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

Abstract

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.

In Vitro Metabolism of the Novel, Highly Selective Oral Angiogenesis Inhibitor Motesanib Diphosphate in Preclinical Species and in Humans

“…M1 was isolated and purified from scaled-up incubations of motesanib with human liver microsomes and UDP-GA, and its structure was fully characterized by LC/MS and NMR. M1 was very labile and susceptible to cleavage back to motesanib and glucuronic acid under very mild acidic conditions, which is consistent with reports for other unstable and acid labile primary and secondary amine N-glucuronides (Babu et al, 1992;Green and Tephly, 1998). In general, glucuronidation is considered a key pathway for detoxifying and eliminating drugs; thus, it is important to more fully understand the mechanism of N-glucuronidation, the enzymes involved, and their regulation.…”

AMIDE N-GLUCURONIDATION OF MAXIPOST CATALYZED BY UDP-GLUCURONOSYLTRANSFERASE 2B7 IN HUMANS

“…Subsequently, many primary, secondary, and tertiary amines have been shown to be excreted as N-glucuronide metabolites (Green and Tephly, 1998). N-Glucuronidation of many primary and secondary aromatic amines has been suggested to have implications in the potential carcinogenicity of these compounds (Babu et al, 1992;Kadlubar et al, 1997). N-Glucuronidation of tertiary amines to quaternary ammonium-linked glucuronide metabolites contributes to the elimination of many therapeutic agents like tricyclic antidepressants and antihistamines.…”

“…The glucuronide of BMS-204352 was the first example of glucuronidation linkages at amide nitrogen that was unambiguously identified by NMR. Although N-glucuronide metabolites of the primary/secondary amines and N-glucuronides of N-hydroxylated amines were hydrolyzed to the parent compounds and glucuronic acid under mild, acidic conditions (Babu et al, 1992;Kadlubar et al, 1997), the quaternaryammonium glucuronides were hydrolyzed under basic conditions (Dulik and Fenselau, 1987;Kowalczyk et al, 2000). Among the several quaternary-ammonium glucuronides tested for stability in the presence of ␤-glucuronidase from bovine liver, mollusks (H. pomatia), and bacteria (E. coli), only E. coli enzyme hydrolyzed all Nglucuronides (Kowalczyk et al, 2000).…”

Glucuronidation of Active Tamoxifen Metabolites by the Human UDP Glucuronosyltransferases

“…For optimal resolution of 4-OH-TAM and endoxifen isomers, the HPLC gradient used in the present kinetic study was pH 5.0. It is possible that endoxifen-N- glucuronides were deconjugated during the HPLC elution process at this pH, since N-glucuronides of secondary amines are susceptible to cleavage under mildly acidic conditions (Babu et al, 1992). When assays were run on a HPLC gradient at pH 7.4, only single glucuronide peaks corresponding to peaks 1 and 3 (for trans-and cisendoxifen, respectively) were observed for both HLMs and UGToverexpressing cells, and these glucuronide peaks were stable in 1 M HCl but sensitive to ␤-glucuronidase treatment (data not shown).…”

“…Kinetic Analysis of 4-OH-TAM and Endoxifen Glucuronidation by Human UGTs. In addition to the newly made UGT1A3 and UGT1A8 cell lines described in this report, cell lines overexpressing UGTs 1A1, 1A4, 1A6, 1A7, 1A9, 1A10, 2B4, 2B7, 2B10, 2B11, 2B15, and 2B17 were used for this analysis as described previously (Babu et al, 1992; G. Chen, R. Dellinger, D. Sun, T. Spratt, and P. Lazarus, manuscript submitted for publication). Of the UGTs tested in this study, UGTs 1A1, 1A3, 1A7, 1A8, 1A9, 1A10, 2B7, 2B15, and 2B17 exhibited detectable levels of O-glucuronidating activity against trans-and cis-4-OH-TAM (Table 1).…”