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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.

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.

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.

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.

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.

The mechanism by which benzidine induces bladder cancer in dog was evaluated by assessing metabolism of [3H]benzidine by dog liver slices and microsomes. Slices incubated with 0.05 mM [3H]benzidine exhibited a 32.5 min incubated with 0.05 mM [3H]benzidine exhibited a 32.5 min peak, which was also produced when microsomal incubations were supplemented with UDP-glucuronic acid. In contrast to microsomes, very little of the 32.5 min peak was produced with the 100,000 g supernatant fraction. Microsomal metabolism was increased 5-fold by pretreatment with Triton X-100. Very little activity was observed with rat microsomes in either the presence or absence of Triton X-100. This metabolite was also generated by incubating benzidine with glucuronic acid at 4 degrees C for 3 days. Thermospray MS identified this metabolite as benzidine N-glucuronide. At 37 degrees C, the t1/2 stability of purified N-glucuronide was 99, 25 and 3 min in dog urine adjusted to pH 7.3, 6.3 and 5.3 respectively. The N-glucuronide was quite stable at pH 9.3, in dog plasma, and in aprotic solvents for 4 h at 37 degrees C. Relative to benzidine, its N-glucuronide is weakly bound to plasma proteins but not more reactive with DNA. Thus, detoxification by liver provides a mechanism for accumulation of benzidine in acidic urine, uptake of benzidine into bladder epithelium, and activation of benzidine in bladder. The liver and N-glucuronidation play a potentially important role in the species specificity of benzidine carcinogenesis.