HLMs. The potential for any ion suppression during the mass spectral analysis, especially at higher substrate concentrations, was ruled out by ensuring that each

Figure 6. Enzyme kinetics of APAP glucuronidation by UGT 2B15 in Tris-HCl buffer (pH 7.5) showing substrate inhibition. Eadie-Hofstee plot is shown as an insert to illustrate inhibition kinetics.

Figure 7. Enzyme kinetics of APAP glucuronidation by pooled HLMs in Tris-HCl buffer (pH 7.5) displaying substrate inhibition at higher concentrations of APAP. Eadie-Hofstee plot is shown as an insert to illustrate inhibition kinetics.

component was chromatographically resolved from each other during the analyses. Substrate Inhibition Kinetics Demonstrated by UGTs 1A6 and 2B15. Studies were conducted to investigate whether inhibition of UGTs 1A6 and 2B15 activities was due to high substrate concentrations or as a result of product (APAP glucuronide) formation. Substrate inhibition was inferred after demonstrating APAP glucuronide (as the product) was not an inhibitor of the enzymes responsible for its formation. This was achieved by using a deuterated analogue of APAP, which exhibited a similar rate of glucuronidation by UGT 2B15 as


Figure 8. Relative levels of GSH adduct of APAP in cell media of human hepatocytes from two donors. Hepatocytes were treated for 24 h with 5 mM APAP alone or in combination with PH (0.1 mM), PB (1 mM), or PB (1 mM) + PH (0.1 mM). A similar trend was observed in cell lysates. *p < 0.05 (Student’s t-test) was considered significantly different from the control treatment group (5 mM APAP).

the nonlabeled APAP (data not shown). It appears that binding of the deuterated APAP to the active site of the enzyme was not affected by the presence of deuteriums on the aromatic ring. Introduction of deuteriums on the molecule leads to an increment in the [M + H]+ of d4-APAP glucuronide by 4 amu. Hence, using mass spectrometry, we were able to monitor the formation of d4-APAP glucuronide (from labeled d4-APAP) in the presence of known concentrations of APAP glucuronide. The peak area ratios of d4-APAP glucuronide to internal standard were unchanged in the presence of nonlabeled APAP glucuronide (up to 3 µM). The concentrations of APAP glucuronide typically formed from UGTs 1A6 and 2B15 in our studies were always less than 3 µM. At concentrations greater than 3 µM, a significant drop in peak area ratios was observed. However, further experiments showed that this drop in the ratios was due to ion suppression by an excessive level of APAP glucuronide rather than due to enzyme inhibition. The samples were reanalyzed after adding a sufficient amount of APAP glucuronide to each sample to give a final concentration equivalent to the 300 µM extract. A consistent peak area ratio was obtained for d4-APAP glucuronide/IS in all of the samples suggesting mass spectral ion suppression at high concentrations of APAP glucuronide. Effect of PB and PH, and Substrate Concentration on the Metabolism of APAP to Reactive NAPQI. Incubations conducted with HLMs showed that neither PB nor PH was able to significantly inhibit (or activate) the formation of NAPQI via phase I metabolism. This was demonstrated by incubating HLMs with 1.0 mM APAP in the presence and absence of these inhibitors. The HLMs were able to metabolize APAP to NAPQI, which was subsequently trapped as the GSH adduct and detected by LC/MS. The GSH adduct served as a surrogate marker for reactive NAPQI produced from APAP. At 0.1 and 1.0 mM APAP, PH (0.1 mM) and PB (0.1 and 1.0 mM) did not alter the levels of NAPQI produced by microsomes. The levels of Cys-Gly found in the liver microsomal extracts were also compared among the various treatment groups and found to parallel the pattern observed with the GSH adducts (data not shown). The results from the microsomal study are consistent with the findings in human hepatocytes, which showed that the formation of NAPQI (trapped as GSH adduct) was not compromised in the presence of PB or PH (Figure 8); in fact, there was an increase in the level of GSH adduct as compared

Mutlib et al.

Figure 9. Effect of APAP concentration on the formation of reactive intermediate, NAPQI, which was trapped as the GSH adduct in the HLM. Typical Michaelis-Menten kinetics were observed with Km ) 3.0 ( 0.6 mM.

Figure 10. Effect of acute addition of PB and PH on APAP toxicity to cultured human hepatocytes. Hepatocytes from two human donors were treated for 24 h with 5 mM APAP alone or in combination with 1 mM PB or 0.1 mM PH. Protein synthesis was determined by a pulse labeling with 14C-leucine as described in the Materials and Methods. *The inhibition of protein synthesis in the “APAP + PB + PH” treatment group was considered statistically significant as compared with the control (p < 0.05 using Student’s t-test).

to the control. The effect of APAP concentration on its biotransformation to NAPQI (trapped as the GSH adduct) by phase I metabolism was studied. Under the incubation conditions described, the formation of NAPQI showed an apparent Michaelis-Menten kinetics (Figure 9) with Km ) 3.0 ( 0.6 mM. The relative levels of NAPQI were indirectly obtained by measuring the GSH and cysteinyl-glycine conjugates formed in HLM (Figure 9). There was a parallel increase in the formation of cysteinyl-glycine and GSH conjugates in HLM. The formation of cysteinyl-glycine conjugate demonstrates the presence of γ-glutamyltranspeptidase in the HLM. Effect of PB and PH on APAP Toxicity in Cultured Hepatocytes. Incubations of human hepatocytes for 24 h (prepared from two different donors) with the combination of (i) APAP (5 mM) and PB (1 mM), (ii) APAP (5 mM) and PH (0.1 mM), or (iii) APAP (5 mM), PB (1 mM), and PH (0.1 mM) resulted in an increase in APAP toxicity as compared to cells treated with APAP alone, as measured by a decrease in total protein synthesis (Figure 10). The combination of APAP with PB (1 mM) and PH (0.1 mM) appeared to be fairly toxic to cells in this study, as shown by the appearance of morphological cell death and a dramatic decrease in protein synthesis



Table 2. Mean ((SEM) Concentrations of APAP Glucuronide and APAP Sulfate Metabolites Present in the Media of Cultured Human Hepatocytes from Two Donorsa µg/mL treatment

APAP sulfate

APAP glucuronide

APAP (5 mM) APAP (5 mM) + PB (1 mM) APAP (5 mM) + PH (0.1 mM) APAP (5 mM) + PB (1 mM) + PH (0.1 mM)

29.8 ( 0.4 23.4 ( 0.7* 22.6 ( 0.8* 21.0 ( 0.1*

177.5 ( 1.5 61.1 ( 2.7** 72.5 ( 1.5** 42.6 ( 0.7**

a *p < 0.05 and **p < 0.001, considered statistically significant as compared to the control (APAP, 5 mM).

Figure 11. Relative levels of APAP sulfate and APAP glucuronide found in media of human hepatocytes exposed to APAP (5 mM) administered in combination with PB (1.0 mM), PH (0.1 mM), or PB (1 mM) + PH (0.1 mM). See Table 2 for the actual concentrations of APAP sulfate and APAP glucuronide metabolites in various treatment groups. *p < 0.05 and **p < 0.001, considered statistically significant as compared to the control (APAP, 5 mM).

to approximately 23% of the control value within 24 h of treatment (Figure 10). The 24 h culture media and cell lysates were analyzed for APAP metabolites, including glucuronide, sulfate, cysteine, cysteinylglycine, and glutathione conjugates. As shown in Table 2 and Figure 11, the formation of APAP glucuronide was reduced in the presence of PH (0.1 mM) by almost 60%, consistent with our previous findings (1). Similarly, PB (1 mM) reduced the glucuronide formation to 35% of the control value. However, when the cells were treated with both PH and PB, the glucuronide formation was reduced further to only 20% as compared to cells treated with only APAP (Table 2 and Figure 11). In our previous study, we had shown that PB had no effect on the sulfation of APAP (1). The levels of the GSH-related adducts were also compared in various treatment groups. Analysis of the 24 h cell media showed that the GSH adduct of APAP had slightly increased in the samples that were treated with PB and PH in combination (Figure 8). The relative levels of other GSH-related adducts (cysteine and cysteinyl-glycine) paralleled the GSH adduct in the media of various treatment groups. A similar trend was also observed in the cell lysates, although it appeared that the levels of GSH adduct were much higher in cell pellets than the media data not shown).

Discussion The formation of glucuronide conjugate of APAP has been previously attributed mainly to the UGT 1A family, especially UGTs 1A1, 1A6, and 1A9 (2-6). Despite its ability to

glucuronidate simple phenolic compounds, it was reported that UGT 2B15 could not form APAP glucuronide (6, 14). Our recent study, however, showed that UGT 2B15 was able to form APAP glucuronide effectively (1). This prompted us to investigate further if UGT2B15 was indeed just as important as UGTs 1A6 and 1A9 in metabolizing APAP. The purity and authenticity of rUGT 2B15 was confirmed by the commercial supplier (BD Gentest). We also tested another batch of UGT 2B15 (lot #23953, BD Gentest) and found that it also glucuronidated APAP to almost the same extent as the first lot (#7). Next, we investigated the possibility of differences in incubation conditions that could affect the ability of UGT 2B15 to form the APAP glucuronide. As shown in Figure 2, there were differences in the rate of APAP glucuronide formation in the presence of Tris-HCl or phosphate buffers. In either one of these buffers, the glucuronidating activity of various UGTs toward APAP was consistent at 0.1 and 1.0 mM substrate concentration. The relative activities of the major enzymes in Tris-HCl buffer at 0.1 and 1.0 mM APAP concentration were 1A9 > 2B15 > 1A6 > 1A1, while under the same conditions in phosphate buffer, 1A9 ) 2B15 > 1A6 > 1A1. At 10 mM APAP concentration, the relative activity of UGT 1A1 was significantly pronounced, while the activity of 1A6 became obscured by similar contributions from other UGTs such as 1A7, 1A8, and 2B7. This observation is consistent with what was observed by Court et al. (6) who showed a significant increase and decrease in UGT 1A1 and UGT 1A6 activities, respectively, at a higher APAP concentration (50 mM). The reduced UGT 1A6 activity was perhaps due to low capacity and perhaps as a result of substrate inhibition as shown in this study. From the studies conducted with the rUGTs, it appears that UGTs 1A1, 1A6, 1A9, and 2B15 are the major enzymes involved in glucuronidating APAP (Figure 2). However, it must be stressed that at this stage, it is impossible to say with certainty which of the four UGTs plays a dominant role in glucuronidating APAP. The reasons for not observing the high UGT 2B15 activity toward APAP by earlier investigators are not clear at this stage. Considering that incubation conditions used by other investigators were similar, one possible explanation could be that the previous batches of UGT 2B15 were either inactive or were not stably expressed. The results from studies conducted with rUGTs led us to speculate that UGT 2B15 was perhaps a very significant enzyme in glucuronidating APAP in humans. To support this hypothesis, the kinetics of each rUGT was studied. As shown in Figure 3, UGT 1A1 showed a high affinity (displayed Hill kinetics with S50 ) 5.5 mM) and a high capacity (Vmax ) 2620 pmol/min/ mg) to conjugate APAP relative to other UGTs. The substrate activation exhibited by UGT 1A1 in this study was also observed by Court et al. (6). UGT 1A6 (Figure 4) displayed a high affinity (Km ) 4.0 mM) and low capacity (Vmax ) 204 pmol/min/mg) with a potential for substrate inhibition. These results are consistent with those reported by Court et al. (6) under their experimental conditions. UGT 1A9 showed typical MichaelisMenten kinetics with an intermediate Km of 9.2 mM and high Vmax of 2070 pmol/min/mg (Figure 5). In contrast, the Km and Vmax values obtained in a previous study were reported to be much higher than found in this study (6). UGT 2B15 showed a much lower affinity (Km ) 23 mM) but a much greater capacity (Vmax ) 3040 pmol/min/mg) to glucuronidate APAP than other enzymes (Figure 6). The kinetics of APAP glucuronidation by pooled HLMs was similar to the profiles obtained from UGTs 1A6 and 2B15 (Figure 7).


Studies conducted with human hepatocytes provided further evidence of the importance of UGT 2B15 in APAP metabolism. Previously, we had shown that PB was able to inhibit UGT 2B15 more selectively and potently than other UGTs involved in glucuronidating APAP (1). UGT 2B15 activity was almost completely abolished, while UGT 1A9 was inhibited to only a slight extent at 1 mM PB concentration using these recombinant UGTs (1). Under those conditions, the glucuronidation of APAP in human hepatocytes was reduced by almost 50%. The current study with human hepatocytes exposed to APAP in combination with various inhibitors clearly shows that at 0.1 mM PH or at 1 mM PB, there was greater than 50% reduction in APAP glucuronide formation in cell cultures (Figure 11). A further reduction in APAP glucuronide formation was observed when both PH (0.1 mM) and PB (1 mM) were added together with APAP (5 mM). This significant reduction in glucuronidation is most likely due to PH inhibiting mostly UGT 1A9 activity, while PB inhibited UGT 2B15. Studies are planned to further characterize the relative contributions of each enzyme present in HLM in glucuronidating APAP. The relative amounts of UGTs in human livers are unknown, but from the UDP-glucuronosyltransferase mRNA levels determined previously in diseased livers, it appears that UGT 2B15 and UGT 1A6 could be present in much greater quantities than either UGTs 1A1 or 1A9 (15). The greater contribution of UGT 2B15 toward glucuronidating APAP will be realized if we can demonstrate the high hepatic levels of UGT 2B15 in healthy adults. Furthermore, this enzyme is encoded by a polymorphic gene (16, 17). Genetic variations of human UGTs and its implications in drug glucuronidation have been well-documented (18-20). The genetic polymorphism exhibited by UGT 2B15 could partially explain the large interindividual variability and sex differences in APAP glucuronidation by HLMs (6). We had previously demonstrated APAP-induced hepatotoxicity (in cultured hepatocytes) mediated by UGT inhibition (1). Specifically, it was found that inhibition of UGT activities by PB and PH led to a potentiation of APAP-mediated toxicity in hepatocytes. This increased toxicity in the presence of these UGT inhibitors was thought to be due to a higher level of bioactivation of APAP to NAPQI as evidenced by greater levels of GSH-related adducts in cell lysates from APAP + PB-treated hepatocytes (1). Under those conditions, sulfation was saturated and a reduction in glucuronidation by almost 50% apparently led to a greater degree of bioactivation of APAP. This was consistent with previous reports, which suggested that during accidental overdoses of APAP, sulfation was saturated, while glucuronidation was the major metabolic route of clearance (2123). Obviously, glucuronidation is a very important protective pathway for APAP and any compromise in this metabolic route can potentially lead to an imbalance between this protective pathway and the bioactivation of the molecule to reactive NAPQI intermediate. The results from the in vitro studies with hepatocytes were consistent with previous reports of increased incidence of hepatotoxicities in patients treated with a combination of APAP with antieplileptics, including PB and PH (2427). It was shown in this study that a combination of PB and PH with APAP led to greater toxicity in hepatocytes than if the cells were treated with each compound separately (Figure 10). We have also shown in this study that while glucuronidation of APAP was reduced significantly in the presence of PH and PB, these compounds had no inhibitory effect on the formation of reactive NAPQI (Figure 8). Furthermore, we established that NAPQI formation was not inhibited by higher concentrations of APAP (Figure 9). It was important to demonstrate this,

Mutlib et al.

especially when it was found that UGTs 1A6 and 2B15 as well as HLM showed substrate inhibition kinetics toward glucuronidating APAP at higher substrate concentrations. The studies conducted with hepatocytes to which APAP was added in combination with PB or PH clearly demonstrates the potential for drug-drug interaction through inhibition of glucuronidation. Such an inhibition of glucuronidation has been reported previously (1, 28, 29). Direct inhibition by probenecid on ether glucuronide formation from APAP and lorazepam was observed in vivo (28). Similarly, valproic acid (VPA) was found to inhibit the formation of steroid glucuronides mediated by UGT 2B15, although VPA itself is not a substrate for this enzyme (29). This potent inhibition of UGT 2B15 by VPA probably explains some of the earlier reports of increased incidence of hepatotoxicity when VPA was coadministered with APAP (24). It has been demonstrated in this study that UGT 2B15 is an important contributor toward glucuronidating APAP at therapeutic and subtoxic concentrations. However, as demonstrated by inhibition of UGTs 1A9 and 2B15 by PH and PB, respectively, both of these enzymes are critical for metabolizing APAP. The previously unnoticed but suggested contribution by UGT2B family toward glucuronidating APAP could probably explain why APAP glucuronidation was still observed in cases where its formation was not expected (30, 31). In conclusion, the contribution made by UGT 2B15 in glucuronidating APAP has been previously overlooked and hence underestimated. In this study, we have shown that UGT 2B15 could potentially play an important role in metabolizing APAP. The potential for non-P450-mediated drug-drug interaction, as shown by the effects of PB and PH on APAP glucuronidation, exists for compounds that can either competitively or noncompetitively inhibit glucuronidation of APAP. This can possibly lead to toxicological consequences, as demonstrated by our in vitro model, and needs to be investigated further in the clinic.

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TX050317I

Kinetics of Acetaminophen Glucuronidation by UDP

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