STUDIES OF THE UPTAKE, HEPATIC METABOLISM AND SECRETION OF AZO DYES; CHARACTERIZATION OF AN AZO DYE BINDING PROTEIN FROM RAT BILE

Abstract

The radiolabeled carcinogens, N, N-dimethyl-4-aminoazobenzene (DAB) and its methylated derivative, 3'-MeDAB, are cleared from the circulation of rats within 5 min of i.v. administration. Forty and 65% of the 3'-MeDAB derived radioactivity are recovered in the liver and bile 30 and 120 min after administration, suggesting the existence of an extrahepatic site which sequesters the azo dye derived radioactivity from the circulation and slowly releases it during the course of the experiment. Only 25% of the DAB derived radioactivity is recovered at these two times.;When analyzed by thin layer chromatography, unique patterns of the azo dye metabolites are isolated from the liver and bile of rats administered either carcinogen, indicating that specific metabolites may be transported from the liver to the bile. In studies using the isolated, perfused rat liver, 3'-MeDAB derived radioactivity is found associated with several biliary proteins only after the addition of these proteins to the perfusate, suggesting that biliary proteins may be involved in the metabolite transport process.;One of the biliary proteins binds approximately 40% of the total 3'-MeDAB derived radioactivity as determined by gel filtration chromatography. This azo dye metabolite binding protein, MBP (50,000 daltons), was purified to homogeneity and was characterized by several physical and immunochemical techniques. Only 2% of the azo dye metabolites appear to covalently bind MBP as determined by recovery of radioactivity following electrophoresis of CNBr digests of the protein.;Evidence that suggests that MBP is found in the liver and may be involved in the azo dye metabolite transport from the liver to the bile is that (1) MBP is detected by immunoprecipitation during in vitro translation studies using rat liver MRNA and (2) MBP azo dye metabolite adducts are immunoprecipitable from the liver cytosol of {lcub}('14)C{rcub}3'-MeDAB administered rats. In addition, biliary MBP does not appear to be derived from the circulation within 2 hr following i.v. administration of the iodinated protein, unlike most other biliary proteins. Taken together, these studies are the first to suggest a role for biliary protein in azo dye clearance from the liver.;MBP binds 5 g-atoms Cu/mol. Possible interactions between azo dye metabolite and Cu binding were not determined. A role in Cu disposition by MBP is discussed.