Glucuronidation of benzidine and its metabolites by cDNA- expressed ...

Download PDF
7 downloads 0 Views 169KB Size Report
Comment
Carcinogenesis vol.20 no.10 pp.1963–1969, 1999. Glucuronidation of benzidine and its metabolites by cDNA- expressed human UDP-glucuronosyltransferases ...
Carcinogenesis vol.20 no.10 pp.1963–1969, 1999

Glucuronidation of benzidine and its metabolites by cDNAexpressed human UDP-glucuronosyltransferases and pH stability of glucuronides

Marco Ciotti1, Vijaya M.Lakshmi2,4, Nikhil Basu1, Bernard B.Davis2,4, Ida S.Owens1 and Terry V.Zenser2,3,4,5 1Heritable Disorders Branch, National Institutes of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA and 2VA Medical Center, 3Department of Biochemistry and 4Division of Geriatric Medicine, St Louis University School of Medicine, St Louis, MO 63125, USA 5To whom correspondence should be addressed at: VA Medical Center (11G-JB), St Louis, MO 63125-4199, USA Email: [email protected]

Although glucuronidation is considered a necessary step in aromatic amine-induced bladder cancer, the specific enzymes involved are not known. This study assessed the capacity of five different human recombinant UDP-glucuronosyltransferases expressed in COS-1 cells to glucuronidate benzidine, its metabolites and 4-aminobiphenyl. [14C]UDP-glucuronic acid was used as co-substrate. UGT1A1, UGT1A4 and UGT1A9 each metabolized all of the aromatic amines. UGT1A9 exhibited the highest relative rates of metabolism with preference for the two hydroxamic acids, N-hydroxy-Nacetylbenzidine and N-hydroxy-N,N9-diacetylbenzidine. UGT1A9 metabolized 4-aminobiphenyl ~50% faster than benzidine or N-acetylbenzidine. UGT1A4 N-glucuronidated N9-hydroxy- N-acetylbenzidine at the highest relative rate compared with the other transferases. UGT1A6 was effective in metabolizing only four of the eight aromatic amines tested. UGT1A1 demonstrated more extensive metabolism of the hydroxamic acid, N-hydroxy-N,N9diacetylbenzidine, and the ring oxidation product, 3-OHN,N9-diacetylbenzidine, than it did for the other six amines. UGT2B7 was the only product of the UGT2 gene family examined and it metabolized all the aromatic amines at similar low relative levels compared with a preferred substrate, 4-OH-estrone. The Km values for Nacetylbenzidine metabolism by UGT1A1 and UGT1A4 were 0.37 K 0.14 and 1.8 K 0.4 mM, respectively. The O-glucuronide of 3-OH-N,N9-diacetylbenzidine was not hydrolyzed during a 24 h 37°C incubation at either pH 5.5 or 7.4. Likewise, the O-glucuronide of 3-OH-benzidine was stable at pH 7.4, with 52% remaining at pH 5.5 after 24 h. These results suggest the following relative ranking of transferase metabolism: UGT1A9 > UGT1A4 >> UGT2B7 > UGT1A6 ≈ UGT1A1. The relative pH stability of O-glucuronides is consistent with a role in detoxification and excretion of aromatic amines, while the acid lability of N-glucuronides is consistent with delivery of these amines to the bladder epithelium for activation, resulting in DNA adducts which may lead to mutations.

Abbreviations: PBS, phosphate-buffered saline; UCE, UGT1 common end. Published by Oxford University Press

Introduction Aromatic amines were suspected of causing bladder cancer in workers in the dye industry over 100 years ago (1). Subsequent studies suggested that aromatic amines were also responsible for the high incidence of bladder cancer in cigarette smokers and in workers in the rubber and chemical industries (2,3). Workers exposed to high levels of benzidine, an aromatic amine, have as much as a 100-fold increased risk for bladder cancer (4). Bladder cancer represents ~7% of human malignancies and is the third most prevalent cancer type in men 60 years and older (5,6). The mechanism by which aromatic amines, including benzidine, initiate bladder cancer is thought to involve multiple pathways and organ systems (7,8). Peroxidation of aromatic amines results in their direct activation to form DNA adducts (8–10). N-Oxidation of aromatic amines generates N-OH products which, following O-acetylation, react to form DNA adducts (11–13). In contrast, ring oxidation products are considered inactive. Aromatic amines and their N- and ring oxidation products can be glucuronidated to facilitate excretion (14,15), N-glucuronides are acid labile (17). N-Glucuronides have been hypothesized to contribute to the carcinogenic process by undergoing hydrolysis to form the parent amines in acidic urine, by accumulation of the amines in bladder epithelium and by activation of amines to form DNA adducts. This hypothesis is supported with recent experiments evaluating benzidine and N-acetylbenzidine metabolism in workers exposed to benzidine: (i) at 37°C, the half-lives of benzidine and N-acetylbenzidine N-glucuronides at pH 7.4 are 104 and 150 min, respectively, which decreases to 4 and 5 min at pH 5.3 (18,19); (ii) in post-workshift urine, pH was inversely correlated with the proportions of benzidine and N-acetylbenzidine present as free compounds (20); (iii) when controlling for internal dose, individuals with urine pH , 6 had a 10-fold higher DNA adduct level, N9-(39-monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine, in their exfoliated bladder cells compared with subjects with urine pH . 7. These results support a role for N-glucuronides of aromatic amines in the carcinogenic process. Although benzidine is glucuronidated by human UGT1.4 (21), studies with human liver microsomes have suggested that more than one UDP-glucuronosyltransferase may be responsible for metabolizing benzidine and N-acetylbenzidine (22,23). UDP-glucuronosyltransferases are separated into two families, designated UGT1 and UGT2, on the basis of evolutionary divergence (24). High interindividual variations in specific transferases (25) and, thus, modulation of the carcinogenic response may be attributed to modification by drugs, diet, environmental chemicals and/or genetic factors (25–28). In general, the UGT1 rather than the UGT2 gene family has been suggested to contribute more to metabolism of aromatic amines (21,23). This study assesses the metabolism of benzidine and its metabolites by several cDNA-expressed human UDP-glucuronosyltransferases to determine their substrate 1963

M.Ciotti et al.

specificity. Kinetic parameters for N-glucuronidation of Nacetylbenzidine by selected transferases were determined along with the pH stability of two O-glucuronides. These experiments are aimed at increasing our understanding of the role of glucuronidation in benzidine-induced bladder cancer. Materials and methods Materials [14C]UDP-glucuronic acid (279 mCi/mmol), [35S]methionine (1175 Ci/mmol) and [3H]benzidine (180 mCi/mmol) were purchased from Amersham (Arlington Heights, IL), ICN Pharmaceuticals (Costa Mesa, CA) and Chemsyn (Lenexa, KS), respectively. N-Acetylbenzidine and [3H]N-acetylbenzidine were synthesized by acetylation of benzidine in glacial acetic acid (29); N9hydroxy-N-acetylbenzidine, N-hydroxy-N-acetylbenzidine, N-hydroxy-N,N9diacetylbenzidine, 3-OH-benzidine, 3-OH-N,N9-diacetylbenzidine were synthesized by Dr Shu Wen Li (Department of Biochemistry, St Louis University Medical School, St Louis, MO) and were .95% pure (13,17,30). Benzidine free base and hydrochloride salt, 4-aminobiphenyl, 4-OH-estrone, UDP-glucuronic acid, β-glucuronidase type VII-A from Escherichia coli and ascorbic acid were from Sigma Chemical Co. (St Louis, MO). The sources of molecular biologicals have been previously identified (26,31,32). Human recombinant UDP-glucuronosyltransferase cDNAs: isolation, sequencing, and construction of plasmids Isolation of UGT1A1, UGT1A4, UGT2B7 and UGT1A6 has been described (26,28,31). UGT1A9 (GenBank accession no. AF056188) has been isolated from a λZAP cDNA library, which was custom synthesized by Invitrogen (San Diego, CA) using mRNA isolated from a specimen of a normal human liver prepared for transplant surgery. UGT1A1, UGT1A4, UGT2B7 and UGT1A6 have been sequenced (26,28,31). Sequencing of the pSKUGT1A9 unit isolated from the λZAP library was carried out with T3, T7 and UGT1A9 specific primers. The specific sense primers were: 59-GCAGAGGTGAGTTGGCA-39, 59-GCAGGAGTTTGTTTAAA-39 and 59-CAAGGAGAGAGTACGGA-39. The specific antisense primer was: 59-TCCAAGCCATTCATTCA-39. Construction of the pSVL-based expression unit for UGT1A1, UGT1A4, UGT2B7 and UGT1A6 has been described (28,31). UGT1A9 was cloned in pSVL as follows. Both the UGT1A1 cDNA-based pSVL and cDNA-based pSK1 units were digested with XhoI and BstEII to generate a 1099 and a 1140 bp fragment, respectively. The XhoI–BstEII UGT1A9 fragment replaced the XhoI–BstEII UGT1A1 fragment in the XhoI–BstEII UGT1A1 cDNA-based pSVL. The XhoI and BstEII digested pSVL contained the 1099– 2100 bp of the common 39-end of the two cDNAs. COS-1 cells were plated in 100 mm dishes at 106 cells and grown to 90% confluency in 24 h in Dulbecco’s modified Eagle’s medium with the HEPES buffer and 4% fetal calf serum. pUGT1A1, pUGT1A4, pUGT1A6, pUGT1A9 and pUGT2B7 were transfected into cells using DEAE–dextran as the carrier (33). Assay for UDP-glucuronosyltransferase activities expression by cDNAs Transferase activity was measured using COS-1 cells transfected with either UGT1A1, UGT1A4, UGT1A6, UGT1A9 or UGT2B7. Cell homogenates [used after storage at –80°C in phosphate-buffered saline (PBS)] were spun at 10 000 r.p.m. in a microfuge for 10 min to remove storage buffer, resuspended in the reaction buffer containing MgCl2 and saccharic acid-1,4lactone and treated with CHAPS (0.7 mg/mg protein). The glucuronidation assay was conducted as previously described (28,32,33) and contained the following: 1.41 mM [14C]UDP-glucuronic acid (1.41 mCi/ mM), 0.2 mM substrate (except for N9-hydroxy-N-acetylbenzidine, which was 0.1 mM), 5.0 mM MgCl2, 16.6 mM saccharic acid-1,4-lactone and 33 mM triethanolamine, pH 7.6, in a total volume of 100 µl. Detergent-treated cell homogenate was added to the reaction tube followed by vitamin C (2 mM) and [14C]UDP-glucuronic acid. Two microliters of each substrate (10 mM) dissolved in methanol, with the exception of N9-OH-N-acetylbenzidine (5 mM), which was in DMF, were added to the 98 µl reaction mixture and incubated for 2 h at 37°C. Incubation conditions were optimized with respect to substrate concentration, protein and time for metabolism of N-acetylbenzidine by UGT1A1, UGT1A4 and UGT1A9 and for N-hydroxy-N,N9-diacetylbenzidine metabolism by UGT1A9. Glucuronides were identified by TLC using a basic solvent system (21) in which the N-glucuronides are stable (17). TLC plates were scanned and products quantitated with an Ambis Radioanalytical Imaging System II (26). Blank values were obtained by incubation of cell homogenates in the absence of aglycone and were subtracted from experimental values. The limit for detection was considered to be twice the blank value. All substrates were assessed with mock-transfected COS-1 cells, which exhibited no activity above the blank. Assays for all substrates were carried out on the same transfec-

1964

tion batch for each cDNA to eliminate problems with inter-transfection variability. For kinetic analysis, the concentration of N-acetylbenzidine substrate was varied from 0.05 to 0.8 mM, with higher concentrations prohibited by solubility. Reactions were linear during the 2 h incubation. Data were analyzed by Sigma Plot (SPSS Inc., Chicago, IL) and GraphPad Prism (GraphPad Software Inc., San Diego, CA). Deacetylation of [3H]N-acetylbenzidine was evaluated using the assay conditions described above. The conversion of N-acetylbenzidine to benzidine was assessed by HPLC (34) and was demonstrated with human liver microsomes. However, COS-1 cells and cDNA-transfected COS-1 cells did not demonstrate detectable deacetylation of N-acetylbenzidine. Activity of the different transferases was normalized as described below. Production of the UGT1 common end antibody and western blot analysis for normalization of isozyme levels In order to establish the relative amounts of each protein, an antibody preparation was elicited against the common 245 amino acid C-terminal peptide that is present in each isoform encoded by the UGT1 complex (35). The 865–1605 bp cDNA fragment of UGT1A1 (HUG-Brl) (31) was adapted and subcloned into the pGEX-2T vector to generate a GST–UGT common end fusion protein from amino acid 289 to 533. The fusion protein was synthesized in E.coli and purified by Veritas Inc. (Rockville, MD) as directed by the supplier (Amersham/Pharmacia Biotech, Piscataway, NJ). The purified peptide was used to immunize prebled rabbits. The UGT1 common end (UCE) antiserum was preabsorbed with normal bacterial extract before use. Each COS-1 cell system transfected with a specific cDNA was harvested after 72 h and pooled extracts for each expression unit were analyzed for specific protein amount using western blotting. In each case, 100 µg of cell homogenate was spun down, solubilized with the SDS sample buffer, applied to a 15% polyacrylamide–SDS gel and electrophoresed (26). The proteins were electrotransblotted onto a nitrocellulose membrane and blocked with reconstituted non-fat dried milk according to the ECL protocol (Amersham Life Science, Arlington Heights, IL). The membranes were exposed consecutively to a 1200 dilution of the UCE antibody for 1 h, five washes in PBS/0.1% Tween detergent for 10 min and to goat anti-rabbit IgG–HRP conjugate for 1 h. Proteins were visualized according to the ECL protocol and the fluorescent conjugate–immunoUGT complexes were exposed to X-ray film for between 3 and 10 s and developed. X-ray films with comparable exposures were scanned on the Umax Power Link and quantitated using Adobe Photoshop III software. The expression units for UGT1A1, UGT1A4, UGT1A6, UGT1A9 and UGT2B7 were each transfected into COS-1 cells and stored at –80°C for substrate determinations and western blot analysis. The relative amounts of UGT1A1, UGT1A4, UGT1A6, UGT1A9 and UGT2B7 were 1.04, 1.11, 1.0, 1.0 and 1.0, respectively. Transferase product was corrected for UGT1A1 and UGT1A4 for protein differences. Stability studies To determine the stability of 3-OH-benzidine and 3-OH-N,N9-diacetylbenzidine O-glucuronides, 14C-labeled glucuronides were prepared with hepatic microsomes (17). The O-glucuronide of 3-OH-N,N9-diacetylbenzidine was prepared with human hepatic microsomes (17) while 3-OH-benzidine O-glucuronide was prepared with microsomes from β-naphthoflavone-treated rats (13). Reaction mixtures were extracted with ethyl acetate to remove the unreacted substrate and then with n-butanol to remove the glucuronide. These partially purified 14C-labeled glucuronides were dissolved in phosphate buffer at pH 5.5 or 7.4 with 3 mM ascorbic acid and incubated at 37°C (17–19). Incubations were stopped by freezing the sample at –70°C and analyzed by HPLC. The percent of radioactivity remaining as glucuronide conjugate was plotted versus time and the t1 calculated. When the glucuronide was stable (t1 . 24 h), the 2 2 data were reported as percent remaining. Microsomal incubations yielded a single glucuronide product for each substrate which was identified by selective hydrolysis with human and E.coli β-glucuronidase (36) and UV detection of the aglycone by HPLC (17).

Results Five different human UDP-glucuronosyltransferase enzymes were assessed for ability to metabolize benzidine and its metabolites (Table I). 4-Aminobiphenyl, a structural analog of benzidine, was included as a substrate to further assess transferase substrate specificity. All of the aromatic amine substrates used in this study have been incubated with liver microsomes and shown to yield a single N- or O-glucuronide product that was rigorously characterized (17–19,36). The glucuronides made by these recombinant human enzymes were not similarly characterized. The highest relative rates of metabolism were

Benzidine glucuronidation

Table I. Glucuronidation of different benzidine analogs with human UDP-glucuronosyltransferases Substrate

UGT1A1

UGT1A4

UGT1A6

Benzidine N-Acetylbenzidine N9-Hydroxy-N-acetylbenzidine N-Hydroxy-N-acetylbenzidine N-Hydroxy-N,N9-diacetylbenzidine 3-OH-N,N9-Diacetylbenzidine 3-OH-Benzidine 4-Aminobiphenyl 4-t-butylphenol 4-OH-Estrone

18 6 2 21 6 5 11 6 1 11 6 1 87 6 1 90 6 5 29 6 2 22 6 1

1114 6 36 889 6 84 1417 6 6 997 6 42 240 6 24 200 6 9 144 6 9 628 6 60

220 NDa ND 400 280 290 ND ND 6867

UGT1A9

6 40

UGT2B7

1327 6 60 1193 6 13 633 6 47 9667 6 600 38 467 6 1270 2947 6 33 5133 6 213 2093 6 53

6 40 6 20 6 20 6 267

150 123 137 170 187 173 193 163

6 6 6 6 6 6 6 6

17 3 7 3 10 7 10 7

1057 6 127

Values are expressed as pmol/2 h/mg protein and represent means 6 SEM for triplicate determinations. Incubation conditions were optimized with respect to substrate concentration, protein and time for metabolism of N-acetylbenzidine metabolism by UGT1A1, UGT1A4 and UGT1A9 and for N-hydroxy-N,N9diacetylbenzidine metabolism by UGT1A9. aND, not detected.

observed with UGT1A9. This transferase metabolized all the aromatic amines with preference for the two hydroxamic acids, N-hydroxy-N-acetylbenzidine and N-hydroxy-N,N9-diacetylbenzidine. These hydroxamic acids exhibited metabolism that was 7- to 30-fold higher than that observed for benzidine or N-acetylbenzidine. For UGT1A4, all the aromatic amines were metabolized, with the primary aromatic amines being metabolized at a relatively higher rate. Compared with the relative metabolism of the other aromatic amines, UGT1A4 was ~2.3-fold better than UGT1A9 in metabolizing N9-hydroxy-Nacetylbenzidine. With UGT1A6, only four of the eight amines tested were metabolized. The extent of metabolism of these compounds was small compared with a preferred substrate, 4t-butylphenol (28). For UGT1A1, all the aromatic amines were metabolized without selection for primary amines. The hydroxamic acid, N-hydroxy-N,N9-diacetylbenzidine, and the ring oxidation product, 3-OH-N,N9-diacetylbenzidine, were metabolized at the highest rate. UGT2B7 was the only product of the UGT2 gene family tested. While UGT2B7 metabolized all the aromatic amines, the level of metabolism was only ~15% that observed for its preferred substrate, 4-OH-estrone. Kinetic parameters of N-acetylbenzidine metabolism were assessed for UGT1A1 and UGT1A4. Kinetic values for the two transferase enzymes were very different from each other. For UGT1A1, the apparent Km and Vmax were 0.37 6 0.14 mM and 0.63 6 0.03 nmol/2 h/mg protein, respectively. Higher Km (1.8 6 0.4 mM) and Vmax (13.5 6 1.6 nmol/2 h/mg protein) values were observed with UGT1A4. The pH stability of the O-glucuronides of 3-OH-benzidine and 3-OH-N,N9-diacetylbenzidine was determined and compared with previous values for glucuronides of benzidine and its metabolites (Table II). The O-glucuronide of 3-OH-N,N9diacetylbenzidine was very stable. After 24 h incubation at either pH 5.5 or 7.4, at least 97% of 3-OH-N,N9-diacetylbenzidine O-glucuronide remained intact. For the O-glucuronide of 3-OH-benzidine, 97% remained intact during a 24 h incubation at pH 7.4, while only 52% remained after 24 h at pH 5.5. Discussion These results provide an extensive comparison of the glucuronidation of benzidine and its analogs by five different human cDNA-expressed UDP-glucuronosyltransferases. UGT1A1, UGT1A9 and UGT1A4 each metabolized all eight of the amines examined. UGT1A9 exhibited preferential meta-

Table II. pH Stability of glucuronide conjugates of benzidine and its metabolites Compound

t1 2

3-OH-N,N9-Diacetylbenzidine pH 5.5 NDa pH 7.4 ND 3-OH-Benzidine pH 5.5 ND pH 7.4 ND Benzidine pH 5.3 5 minb pH 7.4 1.7 hb N-Acetylbenzidine pH 5.3 4 minb pH 5.5 7.5 minc pH 7.4 2.3 hb N9-OH-N-Acetylbenzidine pH 5.5 3.5 hc pH 6.5 14 hc pH 7.4 ND N-OH-N-Acetylbenzidine pH 5.5 1.8 hc pH 7.4 2.0 hc N-OH-N,N9-Diacetylbenzidine pH 5.5 ND pH 7.4 ND

Remaining after 24 h (%) 97 99 52 97 ND ND ND ND ND ND ND 75c ND ND 93c 90c

[14C]Glucuronide conjugates were incubated at 37°C with the indicated pH in phosphate buffer with 3 mM ascorbic acid and analyzed by HPLC. Data were plotted as percent remaining versus time and t1 estimated (n 5 3–6). 2 aNot determined. bValue from Babu et al. (19). cValue from Babu et al. (17).

bolism of the hydroxamic acids, N-hydroxy-N-acetylbenzidine and N-hydroxy-N,N9-diacetylbenzidine, while UGT1A1 demonstrated more metabolism of the hydroxamic acid, Nhydroxy-N,N9-diacetylbenzidine, and the ring oxidation product, 3-OH-N,N9-diacetylbenzidine, than the primary amines. For UGT1A4, a relatively high rate of N-glucuronidation was observed. This transferase metabolized N9-hydroxyN-acetylbenzidine at a higher relative rate than the other transferases examined. UGT1A6 was effective in metabolizing only four of the eight amines tested; metabolism of these aromatic amines was only ~5% of that observed for a preferred substrate, 4-t-butylphenol (28). Thus, while family 1 isozymes glucuronidate benzidine and its metabolites, a significant 1965

M.Ciotti et al.

degree of substrate specificity exists between the individual transferases, with some amines not metabolized. UGT2B7 was the only product of the UGT2 gene family examined and it metabolized all the aromatic amines at similar low relative levels compared with a preferred substrate, 4-OH-estrone. Studies with human liver microsomes have suggested that more than one transferase may be responsible for glucuronidation of benzidine and its metabolites (22,23). 4-Aminobiphenyl metabolism was also examined with each transferase. In general, the metabolism of this primary aromatic amine was similar to benzidine and N-acetylbenzidine. With UGT1A9, the metabolism of 4-aminobiphenyl was ~50% greater than either benzidine or N-acetylbenzidine. The relative overall transferase metabolism of these three structurally similar aromatic amines seems to be comparable. These results suggest the following relative ranking of transferase metabolism for all the aromatic amines examined: UGT1A9 . UGT1A4 .. UGT2B7 . UGT1A6 µ UGT1A1. Other studies have also assessed metabolism of these aromatic amines by specific human UDP-glucuronosyltransferases. UGT1A4 metabolized several aromatic amines, including benzidine and 4-aminobiphenyl (21,37). Kinetic analysis indicated that both amines have a similar Vmax, but that 4-aminobiphenyl has a much lower Km value than benzidine (37). Results from previous studies and the current study suggest that UGT1A4 is important in aromatic amine metabolism. UGT1A1 glucuronidation of several aromatic amines, including 4-aminobiphenyl, was not previously demonstrated (38). This is consistent with the low level of UGT1A1 glucuronidation observed with the different compounds tested in the current study. UGT1A6 metabolism of 4-aminobiphenyl and its N-OH metabolite was demonstrated, with significantly more N-glucuronidation observed for the N-OH metabolite than the primary amine (39). Metabolism of both compounds was at a low level compared with other substrates examined (39). UGT1A6 and UGT2B7 have been shown to glucuronidate the hydroxamic acid N-OH-2-acetylaminofluorene and ring oxidation products of 2-acetylaminofluorene (40). UGT2B7 preferred the hydroxamic acid to the ring oxidation products, while UGT1A6 preferred the 5-OH analog to the hydroxamic acid (40). Thus, previous studies assessing specific human transferase metabolism of aromatic amines have observed similar results to those reported in the current study. Kinetic analysis with N-acetylbenzidine demonstrated different apparent Km values for UGT1A1 and UGT1A4. The Km values for UGT1A1 and UGT1A4 were 0.37 6 0.14 and 1.8 6 0.4 mM, respectively. A previous study with human liver microsomes demonstrated high and low affinity Km values for N-acetylbenzidine of 0.36 6 0.02 and 1.07 6 0.12 mM (22). The Km data obtained with microsomal incubations are similar to those observed in the current study for UGT1A1 and UGT1A4. The enzymatic efficiency for glucuronidating N-acetylbenzidine (Vmax/Km) was 1.7 for UGT1A1 and 7.5 for UGT1A4. For UGT1A4, the Km value for benzidine is reported to be 0.46 mM and the efficiency is 97 (37). Preliminary results with UGT1A9 suggest that the Km for N-acetylbenzidine may be ,0.01 mM (not shown). The existence of low and high capacity transferases for N-glucuronidating benzidine and N-acetylbenzidine is consistent with the extensive glucuronidation of these compounds observed in human liver slice experiments and in urine from workers exposed to benzidine (20,22,23). Because the current hypothesis emphasizes glucuronidation 1966

of benzidine and its metabolites in liver, tissue expression of these transferases is important to consider. Recent studies, using RT–PCR, have examined expression of mRNAs encoding the isozymes of the UGT1A genes in human liver, biliary and gastric tissue (41,42). In liver, UGT1A1, UGT1A4, and UGT1A9 are expressed and are all down-regulated in malignant hepatocellular carcinoma and its premalignant precursor, hepatic adenoma, but not in benign focal nodular hyperplasia. While UGT1A6 is expressed in liver, it is not regulated in liver tumors (41). The mRNAs encoding UGT1A1, UGT1A4 and UGT1A6 are also expressed in biliary tissue, while only those encoding UGT1A6 are present in gastric tissue. Results in our laboratory indicate that UGT1A6 is low in liver when analyzed by conventional northern blot rather than RT–PCR (35). UGT1A9 has been shown by Burchell’s group to be expressed in both liver and kidney (43). Thus, all of the 1A family isozymes studied are expressed in liver. UGT2B7 has been shown to be expressed in human liver and kidney, but not lung, cortex or skin (26,44). This is the first study to assess the stability of O-glucuronides of ring oxidation products. 3-OH-benzidine O-glucuronide has been observed in urine from workers manufacturing benzidine (45). 3-OH-N,N9-diacetylbenzidine O-glucuronide is a product of rat liver metabolism (46), but has not been reported in human. While the O-glucuronide of 3-OH-N,N9-diacetylbenzidine is very stable and not acid labile, this was not the case for 3-OH-benzidine O-glucuronide (Table II). For the latter at pH 7.4 little hydrolysis was observed after 24 h. However, at pH 5.5 only 52% of 3-OH-benzidine O-glucuronide remained after 24 h. These results suggest that a free amino group, 3OH-benzidine, adjacent to an O-glucuronide may increase acid lability relative to an amide functional group, 3-OH-N,N9diacetylbenzidine. With a voiding frequency of 4–6 h, little hydrolysis of 3-OH-benzidine O-glucuronide would be expected at acidic pH. Thus, even though acid lability of 3-OHbenzidine O-glucuronide was detected, it appears sufficiently stable at pH 5.5 to be considered a detoxification product. Extensive data have now accumulated relative to the pH stability of the glucuronides of benzidine and its metabolites and are summarized in Table II. O-glucuronides of the hydroxamic acids, N-hydroxy-N-acetylbenzidine and Nhydroxy-N,N9-diacetylbenzidine, were not acid labile (17). In contrast, the N-glucuronides of benzidine, N-acetylbenzidine and N9-hydroxy-N-acetylbenzidine were acid labile, with the latter having a much shorter t12 than the former two Oglucuronides (17,19). Similar results have been observed for glucuronide conjugates of 4-aminobiphenyl and its metabolites (47). Thus, glucuronide conjugates of primary aromatic amines are much more likely to be hydrolyzed in acidic urine than their N-OH metabolites. In urine samples from workers in India manufacturing benzidine or benzidine-based dyes, postworkshift urine pH was inversely correlated with the proportions of benzidine and N-acetylbenzidine present as free (unconjugated) compounds (20). In addition, individuals with urine pH , 6 had 10-fold higher levels of an N-acetylbenzidinespecific adduct than subjects with pH . 7. This adduct has been shown to cause genotoxic lesions, resulting in mutations in various bacterial and mammalian test systems in vitro and in oncogenes of tumors (48–52), indicating its potential to participate in initiation of bladder tumors. Diet is an important determinant of urine pH (53). These results suggest that urine pH may be a risk factor in bladder cancer. Two of the eight aromatic amines selected for study, N-

Benzidine glucuronidation

Fig. 1. Model illustrating pathways of aromatic amine metabolism and their involvement in human bladder cancer. Aromatic amines (Ar-NH2), such as benzidine, N-acetylbenzidine or 4-aminobiphenyl, are metabolized by oxidation (O), N-glucuronidation using UDP-glucuronic acid (UDPGA) as co-substrate or N-acetylation using acetyl CoA (AcCoA) as co-substrate. Glucuronidation, acetylation and oxidation are competing pathways. Aromatic amines can undergo N- or ring oxidation. N-acetylated aromatic amines (Ar-NH-Ac) are not glucuronidated, but can be oxidized to hydroxamic acids (Ar-N-Ac-OH). Hydroxamic acids and ring oxidation products can be metabolized to O-glucuronides (Ar-N-Ac-OGl or GlO-Ar-NH2) which are relatively stable to acid hydrolysis. Hepatic O-glucuronidation results in detoxification and excretion. N-glucuronides of aromatic amines (Ar-NH-Gl) and N-hydroxy aromatic amines (Ar-N-OH-Gl) are acid labile and can be hydrolyzed by acidic urine to their corresponding arylamines which can then enter the bladder and undergo further metabolism by peroxidation and/or O-acetylation to form DNA adducts. These adducts may initiate carcinogenesis by producing mutations which become fixed in the genome and eventually contribute to tumor formation.

hydroxy-N-acetylbenzidine (a hydroxamic acid) and 3-OHbenzidine, appear capable of forming multiple glucuronidation products. Extensive characterization of each reaction indicated that only a single product was formed (17–19,36). In addition, the stability of these glucuronides reflects their structural assignment (Table II), i.e. if N-hydroxy-N-acetylbenzidine and 3-OH-benzidine formed N- rather than O-glucuronides, their stability would be expected to be measured in minutes rather than hours. The results can be summarized in an overall model for aromatic amine metabolism and initiation of bladder carcinogenesis (Figure 1). According to this model, aromatic amine bladder carcinogens (i.e. benzidine, N-acetylbenzidine and 4aminobiphenyl) undergo a combination of hepatic oxidation, glucuronidation (i.e. UGT1A9 and UGT1A4) and/or acetylation. Aromatic amines can undergo N- or ring oxidation. N-acetylated aromatic amines are not glucuronidated, but can be oxidized to hydroxamic acids. Hydroxamic acids and ring oxidation products can be metabolized to O-glucuronides, which are relatively stable to acid hydrolysis.

Hepatic O-glucuronidation results in detoxification and excretion. N-glucuronides are acid labile and are converted to their carcinogenic aromatic amines in acidic urine. These amines are further activated in the epithelium (i.e. prostaglandin H synthase by a peroxygenation reaction involving N9-hydroxyN-acetylbenzidine; 54) to form DNA adducts [i.e. N9-(39monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine; 8,55]. These adducts may initiate carcinogenesis by producing mutations which become fixed in the genome and eventually contribute to tumor formation. In vivo support for this hypothesis is provided by a study assessing the influence of urine pH on metabolites from workers exposed to benzidine (20). Acknowledgements This work was supported by the Department of Veterans Affairs (T.V.Z.) and National Cancer Institute grant CA-72613 (T.V.Z.).

References 1. Rehn,L. (1895) Blasengeschwulste bei fuchsinarbeitern. Arch. Klin. Chir., 50, 588–600.

1967

M.Ciotti et al. 2. Case,R.A.M., Hosker,M.W., McDonald,D.B. and Pearson,J.T. (1954) Tumors of the urinary bladder in workmen engaged in the manufacture and use of certain dyestuff intermediates in the British chemical industry. Br. J. Ind. Med., 11, 75–104. 3. Doll,R. and Peto,R. (1981) The Causes of Cancer. Oxford University Press, New York, NY. 4. Bi,W., Hayes,R.B., Feng,P., Qi,Y., You,X., Zhen,J., Zhang,M., Qu,B., Fu,Z., Chen,M., Co Chien,H.T. and Blot,W.J. (1992) Mortality and incidence of bladder cancer in benzidine-exposed workers in China. Am. J. Ind. Med., 21, 481–489. 5. Silverberg,E. and Lubera,J. (1987) Cancer statistics, 1987. CA Cancer J. Clin., 37, 2–19. 6. Parker,S.L., Tong,T., Bolden,S. and Wingo,P. (1996) Cancer statistics, 1996. CA Cancer J. Clin., 46, 5–27. 7. Kadlubar,F.F., Miller,J.A. and Miller,E.C. (1977) Hepatic microsomal Nglucuronidation and nucleic acid binding of N-hydroxy arylamines in relation to urinary bladder carcinogenesis. Cancer Res., 37, 805–814. 8. Lakshmi,V.M., Zenser,T.V. and Davis,B.B. (1998) N9-(39-monophosphodeoxyguanosin-8-yl)-N-acetylbenzidine formation by peroxidative metabolism. Carcinogenesis, 19, 911–917. 9. Zenser,T.V., Mattammal,M.B., Armbrecht,H.J. and Davis,B.B. (1980) Benzidine binding to nucleic acids mediated by the peroxidative activity of prostaglandin endoperoxide synthetase. Cancer Res., 40, 2839–2845. 10. Zenser,T.V., Mattammal,M.B., Wise,R.W., Rice,J.R. and Davis,B.B. (1983) Prostaglandin H synthase-catalyzed activation of benzidine: a model to assess pharmacologic intervention of the initiation of chemical carcinogenesis. J. Pharmacol. Exp. Ther., 227, 545–550. 11. Miller,J.A. and Miller,E.C. (1977) The concept of reactive electrophilic metabolites in chemical carcinogenesis: recent results with aromatic amines, safrole, and aflatoxin B1. In Jollow,D.J., Kocsis,J.J., Snyder,R. and Vainio,H. (eds) Biological Reactive Intermediates. Plenum Publishing, New York, NY, pp. 6–24. 12. Frederick,C.B., Weis,C.C., Flammang,T.J., Martin,C.N. and Kadlubar,F.F. (1985) Hepatic N-oxidation, acetyl-transfer and DNA-binding of the acetylated metabolites of the carcinogen benzidine. Carcinogenesis, 6, 959–965. 13. Lakshmi,V.M., Zenser,T.V. and Davis,B.B. (1997) Rat liver cytochrome P450 metabolism of N-acetylbenzidine and N,N9-diacetylbenzidine. Drug Metab. Dispos., 25, 481–488. 14. Dutton,G.J. (1980) Glucuronidation of Drugs and Other Compounds. CRC Press, Boca Raton, FL. 15. Mulder,G.J., Coughtrie,M.W.H. and Burchell,B. (1990) Glucuronidation. In Mulder,G.J. (ed.) Conjugation Reactions in Drug Metabolism: An Integrated Approach. Taylor & Francis, New York, NY, pp. 51–105. 16. Irving,C.C. (1977) Influence of the aryl group on the reaction of glucuronides of N-arylacethydroxamic acids with polynucleotides. Cancer Res., 37, 524–528. 17. Babu,S.R., Lakshmi,V.M., Hsu,F.F., Zenser,T.V. and Davis,B.B. (1995) Glucuronidation of N-hydroxy metabolites of N-acetylbenzidine. Carcinogenesis, 16, 3069–3074. 18. Babu,S.R., Lakshmi,V.M., Hsu,F.F., Zenser,T.V. and Davis,B.B. (1992) Role of N-glucuronidation in benzidine-induced bladder cancer in dog. Carcinogenesis, 13, 1235–1240. 19. Babu,S.R., Lakshmi,V.M., Hsu,F.F., Kane,K.E., Zenser,T.V. and Davis,B.B. (1993) N-Acetylbenzidine-N9-glucuronidation by human, dog, and rat liver. Carcinogenesis, 14, 2605–2611. 20. Rothman,N., Talaska,G., Hayes,R. et al. (1997) Acidic urine pH is associated with elevated levels of free urinary benzidine and Nacetylbenzidine and urothelial cell DNA adducts in exposed workers. Cancer Epidemiol. Biomarkers Prevent., 6, 1039–1042. 21. Green,M.D., Bishop,W.P. and Tephly,T.R. (1995) Expressed human UGT1.4 protein catalyzes the formation of quaternary ammonium-linked glucuronides. Drug Metab. Dispos., 23, 299–302. 22. Babu,S.R., Lakshmi,V.M., Zenser,T.V. and Davis,B.B. (1994) Glucuronidation of N-acetylbenzidine by human liver. Drug Metab. Dispos., 22, 922–927. 23. Babu,S.R., Lakshmi,V.M., Owens,I.S., Zenser,T.V. and Davis,B.B. (1994) Human liver glucuronidation of benzidine. Carcinogenesis, 15, 2003–2007. 24. Burchell,B., Nebert,D.W., Nelson,D.R. et al. (1991) The UDPglucuronosyltransferase gene superfamily: suggested nomenclature based on evolutionary divergence. DNA Cell Biol., 10, 487–494. 25. Munzel,P.A., Bookjans,G., Mehner,G., Lehmkoster,T. and Bock,K.W. (1996) Tissue-specific 2,3,7,8-tetrachlorodibenzo-p-dioxin-inducible expression of human UDP-glucuronosyltransferase UGT1A6. Arch. Biochem. Biophys., 335, 205–210. 26. Ritter,J.K., Sheen,Y.Y. and Owens,I.S. (1990) Cloning and expression of

1968

human liver UDP-glucuronosyltransferase in COS-1 cells. J. Biol. Chem., 265, 7900–7906. 27. Jin,C., Miners,J.O., Lillywhite,K.J. and Mackenzie,P.I. (1993) Complementary deoxyribonucleic acid cloning and expression of a human liver uridine diphosphate-glucuronosyltransferase glucuronidating carboxylic acid-containing drugs. J. Pharmacol. Exp. Ther., 264, 475–479. 28. Ciotti,M., Marrone,A., Potter,C. and Owens,I.S. (1997) Genetic polymorphism in the human UGT1A6 (planar phenol) UDPglucuronosyltransferase: pharmacological implications. Pharmacogenetics, 7, 485–495. 29. Lakshmi,V.M., Mattammal,M.B., Spry,L.A., Kadlubar,F.F., Zenser,T.V. and Davis,B.B. (1990) Metabolism and disposition of benzidine in the dog. Carcinogenesis, 11, 139–144. 30. Lakshmi,V.M., Zenser,N.T., Hsu,F.F., Mattammal,M.B., Zenser,T.V. and Davis,B.B. (1996) NADPH-dependent oxidation of benzidine by rat liver. Carcinogenesis, 17, 1941–1947. 31. Ritter,J.K., Crawford,J.M. and Owens,I.S. (1991) Cloning of two human liver bilirubin UDP-glucuronosyltransferase cDNAs with expression in COS-1 cells. J. Biol. Chem., 266, 1043–1047. 32. Ciotti,M. and Owens,I.S. (1996) Evidence for overlapping active sites for 17α-ethynylestradiol and bilirubin in the human major bilirubin UDPglucuronosyltransferase. Biochemistry, 35, 10119–10124. 33. Ritter,J.K., Yeatman,M.T., Kaiser,C., Gridelli,B. and Owens,I.S. (1993) A phenylalanine codon deletion at the UGT1 gene complex locus of a Crigler–Najjar Type 1 patient generates a pH-sensitive bilirubin UDPglucuronosyltransferase. J. Biol. Chem., 268, 23573–23579. 34. Lakshmi,V.M., Bell,D.A., Watson,M.A., Zenser,T.V. and Davis,B.B. (1995) N-Acetylbenzidine and N,N9-diacetylbenzidine formation by rat and human liver slices exposed to benzidine. Carcinogenesis, 16, 1565–1571. 35. Ritter,J.K., Chen,F., Sheen,Y.Y., Tran,H.M., Kimura,S., Yeatman,M.T. and Owens,I.S. (1992) A novel complex locus UGT1 encodes human bilirubin, phenol, and other UDP-glucuronosyltransferase isozymes with identical carboxyl termini. J. Biol. Chem., 267, 3257–3261. 36. Zenser,T., Lakshmi,V. and Davis,B. (1999) Human and E.coli βglucuronidase hydrolysis of glucuronide conjugates of benzidine and 4aminobiphenyl, and their hydroxy metabolites. Drug Metab. Dispos., 27, in press. 37. Green,M.D. and Tephly,T.R. (1996) Glucuronidation of amines and hydroxylated xenobiotics and endobiotics catalyzed by expressed human UGT1.4 protein. Drug Metab. Dispos., 24, 356–363. 38. King,C.D., Green,M.D., Rios,G.R., Coffman,B.L., Owens,I.S., Bishop,W.P. and Tephly,T.R. (1996) The glucuronidation of exogenous and endogenous compounds by stably expressed rat and human UDPglucuronosyltransferase 1.1. Arch. Biochem. Biophys., 332, 92–100. 39. Orzechowski,A., Schrenk,D., Bock-Hennig,B.S. and Bock,K.W. (1994) Glucuronidation of carcinogenic arylamines and their N-hydroxy derivatives by rat and human phenol UDP-glucuronosyltransferases of the UGT1 gene complex. Carcinogenesis, 15, 1549–1553. 40. Jin,C.-J., Miners,J.O., Burchell,B. and Mackenzie,P.I. (1993) The glucuronidation of hydroxylated metabolites of benzo[a]pyrene and 2acetylaminofluorene by cDNA-expressed human UDP-glucuronosyltransferases. Carcinogenesis, 14, 2637–2639. 41. Strassburg,C.P., Manns,M.P. and Tukey,R.H. (1997) Differential downregulation of the UDP-glucuronosyltransferase 1A locus is an early event in human liver and biliary cancer. Cancer Res., 57, 2979–2985. 42. Strassburg,C.P., Oldhafer,K., Manns,M.P. and Tukey,R.H. (1997) Differential expression of the UGT1A locus in human liver, biliary, and gastric tissue: identification of UGT1A7 and UGT1A10 transcripts in extrahepatic tissue. Mol. Pharmacol., 52, 212–220. 43. McGurk,K.A., Brierley,C.H. and Burchell,B. (1998) Drug glucuronidation by human renal UDP-glucuronosyltransferases. Biochem. Pharmacol., 55, 1005–1012. 44. Ritter,J.K., Chen,F., Sheen,Y.Y., Lubet,R.A. and Owens,I.S. (1992) Two human liver cDNAs encode UDP-glucuronosyltransferases with two log differences in activity toward parallel substrates including hyodeoxycholic acid and certain estrogen derivatives. Biochemistry, 31, 3409–3414. 45. Sciarini,L.J. and Meigs,J.W. (1961) The biotransformation of benzidine. II. Studies in mouse and man. Arch. Environ. Health, 2, 423–428. 46. Lynn,R.K., Garvie-Gould,C., Milam,D.F., Scott,K.F., Eastman,C.L. and Rodgers,R.M. (1983) Metabolism of the human carcinogen, benzidine, in the isolated perfused rat liver. Drug Metab. Dispos., 11, 109–114. 47. Babu,S.R., Lakshmi,V.M., Huang,G.P.-W., Zenser,T.V. and Davis,B.B. (1996) Glucuronide conjugates of 4-aminobiphenyl and its N-hydroxy metabolites: pH stability and synthesis by human and dog liver. Biochem. Pharmacol., 51, 1679–1687. 48. Beland,F.A., Beranek,D.T., Dooley,K.L., Heflich,R.H. and Kadlubar,F.F. (1983) Arylamine–DNA adducts in vitro and in vivo: their role in bacterial

Benzidine glucuronidation mutagenesis and urinary bladder carcinogenesis. Environ. Health Perspect., 49, 125–134. 49. Melchior,W.B.Jr, Marques,M.M. and Beland,F.A. (1994) Mutations induced by aromatic amine DNA adducts in pBR322. Carcinogenesis, 15, 889–899. 50. Heflich,R.H., Morris,S.M., Beranek,D.T., McGarrity,L.J., Chen,J.J. and Beland,F.A. (1986) Relationships between the DNA adducts and the mutations and sister-chromatid exchanges produced in Chinese hamster ovary cells by N-hydroxy-2-aminofluorene, N-hydroxy-N9-acetylbenzidine and 1-nitrosopyrene. Mutagenesis, 1, 201–206. 51. Fox,T.R., Schumann,A.M., Watanabe,P.G., Yano,B.L., Maher,V.M. and McCormick,J.J. (1990) Mutational analysis of the H-ras oncogene in spontaneous C57BL/63C3H/He mouse liver tumors and tumors induced with genotoxic and nongenotoxic hepatocarcinogens. Cancer Res., 50, 4014–4019. 52. Talaska,G., Au,W.W., Ward,J.B.Jr, Randerath,K. and Legator,M.S. (1987)

The correlation between DNA adducts and chromosomal aberrations in the target organ of benzidine exposed, partially-hepatectomized mice. Carcinogenesis, 8, 1899–1905. 53. Remer,T. and Manz,F. (1995) Potential renal acid load of foods and its influence on urine pH. J. Am. Diet. Assoc., 95, 791–797. 54. Zenser,T.V., Lakshmi,V.M., Hsu,F.F. and Davis,B.B. (1999) Peroxygenase metabolism of N-acetylbenzidine by prostaglandin H synthase. J. Biol. Chem., 274, 14850–14856. 55. Rothman,N., Bhatnagar,V.K., Hayes,R.B. et al. (1996) The impact of interindividual variation in NAT2 activity on benzidine urinary metabolites and urothelial DNA adducts in exposed workers. Proc. Natl Acad. Sci. USA, 93, 5084–5089. Received January 12, 1999; revised June 8, 1999; accepted June 25, 1999

1969