Structure elucidation and in vitro reactivity of the major metabolite of 4,4'-methylenebis(2-chloroaniline) (MBOCA) in canine urine.
The structure of the major urinary metabolite of 4,4'-methylenebis(2-chloroaniline) (MBOCA) in dogs was identified and the reactivity of the metabolite was characterized in vitro. Arylsulfatase but not beta-glucuronidase hydrolyzed the metabolite in a time- and enzyme concentration-dependent manner. Electron impact mass spectrometry following derivatization and transesterification indicated that the major metabolite was ring hydroxylated and fast atom bombardment mass spectrometry confirmed the molecular weight as a sulfate ester. Proton nuclear magnetic resonance studies indicated that the ring substitution was ortho to an amine. These analytical and enzymatic data supported the proposed structure of the major urinary metabolite of MBOCA in dogs as 5-hydroxy-3,3'-dichloro-4,4'diaminodiphenylmethane-5-sulfate. Protein and DNA binding in vitro and mutagenicity were investigated. During hydrolysis with arylsulfatase, time- and enzyme concentration-dependent protein binding and time-dependent DNA binding were observed. Mutagenicity during enzymatic hydrolysis in the presence of Salmonella typhimurium TA1538 with up to 20 micrograms metabolite/plate was negative and at 50 micrograms/plate the metabolite was cytotoxic. These results indicated that the metabolite was the sulfate conjugate of a reactive molecule. This study demonstrated that the major metabolite of MBOCA in canine urine is an orthohydroxysulfate and thus is similar to the major metabolites of benzidine, 2-naphthylamine, and 4-aminobiphenyl.
D000818:Animals; D001192:Arylsulfatases / Q000378:metabolism; D001559:Benzhydryl Compounds / Q000302:isolation & purification* / Q000652:urine*; D001711:Biotransformation; D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000378:metabolism; D004285:Dogs; D006868:Hydrolysis; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008753:Methylenebis(chloroaniline) / Q000031:analogs & derivatives / Q000302:isolation & purification* / Q000633:toxicity / Q000652:urine*; D009152:Mutagenicity Tests; D011485:Protein Binding
D001559:Benzhydryl Compounds; D008753:Methylenebis(chloroaniline); D004247:DNA; C044313:5-hydroxy-3,3'-dichloro-4,4'-diaminodiphenylmethane-5-sulfate; D001192:Arylsulfatases