Glutathione Depletion Exacerbates Methylenedianiline Toxicity to Biliary Epithelial Cells and Hepatocytes in Rats

Abstract

Methylenedianiline (DAPM) initially injures epithelial cells of major bile ducts, which is followed by cholestasis, cholangitis, and hepatocellular damage. This pattern of biliary injury resembles that produced by alpha-naphthylisothiocyanate (ANIT), a classic bile duct toxicant. Our goal was to determine whether prior depletion of hepatic total glutathione (GSx), a condition reported to protect against biliary tract injury by ANIT, would also protect against DAPM-induced bile duct injury. A new protocol for extensive, sustained depletion of GSx was established. We found that administration of 1-bromoheptane followed 1 h later by buthionine sulfoximine resulted in an approximately 96% depletion of hepatic GSx that persisted through 6 h without biochemical or morphological signs of hepatic injury. Treatment of rats with a minimally hepatotoxic dose of DAPM (without GSx depletion) produced at 6 h injury similar to previous studies: moderate oncosis of biliary epithelial cells (BEC), mild edema of portal triads, and increases in glutathione S-transferase (GST) activities without alterations in hepatic GSx/glutathione disulfide (GSSG), coenzyme A (CoASH)/coenzyme A-glutathione disulfide (CoASSG), or thiobarbituric acid-reactive substances (TBARS). In contrast, DAPM treatment of GSx-depleted rats produced severe oncosis of BEC, marked inflammatory and edematous alterations to portal tracts, and oncosis/apoptosis in scattered hepatocytes. The observed acceleration and enhancement of DAPM-induced liver injury by GSx depletion was associated with a concurrent sevenfold increase in hepatic CoASSG and a fourfold decrease in the ratio of CoASH to CoASSG, compounds presumably localized to mitochondria and a purported index of mitochondrial thiol/disulfide status. These results indicate that: (1) GSx depletion exacerbates BEC and hepatocellular injury induced by DAPM, and (2) the mechanism by which DAPM causes liver injury is likely different from that of the classic bile duct toxicant, ANIT.

4,4′-Methylenedianiline-Induced Hepatotoxicity Is Modified by N-Acetyltransferase 2 (NAT2) Acetylator Polymorphism in the Rat

“…Other studies have found that metabolic activation is required and that oxidation plays an important and complex role in MDA toxicity (Bailie et al, 1993;Kautiainen et al, 1998). Glutathione depletion exacerbates MDA toxicity in rat biliary epithelial cells and hepatocytes, which suggest that glutathione might play an important cytoprotection role in MDA hepatotoxicity (Kanz et al, 2003). It has been suggested (Kautiainen et al, 1998) that, in analogy with oxidation of other diamines such as benzidine, a reactive intermediate [(4-imino-2,5-cyclohexadien-1-ylidene)-methyl]-4-amino-benzene for hemoglobin adducts is formed through peroxidative oxidation.…”

Inchinkoto, a herbal medicine, and its ingredients dually exert Mrp2/MRP2-mediated choleresis and Nrf2-mediated antioxidative action in rat livers

“…The maintenance of relevant GSH levels in the liver and bile duct is critical for hepatobiliary function. The depletion of GSH in the liver and bile duct epithelial cells has been known to induce severe liver injury and cholestasis (23,24); and 3) GSH is the main driving force of bile acid-independent bile formation, which occupies a large part of bile formation, and therefore the dysfunction of Mrp2/MRP2 results in the loss of bile acid-independent bile formation. The lack of bile acidindependent bile formation may not induce severe cholestasis, at least in naive animals.…”

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

“…Since DTT was able to completely strip our radiolabel from the observed bands (Figure 8), it is reasonable to assume that proteins identified on the gel are bound through thiol residues. Taken together with the preponderance of conjugates derived from glutathione or related pathways, our prior studies suggesting the importance of glutathione levels in protecting against DAPM-induced hepatobiliary toxicity (Kanz et al, 2003), as well as the fact that the expression levels of many isoforms of glutathione- S -transferases are 2–3-fold higher in males than in females (Srivastava, 1993), it is reasonable to assume that the sex-dependent differences in sensitivity to DAPM toxicity may be due to a diminished capacity to form glutathione and other conjugates. Both acetylated (M5) and glucuronyl metabolites (M6 and M7) were shown to be present in larger concentrations in the bile of males as compared to females, suggesting that deficiencies in these conjugation pathways might also be critical for the increased sensitivity of female rats to DAPM.…”