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 (Bailie et at, Env iron. Health Perspect., 124, 25-30, 1993). Our objectives were to dete rmine if a minimally toxic dose of DAPM provided a model system for st udies of selective injury to biliary epithelial cells (BEC) in vivo. T hus, we examined the effects of 50 mg DAPM/kg on (1) biliary constitue nts, (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. Bi le was collected for another 6 h. Histochemical, ultrastructural, and biochemical liver alterations were assessed at 3 h or at 3 and 6 h. DA PM had minimal effects on biliary bile salt and bilirubin excretion ov er 6 h. Biliary glucose and protein excretion were increased similar t o 2-fold starting in Hour 1, while inorganic phosphate excretion was n ot increased until Hour 2. Biliary glutathione excretion initially inc reased (Hour 1) but then declined steadily for 5 h. Microsomal cytochr ome P-450 activities were transiently decreased at 3 h but had returne d to control values by 6 h. Liver glutathione (GSH and GSSG) was not a ffected by DAPM at 3 or 6 h. Necrosis of intrahepatic bile ducts was s evere at 6 h with moderate injury in smaller bile ducts. Ultrastructur al alterations were observed in BEC mitochondria and microvilli at 3 h with no apparent alterations in hepatocyte mitochondria or tight junc tions between cells. In addition, histochemical staining of liver sect ions and assays of mitochondrial enzyme activities in vitro at 3 h rev ealed no loss of mitochondrial function in hepatocytes. These results provide strong evidence for defining DAPM as a selective bile duet tox icant. (C) 1998 Academic Press.