Characterization of biliary conjugates of 4,4′-methylenedianiline in male versus female rats

Abstract

4,4’-Methylenedianiline (4,4’-diaminodiphenylmethane; DAPM) is an aromatic diamine used in the production of numerous polyurethane foams and epoxy resins. Previous studies in rats revealed that DAPM initially injures biliary epithelial cells of the liver, that the toxicity is greater in female than in male rats, and that the toxic metabolites of DAPM are excreted into bile. Since male and female rats exhibit differences in the expression of both phase I and phase II enzymes, our hypothesis was that female rats either metabolize DAPM to more toxic metabolites or have a decreased capacity to conjugate metabolites to less toxic intermediates. Our objective was thus to isolate, characterize, and quantify DAPM metabolites excreted into bile in both male and female bile duct-cannulated Sprague Dawley rats. The rats were gavaged with [14C]-DAPM, and the collected bile was subjected to reversed-phase HPLC with radioisotope detection. Peaks eluting from HPLC were collected and analyzed using electrospray MS, NMR and FT-IR spectroscopy. HPLC analysis indicated numerous metabolites in both sexes, but male rats excreted greater amounts of glutathione and glucuronide conjugates than females. Electrospray MS and NMR spectra of HPLC fractions revealed that the most prominent metabolite found in bile of both sexes was a glutathione conjugate of an imine metabolite of a 4’-nitroso-DAPM. Seven other metabolites were identified, including acetylated, cysteinyl-glycine, glutamyl-cysteine, glycine, and glucuronide conjugates. While our prior studies demonstrated increased covalent binding of DAPM in the liver and bile of female compared to male rats, in these studies, SDS-PAGE with autoradiography revealed 4–5 radiolabeled protein bands in the bile of rats treated with [14C]-DAPM. In addition, these bands were much more prominent in female than in male rats. These studies thus suggest that a plausible mechanism for the increased sensitivity of female rats to DAPM toxicity may be decreased conjugation of reactive DAPM metabolites, leading to greater levels of protein adduct formation.

Liquid chromatography–electrospray tandem mass spectrometry investigations of fragmentation pathways of biliary 4,4′-methylenedianiline conjugates produced in rats

“…As described previously, [13] biliary metabolites of DAPM have been observed using LCradioisotope detection. Either off-line (fraction collection) or on-line LC/MS was carried out with mass spectrometric characterization of the metabolites.…”

“…Both involve N-oxidation and hydroxylation of the parent DAPM compound, along with glutathione conjugation on M2, or glucuronic acid and glycine adduction on M7. Although structural characterizations were accomplished on both metabolites via various spectroscopic techniques, [13] their detailed fragmentation mechanisms based upon MS n data are presented here for the first time.…”

“…The existence of phase I reactive metabolites of DAPM supports the postulation that reactive intermediates are involved in the toxicity of DAPM, possibly implicating covalent protein adducts that were also found in rat bile. [13] Further studies are in progress to characterize those DAPM modified proteins. Proposed fragmentation pathways leading to the MS 3 product ions at m/z 241 and 267, and the MS 4 product ions at m/z 106, 132 and 148…”

“…[12] This biotransformation of DAPM into a reactive imine was speculated to be catalyzed by extrahepatic peroxidase enzyme. We recently reported the characterization of biliary metabolites of DAPM using various spectroscopic techniques [13]. DAPM was found to be converted by phase I and II biotransformation generating N-oxidation, hydroxylation, acetylation, glucuronic acid and glutathione conjugation products.…”

Comparative Proteomic Analysis of Liver Steatosis and Fibrosis after Oral Hepatotoxicant Administration in Sprague-Dawley Rats

“…Liver toxicity resulting from 4,4 0 -MDA exposure is well-documented. Occupational and accidental human 4,4 0 -MDA exposures have resulted in jaundice, toxic hepatitis, and cholestasis (Chen et al 2008). Rats administered 25 mg/kg/ week 4,4 0 -MDA subchronically developed portal vein hyperplasia, bile duct fibrosis, and necrosis of biliary epithelial cells (Dugas et al 2004).…”