Vol. 27, Issue 9, 1064-1067, September 1999

Human and Escherichia coli -Glucuronidase Hydrolysis of Glucuronide Conjugates of Benzidine and 4-Aminobiphenyl, and their Hydroxy Metabolites

Terry V. Zenser, Vijaya M. Lakshmi, and Bernard B. Davis

Veterans Administration Medical Center and Edward A. Doisy Department of Biochemistry and Division of Geriatric Medicine, St. Louis University School of Medicine, St. Louis, Missouri

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 -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 ³H- and ¹⁴C-labeled glucuronides were prepared with human or rat liver microsomes using UDP-glucuronic acid as cosubstrate. Each of the 10 glucuronides (6-12 µM) was incubated at pH 5.5 or 7.0 with either human recombinant (pure) or E. coli (commercial preparation) -glucuronidase for 30 min at 37°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 -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 -glucuronidase inhibitor saccharic acid-1,4-lactone (0.5 mM). The concentration of human -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.