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N'-(3'-Monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine (dGp-ABZ) is thought to play an important role in initiation of benzidine-induced bladder cancer in humans. This report assesses the possible formation of this adduct by peroxidatic activation of N-acetylbenzidine (ABZ). Adduct formation was measured by 32P-post-labeling. Ram seminal vesicle microsomes were used as a source of prostaglandin H synthase (PHS). The peroxidatic activity of PHS was compared with that for horseradish peroxidase. Both peroxidases converted ABZ to dGp-ABZ whether DNA or 2'-deoxyguanosine 3'-monophosphate (dGp) was present. Following 32P-post-labeling, the enzymatic and synthetic adduct were extracted from PEI-cellulose plates and were shown to have the same HPLC elution profiles for the bisphosphate adduct (32P-dpGp-ABZ). Treatment of the enzymatic and synthetic bisphosphate adduct with nuclease P1 yielded a product that eluted at the same time from HPLC (32P-dpG-ABZ). Additional experiments demonstrated that the PHS-derived 5'-monophosphate (dpG-ABZ) and 3'-monophosphate (dGp-ABZ) adducts were also identical to their corresponding synthetic standard. With comparable amounts of total ABZ metabolism, PHS produced 40-fold more dGp-ABZ than horseradish peroxidase (1943 ± 339 versus 49 ± 7.8 fmol/mg dGp). Adduct formation was dependent upon the presence of peroxidase and the specific substrate, i.e. arachidonic acid or H2O2. Adduct formation by PHS was inhibited by indomethacin (0.1 mM), ascorbic acid (1 mM) and glutathione (10 mM), but not by 5,5-dimethyl-1-pyrroline N-oxide (DMPO) (100 mM), a radical scavenger. Horseradish peroxidase adduct formation was also inhibited by ascorbic acid and glutathione. In addition, DMPO elicited greater than a 96% inhibition. Results demonstrate peroxidatic metabolism of ABZ to form dGp-ABZ. The mechanism of dGp-ABZ formation by PHS and horseradish peroxidase may be different. [ABSTRACT FROM PUBLISHER]

Individuals with ulcerative colitis face an increased risk of developing colorectal cancer and would benefit from early chemopreventive intervention. Results from preclinical studies in the mouse model of dextran sulfate sodium-induced colitis demonstrate that flat and polypoid colitis-associated dysplasias arise via distinct genetic pathways, impacted by the allelic status of p53. Furthermore, flat and polypoid dysplasias vary in their response to induction by the heterocyclic amine 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and inhibition by 5-aminosalicylic acid, a common therapy for the maintenance of colitis patients. These data suggest that use of combination therapy is essential for the optimal inhibition of colitis-associated colorectal cancer., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

In studying the metabolic pathways underlying the mechanism of carcinogenesis of the heterocyclic amine of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), we recently found a new metabolite which gave an [M + H](+) ion of m/z 217 when subjected to electrospray ionization (ESI) in positive-ion mode. Following i.p. injection of this metabolite of m/z 217 (designated as m/z 217) to beta-naphthoflavone-treated mice, 57% of the total radioactivity was recovered in a 24-h mouse urine sample. HPLC separation followed by MS analysis indicates that the urine sample contained m/z 217 (36 +/- 3% of total recovered radioactivity) and two other peaks that gave rise to the [M + H](+) ions of m/z 393 (31 +/- 4%, designated as m/z 393) and m/z 233 (14 +/- 1%, designated as m/z 233). Beta-glucuronidase treatment of m/z 393 resulted in a radioactive peak corresponding to m/z 217. ESI in combination with various mass spectrometry techniques, including multiple-stage mass spectrometry, exact mass measurements and H/D exchange followed by tandem mass spectrometry, was used for structural characterization. The urinary metabolites of m/z 217, 393 and 233 were identified as 1,2-dihydro-2-amino-5-hydroxy-3-methylimidazo[4,5-f]quinoline, 1,2-dihydro-2-amino-5-O-glucuronide-3-methylimidazo[4,5-f]quinoline and 1,2-dihydro-2-amino-5,7-dihydroxy-3-methylimidazo[4,5-f]quinoline, respectively. Our results demonstrated that m/z 217 is biotransformed in vivo to m/z 393 by O-glucuronidation and to m/z 233 by oxidation. The observation of these more polar metabolites relative to IQ suggests that they may arise from a previously undescribed detoxification pathway., (2009 John Wiley & Sons, Ltd.)

Metabolism of the heterocyclic amine carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) was evaluated in mice with and without 40 mg/kg beta-naphthoflavone (BNF). Following an oral dose of 40 mg/kg (14)C-IQ, a 24-h urine sample was collected. Metabolism was assessed by high-performance liquid chromatography, and metabolites were identified by electrospray ionization mass spectrometry. Three new metabolites were identified as 1,2-dihydro-2-amino-5-hydroxy-3-methylimidazo[4,5-f]quinoline (m/z 217, [M + H](+)), 1,2-dihydro-2-amino-5-O-glucuronide-3-methylimidazo[4,5-f]quinoline (m/z 393, [M + H](+)), and 1,2-dihydro-2-amino-5,7-dihydroxy-3-methylimidazo[4,5-f]quinoline (m/z 233, [M + H](+)). These metabolites represented 21% of the total urinary radioactivity recovered. For BNF-treated mice, the abundance of metabolites observed was 5-O-glucuronide > m/z 217 > m/z 393 > 5-sulfate > m/z 233 > N-glucuronide > demethyl-IQ > sulfamate. In control mice, metabolite urinary abundance was 5-O-glucuronide > demethyl-IQ > sulfamate > N-glucuronide > m/z 217 > 5-sulfate. In liver slices from BNF-treated mice, synthesis of m/z 217 and 5-O-glucuronide was significantly reduced by ellipticine, a cytochrome P450 (P450) inhibitor, whereas sulfamate synthesis was significantly increased and demethyl-IQ was unchanged. Liver microsomes from BNF-treated mice produced m/z 217 and demethyl-IQ, with the former inhibited by ellipticine and furafylline, a selective 1A2 inhibitor, and the latter by ellipticine only. Injection (intraperitoneal) of demethyl-IQ into BNF-treated mice resulted in only a 30% conversion to three metabolites that were not observed in urine from animals receiving IQ. Results from BNF-treated mice showed significant IQ metabolism by hepatic P450s. Therefore, differences in metabolism between mice treated with and without BNF may affect IQ tumorigenicity.

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline(MeIQx) are heterocyclic amines (HCAs) derived from high temperature cooking of meat and thought to cause colon cancer in humans. Reactive nitrogen oxygen species, which are mediators of the inflammatory response, can convert these amines to the corresponding N-nitrosamines, N-NO-IQ and N-NO-MeIQx. This study was designed to evaluate whether these N-nitrosamines are genotoxic and could be responsible, in part, for the high incidence of colon cancer in individuals with colitis. Such an association would counsel reduced intake of well-done red meat by colitis patients. Mutagenicity was evaluated by reversion of a lacZ frameshift allele in three different E. coli strains. Strains DJ701 and DJ702 express recombinant(S. typhimurium) aromatic amine N-acetyltransferase (NAT); DJ702 also expresses recombinant human cytochrome P450 1A2 and NADPH-P450 reductase; and DJ2002 served as an N-acetyltransferase negative control. In strain DJ701, N-NO-IQ and N-NO-MeIQx elicited dose-dependent mutagenicity,which was not further increased in DJ702. Neither nitrosamine was mutagenic in strain DJ2002. While both N-nitrosamines are stable for >4 h (pH 7.4, 37 degrees C), they react with DNA or 2'-deoxyguanosine 3'-monophosphate at lower pH (5.5) to form adducts. HOCl, a component of the inflammatory response,increased adduct formation, as measured by 32P-postlabeling. Following treatment with nuclease P1and separation by two-dimensional thin-layer chromatography and then HPLC, N-NO-IQ and N-NOMeIQxwere shown to form the same adducts as those formed by N-OH-MeIQx or N-OH-IQ, namely N-(deoxyguanosin-8-yl) adducts. In summary, these N-nitrosamines are genotoxic and might be alternatives to their hydroxylamine analogues as activated intermediates leading to initiation of colon cancer in individuals with colitis.

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) metabolism was evaluated in mouse to better understand its tumorigenicity. Urinary metabolites from mice orally administered 40 mg/kg [(14)C]IQ were compared with those from similarly treated rats. The recovery of radioactivity was significantly greater in mouse urine. The relative proportion of metabolites was significantly different, and a new rodent metabolite was detected. For rat, the proportion of previously identified metabolites excreted was 5-O-glucuronide > sulfamate > 5-sulfate > N-glucuronide. In mouse urine, a new metabolite, demethyl-IQ, represented approximately 26% of IQ metabolism with the proportion of metabolites as follows: 5-O-glucuronide > demethyl-IQ > sulfamate > N-glucuronide > 5-sulfate. Mouse metabolites were identified by electrospray ionization mass spectrometry. Demethyl-IQ was shown to be 2-aminoimidazo[4,5-f]quinoline. N-Acetyl-2-amino-3-methylimidazo[4,5-f]quinoline was not detected with mice. Mouse liver slices produced 5-O-glucuronide, demethyl-IQ, and sulfamate with the former two being significantly reduced by ellipticine. Liver microsomes only produced demethyl-IQ. Ellipticine, a cytochrome P450 1A inhibitor, but not furafylline, an 1A2 selective inhibitor, prevented microsomal N-demethylation. Inhibitors had similar effects on 7-ethoxyresorufin O-deethylation activity. Demethyl-IQ was not further metabolized by an intact mouse or liver microsomes. Thus, mouse IQ metabolism is significantly different from that in rat, and these differences may affect IQ tumorigenicity. N-Demethylation of IQ-like heterocyclic amines occurs in mouse, monkey, and human but not in rat.

The incidence of colon cancer increases with age, and this may be related to altered metabolism and disposition of carcinogens. One such carcinogen implicated in colon cancer is the heterocyclic amine found in well done meat, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). The purpose of these studies was to determine whether the disposition and metabolism of IQ changes with age, comparing young (3-month) and old (22- to 24-month) male F344 rats. Animals were treated with vehicle or beta-naphthoflavone (BNF), an inducer of drug-metabolizing cytochromes P450. Disposition and metabolism of IQ were determined after i.p. injection of radiolabeled IQ. Urinary IQ metabolites were identified and quantitated by high-performance liquid chromatography and mass spectroscopy. In BNF-treated animals, total radiolabeled IQ excretion by old rats was less than half that of young rats. Binding of radiolabeled IQ metabolites by the old kidney was 10 times higher than that of the young. There were no age differences in intestinal and hepatic binding. There was a significant age-related increase in IQ conjugation to glucuronic acid and a decrease in conjugation to sulfate regardless of treatment. The induction of renal CYP1A1, a major P450 involved in IQ metabolism, by BNF did not change with age. Changes in IQ metabolism with age along with altered renal function may contribute to the decreased urinary excretion and increased renal binding of IQ and/or its metabolites seen in the old animals.

Although nitrosation plays an important role in initiation of carcinogenesis, the reactive nitrogen oxygen species (RNOS) mediating this reaction by multiple pathways have not been determined. The heterocyclic amine carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) was used as a target to investigate RNOS and pathways for potentiation of nitric oxide (NO)-mediated nitrosation. 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO) oxidizes NO to NO(2)(.) and was used as a tool to investigate NO(2)(.) potentiation of nitrosation. The IQ nitrosation product, 2-nitrosoamino-3-methylimidazo[4,5-f]quinoline ((14)C-N-NO-IQ), was monitored by HPLC. Autoxidation of NO, generated by spermine NONOate (2.4 microM NO/min) for 7.5 min, did not convert 10 microM (14)C-IQ to N-NO-IQ. However, the presence of 15 muM CPTIO resulted in 3 microM N-NO-IQ formation. Potentiation by CPTIO occurred at low and high fluxes of NO, 0.075 to 1.2 microM/min, and over a range of IQ to CPTIO ratios of 0.5 to 10. A significant portion of N-NO-IQ formation was insensitive to azide (10 mM) inhibition, suggesting oxidative nitrosylation. NADH (0.02 mM) did not alter nitrosation by autoxidation, but effectively inhibited potentiation by CPTIO. Ascorbic acid (0.2 mM) and 5,5-dimethyl-1-pyrroline N-oxide (30 mM) inhibited nitrosation with or without CPTIO, while superoxide dismutase was not inhibitory. The RNOS produced by CPTIO had a 27-fold greater affinity for IQ than those produced by autoxidation. Results are consistent with NO(2)(.) or a RNOS like NO(2)(.) potentiating IQ oxidative nitrosylation. Nitrosation occurring at both low and high fluxes of NO can contribute to carcinogenesis.

2-Nitrosoamino-3,8-dimethylimidazo[4,5-f]quinoxaline (N-NO-MeIQx) is a nitrosation product of the food carcinogen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and is proposed to form in vivo under inflammatory conditions. This study evaluated the stability and reactivity of N-NO-MeIQx to assess its possible role in the initiation of colon cancer by MeIQx. 14C-N-NO-MeIQx (4 microM) was incubated for 4 h over a range of pH values, and its stability was monitored by HPLC. At pH values from pH 7.4 to 9.0, N-NO-MeIQx was very stable with no detectable change observed. Glutathione (1 mM) did not alter stability at pH 7.4. As the pH decreased, this nitrosamine was less stable with only 48 +/- 1% remaining at pH 5.5 and none remaining at pH 3.5 or 2.0. Major products identified by electrospray ionization mass spectrometry were 3,8-dimethylimidazo[4,5-f]quinoxaline and 2-hydroxy-3,8-dimethylimidazo[4,5-f]quinoxaline. MeIQx was a minor product. At pH 2.0, the t(1/2) for N-NO-MeIQx was reduced from 2.1 +/- 0.2 to 1.2 +/- 0.1 min with 10 mM NaN3. This effect of azide was due to the formation of 2-azido-MeIQx. The binding of 14C-N-NO-MeIQx to DNA increased with decreasing pH. The 10-fold increase in binding observed at pH 2.0 as compared to pH 5.5 was completely inhibited by 10 mM NaN3 due to 2-azido-MeIQx formation. The reactivity of N-NO-MeIQx was compared to N-OH-MeIQx by evaluating adduct formation with 2'-deoxyguanosine 3'-monophosphate (dGp) by 32P-postlabeling. N-OH-MeIQx formed a single major adduct, N-(deoxyguanosin-8-yl)-MeIQx (dG-C8-MeIQx). Incubation of N-NO-MeIQx under inflammatory conditions (pH 5.5 +/- HOCl) produced dG-C8-MeIQx along with 4-6 other adducts. dG-C8-MeIQx formation increased in the presence of HOCl. Liver from a MeIQx-treated mouse contained dG-C8-MeIQx and two other adducts detected with N-NO-MeIQx but not N-OH-MeIQx. These results suggest that N-NO-MeIQx could be genotoxic, is activated by conditions that mediate inflammatory responses, and is a possible cancer risk factor for individuals with inflammation of the colon.

Heterocyclic amines and inflammation have been implicated in the etiology of colon cancer. We have recently demonstrated that during autoxidation of the inflammatory mediator nitric oxide 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) undergoes nitrosation to form 2-nitrosoamino-3-methylimidazo[4,5-f]quinoline (N-NO-IQ). This study evaluates the genotoxicity of N-NO-IQ and compares the adducts it forms to those of 2-hydroxyamino-3-methylimidazo[4,5-f]quinoline (N-OH-IQ). N-NO-IQ was incubated with 2'-deoxyguanosine 3'-monophosphate (dGp) under a variety of inflammatory conditions. 32P-Postlabeling demonstrated the presence of multiple adducts. Incubation of N-OH-IQ with dGp at pH 7.4, 5.5, or 2.0 resulted in the formation of a single major adduct, N-(deoxyguanosin-8-yl)-IQ (dG-C8-IQ). Using a combination of 32P-postlabeling, HPLC, and nuclease P1 treatment, N-NO-IQ was shown to produce dG-C8-IQ under several different conditions. HOCl oxidation of N-NO-IQ increased dG-C8-IQ formation, and this was further increased as pH decreased from 7.4 to 5.5. Oxidation of N-NO-IQ formed a new adduct, adduct 2, while in the absence of oxidants adduct m was the major adduct. Adducts 2 and m were not formed by N-OH-IQ and not further identified. The results demonstrate that N-NO-IQ forms N-(deoxyguanosin-8-yl)-IQ, is genotoxic, is activated by conditions that mediate inflammatory responses, and is a possible cancer risk factor for individuals with colitis, inflammation of the colon.

N-Nitrosamines formed by nitrosation of heterocyclic amines might initiate colon cancer in individuals consuming well-done red meat diets and with inflammatory conditions in their colon. This study investigates nitric oxide (NO)-mediated nitrosation of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and the influence of dietary (hemin) and inflammatory [NO, myeloperoxidase (MPO), and H(2)O(2)] components on nitrosation. Using the NO donor spermine NONOate (1.2 microM NO/min) at pH 7.4 with 0.005 mM MeIQx, a product due to NO autoxidation was at the limit of detection (1% of total radioactivity recovered by HPLC). Product formation increased 13- or 16-fold in the presence of 10 microM hemin or 85 nM MPO, respectively, with an in situ system for generating H(2)O(2) (glucose oxidase/glucose). The nitrosation product and its chloro derivative were analyzed by electrospray ionization mass spectrometry, and the product was determined to be 2-nitrosoamino-3,8-dimethylimidazo[4,5-f]quinoxaline (N-NO-MeIQx). Nitrosation by NO autoxidation was only detected at > or =1.2 microM NO/min and was not affected by H(2)O(2). Investigations with hemin determined minimum effective components necessary for potentiation: 1 microM hemin, 1 microM H(2)O(2)/min, and 0.012 microM NO/min. The reactive nitrogen oxygen species (RNOS) produced by hemin and MPO had a 4- and 3-fold, respectively, greater affinity for MeIQx than those produced by NO autoxidation. Test agents were used to characterize nitrosation. Results with catalase, SOD, azide, and NADH are consistent with multiple RNOS, the lack of peroxynitrite involvement in nitrosation, and peroxidatic potentiation by oxidative nitrosylation rather than nitrosation. Using phorbol ester stimulated human neutrophils, the formation of N-NO-MeIQx and its modification by test agents was consistent with MPO and not peroxynitrite. Thus, nitrosation of MeIQx and its potentiation by hemin and MPO provide a mechanism by which well-done red meat consumption and inflammation can generate N-nitroso compounds and initiate colon cancer under inflammatory conditions, such as colitis.

Heme has been reported to be an important contributor to endogenous N-nitrosation within the colon and to the enhanced incidence of colon cancer observed with increased intake of red meat. This study uses the heterocyclic amine 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) as a target to evaluate hemin potentiation of nitric oxide (NO)-mediated nitrosation. Formation of 14C-2-nitrosoamino-3-methylimidazo[4,5-f]quinoline (N-NO-IQ) was monitored by HPLC following incubation of 10 microM IQ with the NO donor spermine NONOate (1.2 microM NO/min) at pH 7.4 in the presence or absence of hemin. N-NO-IQ formation due to autoxidation of NO was at the limit of detection (0.1 microM) and increased 22-fold in the presence of 10 microM hemin and an in situ system for generating H2O2 (glucose oxidase/glucose). A linear increase in N-NO-IQ formation was observed from 1 to 10 microM hemin. Significant nitrosamine formation occurred at fluxes of NO and H2O2 as low as 0.024 and 0.25 microM/min, respectively. Potentiation by hemin was not affected by a 400-fold excess flux of H2O2 over NO or a 4.8-fold excess flux of NO over H2O2. Reactive nitrogen species produced by hemin potentiation had a 46-fold greater affinity for IQ than those produced by autoxidation. Azide inhibited autoxidation, suggesting involvement of the nitrosonium ion, NO+. Hemin potentiation was inhibited by NADH, but not azide, suggesting oxidative nitrosylation with NO2* or a NO2*-like species. IQ and 2,3-diaminonaphthylene were much better targets for nitrosation than the secondary amine morpholine. Apc(min) mice with dextran sulfate sodium-induced colitis demonstrated increased levels of urinary nitrite and nitrate consistent with increased expression of iNOS and NO synthesis. As reported previously, identical conditions increased fecal N-nitroso compounds. Thus, hemin potentiation of NO-mediated nitrosation of heterocyclic amines provides a testable mechanism by which red meat consumption can generate N-nitroso compounds and initiate colon cancer under inflammatory conditions, such as colitis.

Nitrosation is an important reaction elicited by nitric oxide (NO). To better understand how nitrosation occurs in biological systems, we assessed the effect of myeloperoxidase (MPO), a mediator of inflammation, on nitrosation observed during NO autoxidation. Nitrosation of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ; 10 mum) to 2-nitrosoamino-3-methylimidazo[4,5-f]quinoline (N-NO-IQ) was monitored by HPLC. Using the NO donor spermine NONOate at pH 7.4, MPO potentiated N-NO-IQ formation. The minimum effective quantity of necessary components was 8.5 nm MPO, 0.25 mum H(2)O(2)/min, and 0.024 mum NO/min. Autoxidation was only detected at >/=1.2 mum NO/min. MPO potentiation was not affected by a 40-fold excess flux of H(2)O(2) over NO or less than a 2.4-fold excess flux of NO over H(2)O(2). Potentiation was due to an 8.8-fold increased affinity of MPO-derived nitrosating species for IQ. Autoxidation was inhibited by azide, suggesting involvement of the nitrosonium ion, NO(+). MPO potentiation was inhibited by NADH, but not azide, suggesting oxidative nitrosylation with NO(2)(.) or an NO(2)(.)-like species. MPO nonnitrosative oxidation of IQ with 0.3 mm NO(2)(-) at pH 5.5 was inhibited by azide, but not NADH, demonstrating differences between MPO oxidation of IQ with NO compared with NO(2)(-). Using phorbol ester-stimulated human neutrophils, N-NO-IQ formation was increased with superoxide dismutase and inhibited by catalase and NADH, but not NaN(3). This is consistent with nitrosation potentiation by MPO, not peroxynitrite. Increased N-NO-IQ formation was not detected with polymorphonuclear neutrophils from two unrelated MPO-deficient patients. Results suggest that the highly diffusible stable gas NO could initiate nitrosation at sites of neutrophil infiltration.

N-Nitrosamines and nitrosamides can initiate cancer. These studies evaluated the stability and reactivity of 2-nitrosoamino-3-methylimidazo[4,5-f]quinoline (N-NO-IQ) to assess its possible role in the initiation of colon cancer by 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). (14)C-N-NO-IQ was incubated with different solvents and pHs in the presence and in the absence of nucleophiles and analyzed by HPLC. The products identified by electrospray ionization mass spectrometry include 2-chloro-3-methylimidazo[4,5-f]quinoline (2-Cl-IQ), 2,2'-azo-3,3'-dimethylimidazo[4,5-f]quinoline (AZO-IQ), 2-azido-IQ (2-N(3)-IQ), 3-methylimidazo[4,5-f]quinoline (deamino-IQ), and IQ. A variety of organic solvents were tested with 0.1 N HCl. 2-Cl-IQ and IQ were formed following acidification of all solvents. AZO-IQ was only formed in methanol. Deamino-IQ was the major product formed in all of the alcohols tested, except for methanol. Under acidic conditions that completely convert N-NO-IQ in 5 min (acetonitrile with 0.1 N HCl), 62% of N-NO-IQ remains after 30 min if dimethyl sulfoxide is substituted for acetonitrile. N-NO-IQ was stable in the physiologic pH range of 5.5-9.0 and did not react with nucleophiles over a 4 h period at pH 7.4 and 37 degrees C. At acidic pH (pH < or =2.0) for 30 min and 37 degrees C, N-NO-IQ becomes labile forming electrophile(s), which combine with biologically relevant nucleophiles. The reaction of N-NO-IQ at pH 2.0 followed first-order kinetics (t(1/2) = 10 +/- 2 min) and was significantly increased in 10 mM NaN(3) (t(1/2) = 2 +/- 0.1 min). 2-N(3)-IQ was the major product observed in the latter incubation. N-NO-IQ binding to DNA at pH 2.0 is 100-fold more than that at pH 7.4. At pH 2.0, greater than 90% of the binding was inhibited by 10 mM NaN(3). Thus, N-NO-IQ forms a reactive electrophile(s) at acidic pH, which binds DNA. N-NO-IQ reaction products may depend on the pH and the hydrophobic milieu of cells or tissues.

Aromatic amines, such as benzidine (BZ), initiate bladder cancer in humans. Inflammation/infection play an important role in this cancer. This study was designed to assess the influence of inflammatory oxidants, including reactive nitrogen oxygen species (RNOS), on BZ transformation and activation. RNOS were generated under various conditions and reacted with BZ, and the products were examined by HPLC. Conditions that generate nitrogen dioxide radical, NO(2)(-) + myeloperoxidase + H(2)O(2) and ONOO(-), produced primarily a single new product, which was identified by MS as azobenzidine (AZO-BZ). The myeloperoxidase-catalyzed reaction was inhibited by 1 mM cyanide and did not require NO(2)(-). Chloride (100 mM) reduced the myeloperoxidase reaction by 30% with taurine having little effect. In contrast, conditions that generate N(2)O(3), i.e., NO donor diethylamine (DEA) NONOate, produced two products, which were identified by MS as 4'-OH-4-aminobiphenyl (4'-OH-ABP) and 4-aminobiphenyl (ABP). 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, an oxidant of NO thought to produce NO(2)(*), had a biphasic effect on product formation. At a concentration equal to DEA NONOate, a 5-fold increase in BZ nitrosation was observed, while at higher concentrations nitrosation was greatly diminished and formation of AZO-BZ occurred. Glutathione prevented RNOS transformation of BZ. With MPO and ONOO(-), a new product was formed that cochromatographed with 3-(glutathione-S-yl)BZ. Glutathione also prevented nitrosation of BZ but did not form additional BZ products. HOCl-mediated activation of BZ, 4'-OH-ABP, and ABP to bind DNA was assessed. A higher level of binding was observed at pH 5.5 than pH 7.4. BZ elicited the most binding. More binding was observed at both pH values with 4'-OH-ABP than ABP. Thus, components of the inflammatory response are capable of BZ transformation and activation.

A 100-fold increased incidence of bladder cancer is observed with workers exposed to high levels of benzidine (BZ). This review evaluates the overall metabolism of BZ to determine pathways involved in initiation of carcinogenesis. Enzymatic and liver slice incubations demonstrated N-acetylation and N-glucuronidation of BZ and N-acetylbenzidine (ABZ). With rat, N,N'-diacetylbenzidine (DABZ) is the major slice metabolite. With human, ABZ is the major metabolite along with N-glucuronides. Differences between rat and human are attributed to preferential acetylation of BZ and deacetylation of DABZ, resulting in N-glucuronide formation by human liver. Glucuronidation of BZ and its analogues exhibited the following relative ranking of UDP-glucuronosyltransferase (UGT) metabolism: UGT1A9>UGT1A4>>UGT2B7>UGT1A6 approximately UGT1A1. N-Glucuronides of BZ, ABZ, and N'-hydroxy-N-acetylbenzidine (N'HA) are acid labile with the latter having a much longer t(1/2) than the former two glucuronides. O-Glucuronides are not acid labile. In urine from BZ-exposed workers, an inverse relationship between urine pH and levels of free (unconjugated) BZ and ABZ is observed. This is consistent with the presence of labile urinary N-glucuronides. Cytochrome P-450 oxidizes BZ to an inactive product (3-OHz.sbnd;BZ) and ABZ to N'HA and N-hydroxy-N-acetylbenzidine (NHA). Cytochrome P-450, PHS, and horseradish peroxidase activate ABZ to bind DNA forming N'-(3'-monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine (dGp-ABZ). This is the major adduct detected in bladder cells from workers exposed to BZ. An inverse relationship exists between urine pH and levels of bladder cell dGp-ABZ. Bladder epithelium contains relatively high levels of prostaglandin H synthase (PHS) and low levels of cytochrome p-450, suggesting activation by PHS. Activation by PHS involves a peroxygenase oxidation of ABZ to N'HA, while horseradish peroxidase activates ABZ to a diimine monocation. Reactive nitrogen oxygen species (RNOS) offer a new pathway for metabolism and potential activation. Results suggest BZ initiation of bladder cancer is complex, involving multiple organs (i.e. liver, kidney, and bladder) and metabolic pathways (i.e. N-acetylation, N-glucuronidation, peroxidation, and RNOS).

Both cooked red meat intake and chronic inflammation/infection are thought to play a role in the etiology of colon cancer. The heterocyclic amine 2-amino-3-methylimidazo[4,5-f ]quinoline (IQ) is formed during cooking of red meat and may be involved in initiation of colon cancer. Reactive nitrogen oxygen species (RNOS), components of the inflammatory response, contribute to the deleterious effects attributed to inflammation on normal tissues. This study assessed the possible chemical transformation of IQ by RNOS. RNOS were generated by various conditions to react with (14)C-IQ, and samples were evaluated by HPLC. Myeloperoxidase (MPO)-catalyzed reaction was dependent upon both H(2)O(2) and NO(2)(-). This reaction produced an azo-IQ dimer and IQ dimer along with two nitrated IQ products identified by ESI/MS. 2-Nitro-IQ was not detected. Product formation was inhibited by 2 mM cyanide. Reduction in nitrated products observed with 100 mM chloride was not altered with 0.5 mM taurine. Nitrated products were also produced by other conditions, ONOO(-) and NO(2)(-) + HOCl, which generate nitrogen dioxide radical. In contrast, conditions which generate N(2)O(3), such as diethylamine NONOate, produced only small amounts of nitrated products with the major product identified by MS and NMR as N-nitroso-IQ. MPO activation of IQ to bind DNA was dependent upon both H(2)O(2) and NO(2)(-). RNOS generated by ONOO(-) and DEA NONOate also activated IQ DNA binding. The nitrated IQ products were not activated by MPO to bind DNA. In contrast, N-nitroso-IQ was activated to bind DNA by MPO +/- NO(2)(-). HOCl activated N-nitroso-IQ, but not IQ. RAW cells produced N-nitroso-IQ and increased amounts of NO(2)(-)/NO(3)(-), when incubated with 0.1 mM IQ and stimulated with lipopolysaccharide and interferon gamma. Results demonstrate chemical transformation and activation of IQ by RNOS and activation of its N-nitroso product by biological oxidants, events which may contribute to initiation of colon cancer.

Aromatic amine sulfinamide adducts of hemoglobin are biomarkers of exposure and evidence for cytochrome P-450 N-hydroxylation. The possible peroxidatic formation of an N-acetylbenzidine (ABZ) sulfinamide adduct by methemoglobin was examined. Following addition of H2O2, 0.06 mM [3H]ABZ was metabolized by methemoglobin. With 0.3 mM glutathione, a new peak was observed, ABZ-SG, representing 17% of the total radioactivity. N'-Hydroxy-N-acetylbenzidine and 4'-nitro-4-acetylaminobiphenyl were not detected. Optimal ABZ-SG formation was observed with 3 uM methemoglobin, 0.1 to 0.3 mM glutathione, and pH 5.5. Higher concentrations of glutathione were inhibitory. Without glutathione, an H2O2-to-ABZ molar ratio of 1:1 resulted in complete metabolism of ABZ. This ratio increased to greater than 2:1 with 0.3 mM glutathione. Nearly complete inhibition of ABZ-SG formation by cyanide (10 mM), ascorbic acid (0.1 mM), 5,5-dimethyl-1-pyrroline N-oxide (50 mM), thiourea (1 mM), and azide (0.3 mM), and the lack of inhibition by mannitol (50 mM) and superoxide dismutase (2 microg) is consistent with a methemoglobin-mediated peroxidatic reaction, which does not involve hydroxyl radical or superoxide. ABZ-SG was identified by electrospray ionization/mass spectrometry as N'-(glutathion-S-yl)-N-acetylbenzidine S-oxide. Conjugate was hydrolyzed by 0.1 N HCl and NaOH, was relatively stable at pH 5.5 and 7.4, and was susceptible to gamma-glutamyltranspeptidase treatment. Formation of an ABZ sulfinamide conjugate with hemoglobin was demonstrated. The results demonstrate that methemoglobin can catalyze the peroxidatic formation of an ABZ sulfinamide adduct, perhaps by a diimine monocation intermediate.

A close association has been reported for certain types of cancers influenced by aromatic amines and infection/inflammation. Reactive nitric oxygen species (RNOS), components of the inflammatory response, are bactericidal and tumoricidal, and contribute to the deleterious effects attributed to inflammation on normal tissues. This study assessed the possible transformation of the aromatic amine N-acetylbenzidine (ABZ) by RNOS. RNOS were generated by various conditions to react with ABZ, and samples were evaluated by HPLC. Conditions which generate nitrogen dioxide radical (NO(2)(-) + myeloperoxidase + H(2)O(2), ONOO(-), and NO(2)(-) + HOCl) produced primarily a single new product termed 3'-nitro-ABZ. The myeloperoxidase-catalyzed reaction with 0.3 mM NO(2)(-) was completely inhibited by 1 mM cyanide, and not effected by 100 mM chloride with or without 1 mM taurine. In contrast, conditions which generate N(2)O(3), such as spermine NONOate, did not produce 3'-nitro-ABZ, but rather two compounds termed 4'-OH-AABP and AABP. (1)H NMR and mass spectrometry identified 3'-nitro-ABZ as 3'-nitro-N-acetylbenzidine, 4'-OH-AABP as 4'-OH-4-acetylaminobiphenyl, and AABP as 4-acetylaminobiphenyl. Human polymorphonuclear neutrophils incubated with [(3)H]ABZ and stimulated with beta-phorbol 12-myristate 13-acetate produced 3'-nitro-ABZ in the presence of NO(2)(-) (0.1-1 mM). Neutrophil 3'-nitro-ABZ formation was verified by mass spectrometry and was consistent with myeloperoxidase oxidation of NO(2)(-). The results demonstrate that ABZ forms unique products in the presence of nitrosating and nitrating RNOS, which could influence the carcinogenic process and serve as biomarkers for these reactive species.

Nitrating reactive nitric oxygen species (RNOS) elicit many of the deleterious effects of the inflammatory response. Their high reactivity and short half-life make RNOS analysis difficult. Reaction of acetaminophen (APAP) with RNOS generated by various conditions was evaluated by HPLC. When [(14)C]APAP was incubated at pH 7.4, the same new product (3NAP) was produced by at least three separate pathways represented by the following conditions: myeloperoxidase oxidation of NO(2)(-), NO(2)Cl, and ONOO(-) or Sin-1. Diethylamine NONO and spermine NONO did not convert APAP to 3NAP. 3NAP was stable at pH 5, 7.4, or 9, and at pH 7.4 with ONOO(-), spermine NONO, Sin-1, or H(2)O(2). HOCl transformed 3NAP, which was prevented by APAP, ascorbic acid, taurine, or NO(2)(-). ONOO(-)-derived 3NAP was identified by (1)H NMR as 3-nitroacetaminophen or 3-nitro-N-acetyl-p-aminophenol, and the product mass was verified by EI/ESI mass spectrometry. Human polymorphonuclear neutrophils incubated with [(14)C]APAP and stimulated with beta-phorbol 12-myristate 13-acetate produced 3NAP in the presence of NO(2)(-). Neutrophil 3NAP formation was verified by mass spectrometry and was consistent with myeloperoxidase oxidation of NO(2)(-). Spermine NONO supported 3NAP formation by stimulated cells in the absence of NO(2)(-). Results demonstrate that 3NAP is a product of nitrating RNOS generated by at least three separate pathways and may be a biomarker for nitrating mediators of inflammation.

N'-(3'-Monophosphodeoxyguanosin-8-yl)-N-acetylbenzidine (dGp-ABZ) is the major adduct in exfoliated urothelial cells and in peripheral white blood cells of workers exposed to benzidine. This study was designed to assess the metabolic pathways leading to dGp-ABZ formation in human peripheral white blood cells. [(3)H]-N-Acetylbenzidine (ABZ) transformation was assessed using myeloperoxidase (MPO), hypochlorous acid (HOCl), and human peripheral white blood cells in the absence and presence of DNA or dGp. MPO metabolism required H(2)O(2), but not NaCl. While transformation by HOCl was completely inhibited by 10 mM taurine, the level of metabolism of ABZ by MPO was only reduced 56%. Transformation by either MPO or HOCl was inhibited by 100 mM DMPO, 1 mM glutathione, and 1 mM ascorbic acid. Glutathione formed a new product with MPO, but not with HOCl. Previously identified oxidation products of ABZ, N'-hydroxy-N-acetylbenzidine or 4'-nitro-4-acetylaminobiphenyl, were not detected. With DNA or dGp present, a new product was observed that corresponded to synthetic dGp-ABZ in its HPLC elution profile, in nuclease P(1) hydrolysis to dG-ABZ, and in (32)P-postlabeling analysis. The HOCl-derived adduct was identified by electrospray ionization mass spectrometry, with collision-activated dissociation, as dGp-ABZ. Metabolism of [(3)H]ABZ by peripheral blood cells was stimulated about 3-fold with 30 ng/mL beta-phorbol 12-myristate 13-acetate (PMA). Using (32)P-postlabeling, dGp-ABZ was detected only in the presence of PMA and its level was increased more than 300-fold if either 0.7 mg/mL DNA or dGp was present. Indomethacin (0.1 mM) did not alter adduct formation. With dGp, dGp-ABZ formation could be detected with as little as 0.12 x 10(6) neutrophils. Using specific chromatographic and enzymatic techniques, neutrophil-derived dGp-ABZ was identical to the synthetic standard. Thus, these results are consistent with human polymorphonuclear neutrophils forming dGp-ABZ by a peroxidatic mechanism involving MPO.

Arylamine-hemoglobin conjugates identified as sulfinamides are considered dosimeters for the bioavailability of metabolically formed N-oxidation products. This report considers peroxidation as an alternative pathway for aromatic amine metabolism and examines horseradish peroxidase metabolism of N-acetylbenzidine (ABZ) in the presence of glutathione. When 0.06 mM [(3)H]ABZ was incubated with 1 mM glutathione, a decrease in the total extent of metabolism was observed along with detection of a new metabolite (ABZ-SG), representing 12% of the total radioactivity. Optimum ABZ-SG formation occurred at 0.3 mM glutathione with higher concentrations (10 mM) being inhibitory. In the absence of glutathione, a molar ratio of H(2)O(2) to ABZ of 1:1 resulted in complete metabolism of ABZ. This ratio increased to >2:1 in the presence of 0.3 mM glutathione. N-Oxidation products of ABZ metabolism, such as N'-hydroxy-N-acetylbenzidine, were not detected using a variety of incubation conditions. ABZ-SG was sensitive to gamma-glutamyltranspeptidase, and completely hydrolyzed by 0.1 N HC1 or 0.1 N NaOH in 10 min at room temperature. ABZ-SG was identified by mass spectrometry and NMR to be N'-(glutathion-S-yl)-N-acetylbenzidine S-oxide. ABZ-SG formation, but not total ABZ metabolism, was prevented by 0.3 mM NaN(3), 50 mM DMPO, 1.0 mM thiourea, and 1.0 mM histidine. Cyanide (50 mM) and ascorbic acid (0.1 mM) completely inhibited ABZ metabolism. The lack of effect of 50 mM mannitol and 2 microgram of superoxide dismutase suggests that neither hydroxyl radical nor superoxide is involved in the reaction. Studies also indicated that molecular oxygen is not a source of the sulfinamide oxygen. Formation of an ABZ sulfinamide conjugate with hemoglobin was demonstrated. The proposed mechanism for sulfinamide formation, involving two consecutive one-electron oxidations with subsequent rearrangement to a sulfur-stabilized nitrenium ion, suggests that oxygen may be derived from water. The results demonstrate that while arylamine-hemoglobin conjugates serve as useful biomarkers of exposure, their mechanism of formation may be complex, perhaps involving peroxidation as in the case of N'-(glutathion-S-yl)-N-acetylbenzidine S-oxide.

Hypochlorous acid (HOCl), a chemically reactive oxidant, is an important component of the inflammatory response and may contribute to carcinogenesis. This study assessed the possible activation of N-acetylbenzidine (ABZ) by HOCI to form a specific DNA adduct, N'-(3'-monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine. HOCl was incubated with 0.06 mM 3H-ABZ, and transformation assessed by HPLC. Similar results were observed at pH 5.5 or 7.4. A linear increase in transformation was observed from 0.025 to 0.1 mM HOCl with up to 80% of ABZ changed. Approximately, 2 nmoles of HOCI oxidized 1 nmole of ABZ. N-oxidation products of ABZ metabolism, such as N'-hydroxy-N-acetylbenzidine, were not detected. Oxidation of ABZ was prevented by taurine, DMPO, glutathione, and ascorbic acid, whereas mannitol was without effect. Results are consistent with a radical mechanism. In the presence of 2'-deoxyguanosine 3'-monophosphate (dGp), a new product (dGp-ABZ) was observed. The same adduct was observed with DNA. dGp-ABZ was found to be quite stable (>80% remaining) at 70 degrees C in pH 5.5 (60 min) and 7.4 (240 min). Electrospray mass spectrometry indicated that dGp-ABZ was N'-(3'-monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine, and this was confirmed by NMR. 32P-postlabeling in combination with TLC and HPLC determined that the adduct made by either HOCl or prostaglandin H synthase oxidation of ABZ in the presence of dGp or DNA was dGp-ABZ. Thus, HOCI activates ABZ to form dGp-ABZ and may be responsible for the presence of this adduct in peripheral white blood cells from workers exposed to benzidine. Reaction of ABZ with HOCl provides an easy, convenient method for preparing dGp-ABZ.

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. [(14)C]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-N-acetylbenzidine and N-hydroxy-N,N'-diacetylbenzidine. UGT1A9 metabolized 4-aminobiphenyl approximately 50% faster than benzidine or N-acetylbenzidine. UGT1A4 N-glucuronidated N'-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,N'-diacetylbenzidine, and the ring oxidation product, 3-OH-N,N'-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 K(m) values for N-acetylbenzidine metabolism by UGT1A1 and UGT1A4 were 0.37 +/- 0.14 and 1.8 +/- 0.4 mM, respectively. The O-glucuronide of 3-OH-N,N'-diacetylbenzidine was not hydrolyzed during a 24 h 37 degrees 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 approximately 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.

Individuals exposed to carcinogenic aromatic amines excrete arylamine N- and O-glucuronide metabolites. This study assessed the susceptibility of selected glucuronides to hydrolysis by human and Escherichia coli beta-glucuronidase. N- or O-glucuronides were prepared with the following aglycones: benzidine, N-acetylbenzidine, N'-hydroxy-N-acetylbenzidine, N-hydroxy-N-acetylbenzidine, N-hydroxy-N,N'-diacetylbenzidine, 3-hydroxy-N,N'-diacetylbenzidine, 3-hydroxy-benzidine, 4-aminobiphenyl, N-hydroxy-4-aminobiphenyl, and N-hydroxy-N-acetyl-4-aminobiphenyl. The (3)H- and (14)C-labeled glucuronides were prepared with human or rat liver microsomes using UDP-glucuronic acid as cosubstrate. Each of the 10 glucuronides (6-12 microM) was incubated at pH 5.5 or 7.0 with either human recombinant (pure) or E. coli (commercial preparation) beta-glucuronidase for 30 min at 37 degrees C. Hydrolysis was measured by HPLC. Reaction conditions were optimized, using the O-glucuronide of N-hydroxy-N,N'-diacetylbenzidine. Both enzymes preferentially hydrolyzed O-glucuronides over N-glucuronides and distinguished between structural isomers. With E. coli beta-glucuronidase at pH 7.0, selectivity was demonstrated by the complete hydrolysis of N-hydroxy-N-acetyl-4-aminobiphenyl O-glucuronide in the presence of N-acetylbenzidine N-glucuronide, which was not hydrolyzed. Metabolism by both enzymes was completely inhibited by the specific beta-glucuronidase inhibitor saccharic acid-1,4-lactone (0.5 mM). The concentration of human beta-glucuronidase necessary to achieve significant hydrolysis of glucuronides was substantially more than the amount of enzyme reported previously to be present in urine under either normal or pathological conditions. The bacterial enzyme may hydrolyze O-glucuronides, but not N-glucuronides, in urine at neutral pH. Thus, the nonenzymatic hydrolysis of N-glucuronides by acidic urine is likely a more important source of free amine than enzymatic hydrolysis.

Synthesis of prostaglandin H2 by prostaglandin H synthase (PHS) results in a two-electron oxidation of the enzyme. An active reduced enzyme is regenerated by reducing cofactors, which become oxidized. This report examines the mechanism by which PHS from ram seminal vesicle microsomes catalyzes the oxidation of the reducing cofactor N-acetylbenzidine (ABZ). During the conversion of 0.06 mM ABZ to its final end product, 4'-nitro-4-acetylaminobiphenyl, a new metabolite was observed when 1 mM ascorbic acid was present. Similar results were observed whether 0.2 mM arachidonic acid or 0.5 mM H2O2 was used as the substrate. This metabolite co-eluted with synthetic N'-hydroxy-N-acetylbenzidine (N'HA), but not with N-hydroxy-N-acetylbenzidine. The new metabolite was identified as N'HA by electrospray ionization/MS/MS. N'HA represented as much as 10% of the total radioactivity recovered by high pressure liquid chromatography. When N'HA was substituted for ABZ, PHS metabolized N'HA to 4'-nitro-4-acetylaminobiphenyl. Inhibitor studies demonstrated that metabolism was due to PHS, not cytochrome P-450. The lack of effect of 5,5-dimethyl-1-pyrroline N-oxide, mannitol, and superoxide dismutase suggests the lack of involvement of one-electron transfer reactions and suggests that hydroxyl radicals and superoxide are not sources of oxygen or oxidants. Oxygen uptake studies did not demonstrate a requirement for molecular oxygen. When [18O]H2O2 was used as the substrate, 18O enrichment was observed for 4'-nitro-4-acetylaminobiphenyl, but not for N'HA. A 97% enrichment was observed for one atom of 18O, and a 17 +/- 7% enrichment was observed for two 18O atoms. The rapid exchange of 18O-N'HA with water was suggested to explain the lack of enrichment of N'HA and the low enrichment of two 18O atoms into 4'-nitro-4-acetylaminobiphenyl. Results demonstrate a peroxygenase oxidation of ABZ and N'HA by PHS and suggest a stepwise oxidation of ABZ to N'-hydroxy, 4'-nitroso, and 4'-nitro products.

Workers exposed to high levels of benzidine have a 100-fold increased incidence of bladder cancer. This review evaluates the overall metabolism of benzidine to determine pathways important to initiation of bladder cancer. Upon incubation of benzidine with liver slices from rats, dogs, and humans, different proportions of this diamine were N-acetylated and N-glucuronidated. With dogs, a non-acetylator species, N-glucuronidation was the major pathway. In contrast, little glucuronidation was observed in rats with N, N'-diacetylbenzidine, the major metabolite of benzidine. Human liver slices demonstrated both extensive N-acetylation and N-glucuronidation. Differences between rats and humans were attributed to rapid deacetylation by human liver with N-acetylbenzidine rather than an accumulation of N, N'-diacetylbenzidine. N-Acetylbenzidine oxidative metabolism was also observed. The acid lability of glucuronide products of benzidine, N-acetylbenzidine, and oxidation products of N-acetylbenzidine metabolism was assessed. N-Glucuronides of benzidine, N-acetylbenzidine, and N'-hydroxy-N-acetylbenzidine were acid-labile, with the latter having a much longer half-time than the former two glucuronides. Because bladder epithelium contains relatively high levels of prostaglandin H synthase and not cytochrome P450, the peroxidative metabolism of N-acetylbenzidine was assessed. N'-(3'-Monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine was the only DNA adduct detected. This adduct is also the major adduct detected in bladder cells from workers exposed to benzidine. In urine from these workers, an inverse relationship between urine pH and levels of free (unconjugated) benzidine and N-acetylbenzidine was observed. A similar inverse relationship was observed for urine pH and levels of bladder cell N'-(3'-monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine. These results suggest multiple pathways (acetylation, glucuronidation, peroxidation) in multiple organs (liver, blood, kidney, bladder) are important in benzidine-induced bladder cancer.

We evaluated the influence of urine pH on the proportion of urinary benzidine (BZ) and N-acetylbenzidine present in the free, unconjugated state and on exfoliated urothelial cell DNA adduct levels in 32 workers exposed to BZ in India. Postworkshift urine pH was inversely correlated with the proportions of BZ (r = -0.78; P < 0.0001) and N-acetylbenzidine (r = -0.67; P < 0.0001) present as free compounds. Furthermore, the average of each subject's pre- and postworkshift urine pH was negatively associated with the predominant urothelial DNA adduct (P = 0.0037, adjusted for urinary BZ and metabolites), which has been shown to cochromatograph with a N-(3'-phosphodeoxyguanosin-8-yl)-N'-acetylbenzidine adduct standard. Controlling for internal dose, individuals with urine pH < 6 had 10-fold higher DNA adduct levels compared to subjects with urine pH > or = 7. As reported previously, polymorphisms in NAT1, NAT2, and GSTM1 had no impact on DNA adduct levels. This is the first study to demonstrate that urine pH has a strong influence on the presence of free urinary aromatic amine compounds and on urothelial cell DNA adduct levels in exposed humans. Because there is evidence that acidic urine has a similar influence on aromatic amines derived from cigarette smoke, urine pH, which is influenced by diet, may be an important susceptibility factor for bladder cancer caused by tobacco in the general population.

Urinary mutagenicity has been used in occupational and epidemiological studies for over two decades as a cost-effective, general biomarker of exposure to genotoxic agents. However, few studies have compared urinary mutagenicity to additional biomarkers determined among low- and high-exposed groups. To address this issue, we evaluated the relationship between urinary mutagenicity and other types of biomarkers in a cross-sectional study involving 15 workers exposed to the urinary bladder carcinogen benzidine (BZ, high exposure), 15 workers exposed to BZ-dyes (low exposure), and 13 unexposed controls in Ahmedabad, India. Urinary organics were extracted by C18/methanol and evaluated for mutagenicity in the presence of S9 in the Salmonella strain YG1024, which is a frameshift strain that overproduces acetyltransferase. The results were compared to biomarker data reported recently from the same urine samples (Rothman et al., Proc. Natl Acad. Sci. USA, 93, 5084-5089, 1996) that included a metabolite biomarker (the sum of the urinary levels of BZ + N-acetylbenzidine + N,N'-diacetylbenzidine) and a DNA adduct biomarker [a presumptive N-(3'-phosphodeoxyguanosin-8-yl)-N'-acetylbenzidine (C8dG-ABZ) DNA adduct in exfoliated urothelial cells]. The mean +/- SE urinary mutagenicity (revertants/micromol of creatinine) of the low-exposure (BZ-dye) workers was 8.2 +/- 2.4, which was significantly different from the mean of the controls (2.8 +/- 0.7, P = 0.04) as was that of the mean of the high-exposure (BZ) workers (123.2 +/- 26.1, P < 0.0001). Urinary mutagenicity showed strong, positive correlations with urinary metabolites (r = 0.88, P < 0.0001) and the level of the presumptive C8dG-ABZ urothelial DNA adduct (r = 0.59, P = 0.0006). A strong association was found between tobacco use (bidi smoking) and urinary mutagenicity among the controls (r = 0.68, P = 0.01) but not among the exposed workers (r = 0.18, P = 0.11). This study confirms the ability of a biomarker such as urinary mutagenicity to detect low-dose exposures, identify additional genotoxic exposures among the controls, and correlate strongly with urinary metabolites and DNA adducts in the target tissue (urinary bladder epithelia) in humans.

To provide the information necessary for assessing risk and preventing tumorigenesis, the metabolism of N-acetylbenzidine and N,N'-diacetylbenzidine was assessed with rat liver microsomes from control and beta-naphthoflavone-treated rats. The oxidation of [3H]N-acetylbenzidine to [3H]N'-hydroxy-N-acetylbenzidine (N'HA), [3H]N-hydroxy-N-acetylbenzidine (NHA), and 3H-ring oxidation products was assessed. For [3H]N,N'-diacetylbenzidine, the formation of [3H]N-hydroxy-N,N'-diacetylbenzidine (NHDA) and the 3H-ring oxidation product was assessed. With beta-naphthoflavone-treated microsomes, the rate of NHA formation was 8-fold more than observed with control. Although significant formation of ring-oxidation products was demonstrated, the formation of N'HA was at the limit of detection. With control microsomes, N'HA was a major metabolite with more N'HA (49 +/- 6 pmol/mg protein/min) produced than NHA (38 +/- 5). Whereas the oxidation of N,N'-diacetylbenzidine was not observed with control microsomes, significant formation of NHDA (421 +/- 49 pmol/mg protein/min) and ring-oxidation (182 +/- 28) product was observed with beta-naphthoflavone-treated microsomes. Metabolism of [3H]N-acetylbenzidine and [3H]N,N'-diacetylbenzidine by beta-naphthoflavone-treated microsomes was completely inhibited by the specific cytochrome P4501A1/1A2 inhibitors alpha-naphthoflavone and ellipticine at 10 microM. Except for the < 30% inhibition observed with the cytochrome P4502E1 inhibitor (disulfiram), inhibitors of cytochrome P4503A1/3A2 (troleandomycin) and P4502C6 (sulfinpyrazone) were not effective at 10 microM. N'HA formation by control microsomes was not prevented by any of these inhibitors. Conditions that inhibit flavin-dependent monooxygenase metabolism, methimazole (1 mM), and heat treatment (37 degrees C for 60 min) were also ineffective in preventing N'HA formation. The nonspecific cytochrome P450 inhibitor SKF-525A (10 microM) exhibited a partial dose-response inhibition (maximum 41% of complete reaction mixture) of N'HA formation, but did not alter NHA formation. In contrast, the nonspecific cytochrome P450 inhibitor, 2,4-dichloro-6-phenylphenoxyethylamine prevented formation of both N'HA and NHA. beta-Naphthoflavone treatment increased [3H]N-acetylbenzidine binding to DNA, but not [3H]N,N'-diacetylbenzidine. Binding of both compounds to DNA was inhibited by ellipticine. N'-(3'-monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine was detected by 32P-postlabeling in microsomal incubations with N-acetylbenzidine, but not N,N'-diacetylbenzidine. More adduct was detected with control than beta-naphthoflavone-treated microsomes. Results are consistent with cytochrome P4501A1/1A2 playing the major role in N-acetylbenzidine and N,N'-diacetylbenzidine metabolism by liver microsomes from control and beta-naphthoflavone-treated rats. The formation of N'HA by control, but not by beta-naphthoflavone-treated, rats and its insensitivity to inhibition by cytochrome P4501A1/1A2 inhibitors were unexpected.

Multiple studies in the general population have suggested that subjects with the glutathione S-transferase M1 (GSTM1)-null genotype, who lack functional GSTM1, are at higher risk for bladder cancer. To evaluate the impact of the GSTM1-null genotype on bladder cancer caused by occupational exposure to benzidine and to determine its influence on benzidine metabolism, we carried out three complementary investigations: a case-control study of bladder cancer among workers previously exposed to benzidine in China, a cross-sectional study of urothelial cell DNA adducts and urinary mutagenicity in workers currently exposed to benzidine in India, and a laboratory study of the ability of human GSTM1 to conjugate benzidine and its known metabolites in vitro. There was no overall increase in bladder cancer risk for the GSTM1-null genotype among 38 bladder cancer cases and 43 controls (odds ratio, 1.0; 95% confidence interval, 0.4-2.7), although there was some indication that highly exposed workers with the GSTM1-null genotype were at greater risk of bladder cancer compared to similarly exposed workers without this allele. However, the GSTM1 genotype had no impact on urothelial cell DNA adduct and urinary mutagenicity levels in workers currently exposed to benzidine. Furthermore, human GSTM1 did not conjugate benzidine or its metabolites. These results led us to conclude that the GSTM1-null genotype does not have an impact on bladder cancer caused by benzidine, providing a contrast to its association with elevated bladder cancer risk in the general population.

This study used liver microsomes from control an naphthoflavone-treated rats to evaluate NADPH-dependent oxidation of benzidine. With microsomes from beta-naphthoflavone-treated rats, the rates of formation of aqueous soluble metabolite (HPLC analysis) and protein and DNA binding were 835 +/- 81, 14.5 +/- 1.8 and 0.71 +/- 0.14 pmol/mg/min respectively. beta-Naphthoflavone treatment elicited 12.3-, 1.8- and 14.2-fold increases in benzidine metabolism compared with controls as judged by HPLC and protein and DNA binding respectively. For microsomes from treated animals, Km and Vmax values were 47 +/- 6 micromol and 1.13 +/- 0.16 nmol/mg protein/min respectively. All of the metabolic parameters were inhibited to varying degrees by glutathione (1 or 10 mM), N-acetylmethionine (10 mM) and ascorbic acid (10 mM). Following glutathione addition, at least two new metabolite peaks were observed, representing -6% of the total radioactivity recovered by HPLC. Neither metabolite was 3-(glutathion-S-yl)benzidine. Cytochrome P450 inhibitors (10 micro) specific for different members of cytochrome gene families 1-3 indicated that benzidine was metabolized by cytochrome P450 1A1/1A2. Ellipticine and alpha-naphthoflavone, specific 1A1/1A2 inhibitors, elicited 50% inhibition at -0.2 and 0.5 micro respectively. Electron impact and negative ion chemical ionization mass spectro- metry identified the aqueous soluble metabolite as 3-hydroxybenzidine. The lability of 3-hydroxybenzidine observed at pH > 7.0 was prevented by ascorbic acid. Thus, cytochrome P450 1A1/1A2 NADPH-dependent metabolism of benzidine to 3-hydroxybenzidine was demonstrated.

These studies were designed to assess metabolism of benzidine and N-acetylbenzidine by N-acetyltransferase (NAT) NAT1 and NAT2. Metabolism was assessed using human recombinant NAT1 and NAT2 and human liver slices. For benzidine and N-acetylbenzidine, Km and Vmax values were higher for NAT1 than for NAT2. The clearance ratios (NAT1/NAT2) for benzidine and N-acetylbenzidine were 54 and 535, respectively, suggesting that N-acetylbenzidine is a preferred substrate for NAT1. The much higher NAT1 and NAT2 Km values for N-acetylbenzidine (1380 +/- 90 and 471 +/- 23 microM, respectively) compared to benzidine (254 +/- 38 and 33.3 +/- 1.5 microM, respectively) appear to favor benzidine metabolism over N-acetylbenzidine for low exposures. Determination of these kinetic parameters over a 20-fold range of acetyl-CoA concentrations demonstrated that NAT1 and NAT2 catalyzed N-acetylation of benzidine by a binary ping-pong mechanism. In vitro enzymatic data were correlated to intact liver tissue metabolism using human liver slices. Samples incubated with either [3H]benzidine or [3H]N-acetylbenzidine had a similar ratio of N-acetylated benzidines (N-acetylbenzidine + N',N'-diacetylbenzidine/ benzidine) and produced amounts of N-acetylbenzidine > benzidine > N,N'-diacetylbenzidine. With [3H]benzidine, p-aminobenzoic acid, a NAT1-specific substrate, increased the amount of benzidine and decreased the amount of N-acetylbenzidine produced, resulting in a decreased ratio of acetylated products. This is consistent with benzidine being a NAT1 substrate. N-Acetylation of benzidine or N-acetylbenzidine by human liver slices did not correlate with the NAT2 genotype. However, a higher average acetylation ratio was observed in human liver slices possessing the NAT1*10 compared to the NAT1*4 allele. Thus, a combination of human recombinant NAT and liver slice experiments has demonstrated that benzidine and N-acetylbenzidine are both preferred substrates for NAT1. These results also suggest that NAT1 may exhibit a polymorphic expression in human liver.

Glucuronide conjugates of arylamines are thought to be important in the carcinogenic process. This study investigated the pH stability and synthesis of glucuronide conjugates of 4-aminobiphenyl and its N-hydroxy metabolites by human and dog liver. Both dog and human liver slices incubated with 0.06 mM [3H]-4-aminobiphenyl produced the N-glucuronide of 4-aminobiphenyl as the major product. After 2 hr of incubation, the N-glucuronide of 4-aminobiphenyl represented 52 and 27% of the total radioactivity recovered by HPLC in dog and human, respectively. When 4-aminobiphenyl, N-hydroxy-4-aminobiphenyl, or N-hydroxy-N-acetyl-4-aminobiphenyl was added to human microsomes containing [14C]UDP-glucuronic acid, a new product peak was detected by HPLC. At 0.5 mM, the rate of glucuronidation was N-hydroxy-N-acetyl-4-aminobiphenyl > N-hydroxy-4-aminobiphenyl > 4-aminobiphenyl. The rate of formation of the N-glucuronide of 4-aminobiphenyl was similar to that observed with benzidine and N-acetylbenzidine. The glucuronides of 4-aminobiphenyl and N-hydroxy-4-aminobiphenyl were both acid labile with T1/2 values of 10.5 and 32 min, respectively, at pH 5.5. The glucuronide of N-hydroxy-N-acetyl-4-aminobiphenyl was not acid labile with T1/2 values at pH 5.5 and 7.4 of 55 and 68 min, respectively. The glucuronide of 4-aminobiphenyl was the most acid labile conjugate examined. Thus, the glucuronide of 4-aminobiphenyl is a major product of dog and human liver slice metabolism and likely to play an important role in the carcinogenic process.

Several epidemiologic studies indicate that NAT2-related slow N-acetylation increases bladder cancer risk among workers exposed to aromatic amines, presumably because N-acetylation is important for the detoxification of these compounds. Previously, we showed that NAT2 polymorphisms did not influence bladder cancer risk among Chinese workers exposed exclusively to benzidine (BZ), suggesting that NAT2 N-acetylation is not a critical detoxifying pathway for this aromatic amine. To evaluate the biologic plausibility of this finding, we carried out a cross-sectional study of 33 workers exposed to BZ and 15 unexposed controls in Ahmedabad, India, to evaluate the presence of BZ-related DNA adducts in exfoliated urothelial cells, the excretion pattern of BZ metabolites, and the impact of NAT2 activity on these outcomes. Four DNA adducts were significantly elevated in exposed workers compared to controls; of these, the predominant adduct cochromatographed with a synthetic N-(3'- phosphodeoxyguanosin-8-yl)-N'-acetylbenzidine standard and was the only adduct that was significantly associated with total BZ urinary metabolites (r = 0.68, P < 0.0001). To our knowledge this is the first report to show that BZ forms DNA adducts in exfoliated urothelial cells of exposed humans and that the predominant adduct formed is N-acetylated, supporting the concept that monofunctional acetylation is an activation, rather than a detoxification, step for BZ. However, because almost all BZ-related metabolites measured in the urine of exposed workers were acetylated among slow, as well as rapid, acetylators (mean +/- SD 95 +/- 1.9% vs. 97 +/- 1.6%, respectively) and NAT2 activity did not affect the levels of any DNA adduct measured, it is unlikely that interindividual variation in NAT2 function is relevant for BZ-associated bladder carcinogenesis.

Glucuronidation of N-hydroxy arylamines is thought to be a necessary step in their initiation of bladder cancer. This was evaluated for the N-hydroxy metabolites of N-acetylbenzidine (ABZ). N'-Hydroxy-N-acetylbenzidine (N'-HA), N-hydroxy-N-acetylbenzidine (N-HA) and N-hydroxy- N,N'-diacetylbenzidine (N-HDA) were synthesized. Except for N'-HA, these compounds were quite stable. Ascorbic acid and/or acidic pH increased the stability of N'-HA. When each N-hydroxy compound was added to reaction mixtures containing [14C]UDP-glucuronic acid, 3 mM ascorbic acid and human liver microsomes a new product was detected by HPLC. Emulgen 911 was a better detergent than Triton X-100 for expressing microsomal activity, with maximal glucuronidation observed with 0.3% Emulgen 911. At 0.125 mM amine the rate of glucuronidation was N-HDA >> N'-HA = benzidine > ABZ > N-HA. In contrast, at 0.5 mM amine the rate of glucuronidation of N-HA was only exceeded by N-HDA. At pH 5.5 and 37 degrees C the t1/2 for the enzymatically prepared glucuronide conjugates of ABZ, N'-HA and N-HA were 7.5 min and 3.5 and 1.8 h respectively. For N-HDA > 90% of this glucuronide remained after 24 h. At pH 7.4 and 37 degrees C the t1/2 for the glucuronide conjugates of ABZ and N-HA were 2.3 and 2 h respectively, with the amounts remaining after 24 h for N'-HA and N-HDA being 75 and 90% respectively. At pH 6.5 the t1/2 for N'-HA was 14 h. Thus only glucuronides of ABZ and N'-HA exhibit pH-dependent changes in t1/2. Compared with ABZ, glucuronides the N-hydroxy metabolites are more stable at acidic pH. Acidic urine would be more likely to hydrolyze the glucuronide conjugate of ABZ than those of its N-hydroxy metabolites. Because these results are different from that hypothesized for arylmonoamines, a new model was developed to explain the role of N-oxidation, N-glucuronidation and N-acetylation in the carcinogenesis of benzidine, an aryldiamine.

The extent to which N-acetylbenzidine and N,N'-diacetylbenzidine are formed may influence benzidine-induced carcinogenesis. This study compared the formation of these metabolites by rat and human liver slices. The relationship between the NAT2 genotype and the formation of these acetylated products was also evaluated in humans. In rat liver slices incubated with 0.05 mM [3H]benzidine for 1 h (n = 3), N-acetylbenzidine and N,N'-diacetylbenzidine represented 8.8 +/- 3.6 and 73 +/- 2.5% respectively of the total radioactivity recovered by HPLC. No unmetabolized benzidine was observed. This suggests that an equilibrium exists between benzidine, N-acetylbenzidine and N,N'-diacetylbenzidine in rat liver slice incubations which favors N,N'-diacetylbenzidine formation. In the presence of 0.1 mM paraoxon, a deacetylase inhibitor, N-acetylbenzidine and N,N'-diacetylbenzidine increased to 13 +/- 0.6 and 79 +/- 0.3% respectively. Within 2 h after incubating human liver slices with 0.014 mM [3H]benzidine (n = 8), benzidine, N-acetylbenzidine and N,N'-diacetylbenzidine represented 19 +/- 5, 34 +/- 4 and 1.6 +/- 0.5%, respectively, of the total radioactivity recovered by HPLC. Thus in the human, conditions in liver slices favor N-acetylbenzidine rather than N,N'-diacetylbenzidine formation. With paraoxon, benzidine, N-acetylbenzidine and N,N'-diacetylbenzidine represented 2 +/- 0.4, 24 +/- 4 and 51 +/- 3%, respectively. This resulted in a 32-fold increase in N,N'-diacetylbenzidine formation. Individuals with rapid NAT2 genotypes formed 1.4-fold more N-acetylbenzidine than slow acetylators. However, this increase was not significant. There was no apparent correlation of N,N'-diacetylbenzidine formation with NAT2 genotype. Similar results were observed when human slices were incubated with 0.09 mM [3H]benzidine. Deacetylase, perhaps more than N-acetyltransferase, influences hepatic metabolism and subsequent carcinogenesis of benzidine in man. These results help explain the species and organ specificity of benzidine carcinogenesis.

To determine whether benzidine is acetylated in dog, like rat, the metabolism of benzidine was assessed with dog and rat liver slices. Slices were incubated with 0.05 mM [3M]benzidine for 4 h. Media and cellular DNA were analyzed for acetylated benzidine metabolites and adducts. In rat, benzidine was rapidly converted to acetylated metabolites. At 1 h, benzidine, N-acetylbenzidine, and N,N'-diacetylbenzidine represented 5%, 23%, and 54%, respectively, of the total radioactivity in media. Within 2 h, 75% of the radioactivity was N,N'-diacetylbenzidine. In dog, 45% of the radioactivity was present in metabolites more polar than benzidine by 4 h. No N-acetylated metabolites were observed in dog liver slice media. To identify acetylated benzidine DNA adducts, N-(deoxyguanosin-8-yl)-N,N'-diacetylbenzidine was prepared and identified by FAB MS. This nucleoside adduct was used to synthesize N-(deoxyguanosin-8-yl)-N-acetylbenzidine and N'-(deoxyguanosin-8-yl)-N-acetylbenzidine. Nucleoside adducts from slices incubated with [3H]benzidine were analyzed by HPLC. With this method of analysis, the 3H-material did not correlate with the synthetic adduct standards. To improve sensitivity and identify liver adducts, a 32P-postlabeling method was developed. 2'-Deoxyguanosine 3'-monophosphate adduct standards of acetylated benzidine were prepared. 32P-Postlabeling analysis demonstrated that rat liver contained only N'-(3'-monophosphodeoxyguanosine-8-yl)-N-acetylbenzidine after a 1- or 4-h exposure to benzidine. In contrast, no acetylated adducts were detected in dog. Results indicate that dog is a nonacetylator with respect to benzidine. The availability of acetylated benzidine nucleotide standards allowed unambiguous identification of N'-(3'-monophosphodeoxyguanosin-8-yl)-N-acetylbenzidine as the adduct present in rat liver slices. These nucleotide adduct standards will be useful in subsequent studies in animals and human.

N-Glucuronidation is an important pathway in aromatic amine metabolism. This study assessed N-glucuronidation of N-acetylbenzidine by human liver slices and microsomes. With slices, considerable metabolism of [3H]N-acetylbenzidine (0.2 mM) was observed during a 2-hr incubation. N-Acetylbenzidine N'-glucuronide represented significant metabolism in four different human liver samples (6-33% of the total recovered radioactivity following HPLC). Benzidine (11-43%), benzidine N-glucuronide (8-11%), and N,N'-diacetylbenzidine (0-2%) were also formed. The kinetics of N-acetylbenzidine N'-glucuronide formation were investigated using Triton X-100-pretreated microsomes. Data were best described by a two-component Michaelis-Menten model composed of both high-affinity (low KM) and low-affinity (high KM) UDP-glucuronsyltranosferases. The high- and low-affinity KMs were 0.36 +/- 0.02 and 1.07 +/- 0.12 mM, respectively. To help identify the UDP-glucuronosyltransferases metabolizing N-acetylbenzidine, 23 transferase substrates were tested for their ability to inhibit glucuronidation. At 0.25 mM, bilirubin, estriol, and 17-epiestriol were good inhibitors (< 50% of control). Dose-response inhibition studies with estriol and 4-aminobiphenyl demonstrated that each agent reached a plateau as its concentration was increased. IC50 for estriol and 4-aminobiphenyl was 0.15 +/- 0.03 and 0.57 +/- 0.06 mM, respectively. Complimentary inhibition was observed when these agents were combined at maximal inhibitory concentrations. These results suggest that more than one UDP-glucuronosyltransferase metabolizes N-acetylbenzidine. N-Glucuronidation represents a major pathway for N-acetylbenzidine metabolism in humans.

Although glucuronidation is considered an important pathway in aromatic amine-induced bladder cancer, benzidine glucuronidation has not been assessed in humans. Glucuronidation of benzidine was assessed with human liver microsomes and slices. Emulgen 911-treated microsomes exhibited a Km for benzidine of 0.8 +/- 0.06 mM and a Vmax of 4.2 +/- 0.7 nmol/mg protein/min. A variety of agents were tested for their ability to inhibit benzidine N-glucuronide formation. At 0.25 mM, estriol, 17-epiestriol, bilirubin, hyodeoxycholic acid and cyproheptadine were good inhibitors (< 50% of control). Dose-dependent inhibition studies with estriol, testosterone and 4-aminobiphenyl demonstrated that each agent reached a plateau as its concentration was increased. When these agents were combined at maximal inhibitory concentrations, additive inhibition was observed. These results suggest that more than one UDP-glucuronosyltransferase metabolizes benzidine. The cDNA clones pUDPGTh-1 and -2 encode transferases which metabolize hyodeoxycholic acid and estrogen derivatives, but neither transferase catalyzed benzidine glucuronidation. Slices were used to assess metabolism by intact tissue and converted [3H]benzidine (0.09 mM) to N-acetyl-benzidine. N-Glucuronides of both benzidine and N-acetylbenzidine were observed and represented 14-37% of the total recovered radioactivity. The amount of N-acetylbenzidine N'-glucuronide observed was proportional to the amount of N-acetylbenzidine produced. Thus, N-glucuronidation appears to represent a major pathway for metabolism of benzidine in humans. The extent of N-acetylation affects the proportion of benzidine and N-acetylbenzidine glucuronidated by human liver slices.

The formation of thioether conjugates is an important mechanism for inactivation of carcinogens. 3-(Glutathion-S-yl)-benzidine (BZ-SG) formation prevents benzidinediimine and peroxidase-mediated benzidine binding to DNA. Benzidinediimine is the two-electron oxidized product of benzidine thought to be the reactive intermediate involved in peroxidase-mediated binding of benzidine to DNA. Diimine interacts with benzidine to form a dimeric complex known as the charge-transfer complex. The latter is in equilibrium with the cation radical. This study evaluated the mechanism by which BZ-SG forms. Benzidinediimine was synthesized and used to study the formation of BZ-SG. With 0.05 mM benzidinediimine, BZ-SG formation was optimum at pH 4.5 and with glutathione at 0.05 to 0.1 mM. By monitoring specific absorption spectra, the reduction of benzidinediimine at pH 4.5 was evaluated. The t1/2 for diimine decay (425 nm) and maximum absorbance of the charge-transfer complex (600 nm) were each at approximately 5 min. Within 10 min, the maximum amount of benzidine had formed from diimine. BZ-SG formation followed the decay of diimine. The relationship between benzidinediimine and benzidine, with respect to BZ-SG formation, was assessed at a fixed concentration of glutathione (0.05 mM) and a fixed total concentration of amine and diimine (0.05 mM). In three separate experiments, each of these three components was radiolabeled independent of the other two components. Experiments with [3H]glutathione indicated that conjugate formation was dependent upon diimine, and not benzidine. With [3H]benzidinediimine or [3H]benzidine, two different calculations were necessary to assess conjugate formation. For [3H]benzidinediimine, the calculation considered that only the radiolabeled diimine formed conjugate, while with [3H]benzidine, a specific activity calculation was necessary to demonstrate that conjugate formation was dependent upon diimine. With 0.05 mM [3H]benzidine, horseradish peroxidase-catalyzed formation of BZ-SG was optimum between 0.05 and 0.0625 mM H2O2. The latter is consistent with conversion of benzidine to diimine before formation of BZ-SG. Specific inhibitors and the absence of oxygen uptake indicated the lack of involvement of cation, thiyl, and carbon-centered radicals. The results are consistent with the existence of the charge-transfer complex and with benzidinediimine reacting with glutathione to form BZ-SG.

While N-glucuronidation is an important pathway for metabolism of aromatic amines, it has not been demonstrated for N-acetylbenzidine. A glucuronide of N-acetylbenzidine was synthesized and identified by mass spectrometry as N-acetylbenzidine-N'-glucuronide. This N'-glucuronide is acid labile with a t1/2 of 4 min at pH 5.3. A similar acid lability was also observed with benzidine-N-glucuronide. The formation of N-acetylbenzidine-N'-glucuronide was assessed with liver slices and microsomes prepared from human, dog and rat. When 0.014 mM [3H]N-acetylbenzidine was incubated with human liver slices a significant amount of N-acetylbendizine-N'-glucuronide was produced (8-26% of the total radioactivity recovered). With higher concentrations of [3H]N-acetylbenzidine (1 mM) rat slices also produced N-acetylbenzidine-N'-glucuronide. However, N'-glucuronide formation was not detected with dog liver slices incubated with either 0.014 or 1 mM [3H]N-acetylbenzidine. N-Acetylbenzidine-N'-glucuronide formation was observed with microsomes prepared from human, dog and rat. To assess maximum activity four detergents were used at two concentrations. With or without detergent activation the relative amount of glucuronidation was human > > dog > rat. The rate of benzidine N-glucuronide formation was 4.3- and 1.6-fold greater than N-acetylbenzidine-N'-glucuronide in dog and rat respectively, while in human both rates were similar (1.1-fold). With or without detergent activation the relative amount of benzidine-N-glucuronide formation was human > dog > > rat. N-Glucuronidation of [3H]N,N'-diacetylbenzidine was not observed. Thus N-actylbenzidine-N'-glucuronide formation appears to be an important pathway for metabolism of N-acetylbenzidine, especially in humans. Due to their acid lability, formation of the N-glucuronides of N-acetylbenzidine and benzidine provides a mechanism for hepatic detoxification and accumulation of these carcinogens in the bladder. A new model is described illustrating the effect of N-glucuronidation and the influence of N-acetylation on arylmono- and aryldiamine-induced bladder carcinogenesis.

Phenylbutazone, a nonsteroidal anti-inflammatory drug, elicits therapeutic as well as toxic effects by unknown pathways. Phenylbutazone was shown to form a conjugate with the heterocyclic amine bladder carcinogen 2-amino-4-(5-nitro-2-furyl)-thiazole (ANFT). To understand further the reactivity of these compounds, this study was conducted to identify the conjugate formed and determine the mechanism of conjugate formation. Both prostaglandin H synthase and horseradish peroxidase catalyzed conjugate formation. This conjugate was identified by 1H-NMR to be 4-[2-amino-4-(5-nitro-2-furyl)-5-thiazolyl]-4-butyl-1,2-diphenyl-3,5- pyrazolidinedione. Phenylbutazone-mediated oxygen uptake was inhibited by ANFT (0.1 mM) and the spin traps 5,5-dimethyl-1-pyrroline-N-oxide (200 mM) and tert-nitrosobutane (4 mM). By contrast, phenol (0.005 to 0.25 mM) and aminopyrine (0.4 mM) stimulated oxygen uptake. None of these agents mediated oxygen uptake in the absence of phenylbutazone. Conjugate formation was significantly increased by phenol (0.005-0.25 mM) and aminopyrine (0.4 mM), as well as in the absence of oxygen. Conjugate formation was inhibited by 5,5-dimethyl-1-pyrroline-N-oxide (200 mM), tert-nitrosobutane (4 mM), ascorbic acid (2 mM), and 95% oxygen. Horseradish peroxidase initiated conjugate formation at much lower concentrations than it metabolized ANFT. The stoichiometric relationship between phenylbutazone and ANFT, with respect to conjugate formation, was complex. With the concentration of ANFT fixed at 0.05 mM, phenylbutazone exhibited saturation kinetics with a Km of 0.2 mM. In contrast, saturation kinetics were not observed with ANFT.Km values for ANFT varied with the concentration of phenylbutazone used.(ABSTRACT TRUNCATED AT 250 WORDS)

Dog is an animal model for assessing aromatic amine-induced bladder cancer, and hepatic N-glucuronidation is proposed as an important pathway leading to initiation of carcinogenesis. Therefore, benzidine N-glucuronidation was evaluated with dog liver microsomes and slices. Microsomal benzidine UDP-glucuronosyltransferase activity was increased with a variety of detergents. For kinetic analysis, native microsomal preparations were separated into treated (detergent treated, not centrifuged) or soluble (detergent treated, centrifuged) fractions. The detergents Triton X-100, Lubrol PX, Emulgen 911 and CHAPS increased the specific activity of treated fractions relative to the native microsomes 3- to 6-fold. The specific activities of the soluble fractions were highest with Emulgen 911 and CHAPS at a detergent-to-protein ratio of 1. Subsequent studies used Emulgen 911 or CHAPS. Similar results were observed with either preparation. For treated preparations, the Km and Vmax values were 0.142 +/- 0.006 mM and 0.65 +/- 0.1 nmol/mg protein/min respectively. A variety of chemicals were tested for their effect on benzidine N-glucuronide formation. At 0.1 mM, the only effective inhibitors (< 50% of control) were 2-aminofluorene, estriol, 17-epiestriol, 2-OH-estrone, and 4-OH-estrone. With Emulgen-treated microsomes, the Ki values for 2-aminofluorene, 4-aminobiphenyl and estriol were 0.114 +/- 0.014, 0.347 +/- 0.032 and 0.047 +/- 0.003 mM respectively. 2-Aminofluorene and estriol were non-competitive inhibitors, while 4-aminobiphenyl was a competitive inhibitor. Slices incubated with these chemicals exhibited an inhibition profile similar to that observed with microsomes. Thus, N-glucuronidation of benzidine may be an important metabolic pathway in dog. Inhibition of benzidine N-glucuronidation by estriol and catechol estrones may be important in vivo events in aromatic amine-induced carcinogenesis.

Evaluating the diluting and concentrating capacity of the kidney can be accomplished with three basic laboratory tests, urine and plasma osmolalities, and circulating levels of antidiuretic hormone. When used with simple techniques to stimulate dilution and concentration, these tests will provide critical clinical information in patients with defects in either dilution or concentration of the urine.