Satram R. Babu

Saint Louis University

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Terry V. Zenser

Saint Louis University

Bernard B. Davis

University of Pittsburgh

Vijaya M. Lakshmi

North Eastern Indira Gandhi Regional Institute of Health and Medical Sciences

Fong‐Fu Hsu

Washington University in St. Louis

Robert E. Kane

Saint Louis University

Ida S. Owens

Eunice Kennedy Shriver National Institute of Child Health and Human Development

George Pen-Wen Huang

Saint Louis University

V.Jon Wongsurawat

Saint Louis University

Cooperative Institutions

Saint Louis University

St. Louis VA Medical Center

Geriatric Research Education and Clinical Center

Veterans Health Administration

Saint Louis University Hospital

UCLouvain Saint-Louis Brussels

United States Department of Veterans Affairs

University of Cincinnati

Gemological Institute of America

Cardinal Glennon Children’s Medical Center

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N-Acetylbenzidine-N'-glucuronidation by human, dog and rat liver

Carcinogenesis (1993)

Satram R. Babu Vijaya M. Lakshmi Fong‐Fu Hsu Robert E. Kane Terry V. Zenser

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 t½ 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-acetylbenzidine-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-acetylbenzidine-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.

Glucuronide

Benzidine

10.1093/carcin/14.12.2605

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Citations (30)

Human liver glucuronidation of benzidine

Carcinogenesis (1994)

Satram R. Babu Vijaya M. Lakshmi Ida S. Owens Terry V. Zenser Bernard B. Davis

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.

Benzidine

Glucuronide

Glucuronosyltransferase

10.1093/carcin/15.9.2003

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Citations (17)

Glucuronide conjugates of 4-aminobiphenyl and its N-hydroxy metabolites

Biochemical Pharmacology (1996)

Satram R. Babu Vijaya M. Lakshmi George Pen-Wen Huang Terry V. Zenser Bernard B. Davis

Glucuronide

Benzidine

Conjugate

10.1016/0006-2952(96)00165-7

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Citations (57)

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

Polycyclic aromatic compounds (1994)

Terry V. Zenser Vijaya M. Lakshmi Satram R. Babu Fong‐Fu Hsu Robert E. Kane

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 is presented illustrating the effect of N-glucuronidation and the influence of N-acetylation on arylmono- and aryldi- amine-induced bladder carcinogenesis.

Benzidine

Glucuronide

10.1080/10406639408014724

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Citations (0)

Glucuronidation of N-hydroxy metabolites of N-acetylbenzidine

Carcinogenesis (1995)

Satram R. Babu Vijaya M. Lakshmi Fong‐Fu Hsu Terry V. Zenser Bernard B. Davis

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.

Glucuronide

10.1093/carcin/16.12.3069

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Citations (36)

Benzidine glucuronidation in dog liver

Carcinogenesis (1993)

Satram R. Babu V.Jon Wongsurawat Terry V. Zenser Bernard B. Davis

Journal Article Benzidine glucuronidation in dog liver Get access Satram R. Babu, Satram R. Babu VA Medical Center, and Department of Biochemistry and Division of Geriatric Medicine, St Louis University School of MedicineSt Louis, MO, USA Search for other works by this author on: Oxford Academic PubMed Google Scholar V.Jon Wongsurawat, V.Jon Wongsurawat VA Medical Center, and Department of Biochemistry and Division of Geriatric Medicine, St Louis University School of MedicineSt Louis, MO, USA Search for other works by this author on: Oxford Academic PubMed Google Scholar Terry V. Zenser, Terry V. Zenser 1 VA Medical Center, and Department of Biochemistry and Division of Geriatric Medicine, St Louis University School of MedicineSt Louis, MO, USA 1To whom correspondence should be addressed Search for other works by this author on: Oxford Academic PubMed Google Scholar Bernard B. Davis Bernard B. Davis VA Medical Center, and Department of Biochemistry and Division of Geriatric Medicine, St Louis University School of MedicineSt Louis, MO, USA Search for other works by this author on: Oxford Academic PubMed Google Scholar Carcinogenesis, Volume 14, Issue 5, May 1993, Pages 893–897, https://doi.org/10.1093/carcin/14.5.893 Published: 01 May 1993 Article history Received: 17 November 1992 Revision received: 15 January 1993 Accepted: 03 February 1993 Published: 01 May 1993

Benzidine

Estriol

Glucuronide

2-Acetylaminofluorene

Microsoma

Chaps

10.1093/carcin/14.5.893

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Citations (13)

Role of N-ghicuronidation in benzidine-induced bladder cancer in dog

Carcinogenesis (1992)

Satram R. Babu Vijaya M. Lakshmi Fong‐Fu Hsu Terry V. Zenser Bernard B. Davis

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.

Benzidine

Glucuronide

10.1093/carcin/13.7.1235

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Citations (26)