title: Mitochondrial dysfunction occurs before transport or tight junction deficits in biliary epithelial cells exposed to bile from methylenedianiline-treated rats.
abstract: 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 approximately 75%, and transepithelial resistance decreased approximately 16% in agreement with an approximately 65% increase in leakage of a glucose analog, methyl-alpha-D-glucopyranoside (AMG), from apical to basolateral media. By 60 min, AMG uptake was decreased approximately 40%. Mitochondrial function was very rapidly compromised, with approximately 120% increases in the green-to-red fluorescence ratio of JC-1 (mitochondrial membrane potential dye) at 15 min and approximately 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.
authors.affiliation: Department of Pathology, University of Texas Medical Branch, Galveston, Texas 77555, USA.
authors.forename: Vicente
authors.initials: V
authors.lastname: Santa Cruz
authors.forename: Tammy R
authors.initials: TR
authors.lastname: Dugas
authors.forename: Mary F
authors.initials: MF
authors.lastname: Kanz
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000814:Aniline Compounds / Q000378:metabolism / Q000633:toxicity*; D000818:Animals; D001646:Bile / Q000737:chemistry* / Q000187:drug effects; D001659:Biliary Tract / Q000166:cytology / Q000378:metabolism*; D002463:Cell Membrane Permeability / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004247:DNA / Q000032:analysis / Q000096:biosynthesis; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D008854:Microscopy, Electron; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012964:Sodium / Q000502:physiology; D019108:Tight Junctions / Q000187:drug effects / Q000378:metabolism*; D001559:Benzhydryl Compounds
chemical_list: D000255:Adenosine Triphosphate; D000814:Aniline Compounds; D004247:DNA; D005947:Glucose; D012964:Sodium; C009505:4,4'-diaminodiphenylmethane; D001559:Benzhydryl Compounds