Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.24.3 (collagenase)
18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Recent studies have shown that Cd-induced testicular interstitial cell (TIC) tumors can be prevented by low-dose Cd pretreatment. However, the mechanism by which low-dose Cd induces such tolerance is unclear. Thus, in this study we assessed the effects of in vivo Cd pretreatment (3 mumol/kg) on Cd uptake, cytotoxicity, metal content (Zn, K, and Ca), and low molecular weight testicular Cd-binding proteins (low Mr TC-BPs) of isolated TICs exposed to Cd in vitro. TICs were isolated by collagenase dispersion of Wistar (WF/NCr) rat testes and incubated with Cd (1.0 mM) for 15 to 60 min. In vivo Cd pretreatment decreased in vitro Cd uptake by 24% after 1 hr of incubation with Cd. In vivo Cd pretreatment also resulted in a marked reduction of in vitro Cd-induced cytotoxicity, as reflected by reduced loss of cellular K, glutamic-oxaloacetic transaminase, as well as reduced lipid peroxidation and decreased Cd-induced Ca influx into TICs in vitro. These cytotoxic effects were not attributed solely to cell death as TIC viability remained high even after 1 hr of in vitro Cd exposure. Cd-induced inhibition of intercellular enzymes, as assessed by cellular lactate dehydrogenase activity, was also reduced by low-dose Cd pretreatment. Cd pretreatment did not alter basal levels of Zn, Ca, or K. Neither low-dose in vivo Cd pretreatment nor in vitro Cd exposure appeared to greatly alter levels of the low Mr TCBPs as assessed by electrophoresis. In vivo Zn pretreatment, which also effectively inhibits Cd-induced testicular tumors, results in a similar reduction in Cd-induced cytotoxicity in TICs. This indicates that treatments which result in reduced Cd-induced TIC tumors are consistently capable of reducing Cd-induced cytotoxicity in isolated TICs in vitro.
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PMID:Effect of in vivo low-dose cadmium pretreatment on the in vitro interactions of cadmium with isolated interstitial cells of the rat testes. 208 10

The ethanol-inducible form of cytochrome P-450 (IIE1) is expressed and induced by ethanol, predominantly in the centrilobular region. Because this isoenzyme has a particularly high capacity to convert carbon tetrachloride and several other hepatotoxins into reactive intermediates, its role in producing damage was studied by comparing the effect of carbon tetrachloride exposure on hepatocytes isolated from either the periportal or the perivenous region by digitonin-collagenase perfusion. After exposure for 18 hr of primary culture to 600 mumol/L of carbon tetrachloride, periportal cells were only slightly damaged, as estimated from dye exclusion and lactate dehydrogenase leakage. In marked contrast, perivenous cells, which contained a several-fold higher amount of immunoreactive P-450 IIE1 apoprotein, were partly damaged after exposure to 60 to 150 mumol/L of carbon tetrachloride and severely damaged after 600 mumol/L. Similarly, lipid peroxidation after carbon tetrachloride was much more prominent in perivenous cells. The differences between perivenous and periportal cells in carbon tetrachloride-induced injury were larger when cells were isolated from chronically ethanol-treated rats. Isoniazid, an efficient inhibitor of P-450 IIE1, protected against damage by carbon tetrachloride more efficiently than the general P-450 inhibitor cimetidine. Our results suggest that the greater susceptibility of the perivenous hepatocytes to carbon tetrachloride-induced damage is associated with the high expression of P-450 IIE1 in these cells. This enzyme may also be involved in damage elicited by several other typical centrilobular hepatotoxins.
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PMID:Role of ethanol-inducible cytochrome P-450 IIE1 in carbon tetrachloride-induced damage to centrilobular hepatocytes from ethanol-treated rats. 222 5

Rat livers were perfused and stored for 48 hr in cold University of Wisconsin solution before dissociation by the two-step collagenase method. At that time, glycogen content was significantly reduced, but no obvious changes in albumin, beta-actin and aldolase B mRNAs and in glutathione levels were observed. Enzymatic perfusion yielded 280 +/- 30 x 10(6) viable hepatocytes vs. 520 +/- 40 x 10(6) viable hepatocytes from unstored organs. Cell viability determined by trypan blue exclusion was 74% and 90%, respectively. Hepatocytes from University of Wisconsin-preserved livers had a 29% reduced adenosine triphosphate content, but glutathione levels did not significantly differ from those found in unstored cells. When put into culture, hepatocytes formed typical monolayers of granular epithelial cells and did not exhibit alteration of their fine structure when compared with cells from unstored organs. After 24 and 48 hr, they showed variations in cytochrome P-450 content and ethoxyresorufin O-deethylase activity similar to those observed with unstored cells. By contrast, overall protein synthesis and albumin secretion rate were 40% and 30% lower, respectively. Hepatocytes from University of Wisconsin-preserved organs could be cryopreserved and further cultured as unstored cells. The University of Wisconsin solution was also used to preserve isolated hepatocytes. Viability of freshly isolated hepatocytes was decreased by only 10% after 48 hr of hypothermic liver storage when assayed by intracellular lactate dehydrogenase content. However, after 4 hr of storage, in contrast with hepatocytes preserved in L15 Leibovitz medium, the cells attached poorly to plastic and exhibited morphological alterations.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Primary culture of adult rat hepatocytes after 48-hour preservation of the liver with cold UW solution. 225 48

The aim of this study was to investigate direct cytotoxicity to human and rat hepatocytes in primary culture from halothane and compare it with that of isoflurane, which is known to be minimally metabolized and less toxic in vivo. Both human and rat parenchymal cells were isolated by the two-step collagenase perfusion method and after attachment to plastic were incubated with either volatile anesthetic for 24 h. All the cultures were maintained in 20% O2 condition and were not induced prior to anesthetic treatment. Temperature, atmosphere conditions, and anesthetic concentrations were kept constant during the study period. Evaluation of cytotoxicity was based on morphologic, biologic (determination of both extracellular and intracellular lactate dehydrogenase activity), and metabolic (protein synthesis and secretion) end points. Protein synthesis and secretion rates were found to be the most sensitive parameters in hepatocyte cultures from both species. Protein synthesis was inhibited by 18% and protein secretion by 50% in the presence of 1 and 1.25 mM halothane, respectively, in human cell cultures (P less than 0.05). With 1.25 mM halothane intracellular lactate dehydrogenase was also decreased; lactate dehydrogenase leakage and morphologic alterations were detected only beyond 5 mM halothane. By contrast, in rat hepatocyte cultures protein secretion was inhibited by 26% and protein synthesis by 20% in the presence of 0.1 and 0.75 mM halothane, respectively, whereas morphologic alterations and a 37% lactate dehydrogenase leakage increase were observed with the concentration of 1 mM (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effects of halothane on human and rat hepatocyte cultures. 231 35

Matrix vesicles are membrane-invested vesicles that initiate mineralization in the extracellular matrix of calcifying tissues. The epiphyseal cartilages of young-rat rib bones were divided into the growth zone and the resting zone, followed by the isolation of matrix vesicles after collagenase treatment. Matrix vesicles with both alkaline phosphatase and lactate dehydrogenase were detected in the growth cartilage found in the epiphyseal growth plates of young rabbits [Hosokawa, Uchida, Fujiwara & Noguchi (1988) J. Biol. Chem. 263, 10045-10047], but were not detected in the resting zone. By contrast, and surprisingly, lactate dehydrogenase-containing vesicles without alkaline phosphatase were found in the resting zone, but not in the growth zone. In both the growth and resting zones, isoenzyme patterns of lactate dehydrogenase in the two different vesicles were identical with those of cytosolic lactate dehydrogenase of chondrocytes, suggesting the presence of a mechanism for specific uptake of cytosolic lactate dehydrogenase. The same results as for young-rat rib bones were obtained with the resting and growth cartilages of young-dog and monkey rib bones.
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PMID:Vesicles with lactate dehydrogenase and without alkaline phosphatase present in the resting zone of epiphyseal cartilage. 231 Mar 80

Hepatocytes from adult male Sprague-Dawley rats were isolated by the two-stage collagenase perfusion technique; 1 x 10(6) cells/plate were incubated in primary cell culture in Leibovitz's L-15 medium for 24 hr with or without various concentrations (12.5 to 400 mumol/L) of cardioactive cationic amphiphilic compounds such as propranolol, verapamil, sotalol, atenolol and procainamide. Propranolol and verapamil caused a significant release of lactate dehydrogenase (used as cytotoxic index in this study) in the culture media in a concentration-dependent manner, with LC50 values of 220 +/- 10 and 224 +/- 7 mumol/L, respectively. Atenolol, sotalol and procainamide had no effect on lactate dehydrogenase release. Electron microscopy of the hepatocytes showed that subtoxic concentrations of propranolol (12.5 to 125 mumol/L) and verapamil (12.5 to 100 mumol/L) induced multilamellar inclusion bodies after 24 hr of incubation. The two higher concentrations of propranolol (50 and 125 mumol/L) and 100 mumol/L of verapamil produced a significant decrease in the percentage of volume density of the mitochondria as quantitated by morphometrical analysis. An unusual feature of the electron microscopical changes with propranolol and verapamil was the presence of mitochondria within the multilamellar inclusion bodies. When these two drugs were used together or with subtoxic concentrations of amiodarone or desethylamiodarone, release of lactate dehydrogenase was significantly enhanced. No correlation was evident between the cytotoxic response and the volume density of cellular inclusions in hepatocytes treated with different concentrations of propranolol, verapamil, amiodarone or desethylamiodarone. Sotalol, atenolol and procainamide in concentrations up to 400 mumol/L did not produce any ultrastructural changes in hepatocytes after 24 hr of incubation. These results show that (a) cationic amphiphilic structure per se is not the only requirement for induction of multilamellar inclusions, (b) propranolol and verapamil can induce the formation of multilamellar inclusion bodies and cause a concentration-dependent release of lactate dehydrogenase from hepatocytes and (c) combination of different cationic amphiphiles in subtoxic concentrations can enhance cytotoxicity and increase the volume density of multilamellar inclusions.
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PMID:Cytotoxic interactions of cardioactive cationic amphiphilic compounds in primary rat hepatocytes in culture. 237 84

Primary human hepatocytes were used to study bile salt hepatotoxicity and the hepatoprotective potential of ursodeoxycholate in vitro. Hepatocytes were obtained by collagenase perfusion of healthy human liver tissue and were treated with glycochenodeoxycholate for 24 hr 1 day after plating. Clear signs of cytotoxicity were observed at concentrations of about 100 mumol/L glycochenodeoxycholate. Toxicity was determined by release of alkaline phosphatase, gamma-glutamyl transferase, AST, ALT or lactate dehydrogenase into the culture medium, by measuring DNA synthesis of the cultured liver cells and by testing the viability of the hepatocytes using trypan-blue dye exclusion. Addition of ursodeoxycholate, which by itself proved to be of little toxicity, significantly reduced the hepatotoxic effects of glycochenodeoxycholate: 72% +/- 6% of the cells survived treatment with 500 mumol/L glycochenodeoxycholate alone, but addition of 100 mumol/L ursodeoxycholate increased the survival rate to 87% +/- 4% (p less than 0.05). Moreover, all enzymes tested were secreted at a significantly lower level when ursodeoxycholate was present. Similarly, the cellular DNA synthesis was maintained at significantly higher levels as a result of ursodeoxycholate treatment. We conclude that (a) primary human hepatocytes are a suitable model for studying hepatotoxicity of bile salts in vitro, (b) ursodeoxycholate reduces hepatotoxicity of other bile salts and (c) ursodeoxycholate can act hepatoprotectively by itself (i.e., alteration of the metabolism of other bile salts is not necessarily required).
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PMID:Ursodeoxycholate reduces hepatotoxicity of bile salts in primary human hepatocytes. 240 54

The aim of this study was to develop a method by which colonic epithelial cells can be isolated from resected mucosa or colonoscopic biopsy specimens and viability maintained in the short term. The principles of the technique are to digest the lamina propria from the epithelium with Dispase and collagenase, to disrupt the epithelium by trituration, and to purify the epithelial cells by seiving and differential sedimentation. Whole and partial crypts were isolated with consistently high purity of 93.5% +/- 1.2% (excluding red cells). Structural integrity was confirmed by light and electron microscopy, exclusion of trypan blue, minimal leakage of lactic dehydrogenase over 5 h (4.1% +/- 1.7%), and 51Cr leakage of less than 2% per hour over 16 h. Functional integrity was supported by continued deoxyribonucleic acid synthesis [( 3H]thymidine uptake) over 16 h and the formation of epithelial monolayer cultures on plastic. Thus, this simple method yields a highly enriched cell population that maintains high viability in vitro for at least 16 h. Such cells may be useful for the study of the biology of colonic epithelial cells.
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PMID:Isolation of colonic crypts that maintain structural and metabolic viability in vitro. 264 74

The effects of culture duration on primary cultured mouse hepatocyte antioxidant levels (superoxide dismutase, catalase, glutathione peroxidase, vitamin E, and glutathione) and susceptibility to glucose oxidase (GO)- and hydrogen peroxide (H2O2)-induced cell killing and lipid peroxidation were examined. Membrane fatty acid composition was also evaluated. Adult male B6C3F1/CrlBR mouse hepatocytes were isolated by collagenase perfusion of the liver and cultured on 60-mm plastic dishes in Leibovitz's L-15 medium supplemented with glucose (1 mg/ml), dexamethasone (1 microM), fetal bovine serum (10%, v/v), and gentamicin sulfate (50 micrograms/ml) for 0 hr (freshly isolated cells) to 96 hr. Hepatocyte toxicity (determined by lactate dehydrogenase release and lipid peroxidation) after a 2-hr exposure to GO (0.8-80 micrograms/ml) or H2O2 (1-5 mM) decreased with increased time in culture. This decreased hepatocyte sensitivity to GO and H2O2 toxicity was not related to antioxidant enzyme activity since superoxide dismutase, catalase, and glutathione peroxidase declined during the 96-hr culture period. In contrast, glutathione and vitamin E levels in the cultured hepatocytes rose to 274.9 +/- 8.3% and 220.6 +/- 18.6% of the levels in freshly isolated cells (129.6 +/- 11.5 nmol and 0.10 +/- 0.01 nmol per 10(6) hepatocytes, respectively). The percentage of polyunsaturated fatty acids in hepatocyte phospholipids and triglycerides decreased with culture duration while the percentage of oleic acid increased in esterified and free fatty acid pools after 2 hr in culture. Total fatty acids were not affected by time in culture. These results suggest that the decreased hepatocyte susceptibility to the toxic effects of hydrogen peroxide may have been due to elevations in cellular GSH and vitamin E levels and decreases in membrane polyunsaturated fatty acids. The data also indicate that hepatocytes in primary culture undergo changes in antioxidant levels and fatty acid composition that may affect free radical toxicity at different times in culture.
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PMID:Effects of culture duration on hydrogen peroxide-induced hepatocyte toxicity. 278 69

The mechanism of the periportal (p.p.) toxicity of allyl alcohol (AlOH) was investigated in p.p. and perivenous (p.v.) hepatocytes isolated by digitonin-collagenase perfusion. The distinct origin of the cell preparations was confirmed by the p.p./p.v. ratios of alanine aminotransferase (p.p./p.v. = 1.8), lactate dehydrogenase (1.3) and glutamine synthetase (0.10). The activity of alcohol dehydrogenase (ADH) was not markedly different in p.p. and p.v. cells. Both types of cells oxidized AlOH at a high but equal rate of about 3 mumol/(min.g cells). Concomitantly with rapid oxidation of 0.7 mM AlOH, glutathione (GSH) was depleted by about 95% and its secretion was completely inhibited in both cell types. Although the GSH content was partially restored during a subsequent 3-h incubation, cellular ATP and K+ content gradually decreased and the leakage of lactate dehydrogenase increased in both types of cells. However, the p.p. cells tended to resist AlOH in vitro better, probably due to their 26% higher GSH content after preincubation with L-methionine. Altering the partial pressure of oxygen in physiological range had no effect on the toxicity of AlOH. The results are contrary to the suggestions that the p.p. location of AlOH liver injury is caused by higher ADH activity or higher oxygen tension in the p.p. zone. Rather, the regiospecificity of the injury may be due to rapid uptake and oxidation of AlOH in the p.p. region.
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PMID:Allyl alcohol cytotoxicity and glutathione depletion in isolated periportal and perivenous rat hepatocytes. 283 85


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