ToxSci Advance Access originally published online on December 15, 2004
Toxicological Sciences 2005 84(1):129-138; doi:10.1093/toxsci/kfi061
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Toxicological Sciences vol. 84 no. 1 © Society of Toxicology 2005; all rights reserved.
Mitochondrial Dysfunction Occurs before Transport or Tight Junction Deficits in Biliary Epithelial Cells Exposed to Bile from Methylenedianiline-Treated Rats
* Department of Pathology, University of Texas Medical Branch, Galveston, Texas 77555; Department of Pharmacology, Louisiana State University Health Science Center–Shreveport, Shreveport, Louisiana 71130; and Chevron Phillips Chemical Co. LP, The Woodlands, Texas 77380
Received October 26, 2004; accepted December 8, 2004
Methylenedianiline (DAPM) rapidly injures biliary epithelial cells (BEC) in vivo. Prior to evident BEC injury, biliary glucose and inorganic phosphate appreciably rise, which could stem from loosened tight junctions (TJ). Concurrently, ultrastructural abnormalities in BEC mitochondria of DAPM-treated animals are observed, suggesting other impairments. Our objective was to develop an in vitro BEC model to assess the time course of impairments in TJ integrity, glucose uptake, and mitochondrial function following DAPM exposure. We exposed monolayers of primary, polarized rat BEC to bile collected from rats prior to (Basal Bile) or after oral treatment (DAPM-Bile) with 50 mg DAPM/kg. DAPM-Bile collected during 0–60 min (1st Hr) and during 61–120 min (2nd Hr) after treatment was pooled from four to six rats. When monolayers were exposed to 1st Hr DAPM-Bile for 120 min, metabolic activity (XTT assay) decreased 
75%, and transepithelial resistance decreased 16% in agreement with an 65% increase in leakage of a glucose analog, methyl-
-D-glucopyranoside (AMG), from apical to basolateral media. By 60 min, AMG uptake was decreased 40%. Mitochondrial function was very rapidly compromised, with 120% increases in the green-to-red fluorescence ratio of JC-1 (mitochondrial membrane potential dye) at 15 min and 55% decreases in ATP levels at 30 min. This sequence of events indicates that DAPM impairs BEC mitochondria prior to impairments in glucose uptake or TJ integrity. Thus, our in vitro primary rat BEC/bile exposure model mimics in vivo observations and yields basic information about the time course of events that occur during DAPM-induced injury.
Key Words: methylenedianiline; biliary epithelial cells; bile; mitochondria; tight junctions; glucose transport.