UDP-Glucuronosyltransferase-mediated Metabolic Activation of the Tobacco Carcinogen 2-Amino-9H-pyrido[2,3-b]indole*

Background: 2-Amino-9H-pyrido[2,3-b]indole (AαC) is a carcinogen formed in tobacco smoke, but little is known about its metabolism in humans. Results: UDP-Glucuronosyltransferases catalyze the binding of N-oxidized-AαC to DNA. Conclusion: Glucuronidation, normally a detoxication pathway, contributes to the genotoxicity of AαC. Significance: The exposure to and UGT bioactivation of AαC provides a biochemical mechanism for the elevated risk of liver and digestive tract cancers in smokers. 2-Amino-9H-pyrido[2,3-b]indole (AαC) is a carcinogenic heterocyclic aromatic amine (HAA) that arises in tobacco smoke. UDP-glucuronosyltransferases (UGTs) are important enzymes that detoxicate many procarcinogens, including HAAs. UGTs compete with P450 enzymes, which bioactivate HAAs by N-hydroxylation of the exocyclic amine group; the resultant N-hydroxy-HAA metabolites form covalent adducts with DNA. We have characterized the UGT-catalyzed metabolic products of AαC and the genotoxic metabolite 2-hydroxyamino-9H-pyrido[2,3-b]indole (HONH-AαC) formed with human liver microsomes, recombinant human UGT isoforms, and human hepatocytes. The structures of the metabolites were elucidated by 1H NMR and mass spectrometry. AαC and HONH-AαC underwent glucuronidation by UGTs to form, respectively, N2-(β-d-glucosidurony1)-2-amino-9H-pyrido[2,3-b]indole (AαC-N2-Gl) and N2-(β-d-glucosidurony1)-2-hydroxyamino-9H-pyrido[2,3-b]indole (AαC-HON2-Gl). HONH-AαC also underwent glucuronidation to form a novel O-linked glucuronide conjugate, O-(β-d-glucosidurony1)-2-hydroxyamino-9H-pyrido[2,3-b]indole (AαC-HN2-O-Gl). AαC-HN2-O-Gl is a biologically reactive metabolite and binds to calf thymus DNA (pH 5.0 or 7.0) to form the N-(deoxyguanosin-8-yl)-AαC adduct at 20–50-fold higher levels than the adduct levels formed with HONH-AαC. Major UGT isoforms were examined for their capacity to metabolize AαC and HONH-AαC. UGT1A4 was the most catalytically efficient enzyme (Vmax/Km) at forming AαC-N2-Gl (0.67 μl·min−1·mg of protein−1), and UGT1A9 was most catalytically efficient at forming AαC-HN-O-Gl (77.1 μl·min−1·mg of protein−1), whereas UGT1A1 was most efficient at forming AαC-HON2-Gl (5.0 μl·min−1·mg of protein−1). Human hepatocytes produced AαC-N2-Gl and AαC-HN2-O-Gl in abundant quantities, but AαC-HON2-Gl was a minor product. Thus, UGTs, usually important enzymes in the detoxication of many procarcinogens, serve as a mechanism of bioactivation of HONH-AαC.

O-Gl (77.1 l⅐min ؊1 ⅐mg of protein ؊1 ), whereas UGT1A1 was most efficient at forming A␣C-HON 2 -Gl (5.0 l⅐min ؊1 ⅐mg of protein ؊1 ). Human hepatocytes produced A␣C-N 2 -Gl and A␣C-HN 2 -O-Gl in abundant quantities, but A␣C-HON 2 -Gl was a minor product. Thus, UGTs, usually important enzymes in the detoxication of many procarcinogens, serve as a mechanism of bioactivation of HONH-A␣C.
Epidemiologic studies conducted over the past two decades have consistently shown that tobacco smoking is a risk factor for cancers of the gastrointestinal tract (1,2). There is also mounting evidence that tobacco smoke is an independent risk factor for hepatocellular carcinoma, the predominant form of human liver cancer (3,4). However, the causal agents of these cancers in tobacco smoke remain to be determined.
The evaluation of the human health risk of A␣C requires an understanding of the enzymes involved in bioactivation and detoxication of this procarcinogen; however, the metabolism of A␣C has not been well studied in humans (24). Recombinant human cytochrome P450 1A2 catalyzes the N-oxidation of the exocyclic amine group of A␣C to form 2-hydroxyamino-9Hpyrido [2,3-b]indole (HONH-A␣C), a genotoxic metabolite (25). P450 1A2 also catalyzes the detoxication of A␣C by ring oxidation of the C3 or C6 atoms of the heterocyclic ring (25,26) followed by O-glucuronidation (27,28). However, human hepatocytes efficiently bioactivate A␣C to reactive metabolites that form DNA adducts (29). The propensity of A␣C and structurally related compounds in tobacco smoke to undergo bioactivation by enzymes expressed in liver and extrahepatic tissues provides a biochemical mechanism for the elevated risk of liver and digestive cancers in smokers (25, 29 -31).
In this study we have characterized the metabolic products of A␣C and HONH-A␣C that are formed by UGT enzymes present in human liver microsomes, recombinant human UGT1A and UGT2B isoforms, and freshly cultured human hepatocytes. We report a novel pathway of bioactivation of HONH-A␣C where UGTs catalyze the formation an O-linked glucuronide conjugate, A␣C-HN 2 -O-Gl, which binds covalently to DNA. Thus, UGT enzymes, normally viewed as a means of detoxication of many carcinogens, serves as a mechanism of bioactivation and likely transports of HON-A␣C from liver to extrahepatic tissues.

EXPERIMENTAL PROCEDURES
Caution-A␣C and several of its derivatives are potential human carcinogens and should be handled with caution in a well ventilated fume hood with the appropriate protective clothing.
Chemicals and Reagents-A␣C was purchased from Toronto Research Chemicals (Toronto, ON, Canada). Uridine-5Јdiphosphoglucuronic acid (UDPGA), alamethicin, and ␤-glucuronidase type IX-A from Escherichia coli were purchased from Sigma.
General Methods-Mass spectra were acquired on a Finnigan Quantum Ultra triple stage quadrupole mass spectrometer (Thermo Fisher, San Jose, CA) with a Michrom Advance CaptiveSpray TM source (Auburn, CA). Typical instrument tuning parameters were as follows: capillary temperature, 200°C; source spray voltage, 2 kV; tube lens offset, 95 V; capillary offset, 35 V; source fragmentation, 10 V. Argon, set at 1.5 millitorr, was used as the collision gas. There was no sheath or auxiliary gas. All analyses were conducted in the positive ionization mode. Metabolites were also characterized with the Thermo Fisher linear quadrupole ion trap mass spectrometer with the Advance CaptiveSpray TM ion source. The source voltage was 1.5 kV, the capillary voltage was 25 V, and the tube lens was 80 V. The isolation width was set at m/z 4 and 1, respectively, for the MS 2 and MS 3 scan modes, the activation Q was set at 0.35, and the activation time was 10 ms. Helium was used as the collision damping gas in the ion trap. One microscan was used for data acquisition. The automatic gain control settings were full MS target 30,000 and MS n target 10,000, and the maximum injection time was 10 ms.
NMR Studies-1 H NMR resonance assignment experiments for the glucuronide metabolites of A␣C and HNOH-A␣C were conducted at 25°C with a Bruker Avance III 600 MHz spectrometer equipped with a triple resonance cryoprobe (Bruker BioSpin Corp., Billerica, MA). The 1 H chemical shifts were referenced directly from the DMSO-d 6 multiplet at 2.56 ppm. A standard double quantum filtered COSY experiment was employed to collect 1024 t 1 increments over a 7507-Hz spectral window. Standard rotating frame NOESY (ROESY) was employed using 400 ms mixing time. The 2-D NMR data were analyzed with the program SPARKY (University of California, San Francisco).
Synthesis of HONH-A␣C and Biosynthesis of Glucuronide Conjugates of A␣C and HNOH-A␣C-HONH-A␣C was synthesized by reduction of 2-nitro-9H-pyrido[2,3-b]indole as described (31). The glucuronide metabolites were prepared by incubating A␣C or HONH-A␣C (1 mg in 100 l DMSO) with 5 mg of human liver microsomal protein in 5 ml of 100 mM Tris-HCl buffer (pH 7.5) containing 10 mM MgC1 2 , 0.5 mM EDTA, and 5 mM UDPGA for 3 h at 37°C. The microsomal mixture was preincubated with alamethicin (50 g/mg protein) on ice for 30 min to overcome the latency phenomena associated with UGT enzymes before the addition of the A␣C compounds. Ascorbic acid (2 mM) was added to the microsomal incubation containing NOH-A␣C to minimize oxidation of the substrate. The reactions were terminated by the addition of 1 volume of ice-cold CH 3 OH, and the mixtures were placed on ice for 30 min. The precipitated proteins were removed by centrifugation.
UGT Glucuronidation and Enzyme Kinetics of A␣C and HNOH-A␣C with Recombinant UGT Isoforms-UGTs were diluted to a concentration of 0.5 mg protein/ml in 100 mM Tris-HCl buffer (pH 7.5) containing 10 mM MgC1 2 , 0.5 mM EDTA, 2 mM UDPGA, and 25 g of alamethicin per 0.5 mg of protein and incubated on ice for 30 min before the addition of A␣C substrates. The reactions were conducted under an atmosphere of argon at 37°C. Time-dependent studies with HONH-A␣C (10 or 100 M) showed that product formation was linear over 140 min (data not shown). Enzyme kinetics experiments were conducted with HNOH-A␣C at various concentrations between 5 and 500 M, and the concentrations of A␣C ranged from 75 to 1500 M. The time of incubation was 1 h. Aliquots (100 l) were taken and added to 2 volumes of ice-cold CH 3 OH to terminate the reaction followed by centrifugation to remove protein. The methanolic extracts were analyzed by HPLC. The activities of UGT isoforms were assessed with ␤-estradiol (150 M) as a substrate for UGTs 1A1 and 1A3, trifluoperazine (200 M) as a substrate for UGT 1A4, and 7-hydroxy-4-trifluoromethylcoumarin (50 M) as a substrate for UGTs 1A6, 1A8, 1A9, 1A10, and 2B7. The enzyme activities were in good agreement to those values provided by BD Biosciences.
HPLC Analysis of A␣Cand HON-A␣C-glucuronide Conjugates-Metabolites were analyzed with an Agilent model 1100 HPLC Chemstation (Palo Alto, CA) equipped with a photodiode array detector. The metabolites were separated with a Aquasil C18 column (4.6 ϫ 150 mm, 5-m particle size) from Thermo Scientific. The chromatography of the glucuronide conjugates of A␣C began at 10 mM NH 4 CH 3 CO 2 (pH 6.8) for 2 min followed by a linear gradient over 22 min to 100% CH 3 CN at a flow rate of 1 ml/min. The chromatography of the glucuronide conjugates of HNOH-A␣C also commenced at 10 mM NH 4 CH 3 CO 2 (pH 6.8) for 2 min followed by a linear gradient over 16 min to arrive at 40% CH 3 CN and reached 100% CH 3 CN at 25 min. The estimates of formation of glucuronide conjugates of A␣C and HNOH-A␣C were determined by integration of the peak monitored at 338 nm. We assumed that the molar extinction coefficients of the glucuronide conjugates were comparable with the molar extinction coefficient of A␣C Kinetic Studies on A␣C-N 2 -Gl, A␣C-HON 2 -Gl, and A␣C-HN 2 -O-Gl as Function of pH or by Treatment with ␤-Glucuronidase-The stabilities A␣C-and HONH-A␣C glucuronide conjugates were examined in 50 mM potassium phosphate buffer (pH 7.0) or 50 mM citric acid buffer (pH 5.0). The conjugates were also incubated with ␤-glucuronidase (240 units/ml) in 50 mM potassium phosphate buffer (pH 7.0). The compounds (4 g, 10.7 nmol) were incubated under an atmosphere of argon at 37°C for up to 3 h. The conjugates and hydrolysis products were assayed directly by HPLC except for studies with ␤-glucuronidase; the solution was diluted with 2 volumes of ice-cold CH 3 OH, and the protein was removed by centrifugation, prior HPLC (see above). UGT-mediated Binding of HONH-A␣C to DNA-Human liver microsomal protein (0.5 mg) in 0.5 ml of 100 mM Tris-HCl buffer (pH 7.5) containing salts, ascorbic acid, 5 mM UDPGA (see above), and calf thymus DNA (0.3 mg) was preincubated with alamethicin (25 g/0.5 mg protein) on ice for 30 min followed by the addition 1-naphthol (0, 100 or 1000 M) and then HONH-A␣C (10 M). The incubation proceeded at 37°C for 30 min. The reaction was terminated by the addition of CaCl 2 (50 mM final concentration) to precipitate the protein. After centrifugation, the supernatant was retrieved, and 0.1 volumes of 5 M NaCl and 2 volumes C 2 H 5 OH were added to precipitate DNA. The supernatants containing HONH-A␣C glucuronide conjugates were measured by HPLC (see above). The pelleted DNA was washed with C 2 H 5 OH:H 2 O mixture (7:3) and digested enzymatically as described below.

DNA Binding Studies with HONH-A␣C, A␣C-HON 2 -Gl, and A␣C-HN 2 -O-Gl-Calf
Metabolism Studies of A␣C with Human Hepatocytes-Human liver samples were obtained from patients undergoing liver resection for primary or secondary hepatomas through the Biological Resource Center (CHRU Pontchaillou, Rennes, France). The research protocol was conducted under French legal guidelines and fulfilled the requirements of the local institutional ethics committee. This study was approved by the Institutional Review Board at the Wadsworth Center. Hepatocytes were isolated by a two-step collagenase perfusion procedure, and parenchymal cells were seeded in Petri dishes at a density of 3 ϫ 10 6 viable cells/19.5-cm 2 dish in 3 ml of Williams' modified medium before incubation with A␣C (10 or 50 M) as previously described (29). Metabolites and DNA were isolated from the cell extracts (29) and characterized by LC-ESI/MS (see below).
Data Analysis-GraphPad Prism 5 software (La Jolla, CA) was employed to calculate enzyme kinetic values. Apparent K m and V max values for glucuronidation by each enzyme were derived from the Michaelis-Menten equation, where v is the initial velocity, V max is the maximum enzyme velocity (pmol⅐min Ϫ1 ⅐mg Ϫ1 protein), K m is the Michaelis constant, [S] is the initial substrate concentration, K si is the dissociation constant for the substrate from the enzyme inhibitor complex. Catalytic efficiency (V max /K m ) was expressed as l⅐min Ϫ1 ⅐mg of protein Ϫ1 . The data were fitted using nonlinear regression employing the least squares to obtain the best curve.

RESULTS
Glucuronide Metabolites of A␣C and HONH-A␣C Produced by Human Liver Microsomes-The HPLC profile of glucuronide conjugates of A␣C and HONH-A␣C produced by human liver microsomes under elevated substrate concentrations (1 mM) is shown in Fig. 2. One major glucuronide conjugate was formed with A␣C (t R 9.4). The online UV spectrum of the metabolite displayed a chromophore that was very similar to the spectrum of A␣C, suggesting the metabolite was A␣C-N 2 -Gl. Two glucuronide conjugates were formed with HONH-A␣C (t R 14.9 and 15.8 min). The UV spectra of both conjugates strongly resembled the UV spectrum of HONH-A␣C. The ratio of the peak area between the conjugates was about 2.4:1.
1 H NMR Spectroscopy of Glucuronide Conjugates of A␣C and HONH-A␣C-The glucuronide conjugate of A␣C (120 g) and the major (210 g) and minor (80 g) conjugates of HONH-A␣C were produced in sufficient quantities to analyze by 1 H NMR spectrometry (Figs. 3-5). The chemical shift values for the metabolites are summarized in Table 1.
All of the protons of the heterocyclic ring and the endocyclic N-9 atom were observed in the 1 H NMR spectrum of the glucuronide conjugate of A␣C. The resonance signal at 7.18 ppm had the intensity of one proton and occurred as a doublet (J ϭ 8.8 Hz). This signal was assigned as the N 2 -H. It is coupled to the anomeric proton (H-1Ј) of the glucuronide moeity at 5.09 ppm, seen as a "triplet" due to the addition to the passive coupling with the glucuronide H-2Ј proton (Fig. 3). There is also an NOE connectivity between this resonance at 7.18 ppm and the H-3 of the heterocyclic ring. The analogous NOE connectivity is observed in the parental AaC analysis. This conjugate also exhibits an NOE connectivity between H-1Ј of the glucuronic acid and the H-3 proton of the heterocyclic ring. These results provide spectral data to assign this glucuronide conjugate as A␣C-N 2 -Gl.
All of the protons of the heterocyclic ring and the endocyclic N9 atom were observed in the 1 H NMR spectra of the two glucuronide conjugates of HONH-A␣C (Figs. 4 and 5). A clear NOE signal was also observed in the NOESY spectra for the protons attached to the N9 atom and the H8 protons of both conjugates. These spectral data exclude the N9 atom as site of conjugation with glucuronic acid. The NOESY spectra also showed NOE signals between the H3 protons of HONH-A␣C and the H1Ј of the glucuronic acid moieties of both conjugates. These observed NOE connectivities support the proposed sites of conjugation at the exocyclic N 2 -or N 2 -O atoms and not at the more distant N1 pyridinyl nitrogen atom. The similarities among the UV chromophores of both conjugates and HONH-A␣C (Fig. 2) also favor the assignment of the glucuronide conjugation at the exocyclic N 2 or N 2 -O atoms. The minor glucuronide product of HONH-A␣C displayed a pronounced up-field shift in the resonance signal of the H1Ј glucuronide at 4.58 ppm in comparison to the H1Ј resonance signal of the major conjugate, which was situated at 5.64 ppm. The resonance signals of the H1Ј glucuronide proton for O-glucuronide conjugates of several hydroxylamine metabolites occur at 4.5-4.7 ppm (37-40), whereas the resonance signals of the H1Ј glucuronide proton of N-glucuronide conjugates of N-hydroxylated HAA metabolites are situated above 5.0 ppm (41,42). The spectral data suggest that A␣C-HON 2 -Gl is the more abundant glucuronide conjugate and that A␣C-HN 2 -O-Gl is the minor conjugate. However, the 1 H NMR spectra do not permit unequivocal assignment of the structures. The identities of these isomeric metabolites were distinguished by ESI/MS/MS n .  (Fig. 6D) (43) and proves that the linkage formed between HONH-A␣C and glucuronic acid occurred at the oxygen atom of HONH-A␣C. The product ion spectra of O-glucuronide conjugates of arylhydroxamic acids typically display a prominent ion at m/z 193, attributed to the glucuronate, in the negative ion mode (44).

FIGURE 2. HPLC profiles and online UV spectra of glucuronide metabolites of A␣C and HONH-A␣C produced by human liver microsomes, glucuronide metabolite of A␣C (A) and glucuronide metabolites of HONH-A␣C (B).
Different gradient conditions were employed for the resolution of the metabolites, as described under "Experimental Procedures".
2B7 but with different specificities in product formation (supplemental Table 1S). A␣C-HON 2 -Gl was the predominant conjugate formed with UGT2B7, whereas A␣C-HN 2 -O-Gl was the major conjugate produced by UGTs 1A1 and 1A9. UGT1A4 catalyzed the formation A␣C-HON 2 -Gl but did not form A␣C-HN 2 -O-Gl. UGTs 1A1, 1A9, and 2B7 produced both A␣C-HON 2 -Gl and A␣C-HN 2 -O-Gl. The activity observed for UGT1A8 was below the level of metabolite detection by HPLC (Ͻ7 pmol ⅐ min Ϫ1 ⅐ mg of protein Ϫ1 ).

Steady-state Enzyme Kinetic Parameters of A␣C-N 2 -Gl, A␣C-HON 2 -Gl, and A␣C-HN 2 -O-Gl Formation-
The steadystate enzyme kinetic parameters were determined for UGTs 1A1, 1A4, 1A9, and 2B. The data are summarized in Table 2,     nucleophilic substitution reactions with DNA or protein (20). A␣C-HN 2 -O-Gl was a substrate for ␤-glucuronidase (E. coli). The sole, initial product formed was HONH-A␣C (56 pmol⅐min Ϫ1 ⅐unit Ϫ1 ␤-glucuronidase, t1 ⁄ 2 ϭ 2 min) in 50 mM potassium phosphate buffer (pH 7.0), whereas the rates of hydrolysis of A␣C-N 2 -Gl and A␣C-HO-N 2 -Gl were considerably slower (Յ93 fmol⅐min Ϫ1 ⅐unit Ϫ1 ␤-glucuronidase, t1 ⁄ 2 not deter-  NMR spectra (top panel, 11. 78 -3.15 ppm) show the aromatic protons of A␣C moiety and H1,H2 protons of the glucuronide moiety before and after the addition of D 2 O. The small amount of D 2 O served to dilute and shift the resonances of the exchangeable protons. Previous to the addition of D 2 O, the contour plot of a two-dimensional ROESY spectrum (middle panel) showed NOE connectivities between protons through distance coupling around the NH9, the aromatic proton region, and the aliphatic region situated around the H1Ј of the glucuronic acid. The observe dimension is oriented along the y axis of this plot to take advantage of the stronger intensity of the NH-9-H8 cross-peak along this dimension. The portions of the two-dimensional COSY spectrum (bottom panel) focused on the protons having coupling through chemical bonds on the moiety of A␣C. Note: x is an impurity. mined) (supplemental Fig. 2S). The differences in rates of enzymatic hydrolysis of these A␣C conjugates are consistent with the known fact that O-glucuronide conjugates are superior substrates to N-glucuronide conjugates for ␤-glucuronidase (E. coli) (46).

Kinetic Studies on A␣C-N 2 -Gl A␣C-HON 2 -Gl and A␣C-HN 2 -O-Gl as Function of pH or by Treatment with
Reactivity of HONH-A␣C, A␣C-HON 2 -Gl, and A␣C-HN 2 -O-Gl with Calf Thymus DNA-The reactivity of HONH-A␣C and its glucuronide conjugates to DNA under different pH con-ditions was compared with the DNA binding of the N-hydroxy derivative of 4-ABP, a tobacco carcinogen (8), and the N-hydroxy derivative of 2-amino-1-methyl-6-phenylimidazo [4,5b]pyridine (PhIP), a carcinogen formed in cooked meat (12). The amounts of the dG-C8-A␣C, dG-C8 -4-ABP, and dG-C8-PhIP adducts formed by these reactive metabolites were determined by LC-ESI/MS/MS 3 (29). The mass chromatograms are  UGT-mediated Binding of HONH-A␣C to DNA-UGT isoforms expressed in human liver microsomes produced A␣C-HON 2 -Gl and A␣C-HN 2 -O-Gl and catalyzed the binding of HONH-A␣C to calf thymus DNA (Fig. 8, A and B). The presence of 1-naphthol, a substrate for multiple UGTs (18), in the incubation medium led to dose-dependent decreases in A␣C-HN 2 -O-Gl and A␣C-HON 2 -Gl and dG-C8-A␣C adduct formation. These findings show that UGTs catalyze the binding of HONH-A␣C to DNA.

A␣C-N 2 -Gl, A␣C-HON 2 -Gl, and A␣C-HN 2 -O-Gl Formation in Human
Hepatocytes-We have shown that A␣C undergoes extensive metabolism and forms DNA adducts at high levels in hepatocytes (29). In this study we have characterized several glucuronide conjugates of A␣C produced in hepatocytes. The product ion spectra of A␣C-HON 2 (Fig. 9, B and C). These findings show that O-glucuronidation is a principal pathway of conjugation of HONH-A␣C under low exposure conditions to A␣C (Fig. 9, A). We surmise that a portion of A␣C-HN 2 O-Gl reacts with DNA and possibly protein.

DISCUSSION
The N-glucuronidation of HAAs, aromatic amines, and their genotoxic N-hydroxylated metabolites by UGTs is an important mechanism of detoxication of these structurally related chemicals. The UGT enzyme pathways compete with P450 and phase II enzyme pathways, which bioactivate these procarcinogens (Scheme 1) (17, 19 -21, 23).
To date, the direct O-glucuronidation of carcinogenic N-hydroxy-HAAs or arylhydroxylamines by UGTs (14,17,20,47,48) has not been reported. The formation of A␣C-HN 2 -O-Gl by human UGTs is the first example of the occurrence of an O-glucuronide conjugate from this group of structurally related carcinogens. A␣C-HN 2 -O-Gl is a biologically reactive metabolite that binds to DNA (Fig. 7). Moreover, the binding of HONH-A␣C to DNA is catalyzed by UGTs present in human liver microsomes (Fig. 8).
The kinetic parameters of UGT enzymes were characterized to determine which isoforms catalyze this unique pathway of bioactivation of HONH-A␣C. UGT1A9 is the most catalytically active isoform in O-glucuronidation of HONH-A␣C (Table 2). UGT1A9 is also the principal UGT isoform involved in the O-linked glucuronidation of simple phenols (18) and a major isoform involved in the detoxication by N3-glucuronidation of HONH-PhIP, the genotoxic metabolite of the cooked meat carcinogen PhIP (21). UGT1A9 is present in human liver, colon, prostate, and breast among other tissues (16). Given the low K m (0.9 M) value of UGT1A9-mediated A␣C-HN 2 -O-Gl formation, we may expect that UGT1A9 catalyzes the O-glucurionidation of HONH-A␣C and its binding to DNA in vivo.
O-Glucuronide conjugates of several arylhydroxamic acids are produced by UGT enzymes (20,49), including the O-glucuronide conjugate of N-hydroxy-2-acetylaminofluorene, which was identified in urine of rats treated with 2-acetylaminofluorene (50). The metabolite was stable in urine, but it was labile in vitro under slightly alkaline pH. The investigators proposed that the N-acetyl group of the AAF moiety had migrated to a hydroxyl group of the glucuronic acid (20). The resulting O-glucuronide of 2-hydroxyamino-  fluorene underwent reaction with nucleophiles (Scheme 2). This metabolite may have contributed to 2-acetylaminofluorene-DNA adduct formation in vivo (13,20). However, the O-glucuronide conjugate of 2-hydroxyaminofluorene or O-glucuronide conjugates of other arylhydroxylamines have not been detected in vivo probably because the conjugates are unstable in aqueous solution and decompose within several minutes (48).
The stability of the O-glucuronide linkage of A␣C-HN 2 -O-Gl is considerably greater than that of the O-glucuronide conjugate of 2-hydroxyaminofluorene. The half-life of A␣C-HN 2 -O-Gl exceeds 6 h at pH 7.0, but A␣C-HN 2 -O-Gl undergoes a facile nucleophilic displacement reaction with dG to form the dG-C8-A␣C adduct. Electrophiles of intermediate reactivity have been viewed as the most genotoxic species because highly reactive electrophiles will react with weaker nucleophiles or undergo solvolysis with water before they can react with DNA (51). Thus, A␣C-HN-O-Gl is an ideal genotoxic electrophile that can react with DNA.
UGTs play several different roles in the metabolism of A␣C that impact its biological activity. N 2 -Glucuronidation of A␣C leads to the formation of a detoxicated product. However, HONH-A␣C can undergo either N 2 -or O-glucuronidation. The enzyme kinetic studies with recombinant human UGT isoforms and metabolism studies with human hepatocytes reveal that A␣C-HN-O-Gl is the predominant glucuronide conjugate of HONH-A␣C formed under low substrate concentrations ( Fig. 9 and supplemental Table 1S). Moreover, because of its long half-life, A␣C-HN-O-Gl is likely to be exported from liver to extrahepatic tissues, where it can react with DNA (Scheme 2). The role of UGTs in the genotoxicity of A␣C, as opposed to N-acetyltransferase and sulfotransferase enzymes that are normally associated with the bioactivation of HAAs (17), warrants further study.