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)

Short-term culture of rainbow trout (Onchorhynchus mykiss) hepatocytes was used to examine the effect of dexamethasone (DEX) on microsomal CYP 1A1 protein content and 7-ethoxyresorufin-O-deethylase (EROD) activity in vitro. Hepatocytes prepared by controlled collagenase digestion and plated at a density of 0.25 x 10(6) cells/cm2 in plastic culture dishes precoated with trout skin extract (7.6 micrograms skin protein/cm2) to facilitate cell attachment were maintained at 16 degrees C. Cells were treated with DEX (10(-9) to 10(-7) M) or vehicle (dimethyl sulfoxide, DMSO) at 24 h. Microsomal CYP 1A1 protein content and EROD activities were measured at 72 h. Both CYP 1A1 protein as measured by Western blots using CYP 1A1 specific anti-sera and EROD activity were significantly lower in DEX (10(-8) to 10(-7) M)-treated hepatocytes compared to untreated (control) or DMSO-treated cells. The effect was dose dependent in that a gradual decrease of CYP 1A1 protein and EROD activities were seen with increasing doses of DEX (10(-8) to 10(-7) M). DEX at 10(-9) M was ineffective. Concomitant addition of 10(-6) M RU486, a type II specific glucocorticoid receptor antagonist, to hepatocytes treated with 10(-7) M DEX abolished the DEX effect. RU486 at 10(-8) M was ineffective. Spironolactone (10(-8) to 10(-6) M), a type I specific glucocorticoid receptor antagonist, did not counteract the DEX effect. RU486 or spironolactone (10(-6) M) alone had no effect on CYP 1A1 under similar conditions. DEX thus down regulates CYP 1A1 in fish cultured hepatocytes and this regulation is mediated through the type II glucocorticoid receptor(s).
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PMID:Down regulation of CYP 1A1 by glucocorticoids in trout hepatocytes in vitro. 839 84

Alcohol affects the liver through metabolic disturbances associated with its oxidation. Redox changes produced by the hepatic alcohol dehydrogenase pathway affect lipid, carbohydrate and protein metabolism. Ethanol is also oxidized in liver microsomes by the ethanol-inducible cytochrome P4502E1, resulting in ethanol tolerance and selective hepatic perivenular damage. Furthermore, P4502E1 activates various xenobiotics, explaining the increased susceptibility of the heavy drinker to the toxicity of anesthetics, commonly used medications (i.e. isoniazid), analgesics (i.e. acetaminophen), and chemical carcinogens. Induction of microsomal enzymes also contributes to vitamin A depletion, enhances its hepatotoxicity and results in increased acetaldehyde generation from ethanol, with formation of protein adducts, glutathione depletion, free-radical-mediated toxicity, and lipid peroxidation. Chronic ethanol consumption strikingly enhances the number of hepatic collagen-producing activated lipocytes. Both in vivo (in our baboon model of alcoholic cirrhosis) and in vitro (in cultured myofibroblasts and activated lipocytes) ethanol and/or its metabolite acetaldehyde increase collagen accumulation and mRNA for collagen. Gender differences are related, in part, to lower gastric ADH activity (with consequent reduction of first pass ethanol metabolism) in young women, decreased hepatic fatty acid binding protein and increased free-fatty acid levels as well as lesser omega-hydroxylation, all of which result in increased vulnerability to ethanol. Elucidation of the biochemical effects of ethanol are now resulting in improved therapy: in baboons, S-adenosyl-L-methionine attenuates the ethanol-induced glutathione depletion and associated mitochondrial lesions, and polyenylphosphatidylcholine opposes the ethanol-induced hepatic phospholipid depletion, the decrease in phosphatidylethanolamine methyltransferase activity and the activation of hepatic lipocytes, with full prevention of ethanol-induced septal fibrosis and cirrhosis; its dilinoleoyl species also increases collagenase activity in lipocytes. The efficacy of this compound in man is now being studied in randomized multicenter clinical trials.
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PMID:Susceptibility to alcohol-related liver injury. 897 51

Alcohol-induced tissue damage results from associated nutritional deficiencies as well as some direct toxic effects, which have now been linked to the metabolism of ethanol. The main pathway involves liver alcohol dehydrogenase which catalyzes the oxidation of ethanol to acetaldehyde, with a shift to a more reduced state, and results in metabolic disturbances, such as hyperlactacidemia, acidosis, hyperglycemia, hyperuricemia and fatty liver. More severe toxic manifestations are produced by an accessory pathway, the microsomal ethanol oxidizing system involving an ethanol-inducible cytochrome P450 (2E1). After chronic ethanol consumption, there is a 4- to 10-fold induction of 2E1, associated not only with increased acetaldehyde generation but also with production of oxygen radicals that promote lipid peroxidation. Most importantly, 2E1 activates many xenobiotics to toxic metabolites. These include solvents commonly used in industry, anaesthetic agents, medications such as isoniazid, over the counter analgesics (acetaminophen), illicit drugs (cocaine), chemical carcinogens, and even vitamin A and its precursor beta-carotene. Furthermore, enhanced microsomal degradation of retinoids (together with increased hepatic mobilization) promotes their depletion and associated pathology. Induction of 2E1 also yields increased acetaldehyde generation, with formation of protein adducts, resulting in antibody production, enzyme inactivation, decreased DNA repair, impaired utilization of oxygen, glutathione depletion, free radical-mediated toxicity, lipid peroxidation, and increased collagen synthesis. New therapies include adenosyl-L-methionine which, in baboons, replenishes glutathione, and attenuates mitochondrial lesions. In addition, polyenylphosphatidylcholine (PPC) fully prevents ethanol-induced septal fibrosis and cirrhosis, opposes ethanol-induced hepatic phospholipid depletion, decreased phosphatidylethanolamine methyltransferase activity and activation of hepatic lipocytes, whereas its dilinoleoyl species increases collagenase activity. Current clinical trials with PPC are targeted on susceptible populations, namely heavy drinkers at precirrhotic stages.
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PMID:Ethanol metabolism, cirrhosis and alcoholism. 902 26

1. We have investigated interactions between intracellular pH (pHi) and the intracellular free calcium concentration ([Ca2+]i) in collagenase-isolated rat lacrimal acinar cells. The fluorescent dyes fura-2 and 2',7'-bis(carboxyethyl)-5-carboxyfluorescein (BCECF) were used to measure [Ca2+]i and pHi, respectively. 2. Application of the weak base NH4Cl alkalinized the cytosol and caused a dose-dependent increase in [Ca2+]i. Trimethylamine (TMA) also alkalinized the cytosol and increased [Ca2+]i. The increase in [Ca2+]i evoked by NH4Cl or TMA was much smaller than that evoked by the secretory agonist acetylcholine (ACh). 3. Application of NH4Cl also increased [Ca2+]i in cells bathed in Ca(2+)-free medium, indicating that NH4Cl released Ca2+ from an intracellular pool. 4. Ammonium chloride had no effect on [Ca2+]i in cells bathed in Ca(2+)-free medium if agonist-sensitive intracellular Ca2+ pools had been depleted with either ACh or the microsomal Ca(2+)-ATPase inhibitor 2,5-di(tert-butyl)hydroquinone. Treatment of cells with NH4Cl in Ca(2+)-free medium reduced the amount of Ca2+ released by ACh. These results suggest that NH4Cl released Ca2+ from the same intracellular pool released by ACh. 5. Calcium release from the agonist-sensitive pool was also triggered when the cytosol was alkalinized by removing the weak acid acetate. 6. Ammonium chloride caused a modest increase in inositol phosphate production, suggesting that NH4Cl may have released stored Ca2+ via an increase in the intracellular inositol 1,4,5-trisphosphate concentration. 7. The increase in [Ca2+]i evoked by NH4Cl was not sustained even in the presence of extracellular Ca2+. In contrast, when a low dose of ACh was used to evoke intracellular Ca2+ release of similar magnitude, sustained Ca2+ entry was observed. 8. Alkalinizing the cytosol appeared to partially inhibit Ca2+ entry triggered by thapsigargin or by ACh. 9. We suggest that alkalinizing the cytoplasm in unstimulated lacrimal acinar cells can release Ca2+ from the intracellular agonist-sensitive Ca2+ pool. However, releasing stored Ca2+ via alkalinization does not appear to trigger significant Ca2+ entry, perhaps because intracellular alkalinization inhibits either the Ca2+ entry pathway or the mechanism which couples the entry pathway to store depletion.
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PMID:Intracellular alkalinization mobilizes calcium from agonist-sensitive pools in rat lacrimal acinar cells. 913 Jan 57

The expression of CYP1B1 in human mammary fibroblasts (HMFs) was characterized as a potential modulator of their individual function as well as effects on adjacent mammary epithelia. We have used these characteristics to explore the diversity of fibroblast cells isolated from reduction mammoplasty patients and from different breast locations in breast cancer patients (tumors, peripheral to tumor and skin). These parameters have also been used to examine differences between two donors. The results have shown that while none of these HMFs expressed a detectable CYP1A1 protein basally or in response to TCDD, they all expressed CYP1B1 constitutively at similar levels (0.5-0.9 pmol/mg microsomal proteins) and they were induced by TCDD (up to 5-fold) consistent with mediation by the Ah receptor (AhR). DMBA metabolism by HMFs exhibited high proportions of 5,6-, 10,11- and 3,4-dihydrodiols, a profile that is typical of human CYP1B1 regioselectivity. RT-PCR followed by Southern blot analyses demonstrated that CYP1B1 mRNA expression in HMFs parallels levels of respective microsomal proteins. The AhR is expressed in these HMFs as two cytosolic forms (approximately 106 and 104 kDa) and a substantial proportion of the 104 kDa form was localized to the nucleus even prior to TCDD treatment. In all HMFs isolated directly from collagenase digested breast tissues the AhR is expressed at levels 10-fold lower than in breast epithelial cells. However, HMFs that were isolated after serial passaging of mammary epithelial cultures had shown much higher levels of the AhR expression and more dramatic TCDD-induced down-regulation (>80% in 24 h) associated with more efficient nuclear translocation. These differences suggested the presence of two functionally distinct subtypes of HMFs: interstitial stromal fibroblasts that are readily released by collagenase digestion of breast tissues, and lobular stromal fibroblasts which are more tightly associated with the breast epithelia.
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PMID:Expression of CYP1B1 but not CYP1A1 by primary cultured human mammary stromal fibroblasts constitutively and in response to dioxin exposure: role of the Ah receptor. 974 40

To investigate the hormonal control of the expression of flavin-containing monooxygenase (FMO; EC 1.14.13.8) under defined in vitro conditions, adult male rat hepatocytes were isolated by collagenase perfusion and co-cultured with rat liver epithelial cells of primitive biliary origin. The direct effect of 17beta-estradiol, testosterone, 5alpha-dihydrotestosterone (5alpha-DHT) and human growth hormone (hGH) on FMO activity was studied using this in vitro model. Optimal, non-cytotoxic hormonal concentrations were determined by measuring the lactate dehydrogenase (LDH) index. In addition, the microsomal protein content of the cultured hepatocytes was determined as a function of culture time. The female sex hormone 17beta-estradiol caused a significant decrease in FMO as a function of culture time. After 14 days of exposure, FMO activity decreased by 56%. Neither of the male sex hormones or human growth hormone had an effect on FMO activity. These results in co-cultured male rat hepatocytes support in vivo observation that 17beta-estradiol is a potent hormone involved in the negative regulation of the expression of FMO in male rat liver.
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PMID:Hormonal regulation of microsomal flavin-containing monooxygenase activity by sex steroids and growth hormone in co-cultured adult male rat hepatocytes. 977 17

The ultraviolet A (UVA, 320-400 nm) component of sunlight has the potential to generate an oxidative stress in cells and tissue so that antioxidants (both endogenous and exogenous) strongly influence the biological effects of UVA. The expression of several genes (including heme oxygenase-1, HO-1; collagenase; the CL100 phosphatase and the nuclear oncogenes, c-fos and c-jun) is induced following physiological doses of UVA to cells and this effect can be strongly enhanced by removing intracellular glutathione or enhancing singlet oxygen lifetime. We have observed that heme is released from microsomal heme-containing proteins by UVA and other oxidants and that activation of HO-1 expression by UVA correlates with levels of heme release. UVA radiation also leads to an increase in labile iron pools (either directly or via HO-1) and eventual increases in ferritin levels. The role of heme oxygenase in protection of skin fibroblasts is probably an emergency inducible defense pathway to remove heme liberated by oxidants. The slower increase in ferritin levels is an adaptive response which serves to keep labile iron pools low and thereby reduce Fenton chemistry and oxidant-induced chain reactions involving lipid peroxidation. In keratinocytes, the primary target of UVA radiation, heme oxygenase levels are constitutively high (because of HO-2 expression). Since there is a corresponding increase in basal levels of ferritin the epidermis appears to be well protected constitutively against the oxidative stress generated by UVA.
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PMID:Redox regulation and oxidant activation of heme oxygenase-1. 1051 38

In the past, alcoholic liver disease was attributed exclusively to dietary deficiencies, but experimental and judicious clinical studies have now established alcohol's hepatotoxicity. Despite an adequate diet, it can contribute to the entire spectrum of liver diseases, mainly by generating oxidative stress through its microsomal metabolism via cytochrome P4502E1 (CYP2E1). It also interferes with nutrient activation, resulting in changes in nutritional requirements. This is exemplified by methionine, one of the essential amino acids for humans, which needs to be activated to S-adenosylmethionine (SAMe), a process impaired by liver disease. Thus, SAMe rather than methionine is the compound that must be supplemented in the presence of significant liver disease. In baboons, SAMe attenuated mitochondrial lesions and replenished glutathione; it also significantly reduced mortality in patients with Child A or B cirrhosis. Similarly, decreased phosphatidylethanolamine methyltransferase activity is associated with alcoholic liver disease, resulting in phosphatidylcholine depletion and serious consequences for the integrity of membranes. This can be offset by polyenylphosphatidylcholine (PPC), a mixture of polyunsaturated phosphatidylcholines comprising dilinoleoylphosphatidylcholine (DLPC), which has high bioavailability. PPC (and DLPC) opposes major toxic effects of alcohol, with down-regulation of CYP2E1 and reduction of oxidative stress, deactivation of hepatic stellate cells, and increased collagenase activity, which in baboons, results in prevention of ethanol-induced septal fibrosis and cirrhosis. Corresponding clinical trials are ongoing.
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PMID:ALCOHOL: its metabolism and interaction with nutrients. 1094 Mar 40

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 present work describes an isozyme-related effect of collagenase perfusion on hepatocyte microsomal cytochrome (CYP)-dependent monooxygenase activities: CYP 1A1/2-, 2B1/2-, 3A1/2- and 2E1-dependent activities in microsomes from rat hepatocytes after isolation were about 60% of that of liver microsomes, and CYP 4A1-dependent activity was equivalent to liver microsomes. In contrast, the microsomal protein content of the various CYP isoforms was not affected by hepatocyte isolation. This is in accordance with the hypothesis of CYP inactivation during the process of hepatocyte isolation by collagenase digestion. L-NAME (1 mM) was found unable to protect from the decline of CYP-dependent monooxygenase activities following hepatocyte isolation. It is possible that the decrease in glutathione peroxidase activity observed in the presence of L-NAME, namely depression of defense against peroxynitrite, could counteract the beneficial effect of L-NAME on nitric oxide synthesis inhibition. The present work also shows that L-NAME could not avoid the progressive, isoform-specific, most probably turnover-related, decline of CYP proteins and related monooxygenase activities in cultured hepatocytes. Dysregulations in the mechanisms of CYP expression in rat hepatocyte cultures, presently unknown but nitric oxide independent, thus appear to occur in cultured rat hepatocytes.
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PMID:Time course of cytochromes P450 decline during rat hepatocyte isolation and culture: effect of L-NAME. 1253 63


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