Isolation and partial characterizations of oval and hyperplastic bile ductular cell-enriched populations from the livers of carcinogen and noncarcinogen-treated rats.
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An oval cell-enriched population was isolated using two isopyknic centrifugation steps in Percoll gradients from the livers of young adult male rats maintained for 6 to 12 weeks on a choline-deficient diet containing 0.05% DL-ethionine. This cell population equilibrated sharply at densities ranging between 1.07 and 1.08 g/ml, possessed a mean cell diameter in fixed-cell smears of 13.6 micron, and showed viabilities of greater than 95% as judged by trypan blue dye exclusion. Contamination of this population by hepatocytes and Kupffer cells was determined to be less than 1% and between 4 and 14%, respectively. gamma-Glutamyl transpeptidase activity was demonstrated both biochemically and histochemically to be the most constant marker for evaluating the oval cell-enriched population isolated at various times over the 6 to 12 weeks of the choline-deficient/DL-ethionine dietary regimen. In contrast, the percentages of nonhepatocytic cells showing labeling for DNA synthesis and for alpha-fetoprotein were both found to be the highest in the oval cell-enriched population isolated at 6 weeks and lowest in that obtained at 12 weeks of dietary treatment. Furthermore, at 10 to 11 weeks, 19.2% of the nonhepatocytic cells in this population were positive for albumin, while 2.1% were positive for glucose-6-phosphatase activity, indicating some cells to be intermediate in function between the oval cell and the hepatocyte. In comparison, hyperplastic bile ductular epithelial cells in tissue preparations isolated from the livers of rats previously subjected to 13 weeks of chronic feeding of the noncarcinogenic cholestatic agent, 1-naphthyl isothiocyanate, or at 8 to 13 weeks following bile duct ligation were found to be strongly positive for gamma-glutamyl transpeptidase activity, as well as to be positive for alkaline phosphatase activity, but to be essentially negative for glucose-6-phosphatase activity, glycogen content, and albumin production. However, an occasional bile ductular cell in these preparations was found to exhibit a strong cytoplasmic binding of [6,7-3H]estradiol, an indirect measure of alpha-fetoprotein production. Also, a low, but demonstrable amount of DNA synthesis was noted in the bile ductular cells present in these preparations. Furthermore, a viable cell population highly enriched in bile ductular epithelial cells was isolated by isopyknic centrifugation in Percoll following enzymatic dissociation of the hyperplastic tissue preparation from bile duct ligated rats.(ABSTRACT TRUNCATED AT 400 WORDS) (+info)
Alterations of high density lipoproteins in experimental intrahepatic cholestasis in the rat induced by administration of alpha-naphthylisothiocyanate.
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It has previously been reported that abnormally enlarged high density lipoproteins (HDL) appear in rats with extrahepatic cholestasis induced by ligation of the common bile duct. To see whether similar changes in HDL occur in intrahepatic cholestasis in rats, we studied HDL alterations in rats treated with alpha-naphthylisothiocyanate (ANIT), which is known to produce a cholestatic response in rats similar to intrahepatic cholestasis in man. Findings were obtained which indicated changes in HDL similar to those in bile duct-ligated rat serum: HDL from ANIT-treated rats were separated into two subfractions, enlarged particles and smaller ones, on Bio-Gel A5m column chromatography. In electron micrographs, the two subfractions appeared spherical and the diameters of the enlarged particles and the other ones were 15.0 +/- 2.6 nm and 11.5 +/- 2.2 nm, respectively. Both subfractions showed slow alpha-mobility in agarose gel electrophoresis. The enlarged HDL had apoE as their major apoprotein, while apoA-I was the major apoprotein in the other HDL subfraction. The enlarged HDL contained less protein and more cholesterol than the other HDL subfraction. The two HDL subfractions were also separated by heparin-Sepharose affinity chromatography. (+info)
Bile acid assays as an index of cholestasis.
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Four bile acid profiles, bilirubin, alkaline phosphatase, alanine aminotransferase, glutamate dehydrogenase, and isocitrate dehydrogenase were measured in rats in which normal bile flow had been disturbed either by administration of alpha-naphthylisothiocyanate or by surgical ligation of the bile duct. The most sensitive index to the early onset of cholestasis was an increase in total cholate. This increase was measurable before morphological changes could clearly be identified. (+info)
Oxidative conversion of isothiocyanates to isocyanates by rat liver.
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This report describes the oxidative metabolism of isothiocyanates to isocyanates catalyzed by rat liver microsomes. Incubation of 2-naphthylisothiocyanate, microsomes, and NADPH yielded either N,N'-di-naphthylurea or, on inclusion of 2-aminofluorene in the incubations, N-2-naphthyl-N'-2-fluorenylurea. These ureas were formed by the production of the known genotoxicant, 2-naphthylisocyanate, which reacted with its hydrolysis product, 2-aminonaphthalene, to yield the symmetrical urea, or with 2-aminofluorene to form the mixed urea. Formation of N,N'-di-2-naphthylthiourea was also observed because 2-aminonaphthalene reacted with the substrate. Urea formation was dependent on the microsomes, NADPH, and oxygen. Use of microsomes from rats previously treated with Aroclor 1254 increased urea formation greater than 10-fold. The enzyme activity was inhibited by alpha-napthoflavone, flavone, or CO, and slightly inhibited by metyrapone, 7-ethoxycoumarin, or SKF-525A. It was not inhibited by methimazole or paraoxon, suggesting that neither flavin-containing monooxygenase nor hydrolytic enzyme was involved. These data are consistent with a cytochrome P450-dependent, oxidative desulfuration of 2-naphthylisothiocyanate to yield 2-naphthylisocyanate. Further studies with the isomeric 1-naphthylisothiocyanate and the dietary benzylisothiocyanate showed that they can also be metabolized to their isocyanates, as evidenced by the trapping of isocyanates with 2-aminofluorene to form the mixed ureas. (+info)
Subpopulations of rat hepatocytes separated by Percoll density-gradient centrifugation show characteristics consistent with different acinar locations.
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Freshly isolated viable rat hepatocytes were separated into five subpopulations on shallow discontinuous Percoll density gradients. The periportal marker enzymes alanine aminotransferase (ALT), malate dehydrogenase (MDH) and lactate dehydrogenase (LDH) showed gradients of increasing activity from the subpopulation of least density (band 1, rho = 1.07 g/ml) to the subpopulation of greatest density (band 5, rho = 1.09 g/ml). The perivenous marker enzymes pyruvate kinase (PK) and glutamate dehydrogenase (GDH) showed gradients of decreasing activity from band-1 cells to band-5 cells. Glutamine synthetase (GS), which is confined to the two or three cell layers around the hepatic venule, was almost entirely restricted to band-1 hepatocytes. Band-5: band-1 ratios of enzyme activity were as follows: ALT, 8.0; LDH, 2.1; MDH, 1.6; GDH, 0.7; PK, 0.2; GS, 0.01. Band-5:band-1 ratios for ALT, LDH, PK and GS were maintained after culture of subpopulations in identical conditions for up to 72 h, whereas the ratios for MDH and GDH decreased and increased respectively towards unity. Band-1 hepatocytes exhibited greater cytotoxicity than band-5 cells after incubation with carbon tetrachloride or paracetamol. These perivenous-selective toxins produced greater decreases in cell viability and greater release of ALT and LDH from band-1 hepatocytes than from band-5 hepatocytes. Conversely, band-5 hepatocytes were more susceptible than band-1 hepatocytes to the cytotoxic effects of 1-naphthylisothiocyanate and methotrexate (known periportal-selective toxins). It is concluded that band-5 hepatocytes are enriched in periportal cells, whereas band-1 hepatocytes are enriched in perivenous cells. Isolation of hepatocyte subpopulations by Percoll density-gradient centrifugation has the considerable advantage that periportal and perivenous cells can be obtained from the same liver. (+info)
Novel mechanisms in chemically induced hepatotoxicity.
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This review focuses on cellular events that modulate hepatotoxicity subsequent to initial liver insult. Cellular events that determine the nature and extent of hepatotoxic injury and the ultimate outcome of that injury are also discussed. The roles of cell types other than hepatocytes, hepatocyte organelle-specific processes, and regeneration in progression or recovery from liver injury are emphasized. Leukocyte activities are key events in two distinct hepatotoxicities. Neutrophil-mediated, periportal inflammation appears to play a primary role in progression of alpha-naphthylisothiocyanate-induced cholangiolitic hepatitis. However, a humorally mediated autoimmune response to protein adducts that occurs after anesthesia is critical in onset of halothane-induced hepatitis. New insights into specific events at the hepatocyte level are also emerging. Although reducing gap junctional communication between hepatocytes can protect against progression of liver injury, down-regulation of the subunit proteins (connexins) can isolate neoplastic cells from growth regulation. Acidic intracellular pH characteristic of hypoxia is protective against both hypoxic and toxicant-induced cell injury. In oxidative injury, a pH-mediated mitochondrial permeability transition causes mitochondrial uncoupling and ATP loss and leads to cell death. The ultimate outcome of hepatotoxic injury depends on the extent of tissue repair. Stimulation of tissue repair after a sublethal dose of CCl4 appears to be the central mechanism in protection against death from a subsequent large dose. Taken together, these examples illustrate the importance of events subsequent to initial liver injury as determinants of extent of liver damage. (+info)
Protection against alpha-naphthylisothiocyanate-induced acute cholestasis in mice by a novel taurine conjugate of ursodeoxycholate (UR-906).
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We investigated the effects of tauroursodeoxycholate (UR-906) and ursodeoxycholic acid (UDCA) on alpha-naphthylisothiocyanate (ANIT)-induced cholestasis in mice. UR-906 and UDCA were given intravenously 2 hr prior to and 2 hr after ANIT (80 mg/kg, p.o.) treatment. The animals were sacrificed 48 hr after ANIT administration, and serum markers of liver injury were examined. UR-906 prevented significant elevations in total bilirubin, bile acids and LDH. Furthermore, this drug reduced significant elevations in ALP and LAP. UDCA also prevented significant elevations in total bilirubin and LAP. These results indicate that UR-906 as well as UDCA has a beneficial effect against ANIT-induced cholestasis in mice. (+info)
Nuclear magnetic resonance identification of the taurine conjugate of 3 alpha,6 beta,7 beta-trihydroxy-5 beta,22-cholen-24-oic acid (tauro-delta 22-beta-muricholate) in the serum of female rats treated with alpha-naphthylisothiocyanate.
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Recently developed 1H nuclear magnetic resonance methods, including 2-dimensional, 1H-detected-[13C] shift correlation spectroscopy and 1-dimensional HOHAHA spectroscopy at 500 MHz have been used to identify the major bile acid in the serum of rats treated with alpha-naphthylisothiocyanate as the taurine conjugate of 3 alpha,6 beta,7 beta-trihydroxy-5 beta,22-cholen- 24-oic acid (tauro-delta 22-beta-muricholate), a derivative of beta-muricholate having an unsaturated bond in the acyclic side chain. Complete stereospecific assignments of the 1H and protonated 13C spectra of the title compound and beta-muricholate are reported. The assignments were based entirely on 1H-1H and 1H-13C scalar connectivities and were made using approximately 0.5-2.0 mg of material. It is suggested that these new methods will be of general value for identifying the structure and assigning the spectra of other scarce steroid-like molecules. (+info)