Human and Escherichia coli beta-glucuronidase hydrolysis of glucuronide conjugates of benzidine and 4-aminobiphenyl, and their hydroxy metabolites

Individuals exposed to carcinogenic aromatic amines excrete arylamine N- an d O-glucuronide metabolites. This study assessed the susceptibility of sele cted glucuronides to hydrolysis by human and Escherichia coil beta-glucuron idase. N- or O-glucuronides were prepared with the following aglycones: ben zidine, N-acetylbenzidine , N'-hydroxy-N-acetylbenzidine, N-hydroxy-N-acety lbenzidine, N-hydroxy-N,N'-diacetylbenzidine, 3-hydroxy-N,N'-diacetylbenzid ine, a-hydroxy-benzidine, 4-aminobiphenyl, N-hydroxy-4-aminobiphenyl, and N -hydroxy-N-acetyl-4-aminobiphenyl. The H-3- and C-14-labeled glucuronides w ere prepared with human or rat liver microsomes using UDP-glucuronic acid a s cosubstrate. Each of the 10 glucuronides (6-12 mu M) was incubated at pH 5.5 or 7.0 with either human recombinant (pure) or E. coil (commercial prep aration) beta-glucuronidase for 30 min at 37 degrees C. Hydrolysis was meas ured by HPLC. Reaction conditions were optimized, using the O-glucuronide o f N-hydroxy-N,N'-diacetylbenzidine. Both enzymes preferentially hydrolyzed O-glucuronides over N-glucuronides and distinguished between structural iso mers. With E. coil beta-glucuronidase at pH 7.0, selectivity was demonstrat ed by the complete hydrolysis of N-hydroxy-N-acetyl-4-aminobiphenyl O-glucu ronide in the presence of N-acetylbenzidine N-glucuronide, which was not hy drolyzed. Metabolism by both enzymes was completely inhibited by the specif ic beta-glucuronidase inhibitor saccharic acid-1,4-lactone (0.5 mM). The co ncentration of human beta-glucuronidase necessary to achieve significant hy drolysis of glucuronides was substantially more than the amount of enzyme r eported previously to be present in urine under either normal or pathologic al 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 a mine than enzymatic hydrolysis.