Home About us Contact
|
Home About us Contact | ||
|
|
||
Hepatic Microsomes (hepatic + microsome)
Selected AbstractsInteraction of tributyltin with hepatic cytochrome P450 and uridine diphosphate-glucuronosyl transferase systems of fish: In vitro studiesENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 4 2004Yolanda Morcillo Abstract Hepatic microsomes of red mullet (Mullus barbatus) and flounder (Platichthys flesus) were preincubated in the presence of a concentration range of the antifouling agent tributyltin (TBT) chloride, and the interactions of TBT with cytochrome P450 and uridine diphosphate,glucuronyl transferase systems were investigated. The enzyme systems were examined in terms of cytochrome P4501A (CYP1A)-catalyzed 7-ethoxyresorufin O -deethylase (EROD) activity and benzo[a]pyrene (BaP) metabolism and in terms of glucuronidation of testosterone and 17,-estradiol, respectively. Ethoxyresorufin O -deethylase and BaP hydroxylase (BPH) activities of both fish species were progressively inhibited by increasing concentrations of TBT, and the effects were more pronounced for EROD than for BPH (maximal inhibition at 100 ,M TBT for EROD and 250,500 ,M TBT for BPH). Hydroxylated metabolites of BaP (3-hydroxy-, 7,8-dihydrodiol, and 9,10-dihydrodiol), representing 95% of the total metabolites formed, were reduced up to 75 % in the presence of 100 to 500 ,M TBT, whereas the formation of other metabolites was less affected. This may alter BaP toxicity and carcinogenicity. Overall, the results were consistent with a specific inhibitory effect of TBT on CYP1A in the two fish species. Additionally, the conjugation of testosterone was significantly inhibited (20%) at low TBT doses (5 ,M), with no effect on the glucuronidation of estradiol. [source] Hepatic microsomal cytochrome P450 enzyme activity in relation to in vitro metabolism/inhibition of polychlorinated biphenyls and testosterone in Baltic grey seal (Halichoerus grypus)ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 3 2003Hongxia Li Abstract Among other factors, cytochrome P450 (CYP) enzyme activity determines polychlorinated biphenyl (PCB) bioaccu-mulation, biotransformation, and toxicity in exposed species. We measured the oxidative metabolism in vitro of 12 PCB congeners, representing structural groups based on the number and position of the chlorine atoms, by the hepatic microsomes of one Baltic grey seal (Halichoerus grypus). Microsomal metabolism was observed for several PCBs with vicinal H atoms exclusively in the ortho and meta positions and without any ortho -Cl substituents (CB-15 [4,4,-Cl2] and CB-77 [3,3,,4,4,-Cl4]), vicinal meta and para -H atoms (CB-52 [2,2,,5,5,-Cl4], and ,101 [2,2,,4,5,5,-Cl5]) or with both characteristics in combination with either only one ortho -Cl (CB-26 [2,3,,5-Cl3], CB-31 [2,4,,5-Cl3]) or two ortho -Cl substituents (CB-44 [2,2,,3,5,-Cl4]). To allocate PCB biotransformation to specific CYPs, the inhibitive effect of compounds with known CYP-specific inhibition properties was assessed on in vitro PCB metabolism and on regio- and stereospecific testosterone hydroxylase activities. Metabolic inhibition was considered relevant at concentrations ,1.0 ,M because these inhibitors became decreasingly selective at higher concentrations. At <1.0 ,M, ellipticine (CYP1A1/2 inhibitor) selectively inhibited CB-15, ,26, ,31, and ,77 metabolism, with no significant inhibition of CB-44, ,52, and ,101 metabolism. Inhibition of CB-52 and ,101 metabolism by chloramphenicol (CYP2B inhibitor) started at 1.0 ,M and maximized at about 100% at 10 ,M. Ketoconazole (CYP3A inhibitor) appeared to selectively inhibit CB-26, ,31, and ,44 metabolism relative to CB-15, ,77, and ,52 at concentrations ,1.0 ,M. Major testosterone metabolites formed in vitro were 2,-(CYP3A), 6,- (CYP3A, CYP1A), and 16,- (CYP2B) hydroxytestosterone and androstenedione (CYP2B, CYP2C11). The CYP forms indicated are associated with the specific metabolism of testosterone in laboratory animals. Inhibition of 2,- and 6,-hydroxytestosterone formation at ellipticine and ketoconazole concentrations ,1.0,M suggested that both inhibitors were good substrates of CYP3A-like enzymes in grey seal. Chloramphenicol (model for CYP2B) is apparently not a good inhibitor of CYP1A and CYP3A activities in grey seal because the chemical did not inhibit any metabolic route of testosterone at concentrations from 0.1 to 10 ,M. Our findings demonstrated that at least CYP1A- and CYP3A-like enzymes in the liver of grey seals are capable of metabolizing PCBs with ortho - meta and/or meta - para vicinal hydrogens. A CYP2B form might also be involved, but this could not be proven by the results of our experiments. Defining the profiles of CYP enzymes that are responsible for PCB biotransformation is necessary to fully understand the bioaccumulation, toxicokinetics, and risk of PCB exposure in seals and other free-ranging marine mammals. [source] Immunohistochemical localization of cytochrome P4501A induced by 3,3,,4,4,,5-pentachlorobiphenyl (PCB 126) in multiple organs of northern leopard frogs, Rana pipiensENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 1 2001Yue-Wern Huang Abstract Monoclonal antibody 1 12,3(MA1 12,3)recognizesanepitopeexclusivetocytochromeP450sinsubfamily1A (CYP1A) from all vertebrates tested so far, including one amphibian species. In this study, we first tested the utility of MAb 1,12-3 for detection of presumed CYP1A proteins in hepatic microsomes of northern leopard frogs treated without or with 3,3,,4,4,,5-pentachlorobiphenyl (PCB 126). Statistical analysis showed that ethoxyresorufin- O -deethylase (EROD) activities and CYP1A equivalents in treated groups were significantly increased at doses >2.3 mg/kg compared with the control groups (p < 0.05), and the increases were maintained for at least four weeks. This result confirmed that MAb 1,12-3 can be used for detection of CYP1A in northern leopard frogs and indicated that CYP1A is the primary catalyst for EROD in this species. In a subsequent experiment, sections of organs of PCB 126-treated frogs were immunohistochemically stained with MAb 1,12-3 to identify localization of the CYP1A in different cell types. The CYP1A staining was seen prominently in hepatocytes and epithelium of nephronic duct, while capillaries close to gastric epithelium and submucosal vascular epithelium in both stomach and intestine exhibited moderate to strong staining. The CYP1A was immunodetected in coronary endothelium and the vascular endothelium of lung and gonad. In skin, mild staining was seen in epithelial cells of mucous glands and serous glands and in vascular endothelium, demonstrating induction of CYP1A in the dermal layer. [source] Inhibition of microsomal triglyceride transfer protein: Another mechanism for drug-induced steatosis in miceHEPATOLOGY, Issue 1 2003Philippe Lettéron Although many steatogenic drugs inhibit mitochondrial fatty acid ,-oxidation, limited information is available on possible effects on hepatic lipoprotein secretion. In the endoplasmic reticulum (ER) lumen, microsomal triglyceride transfer protein (MTP) lipidates apolipoprotein B (Apo B), to form triglyceride (TG)-rich very low density lipoprotein (VLDL) particles, which follow vesicular flow to the plasma membrane to be secreted, whereas incompletely lipidated Apo B particles are partly degraded. We studied hepatic MTP activity, the lipoproteins present in the ER lumen, and hepatic lipoprotein secretion 4 hours after administration of a single dose of amineptine (1 mmol/kg), amiodarone (1 mmol/kg), doxycycline (0.25 mmol/kg), tetracycline (0.25 mmol/kg), tianeptine (0.5 mmol/kg), or pirprofen (2 mmol/kg) in mice. These various doses have been shown previously to markedly inhibit fatty acid oxidation after a single dose, and to trigger steatosis either after repeated doses (doxycycline) or a single dose (other compounds) in mice. In the present study, amineptine, amiodarone, pirprofen, tetracycline, and tianeptine, but not doxycycline, inhibited MTP activity in vitro, decreased ex vivo MTP activity in the hepatic homogenate of treated mice, decreased TG in the luminal VLDL fraction of hepatic microsomes of treated mice, and decreased in vivo hepatic lipoprotein secretion (TG and Apo B). In conclusion, several steatogenic drugs inhibit not only mitochondrial ,-oxidation, as previously shown, but also MTP activity, Apo B lipidation into TG-rich VLDL particles, and hepatic lipoprotein secretion. Drugs with these dual effects may be more steatogenic than drugs acting only on ,-oxidation or only MTP. [source] CYP3A4 is a Human Microsomal Vitamin D 25-Hydroxylase,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 4 2004Ram P Gupta Abstract The human hepatic microsomal vitamin D 25-hydroxylase protein and gene have not been identified with certainty. Sixteen hepatic recombinant microsomal enzymes were screened for 25-hydroxylase activity; 11 had some 25-hydroxylase activity, but CYP3A4 had the highest activity. In characterized liver microsomes, 25-hydroxylase activity correlated significantly with CYP3A4 testosterone 6,-hydroxylase activity. Activity in pooled liver microsomes was inhibited by known inhibitors of CYP3A4 and by an antibody to CYP3A2. Thus, CYP3A4 is a hepatic microsomal vitamin D 25-hydroxylase. Introduction: Studies were performed to identify human microsomal vitamin D-25 hydroxylase. Materials and Methods: Sixteen major hepatic microsomal recombinant enzymes derived from cytochrome P450 cDNAs expressed in baculovirus-infected insect cells were screened for 25-hydroxylase activity with 1,-hydroxyvitamin D2 [1,(OH)D2], 1,-hydroxyvitamin D3 [1,(OH)D3], vitamin D2, and vitamin D3 as substrates. Activity was correlated with known biological activities of enzymes in a panel of 12 characterized human liver microsomes. The effects of known inhibitors and specific antibodies on activity also were determined. Results: CYP3A4, the most abundant cytochrome P450 enzyme in human liver and intestine, had 7-fold greater activity than that of any of the other enzymes with 1,(OH)D2 as substrate. CYP3A4 25-hydroxylase activity was four times higher with 1,(OH)D2 than with 1,(OH)D3 as substrate, was much less with vitamin D2, and was not detected with vitamin D3. 1,(OH)D2 was the substrate in subsequent experiments. In a panel of characterized human liver microsomes, 25-hydroxylase activity correlated with CYP3A4 testosterone 6,-hydroxylase activity (r = 0.93, p < 0.001) and CYP2C91 diclofenac 4,-hydroxylase activity (r = 0.65, p < 0.05), but not with activity of any of the other enzymes. Activity in recombinant CYP3A4 and pooled liver microsomes was dose-dependently inhibited by ketoconazole, troleandomycin, isoniazid, and ,-naphthoflavone, known inhibitors of CYP3A4. Activity in pooled liver microsomes was inhibited by antibodies to CYP3A2 that are known to inhibit CYP3A4 activity. Conclusion: CYP3A4 is a vitamin D 25-hydroxylase for vitamin D2 in human hepatic microsomes and hydroxylates both 1,(OH)D2 and 1,(OH)D3. [source] In vivo pharmacology and antidiarrheal efficacy of a thiazolidinone CFTR inhibitor in rodentsJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 1 2005N.D. Sonawane Abstract A small-molecule inhibitor of the cystic fibrosis transmembrane conductance regulator (CFTR), 3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-thiazolidinone (CFTRinh -172), reduces enterotoxin-induced intestinal fluid secretion in rodents. Here, we study CFTRinh -172 pharmacology and antidiarrheal efficacy in rodents using 14C-labeled CFTRinh -172, liquid chromatography/mass spectrometry, and a closed intestinal loop model of fluid secretion. CFTRinh -172 was cleared primarily by renal glomerular filtration without chemical modification. CFTRinh -172 accumulated in liver within 5 min after intravenous infusion in mice, and was concentrated fivefold in bile over blood. At 30,240 min, CFTRinh -172 was found mainly in liver, intestine, and kidney, with little detectable in the brain, heart, skeletal muscle, or lung. Pharmacokinetic analysis in rats following intravenous bolus infusion showed a distribution volume of 770 mL with redistribution and elimination half-times of 0.14 h and 10.3 h, respectively. CFTRinh -172 was stable in hepatic microsomes. Closed-loop studies in mice indicated that a single intraperitoneal injection of 20 ,g CFTRinh -172 inhibited fluid accumulation at 6 h after cholera toxin by >90% in duodenum and jejunum, ,60% in ileum and <10% in colon. No toxicity was seen after high-dose CFTRinh -172 administration (3 mg/kg/day in two daily doses) in mice over the first 6 weeks of life. The metabolic stability, enterohepatic recirculation, slow renal elimination, and intestinal accumulation of CFTRinh -172 account for its efficacy as an antidiarrheal. © 2004 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 94:134,143, 2005 [source] Inhibition of CYP3A-mediated oxidation in human hepatic microsomes by the dietary derived complex phenol, gallic acidJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 2 2002Ieva Stupans Plant polyphenols, such as gallic acid, have been reported to have a range of biological activities including antimutagenic effects. Previously, we reported that gallic acid (3,4,5-trihydroxy-benzoic acid), an agent found in wine and tea, inhibits androstenedione 6,-hydroxylase activity (Ki 70 ,M), a cytochrome P450 (CYP3A) marker in human liver microsomes. The preincubation of gallic acid (100 ,M) with human liver microsomes in the absence of NADPH, as compared with the presence of NADPH, before assay of androstenedione 6,-hydroxylase activity significantly increased the inhibitory effects of the gallic acid (0.03 ± 0.03 nmol (mg microsomal protein),1 min,1 compared with 0.20 ± 0.06 nmol (mg microsomal protein),1 min,1 (P< 0.05)). The antioxidant ascorbic acid and the radical scavenger glutathione prevented this observed increase in inhibition. Removal of gallic acid-derived products from the incubation completely restored CYP3A activity. In contrast, the activities of CYP1A and CYP2E, and non-CYP mediated reductive microsomal 17,-hydroxysteroid dehydrogenase activity were refractory to inhibition by gallic acid. [source] Acetaminophen UDP-glucuronosyltransferase in ferrets: species and gender differences, and sequence analysis of ferret UGT1A6JOURNAL OF VETERINARY PHARMACOLOGY & THERAPEUTICS, Issue 6 2001M. H. Court The principal objective of this study was to determine whether ferrets glucuronidate acetaminophen more slowly compared with other species, and if so investigate the molecular basis for the difference. Acetaminophen-UDP-glucuronosyltransferase (UGT) activities were measured using hepatic microsomes from eight ferrets, four humans, four cats, four dogs, rat, mouse, cow, horse, monkey, pig and rabbit. Gender differences between male and female ferret livers were explored using enzyme kinetic analysis. Immunoblotting of microsomal proteins was also performed using UGT-specific antibodies. Finally, the exon 1 region of UGT1A6, a major acetaminophen-UGT, was sequenced. Glucuronidation of acetaminophen was relatively slow in ferret livers compared with livers from all other species except cat. Gender differences were also apparent, with intrinsic clearance (Vmax/Km) values significantly higher in male compared with female ferret livers. Furthermore, Vmax values correlated with densitometric measurements of two protein bands identified with a UGT1A subfamily-specific antibody. No deleterious mutations were identified in the exon 1 or flanking regions of the ferret UGT1A6 gene. In conclusion, like cats, ferret livers glucuronidate acetaminophen relatively slowly. However, unlike cats, in which UGT1A6 is encoded by a pseudogene and dysfunctional, there are no defects in the ferret UGT1A6 gene which could account for the low activity. [source] Influence of verapamil on the efflux and metabolism of 14C moxidectin in cultured rat hepatocytesJOURNAL OF VETERINARY PHARMACOLOGY & THERAPEUTICS, Issue 3 2001J. Dupuy Moxidectin (MOX) is an antiparasitic drug widely used in cattle, sheep and companion animals. As a result of the implication of cytochrome P450 3 A in the metabolism of MOX and the role of competitor substrates of P-glycoprotein (Pgp) in modification of the bioavailability of endectocides, we studied the influence of verapamil (a multidrug-resistance reversing agent) on the metabolism of 14C moxidectin in cultured rat hepatocytes over 72 h. The metabolism of MOX remained low: 10.79 ± 1.99% of the total 14C moxidectin for the main detected metabolite in verapamil-treated cells and 7.17 ± 0.74% for the control cells after 24 h. The main detected metabolite in rat hepatocytes was the same as that detected in rat hepatic microsomes (the C29 monohydroxymethyl metabolite). Verapamil increased the quantity of MOX in the cells after 24, 48 and 72 h. Examination of the Area Under the concentration time Curve (AUC) of the main detected metabolite revealed a significant increase in the exposure of cells to MOX after verapamil treatment throughout the experiment. It is hypothesized that verapamil interfered with MOX as a substrate for Pgp during the initial incubation period. After this initial interaction, verapamil metabolites were able to interfere with Pgp. This experiment demonstrated the implication of Pgp in the transport of MOX and allowed prediction of the drug,drug interactions which might modify the bioavailability of endectocides. [source] Genotoxic potential of xenobiotic growth promoters and their metabolites,APMIS, Issue 2 2001Review article This paper reviews data reported in the literature as well as recent and unpublished studies from our laboratory on the metabolism and genotoxicity of the xenobiotic growth promoters 17,-trenbolone, melengestrol acetate and zeranol. In our metabolic study, the oxidative in vitro metabolites generated by hepatic microsomes from rats, bovine and humans were analyzed by HPLC and GC/MS. 17,-Trenbolone gave rise to at least 13 monohydroxylated products, whereas 12 mono- and dihydroxylated metabolites were obtained with melengestrol acetate and at least 5 with zeranol. The genotoxic potential of the parent compounds was studied using the following endpoints: induction of HPRT mutations in cultured V79 cells and of lacI mutations in E. coli; induction of micronuclei in V79 cells; and formation of DNA adducts in cultured primary rat hepatocytes. Negative results were obtained in most of these assay systems. Only the micronucleus induction was marginally positive with 17,-trenbolone and zeranol at near-cytotoxic concentrations. Commercial melengestrol acetate was found to contain an impurity causing apoptosis in V79 cells. The genotoxic potential of the numerous oxidative metabolites of the xenobiotic growth promoters remains to be studied. [source] Pharmacokinetics of omeprazole in rats with water deprivation for 72 hoursBIOPHARMACEUTICS AND DRUG DISPOSITION, Issue 8 2006Dae Y. Lee Abstract Dehydration can occur by excessive sweating, polyuria, severe diarrhea and hyperthermia. Previous studies reported that the expressions of CYP1A1/2 and 3A1(23)/2 were not changed in male Sprague,Dawley rats with 72 h water deprivation (dehydrated rats), and that the metabolism of omeprazole is mainly catalysed via CYP1A1/2, 2D1 and 3A23/2 in rats. Hence, it could be expected that the hepatic metabolism of omeprazole would not be changed considerably in dehydrated rats, if the contribution of CYP2D1 to the metabolism of omeprazole in dehydrated rats is not considerable. Therefore, the pharmacokinetics of omeprazole were compared after intravenous (20 mg/kg) and oral (40 mg/kg) administration in control rats and in dehydrated rats. After intravenous administration, the time-averaged nonrenal clearance (Clnr) values of omeprazole were comparable between the two groups of rats. This could be supported by comparable in vitro intrinsic clearance (Clint) values for the disappearance of omeprazole in rat hepatic microsomes and the comparable free (unbound to plasma proteins) fractions of omeprazole in plasma in the two groups of rats. After oral administration, the AUC values of omeprazole were also comparable in the two groups of rats. The above data suggest that the dehydration state did not affect considerably the pharmacokinetics of omeprazole in rats. Copyright © 2006 John Wiley & Sons, Ltd. [source] CYP3A inductive potential of the rifamycins, rifabutin and rifampin, in the rabbitBIOPHARMACEUTICS AND DRUG DISPOSITION, Issue 4 2001Allan Weber Abstract Rifabutin is effective in the treatment and prevention of Mycobacterium avium infection in people with HIV infection. Rifabutin is structurally related to another rifamycin, rifampin, a well-known inducer of the human P-450 isoform 3A. The rabbit isoform CYP3A6 and the human isoform CYP3A4 have similar P-450 predominance and substrate specificity and are both induced by rifampin. Our goal was to predict the CYP3A induction capacity of rifabutin and to determine if ex vivo CYP3A induction potential of rifamycins is predictive of that obtained in vivo. We determined the in vivo and ex vivo CYP3A6 induction by 4 days of treatment with rifabutin (100 mg/kg), rifampin (100 mg/kg), or vehicle (DMSO) in the rabbit. The ex vivo measures were CYP3A6 activity (N-demethylation of erythromycin and hydroxylation of triazolam) and CYP3A content in rabbit hepatic microsomes preparations. The in vivo measures were oral clearance of triazolam and its formation clearance to its hydroxylated metabolites, , -hydroxytriazolam and 4-hydroxytriazolam. Rifampin increased CYP3A6 activity by 2- to 3-fold in hepatic microsomes compared to vehicle. Rifabutin increased CYP3A content 1.7-fold, but did not significantly increase microsomal CYP3A6 activity. Oral triazolam clearance and formation clearances to the two hydroxylated metabolites were 2- to 3-fold greater in rabbits treated with rifampin. These clearances were unaffected by rifabutin administration. Ex vivo enzyme activities correlated with in vivo changes in clearance of triazolam and the formation clearance to its hydroxylated metabolites. Rifabutin is a weaker inducer of CYP3A6 than rifampin. These data suggest that ex vivo enzyme activity is a viable approach to predict in vivo inductive potential of CYP3A inducers. Copyright © 2001 John Wiley & Sons, Ltd. [source] Arachidonic acid-mediated cooxidation of all- trans -retinoic acid in microsomal fractions from human liverBRITISH JOURNAL OF PHARMACOLOGY, Issue 4 2000Louise Nadin The quantitative importance of prostaglandin H synthase (PGHS)-mediated cooxidation of all- trans -retinoic acid (ATRA) was evaluated in human liver microsomes (n=17) in relation to CYP-dependent ATRA 4-hydroxylation. Observed rates of ATRA cooxidation (4.6,20 pmol mg protein,1 min,1) and 4-hydroxylation (8.7,45 pmol mg protein,1 min,1) were quantitatively similar and exhibited similar individual variation (4 and 5 fold, respectively). From kinetic studies cooxidation was an efficient process in human hepatic microsomes (VmaxKm,1=0.25) compared with NADPH- and NADH-mediated 4-hydroxylation by CYP (VmaxKm,1=0.14 and 0.02, respectively). The capacity of lipid hydroperoxide metabolites of arachidonic acid to mediate ATRA oxidation was established directly, but downstream products (D, E, F and I-series prostaglandins) were inactive. cDNA-expressed CYPs supported ATRA oxidation by lipid hydroperoxides. Whereas CYPs 2C8, 2C9 and 3A4, but not CYPs 1A2 or 2E1, were effective catalysts of the NADPH-mediated reaction, cooxidation supported by 15(S)-hydroperoxyeicosatetraenoic acid was mediated by all five CYPs. The cooxidation reaction in human hepatic microsomes was inhibited by the CYP inhibitor miconazole. These findings indicate that ATRA oxidation is quantitatively significant in human liver. Lipid hydroperoxides generated by intracellular enzymes such as prostaglandin synthase and lipoxygenases are sources of activated oxygen for CYP-mediated deactivation of ATRA to polar products. British Journal of Pharmacology (2000) 131, 851,857; doi:10.1038/sj.bjp.0703579 [source] | ||||||||||