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Catechol-O-methyltransferase (COMT) plays an important role in the inactivation of biologically active and toxic catechols. It has been shown that COMT is genetically polymorphic with a wild-type and variant form where a valine has been... more
Catechol-O-methyltransferase (COMT) plays an important role in the inactivation of biologically active and toxic catechols. It has been shown that COMT is genetically polymorphic with a wild-type and variant form where a valine has been substituted with a methionine. Several, but not all, epidemiological studies have shown that women, homozygous with the variant form, have an increased risk of developing breast cancer. Previously, we showed that 4-hydroxyequilenin (4-OHEN), a cytotoxic/genotoxic equine catechol estrogen metabolite, is both a substrate of COMT and an irreversible inhibitor of the methylation activity of COMT in vitro. To further understand the mechanism(s) of the association between the breast cancer risk and the COMT polymorphism, it was of interest to study the effect of the Val/Met polymorphism on COMT-catalyzed catechol estrogen methylation and 4-OHEN-mediated inhibition. In the present study, Michaelis-Menten analysis showed no difference between the relative ability of each form to methylate 4-OHEN. However, we found that the COMT variant form was more susceptible to 4-OHEN-mediated irreversible inactivation. Electrospray ionization mass spectrometry and SDS-gel analysis of COMT modified by 4-OHEN revealed that inhibition mechanisms include alkylation and/or oxidation of certain amino acids. In addition, site-directed mutagenesis experiments showed that Cys33 played a more important role in the variant form of COMT demonstrated by the fact that the C33A mutant of the variant form of COMT decreased its catalytic capability more dramatically as compared with that of wild type. Furthermore, thermotropic studies indicated that the variant form was more thermolabile, which suggested that the valine to methionine substitution may have changed the secondary/tertiary structure of the variant form of COMT, making it more susceptible to 4-OHEN and heat inactivation. These data suggest that 4-OHEN-mediated inhibition of the variant form of COMT in vivo might affect the detoxification efficiency of endogenous and/or exogenous catechol estrogens and play a role in the association between breast cancer risk and COMT polymorphism.
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1Table 1. I.Introduction76II.Screening Applications77 A. Ligand-Screening Assays77 B. Characterization of Ligand–Protein Binding81 C. Metabolic Screening81 D. Screening for Electrophilic... more
1Table 1. I.Introduction76II.Screening Applications77 A. Ligand-Screening Assays77 B. Characterization of Ligand–Protein Binding81 C. Metabolic Screening81 D. Screening for Electrophilic Metabolites83III.Conclusions84Acknowledgments85References85Pulsed ultrafiltration-mass spectrometry (PUF-MS) is a method with a variety of uses for the discovery and development of biologically active small molecules, including the screening of combinatorial libraries and natural product extracts for biologically active compounds, investigation of thermodynamic and kinetic ligand–receptor binding parameters, high-throughput metabolic screening, and the screening of combinatorial libraries and botanical extracts for electrophilic metabolites. Solution-phase ligand-screening assays that use pulsed ultrafiltration-mass spectrometry are useful for “reverse pharmacology” studies in which a macromolecular receptor of interest has been isolated, but ligands for the receptor are needed. Protein-binding studies that involve pulsed ultrafiltration can be used to rapidly determine classical binding parameters for interactions between a macromolecular receptor and a compound of interest. Metabolic screening assays can identify substrates for cytochromes P450, and should be capable of characterizing phase I metabolites with a throughput of at least 60 compounds/hr. Pulsed ultrafiltration can also be used in conjunction with LC-MS-MS to screen mixtures for compounds that might be activated metabolically to electrophilic quinoid and epoxide metabolites by cytochrome P450; that screening can provide early warning of compounds likely to be toxic when administered in large doses. The combination of pulsed-ultrafiltration extraction and mass spectrometric detection provides the sensitivity and selectivity necessary to characterize compounds present at low concentrations in complex chemical mixtures, and is applicable to the analysis of biologically active compounds from combinatorial libraries and botanical extracts. © 2002 Wiley Periodicals, Inc., Mass Spec Rev 21:76–86, 2002; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/mas.10020
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Complementary and Alternative Medicine, Cell Division, Humans, Female, Alternative, and 8 moreAlkaline phosphatase, Complementary and Alternative Medicine in the Health Service, High Pressure Liquid Chromatography, Plant extracts, Trifolium, Phytoestrogens, Estrogen receptor-beta, and Estrogen receptor alpha
Estrogenicity-directed fractionation of a methanol extract of the strobiles of Humulus lupulus that had been extracted previously with supercritical CO(2), known as... more
Estrogenicity-directed fractionation of a methanol extract of the strobiles of Humulus lupulus that had been extracted previously with supercritical CO(2), known as "spent hops", led to the isolation and identification of 22 compounds including 12 prenylated chalcones (1-8, 10-13), five prenylflavanones (14-17), 4-hydroxybenzaldehyde (18), sitosterol-3-O-beta-glucopyranoside (19), humulinone (20), and cohumulinone (21). In addition, the prenylated chalcone xanthohumol C (9a) was obtained as a 6:1 mixture along with its 1' ',2' '-dihydro derivative (9b). Three new chalcones (4, 11, 12) and four previously unreported constituents of hops (5, 6, 9b, 13) are reported. The structures of the new compounds were determined through a combination of spectrometric techniques including 1D and 2D NMR, HRESIMS, and ESIMS-MS. Full 1H NMR spin system analyses were performed to characterize the higher-order glucopyranosyl, prenyl, and chalcone B-ring spectra of the isolates. The principle estrogen 8-prenylnaringenin (15) from hops is an artifact formed along with its positional isomer 6-prenylnaringenin (16) through the spontaneous isomerization of the pro-estrogenic chalcone DMX (7).
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The female flowers of hops (Humulus lupulus L.) used to flavor beer contain the prenylated flavonoids xanthohumol (XN) and isoxanthohumol (IX). IX is moderately estrogenic in vitro and XN has pharmacological properties that might make it... more
The female flowers of hops (Humulus lupulus L.) used to flavor beer contain the prenylated flavonoids xanthohumol (XN) and isoxanthohumol (IX). IX is moderately estrogenic in vitro and XN has pharmacological properties that might make it useful as a cancer chemopreventive agent. The metabolism of these dietary flavonoids was investigated in vitro using human liver microsomes. Hydroxylation of a prenyl methyl group was the primary route of oxidative metabolism forming either cis or trans hydroxylated metabolites of IX but only the trans isomer of XN. The double bond on the prenyl group of both compounds formed an epoxide which was opened by an intramolecular reaction with the neighboring hydroxyl group. The potent phytoestrogen 8-prenylnaringenin (8-PN) was detected as a demethylation product of IX. However, the analogous demethylation reaction was not observed for XN. Since XN can be converted to IX through acid-catalyzed cyclization in the stomach, XN might contribute to the in vivo levels of estrogenic 8-PN following consumption of hops extracts. Copyright © 2005 John Wiley & Sons, Ltd.
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Kinetics, Mass Spectrometry, Drug metabolism, Enzyme Inhibitors, Humans, and 15 moreLiver, Animals, Male, Monoclonal Antibodies, High Performance Liquid Chromatography, Enzyme, Theophylline, Rats, Xenobiotics, Quercetin, Biotransformation, Porphyrias, Hydroxylation, Recombinant Proteins, and N-dealkylation
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The N-7 and O-6 positions of 2'-deoxyguanosine are the predominant sites of methylation by... more
The N-7 and O-6 positions of 2'-deoxyguanosine are the predominant sites of methylation by N-methyl-N-nitrosourea (MNU), which is used to produce a variety of experimental cancers in animal models. Here we report the development of a highly sensitive quantitative assay based on high-performance liquid chromatography-UV-tandem mass spectrometry (LC-UV-MS-MS) to measure N7-methyl-2'-deoxyguanosine (N7-MedG) and O6-methyl-2'-deoxyguanosine (O6-MedG) in DNA hydrolysates. Since this assay was selective for deoxyribonucleosides, potential interference from methylated RNA was eliminated. Isotopically labeled analogues, [2H3]N7-MedG and [2H3]O6-MedG, were synthesized and added to the DNA hydrolysates as internal standards. In-line UV absorbance detection was used for the quantitative analysis of the native deoxyribonucleoside dG, and MS-MS was used for the determination of N7-MedG and O6-MedG. The limits of detection for N7-MedG and O6-MedG were determined to be 64 and 43 fmol, respectively. The limits of quantification were 0.13 pmol for N7-MedG and 0.085 pmol for O6-MedG. The stabilities of N7-MedG and O6-MedG were also investigated. Although O6-MedG was stable at room temperature for at least 11 days, the half-life of N7-MedG at room temperature was 2 days. Both adducts were stable at -20 degrees C. Calf thymus DNA and DNA from the livers of MNU-treated Sprague-Dawley rats were assayed using LC-UV-MS-MS, which was optimized for speed as well as for sensitivity. The levels of N7-MedG and 06-MedG in calf thymus DNA increased with MNU concentration and incubation time. The levels of N7-MedG and O6-MedG in the rat livers 2 h after treatment with a single dose of 50 mg/kg MNU were 95.2 N7-MedG/105 dG and 14.8 O6-MedG/105 dG. This LC-UV-MS-MS assay provides the sensitivity and speed required to evaluate the extent of methylated DNA lesions in animal models of cancer induced by the methylating agent MNU.
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A method for the quantitative analysis of the vitamin A compounds all-trans-retinol and all-trans-retinyl palmitate was developed using high-performance liquid chromatography–atmospheric pressure chemical ionization–mass spectrometry... more
A method for the quantitative analysis of the vitamin A compounds all-trans-retinol and all-trans-retinyl palmitate was developed using high-performance liquid chromatography–atmospheric pressure chemical ionization–mass spectrometry (APCI–LC–MS). Unlike previous quantitative mass spectrometric methods for vitamin A, HPLC separations were carried out using a C30 reversed-phase column instead of GC separation. Because no sample hydrolysis or derivatization was necessary, retinyl palmitate was preserved for analysis instead of being hydrolyzed to retinol. Human serum was analyzed following simple hexane extraction without saponification or any additional purification. A comparison of APCI and electrospray ionization showed that only APCI produced a linear response over all four orders of magnitude of retinol and three orders of magnitude of retinyl palmitate concentrations. Selected ion monitoring of the fragment ion of m/z 269 was used for APCI quantitation of both retinol and retinyl palmitate, since it was the base peak and the only abundant ion in the mass spectra of both compounds and the internal standard, retinyl acetate. The ion of m/z 269 corresponded to loss of water, loss of palmitic acid, or elimination of acetic acid from the protonated molecules of retinol, retinyl palmitate and retinyl acetate, respectively. The limit of detection of APCI–LC–MS for all-trans-retinol and all-trans-retinyl palmitate was determined to be approximately 34 fmol/μl and 36 fmol/μl (0.670 pmol all-trans-retinol and 0.720 pmol all-trans-retinyl palmitate injected in 20 μl on-column), respectively. The limit of quantitation was approximately 500 fmol/μl and 250 fmol/μl (10 pmol and 5 pmol injected in 20 μl on-column) for retinol and retinyl palmitate, respectively.
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A methanol extract of chaste-tree berry (Vitex agnus-castus L.) was tested for its ability to displace radiolabeled estradiol from the binding site of estrogen receptors alpha (ERα) and beta (ERβ). The extract at 46 ± 3 μg/ml displaced... more
A methanol extract of chaste-tree berry (Vitex agnus-castus L.) was tested for its ability to displace radiolabeled estradiol from the binding site of estrogen receptors alpha (ERα) and beta (ERβ). The extract at 46 ± 3 μg/ml displaced 50% of estradiol from ERα and 64 ± 4 μg/ml from ERβ. Treatment of the ER+ hormone-dependent T47D:A18 breast cancer cell line with the extract induced up-regulation of ERβ mRNA. Progesterone receptor (PR) mRNA was upregulated in the Ishikawa endometrial cancer cell line. However, chaste-tree berry extract did not induce estrogen-dependent alkaline phosphatase (AP) activity in Ishikawa cells. Bioassay-guided isolation, utilizing ER binding as a monitor, resulted in the isolation of linoleic acid as one possible estrogenic component of the extract. The use of pulsed ultrafiltration liquid chromatography-mass spectrometry, which is an affinity-based screening technique, also identified linoleic acid as an ER ligand based on its selective affinity, molecular weight, and retention time. Linoleic acid also stimulated mRNA ERβ expression in T47D:A18 cells, PR expression in Ishikawa cells, but not AP activity in Ishikawa cells. These data suggest that linoleic acid from the fruits of Vitex agnus-castus can bind to estrogen receptors and induce certain estrogen inducible genes.
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In response to the need for rapid screening of combinatorial libraries to identify new lead compounds during drug discovery, we have developed an on-line combination of ultrafiltration and electrospray mass spectrometry, called pulsed... more
In response to the need for rapid screening of combinatorial libraries to identify new lead compounds during drug discovery, we have developed an on-line combination of ultrafiltration and electrospray mass spectrometry, called pulsed ultrafiltration mass spectrometry, which facilitates the identification of solution-phase ligands in library mixtures that bind to solution-phase receptors. After ligands contained in a library mixture were bound to a macromolecular receptor, e.g., human serum albumin or calf intestine adenosine deaminase, the ligand-receptor complexes were purified by ultrafiltration and then dissociated using methanol to elute the ligands into the electrospray mass spectrometer for detection. Ligands with dissociation constants in the micromolar to nanomolar range were successfully bound, released, and detected using this method, including warfarin, salicylate, furosemide, and thyroxine binding to human serum albumin, and erythro-9-(2-hydroxy-3-nonyl)adenine binding to calf intestine adenosine deaminase. Repetitive bind- and-release experiments demonstrated that the receptor could be reused. Thus, pulsed ultrafiltration mass spectrometry was shown to provide a simple and powerful new method for the screening of combinatorial libraries in support of new drug discovery.
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Chemical Engineering, Analytical Chemistry, Mass Spectrometry, Enzyme Inhibitors, Ultrafiltration, and 19 moreHumans, Animals, Methanol, Drug Design, Analytical, Thyroxine, Online Systems, Cattle, Intestines, Warfarin, Molecular weight, Salicylic Acid, Furosemide, Adenosine Deaminase, Adenine, Ligands, Pulsatile Flow, Salicylates, and Serum albumin
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The photocatalytic degradation of the organophosphorus insecticide diazinon in aqueous suspensions has been studied by using titanium dioxide as a photocatalyst. The degradation of the insecticide was a fast process and included the... more
The photocatalytic degradation of the organophosphorus insecticide diazinon in aqueous suspensions has been studied by using titanium dioxide as a photocatalyst. The degradation of the insecticide was a fast process and included the formation of several intermediates that were identified using GC/ion-trap mass spectrometry with EI or CI in positive and negative ionization mode and HPLC/electrospray-QqTOF mass spectrometry. Since primarily hydroxy derivatives were identified in these aqueous suspensions, the mechanism of degradation was probably based on hydroxyl radical attack. The initial oxidative pathways of the degradation of diazinon involved the substitution of sulfur by oxygen on the P=S bond, cleavage of the pyrimidine ester bond, and oxidation of the isopropyl group. Exact mass measurements of the derivatives allowed the elemental formula of the molecules to be determined confidently. Similarities to the metabolic pathways occurring in living organisms were observed.
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Analytical Chemistry, Catalysis, Mass Spectrometry, Titanium, Time-of-flight mass spectrometry, and 16 moreElectrospray, Insecticides, Photocatalysis, Pesticide, Pesticides, Titanium dioxide, Gas Chromatography, Chemical Analysis, High Pressure Liquid Chromatography, Liquid Chromatography / Electrospray Ionization Mass Spectrometry, Gas Chromatography/mass Spectrometry, Photolysis, Metabolic pathway, Electron Impact Ionization, Diazinon, and Aqueous Solution
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Analytical Chemistry, Kinetics, Pharmaceutical Chemistry, Quantitative analysis, Humans, and 14 moreSheep, Animals, Tandem Mass Spectrometry, Inflammatory disease, High Performance Liquid Chromatography, Enzyme, Liquid Chromatography, Reproducibility of Results, Quantitative Analysis, Side Effect, Species Specificity, Ligands, Arachidonic Acid, and Prostaglandin E2
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From the anti-TB active fractions of the inner stem bark of Oplopanax horridus, two new heterocyclic nerolidol derivatives, 3,10-epoxy-3,7,11-trimethyldodeca-1,6-dien-11-ol, named neroplomacrol (1), and... more
From the anti-TB active fractions of the inner stem bark of Oplopanax horridus, two new heterocyclic nerolidol derivatives, 3,10-epoxy-3,7,11-trimethyldodeca-1,6-dien-11-ol, named neroplomacrol (1), and rel-(3S,6R,7S,10R)-7,10-epoxy-3,7,11-trimethyldodec-1-ene-3,6,11-triol, named neroplofurol (2), were isolated together with oplopandiol (3), falcarindiol (4), and sesamin (5). Extensive spectroscopic analysis revealed that 1 possesses a novel 3,10-oxanonacyclic ring system. Computer-iterated full spin system analysis led to the generation of (1)H NMR fingerprints that will facilitate future dereplication of analogues by providing characteristic spin-spin coupling patterns. The full spin analysis of 5 revealed asymmetric coupling patterns among the chemically equivalent spins, thus confirming the magnetic asymmetry of 5. It was further demonstrated that (1)H NMR fingerprints and MS data enable dereplication of isolates at a submilligram levels including their relative configuration. Countercurrent concentration of the anti-TB activity of the ethnobotanical O. horridus versus the Mycobacterium tuberculosis Erdman strain led to polyynes 3 and 4 as main anti-TB active principles. Their synergistic behavior is linked to a complex fraction containing the new nerolidiol sesquiterpenes, 1 and 2, as phytochemical marker compounds.
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Natural Products, Pharmacognosy, Medicinal Plants, Structural Analysis, Biological Sciences, and 14 moreMycobacterium tuberculosis, Alcohol, Bacteria, Sesquiterpenes, CHEMICAL SCIENCES, Bark, Isolation, Natural, Microbial Sensitivity Tests, Biological activity, Structure activity Relationship, Molecular Structure, Alaska, and Terpenoid
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Complementary and Alternative Medicine, Plant Biology, Pharmacognosy, Magnetic Resonance Spectroscopy, Flavonoids, and 13 moreLignans, Mu Opioid Receptor, Fruit, Terpenes, Liquid Chromatography / Electrospray Ionization Mass Spectrometry, Phenols, Phenolic compound, Diterpenes, Fitoterapia, Vitex, Polyphenol, Molecular Structure, and Terpenoid
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Trimethylamine dehydrogenase (TMADH) from the bacterium Methylophilus methylotrophus (sp. W(3)A(1)) and its C30A mutant were inactivated with three known inactivators of monoamine oxidase, namely, phenylhydrazine,... more
Trimethylamine dehydrogenase (TMADH) from the bacterium Methylophilus methylotrophus (sp. W(3)A(1)) and its C30A mutant were inactivated with three known inactivators of monoamine oxidase, namely, phenylhydrazine, N-cyclopropyl-alpha-methylbenzylamine, and 1-phenylcyclopropylamine. All three compounds irreversibly inactivated both the wild-type and C30A mutant enzymes, although phenylhydrazine was 10 times more potent than N-cyclopropyl-alpha-methylbenzylamine, which was much more potent than 1-phenylcyclopropylamine. The change in the UV--visible absorption spectra upon modification indicated that the flavin was modified. In the case of the C30A mutant, the absence of a covalent attachment of the flavin to the polypeptide has permitted LC-electrospray mass spectrometry of the reaction product to be undertaken, demonstrating new mass peaks corresponding to various chemically modified forms of the flavin cofactor. In the case of N-cyclopropyl-alpha-methylbenzylamine, masses corresponding to hydroxy-FMN and hydroxyriboflavin were detected. 1-Phenylcyclopropylamine inactivation of the C30A mutant produced three modified flavins, as evidenced by the electrospray mass spectrum: hydroxy-FMN, FMN plus C(6)H(5)COCH(2)CH(2), and hydroxy-FMN plus C(6)H(5)COCH(2)CH(2). Phenylhydrazine inactivation of the C30A mutant gave at least seven different modified flavins: hydroxyriboflavin, hydroxy-FMN, two apparently isomeric compounds corresponding to hydroxy-FMN plus one phenyl group, two apparently isomeric compounds corresponding to FMN plus one phenyl group, and FMN plus two phenyl groups. Covalent flavin adduct formation appears to be the only modification because dialysis of the inactive enzyme followed by reconstitution with FMN restores the enzyme activity to that of a noninactivated control.
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In response to the need for rapid screening of combinatorial libraries to identify new lead compounds during drug discovery, we have developed an on-line combination of ultrafiltration and electrospray mass spectrometry, called pulsed... more
In response to the need for rapid screening of combinatorial libraries to identify new lead compounds during drug discovery, we have developed an on-line combination of ultrafiltration and electrospray mass spectrometry, called pulsed ultrafiltration mass spectrometry, which facilitates the identification of solution-phase ligands in library mixtures that bind to solution-phase receptors. After ligands contained in a library mixture were bound to a macromolecular receptor, e.g., human serum albumin or calf intestine adenosine deaminase, the ligand-receptor complexes were purified by ultrafiltration and then dissociated using methanol to elute the ligands into the electrospray mass spectrometer for detection. Ligands with dissociation constants in the micromolar to nanomolar range were successfully bound, released, and detected using this method, including warfarin, salicylate, furosemide, and thyroxine binding to human serum albumin, and erythro-9-(2-hydroxy-3-nonyl)adenine binding to calf intestine adenosine deaminase. Repetitive bind- and-release experiments demonstrated that the receptor could be reused. Thus, pulsed ultrafiltration mass spectrometry was shown to provide a simple and powerful new method for the screening of combinatorial libraries in support of new drug discovery.
Research Interests:
Chemical Engineering, Analytical Chemistry, Mass Spectrometry, Enzyme Inhibitors, Ultrafiltration, and 19 moreHumans, Animals, Methanol, Drug Design, Analytical, Thyroxine, Online Systems, Cattle, Intestines, Warfarin, Molecular weight, Salicylic Acid, Furosemide, Adenosine Deaminase, Adenine, Ligands, Pulsatile Flow, Salicylates, and Serum albumin
The N-7 and O-6 positions of 2'-deoxyguanosine are the predominant sites of methylation by... more
The N-7 and O-6 positions of 2'-deoxyguanosine are the predominant sites of methylation by N-methyl-N-nitrosourea (MNU), which is used to produce a variety of experimental cancers in animal models. Here we report the development of a highly sensitive quantitative assay based on high-performance liquid chromatography-UV-tandem mass spectrometry (LC-UV-MS-MS) to measure N7-methyl-2'-deoxyguanosine (N7-MedG) and O6-methyl-2'-deoxyguanosine (O6-MedG) in DNA hydrolysates. Since this assay was selective for deoxyribonucleosides, potential interference from methylated RNA was eliminated. Isotopically labeled analogues, [2H3]N7-MedG and [2H3]O6-MedG, were synthesized and added to the DNA hydrolysates as internal standards. In-line UV absorbance detection was used for the quantitative analysis of the native deoxyribonucleoside dG, and MS-MS was used for the determination of N7-MedG and O6-MedG. The limits of detection for N7-MedG and O6-MedG were determined to be 64 and 43 fmol, respectively. The limits of quantification were 0.13 pmol for N7-MedG and 0.085 pmol for O6-MedG. The stabilities of N7-MedG and O6-MedG were also investigated. Although O6-MedG was stable at room temperature for at least 11 days, the half-life of N7-MedG at room temperature was 2 days. Both adducts were stable at -20 degrees C. Calf thymus DNA and DNA from the livers of MNU-treated Sprague-Dawley rats were assayed using LC-UV-MS-MS, which was optimized for speed as well as for sensitivity. The levels of N7-MedG and 06-MedG in calf thymus DNA increased with MNU concentration and incubation time. The levels of N7-MedG and O6-MedG in the rat livers 2 h after treatment with a single dose of 50 mg/kg MNU were 95.2 N7-MedG/105 dG and 14.8 O6-MedG/105 dG. This LC-UV-MS-MS assay provides the sensitivity and speed required to evaluate the extent of methylated DNA lesions in animal models of cancer induced by the methylating agent MNU.