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)

Cocaine remains a widely abused illicit substance in our society. Cocaine hepatotoxicity has been linked to cocaine metabolism. Cocaine can undergo hydrolytic inactivation via plasma and hepatic esterases or it can be N-oxidized by cytochrome P-450 and FAD-containing monooxygenases. Ethanol is frequently used in combination with cocaine. The presence of ethanol can affect the metabolism of other agents, depending on the dose and duration of exposure. In this investigation, hepatocytes isolated from male Sprague-Dawley rats were utilized to study the effect of ethanol exposure on cocaine metabolism. Hepatocytes were isolated using a two-step collagenase perfusion system. Hepatocytes (2 x 10(6) cells ml(-1)) were exposed to cocaine, ethanol or the combination of cocaine and ethanol for a 2-h period in a shaking water-bath at 30 oscillations per minute maintained at 37 degrees C. Sodium fluoride (NaF) was added to aliquots of cells which were removed from the incubation following 30, 60 and 120 min. The cells were homogenized on ice and immediately extracted for the quantification of cocaine, benzoylecognine, norcocaine and ethylcocaine by HPLC. Quantitative analysis revealed that there was a time-dependent increase in the disappearance of cocaine from hepatocytes. The rate of cocaine disappearance was not changed when ethanol was included in incubations containing cocaine. However, in the presence of ethanol there was a difference in the quantities of cocaine metabolites produced. When ethanol was included in incubations containing cocaine, the formation of norcocaine and benzoylecognine was less than that formed in hepatocytes exposed to cocaine alone. Additionally, when hepatocytes were exposed to cocaine in combination with ethanol, the formation of ethylcocaine was linear with time. This study revealed that in the presence of ethanol, cocaine qualitative metabolism is altered.
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PMID:Role of ethanol exposure on cocaine metabolism in rat hepatocytes. 918 53

Freshly isolated and primary cultures of rat kidney cells derived from specific nephron segments can be useful in vitro models for studying processes such as drug metabolism, membrane transport, and biochemical mechanisms of chemically induced toxicity. Proximal tubular (PT) and distal tubular (DT) cells were isolated from rat renal cortex by collagenase perfusion and Percoll density-gradient centrifugation. Oxidants produced glutathione (GSH) oxidation and lipid peroxidation and were markedly more cytotoxic to DT cells than to PT cells. Similarly, alkylating agents that target soft nucleophiles such as GSH and protein sulfhydryls were more toxic to DT cells than to PT cells, whereas an alkylating agent that targets hard nucleophiles was equally cytotoxic in the 2 cell types. DT cells were also more sensitive to brief periods of oxygen deprivation and were markedly more susceptible to ATP depletion by treatment with iodoacetate and cyanide than were PT cells. Serum-free, hormonally defined conditions have been optimized for primary culture of rat renal PT and DT cells to maintain differentiated function for up to 9 days. Primary cultures exhibited similar susceptibilities as freshly isolated cells to acute injury from chemical toxicants and the cultures express several isoforms of cytochrome P-450. These studies show that freshly isolated and primary cultures of rat renal PT and DT cells can be used to study both short-term and long-term responses to toxic chemicals.
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PMID:In vitro methods of assessing renal damage. 950 85

Troglitazone (TRO) is an insulin sensitizer used in the treatment of type II diabetes. TRO is known to increase the activity of cytochrome P-450 (CYP) 3A in vivo. We have investigated the effect of TRO on CYP3A protein content and the activity of CYP3A (as measured by the formation of 6beta-hydroxytestosterone formation) in primary cultures of human hepatocytes in comparison with rifampicin (RIF). Hepatocytes were isolated from four human livers by perfusion with collagenase, plated on collagen-coated plates, and maintained in William's E medium. After 48 h in culture, cells were exposed to RIF (10 microM) or TRO (0-50 microM) twice, each over a period of 24 h, and the activity of CYP3A was measured. TRO increased the activity of CYP3A in a concentration-dependent manner, reaching a maximal response at 5 microM. Pretreatment of the hepatocytes with 10 microM TRO or 10 microM RIF resulted in a 4- to 15-fold increase in the activity of CYP3A. Maximum increase in CYP3A protein was observed at 5 microM TRO. There was a significant correlation (R(2) = 0.89) between the content of immunoreactive CYP3A protein in the hepatocytes and the rate of formation of 6beta-hydroxytestosterone. These results indicate that TRO is a potent inducer of CYP3A and is similar to RIF in inducing CYP3A in human hepatocytes. At concentrations of 25 microM and above, TRO was toxic to the cells, as determined by a decrease in the activity of CYP3A, a reduction in the amount of immunoreactive protein, and changes in the morphology of the cells.
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PMID:Troglitazone increases cytochrome P-450 3A protein and activity in primary cultures of human hepatocytes. 1049 47

The arachidonate signaling pathways comprise prostanoids formed by cyclooxygenases, EETs, and HETEs formed by cytochrome P-450 (CYP) enzymes and HETEs and leukotrienes generated by lipoxygenases. Whereas the intrarenal localization of cyclooxygenases and of some CYP enzymes along the nephron has already been determined, the localization of lipoxygenases and leukotriene-forming enzymes together with leukotriene receptors in the kidney is less clear. This study therefore aimed to determine the expression of 5-, 12-, and 15-lipoxygenases as well as the leukotriene receptors along the rat nephron. The kidneys were dissected into cortex and outer and inner medulla, and the microdissected nephron segments were collected after a collagenase digestion. mRNA abundance was determined by RT-PCR and real-time PCR. 15-LOX mRNA showed a characteristic expression pattern along the distal nephron. 12-LOX mRNA was only found in the glomerulus. Similarly, 5-LOX mRNAs together with 5-LOX-activating protein mRNAs were expressed in the glomerulus and also in the vasa recta. The leukotriene A4 hydrolase was found in all nephron segments, whereas leukotriene C4 synthase mRNA could not be found in any nephron segment. The leukotriene receptor B4 and the cysteinyl leukotriene receptor type 1 were selectively expressed in the glomerulus, whereas cysteinyl receptor type 2 was not found in any nephron segment. Our data suggest that the glomerulus is a major source and target for 5- and 12-HETE and for leukotrienes. The collecting duct system, on the other hand, appears to be a major source of 15-HETE.
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PMID:Gene expression of 5-, 12-, and 15-lipoxygenases and leukotriene receptors along the rat nephron. 1621 16

It was previously reported that in vivo exposure of fish to combined aryl hydrocarbon receptor agonist (AhR; 3,3',4,4'-tetrachlorobiphenyl, PCB-77) and estrogen receptor agonist (ER; nonylphenol, NP) resulted in potentiation and inhibition (depending on dose ratio, sequential order of exposure, and seasonal changes) of NP-induced responses by PCB-77. The experiments described in this report extend this study by testing whether the effects of PCB-77 on NP-induced ER signaling are mediated through AhR-induced transcriptional suppression of target genes. Trout hepatocytes were isolated by a two-step collagenase perfusion method. After 48-h culture, hepatocytes were exposed to 5 or 10 microM nonylphenol (NP) singly and in combination with PCB-77 at 0.1, 1, and 10 microM. Cells were harvested after 96-h exposure and processed for RNA isolation. Gene expression patterns were quantified using real-time polymerase chain reaction (PCR) with specific primer sets and by Northern blot. Exposure of cells to NP caused significant elevation of ERalpha, ERbeta, Vtg, and Zrp mRNA expressions, while combined exposure with PCB-77 concentration inhibited NP-induced ERs and their target gene expressions. Exposure of trout hepatocytes to PCB-77 alone caused a rapid induction of cytochrome P-450 (CYP) 1A1 mRNA, and combined exposure with NP caused significant reduction in PCB-77 induced CYP1A1 gene expression. Exposure of cells to PCB-77 concentrations induced significant reduction in AhRalpha mRNA (except 1 microM PCB-77, which caused the induction of AhRalpha mRNA levels). AhRbeta mRNA levels in the cells were inhibited after 96-h exposure to PCB-77, while combined exposure with 5 microM NP restored the PCB-77-inhibited AhRbeta mRNA levels to baseline. Taken together, the overall results in this study show that PCB-77 suppresses the gene expression of the ERs and their target genes by transcription mechanism(s). The roles of AhRs in mediating these responses seem to involve the ligand-activated AhR transcriptional induction of CYP1A1. In addition to their frequently described functions as activators of metabolic potentiation and detoxification of various foreign chemicals, data presented in the present study point to other endogenous functions of AhRs that need to be studied further.
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PMID:Gene expression patterns in estrogen (nonylphenol) and aryl hydrocarbon receptor agonists (PCB-77) interaction using rainbow trout (Oncorhynchus Mykiss) primary hepatocyte culture. 1629 59


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