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PubMed1984

Hexose phosphate binding sites of fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase. Interaction with N-bromoacetylethanolamine phosphate and 3-bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate.

N-Bromoacetylethanolamine phosphate and 3-bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate have been tested in order to study the hexose phosphate binding sites of a bifunctional enzyme, fructose-6-P,2-kinase:fructose-2,6-bisphosphatase. N-Bromoacetylethanolamine phosphate is a competitive inhibitor with respect to fructose-6-P (Ki = 0.24 mM) and a noncompetitive inhibitor with ATP (Ki = 0.8 mM). The reagent inactivates fructose-6-P,2-kinase but not fructose-2,6-bisphosphatase, and the inactivation is prevented by fructose-6-P. The inactivation reaction follows pseudo first-order kinetics to completion and with increasing concentrations of N-bromoacetylethanolamine phosphate a rate saturation effect is observed. The concentration of the reagent giving the half-maximum inactivation is 2.2 mM and the apparent first order rate constant is 0.0046 s-1. The enzyme alkylated by N-bromoacetylethanolamine-P has lost over 90% of the kinase activity, retains nearly full activity of fructose-2,6-bisphosphatase, and its inhibition by fructose-6-P is not altered. 3-Bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate is also a competitive inhibitor of fructose-6-P,2-kinase with respect to fructose-6-P in the forward reaction and fructose-2,6-P2 in the reverse direction. This reagent inhibits 93% of fructose-6-P,2-kinase but activates fructose-2,6-bisphosphatase 3.7-fold. 3-Bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate alters the fructose-2,6-P2 saturation kinetic curve from negative cooperativity to normal Michaelis-Menten kinetics with K0.5 of 0.8 microM. The reagent, however, has no effect on the fructose-6-P inhibition of the phosphatase. These results strongly suggest that hexose phosphate binding sites of fructose-6-P,2-kinase and fructose-2,6-bisphosphatase are distinct and located in different regions of this bifunctional enzyme.

Adenosine Triphosphate↗
PubMed1999

Electrochemical Behavior and Semiempirical Approach to the Electroadsorption of 3-Buten-2-one and 2-Butanone on Platinum.

The electroadsorption of 3-buten-2-one was studied on polycrystalline and faceted Pt electrodes in aqueous 0.5 M H(2)SO(4) between 20 and 70 degrees C using voltammetric and transient techniques. The largest values of anodic charge densities in stripping experiments were observed at the potential of zero charge of the Pt/acid interface (ca. 0.20 V), that is, 550, 680, and 720 µC cm(-2), on polycrystalline and (111)- and (100)-faceted electrodes, respectively. The anodic and cathodic strippings of 3-buten-2-one showed the formation of at least two residues with different electrochemical behavior. For comparison cyclic voltammetries and current transients of 2-butanone on Pt were also studied. To get a deeper insight into the adsorption configurations of 3-buten-2-one and 2-butanone, the extended Hückel molecular orbital methodology was used to predict adsorption geometries and binding energies on simulated Pt(100) and Pt(111) single-crystal surfaces. At equilibrium potentials, two types of adsorbed species were recognized, a planar 3-buten-2-one formed by the interaction of carbonyl and alkene groups and adsorbates coming from the single interaction of the >C&dbond;O moiety for both 3-buten-2-one and 2-butanone. At simulated positive potentials, 3-buten-2-one adsorption occurred on Pt(111) and Pt(100) clusters through the pi-orbitals of the >C&dbond;C< moiety, whereas at negative potentials 3-buten-2-one was adsorbed via µ-bridging or di-varsigma configuration(s). Copyright 1999 Academic Press.

Journal Article↗
PubMed1995

Inhibition of metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone by dietary benzaldehydes.

As part of a routine screening assay, benzaldehyde was found to inhibit 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) metabolism. Consequently, the effects of benzaldehyde and several structurally related compounds on NNK metabolism were examined in murine hepatic and pulmonary microsomes. All test compounds inhibited formation of the metabolites 4-oxo-4-(3-pyridyl)butyric acid (OPBA), 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in hepatic microsomes and inhibited formation of 4-(methylnitrosamino)-1-(3-pyridyl-N-oxide)-1-butanone (NNK N-oxide), HPB, and NNAL in pulmonary microsomes. m-Anisaldehyde was the most potent inhibitor, and p-hydroxybenzaldehyde and syringaldehyde were less potent than benzaldehyde and vanillin in inhibiting the formation of OPBA and HPB, NNK metabolites that reflect metabolic activation (alpha-hydroxylation). Vanillin was essentially as potent as benzaldehyde. The mechanism of inhibition exhibited by these compounds appears to be competitive in nature. The ability of these compounds to inhibit NNK activation suggests that these compounds may be effective blocking agents (anti-initiating agents) for NNK lung tumorigenesis.

Animals↗
PubMed2004

Potential anti-infective targets in pathogenic yeasts: structure and properties of 3,4-dihydroxy-2-butanone 4-phosphate synthase of Candida albicans.

A synthetic gene specifying a putative 3,4-dihydroxy-2-butanone 4-phosphate synthase of Candida albicans directed the synthesis of a 22.5 kDa peptide in a recombinant Escherichia coli strain. The recombinant protein was purified to apparent homogeneity by two chromatographic steps and was shown to catalyze the formation of L-3,4-dihydroxy-2-butanone 4-phosphate from ribulose 5-phosphate at a rate of 332 nmol mg(-1) min(-1). Hydrodynamic studies indicated a native molecular mass of 41 kDa in line with a homodimer structure. The protein was crystallized in its apoform. Soaking yielded crystals in complex with the substrate ribulose 5-phosphate. The structures were solved at resolutions of 1.6 and 1.7 angstroms, respectively, using 3,4-dihydroxy-2-butanone 4-phosphate synthase of E. coli for molecular replacement. Structural comparison with the orthologs of Magnaporthe grisea and Methanococcus jannaschii revealed a hitherto unknown conformation of the essential acidic active-site loop.

Amino Acid Sequence↗
PubMedDate not supplied

Analysis for N2-(pyridyloxobutyl)deoxyguanosine adducts in DNA of tissues exposed to tritium-labeled 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N'-nitrosonornicotine.

The tobacco-specific carcinogens 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) are metabolically activated to DNA binding intermediates, partially via 4-(3-pyridyl)-4-oxobutanediazohydroxide (7) or related carbonium ions. Previous studies have shown that generation of 7 from 4-(carbethoxynitrosamino)-1-(3-pyridyl)-1-butanone (11) in the presence of deoxyguanosine yields a major adduct identified as 2'-deoxy-N-[1-methyl-3-oxo-3-(3-pyridyl)propyl]guanosine (adduct 1). These results suggested that adduct 1 should be present in DNA of tissues that can metabolically activate NNK and NNN. In the present study, we evaluate the formation of adduct 1 and its structurally related straight-chain analogue 2'-deoxy-N-[4-oxo-4-(3-pyridyl)butyl]guanosine (adduct 2) in DNA of tissues of rats treated with [5-3H]NNK or [5-3H]NNN, and in DNA of nasal mucosa that had been cultured in medium containing [5-3H]NNK or [5-3H]NNN. Hepatic DNA from rats treated with [5-3H]NNK was enzymatically hydrolyzed to deoxyribonucleosides and analyzed by HPLC. One of the radioactive peaks, peak E, coeluted with adduct 1. However, treatment of peak E with NaBH4 resulted in the formation of products different from those produced by NaBH4 treatment of adduct 1, demonstrating that adduct 1 could not be detected under these conditions. Hydrolysis of peak E with acid produced 4-hydroxy-1-(3-pyridyl)-1-butanone (9), suggesting that peak E might be adduct 2. Therefore, adduct 2 was synthesized by reaction of deoxyguanosine with 1-(3-pyridyl)butane-1,4-dione (5) in the presence of NaCNBH3.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2003

The pyridyloxobutyl DNA adduct, O6-[4-oxo-4-(3-pyridyl)butyl]guanine, is detected in tissues from 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-treated A/J mice.

The tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is a potent pulmonary carcinogen. This unsymmetric nitrosamine can be metabolically activated to lung DNA methylating and pyridyloxobutylating intermediates. The methyl DNA adducts are well characterized. The pyridyloxobutyl adducts are unstable under DNA hydrolysis conditions and decompose to release 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB). One of the HPB-releasing adducts,O6-[4-oxo-4-(3-pyridyl)butyl]guanine (O6-pobG), has been detected in DNA reacted in vitro with the model pyridyloxobutylating agent, 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc). To determine whether this adduct was formed in vivo, A/J mice were treated with 10 mumol of [5-3H]NNK and sacrificed 24 h postinjection. The mutagenic O6-pobG was detected in liver but not lung DNA from these animals. Since 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) is a major metabolite of NNK, it is also possible that these animals are activating NNAL to a pyridylhydroxybutylating agent. Therefore, we also measured the levels of O6-[4-hydroxy-4-(3-pyridyl)butyl]guanine (O6-phbG) in these DNA samples. While radioactivity did coelute with synthetic standard for this potential NNAL adduct in one lung DNA sample, significant levels of O6-phbG were not detected in any other lung or liver DNA samples. The pyridyloxobutyl adduct, O6-pobG, was also observed in lung and liver DNA from mice treated with 4.2 mumol of [5-3H]NNKOAc in the presence but not absence of 2.5 mumol of O6-benzylguanine, a known depletor of the repair protein O6-alkylguanine-DNA alkyltransferase (AGT). These data indicate that this adduct is formed in vivo but is repaired in part by AGT. Cell-free extracts from A/J mouse lung and liver were used to determine the relative rate of O6-alkylguanine repair. O6-mG and O6-pobG were removed from DNA to the same extent in a competitive assay, suggesting that low levels of O6-pobG in lungs of NNK-treated mice did not result from preferential repair of O6-pobG by AGT. It is more likely that initial levels of O6-pobG are much lower than initial levels of O6-mG in lung DNA from NNK-treated A/J mice. These data are consistent with previous studies, which indicate that DNA methylation is the critical pathway for NNK-induced lung carcinogenesis in A/J mice.

Animals↗
PubMed2003

Identification of adducts formed by pyridyloxobutylation of deoxyguanosine and DNA by 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone, a chemically activated form of tobacco specific carcinogens.

The tobacco specific carcinogens 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) are metabolically activated to 4-oxo-4-(3-pyridyl)-1-butanediazohydroxide (7), which is known to pyridyloxobutylate DNA. A substantial proportion of the adducts in this DNA releases 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB, 11) under various hydrolysis conditions, including neutral thermal hydrolysis. These HPB-releasing DNA adducts have been detected in target tissues of animals treated with NNK and NNN as well as in lung tissue from smokers. Although their presence in pyridyloxobutylated DNA was conclusively demonstrated 15 years ago, their structures have not been previously determined. We investigated this question in the present study by determining the structures of products formed in reactions with dGuo and DNA of 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKCH(2)OAc, 3), a stable precursor to 7. Reaction mixtures from NNKCH(2)OAc and dGuo were analyzed by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) with selected ion monitoring at m/z 415. A major peak was detected and identified as 7-[4-oxo-4-(3-pyridyl)but-1-yl]dGuo (37) by its ESI-MS fragmentation pattern and by neutral thermal hydrolysis, which converted it to 11 and 7-[4-oxo-4-(3-pyridyl)but-1-yl]Gua (26). The latter was identified by comparison to synthetic 26 using LC-ESI-MS with selected ion monitoring at m/z 299, M + 1 of 26. Further evidence was obtained by NaBH(4) reduction of 26 to 7-[4-hydroxy-4-(3-pyridyl)but-1-yl]Gua, which was also matched with a standard. Adduct 37 was similarly identified in enzyme hydrolysates of DNA reacted with NNKCH(2)OAc, accounting for 30-35% of the HPB-releasing adducts in this DNA. Several other adducts resulting from pyridyloxobutylation of the N(2)- and O(6)-positions of Gua were also identified as products in the dGuo or DNA reactions by comparison to standards; their concentrations were considerably less than that of 37. These adducts were N(2)-[4-oxo-4-(3-pyridyl)but-1-yl]dGuo (23), N(2)-[4-oxo-4-(3-pyridyl)but-2-yl]dGuo (25), N(2)-[2-(3-pyridyl)tetrahydrofuran-2-yl]dGuo (31a) (or its open chain tautomer 31b), and O(6)-[4-oxo-4-(3-pyridyl)but-1-yl]dGuo (10). Adducts 23, 25, and 10 did not release HPB upon neutral thermal hydrolysis. The results of this study provide the first structural identification of an HPB-releasing DNA adduct of the tobacco specific nitrosamines NNK and NNN.

Carcinogens↗
PubMed2005

Mass spectrometric analysis of relative levels of pyridyloxobutylation adducts formed in the reaction of DNA with a chemically activated form of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

Exposure to the tobacco-related nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine is carcinogenic to humans. Metabolic activation of NNK leads to the formation of DNA adducts, which play a critical role in NNK carcinogenesis. Adducts specific to NNK result from covalent linkage of a pyridyloxobutyl (POB-1-yl) group to DNA. Furthermore, some such adducts are unstable, releasing the degradation product 4-hydroxy-1-(3-pyridyl)-1-butanone (4-HPB). Previous qualitative reports from our laboratory have established the chemical structures of the major POB-1-yl-DNA adducts. In this study, we have quantitated the levels of each of these adducts in vitro, as well as their contribution to the biomarker of DNA pyridyloxobutylation, 4-HPB. Standards for the POB-DNA adducts O(6)-(POB-1-yl)dGuo, 7-(POB-1-yl)Gua, O(2)-(POB-1-yl)dThd, and O(2)-(POB-1-yl)Cyt were synthesized and used to determine standard responses by reverse phase HPLC-electrospray ionization-tandem mass spectrometry (ESI-MS/MS). DNA was incubated with varying amounts of 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone in the presence of an esterase, conditions favorable to the formation of an active pyridyloxobutylating agent. After sequential enzymatic and neutral thermal hydrolysis, isolation, and purification, the pyridyloxobutylated mixture was analyzed by HPLC-ESI-MS/MS to quantify the relative level of each of these four adducts as well as the released 4-HPB. The most abundant product was 4-HPB, which accounted for two-thirds of the analyzed mixture. The highest adduct levels measured were those of bases that result from loss of deoxyribose upon neutral thermal hydrolysis. These adducts, 7-(POB-1-yl)Gua and O(2)-(POB-1-yl)Cyt, comprised an average of 23 and 6% of the analyzed mixture, respectively. O(2)-(POB-1-yl)dThd and the mutagenic adduct O(6)-(POB-1-yl)dGuo were detected at the lowest levels, 4 and 2%, respectively. The relative levels of adducts determined in this study provide further insight regarding the chemical reactivity of the activated form of NNK with respect to DNA bases. Furthermore, the analytical standards and mass spectrometric methods used lay the groundwork for establishing a representative array of pyridyloxobutylation adducts as biomarkers of tobacco exposure in further biochemical and in vivo studies.

Animals↗
PubMed1997

Pyridyloxobutyl adduct O6-[4-oxo-4-(3-pyridyl)butyl]guanine is present in 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone-treated DNA and is a substrate for O6-alkylguanine-DNA alkyltransferase.

The lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is activated to reactive metabolites that methylate or pyridyloxobutylate DNA. Previous studies demonstrated that pyridyloxobutylated DNA interferes with the repair of O6-methylguanine (O6-mG) by O6-alkylguanine-DNA alkyltransferase (AGT). The AGT reactivity of pyridyloxobutylated DNA was attributed to (pyridyloxobutyl)guanine adducts. One potential AGT substrate adduct, 2'-deoxy-O6-[4-oxo-4-(3-pyridyl)butyl]guanosine (O6-pobdG), was prepared. This adduct was stable at pH 7.0 for greater than 13 days and to neutral thermal hydrolysis conditions (pH 7.0, 100 degrees C, 30 min). Under mild acid hydrolysis conditions (0.1 N HCl, 80 degrees C), O6-pobdG was depurinated to yield O6-[4-oxo-4-(3-pyridyl)butyl]guanine (O6-pobG). O6-pobdG was hydrolyzed to 4-hydroxy-1-(3-pyridyl)-1-butanone and guanine under strong acid hydrolysis conditions (0.8 N HCl, 80 degrees C). O6-pobG was detected in 0.1 N HCl hydrolysates of DNA alkylated with the model pyridyloxobutylating agent 4-(acetoxymethylnitrosamino)-1-(3-[5-3H]pyridyl)-1-butanone ([5-3H]NNKOAc). When [5-3H]NNKOAc-treated DNA was incubated with either rat liver or recombinant human AGT, O6-pobG was removed, presumably a result of transfer of the pyridyloxobutyl group from the O6-position of guanine to AGT's active site.

Animals↗
PubMed1990

Investigations of metabolic precursors to hemoglobin and DNA adducts of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

Levels of DNA and/or hemoglobin pyridyloxobutylation were compared in A/J mice or F344 rats treated with a single dose of [5-3H]4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone ([5-3H]NNK), [5-3H]4-hydroxy-1-(3-pyridyl)-1-butanone ([5-3H]4-HPB) or [5-3H]4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone ([5-3H]NNKOAc), a compound that generates the proposed pyridyloxobutylating agent in situ upon esterase hydrolysis. The lung and liver DNA samples isolated from A/J mice treated with the various compounds were subjected to acid hydrolysis and the hydrolysates were analyzed for the presence of [5-3H]4-HPB. No detectable levels were found in the lung DNA isolated from [5-3H]4-HPB-treated animals, whereas significant amounts of [5-3H]4-HPB were released from lung and liver DNA isolated from [5-3H]NNK- and [5-3H]NNKOAc-treated mice. The levels of total binding and [5-3H]4-HPB released from the globin isolated from these animals showed a similar trend. That is, low binding levels were detected in the globin isolated from [5-3H]4-HPB-treated animals and significantly higher levels of binding were detected in the globin from the [5-3H]NNKOAc- and [5-3H]NNK-treated animals. Comparable findings were obtained in the rat experiments. These studies clearly demonstrate that methyl hydroxylation of NNK leads to a species that is capable of reacting covalently with nucleophiles in DNA and protein. Thus, the levels of 4-HPB released from DNA and globin can be attributed to the activation of NNK and not to the direct binding of 4-HPB.

Animals↗
PubMed1996

Metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) by human cytochrome P450 1A2 and its inhibition by phenethyl isothiocyanate.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent tobacco-specific nitrosamine in animals and has been suggested to play a role in human tobacco-related cancers. Our previous study demonstrated that cytochrome P450 (P450) 1A2 catalyzes the formation of 4-hydroxy-1-(3-pyridyl)-1-butanone (keto alcohol) (an alpha-hydroxylation product) from NNK in human liver microsomes. Phenethyl isothiocyanate (PEITC) inhibits NNK tumorigenesis by blocking the activation of NNK. The purpose of the present study was to elucidate the mechanism of inhibition of P450 1A2-catalyzed NNK activation by PEITC. Human P450 1A2 was expressed in Escherichia coli and purified to homogeneity. In a reconstituted system, P450 1A2 catalyzed the formation of keto alcohol and 4-oxo-1-(3-pyridyl)-1-butanone (keto aldehyde) from NNK, with the keto alcohol being the major metabolite. The apparent Km and Vmax values for keto alcohol formation was 380 microM and 1.7 nmol/min/nmol P450, respectively. For the tobacco-specific nitrosamine N-nitrosonornicotine (NNN), P450 1A2 catalyzed the formation of the derived 4-hydroxy-4-(3-pyridyl)butyric acid (hydroxy acid),4-oxo-4-(3-pyridyl)butyric acid (keto acid) and keto alcohol. In comparison to NNK, NNN had a lower rate of oxidation with P450 1A2. PEITC decreased the formation of the NNK-derived keto alcohol in a concentration-dependent manner, with an IC50 value of 0.14 microM. PEITC was a competitive inhibitor of P450 1A2, exhibiting a Ki value of 0.18 microM. Preincubation of PEITC with NADPH in the reconstituted system resulted in a further decrease (25%) in the catalytic activity of P450 1A2, suggesting that there is a slow metabolism-dependent inhibition of P450 1A2 by PEITC. The formation of keto aldehyde and keto alcohol was inhibited by PEITC in human liver microsomes with IC50 values of 9.5 and 4.6 microM respectively. Methoxyresorufin O-dealkylase activity, a marker for P450 1A2, was decreased by PEITC in a concentration-dependent manner, with an IC50 of 0.34 microM. The results suggest that PEITC itself is a potent inhibitor of P450 1A2 and that a metabolite(s) of PEITC can also inhibit P450 1A2. We conclude that PEITC may be an effective inhibitor of the carcinogenicity or toxicity of chemicals that are activated by P450 1A2.

Carcinogens↗
PubMed1987

Effects of alpha-deuterium substitution on the tumorigenicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in F344 rats.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its analogues substituted with deuterium at the methylene carbon, 4,4-dideutero-4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone [4,4-D2)NNK], and the methyl carbon, 4-(trideuteromethylnitrosamino)-1-(3-pyridyl)-1-butanone [(CD3)NNK], adjacent to the N-nitroso group were tested for tumorigenicity in F344 rats. Each compound was administered by 60 s.c. injections over a 20-week period such that the total doses were either 1.0 or 0.33 mmol/kg. The experiment was terminated after 104 weeks. Survival of the rats treated with the higher dose of (4,4-D2)NNK was significantly less than survival in the groups treated with the same doses of NNK or (CD3)NNK. Target tissues were liver, lung and nasal cavity for all three compounds. The higher dose of (4,4-D2)NNK induced higher numbers of nasal tumors and malignant nasal tumors than did NNK. The lower dose of (4,4-D2)NNK induced a higher number of nasal tumors than did NNK. No other significant differences in tumor incidence were observed. The results suggest that 4-(3-pyridyl)-4-oxobutylation of DNA might be important in induction of nasal cavity tumors by NNK.

Animals↗
PubMed1988

DNA and hemoglobin alkylation by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and its major metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in F344 rats.

Alkylation of DNA and hemoglobin was compared in male F344 rats given a single s.c. injection of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), or its major metabolite formed by carbonyl reduction, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL). In hepatic DNA, levels of 7-methylguanine and O6-methylguanine formed from NNK 1-48 h after treatment were similar to those formed from NNAL. In nasal mucosa and lung DNA, levels of 7-methylguanine and O6-methylguanine were somewhat higher after treatment with NNK than with NNAL. Acid hydrolysis of hepatic DNA, isolated from rats treated with either [5-3H]NNK or [5-3H]NNAL, gave 180 +/- 48 or 120 +/- 23 mumol/mol guanine, respectively, of 4-hydroxy-1-(3-pyridyl)-1-butanone. Basic hydrolysis of globin isolated from rats treated with either [5-3H]NNK or [5-3H]NNAL gave 4.1 +/- 0.7 or 2.0 +/- 0.1 pmol/mg, respectively of 4-hydroxy-1-(3-pyridyl)-1-butanone. These results indicate that NNAL is not a detoxification product of NNK, since treatment of rats with NNAL results in modifications of DNA which are qualitatively and quantitatively similar to those observed upon treatment with NNK. Alkylation of DNA and globin by NNAL may result mainly from its metabolic reconversion to NNK.

Alkylating Agents↗
PubMed1989

Studies on gastric antiulcer active agents. III. Synthesis of 1-substituted 4-(5-tetrazolyl)thio-1-butanones and related compounds.

Many 1-substituted 4-(5-tetrazolyl)thio-1-butanones were synthesized and tested for antiulcer activity against acetic acid-induced gastric ulcer in rats. These compounds were prepared by the reaction of 5-mercaptotetrazoles and 4-halogeno-1-butanones. Among them, 1-cyclohexyl-4-(1-phenyl-5-tetrazolyl)thio-1-butanone (VIIIp) was found to have the most potent activity. The structure-activity relationships are discussed.

Animals↗
PubMed2001

Interactions between methylating and pyridyloxobutylating agents in A/J mouse lungs: implications for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung tumorigenesis.

The tobacco-specific nitrosamine, 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone, is activated to lung DNA methylating and pyridyloxobutylating intermediates. It is likely that both pathways play a role in lung tumor initiation by this nitrosamine. Previous studies indicated that O(6)-methylguanine (O(6)-mG) persistence is critical for lung tumor formation in A/J mice. The model pyridyloxobutylating agent, 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc), enhanced the tumorigenic activity of a model methylating agent, acetoxymethylmethylnitrosamine (AMMN), presumably by increasing O(6)-mG persistence in lung DNA. We have been testing the hypothesis that the pyridyloxobutylation pathway increases the mutagenic activity of the DNA methylation pathway by preventing the repair of O(6)-mG by O(6)-alkylguanine-DNA alkyltransferase (AGT). In this study, we report that NNKOAc depletes AGT in lungs but not livers of A/J mice. The consequences of AGT depletion by NNKOAc were then compared with those observed with a known AGT inhibitor, O(6)-benzylguanine (O(6)-bG). NNKOAc and O(6)-bG had similar effects on the levels of AMMN-derived O(6)-mG at 4 and 96 h postinjection. This increase in O(6)-mG levels correlated to increased lung tumor multiplicity in animals simultaneously treated with AMMN (0.75 or 1 micromol) and NNKOAc or O(6)-bG. Only NNKOAc significantly increased lung tumor multiplicity at doses of 0.25 or 0.5 micromol AMMN. The results from these studies indicate that the pyridyloxobutylating agent, NNKOAc, can influence the tumorigenic activity of methylating agents in two ways. At low AMMN doses, the increase in tumor multiplicity is dominated by the additive tumorigenic properties of AMMN and NNKOAc. At higher AMMN doses, NNKOAc appears to enhance the tumorigenic activity of AMMN through enhanced depletion of the repair protein, AGT, leading to increased O(6)-mG persistence. It is likely that similar interactions are important for the organospecific effects of 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone.

Alkylating Agents↗
PubMed1992

Metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in human lung and liver microsomes and cytochromes P-450 expressed in hepatoma cells.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a potent tobacco-specific carcinogen in animals, has been linked to tobacco-related cancers in humans. The cytochrome(s) P-450 (P-450) responsible for the metabolic activation of NNK in humans has not been identified. The present work investigated the ability of human lung and liver microsomes and 12 forms of human P-450, expressed in Hep G2 (hepatoma) cells, to metabolize NNK. Of the 12 P-450 forms, P-450 1A2 had the highest activity in catalyzing the conversion of NNK to the keto alcohol, 4-hydroxy-1-(3-pyridyl)-1-butanone. P-450s 2A6, 2B7, 2E1, 2F1, and 3A5 also had measurable activities in the formation of keto alcohol. The apparent Km and Vmax for the formation of keto alcohol in the P-450 1A2-expressed Hep G2 cell lysate were 309 microM and 55 pmol/min/mg protein, respectively. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol, a reductive product, was the major metabolite formed, whereas the formation of keto alcohol and its aldehyde and acid derivatives (all alpha-hydroxylation products) constituted approximately 1% of the initial amount of NNK in P450-expressed Hep G2 cell lysate. A similar metabolite pattern was observed with human lung or liver microsomes. In human lung microsomes, the apparent Kms for the formation of 4-hydroxy-4-(3-pyridyl)butyric acid, 4-oxo-1-(3-pyridyl)-1-butanone, NNK-N-oxide, and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol were 526, 653, 531, and 573 microM, respectively; the formation of keto alcohol was not observed. For human lung microsomes, there was no sex-related difference in NNK metabolism. Carbon monoxide (90% atmosphere) significantly inhibited the metabolism of NNK in human lung and liver microsomes. 7,8-Benzoflavone, an inhibitor of P-450s 1A1 and 1A2, had no effect on NNK metabolism in human lung microsomes but decreased the formation of keto alcohol by 47% in human liver microsomes. Similarly, antibodies against human P-450s 1A2 and 2E1 decreased keto alcohol formation by 42% and 53%, respectively, in human liver microsomes but did not affect NNK metabolism in lung microsomes. Inhibitory antibodies against P-450s 2A1, 2C8, 2D1, or 3A4 had little or no effect on the metabolism of NNK in human liver or lung microsomes.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibodies↗
PubMed1991

Quantitation of microsomal alpha-hydroxylation of the tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is activated to DNA alkylating species via two different alpha-hydroxylation pathways. Methylene hydroxylation leads to DNA methylation, whereas methyl hydroxylation yields DNA pyridyloxobutylation. We have developed a high-pressure liquid chromatography assay utilizing radiochemical detection that permits the determination of the extent of metabolism through each pathway in microsomal preparations. Levels of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) were used to measure the extent of methyl hydroxylation, whereas levels of the aldehyde, 4-oxo-1-(3-pyridyl)-1-butanone (OPB), were used to quantify the extent of methylene hydroxylation. Incubations of [5-3H]NNK with microsomes and cofactors were conducted in the presence of 5 mM sodium bisulfite to trap the reactive OPB. The inclusion of bisulfite did not affect the rate of NNK metabolism. Trapping the aldehyde also inhibited its further oxidation to the corresponding acid or reduction to HPB. Furthermore, the conversion of HPB to OPB made only a minor contribution to the OPB levels under our incubation conditions. Analysis of incubation mixtures containing [5-3H]NNK, cofactors, and either A/J mouse liver or lung microsomes demonstrated that OPB was a significant metabolite of NNK. The OPB:HPB ratio was greater in liver (1.5) than in lung (0.2-1) microsomal preparations. Apparent Km values for OPB and HPB formation in lung microsomes were 23.7 and 3.6 microM, respectively, whereas the corresponding values for liver microsomes were 19.1 and 73.8 microM, respectively. These data are consistent with the involvement of more than one cytochrome P-450 isozyme in the activation of NNK to DNA reactive species.

Animals↗
PubMed1990

Metabolism and DNA single strand breaks induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and its analogues in primary culture of rat hepatocytes.

Previous studies have shown that the tobacco specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is hepatocarcinogenic and results in alkylation of hepatic DNA in F344 rats. In this study, we have characterized the metabolism of NNK in cultured rat hepatocytes and have established the relationship between various metabolic pathways and the induction of DNA single strand breaks. DNA fragmentation by NNK and two other related N-nitrosamines, N'-nitrosonornicotine and nitrosodimethylamine, were compared. Metabolism of [5-3H]NNK (4.5 microM) by carbonyl reduction, alpha-carbon hydroxylation, and pyridine N-oxidation was linear from 0 to 6 h and with 0 to 2 x 10(6) hepatocytes. Using the alkaline elution assay, we observed that NNK induces DNA single strand breaks (SSB) in a dose (1-10 mM) and time (0.5-6 h) dependent manner. SSB induced by NNK (5 mM; rate of elution between 3 and 9 h, 0.117) are rejoined partially within 2 h (rate, 0.039) and totally 12 h after exposure. NNK N-oxide (5 mM) produces a smaller number of SSB (rate, 0.017) than NNK (rate, 0.105) suggesting that pyridine N-oxidation of NNK is a deactivation pathway. Hydrolysis of carbethoxy-nitrosaminomethane and 4-(N-carbethoxy-N-nitrosamino)-1-(3-pyridyl)butanone yields methyldiazohydroxide and 4-(3-pyridyl)-4-oxobutyl-diazohydroxide, respectively. These two alkylating intermediates are generated during alpha-carbon hydroxylation of NNK. After treatment of hepatocytes with 5 microM carbethoxynitrosaminomethane and 1 mM 4-(N-carbethoxyl-N-nitrosamino)-1-(3-pyridyl)butanone, the rates of DNA elution were 0.092 and 0.120, respectively. Carbonyl reduction of NNK leads to 4-(methylnitrosamino)-1-(3-pyridyl)butan-1-ol (NNAl). Reaction of NNK with methyl magnesium iodide gives 1-MeNNAl with 82% yield, NNAl but not 1-MeNNAl can be reoxidized to NNK. Both 5 mM NNAl (rate, 0.073) and 5 mM 1-MeNNAl (rate, 0.054) induce SSB indicating that NNAl does not require reconversion to NNK to be activated to DNA damaging intermediates. alpha-Methylene hydroxylation of NNK results in an equimolar formation of methyldiazohydroxide and 4-oxo-4-(3-pyridyl)-butanal. This aldehyde, at a concentration of 1 mM, induces the same frequency of SSB (rate, 0.116) as 5 mM NNK (0.105) and could possibly play a role in the carcinogenicity of NNK.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗