A Minimally Toxic Dose of Methylene Dianiline Injures Biliary Epithelial Cells in Rats

The threshold for hepatotoxicity and cholestasis induced by methylene dianiline (DAPM) in rats is between 25 and 75 mg/kg (Bailieet al., Environ. Health Perspect.,124, 25–30, 1993). Our objectives were to determine if a minimally toxic dose of DAPM provided a model system for studies of selective injury to biliary epithelial cells (BEC)in vivo.Thus, we examined the effects of 50 mg DAPM/kg on (1) biliary constituents, (2) liver constituents likely involved in DAPM biotransformation/detoxification, and (3) early morphological and histochemical changes in the liver. Male Sprague Dawley rats had biliary cannulas positioned under pentobarbital anesthesia. After 1 h of control bile collection, rats received 50 mg DAPM/kg po in 35% ethanol or 35% ethanol only. Bile was collected for another 6 h. Histochemical, ultrastructural, and biochemical liver alterations were assessed at 3 h or at 3 and 6 h. DAPM had minimal effects on biliary bile salt and bilirubin excretion over 6 h. Biliary glucose and protein excretion were increased ∼2-fold starting in Hour 1, while inorganic phosphate excretion was not increased until Hour 2. Biliary glutathione excretion initially increased (Hour 1) but then declined steadily for 5 h. Microsomal cytochrome P-450 activities were transiently decreased at 3 h but had returned to control values by 6 h. Liver glutathione (GSH and GSSG) was not affected by DAPM at 3 or 6 h. Necrosis of intrahepatic bile ducts was severe at 6 h with moderate injury in smaller bile ducts. Ultrastructural alterations were observed in BEC mitochondria and microvilli at 3 h with no apparent alterations in hepatocyte mitochondria or tight junctions between cells. In addition, histochemical staining of liver sections and assays of mitochondrial enzyme activitiesin vitroat 3 h revealed no loss of mitochondrial function in hepatocytes. These results provide strong evidence for defining DAPM as a selective bile duct toxicant.