Abstract

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 coliβ-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, andN-hydroxy-N-acetyl-4-aminobiphenyl. The³H- 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) β-glucuronidase for 30 min at 37°C. Hydrolysis was measured by HPLC. Reaction conditions were optimized, using the O-glucuronide ofN-hydroxy-N,N′-diacetylbenzidine. Both enzymes preferentially hydrolyzed O-glucuronides overN-glucuronides and distinguished between structural isomers. With E. coli β-glucuronidase at pH 7.0, selectivity was demonstrated by the complete hydrolysis ofN-hydroxy-N-acetyl-4-aminobiphenylO-glucuronide in the presence ofN-acetylbenzidine N-glucuronide, which was not hydrolyzed. Metabolism by both enzymes was completely inhibited by the specific β-glucuronidase inhibitor saccharic acid-1,4-lactone (0.5 mM). The concentration of human β-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 notN-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.