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)

Cytochrome P450-dependent alkoxyphenoxazone dealkylase activity was measured in alveolar type II cells from control and beta-naphthoflavone (ip) treated-rats. Type II cells were isolated from collagenase/elastase-digested lung tissue and purified by centrifugal elutriation. The specificity of the cytochrome P450-dependent activity towards four alkoxyphenoxazones (methoxy-, ethoxy-, pentoxy-, and benzyloxyphenoxazone) was measured under conditions that minimized interference by cytosolic conjugating- and NADPH-dependent quinone reductase activities. Ethoxyphenoxazone dealkylase activity was induced 17-fold following beta-naphthoflavone treatment and was further characterized by its kinetic parameters and sensitivities toward in vitro inhibitors (Km(app) = 0.20 microM, Vmax = 1.74 pmoles resorufin min-1 (10(6) cells)-1 10(6) cells; I50 (alpha-naphthoflavone) = 0.025 microM, and I50 (metyrapone) = 72 microM). beta-Naphthoflavone pretreatment of the rats did not result in statistically significant changes in methoxy-, pentoxy-, or benzyloxyphenoxazone dealkylase activity of alveolar type II cells, although, a trend towards decrease activity was observed for benzyloxyphenoxazone. beta-Naphthoflavone pretreatment had no effect on oxygen consumption or trypan blue exclusion in alveolar type II cells and macrophage ethoxyphenoxazone dealkylase and benzyloxphenoxazone dealkylase activities were not affected by the beta-naththoflavone pretreatment. The results show that exposure to beta-naphthoflavone resulted in an increase in type II cell cytochrome P450-dependent ethoxyphenoxazone dealkylase activity but not in other alveolar type II cell or macrophage alkoxyphenoxazone dealkylase activities or in parameters that monitor viability and cell wall integrity.
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PMID:Cytochrome P450-dependent alkoxyphenoxazone dealkylase activity in rat alveolar type II cells: effect of pretreatment with beta-naphthoflavone. 247 71

The study investigates the influence of different culture conditions on attachment, viability and functional status of rainbow trout (Oncorhynchus mykiss) liver cells in primary culture. Cells were isolated by a two-step collagenase perfusion and incubated in serum-free, chemically defined minimal essential medium (MEM), (a) as a monolayer on uncoated PRIMARIA dishes, (b) as a monolayer on culture dishes coated with calf collagen type 1, and (c) in coculture with the established fish cell lines RTH-149 or RTG-2. Cell attachment was assessed from DNA and protein concentrations per dish, viability was estimated from cellular lactate dehydrogenase release, and the metabolic status was investigated by measuring activities of the phosphoenolpyruvate carboxykinase and biotransformation enzymes as well as the total cytochrome P450 contents. Seeding of hepatocytes on collagen-coated dishes did not alter cell attachment or detachment from the (culture substrate, but had a small, but not significant effect on cell viability and metabolic parameters. Coculture of liver cells and RTG-2 cells reduced hepatocyte detachment from the culture substrate, and it was associated with a significant elevation of 7-ethoxyresorufin-O-deethylase activities in the hepatic cells. Cytochrome P450 contents, however, were not altered. The coculture effect on liver cell physiology clearly depended on the type of cell line, because coculture with RTH-149 cells led to similar, but much weaker effects than obtained in cocultures with RTG-2 cells. Electron microscopical observations revealed the existence of gap junctions and possible exocytosis-like transport between cell lines and hepatocytes. The results point to the potential of coculture systems to improve physiological parameters of trout liver cells in primary culture.
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PMID:Viability and differential function of rainbow trout liver cells in primary culture: coculture with two permanent fish cells. 987 May 25

In the present study, time-dependent variations of drug-metabolising enzyme activities (DMEs) in primary cultures of rabbit hepatocytes, a species of economic importance in Mediterranean countries, were investigated. Cross-bred rabbits were anesthetised and their livers perfused in situ by a two-step collagenase technique; cells suspensions were filtered, seeded in collagen-coated dishes and cultivated at 37 degrees C in a controlled atmosphere for 24 and 72 h. Cytochrome P450 and b(5) contents as well as the catalytic activity of some P450-dependent monooxygenases were measured in subcellular fractions obtained by differential ultracentrifugation; microsomal proteins were also subjected to immunoblotting, using antibodies to rat P4501A, 2B, 2E1 and 3A isoforms. The activity of some microsomal hydrolytic enzymes was also determined. As regards conjugative enzymes, glutathione content and activities of glutathione S-transferase, uridindiphosphoglucuronosyl-transferase, acetyl-transferase and 1,2-epoxibuthane glutathione transferase were assayed. An overall reduction of the catalytic activity was observed 72 h after plating, reaching in certain instances the level of statistical significance. On the whole, our data confirm those previously reported with hepatocytes obtained from other species; however, the evidence that DMEs were still measurable after 72 h supports the usefulness of this in vitro method for drug metabolism studies in the rabbit as well.
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PMID:Time-dependent variations of drug-metabolising enzyme activities (DMEs) in primary cultures of rabbit hepatocytes. 1211 Feb 75

The preparation of hepatocytes using the two-stage perfusion technique usually involves the use of collagenase (CII) alone or in combination with dispase (C/D) or trypsin inhibitor (CA/TI) as digestion enzymes. The effect of CII, C/D and CA/TI on cell viability, yield, cytochrome P450 mediated oxidation of testosterone, glucuronidation and sulfation of 7-hydroxycoumarin, glutathione content, glutathione-S-transferase activity and glutathione-conjugation capacity of hepatocytes has been assessed. Cytochrome P450 mediated oxidation of testosterone was significantly (p < 0.05) decreased with CII isolated hepatocytes (81.7 +/- 3.3 nmol/10(6) cells, mean +/- S.E.M., n = 3), compared with those isolated using CA/TI (96.6 +/- 1.9 nmol/10(6) cells) or C/D (95.1 +/- 2.1 nmol/10(6) cells). In contrast, glutathione conjugation of the non-specific substrate 1-chloro-2,4-dinitrobenzene was significantly (p < 0.05) increased with CII isolated hepatocytes (56.9 +/- 5.9 nmol/10(6) cells, mean +/- S.E.M., n = 3), compared with those isolated using CA/TI (36.0 +/- 3.7 nmol/10(6) cells) or C/D (31.6 +/- 3.7 nmol/10(6) cells). These findings have significant implications for the interpretation of metabolism data derived from hepatocytes in suspension, particularly in terms of glutathione conjugation of potentially toxic reactive intermediates of xenobiotic metabolism. Indeed, data presented show that the presence of trypsin inhibitor in the preparation of isolated rat hepatocytes significantly affects the formation of glutathione conjugates of reactive intermediate products of troglitazone metabolism.
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PMID:The extent of phase I and phase II reactions is affected by the choice of enzyme used to prepare rat hepatocytes. 1933 Aug 83

Acrylamide (ACR) is an important industrial chemical used primarily in the production of polymers and co-polymers. Acrylamide is mainly neurotoxic to experimental animals as well as humans and has also been shown to be mutagenic and carcinogenic. The present study was designed to investigate the toxicity of ACR on isolated rat hepatocytes. The hepatocytes were isolated by collagenase perfusion method and were incubated with different concentrations of ACR (0.1, 1, 10mm) for 2 hours. Cell viability by trypan blue exclusion and leakage of the enzymes such as alanine transaminase (ALT) and aspartate transaminase (AST) were determined. Reduced glutathione (GSH), glutathione S-transferase (GST) activity were also measured. A significant decrease in the cell viability was observed after exposure to 10mm ACR for 30min, while 1mm ACR caused a significant decrease in the viability after 60min. ALT leakage was parallel to the cell viability. AST leakage was significantly increased at 30min of incubation with 10mm ACR, whereas 2 hours of incubation was required for the leakage of AST from rats hepatocytes with 1mm ACR. 10mm ACR decreased significantly GSH as early as 30min, while GSH level was decreased at 60min after exposure to 1mm ACR. Also, the GST activity increased with increasing the dose of ACR. Cytochrome P450 concentration was decreased after exposure to 10mm ACR. The effect of ACR on cell viability, ALT and AST leakage, GSH and GST activity was time and dose dependent.
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PMID:Acrylamide toxicity in isolated rat hepatocytes. 2065 59