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PubMedDate not supplied

Metabolism of the antimammary cancer antiestrogenic agent tamoxifen. I. Cytochrome P-450-catalyzed N-demethylation and 4-hydroxylation.

Previous studies suggested that the therapeutic effect of the antimammary cancer agent tamoxifen might be related to its metabolism. This study examined the cytochrome P-450 enzymes in rat and human liver catalyzing the metabolism of tamoxifen. Incubations of tamoxifen with rat liver microsomes yielded three major polar metabolites identified as the N-oxide, N-desmethyl, and 4-hydroxy derivatives. N-Oxide formation was catalyzed by the flavin-containing monooxygenase (see part II). Carbon monoxide, SKF-525A, metyrapone, and benzylimidazole strongly inhibited N-demethylation and 4-hydroxylation, indicating the participation of P-450 monooxygenase in these reactions. Antibodies to NADPH-P450 reductase inhibited N-demethylation and 4-hydroxylation. Comparison of the metabolism of tamoxifen in untreated male and female rats demonstrated some sexual dimorphism. N-Demethylation was higher in the male rat and 4-hydroxylation was higher in the female. Treatment of rats with phenobarbital (PB), pregnenolone-16 alpha-carbonitrile (PCN), and methylcholanthrene (MC) enhanced N-demethylation, demonstrating the potential participation of multiple P-450s in N-demethylation. Evidence strongly indicates that CYP3A enzyme(s) catalyzes N-demethylation in liver microsomes of PB- and PCN-treated rats (PB and PCN microsomes, respectively): i) N-demethylation was inhibited by cortisol and erythromycin (alternate substrates) and a time-dependent inhibition was observed with troleandomycin (TAO) in vitro; ii) treatment of female rats with TAO, followed by dissociation of the microsomal TAO-P-450 complex, elevated N-demethylation; iii) treatment of PCN-induced female rats with chloramphenicol inhibited N-demethylation; and iv) polyclonal antibodies (PAbs) to CYP3A1 inhibited N-demethylation in PCN- and PB-treated female rats. Although we were unable to reconstitute the N-demethylation activity with purified CYP3A1, which is difficult to reconstitute, collectively the evidence demonstrated that CYP3A enzymes catalyze N-demethylation in PB and PCN microsomes. By contrast, antibodies against CYP2B1/B2 did not inhibit N-demethylation and reconstituted 2B1 did not catalyze N-demethylation of tamoxifen, indicating that 2B1 was not involved. The increase in N-demethylation by MC treatment appears to be due to elevation of CYP1A1/1A2 (P-450c/d). Alternate substrates of CYP1A1/1A2 inhibited N-demethylation and reconstituted rat CYP 1A1-catalyzed N-demethylation. Surprisingly, monoclonal antibodies (MAbs) against CYP1A1/1A2 only partially inhibited, and PAbs against CYP1A1 did not inhibit N-demethylation in MC microsomes, indicating that in MC microsomes, 1A1 does not contribute significantly to that reaction. Mab anti-CYP2C11/2C6 (P-450h/k) inhibited N-demethylation in PB, PCN, and control male rat liver microsomes, suggesting that CYP2C11 and/or CYP2C6 catalyze this reaction to some extent.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1997

Stereoselective N-demethylation of chlorpheniramine by rat-liver microsomes and the involvement of cytochrome P450 isozymes.

Previous studies have suggested that degradation of the two stereoisomers of chlorpheniramine in the liver might be catalysed by different types of cytochrome P450. Stereoselective N-demethylation of chlorpheniramine and the involvement of cytochrome P450 (CYP) isozymes have, therefore, been investigated in the liver microsomes of eight-week-old male rats. Incubation of racemic chlorpheniramine with liver microsomes from the male rat resulted in the formation of both enantiomers of monodesmethylchlorpheniramine (DMChp). Further metabolism of DMChp to didesmethylchlorpheniramine (DDMChp) did not, however, occur. The S/R enantiomeric ratio for intrinsic clearance (Vmax/Km) was approximately 2.0, suggesting that the N-demethylation was stereoselective for S-(+)-chlorpheniramine. On the other hand, although the Vmax/Km value for the formation of S-(+)- and R-(-)-DMChp increased with phenobarbitone-inducible rat-liver microsomes, there was no difference between the rates of N-demethylation of the enantiomers. In contrast, 3-methylcholanthrene reduced the intrinsic clearance of S-(+)-chlorpheniramine by N-demethylation and increased its value for R-(-)-chlorpheniramine, showing no stereoselectivity for the N-demethylation of chlorpheniramine. The difference between the intrinsic clearance of the two enantiomers by N-demethylation was because of differences in affinity for the catalysing enzyme. This is indicative of stereoselective involvement of the main enzyme concerned in the N-demethylation of the enantiomers, considered to be CYP 2C11. Anti-CYP 2C11 also partially inhibited the N-demethylation of racemic chlorpheniramine in rat-liver microsomes exposed to phenobarbitone and 3-methylcholanthrene. That CYP 2B1 was involved in the N-demethylation of both enantiomers was also supported by results from an experiment using phenobarbitone-inducible rat-liver microsomes. CYP1A1 did not, however, catalyse the N-demethylation of either enantiomer. These results indicate that N-demethylation of the S-(+)-enantiomer of chlorpheniramine occurs preferentially in the microsomes, demonstrating the stereoselective contribution of CYP2C11. Immunoinhibition studies suggest, moreover, that the N-demethylation of both chlorpheniramine enantiomers is catalysed by CYP2B1, but not by CYP1A1.

Animals↗
PubMed1999

Evidence that protein binding specifies sites of DNA demethylation.

It has been hypothesized that protein factors may protect CpG islands from methyltransferase during development and that demethylation may involve protein-DNA interactions at demethylated sites. However, direct evidence has been lacking. In this study, demethylation at the EBNA-1 binding sites of the Epstein-Barr virus latent replication origin, oriP, was investigated by using human cells. Several novel findings are discussed. First, there are specific preferential demethylation sites within the oriP region. Second, the DNA sequence of oriP alone is not the target of an active demethylation process. Third, EBNA-1 binding is required for the site-specific demethylation in oriP. Interestingly, CpG sites adjacent to and between the EBNA-1 sites do not become demethylated. Fourth, demethylation of the first DNA strand in oriP at the EBNA-1 binding sites involves a passive (replication-dependent) mechanism. The second-strand demethylation appears to occur through an active mechanism. That is, EBNA-1 protein binding prevents the EBNA-1 binding sites from being remethylated after one round of DNA replication, and it appears that an active demethylase then demethylates these hemimethylated sites. This study provides clear evidence that protein binding specifies sites of DNA demethylation and provides insights into the sequence of steps and the mechanism of demethylation.

Base Sequence↗
PubMed1997

Different effects of inhibitors on the O- and N-demethylation of codeine in human liver microsomes.

OBJECTIVE: The O- and N-demethylation of codeine is catalysed by CYP2D6 and CYP3A4 respectively. The formation rates of morphine by O-demethylation and norcodeine by N-demethylation were studied in two sets of human liver microsomes. RESULTS: Relatively high K(m) values were found for both O- and N-demethylations, suggesting a low affinity to the corresponding enzymes. The inhibitory effects of various drugs were found to be different for O- and N-demethylations. The substrates of CYP2D6 such as thioridazine, amitriptyline and metoprolol inhibited the O-demethylation of codeine preferentially, while the substrates of CYP3A4 such as cyclosporine A, midazolam and erythromycin were all strong inhibitors of the N-demethylation of codeine. Quinidine and lignocaine, although they are substrates of CYP3A, showed preferential inhibition over the O-demethylation of codeine, suggesting a low affinity to the CYP3A. Methadone and dextropropoxyphene showed a preferential inhibition of CYP2D6 over CYP3A, while theophylline did not inhibit the O- or N-demethylation to a greater extent. CONCLUSION: It seems that there was a good correspondence between the capacity of drugs to inhibit the O- and N-demethylation of codeine in human liver microsomes and their apparent metabolism by CYP2D6 or CYP3A4, respectively in vivo in man, suggesting that this in vitro inhibition test may be a useful screen for drugs which interact with these two important drug-metabolising enzymes.

Codeine↗
PubMed2001

Non-growth-associated demethylation of dimethylsulfoniopropionate by (homo)acetogenic bacteria.

The demethylation of the algal osmolyte dimethylsulfoniopropionate (DMSP) to methylthiopropionate (MTPA) by (homo)acetogenic bacteria was studied. Five Eubacterium limosum strains (including the type strain), Sporomusa ovata DSM 2662(T), Sporomusa sphaeroides DSM 2875(T), and Acetobacterium woodii DSM 1030(T) were shown to demethylate DMSP stoichiometrically to MTPA. The (homo)acetogenic fermentation based on this demethylation did not result in any significant increase in biomass. The analogous demethylation of glycine betaine to dimethylglycine does support growth of acetogens. In batch cultures of E. limosum PM31 DMSP and glycine betaine were demethylated simultaneously. In mixed substrates experiments with fructose-DMSP or methanol-DMSP, DMSP was used rapidly but only after exhaustion of the fructose or the methanol. In steady-state fructose-limited chemostat cultures (at a dilution rate of 0.03 h(-1)) with DMSP as a second reservoir substrate, DMSP was biotransformed to MTPA but this did not result in higher biomass values than in cultures without DMSP; cells from such cultures demethylated DMSP at rates of approximately 50 nmol min(-1) mg of protein(-1), both after growth in the presence of DMSP and after growth in its absence. In cell extracts of glycine betaine-grown strain PM31, DMSP demethylation activities of 21 to 24 nmol min(-1) mg of protein(-1) were detected with tetrahydrofolate as a methyl acceptor; the activities seen with glycine betaine were approximately 10-fold lower. A speculative explanation for the demethylation of DMSP without an obvious benefit for the organism is that the DMSP-demethylating activity is catalyzed by the glycine betaine-demethylating enzyme and that a transport-related factor, in particular a higher energy demand for DMSP transport across the cytoplasmic membrane than for glycine betaine transport, may reduce the overall ATP yield of the fermentation to virtually zero.

Acetyl Coenzyme A↗
PubMed2000

Modulation of DNA binding protein affinity directly affects target site demethylation.

It has recently been shown that in Xenopus, DNA demethylation at promoter regions may involve protein-DNA interactions, based on the specificity of the demethylated sites. Utilizing a stable episomal system in human cells, we recently mapped the sites and dissected the steps of demethylation at oriP sites bound by EBNA1 protein. Although it is clear that protein binding is required for demethylation of the oriP sites, it is uncertain whether this is a unique feature of the replication origin or whether it is a general phenomenon for all DNA sequences to which sequence-specific proteins are bound. In the present study, we utilize the well-defined Escherichia coli lac repressor/operator system in human cells to determine whether protein binding to methylated DNA, in a region that is neither a replication origin nor a promoter, can also lead to demethylation of the binding sites. We found that demethylation specified by protein binding is not unique to the replication origin or to the promoter. We also found that transcriptional activity does not influence demethylation of the lac operator. Isopropyl-beta-D-thiogalactopyranoside (IPTG), an inhibitor of the lac repressor, can prevent demethylation of the lac operator DNA sites and can modulate demethylation of the lac operator by affecting the binding affinity of the lac repressor. Using this system, a titration of protein binding can be done. This titration permits one to infer that protein binding site occupancy is the determinant of demethylation at DNA sites and permits a determination of how this process progresses over time.

Bacterial Proteins↗
PubMed1996

Cytochromes P450, 1A2, and 2C9 are responsible for the human hepatic O-demethylation of R- and S-naproxen.

A preliminary report implicated cytochrome P450 (CYP) 2C9 in the human liver microsomal O-demethylation of S-naproxen, suggesting that this pathway may be suitable for investigation of human hepatic CYP2C9 in vitro. Kinetic and inhibitor studies with human liver microsomes and confirmatory investigations with cDNA-expressed enzymes were undertaken here to define the role of CYP2C9 and other isoforms in the O-demethylation of R- and S-naproxen. All studies utilised a newly developed sensitive and specific HPLC assay that measured the respective O-desmethyl metabolites of R- and S-naproxen in incubations of human liver microsomes and in COS cell lysates. Microsomal R- and S-naproxen O-demethylation kinetics followed Michaelis-Menten kinetics, with respective mean apparent Km values of 123 microM and 143 microM. Sulfaphenazole, a specific inhibitor of CYP2C9, reduced the microsomal O-demethylation of R- and S-naproxen by 43% and 47%, respectively, and the CYP1A2 inhibitor furafylline decreased R- and S-naproxen O-demethylation by 38% and 28%, respectively. R,S-Mephenytoin was a weak inhibitor of R- and S-naproxen O-demethylation, but other CYP isoform specific inhibitors (e.g., coumarin, diethyldithiocarbamate, quinidine, troleandomycin) had little or no effect on these reactions. cDNA-expressed CYP2C9 and CYP1A2 were both shown to O-demethylate R- and S-naproxen. Apparent Km values (92-156 microM) for the reactions catalysed by the recombinant enzymes were similar to those observed for human liver microsomal R- and S-naproxen O-demethylation. The data demonstrate that CYP2C9 and CYP1A2 together account for the majority of human liver R- and S-naproxen O-demethylation, precluding the use of either R- or S-naproxen as a CYP isoform-specific substrate in vitro and in vivo.

Aryl Hydrocarbon Hydroxylases↗
PubMed1994

Caffeine-derived N-nitroso compounds. IV: Kinetics of mononitrosocaffeidine demethylation by rat liver microsomes.

The study describes the kinetics of demethylation of mononitrosocaffeidine (MNC), a new asymmetric N-nitrosamine derived from caffeine. The demethylation of its precursor compound caffeidine was also studied. The results presented here suggest (a) that liver microsomes from fasted rats preferentially demethylate the N-methylnitrosamine group in MNC indicating the demethylation by cytochrome P450IIE1, (b) demethylation of MNC shows two apparent Km values, one of 117-166 microM responsible for the demethylation at the N-methylnitrosamino group of MNC, and the other Km of 1.84-2.26 mM for the remaining N-demethylations, (c) in contrast, caffeidine is a low affinity substrate for microsomal demethylation as indicated by a high Km of 14.3-16.3 mM, and (d) the demethylation at amino-N amino-N, and N-1 in both these compounds are mainly catalysed by P450 enzymes induced by Aroclor 1245 in rats.

Animals↗
PubMed1983

Characterization of the N-demethylation reactions catalyzed by horseradish peroxidase.

The hydroperoxide-supported N-demethylation reactions catalyzed by horseradish peroxidase have been characterized in detail. The ethyl hydroperoxide-supported N-demethylation of N,N-dimethylaniline by horseradish peroxidase resulted in the formation of equimolar amounts of N-methylaniline and formaldehyde with no other products detectable by high performance liquid chromatography analysis of the reaction mixture. One molecule of ethyl hydroperoxide was consumed for each molecule of formaldehyde formed in the reaction. Similar results were obtained for the hydrogen peroxide-supported N-demethylation of N,N-dimethylaniline. The horseradish peroxidase-catalyzed N-demethylation reaction could be supported by a variety of hydroperoxides, peroxides, and peracids. The turnover number for the hydrogen peroxide-supported demethylation reaction (7061) was larger than that for the ethyl hydroperoxide-supported reaction (5382) or for chloroperoxidase- or cytochrome P-450-catalyzed dealkylations. The demethylation reaction exhibited normal Michaelis-Menten saturation kinetics with respect to N,N-dimethylaniline (Km = 0.34 mM) and ethyl hydroperoxide (Km = 0.020 mM), as well as hydrogen peroxide (Km = 0.016 mM). The horseradish peroxidase-catalyzed N-demethylation reaction was not significantly inhibited by reagents which react with the superoxide anion, the hydroxyl radical, or singlet oxygen, suggesting that these activated oxygen species are not free intermediates in the reaction. There was no significant inhibition of the reaction by alpha-phenyl-t-butylnitrone, 5,5-dimethylpyrroline-N-oxide, or other free radical trapping agents. Substitution of D2O for H2O resulted in an inhibition of the reaction with a solvent isotope effect (VH2O/VD2O) of 1.6. Horseradish peroxidase did not catalyze the demethylation of N,N-dimethylaniline-N-oxide, indicating that the reaction does not proceed via N-oxidation of the amine. When the concentrations of both N,N-dimethylaniline and ethyl hydroperoxide were varied in a constant ratio a linear double reciprocal plot was obtained, which is consistent with a ping-pong kinetic mechanism for the horseradish peroxidase-catalyzed demethylation reaction.

Horseradish Peroxidase↗
PubMed1995

Promoter demethylation in MMTV/N-rasN transgenic mice required for transgene expression and tumorigenesis.

We studied demethylation within the transgene promoter in transgenic mice carrying the N-ras proto-oncogene driven by the mouse mammary tumor long terminal repeat (MMTV/N-rasN) and the relationship of demethylation to transgene overexpression and tumorigenesis. Demethylation at Fspl or Clal sites correlated with age of the animal and transgene expression in nontumorous mammary gland. Demethylation preceded expression in this tissue. In lymphomas and mammary tumors, the promoter Fspl and Clal sites were significantly more demethylated than in nontumorous control tissues. The Aval, Cfol, and Hpall sites were also found to be undermethylated in older animals and showed differences between tumor and control tissues. Two additional sites (Eagl and Narl) remained fully methylated in all tissues. In contrast with normal tissue, demethylation at the Fspl and Clal sites and expression were not correlated in tumor tissue. An increase in expression in normal tissue initially occurred and was correlated with the level of promoter demethylation; this increase was followed by a further increment in transgene expression when tumors developed. Thus, promoter demethylation leading to transgene overexpression was associated with long-latency tumorigenesis in MMTV/N-rasN transgenic mice. Demethylation of proto-oncogene promoters may therefore be a mechanism of carcinogenesis that requires further investigation in human tumors.

5-Methylcytosine↗
PubMed1995

Cytochromes of the P450 2C subfamily are the major enzymes involved in the O-demethylation of verapamil in humans.

The calcium channel blocker verapamil [2,8-bis-(3,4-dimethoxyphenyl)-6-methyl-2-isopropyl-6-azaoctanitrile+ ++] undergoes extensive biotransformation in man. We have previously demonstrated cytochrome P450 (CYP) 3A4 and 1A2 to be the enzymes responsible for verapamil N-dealkylation (formation of D-617 [2-(3,4-dimethoxyphenyl)-5-methylamino-2-isopropylvaleronitrile], and verapamil N-demethylation (formation of norverapamil [2,8-bis-(3,4-dimethoxyphenyl)-2-isopropyl-6-azaoctanitrile]), while there was no involvement of CYP3A4 and CYP1A2 in the third initial metabolic step of verapamil, which is verapamil O-demethylation. This pathway yields formation of D-703 [2-(4-hydroxy-3-methoxyphenyl)-8-(3,4-dimethoxyphenyl)-6-methyl-2-isopro pyl-6-azaoctanitrile] and D-702 [2-(3,4-dimethoxyphenyl)-8-(4-hydroxy-3-methoxyphenyl)-6-methyl-2-isopro pyl-6-azaoctanitrile]. The enzymes catalyzing verapamil O-demethylation have not been characterized so far. We have therefore identified and characterized the enzymes involved in verapamil O-demethylation in humans by using the following in vitro approaches: (I) characterization of O-demethylation kinetics in the presence of the microsomal fraction of human liver, (II) inhibition of verapamil O-demethylation by specific antibodies and selective inhibitors and (III) investigation of metabolite formation in microsomes obtained from yeast strain Saccharomyces cerevisiae W(R), that was genetically engineered for stable expression of human CYP2C8, 2C9 and 2C18. In human liver microsomes (n=4), the intrinsic clearance (CLint), as derived from the ratio of Vmax/Km, was significantly higher for O-demethylation to D-703 compared to formation of D-702 following incubation with racemic verapamil (13.9 +/- 1.0 vs 2.4 +/- 0.6 ml*min-1*g-1, mean+/-SD; p<0.05), S-verapamil (16.8 +/- 3.3 vs 2.2 +/- 1.2 ml* min-1*g-1, p<0.05) and R-verapamil (12.1 +/- 2.9 vs 3.6 +/- 1.3 ml*min-1*g-1; p<0.05), thus indicating regioselectivity of verapamil O-demethylation process. The CLint of D-703 formation in human liver microsomes showed a modest but significant degree of stereoselectivity (p<0.05) with a S/R-ratio of 1.41 +/- 0.17. Anti-LKM2 (anti-liver/kidney microsome) autoantibodies (which inhibit CYP2C9 and 2C19) and sulfaphenazole (a specific CYP2C9 inhibitor) reduced the maximum rate of formation of D-703 by 81.5 +/- 4.5% and 45%, that of D-702 by 52.7 +/- 7.5% and 72.5%, respectively. Both D-703 and D-702 were formed by stably expressed CYP2C9 and CYP2C18, whereas incubation with CYP2C8 selectively yielded D-703. In conclusion, our results show that enzymes of the CYP2C subfamily are mainly involved in verapamil O-demethylation. Verapamil therefore has the potential to interact with other drugs which inhibit or induce these enzymes.

Calcium Channel Blockers↗
PubMed1998

An embryonic demethylation mechanism involving binding of transcription factors to replicating DNA.

In vertebrates, transcriptionally active promoters are undermethylated. Since the transcription factor Sp1, and more recently NF-kappaB, have been implicated in the demethylation process, we examined the effect of transcription factors on demethylation by injecting in vitro methylated plasmid DNA into Xenopus fertilized eggs. We found that various transactivation domains, including a strong acidic activation domain from the viral protein VP16, can enhance demethylation of a promoter region when fused to a DNA binding domain which recognizes the promoter. Furthermore, demethylation occurs only after the midblastula transition, when the general transcription machinery of the host embryo becomes available. Nevertheless, transcription factor binding need not be followed by actual transcription, since demethylation is not blocked by alpha-amanitin treatment. Finally, replication of the target DNA is a prerequisite for efficient demethylation since only plasmids that carry the bovine papilloma virus sequences which support plasmid replication after the midblastula transition are demethylated. No demethylation is detectable in the oocyte system where DNA is not replicated. These results suggest that, in the Xenopus embryo, promoters for which transcription factors are available are demethylated by a replication-dependent, possibly passive mechanism.

Amanitins↗
PubMed1991

Methylmercury decomposition in sediments and bacterial cultures: involvement of methanogens and sulfate reducers in oxidative demethylation.

Demethylation of monomethylmercury in freshwater and estuarine sediments and in bacterial cultures was investigated with CH(3)HgI. Under anaerobiosis, results with inhibitors indicated partial involvement of both sulfate reducers and methanogens, the former dominating estuarine sediments, while both were active in freshwaters. Aerobes were the most significant demethylators in estuarine sediments, but were unimportant in freshwater sediments. Products of anaerobic demethylation were mainly CO(2) as well as lesser amounts of CH(4). Acetogenic activity resulted in fixation of some CO(2) produced from CH(3)HgI into acetate. Aerobic demethylation in estuarine sediments produced only CH(4), while aerobic demethylation in freshwater sediments produced small amounts of both CH(4) and CO(2). Two species of Desulfovibrio produced only traces of CH(4) from CH(3)HgI, while a culture of a methylotrophic methanogen formed traces of CO(2) and CH(4) when grown on trimethylamine in the presence of the CH(3)HgI. These results indicate that both aerobes and anaerobes demethylate mercury in sediments, but that either group may dominate in a particular sediment type. Aerobic demethylation in the estuarine sediments appeared to proceed by the previously characterized organomercurial-lyase pathway, because methane was the sole product. However, aerobic demethylation in freshwater sediments as well as anaerobic demethylation in all sediments studied produced primarily carbon dioxide. This indicates the presence of an oxidative pathway, possibly one in which methylmercury serves as an analog of one-carbon substrates.

Journal Article↗