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PubMedDate not supplied

In vitro metabolism of phenylacetone, phenyl-2-butanone, and 3-methyl-1-phenyl-2-butanone by rabbit liver preparations.

Phenylacetone, phenyl-2-butanone, and 3-methyl-1-phenyl-2-butanone were incubated with rabbit liver 9000g supernatant fraction for 30 and 60 min to yield primarily the alcohols, plus small amounts of 1,2-glycols and ketols (2-one-1-ols). Phenylacetone incubations produced small amounts of benzoic acid, which was confirmed as a true metabolite by incubation of 2H5 (phenyl-labeled) phenylacetone and corresponding recovery of phenyl-labeled benzoic acid. No benzoic acid was detectable from incubations of phenyl-2-butanone and 3-methyl-1-phenyl-2-butanone. 3-Methyl-1-phenyl-2-butanone yielded eight metabolites in addition to the corresponding alcohol, and structures are proposed for some of these metabolic products.

Acetone↗
PubMedDate not supplied

Glucuronidation of 4-((hydroxymethyl)nitrosamino)-1-(3-pyridyl)-1-butanone, a metabolically activated form of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, by phenobarbital-treated rats.

In the rat, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induces lung tumors independent of the route of administration. To exert its carcinogenic potential, NNK must be metabolically activated. Like most nitrosamines NNK is activated by alpha-hydroxylation. The striking tissue specificity of tumor induction by nitrosamines has been primarily attributed to the efficient alpha-hydroxylation of a particular nitrosamine by its target tissue. Two other factors which may contribute to this are the following: the relative capacity of different tissues to detoxify the alpha-hydroxynitrosamine and the preferential uptake of the active metabolite by the target tissue. In the present study we report the characterization of the O-glucuronide of 4-((hydroxymethyl)nitrosamino)-1-(3-pyridyl)-1-butanone (alpha-hydroxymethylNNK-Gluc). The formation of this glucuronide could either serve as a detoxification pathway or provide a stable transport form of the alpha-hydroxylated metabolite. In addition, the metabolism of NNK to a glucuronide of the alpha-hydroxynitrosamine provides the first definitive evidence for the formation of alpha-hydroxymethylNNK. alpha-HydroxymethylNNK-Gluc was present in the urine of rats treated with phenobarbital (PB) and NNK. It was also formed by hepatocytes from PB-treated rats, accounting for 4% of the total metabolites in the media following incubation with 1 microM NNK. The data that support the identity of this metabolite as alpha-hydroxymethylNNK-Gluc are as follows. (1) Incubation of this metabolite with beta-glucuronidase resulted in the quantitative release of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB), the decomposition product of alpha-hydroxymethylNNK.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylation↗
PubMedDate not supplied

Identification of 4-(methylnitrosamino)-1-[3-(6-hydroxypyridyl)]-1-butanone as a urinary metabolite of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in rodents.

A previously unknown urinary metabolite of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was identified as 4-(methylnitrosamino)-1-[3-(6-hydroxypyridyl)]-1-butanone (6-hydroxyNNK). The metabolite was initially isolated from rat urine. On the basis of its MS and NMR, it was either a 4- or 6-hydroxypyridyl derivative of NNK. Model compounds were synthesized to distinguish between these possibilities; the results indicated that the metabolite was 6-hydroxyNNK. This was confirmed by independent synthesis; the spectral and chromatographic properties of 6-hydroxyNNK were the same as those of the metabolite. F-344 rats and A/J mice treated with 100 mg/kg NNK excreted approximately 1% of urinary metabolites as 6-hydroxyNNK; it was not detected as a sulfate or glucuronide conjugate. This is the first example of a pyridyl-hydroxylated metabolite of a tobacco-specific nitrosamine. On the basis of comparison to published data on other pyridine derivatives, 6-hydroxyNNK may be formed by bacterial metabolism. The potential utility of 6-hydroxyNNK as a dosimeter of human uptake of NNK is discussed.

Animals↗
PubMed2001

Mutagenesis induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N-nitrosonornicotine in lacZ upper aerodigestive tissue and liver and inhibition by green tea.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and nitrosonornicotine (NNN) were administered to lacZ mice (MutaMouse) at equal concentrations in drinking water (2 weeks at 0.1 followed by 2 weeks at 0.2 mg/ml) over a 4 week period, for a total estimated dose of 615 mg/kg) and mutagenesis in a number of organs was measured. For mutagenesis induced by NNK the potency order was: liver > lung> pooled oral tissues kidney > esophagus > tongue. The mutant fraction varied from approximately 6 to 40 mutants per 10(-5) plaque forming units This corresponds to approximately 2-13 times the background levels. A somewhat different pattern was observed with NNN, where the order was liver > esophagus oral tissue approximately tongue > lung > kidney. The potency of NNK was about twice that of NNN in liver and lung, but somewhat less in aerodigestive tract tissue. When compared with results previously obtained for a similar administered dose of benzo[a]pyrene, NNK was approximately 10-100% as mutagenic in the corresponding organs. Reported target organs for carcinogenesis by NNN and NNK in rodents were targets for mutagenesis, but mutagenesis was also observed at other sites, suggesting that these sites are initiated. The effect of green tea consumption on mutagenesis by NNK was also investigated. Green tea reduced mutagenesis by approximately 15-50% in liver, lung, pooled oral tissue and esophagus.

Animals↗
PubMed1979

Potentiation of CCl4 hepatotoxicity in rats by a metabolite of 2-butanone: 2,3-butanediol.

The role of ketaone metabolism in 2-butanone-induced potentiaion of carbon tetrachloride (CCl4) hepatotoxicity was studied in rats. The blood concentrations of 2-butanol, 3-hydroxy-2-butanone and 2,3-butanediol detected 4 h after dosing were 3.2 mg/100 ml, 2.4 mg/100 ml and 8.6 mg/100 ml, respectively. Eighteen hours after 2-butanone, the concentration of 2,3-butanediol rose to 25.6 mg/100 ml, while the concentrations of 2-butanol and 3-hydroxy-2-butanone declined to 0.6 mg/100 ml and 1.4 mg/100 ml, respectively. A 16-h pretreatment with either 2-butanone (2.1 ml/kg, p.o.) or 2,3-butanediol (2.12 ml/kg, p.o.) markedly enhanced the hepatotoxic response to CCl4 (0.1 ml/kg, i.p.), as measured by serum glutamic pyruvic transaminase activity and hepatic triglyceride content. In vivo, limited formation of 3-hydroxy-2-butanone occurred after this dose of 2,3-butanediol. These data suggest that the production of 3-hydroxy-2-butanone and 2,3-butanediol via 2-butanone metabolism may participate in the augmented necrogenic effect of CCl4 seen after pretreatment with 2-butanone.

Alanine Transaminase↗
PubMed1987

Formation of hemoglobin adducts upon treatment of F344 rats with the tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N'-nitrosonornicotine.

[5-3H]4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone ([5-3H]NNK), [C3H3]NNK, and [5-3H]N'-nitrosonornicotine ([5-3H]NNN) were administered to F344 rats by i.p. injection. Levels of tritium present per milligram globin, 24 h after treatment were 720 fmol (0.1% of dose) for [5-3H]NNK, 640 fmol for [C3H3]NNK, and 370 fmol for [5-3H]NNN. Tritium was detectable in globin 7-8 weeks after treatment with [5-3H]NNK or [5-3H]NNN. Approximately 10-15% of the bound tritium in the globin of rats treated with [5-3H]NNK was released upon incubation of the globin with dilute NaOH or HCl. The released material was identified as 4-hydroxy-1-(3-pyridyl)-1-butanone; it was detectable in globin for 6 weeks (t1/2 = 9.1 days) after administration of [5-3H]NNK. 4-Hydroxy-1-(3-pyridyl)-1-butanone was also formed upon NaOH treatment of globin isolated from rats injected with [5-3H]NNN or [5-3H]4-(carbethoxynitrosamino)-1-(3-pyridyl)-1-butanone. The formation of 4-hydroxy-1-(3-pyridyl)-1-butanone under these conditions is consistent with a mechanism by which 4-(3-pyridyl)-4-oxobutyldiazohydroxide is produced upon metabolic alpha-hydroxylation of NNK or NNN and binds to globin of hemoglobin, yielding an adduct which is readily hydrolyzed by acid or base. Support for this mechanism was obtained by in vitro experiments. Levels of 4-hydroxy-1-(3-pyridyl)-1-butanone released upon base treatment of globin were 50 times greater after incubation of rat hemoglobin with [5-3H]4-(carbethoxynitrosamino)-1-(3-pyridyl)-1-butanone than with either [5-3H]NNK or [5-3H]4-hydroxy-1-(3-pyridyl)-1-butanone. The results of this study suggest methods that might be applicable for assessing the molecular dosimetry of NNK and NNN in individuals exposed to tobacco and tobacco smoke.

Animals↗
PubMed1989

Effect of 2-butanol and 2-butanone on rat hepatic ultrastructure and drug metabolizing enzyme activity.

The effect of a single oral dose of 2-butanol (2.2 ml/kg) or 2-butanone (1.87 ml/kg) on hepatic ultrastructure and drug-metabolizing enzyme activity was studied in the rat. A 135-197% increase in acetanilide hydroxylase activity was found in rats sacrificed 12-40 h after dosing with 2-butanol or 2-butanone. A 40-h pretreatment with 2-butanone produced a 155% increase in aminopyrine N-demethylase activity. NADPH-cytochrome c reductase activity and the concentrations of cytochromes P-450 and b5 were largely unaltered 2-40 h after dosing with either agent. Electron microscopic examination of hepatocytes from rats sacrificed 16 h after 2-butanol or 2-butanone revealed a marginal increase in the prevalence of smooth endoplasmic reticulum. However, by 40 h, there was a marked proliferation of the smooth endoplasmic reticulum and reduction in rough endoplasmic reticulum in response to both agents. The most marked potentiation of CCl4 hepatotoxicity occurred when rats were pretreated with 2-butanol or 2-butanone 16 h before CCl4 administration. The coincidental finding of maximal CCl4-induced hepatic injury and elevation of microsomal xenobiotic activity within the same time frame following 2-butanol or 2-butanone supports the hypothesis that aliphatic alcohols and ketones potentiate CCl4 hepatotoxicity by enhancing biotransformation of the halocarbon to cytotoxic metabolites.

Aminopyrine N-Demethylase↗
PubMedDate not supplied

Solvolysis of model compounds for alpha-hydroxylation of N'-nitrosonornicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone: evidence for a cyclic oxonium ion intermediate in the alkylation of nucleophiles.

N'-Nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), two potent tobacco-specific carcinogens, have been previously found to pyridyloxobutylate DNA. The adducts were found to be unstable and have not been fully characterized. In order to gain an understanding of the chemistry of the pyridyloxobutylating species, five model pyridyloxobutylating agents have been solvolyzed and the products identified. 4-[(Acetoxymethyl)-nitrosamino]-1-(3-pyridyl)-1-butanone (3), 4-(carbethoxynitrosamino)-1-(3-pyridyl)-1-butanone (4), 4-oxo-4-(3-pyridyl)-1-butyl p-toluenesulfonate (16), 2-chloro-2-(3-pyridyl)-2,3,4,5-tetrahydrofuran (17), and 4-[(acetoxymethyl)nitrosamino]-1-(3-pyridyl)-1-butanol (20) were solvolyzed in buffer and in buffer containing 20% MeOH. The solvolyses of 16 and 17 in H2O produced only 4-hydroxy-1-(3-pyridyl)-1-butanone (7). In the presence of 20% MeOH, 7 and 2-methoxy-2-(3-pyridyl)-2,3,4,5-tetrahydrofuran (12) were produced from 16 and 17 in a 4:1 ratio. The solvolysis of 3 and 4 in the presence of esterase gave similar products. 4-Methoxy-1-(3-pyridyl)-1-butanone (8) was not detected as a product. In the absence of MeOH, compound 7, 3-pyridyl cyclopropyl ketone (10), and 1-(3-pyridyl)-but-2-en-1-one (18) were observed. In the presence of MeOH, 12 was also formed and the ratio of 7 to 12 was again about 4:1. The esterase-catalyzed hydrolysis of 20 yielded 1-(3-pyridyl)-1,4-butanediol (22), 1-(3-pyridyl)-1,3-butanediol (27), 1-(3-pyridyl)-but-3-en-1-ol (25), 1-(3-pyridyl)but-2-en-1-ol (26), and 2-(3-pyridyl)-2,3,4,5-tetrahydrofuran (24).(ABSTRACT TRUNCATED AT 250 WORDS)

Alkylation↗
PubMed1983

Effects of alpha-deuterium substitution on the mutagenicity of 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK).

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a carcinogenic tobacco specific nitrosamine, can be converted to electrophilic diazohydroxide intermediates by metabolic hydroxylation of either the methylene carbon (carbon 4) or the methyl carbon attached to the nitrosamine group. To investigate the relative importance of these two processes in NNK mutagenesis, we synthesized 4,4-dideutero-4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone([4,4,-D2]NNK) and 4-(trideuteromethylnitrosamino)-1-(3-pyridyl)-1-butanone ([CD3] NNK), and evaluated their mutagenic activities in Salmonella typhimurium tester strains. In the presence of Aroclor induced rat liver 9000 g supernatant, NNK and [4,4-D2]NNK had comparable mutagenic activities towards S. typhimurium TA 1535 and TA 100, but [CD3]NNK was inactive in both strains. These results suggest that hydroxylation of the methyl group of NNK is more important than hydroxylation of carbon 4 in its activation to a mutagen. To test the inherent mutagenicity of 4-oxo-4-(3-pyridyl)butyldiazohydroxide and methyldiazohydroxide which would be formed by methyl hydroxylation or carbon 4 hydroxylation, respectively, we compared the mutagenicities, without activation, of the corresponding model compounds, 4-(carbethoxynitrosamino)-1-(3-pyridyl)-1-butanone and carbethoxynitrosaminomethane (methylnitrosourethane). Both compounds were highly mutagenic toward S. typhimurium TA 1535 and TA 100, but at doses of 4 x 10(-3) to 4 x 10(-4) mumol/plate, only 4-(carbethoxynitrosamino)-1-(3-pyridyl)-1-butanone was mutagenic. These results are consistent with those obtained with the deuterium substituted compounds and indicate the importance of 4-oxo-4-(3-pyridyl)butylation of DNA in NNK mutagenesis.

Deuterium↗
PubMed1976

The participation of 2-butanone in 2-butanol-induced potentiation of carbon tetrachloride hepatotoxicity.

The role of alcohol metabolism in 2-butanol-induced potentiation of carbon tetrachloride (CCl4) hepatotoxicity was studied in rats. Animals were sacrificed at various times after the administration of 2-butanol (2.2 ml/kg p.o.) for the determination of blood 2-butanol and 2-butanone concentrations by gas chromatographic analysis. 2-butanol exhibited an apparent elimination half-life of 2.5 hours. With the decline of 2-butanol concentrations, there was a rise in 2-butanone blood concentrations with 43 mg/100 ml detected at 1 hour and a maximum of 105 mg/100 ml detected 4 hours after the administration of the alcohol. A 16-hour pretreatment with either 2-butanol (2.2 ml/kg p.o.) or 2-butanone (1.87 ml/kg p.o.) markedly enhanced the hepatotoxic response of CCl4 (0.1 ml/kg i.p.) as measured by serum glutamic pyruvic transaminase activity, hepatic glucose-6-phosphatase activity and triglyceride content. The enhanced hepatotoxicity produced by 2-butanol was not significantly different from that produced by 2-butanone. The potentiation of CCl4 hepatotoxicity by both agents was substantiated morphologically. The results indicate that 2-butanone production via the oxidation of 2-butanol appears to contribute to the marked response of 2-butanol.

Alanine Transaminase↗
PubMed1992

Metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone by inducible and constitutive cytochrome P450 enzymes in rats.

The tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induces tumor formation in the liver, lung, nasal cavity, and pancreas of rats. Metabolic activation is required for the tumorigenicity of this compound. The involvement of cytochrome P450 enzymes in NNK bioactivation was investigated in rats by studies with chemical inducers and antibodies against P450s. Liver microsomal enzymes catalyzed the formation of 4-oxo-1-(3-pyridyl)-1-butanone (keto aldehyde), 4-hydroxy-1-(3-pyridyl)-1-butanone (keto alcohol), 4-(methylnitrosamino)-1-(3-pyridyl-N-oxide)-1-butanone (NNK-N-oxide), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) from NNK. When the activity was expressed on a per nanomole P450 basis, treatments of rats with 3-methylcholanthrene (MC), phenobarbital (PB), pregnenolone 16-alpha-carbonitrile (PCN), Aroclor 1254 (AR), safrole (SA), and isosafrole (ISA) increased the keto aldehyde formation in liver microsomes 2.0-, 2.4-, 3.8-, 2.5-, 2.1-, and 1.8-fold, respectively; PB, AR, SA, and ISA increased the keto alcohol formation 1.7-, 1.3-, 2.0-, and 1.3-fold, respectively. The extents of induction were more pronounced when expressed on a per milligram protein basis, due to the higher microsomal P450 contents in the induced microsomes. The formation of NNK-N-oxide was markedly increased by PB and PCN and slightly increased by AR, SA, and ISA. However, the formation of NNAL, the major metabolite due to carbonyl reduction, was not increased by the treatments but was decreased by AR, ISA, and acetone (AC). The kinetic parameters of NNK metabolism by control, MC-, PB-, and PCN-induced liver microsomes were obtained. A panel of monoclonal (anti-1A1, -2B1, -2C11, and -2E1) and polyclonal (anti-1A2, -2A1, and -3A) antibodies were used to assess the involvement of constitutive hepatic P450 enzymes in NNK metabolism. Keto aldehyde formation was inhibited by anti-1A2 and anti-3A (about 15%) but not by others; the formation of keto alcohol was inhibited by anti-1A2, anti-2A1, and anti-3A (by 13-26%). In incubations with lung microsomes, the formation of keto aldehyde, keto alcohol, NNK-N-oxide, and NNAL were observed. With nasal mucosa microsomes, however, only keto aldehyde and keto alcohol formation were appreciable. SA and AC significantly decreased NNK metabolism in lung and nasal mucosa microsomes.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1997

Enzymes involved in the bioactivation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in patas monkey lung and liver microsomes.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent tobacco-specific carcinogen in animals. Our previous studies indicated that there are differences between rodents and humans for the enzymes involved in the activation of NNK. To determine if the patas monkey is a better animal model for the activation of NNK in humans, we investigated the metabolism of NNK in patas monkey lung and liver microsomes and characterized the enzymes involved in the activation. In lung microsomes, the formation of 4-oxo-1-(3-pyridyl)-1-butanone (keto aldehyde), 4-(methylnitrosamino)-1-(3-pyridyl-N-oxide)-1-butanone (NNK-N-oxide), 4-hydroxy-1-(3-pyridyl)-1-butanone (keto alcohol), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) was observed, displaying apparent Km values of 10.3, 5.4, 4.9, and 902 microM, respectively. NNK metabolism in liver microsomes resulted in the formation of keto aldehyde, keto alcohol, and NNAL, displaying apparent Km values of 8.1, 8.2, and 474 microM, respectively. The low Km values for NNK oxidation in the patas monkey lung and liver microsomes are different from those in human lung and liver microsomes showing Km values of 400-653 microM, although loss of low Km forms from human tissue as a result of disease, surgery or anesthesia cannot be ruled out. Carbon monoxide (90%) significantly inhibited NNK metabolism in the patas monkey lung and liver microsomes by 38-66% and 82-91%, respectively. Nordihydroguaiaretic acid (a lipoxygenase inhibitor) and aspirin (a cyclooxygenase inhibitor) decreased the rate of formation of keto aldehyde and keto alcohol by 10-20 % in the monkey lung microsomes. Alpha-Napthoflavone and coumarin markedly decreased the oxidation of NNK in monkey lung and liver microsomes, suggesting the involvement of P450s 1A and 2A6. An antibody against human P450 2A6 decreased the oxidation of NNK by 12-16% and 22-24% in the patas monkey lung and liver microsomes, respectively. These results are comparable to that obtained with human lung and liver microsomes. Coumarin hydroxylation was observed in the patas monkey lung and liver microsomes at a rate of 16 and 4000 pmol/min/mg protein, respectively, which was 5-fold higher than human lung and liver microsomes, respectively. Immunoblot analysis demonstrated that the P450 2A level in the individual patas monkey liver microsomal sample was 6-fold greater than in an individual human liver microsomal sample. Phenethyl isothiocyanate, an inhibitor of NNK activation in rodents and humans, decreased NNK oxidation in the monkey lung and liver microsomes displaying inhibitor concentration resulting in 50% inhibition of the activity (IC50) values of 0.28-0.8 microM and 4.2-6.8 microM, respectively. The results demonstrate the similarities and differences between species in the metabolic activation of NNK. The patas monkey microsomes appear to more closely resemble human microsomes than mouse or rat enzymes and may better reflect the activation of NNK in humans.

Animals↗
PubMed1992

Biosynthesis of riboflavin: cloning, sequencing, and expression of the gene coding for 3,4-dihydroxy-2-butanone 4-phosphate synthase of Escherichia coli.

3,4-Dihydroxy-2-butanone 4-phosphate is biosynthesized from ribulose 5-phosphate and serves as the biosynthetic precursor for the xylene ring of riboflavin. The gene coding for 3,4-dihydroxy-2-butanone 4-phosphate synthase of Escherichia coli has been cloned and sequenced. The gene codes for a protein of 217 amino acid residues with a calculated molecular mass of 23,349.6 Da. The enzyme was purified to near homogeneity from a recombinant E. coli strain and had a specific activity of 1,700 nmol mg-1 h-1. The N-terminal amino acid sequence and the amino acid composition of the protein were in agreement with the deduced sequence. The molecular mass as determined by ion spray mass spectrometry was 23,351 +/- 2 Da, which is in agreement with the predicted mass. The previously reported loci htrP, "luxH-like," and ribB at 66 min of the E. coli chromosome are all identical to the gene coding for 3,4-dihydroxy-2-butanone 4-phosphate synthase, but their role had not been hitherto determined. Sequence homology indicates that gene luxH of Vibrio harveyi and the central open reading frame of the Bacillus subtilis riboflavin operon code for 3,4-dihydroxy-2-butanone 4-phosphate synthase.

Amino Acid Sequence↗
PubMed1990

Evaluation of cysteine adduct formation in rat hemoglobin by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and related compounds.

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) forms hemoglobin adducts in rats. Upon mild base treatment, 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) is released from this globin. HPB has been suggested as a dosimeter for exposure to and metabolic activation of tobacco-specific nitrosamines. The purpose of this study was 2-fold: (a) to determine whether cysteine adducts of NNK were precursors to HPB, and (b) to determine to what extent cysteine adducts accounted for the material bound to globin that is not released upon mild base hydrolysis. The chemistry of cysteine adduct formation was investigated by reacting N-acetyl-L-cysteine with three model compounds for pyridyloxobutylation by metabolically activated NNK: 4-(carbethoxynitrosamino)-1-(3-pyridyl)-1-butanone (1); 4-oxo-4-(3-pyridyl)-1-butylmethanesulfonate (2); and 4-iodo-1-(3-pyridyl)-1-butanone (3). Five adducts were isolated and characterized by their spectral properties and by independent syntheses: two diastereomers of N-acetyl-S-[1-methyl-3-oxo-3-(3-pyridyl)propyl]-L-cysteine (7a,b), N-acetyl-S-[4-oxo-4-(3-pyridyl)-1-butyl]-L-cysteine (9), and two diastereomers of N-acetyl-S-(2-[2-(3-pyridyl)]-2,3,4,5-tetrahydrofuranyl)-L-cystein e (11a,b). Only 11a,b produced HPB upon mild base treatment; however, the chemistry of this adduct did not support its role as a major precursor to HPB released upon base treatment of globin. The formation of adducts in rat hemoglobin was then examined by reacting it with tritium-labeled 1 [( 5-3H]1) or tritium-labeled 4-oxo-4-(3-pyridyl)-1-butyl p-toluenesulfonate [( 5-3H]4). The results demonstrated that the amino acids corresponding to 7a,b were present in hemoglobin reacted with [5-3H]1, accounting for 72% of the bound tritium. Amino acids corresponding to 9 were not detected in this globin. In contrast, hemoglobin reacted with [5-3H]4 contained the amino acid corresponding to 9 (15% of bound tritium), but not those corresponding to 7a,b. These results indicated that the alpha, beta-unsaturated ketone, 1-(3-pyridyl)-2-buten-1-one (5), played a major role in the hemoglobin binding of 1, but not of 4. Cysteine adducts were not detected in globin isolated from rats treated with [5-3H]NNK. The results of this study provide insights into the mechanisms of cysteine adduct formation in vitro by pryidyloxobutylating agents and indicate that these adducts are not formed in NNK-treated rats.

Animals↗
PubMed1990

Metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in mouse lung microsomes and its inhibition by isothiocyanates.

The tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induces lung tumors in rats, mice, and hamsters, and metabolic activation is required for the carcinogenicity. 2-Phenethyl isothiocyanate (PEITC), whose precursor gluconasturtiin (a glucosinolate) occurs in cruciferous vegetables, has been found to inhibit carcinogenesis by NNK. The purpose of the study was to investigate the enzymes involved in the metabolism of NNK in lung microsomes and to elucidate the mechanisms of inhibition of NNK metabolism by isothiocyanates. NNK metabolism in lung microsomes (isolated from female A/J mice) resulted in the formation of formaldehyde, 4-hydroxy-1-(3-pyridyl)-1-butanone (keto alcohol), 4-oxo-4-(3-pyridyl)butyric acid (keto acid), 4-(methylnitrosamino)-1-(3-pyridyl-N-oxide)-1-butanone, and 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanol, displaying apparent Km values of 5.6, 5.6, 9.2, 4.7, and 2540 microM, respectively. Higher Km values in the formation of formaldehyde and keto alcohol were also observed. When cytochrome P-450 inhibitors [2-(diethylamino)ethyl 2,2-diphenylpentenoate] hydrochloride (100 microM), carbon monoxide (90%), and 9-hydroxyellipticine (10 microM) were used, NNK metabolism was inhibited by each 70, 100, and 30%, respectively. Methimazole (1 mM), an inhibitor of the flavin-dependent monooxygenase, inhibited the formation of 4-(methyl-nitrosamino)-1-(3-pyridyl-N-oxide)-1-butanone and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol by 20%, but had no effect on the formation of keto alcohol. Inhibitory antibodies against cytochromes P-450IIB1 and -2, P-450IA1, and P-450IA2 inhibited the formation of keto alcohol by 25, 15, and 0%, respectively. Administration of PEITC at doses of 5 and 25 mumol/mouse 2 h before sacrifice produced a 40 and 70% decrease in microsomal NNK metabolism, respectively. PEITC and 3-phenylpropyl isothiocyanate exhibited a mixed type of inhibition, and the competitive component of inhibition had apparent Ki values of 90 and 30 nM, respectively. Preincubation of PEITC in the presence of a NADPH-generating system did not result in a further decrease in the formation of NNK metabolites, indicating that the metabolism of PEITC was not required for the inhibition. When a series of isothiocyanates with varying alkyl chain length (phenyl isothiocyanate, benzyl isothiocyanate, PEITC, 3-phenylpropyl isothiocyanate, and 4-phenylbutyl isothiocyanate) were used, the potency of the inhibition increased with the increase in chain length.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
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↗