Phospholipid requirement for dimethylnitrosamine demethylation by hamster hepatic microsomal cytochrome P-450 enzyme system.
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Extraction with butan-1-ol of freeze-dried microsomal fractions from livers of 3-methyl-cholarthrene-pre-treated hamsters removed about 90% of the total lipid content, but the lipid remaining proved sufficient for the cytochrome P-450 enzyme system to retain about 15-40% of its original catalytic activity for dimethylnitrosamine demethylation. Addition of butan-1-ol-extracted total phospholipid or phosphatidylcholine could not restore any activity, whereas the addition of the synthetic phospholipid dilauroyl phosphatidylcholine was able to restore almost complete activity. Synthetic dipalmitoyl or distearoyl phosphatidylcholine was ineffective in restoring the activity in this reconstituted system. (+info)
Distribution of microsomal glutathione transferase 1 in mammalian tissues. A predominant alternate first exon in human tissues.
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An extensive Northern blot analysis of microsomal glutathione transferase 1 in human and rat tissues was performed. When normalized against the glyceraldehyde-3-phosphate dehydrogenase or actin expression it was evident that the predominant expression occurs in liver and pancreas. An ontogenetic, as well as a functional, basis for the high levels in these two organs is possible. The relative expression levels in man ranged from: liver and pancreas (100%), to kidney, prostate, colon (30-40%), heart, brain, lung, testis, ovary, small intestine (10-20%), placenta, skeletal muscle, spleen, thymus and peripheral blood leucocytes (1-10%). Liver-enriched expression was detected in human fetal tissues with lung and kidney displaying lower levels (10-20%). No transcripts could be detected in fetal brain or heart. When comparing the expression levels between rat and man it is apparent that human extrahepatic mRNA levels are much higher relative to liver. Rat microsomal glutathione transferase mRNA expression ranges from 0.2 to 10% that of liver, with adrenal, uterus, ovary and stomach displaying the highest levels of the organs tested. Based on these observations, and the fact that the enzyme is encoded by a highly conserved single-copy gene, it is suggested that microsomal glutathione transferase 1 performs essential functions vital to most mammalian cell types. We suggest that protection against oxidative stress constitutes one such function. Human expressed sequence tag (EST) characterization yielded four alternate mRNA transcripts with different 5'-ends (four alternate noncoding exons 1). The predominant exon (based on the observed EST frequency) revealed a tissue distribution similar to that obtained using the reading frame as probe. Thus, it appears that one exon preferentially gives rise to mature mRNA in the human tissues examined. This exon is different from the one reported in the original cDNA characterized. (+info)
Evidence that carnitine palmitoyltransferase I (CPT I) is expressed in microsomes and peroxisomes of rat liver. Distinct immunoreactivity of the N-terminal domain of the microsomal protein.
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Mitochondria, microsomes and peroxisomes all express overt (cytosol-facing) carnitine palmitoyltransferase activity that is inhibitable by malonyl-CoA. The overt carnitine palmitoyltransferase activity (CPTo) associated with the different fractions was measured. Mitochondria accounted for 65% of total cellular CPTo activity, with the microsomal and peroxisomal contributions accounting for the remaining 25% and 10%, respectively. In parallel experiments, rat livers were perfused in situ with medium containing dinitrophenyl (DNP)-etomoxir in order to inhibit quantitatively and label covalently (with DNP-etomoxiryl-CoA) the molecular species responsible for CPTo activity in each of the membrane systems under near-physiological conditions. In all three membrane fractions, a single protein with an identical molecular mass of approximately 88,000 kDa (p88) was labelled after DNP-etomoxir perfusion of the liver. The abundance of labelled p88 was quantitatively related to the respective specific activities of CPTo in each fraction. On Western blots the same protein was immunoreactive with three anti-peptide antibodies raised against linear epitopes of the cytosolic N- and C-domains and of the inter-membrane space loop (L) domain of the mitochondrial enzyme (L-CPT I). However, the reaction of the microsomal protein with the anti-N peptide antibody (raised against epitope Val-14-Lys-29 of CPT I) was an order of magnitude stronger than expected from either microsomal CPTo activity or its DNP-etomoxiryl-CoA labelling. This suggests that the N-terminal domain of the microsomal protein differs from that in the mitochondrial or peroxisomal protein. This conclusion was confirmed using antibody back-titration experiments, in which the binding of anti-N and anti-C antibodies by mitochondria and microsomes was quantified. (+info)
Improved reliability of the rapid microtiter plate assay using recombinant enzyme in predicting CYP2D6 inhibition in human liver microsomes.
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A higher throughput method of screening for the inhibition of recombinant CYP2D6 using a microtiter plate (MTP) assay was evaluated using 62 new chemical entities and compared to data from the dextromethorphan O-demethylase assay in human liver microsomes (HLM). The IC50 values for the two assays closely matched for 53 compounds (85%). Six of the variant nine compounds had higher IC50 values with the recombinant enzyme, whereas three had lower IC50 values with the recombinant enzyme. When the inhibition with the recombinant enzyme was determined at various time points, the IC50 values increased as the duration of the incubation increased for the six compounds with higher IC50 values in the MTP assay. The IC50 values at 10 min matched more closely the IC50 values in HLM (95% compared with 85%). For three compounds that showed comparable IC50 values in the two assays, and the three compounds with lower IC50 values in the MTP assay, the IC50 values did not change over time. These results suggest that the six compounds that showed higher IC50 values in the MTP assay at 45 min are substrates for CYP2D6. Using known CYP2D6 substrates, a similar phenomenon was observed, i.e., inhibition curves shifted to higher IC50 values as incubation time increased. These results indicate that the higher throughput MTP assay is more comparable to HLM if the IC50 values are determined at 10 min rather than the recommended 45 min. Furthermore, data acquisition at multiple time points may indicate if a compound is a potential substrate or metabolism/mechanism-based inhibitor for the enzyme. (+info)
Characterization of metabolites of astaxanthin in primary cultures of rat hepatocytes.
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The metabolism of the nonprovitamin A carotenoid astaxanthin was investigated in primary cultures of rat hepatocytes. In a time course study based on HPLC and gas chromatography-mass spectrometry analyses, one main metabolite, (rac)-3-hydroxy-4-oxo-beta-ionone, was found. This metabolite was conjugated mainly into glucuronides, as demonstrated by glusulase treatment of the conjugates under sulfatase-inhibiting conditions. Within 24 h more than 50% astaxanthin was metabolized and conjugated. Deconjugation of the polar conjugates with glusulase and analyses with HPLC and gas chromatography-mass spectrometry identified two metabolites, (rac)-3-hydroxy-4-oxo-beta-ionone and its reduced form (rac)-3-hydroxy-4-oxo-7,8-dihydro-beta-ionone, indicating that the former was reduced in the conjugated form. We confirmed that the ketocarotenoid astaxanthin induces xenobiotic-metabolizing enzymes in rat liver in vivo. However, there were no differences in the metabolism of astaxanthin in cultured hepatocytes from rats that were pretreated with astaxanthin and, thus, with induced cytochrome P-450 systems compared with control hepatocytes. Neither liver microsomes from astaxanthin-pretreated nor control rats metabolized astaxanthin. These results indicated that the cytochrome P-450 enzymes were not involved in the metabolism of astaxanthin in rat hepatocytes. We conclude that astaxanthin was metabolized in primary cultures of rat hepatocytes into (rac)-3-hydroxy-4-oxo-beta-ionone and its reduced form (rac)-3-hydroxy-4-oxo-7,8-dihydro-beta-ionone independent of the xenobiotic-metabolizing enzymes induced by astaxanthin. (+info)
Monospecific antipeptide antibody to cytochrome P-450 2B6.
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To study cytochrome P-450 (CYP) 2B6 contribution to methoxychlor metabolism within human liver microsomes and to initiate an investigation of CYP2B6 protein expression, we developed a polyclonal antibody targeted to a 20-residue peptide within that protein. The antibody was found to be highly sensitive and monospecific for CYP2B6 on immunoblots. Although many immunological studies have described the absence or low expression of CYP2B6 in human livers, in the present investigation, we have found this not to be the case. We immunoquantified CYP2B6 apoprotein expression in a panel of 28 livers and found concentrations ranging from 2 to 82 pmol/mg protein, with a mean value of 25 pmol/mg protein. Five livers ( approximately 18%) displayed relatively high levels of CYP2B6 (>40 pmol/mg protein). There were no sex-related differences, although the highest level was observed in a 1-week postpartum donor given several medications. A marked diminution in variability was found in individuals aged 56 or older (n = 12), but there were no age-related trends in mean CYP2B6 content. We suggest that CYP2B6 represents a significant portion of total CYP in human liver. The exquisite sensitivity of this antibody (fmol quantities are detected easily on immunoblots) may explain our detection of CYP2B6 in 100% of livers versus its detection in a limited number of livers by certain other investigators. The antibody also was found to immunoinhibit CYP2B6-catalyzed N-demethylation of (S)-mephenytoin in human liver microsomes by 68 to 79%. The utility of this antibody for determining human liver microsomal CYP2B6 contribution to the ortho-hydroxylation of methoxychlor was demonstrated. (+info)
Kinetics of drug metabolism in rat liver slices: IV. Comparison of ethoxycoumarin clearance by liver slices, isolated hepatocytes, and hepatic microsomes from rats pretreated with known modifiers of cytochrome P-450 activity.
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To evaluate the theory that within precision-cut liver slices intercellular transport occurs in parallel with cellular metabolism and to illustrate the constraints this places on clearance predictions, the kinetics of ethoxycoumarin O-deethylation have been determined under varying conditions of hepatic cytochrome P-450 activity. Liver slices, isolated hepatocytes, and microsomes were obtained from rats treated with the inducers phenobarbital (PB) and beta-naphthoflavone (betaNF) and the inhibitor aminobenzotriazole (ABT). In hepatocytes and microsomes, a two-site kinetic model with a high-affinity, low-capacity site and an unsaturated low-affinity, high-capacity site described the hydroxycoumarin formation data. There were marked increases in Vmax (2- to 5-fold and 50- to 70-fold for PB and betaNF, respectively) in both systems and in CLint (3- and 9-fold for PB and betaNF, respectively) in hepatocytes and substantial decreases in both parameters (3-8 and 12-23% of control, respectively) in ABT hepatocytes and microsomes. A qualitatively similar response was evident in slices obtained from livers of rats treated with phenobarbital and ABT, but although slices from betaNF livers produced high metabolic rates (comparable to slices obtained from livers of rats treated with phenobarbital), these showed a linear increase with substrate concentration without indication of a high-affinity site. The intrinsic clearance parameters were scaled to full liver capacity using hepatocellularities and microsomal recovery indices to allow direct comparison of these responses. The slice system consistently underestimated the effects of the modifiers. When compared with hepatocytes, estimates of 30, 15, and 1% for ABT, PB, and betaNF, respectively, were observed and the degree of underestimation was dependent on the magnitude of intrinsic clearance and was consistent with the above theory. (+info)
Role of CYP2B6 and CYP3A4 in the in vitro N-dechloroethylation of (R)- and (S)-ifosfamide in human liver microsomes.
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The central nervous system toxicity of ifosfamide (IFF), a chiral antineoplastic agent, is thought to be dependent on its N-dechloroethylation by hepatic cytochrome P-450 (CYP) enzymes. The purpose of this study was to identify the human CYPs responsible for IFF-N-dechloroethylation and their corresponding regio- and enantioselectivities. IFF exists in two enantiomeric forms, (R) - and (S)-IFF, which can be dechloroethylated at either the N2 or N3 positions, producing the corresponding (R,S)-2-dechloroethyl-IFF [(R, S)-2-DCE-IFF] and (R,S)-3-dechloroethyl-IFF [(R,S)-3-DCE-IFF]. The results of the present study suggest that the production of (R)-2-DCE-IFF and (S)-3-DCE-IFF from (R)-IFF is catalyzed by different CYPs as is the production of (S)-2-DCE-IFF and (R)-3-DCE-IFF from (S)-IFF. In vitro studies with a bank of human liver microsomes revealed that the sample-to-sample variation in the production of (S)-3-DCE-IFF from (R)-IFF and (S)-2-DCE-IFF from (S)-IFF was highly correlated with the levels of (S)-mephenytoin N-demethylation (CYP2B6), whereas (R)-2-DCE-IFF production from (R)-IFF and (R)-3-DCE-IFF production from (S)-IFF were both correlated with the activity of testosterone 6beta-hydroxylation (CYP3A4/5). Experiments with cDNA-expressed P-450 and antibody and chemical inhibition studies supported the conclusion that the formation of (S)-3-DCE-IFF and (S)-2-DCE-IFF is catalyzed primarily by CYP2B6, whereas (R)-2-DCE-IFF and (R)-3-DCE-IFF are primarily the result of CYP3A4/5 activity. (+info)