Effects of methylenedianiline on tight junction permeability of biliary epithelial cells in vivo and in vitro

Abstract

Methylenedianiline (DAPM) is considered a cholangiodestructive toxicant in vivo. Increases in biliary inorganic phosphate (P(i)) and glucose occur prior to biliary epithelial cell (BEC) injury, which could be due to increased paracellular permeability and/or impairment of P(i) and glucose uptake by BEC. To evaluate these possibilities, we induced mild injury [loss of BEC from major bile ducts (6 h), ultrastructural alterations in BEC mitochondria and Golgi cisternae (3 h), and striking increases in biliary P(i) and glucose (3-6 h)] with 25 mg DAPM/kg and then assessed temporal alterations in tight junction (TJ) permeability by measuring bile to plasma (B:P) ratios of [(3)H]-inulin. Parameters maintained by hepatocytes in bile were unchanged (bile flow, bile salts, bilirubin) or only transiently perturbed (protein, glutathione). Minimal elevations in B:P ratios of inulin occurred temporally later (4 h) in DAPM-treated rats than increases in biliary P(i) and glucose. To confirm a direct effect of DAPM on BEC TJs, we measured transepithelial resistance (TER) and bi-ionic potentials of BEC monolayers prior to and after exposure to pooled (4-6) bile samples collected from untreated rats (Basal Bile) or rats treated with 50 mg DAPM/kg (DAPM-Bile). BEC TJs were found to be cation selective. Exposure to DAPM-Bile for 1 h decreased TERs by approximately 35% and decreased charge selectivity of BEC TJs while exposure to Basal Bile had no effects. These observations indicate that DAPM-Bile impairs paracellular permeability of BEC in vitro. Further, our in vivo model suggests that increases in paracellular permeability induced by DAPM are localized to BEC because bile flow and constituents excreted by hepatocytes were unchanged, BEC damage was temporally correlated with increases in biliary P(i) and glucose, and elevations in B:P ratios of inulin were delayed and minimal.

Figure 1

Representative micrographs of liver portal tracts from (A) control and (B, C) DAPM-treated rats. At 6 h after 25 mg DAPM/kg, major bile ducts (BD) were (B) partially or (C) completely denuded of BEC while bile ductules (arrows) were spared. Severe injury to major bile ducts (C) was accompanied by edema, inflammatory infiltrate (arrowheads), and fibrin exudate (asterisks). HA, hepatic artery; PV, portal vein. Hematoxylin and eosin; each bar = 2.5 μm.

Figure 2

Time course of changes in (A) bile flow and biliary excretion of (B) bilirubin, (C) protein, (D) glucose, (E) inorganic phosphate and (F) glutathione of control rats and rats treated with 25 mg DAPM/ kg. Values are means ± SE of four control or six DAPM-treated rats per group. Open symbols represent basal values; symbols without error bars indicate the SE lies within the symbol. Asterisks near symbols of DAPM-treated rats indicate values significantly different from control rats at that time point. * p < 0.05; ** p < 0.01. Note the Y axis is ~7-fold greater in graphs D and E than graphs B, C, and F.

Figure 3

Electron micrographs of BEC from control rat livers. (A) Cross-sections of mitochondria (M) are rounded with dense matrices and typical cristae. Arrows indicate cisternae of the rough endoplasmic reticulum and a single microvilli extends from the lumenal (L) surface of the cell. Original magnification; 82,500X. (B) Regularly spaced cisternae of several Golgi regions (arrows) are seen adjacent to mitochondria that are rounded or elongated. Numerous microvilli extend from the lumenal surface of the cell. Original magnification; 41,250X.

Figure 4

Electron micrographs of BEC from a rat liver 3 h after 25 mg DAPM/kg. The enlarged mitochondrion on the left displays a translucent matrix and areas that appear disrupted (asterisk). Cisternae of the Golgi region (G) are dilated and disorganized (upper right). Inset: Enlarged mitochondrion with translucent matrix, areas that appear disrupted (asterisk) and membrane whorls (arrows). Original magnification; 41,250X; inset; 82,500X.

Figure 5

Electron micrograph of a BEC from a rat liver 3 h after 25 mg DAPM/kg. Regularly spaced cisternae of the Golgi region are replaced by dilated cisternae that show areas of membrane coalescence or disruption (arrows). Original magnification; 82,500X.

Figure 6

Time course of changes in (A) bile flow and biliary excretion of (B) bilirubin, (C) protein, (D) glucose, and (E) inorganic phosphate of control rats and rats treated with 25 mg DAPM/ kg that were continuously infused with [³H]-inulin. Values are means ± SE of four rats per group (B, C) or four control and seven DAPM-treated rats per group (A, C, D). Open symbols represent basal values; symbols without error bars indicate the SE lies within the symbol. Asterisks near symbols of DAPM-treated rats indicate values significantly different from control rats at that time point. * p < 0.05; ** p < 0.01. Note the Y axis is ~6-fold greater in graphs D and E than graphs B and C.

Figure 7

(A) Time course of changes in [³H]-inulin in plasma and bile and (B) ratio of [³H]-inulin in bile to plasma of control rats and rats treated with 25 mg DAPM/ kg. Values are means ± SE of four control and seven DAPM-treated rats per group. Open symbols represent basal values; symbols without error bars indicate the SE lies within the symbol. Asterisks near symbols of DAPM-treated rats indicate values significantly different from control rats at that time point. * p < 0.05. Note the Y axes are log scale.

Figure 8

Linear regression plots of the biliary excretion of (A) inorganic phosphate versus glucose, (B) inorganic phosphate versus B:P ratio of inulin, and (C) glucose versus B:P ratio of inulin in 4 control rats (black symbols) and 7 rats treated with 25 mg DAPM/ kg (gray symbols). Each symbol shape represents the values from one rat.

Figure 9

Representative measurements of (A,C) transepithelial resistance (TER) and (B,D) bi-ionic potentials of BEC monolayers before and after incubation with Basal Bile (left) and DAPM-Bile (1^st Hr) (right). A). The TER increases observed when Na⁺ is replaced by TMA⁺ are equivalent prior to (arrow) and after (double arrows) monolayer exposure to Basal Bile. B). Changes in V_t in the presence of different ions follow the same pattern before and after incubation with Basal Bile. C). Baseline TER declined from ~590 to ~330 Ω * cm² following monolayer exposure to DAPM-Bile (1^st Hr). The TER increase (double arrow) observed when Na⁺ is replaced by TMA⁺ after monolayer exposure to DAPM-Bile (1^st Hr) is one-third of the TER increase (arrow) observed during Na⁺ replacement by TMA⁺ prior to bile exposure. D). V_t change during Na⁺ replacement by TMA⁺ is decreased (arrowhead) while V_t change during Cl⁻ replacement by GA⁻ becomes slightly more negative (double arrowheads) following DAPM-Bile (1^st Hr) exposure. Data are representative of a minimum of 3 monolayers exposed to pooled bile samples of Basal Bile and DAPM-Bile (1^st Hr).