Research will focus on the bile canaliculus, a specialized domain of the hepatocyte plasma membrane. Constituents of the canaliculus include structural elements, a variety of transporters for solute flux in and out of the canalicular lumen, a protease that cleaves plgA before its excretion into bile, and a rich web of cytoskeleton that is necessary for active canalicular contraction. Our approach is to first perturb the canaliculus with a toxicant and then to identify impairments in function, alterations in structure, and changes in canalicular constituents. Our ultimate goal is to clarify the interrelationships among these perturbations. In the prior funding period, we found rats treated with 1,1-dichloroethylene (DCE) to be an excellent in vivo model of canalicular injury. Our working hypothesis is that this target site is a consequence of DCE metabolism to reactive GS-conjugates which are stable enough to congregate in the pericanalicular regions, enroute to excretion in bile. DCE-induced structural changes were confined to zone 3 (centrolobulas, canaliculi, and were accompanied by diminished staining for 4 canalicular components indicating their loss or alteration. Concurrent changes in biliary function were slower organic anion excretion, > 90% decrease in a lgA and a 40% total protein excretion, but no alterations in bile volume, bile salt excretion, or Pi exclusion. Proposed function studies will use freely moving rats with exteriorized cannulas and physiological replacement of bile salt.
AIM 1 will establish the utility of fluorescent markers that allow zonal localization of functional impairments.
AIM 2 will attempt to distinguish injury-associated functional alterations from less relevant alterations due to a secondary depletion of GSH.
AIM 3 will test our protein, by impairing the canalicular-localized, pre-excretion processing of plgA to its secreted form.
AIM 4 will characterize effects on DCE on the pericanalicular cytoskeleton and verify the apparent sparing of the tight junctions between hepatocytes.
AIM 5 will use canalicular membrane vesicles and hepatocyte couplets to address mechanistic questions about the postulated role of the reactive DCE-GS conjugates in the canaliculi injury. New coinvestigator will facilitate this AIM by synthesizing stable and reactive forms of DCE-GS conjugates, by microinjecting these compounds into hepatocyte couplets for toxicity studies, and by providing an antibody to the canalicular membrane GS-conjugate transporter that we predict is a particularly vulnerable target. Information obtained should clarify the pathogenesis of DCE injury to the canaliculus.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
2R01DK034806-04A3
Application #
2139376
Study Section
Toxicology Subcommittee 2 (TOX)
Project Start
1985-09-01
Project End
1997-12-31
Budget Start
1994-01-01
Budget End
1994-12-31
Support Year
4
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of Texas Medical Br Galveston
Department
Pathology
Type
Schools of Medicine
DUNS #
041367053
City
Galveston
State
TX
Country
United States
Zip Code
77555
Jones, Juliet A; Kaphalia, Lata; Treinen-Moslen, Mary et al. (2003) Proteomic characterization of metabolites, protein adducts, and biliary proteins in rats exposed to 1,1-dichloroethylene or diclofenac. Chem Res Toxicol 16:1306-17
Atchison, C R; West, A B; Balakumaran, A et al. (2000) Drug enterocyte adducts: possible causal factor for diclofenac enteropathy in rats. Gastroenterology 119:1537-47
Atchison, C R; Balakumaran, A; West, A B et al. (2000) Aging enhances susceptibility of diclofenac-treated rats to gastric ulceration, while attenuating enteropathy. Dig Dis Sci 45:614-20
Kanz, M F; Gunasena, G H; Kaphalia, L et al. (1998) A minimally toxic dose of methylene dianiline injures biliary epithelial cells in rats. Toxicol Appl Pharmacol 150:414-26
Woodard, S H; Moslen, M T (1998) Decreased biliary secretion of proteins and phospholipids by rats with 1,1-dichloroethylene-induced bile canalicular injury. Toxicol Appl Pharmacol 152:295-301
Moslen, M T; Kanz, M F; Bhatia, J et al. (1994) Biliary glutathione and some amino acids are markedly diminished when biliary pressure is elevated. Exp Mol Pathol 61:1-15
Moslen, M T; Kanz, M F (1993) Biliary excretion of marker solutes by rats with 1,1-dichloroethylene-induced bile canalicular injury. Toxicol Appl Pharmacol 122:117-30
Moslen, M T; Kanz, M F; Bhatia, J et al. (1992) Biliary endogenous inorganic phosphate, D-glucose, IgA and transferrin are differentially altered by hydrostatic pressure. J Hepatol 16:89-97
Kanz, M F; Whitehead, R F; Ferguson, A E et al. (1992) Biliary function studies during multiple time periods in freely moving rats. A useful system and set of marker solutes. J Pharmacol Toxicol Methods 27:7-15
Kanz, M F; Taj, Z; Moslen, M T (1991) 1,1-Dichloroethylene hepatotoxicity: hypothyroidism decreases metabolism and covalent binding but not injury in the rat. Toxicology 70:213-29

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