The threshold for hepatotoxicity and
cholestasis induced by methylene dianiline (
DAPM) in rats is between 25 and 75 mg/kg (Bailie et 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 approximately 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 activities in vitro at 3 h revealed no loss of mitochondrial function in hepatocytes. These results provide strong evidence for defining
DAPM as a selective bile duct toxicant.