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)

Two methods avoiding the widespread technique of collagenase perfusion have been employed to study the regulation of total cytochrome P450 content in rat hepatocyte culture. One technique required the perfusion of the liver with the chelating agent EDTA to dissociate the parenchymal cells prior to culture. Over a period of 48 hr, cultured hepatocytes isolated by EDTA perfusion showed comparable losses of cytochrome P450 as cells isolated by perfusion with collagenase. The second technique involved the culture of 210-240 microns thick "precision cut" liver slices. The results presented here indicate that the liver slices remain viable for 24 hr of culture, but that liver slices also lose their cytochrome P450 content at a comparable rate to collagenase prepared cells in culture. Collectively the results suggest that there is not a direct causal relationship between the loss of cytochrome P450 and one or a combination of the use of collagenase; the loss of cell-cell contacts and the absence of an extracellular matrix.
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PMID:Evidence that the loss of rat liver cytochrome P450 in vitro is not solely associated with the use of collagenase, the loss of cell-cell contacts and/or the absence of an extracellular matrix. 131 Aug 50

The involvement of cytochrome P450 in the liver toxicity of the potent carcinogen, N-nitrosodimethylamine (NDMA) was investigated in hepatocytes isolated from the periportal or perivenous region by digitonin-collagenase perfusion. Exposure of hepatocytes in culture to NDMA (0.5 or 5 mM) for up to 18 hrs caused little damage, but after 42 hr loss of cell viability became evident, and the extent of cell death was higher in perivenous cells than in periportal cells. Pretreatment of rats with ethanol caused a dramatically enhanced cell damage in perivenous cells (80%) compared to periportal cells (45%). This ethanol pretreatment caused a several-fold induction of cytochrome P450 2E1, as determined both with Western blot and as NDMA demethylase activity, and the effect was observed almost exclusively in perivenous cells. Isoniazid, an inhibitor of cytochrome P450 2E1, completely protected against NDMA toxicity. Glutathione dependent cytoprotective mechanisms and lipid peroxidation did not appear to be critical in NDMA toxicity, as evidence by lack of potentiation of toxicity by buthionine sulfoximine, an inhibitor of glutathione synthesis, and by the absence of increased lipid peroxidation. Instead, the higher expression of cytochrome P450 2E1 in the perivenous cells seems to be the main determinant for the regiospecific toxicity of NDMA, and, consequently, probably also for the associated genotoxicity.
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PMID:Evidence for cytochrome P450 2E1-mediated toxicity of N-nitrosodimethylamine in cultured perivenous hepatocytes from ethanol treated rats. 143 24

Local production of estrogen in breast tissue may influence the growth of breast cancers. Peripheral conversion of C19 steroids to estrogens is catalyzed by the aromatase enzyme complex which is comprised of a specific form of cytochrome P450, aromatase cytochrome P450 (P450AROM) and the flavoprotein, NADPH-cytochrome P450 reductase. To evaluate P450AROM mRNA levels in breast tissue, a specific competitive polymerase chain reaction amplification procedure was devised. In this method, a rat P450AROM complementary RNA is coamplified as an internal standard in order to compare amplification reactions. The amplification products are recognized by hybridization with 32P-labeled oligonucleotides specific for each species. Densitometry is used to quantitate autoradiographs. Initial studies using RNA from whole breast tissue obtained from reduction mammoplasty revealed linearity of the relationship between the densitometer signal from the human amplification product and total RNA concentration. Breast tissue was then separated into a floating adipocyte fraction and a pelleted fraction containing the other cellular elements by collagenase digestion and centrifugation. Comparison of specific content of aromatase amplification product per unit weight of RNA extracted from adipocytes and pelleted cells revealed considerably higher levels in the RNA from the nonadipocyte fraction. Immunocytochemical characterization of this fraction revealed the presence of several cell types including macrophages, ductal epithelial cells, and endothelial cells, but primary cells of stromal origin.
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PMID:Determination of aromatase cytochrome P450 messenger ribonucleic acid in human breast tissue by competitive polymerase chain reaction amplification. 159 66

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

In order to facilitate the homogenization of lung tissue it was previously incubated with collagenase during 30 minutes. Morphological observations were performed in order to ascertain the cell integrity. The enzymatically digested tissue was homogenized in a 0.25 M sucrose solution containing 1 mM EDTA, 3 mM imidazole (pH.7.3) and supplemented with 1 mM imipramine in order to stabilize the mitochondria, which otherwise might contaminate the microsomal fraction. The homogenate was then centrifuged and subdivided into four fractions which were analyzed for their content in protein and for the activities of so-called marker enzymes. The cytochrome P450 level was measured in both control and 3-methylcholanthrene preparations. The activities and the kinetic parameters of lung benzpyrene hydroxylase and aldrin epoxidase were measured using the lung microsomal fractions from control and previously 3-methylcholanthrene treated rats; 3-methylcholanthrene pretreatment modified the catalytiac properties of both enzymes.
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PMID:Preparation and analysis of a lung microsomal fraction from control and 3-methylcholanthrene treated rats. 626 52

Ammonium perfluorooctanoate (C8) produced a dose-dependent increase in Leydig cell adenomas in Crl:CD BR (CD) rats fed 0, 30, or 300 ppm for 2 years. Administration of C8 to adult male CD rats, by gavage for 14 days, produced decreased serum and testicular interstitial fluid testosterone levels and increased serum estradiol levels. The C8-mediated decrease in the serum testosterone levels appeared to be due to a lesion at the level of the testis. These endocrine changes may play a role in the C8 induction of Leydig cell tumors. In the present work, C8 was examined for its ability to (1) directly affect Leydig cells in vitro using isolated Leydig cells from untreated rats and ex vivo using Leydig cells isolated from C8-treated rats, (2) affect testicular interstitial fluid hormone levels, and (3) induce aromatase activity. These studies were conducted to investigate the hypothesis that C8 produces an increase in estradiol by inducing cytochrome P450 XIX (aromatase), which converts testosterone to estradiol, and that the elevated estradiol levels ultimately produce Leydig cell hyperplasia and adenoma formation by acting as a mitogen or enhancing growth factor secretion. In the in vivo and ex vivo studies, adult male CD rats were gavaged with either 0 or 25 mg/kg/day C8 for 14 days. In addition to the ad libitum control, a second control group was pair-fed to the 25 mg/kg/day C8 group. In the in vitro and ex vivo studies, Leydig cells were isolated from testes of adult males by collagenase digestion followed by enrichment over Percoll gradients. A dose-dependent decrease in testosterone levels was observed in hCG-stimulated Leydig cells treated in vitro with C8 for 5 hr (IC50 approximately 200 microM). In contrast, ex vivo studies demonstrated an increase in testosterone production in hCG-stimulated Leydig cells from C8-treated rats when compared to Leydig cells isolated from either the ad libitum or pair-fed control rats. The in vitro data demonstrate that C8 directly inhibits testosterone release from Leydig cells, while the ex vivo data demonstrate that this inhibition is reversible.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Effects of ammonium perfluorooctanoate on Leydig cell function: in vitro, in vivo, and ex vivo studies. 767 54

The O-dealkylation of ethylmorphine (EM) and codeine (CD) to morphine (M) co-segregates with debrisoquine/sparteine genetic polymorphism in man. CD O-demethylation is catalysed by cytochrome P450 2D1 (CYP2D1) in rats. In the present study, the O-deethylation of EM was examined and compared with that of CD in suspensions of freshly-isolated hepatocytes prepared by a collagenase method from Wistar rats with and without CYP2D1 inhibitors. Isolated hepatocytes were also prepared from Dark Agouti (DA) rats deficient in CYP2D1, and were incubated with EM or CD. EM, CD and their metabolites were quantified by HPLC with UV detection. EM had a similar pattern of metabolism to that of CD in suspensions of hepatocytes from Wistar rats. Both EM and CD were O-dealkylated to form M plus morphine-3-glucuronide (M3G) and N-demethylated to form norethylmorphine (NEM) or norcodeine (NCD), respectively, which were further metabolized to normorphine (NM) and finally glucuronidated to normorphine-3-glucuronide (NM3G). As compared to hepatocytes from Wistar rats, DA rats were characterized by a markedly decreased formation (70 approximately 75% reduction) of M plus M3G from both EM and CD. Quinine, quinidine, propafenone and sparteine all inhibited EM O-deethylation as well as CD O-demethylation. Quinine was the most potent inhibitor of both these O-dealkylations (Ki = 0.2 microM for both EM and CD, respectively). Quinine as well as the other inhibitors inhibited both EM and CD O-dealkylation competitively and with small differences in Ki versus EM and CD, respectively. The metabolism of EM to M plus M3G and that of CD to M plus M3G was highly correlated when results from the various separate cell suspensions were plotted. In conclusion all findings indicated that the enzyme responsible for O-demethylation of CD, CYP2D1 was also responsible for the O-deethylation of EM to M.
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PMID:Ethylmorphine O-deethylation in isolated rat hepatocytes. Involvement of codeine O-demethylation enzyme systems. 787 51

The effects of cryopreservation and long-term storage on substrate-specific cytochrome P450-dependent activities and unscheduled DNA synthesis were studied in freshly isolated and cryopreserved hepatocytes derived from adult male Fischer 344 and Sprague-Dawley rats. Primary rat hepatocytes were isolated via an in situ collagenase perfusion technique, cryopreserved at -196 degrees C, and thawed at 5 weeks and 104 and 156 weeks post-freezing. In Fischer 344 and Sprague-Dawley rats, cryopreserved hepatocytes were equivalent or similar to freshly isolated hepatocytes in substrate-specific activities for 7-ethoxyresorufin-O-deethylase and dimethylnitrosamine-N-demethylase and unscheduled DNA synthesis responses. No significant differences in activities toward 7-ethoxyresorufin-O-deethylase and dimethylnitrosamine-N-demethylase, the substrate-specific activities for cytochromes P4501A1 and P4501A2 and cytochrome P4502E1, respectively, were observed between freshly isolated and cryopreserved hepatocytes. Similar unscheduled DNA synthesis responses, a measure of DNA damage and repair, were observed after exposure to the genotoxic carcinogens 2-acetylamino-fluorene, 7,12-dimethylbenz[a]anthracene, and dimethylnitrosamine; although some decreases were also observed in Fischer 344 hepatocytes after 104 weeks and Sprague-Dawley hepatocytes after 156 weeks in the highest concentrations tested. These results suggest that cryopreserved hepatocytes, stored for extended periods of time in liquid nitrogen, are metabolically equivalent to freshly isolated hepatocytes in their ability to activate precarcinogens.
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PMID:Cryopreservation and long-term storage of primary rat hepatocytes: effects on substrate-specific cytochrome P450-dependent activities and unscheduled DNA synthesis. 803 11

Three decades of research in ethanol metabolism have established that alcohol is hepatotoxic not only because of secondary malnutrition, but also through metabolic disturbances associated with the oxidation of ethanol. Some of these alterations are due to redox changes produced by the NADH generated via the liver ADH pathway, which in turn affects the metabolism of lipids, carbohydrates, proteins, and purines. Exaggeration of the redox change by the relative hypoxia, which prevails physiologically in the perivenular zone, contributes to the exacerbation of the ethanol-induced lesions in zone III. Gastric ADH also explains first-pass metabolism by ethanol; its activity is low in alcoholics and in females and is decreased by some H2 blockers. In addition to ADH, ethanol can be oxidized by liver microsomes: studies over the last 20 years have culminated in the molecular elucidation of the ethanol-inducible cytochrome P450 (P4502E1) which contributes not only to ethanol metabolism and tolerance, but also to the selective hepatic perivenular toxicity of various xenobiotics. Their activation by P4502E1 now provides an understanding for the increased susceptibility of the heavy drinker to the toxicity of industrial solvents, anesthetic agents, commonly prescribed drugs, over-the-counter analgesics, chemical carcinogens, and even nutritional factors such as vitamin A. Ethanol causes not only vitamin A depletion, but it also enhances its hepatotoxicity. Furthermore, induction of the microsomal pathway contributes to increased acetaldehyde generation, with formation of protein adducts, resulting in antibody production, enzyme inactivation, decreased DNA repair; it is also associated with a striking impairment of the capacity of the liver to utilize oxygen. Moreover, acetaldehyde promotes GSH depletion, free-radical-mediated toxicity, and lipid peroxidation. In addition, acetaldehyde affects hepatic collagen synthesis; both in vivo (in our baboon model of alcoholic cirrhosis) and in vitro (in cultured myofibroblasts and lipocytes); ethanol and its metabolite acetaldehyde were found to increase collagen accumulation and mRNA levels for collagen. This new understanding may eventually improve therapy with drugs and nutrients. Encouraging results have been obtained with some "super" nutrients. On the one hand, SAMe, the active form of methionine, was found to attenuate the ethanol-induced depletion in SAMe and GSH and associated mitochondrial lesions. On the other hand, phosphatidylcholine, purified from polyunsaturated lecithin, was discovered to oppose the ethanol-induced fibrosis by decreasing the activation of lipocytes to transitional cells, and possibly also by stimulating collagenase activity, an effect for which dilinoleoylphosphatidylcholine, its major phospholipid species, was found to be responsible.
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PMID:Biochemical factors in alcoholic liver disease. 833 2

After postnatal day 1 (d1), the hypothalamo-pituitary-adrenal axis of neonatal rats becomes less responsive to certain stimuli for up to 2 weeks. The present study was designed to quantify the development of adrenocortical cell responsiveness to its normal secretagogue, adrenocorticotropic hormone (ACTH), and to better localize intracellular sites of adrenal cell hyporesponsivity. Maximum steroidogenic responses of collagenase-dispersed adrenocortical cells (using two isolation methods) to ACTH varied significantly in the order adult > d1 > d10. The response pattern to dibutyryl cAMP ((Bu)2cAMP) was identical to that observed for ACTH (adult > d1 > d10), suggesting that neonatal adrenal responsiveness is limited by a site distal to cAMP formation. Sensitivity (EC50) of adult cells to ACTH was approximately 3-fold greater than in neonatal cells, but there was no age-dependent shift in sensitivity to (Bu)2cAMP. 20 alpha-Hydroxycholesterol (20 alpha-OHCHOL), a membrane permeable analog of cholesterol, also failed to normalize the d10 adrenal response to ACTH. This result indicates that one site of refractoriness is apparently distal to cholesterol transport, and strongly suggests possible differential cytochrome P450 enzyme expression or activity in neonatal rat adrenal cells. Finally, although stimulated secretion was lower in neonatal cells, basal corticosterone secretion was significantly greater in neonatal adrenals, suggesting that constitutive activity of neonatal adrenal cells is high compared to that of adult cells.
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PMID:Steroidogenesis in isolated adrenocortical cells during development in rats. 838 18


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