Phase 2 metabolites of H-hydroxylated amidines (amidoximes): Synthesis, invitro formation by pig hepatocytes, and mutagenicity testing

A pig hepatocyte culture system was used for phase 2 biotransformation stud ies in vitro. The viability of the cultured hepatocytes was characterized d aily during cultivation by lactate dehydrogenase release into the supernata nt and albumin synthesis of the cells. The metabolic activity of the hepato cyte cultures was measured by 7-ethoxycoumarin (ECOD) and 7-ethoxyresorufin O-deethylation (EROD). The viability and metabolic activity of these pig h epatocytes were preserved for several days by culturing the cells in a mono layer culture system. Besides the known reduction of N-hydroxylated benzami dine (benzamidoxime) (2) to benzamidine (1), glucuronidation and, to a much smaller extent, sulfation of 2 to benzamidoxime O-glucuronide (3) and benz amidoxime O-sulfate (4) by cultured pig hepatocytes were found. The analyse s were performed using HPLC and LC/MS studies. For unequivocal assignment, the hitherto unknown metabolites 3 and 4 were synthesized and characterized by spectroscopic techniques. Examination of benzamidoxime O-glucuronide an d benzamidoxime O-sulfate for mutagenicity by means of the Ames test reveal ed that both phase 2 conjugates have no mutagenic effects in the TA98 and T A100 strains. So the phase 2 conjugation of benzamidoxime is significant in terms of detoxification.