Vijaya M. Lakshmi

Glucuronide conjugates of 4-aminobiphenyl and its N-hydroxy metabolites: pH stability and synthesis by human and dog 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.

Peroxygenase Metabolism of N-Acetylbenzidine by Prostaglandin H Synthase FORMATION OF AN N-HYDROXYLAMINE

Synthesis of prostaglandin H2by 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 mmH2O2 was used as the substrate. This metabolite co-eluted with syntheticN′-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 two18O 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.

Identification of New 2-Amino-3-methylimidazo[4,5-f]quinoline Urinary Metabolites from β-Naphthoflavone-Treated Mice

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 β-naphthoflavone (BNF). Following an oral dose of 40 mg/kg 14C-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.

Characterization of new metabolites from in vivo biotransformation of 2-amino-3-methylimidazo[4,5-f]quinoline in mouse by mass spectrometry.

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.

Liver NADPH-dependent oxidation of the 5-nitrofurans, FANFT and ANFT, by guinea pig and rat.

1. Oxidative metabolism of the bladder carcinogens FANFT/ANFT was examined in vitro in guinea pig (resistant species) relative to rat (susceptible species). 2. The total rate of ANFT hepatic metabolism by guinea pig (soluble metabolites plus protein bound, 354 pmol/min per mg protein) was approx. 4 times that in rat. 3. The total rate of FANFT metabolism was similar in both species and approx. one-quarter that for ANFT in guinea pig. In rat, the rate of total metabolism of FANFT and ANFT was similar. 4. Cytochrome P450 inhibitors, 2,4-dichloro-6-phenylphenoxyethylamine, 7,8-benzoflavone, and n-octylamine largely inhibited metabolism in guinea pig, but had little effect in rat. 5. H.p.l.c. analysis of ANFT metabolites indicated distinctly different products in guinea pig compared to rat. 7,8-Benzoflavone decreased metabolite formation by 80% in guinea pig, but only 30% in rat. 6. Flavin-dependent monooxygenases may participate in metabolism of these carcinogens in rat, but not guinea pig. 7. Because ANFT is thought to be a more proximate carcinogen than FANFT, the increased rate of ANFT metabolism and the formation of different products in guinea pig compared to rat may partially explain the resistance of guinea pig to FANFT-induced bladder cancer.

Benzidine N-Glucuronidation by Human, Rat, and Dog Liver

Abstract Hepatic N-glucuronidation is proposed to be an important pathway for metabolism of aromatic amine carcinogens and was assessed in human, rat, and dog. Benzidine and its monoacetylated product N-acetylbenzidine were glucuronidated. The latter was identified as an N′-glucuronide. Both glucuronides were acid labile with t1/2s of approximately 5 min at pH 5.3 and 37 °C. In plasma, the stability of both glucuronides increased. Irrespective of the detergent used to increase microsomal activity, the relative amount of glucuronidation was human > dog > rat. N-Glucuronidation of N,N′-diacetylbenzidine was not detected. Following incubation with 3H-benzidine, liver slices from all three species produced N-glucuronides. However, only human and rat slices produced N-acetylbenzidine N′-glucuronide. The stability of these N-glucuronides in plasma and their lability in acidic urine provides a mechanism by which these detoxified products are transported to the bladder and then recycled to the arylamine. A model ...