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PubMed1999

Demethylation and expression of methylated plasmid DNA stably transfected into HeLa cells.

In vitro methylation at CG dinucleotides (CpGs) in a transfecting plasmid usually greatly inhibits gene expression in mammalian cells. However, we found that in vitro methylation of all CpGs in episomal or non-episomal plasmids containing the SV40 early promoter/enhancer (SV40 Pr/E) driving expression of an antibiotic-resistance gene decreased the formation of antibiotic-resistant colonies by only approximately 30-45% upon stable transfection of HeLa cells. In contrast, when expression of the antibiotic-resistance gene was driven by the Rous sarcoma virus long terminal repeat or the herpes simplex virus thymidine kinase promoter, this methylation decreased the yield of antibiotic-resistant HeLa transfectant colonies approximately 100-fold. The low sensitivity of the SV40 Pr/E to silencing by in vitro methylation was probably due to demethylation upon stable transfection. This demethylation may be targeted to the promoter and extend into the gene. By genomic sequencing, we showed that four out of six of the transfected SV40 Pr/E's adjacent Sp1 sites were hotspots for demethylation in the HeLa transfectants. High frequency demethylation at Sp1 sites was unexpected for a non-embryonal cell line and suggests that DNA demethylation targeted to certain aberrantly methylated regions may function as a repair system for epigenetic mistakes.

DNA Methylation↗
PubMed1998

A gene-specific promoter in transgenic mice directs testis-specific demethylation prior to transcriptional activation In vivo.

Transcription of the autosomal phosphoglycerate kinase gene, Pgk-2, is initiated at the onset of meiosis during spermatogenesis in mammals. However, in the mouse, the 5' portion of the endogenous Pgk-2 coding sequence undergoes a specific demethylation event that precedes transcriptional activation by 10-12 days. Here we show that transgenes consisting of the Pgk-2 core promoter ligated to the CAT reporter gene undergo a similar tissue-, stage-, and cell type-specific demethylation in the 5' portion of the CAT coding sequence, whereas transgenes consisting of the CAT reporter sequence alone, or of the CAT sequence ligated to the CpG island-containing transferrin gene promoter, demonstrate different patterns of demethylation. These results indicate that specific promoter sequences can influence the pattern of tissue-specific demethylation within different genes and that a signal for spermatogenic cell-specific demethylation resides within the core promoter of the mammalian Pgk-2 gene.

Animals↗
PubMed1998

Relationship between in vivo FK506 clearance and in vitro 13-demethylation activity in living-related liver transplantation.

BACKGROUND: Although it is important to maintain an appropriate blood concentration of FK506 after liver transplantation, significant interindividual variability in the actual FK506 dosage has been observed, presumably due to the wide variability of cytochrome P450 3A4 activity in liver microsomes. METHODS: A study was conducted in patients undergoing living-related liver transplantation and their donors to investigate the relationship between the in vitro FK506 demethylation activity in graft liver microsomes and the in vivo blood clearance of FK506. Liver biopsy tissue was obtained from 17 living donors to measure the in vitro formation rate of 13-demethyl derivative (M-I: the major metabolite of FK506). Erythromycin N-demethylation activity in vitro was also assessed in 11 cases. The FK506 blood clearance (CLss) was calculated from its constant infusion rate and steady-state blood concentration on day 4 after transplantation in 17 recipients. RESULTS: The FK506 infusion rate varied 4.6-fold from 8.3 to 38.4 ng/min/kg. The mean CLss of FK506 was 22.1+/-10.8 ml/min (10.1-45.2 ml/min). The M-I formation rate showed a wide variability, ranging from 0.098 to 0.571 nmol/min/mg protein. A significant correlation was observed between the in vitro estimated total metabolic ability of the graft for FK506 (M-I formation rate x graft weight) and the in vivo CLss of FK506 (r=0.770, P<0.001). Erythromycin N-demethylation (0.066-0.443 nmol/min/mg protein) showed a strong correlation with the M-I formation rate (r=0.891, P<0.01). CONCLUSIONS: The in vivo FK506 clearance can mainly reflect in vitro FK506 demethylation activity.

Adolescent↗
PubMed1993

Cytochrome P450 mediated metabolism of diazepam in human and rat: involvement of human CYP2C in N-demethylation in the substrate concentration-dependent manner.

Metabolism of diazepam (DZP) was studied in vitro to clarify the involvement of different forms of hepatic cytochrome P450 (CYP) in rats, and humans of Japanese and Caucasian origin. Microsomal 3-hydroxylation was the major pathway of DZP metabolism in rats and was inhibited by anti-CYP3A antibodies. Purified CYP3As and CYP2C11 catalysed 3-hydroxylation and N-demethylation, respectively, in the reconstituted systems. The rates of both reactions in human liver microsomes depended on the substrate concentration: the rate of 3-hydroxylation was 3-4 times higher than N-demethylation at 0.2 mM; the two activities were essentially the same at a lower substrate concentration (0.02 mM). Inhibitions of the N-demethylation by anti-CYP2C antibody and S-mephenytoin also depended on the substrate concentration and was detectable only at a low substrate concentration. Kinetic studies revealed the presence of two distinct catalytic activities for the N-demethylation; low Km and low Vmax, and high Km and high Vmax. The former activity seems to be mediated by a CYP2C P450 form. On the other hand, DZP 3-hydroxylation was rather selectively catalysed by a CYP3A P450 at the low and high substrate concentrations. These results were consistent with the observation in vivo that DZP N-demethylation and S-mephenytoin 4'-hydroxylation are closely correlated in humans. These results also suggest that the apparent discrepancy on the role of CYP forms in DZP metabolism in vitro and in vivo may reside in the difference in substrate concentration.

Adolescent↗
PubMed1997

Identification of three cytochrome P450 isozymes involved in N-demethylation of citalopram enantiomers in human liver microsomes.

Using in vitro techniques, the present study demonstrates that CYP2D6, and 3A4 are involved in N-demethylation of citalopram (CIT) enantiomers. Human liver microsome incubations performed with specific inhibitors of these three CYP isozymes have shown up to 60% inhibition of demethylcitalopram production. cDNA expressed human cytochrome P-450 3A4, 2C19 and 2D6 isozymes, but not CYP1A2, were identified to be involved in N-demethylation of CIT enantiomers. Kinetics using cDNA expressed CYP2C19 and CYP3A4 show K(m) values in the same range: 198 microM, 211 microM for CYP2C19 and 169 microM, 163 microM for CYP3A4 for S- and R-CIT demethylation, respectively. In contrast, kinetics using cDNA expressed CYP 2D6 show a K(m) of 18 microM and 22 microM for S- and R-CIT demethylation, respectively. Nevertheless, kinetics using cDNA expressed CYP2C19 and 3A4 have a range of Vmax values ten times higher than that of CYP2D6. For this reason, intrinsic clearance values (Vmax/K(m)) for S- and R-CIT were within a small range for these three isozymes: 0.25 to 0.39 microliter h-1 x pmol-1 of CYP. CYP2D6 has an opposite stereoselectivity in the biotransformation of CIT enantiomers than CYP2C19 and 3A4; the S/R ratios of the intrinsic clearance were 0.71, 1.57 and 1.37, respectively. Taking into account that CYP isozymes are expressed at various levels, CYP2D6, which is expressed at lower levels than CYP2C19 and CYP3A4, plays a minor role in the biotransformation of CIT enantiomers. These results confirm that the use of cDNA expressed CYP isozymes is a potent tool for the measurement of kinetic constants and help to predict clearance modifications of CIT enantiomers, especially in poor metabolizers of mephenytoin (with a CYP2C19 deficiency) or patients comedicated with potent CYP2C19 or 3A4 inhibitor(s). For instance, fluvoxamine (100 microM) inhibits CIT N-demethylation by 64% in microsomes.

Aryl Hydrocarbon Hydroxylases↗
PubMed1990

Demethylation of genes in animal cells.

Tissue-specific animal cell genes are usually fully methylated in the germ line and become demethylated in those cell types in which they are expressed. To investigate this process, we inserted a methylated IgG kappa gene into fibroblasts and lymphocytes at various stages of development. The results show that this gene undergoes demethylation only in the mature lymphocytes and therefore suggest that the ability to demethylate a gene is developmentally regulated. These studies were supported by similar experiments using the rat Insulin I gene, and in this case it appears that the cis-acting elements that control demethylation may be different from those responsible for gene activation. The ability to demethylate the housekeeping gene APRT is also under developmental control, because this occurs only in embryonic cells, both in tissue culture and in transgenic mice.

Adenine Phosphoribosyltransferase↗
PubMed1984

Demethylation of methyl-accepting chemotaxis proteins in Escherichia coli induced by the repellents glycerol and ethylene glycol.

The addition of glycerol or ethylene glycol caused not only severe tumbling but also a drastic decrease in the methylation level of methyl-accepting chemotaxis proteins (MCPs) in Escherichia coli. Experiments with various mutants having defects in their MCPs showed that the demethylation occurred in all three kinds of MCPs, MCPI, II, and III. The addition of an attractant to the glycerol- or ethylene glycol-treated cells resulted in a distinct increase in the methylation level of the relevant MCP, indicating that glycerol and ethylene glycol do not directly damage the methylation-demethylation system in the cell. The time courses of adaptation and MCP demethylation upon addition of these repellents were consistent with each other. Furthermore, both the response time and the extent of MCP demethylation were increased in parallel with increasing concentrations of glycerol or ethylene glycol. These results indicate that the adaptation to these repellents is performed by the demethylation of MCPs. Thus, glycerol and ethylene glycol are novel repellents, which utilize not just one but all three kinds of MCPs for both information processing and adaptation.

Bacterial Proteins↗
PubMed2000

Estimation of minimum sterol 14alpha-demethylation-inhibitory concentration of azoles in Candida yeasts using acetate-mediated growth inhibition: potential utility in susceptibility testing.

We have recently shown that 14alpha-demethylation-deficient cells of Candida albicans are subject to growth arrest by 0.24 M acetate in a yeast extract-peptone-glucose medium and that the minimum concentration of an azole antifungal agent required for total inhibition of sterol 14alpha-demethylation (MDIC for minimum demethylation-inhibitory concentration) is practically identical to its MIC determined in the acetate-supplemented medium (O. Shimokawa and H. Nakayama, Antimicrob. Agents Chemother. 43:100-105, 1999). In the present study we estimated the MDICs of three different azoles (fluconazole, ketoconazole, and itraconazole) for strains of various Candida species using this method and compared them with the MICs determined in the corresponding acetate-free medium. The results demonstrated that the test strains were divided into two classes. One class of strains was characterized by tolerance to 14alpha-demethylation deficiency (MIC > MDIC) and consisted of strains of C. albicans, C. guilliermondii, C. kefyr, and C. tropicalis. The other class was intolerant to 14alpha-demethylation deficiency (MIC approximately MDIC) and comprised strains of C. glabrata, C. krusei, and C. parapsilosis. We also showed that replacement of the yeast extract-peptone-glucose medium with RPMI 1640 medium did not affect the results substantially. Furthermore, the 80% inhibitory concentration (IC(80)) in RPMI 1640 medium, recommended as a substitute for the conventional MIC in susceptibility testing, was found to be close to the MDIC.

Acetates↗
PubMed2005

Thrombopoietin initiates demethylation-based transcription of GP6 during megakaryocyte differentiation.

Glycoprotein VI (GPVI) is an essential platelet receptor for collagens that is exclusively expressed in the megakaryocytic lineage. Transcription of the human gene GP6 is driven largely by GATA-binding protein 1 (GATA-1), specificity protein 1 (Sp1), and Friend leukemia integration 1 (Fli-1). In this report, we show that GPVI expression during megakaryocytic differentiation is dependent on cytosine-phosphate-guanosine (CpG) demethylation that can be initiated by thrombopoietin (TPO). Sodium bisulfite genomic sequencing established that a CpG-rich island within the GP6 promoter region is fully methylated at 10 CpG sites in GPVI-nonexpressive cell lines, such as UT-7/EPO and C8161, but completely unmethylated in GPVI-expressive cell lines, including UT-7/TPO and CHRF288-11. To further confirm the relationship between CpG demethylation and expression of GPVI in primary cells, we treated human cord blood cells with TPO. The GP6 promoter is highly methylated in cord blood mononuclear cells (progenitors) but not in CD41+-enriched cells obtained after TPO differentiation. Furthermore, when UT-7/EPO-Mpl cells, which stably express human C-myeloproliferative leukemia virus ligand (c-Mpl), were treated with TPO, demethylation of the GP6 promoter was induced. In every case, demethylation of the GP6 promoter correlated with an increase in mRNA level. Thus, megakaryocyte-specific expression of the GP6 gene is regulated, in part, by CpG demethylation, which can be directly initiated by TPO.

Base Sequence↗
PubMed2006

Global DNA hypermethylation-associated cancer chemotherapy resistance and its reversion with the demethylating agent hydralazine.

BACKGROUND: The development of resistance to cytotoxic chemotherapy continues to be a major obstacle for successful anticancer therapy. It has been shown that cells exposed to toxic concentrations of commonly used cancer chemotherapy agents develop DNA hypermethylation. Hence, demethylating agents could play a role in overcoming drug resistance. METHODS: MCF-7 cells were rendered adriamycin-resistant by weekly treatment with adriamycin. Wild-type and the resulting MCF-7/Adr cells were analyzed for global DNA methylation. DNA methyltransferase activity and DNA methyltransferase (dnmt) gene expression were also determined. MCF-7/Adr cells were then subjected to antisense targeting of dnmt1, -3a, and -b genes and to treatment with the DNA methylation inhibitor hydralazine to investigate whether DNA demethylation restores sensitivity to adriamycin. RESULTS: MCF-7/Adr cells exhibited the multi-drug resistant phenotype as demonstrated by adriamycin resistance, mdr1 gene over-expression, decreased intracellular accumulation of adriamycin, and cross-resistance to paclitaxel. The mdr phenotype was accompanied by global DNA hypermethylation, over-expression of dnmt genes, and increased DNA methyltransferase activity as compared with wild-type MCF-7 cells. DNA demethylation through antisense targeting of dnmts or hydralazine restored adriamycin sensitivity of MCF-7/Adr cells to a greater extent than verapamil, a known inhibitor of mdr protein, suggesting that DNA demethylation interferes with the epigenetic reprogramming that participates in the drug-resistant phenotype. CONCLUSION: We provide evidence that DNA hypermethylation is at least partly responsible for development of the multidrug-resistant phenotype in the MCF-7/Adr model and that hydralazine, a known DNA demethylating agent, can revert the resistant phenotype.

Journal Article↗
PubMed1995

[Stereoselective N-demethylation of chlorpheniramine in rat liver microsomes: studies on age and sex differences].

We have established a simple method for the chiral stationary-phase liquid chromatographic resolution of racemic chlorpheniramine (Chp) and its two N-demethylated metabolites, monodesmethylchlorpheniramine (DMChp) and didesmethylchlorpheniramine (DDMChp), using an ovomucoid-conjugated column with respective quantitation limits of 5 ng/ml. The assay was used to study the age and sex difference in stereoselective N-demethylation of Chp by rat liver microsomes. The formation rate of each DMChp from racemic Chp was about 2.2 times faster with the S-(+)-enantiomer than the R-(-)-enantiomer in male rats at the age of only 8 weeks, whereas the rate of N-demethylation in male rats was not different between S-(+)- and R-(-)-enantiomers at the age of 3 and 24 weeks. The Vmax/Km value for the formation of S-(+)-DMChp increased with age in male rats, but the value in 8-week-old rats was lower than that in 3-week-old rats in the formation of R-(-)-DMChp. In female rats, on the other hand, the formation rate of DMChp from racemic Chp did not differ between S-(+)- and R-(-)-enantiomers at each age, suggesting a lack of stereoselectivity in the microsomal N-demethylation. Moreover, the Km and Vmax values for N-demethylation of Chp enantiomers were nearly identical between two consecutive ages in female rats. Further metabolism of DMChp to DDMChp was not observed between both enantiomers in male and female liver microsomes at the age of 3, 8 and 24 weeks.

Aging↗
PubMed1979

The involvement of cytochrome P-488 and P-450 in NADH-dependent O-demethylation of p-nitroanisole in rat liver microsomes.

These studies have shown that addition of p-nitroanisole to a reaction mixture containing rat liver microsomes resulted in an increase the reoxidation rate of NADH-reduced cytochrome b5. Fortification of rat liver microsomes with partially purified cytochrome b5 produces an increase in both NADPH-dependent and NADH-dependent p-nitroanisole O-demethylation activity. Antiserum to cytochrome P-450 isolated from phenobarbital-treated rat liver microsomes inhibited the NADH-dependent O-demethylation activity as well as the NADPH-dependent O-demethylation activity seen in rat liver microsomes. Addition of either purified cytochrome P-450 or cytochrome P-448 to an incubation mixture containing phenobarbital-treated rat liver microsomes enhanced the NADH-dependent p-nitroanisole O-demethylation activity. These results suggest that NADH-dependent and, in part, NADPH-dependent O-demethylations are catalyzed by cytochrome P-448 and cytochrome P-450 receiving electrons from cytochrome b5.

Animals↗
PubMed2004

Kinetic analysis of demethylation of 13C-aminopyrine in healthy dogs.

OBJECTIVE: To describe the kinetics of demethylation of 13C-aminopyrine in healthy dogs for use in determining the most appropriate time for collection of blood samples for a 13C-aminopyrine demethylation blood test for evaluation of hepatic function. ANIMALS: 9 healthy dogs. PROCEDURES: A 2-mL baseline blood sample was collected into an evacuated heparinized tube, and 13C-aminopyrine was administered to each dog (2 mg/kg, IV). Additional 2-mL blood samples were collected 15, 30, 45, 60, 75, 90, 105, 120, 135, 150, 180, 240, 300, and 360 minutes after 13C-aminopyrine administration. The CO2 was extracted from blood samples by addition of a strong acid, and the percentage dose of 13CO2 (PCD) in the extracted gas was determined by fractional mass spectrometry. RESULTS: No dogs had gross evidence of adverse effects, and all had an increase in PCD after IV administration of 13C-aminopyrine. The PCD had the least variability among 5 variables used to evaluate hepatic demethylating capacity. Peak PCD was detected at 30 minutes in 1 dog, 45 minutes in 5 dogs, 60 minutes in 2 dogs, and 75 minutes in 1 dog. The mean PCD for the 9 dogs peaked at 45 minutes after 13C-aminopyrine administration. CONCLUSIONS AND CLINICAL RELEVANCE: PCD appears to be the preferable variable for evaluation of hepatic demethylating capacity. Intravenous administration of 13C-aminopyrine leads to a consistent increase in PCD. Mean PCD peaked 45 minutes after administration, suggesting that blood sample collection 45 minutes after 13C-aminopyrine administration may be appropriate for use in estimating hepatic demethylating capacity.

Aminopyrine↗
PubMed1986

Pharmacogenetics of dextromethorphan O-demethylation in man.

The metabolism of dextromethorphan has been investigated from the aspect of genetically determined intersubject differences of oxidative drug metabolism in man. For this purpose, the urinary elimination of dextromethorphan and dextrorphan, which is the major O-demethylated metabolite in urine, has been studied in selected drug hydroxylation phenotypes. Dextromethorphan O-demethylation co-segregates with polymorphic debrisoquine hydroxylation, whereas no such co-segregation exists with the independently controlled mephenytoin polymorphism in man. The urinary dextromethorphan over dextrorphan metabolic ratio was validated for linearity of O-demethylation vs dose administered, and for varying urine collection intervals at different urinary pH values. A 94% repeatability of the dextromethorphan metabolic ratio could be established in extensive and poor metabolizer phenotypes. In a preliminary study, different rates of N-, O- and N,O- demethylation of dextromethorphan to yield D-methoxymorphinane, dextrorphan and D-hydroxymorphinane, respectively, were found in extensive- (Sprague-Dawley) and poor-metabolizer (female dark Agouti) rat strains. The observed interphenotype differences in man and the interstrain variations in an experimental animal model indicate that dextromethorphan O-demethylation is catalysed by the debrisoquine-type cytochrome P-450 isozyme. Therefore, the common genetic control of debrisoquine and dextromethorphan metabolism indicates that dextromethorphan might be used as a safe and innocuous substitute for debrisoquine in future routine phenotyping in the field of human pharmacogenetics of oxidative drug metabolism.

Adult↗
PubMed1991

Thebaine O-demethylation to oripavine: genetic differences between two rat strains.

1. Codeine O-demethylation to morphine is mediated by cytochrome P450 IID1 (rat), or P450 IID6 (man), and exhibits genetic polymorphism. Thebaine is a precursor in the formation of endogenous morphine and codeine in man, being O-demethylated to oripavine. 2. The objective of the present study was to ascertain whether the O-demethylation of thebaine to oripavine was mediated by cytochrome P450 IID1 in rat liver microsomes. 3. Thebaine O-demethylation showed strain differences in female Sprague-Dawley (SD) and female Dark-Agouti (DA) rats, which serve as a model for the human debrisoquine/sparteine metabolism phenotypes. 4. The total intrinsic clearance of thebaine to oripavine was high (19.7 ml/h per mg protein) in SD rats, indicating that oripavine is a major metabolite of thebaine. A 3-fold lower intrinsic clearance was observed in DA rats (6.7 ml/h per mg protein). 5. Thebaine O-demethylation was inhibited by quinine and known substrates of cytochrome P450 IID1/P450 IID6, supporting the major involvement of cytochrome P450 IID1 in oripavine formation in rats.

Animals↗
PubMed1982

Demethylation and auto-oxidation of different cobalt-=bleomycin complexes.

Demethylation of Co-bleomycin A2 by heating yields three different complexes: form I and form II and "orange" Co-bleomycin-demethyl A2. These complexes can be separated by HPLC and show different 1H NMR spectra. Preparation of Co-bleomycin-demethyl A2 by chelation of bleomycin-demethyl A2 with cobalt yields a Co-bleomycin-demethyl A2, which is auto-oxidized into Co-bleomycin A1.

Bleomycin↗
PubMed2002

[Lipoxygenase-mediated N-demethylation of pesticides in vitro].

OBJECTIVE: In order to explore the pathway of dealkylation of pesticides other than cytochrome P450 monocoxygenases, lipoxygenase (LOX)-mediated demethylation of aminocarb and some other pesticides were measured. METHOD: Formaldehyde generated in the reaction was estimated by Nash reaction to express the rate of demethylation of pesticides mediated by soy lipoxygenase (SLO). RESULTS: N-demethylation of aminocarb mediated by SLO was found to depend on the incubation time, concentration of the enzyme, concentration of aminocarb and hydrogen peroxide. Under optimal conditions, Vmax value of 18 nmol of formaldehyde.min-1.nmol-1 of lipoxygenase was observed. The reaction exhibited Km values of 3.4 mmol/L for aminocarb and 235 mumol/L for hydrogen peroxide. A strong inhibition of the reaction by nordihydroguaiaretic acid, gossypol, and phenidone clearly implicated the lipoxygenase involvement as the protein catalyst. A significant decline in the formaldehyde accumulation in the presence of either reduced glutathione or dithiothreitol suggested generation of a free radical species as an initial oxidation intermediate during the demethylation of aminocarb by SLO. The inhibition of formaldehyde generation by butylated hydroxyanisole(BHT) and butylated hydroxy toluene(BHA) further supported this contention. In addition to aminocarb, seven other pesticides were also found to undergo N-demethylation, albeit at relatively low rates. CONCLUSION: Certain pesticides may oxidatively undergo dealkylation via the lipoxygenase pathway in animals and plants.

Butylated Hydroxyanisole↗
PubMed1986

DNA demethylation induced by 5-azacytidine does not affect fragile X expression.

Experiments were performed to determine the role of DNA demethylation in fragile X expression. Fragile X positive lymphoblastoid cells were treated with 5-azacytidine and harvested for analysis of fragile X expression both directly following treatment and after a recovery period in the absence of the drug. The effectiveness of 5-azacytidine treatment in inducing DNA demethylation was concurrently monitored by analysis of methylation changes at random autosomal loci in isolated DNA from treated cells. Under conditions where 5-azacytidine was found to inhibit fragile X expression, no DNA demethylation was observed. At the time when demethylation did occur, fragile X expression was not affected. These results strongly indicate that DNA demethylation is not involved in fragile X expression.

Azacitidine↗