Purification and characterization of oxidoreductases-catalyzing carbonyl reduction of the tobacco-specific nitrosamine 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) in human liver cytosol

1. Four enzymes were purified to homogeneity from human liver cytosol and were demonstrated to be responsible for carbonyl reduction of the tobacco-specific nitrosamine 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK). 2. Carbonyl reductase (EC 1.1.1.184), a member of the short-chain dehydrogenase reductase (SDR) superfamily, was compared with three isoenzymes of the aldo-keto reductase (AKR) superfamily in terms of enzyme kinetics, co-substrate dependence and inhibition pattern. 3. AKR1C1, 1C2 and 1C4, previously designated as dihydrodiol dehydrogenases (DD1, DD2 and DD4), showed lower K_m (0.2, 0.3 and 0.8?mm respectively) than did carbonyl reductase (7 mM), whereas carbonyl reductase exhibited the highest enzyme efficiency (V_max/K_m) for NNK. Multiplication of enzyme efficiencies with the relative quantities of individual enzymes in cytosol resulted in a rough estimate of their contributions to total alcohol metabolite formation. These were ~ 60% for carbonyl reductase, 20% each for AKR1C1 and 1C2, and 1% for AKR1C4. 4. Except for AKR1C4, the enzymes had a strong preference for NADPH over NADH, and the highest activities were measured with an NADPH-regenerating system. Carbonyl reductase activity was extensively inhibited by menadione, rutin and quercitrin, whereas medroxyprogesterone acetate, phenolphthalein and flufenamic acid were potent inhibitors of AKR1C1, 1C2 and 1C4. 5. In conclusion, cytosolic members of the SDR and AKR superfamilies contribute to reductive NNK detoxification in human liver, the enzymes responsible being carbonyl reductase and aldoketo reductases of the AKR1C subfamily.     

Purification and characterization of oxidoreductases-catalyzing carbonyl reduction of the tobacco-specific nitrosamine 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) in human liver cytosol

1. Four enzymes were purified to homogeneity from human liver cytosol and were demonstrated to be responsible for carbonyl reduction of the tobacco-specific nitrosamine 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK). 2. Carbonyl reductase (EC 1.1.1.184), a member of the short-chain dehydrogenase/reductase (SDR) superfamily, was compared with three isoenzymes of the aldo-keto reductase (AKR) superfamily in terms of enzyme kinetics, co-substrate dependence and inhibition pattern. 3. AKR1C1, 1C2 and 1C4, previously designated as dihydrodiol dehydrogenases (DD1, DD2 and DD4), showed lower K(m) (0.2, 0.3 and 0.8 mM respectively) than did carbonyl reductase (7 mM), whereas carbonyl reductase exhibited the highest enzyme efficiency (Vmax/K(m)) for NNK. Multiplication of enzyme efficiencies with the relative quantities of individual enzymes in cytosol resulted in a rough estimate of their contributions to total alcohol metabolite formation. These were approximately 60% for carbonyl reductase, 20% each for AKR1C1 and 1C2, and 1% for AKR1C4. 4. Except for AKR1C4, the enzymes had a strong preference for NADPH over NADH, and the highest activities were measured with an NADPH-regenerating system. Carbonyl reductase activity was extensively inhibited by menadione, rutin and quercitrin, whereas medroxyprogesterone acetate, phenolphthalein and flufenamic acid were potent inhibitors of AKR1C1, 1C2 and 1C4. 5. In conclusion, cytosolic members of the SDR and AKR superfamilies contribute to reductive NNK detoxification in human liver, the enzymes responsible being carbonyl reductase and aldoketo reductases of the AKRIC subfamily.     

Low fidelity bypass of O(2)-(3-pyridyl)-4-oxobutylthymine, the most persistent bulky adduct produced by the tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone by model DNA polymerases

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the most important human carcinogens. It is metabolized to produce a variety of methyl and 4-(3-pyridyl)-4-oxo-butyl (POB) DNA adducts. A potentially important POB adduct is O(2)-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine (O(2)-POB-dT) because it is the most abundant POB adduct in NNK-treated rodents. To evaluate the mutagenic properties of O(2)-POB-dT, we measured the rate of insertion of dNTPs opposite and extension past both O(2)-POB-dT and O(2)-methylthymidine (O(2)-Me-dT) by two model polymerases, E. coli DNA polymerase I (Klenow fragment) with the proofreading exonuclease activity inactivated (Kf) and Sulfolobus solfataricus DNA polymerase IV (Dpo4). We found that the size of the alkyl chain only marginally affected the reactivity and that the specificity of adduct bypass was very low. The k(cat)/K(m) for the Kf catalyzed incorporation opposite and extension past the adducts was reduced ～10(6)-fold when compared to undamaged DNA. Dpo4 catalyzed the incorporation opposite and extension past the adducts approximately 10(3)-fold more slowly than undamaged DNA. The dNTP specificity was less for Dpo4 than for Kf. In general, dA was the preferred base pair partner for O(2)-Me-dT and dT the preferred base pair partner for O(2)-POB-dT. With enzyme in excess over DNA, the time courses of the reactions showed a biphasic kinetics that indicates the formation inactive binary and ternary complexes.     

Nucleophilic reactions between thiols and a tobacco specific nitrosamine metabolite, 4-hydroxy-1-(3-pyridyl)-1-butanone

4-Hydroxy-1-(3-pyridyl)-1-butanone (HPB) is a metabolite of the tobacco specific nitrosamines, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN). HPB is also a breakdown product of covalently bound pyridyloxobutyl adducts resulting from NNK and NNN exposure. HPB released from DNA or hemoglobin has been used as an important dosimeter of tobacco specific nitrosamine exposure in a variety of studies. This compound is not reactive with cellular nucleophiles under biological conditions. We have discovered that HPB reacts with nucleophiles under acidic conditions to form cyclic tetrahydrofuranyl reaction products. Dithiothreitol, 2-mercaptoethanol, and N-acetylcysteine all reacted with HPB under these reaction conditions. In addition, reactions were observed with buffer chemicals such as 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and tris(hydroxymethyl)aminomethane. The resulting cyclic adducts were unstable at room temperature. Their half-lives were significantly longer under neutral conditions than under acidic conditions. NMR studies established that the cyclic form of HPB, 2-hydroxy-2-(3-pyridyl)-2,3,4,5-THF, is present at significant concentrations in acidic solutions. The observation of this cyclic compound suggests that the reaction with nucleophiles may occur via a cyclic oxonium ion intermediate. This reaction was significant in our biological samples; there was up to 40% conversion of [5-(3)H]HPB to cyclic DTT-derived compounds when acidic DNA repair reactions containing [5-(3)H]pyridyloxobutylated DNA were stored overnight at -20 degrees C. Therefore, long-term storage of acid hydrolysates of pyridyloxobutylated DNA or protein for the analysis of HPB-releasing adducts could result in an underestimation of HPB-releasing adduct in those samples. In addition, these observations provide a mild synthetic method to prepare large quantities of cyclic 2-(3-pyridyl)-2,3,4,5-THF adducts predicted to result from pyridyloxobutylation of important cellular nucleophiles as a result of NNK and/or NNN exposure.     

Effect of nicotine or cotinine on metabolism of 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) in isolated rat lung and liver

The scope of the present study was to investigate whether nicotine or cotinine will affect the metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in isolated perfused rat lungs and livers and to study the effect of starvation on pulmonary metabolism of NNK. NNK metabolism was investigated in isolated perfused liver and lung of male F344 rats perfused with 35 nM [5-³H]NNK in presence of a 1400-fold excess of the main tobacco alkaloid nicotine and its metabolite cotinine. In perfused rat livers, nicotine and cotinine inhibited NNK elimination and metabolism and led to a substantial increase of elimination half-life from 14.6 min in controls to 25.5 min after nicotine and 36.6 min after cotinine co-administration, respectively. In parallel, the pattern of NNK metabolites was changed by nicotine and cotinine. The pathway of α-hydroxylation representing the metabolic activation of NNK was decreased to 77% and 85% of control values, whereas N-oxidation of NNK and glucuronidation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) was increased 2.6- and 1.2-fold in presence of nicotine and cotinine, respectively. When isolated rat lungs were perfused with 35 nM NNK for 3 h neither the elimination nor the pattern of metabolites were substantially affected due to co-administration of 50 μM nicotine or cotinine. Cytochrome P450 2E1 is known to participate in the activation of NNK and can be induced by starvation. However, isolated rat lungs from male Sprague Dawley rats perfused with [1-¹⁴C]NNK at about 2 μM for 3 h, revealed only small differences in pulmonary elimination and pattern of NNK metabolites between fed and starved animals. These results suggest that nicotine and its main metabolite cotinine inhibit the metabolic activation of NNK predominantly in the liver whereas activation in lung, a main target organ of NNK induced carcinogenesis, remained almost unaffected. Received: 13 March 1997 / Accepted: 21 November 1997     

Stereospecific deuterium substitution attenuates the tumorigenicity and metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

Stereochemical determinants of the tumorigenicity and metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were investigated using the stereospecifically deuterated isotopomers (4R)-[4-(2)H(1)]NNK and (4S)-[4-(2)H(1)]NNK. Upon ip administration to groups of 20 female A/J mice, NNK and (4S)-[4-(2)H(1)]NNK exhibited similar lung tumorigenicity at three different doses, whereas (4R)-[4-(2)H(1)]NNK was 2-fold less tumorigenic at all three doses. In a parallel experiment, levels of O(6)-methylguanine and 7-methylguanine were 2-fold lower in lung DNA of mice treated with (4R)-[4-(2)H(1)]NNK than in mice treated with NNK or (4S)-[4-(2)H(1)]NNK. To corroborate these in vivo data, the in vitro metabolism of these compounds was investigated using A/J mouse lung microsomes and Spodoptera frugiperda (Sf9)-expressed mouse cytochrome p450s 2A4 and 2A5. Kinetic isotope effects on the apparent V(max) ((D)V) for the product of NNK 4-hydroxylation, OPB, were 2.7 +/- 0.2 and 2.8 +/- 0.4 when (4R)- and (4S)-[4-(2)H(1)]NNK were incubated with mouse lung microsomes, respectively. The (D)V values for OPB formation were 3.2 +/- 0.2 and 2.2 +/- 0.2 when (4R)-[4-(2)H(1)]NNK was the substrate for p2A4 and 2A5, respectively, whereas they were 1.3 +/- 0.1 and 1.1 +/- 0.1 when (4S)-[4-(2)H(1)]NNK was the substrate for these respective enzymes. Analysis of an OPB derivative (10) for deuterium content by LC/MS confirmed the results from the kinetic assays and indicated that p450s 2A4 and 2A5 preferentially abstract the pro-R 4-hydrogen of NNK. The results obtained using Sf9-expressed p450s provide a rationale for the differences observed in the lung tumor and DNA adduct experiments, namely, that the attenuated tumorigenicity of (4R)-[4-(2)H(1)]NNK relative to (4S)-[4-(2)H(1)]NNK is due to prochiral selectivity during p450-catalyzed metabolic activation.     

Enhancements of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) metabolism and carcinogenic risk via NNK/arsenic interaction

Epidemiological studies indicated an enhancement of cigarette smoke-induced carcinogenicity, including hepatocellular carcinoma, by arsenic. We believe that arsenic will enhance the expression of hepatic CYP2A enzyme and NNK metabolism (a cigarette smoke component), thus its metabolites, and carcinogenic DNA adducts. Male ICR mice were exposed to NNK (0.5 mg/mouse) and sodium arsenite (0, 10, or 20 mg/kg) daily via gavaging for 10 days and their urine was collected at day 10 for NNK metabolite analysis. Liver samples were also obtained for CYP2A enzyme and DNA adducts evaluations. Both the cyp2a4/5 mRNA levels and the CYP2A enzyme activity were significantly elevated in arsenic-treated mice liver. Furthermore, urinary NNK metabolites in NNK/arsenic co-treated mice also increased compared to those treated with NNK alone. Concomitantly, DNA adducts (N(7)-methylguanine and O(6)-methylguanine) were significantly elevated in the livers of mice co-treated with NNK and arsenic. Our findings provide clear evidence that arsenic increased NNK metabolism by up-regulation of CYP2A expression and activity leading to an increased NNK metabolism and DNA adducts (N(7)-methylguanine and O(6)-methylguanine). These findings suggest that in the presence of arsenic, NNK could induce greater DNA adducts formation in hepatic tissues resulting in higher carcinogenic potential.     

Molecular modelling of CYP2A enzymes: application to metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

1.?Tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a lung carcinogen in a variety of animal models and a putative human lung carcinogen. Its tumorigenic potential is unmasked via cytochrome P450 (CYP)-mediated hydroxylation of the carbon atoms adjacent to the nitroso moiety (i.e.?α-hydroxylation). Therefore, elucidation of enzyme–substrate interactions that facilitate?α-hydroxylation is important to gain insight into the tumorigenic mechanism of NNK and to develop potent inhibitors of this detrimental reaction.

2.?Molecular models of CYP2A enzymes from mice, rats and humans that are catalysts of NNK bioactivation were constructed and used, in conjunction with docking experiments, to identify active-site residues that make important substrate contacts.

3.?Docking studies revealed that hydrophobic residues at positions 117, 209, 365 and 481, among others, play critical roles in orienting NNK in the active site to effect?α-hydroxylation. These molecular models were then used to rationalize the stereo- and regioselectivity, as well as the efficiency, of CYP2A-mediated NNK metabolism.     

Molecular modelling of CYP2A enzymes: application to metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

1. Tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a lung carcinogen in a variety of animal models and a putative human lung carcinogen. Its tumorigenic potential is unmasked via cytochrome P450 (CYP)-mediated hydroxylation of the carbon atoms adjacent to the nitroso moiety (i.e. alpha-hydroxylation). Therefore, elucidation of enzyme-substrate interactions that facilitate alpha-hydroxylation is important to gain insight into the tumorigenic mechanism of NNK and to develop potent inhibitors of this detrimental reaction. 2. Molecular models of CYP2A enzymes from mice, rats and humans that are catalysts of NNK bioactivation were constructed and used, in conjunction with docking experiments, to identify active-site residues that make important substrate contacts. 3. Docking studies revealed that hydrophobic residues at positions 117, 209, 365 and 481, among others, play critical roles in orienting NNK in the active site to effect alpha-hydroxylation. These molecular models were then used to rationalize the stereo- and regioselectivity, as well as the efficiency, of CYP2A-mediated NNK metabolism.     

Metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in isolated rat lung and liver

The tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a strong lung carcinogen in all species tested. To elicit its tumorigenic effects NNK requires metabolic activation which is supposed to take place via α-hydroxylation, whereas N-oxidation is suggested to be a detoxification pathway. The differences in the organ specific metabolism of NNK may be crucial for the organotropy in NNK-induced carcinogenesis. Therefore, metabolism of NNK was investigated in the target organ lung and in liver of Fischer 344 (F344) rats using the model of isolated perfused organs. High activity to metabolize 35 nM [5-³H]NNK was observed in both perfused organs. NNK was eliminated by liver substantially faster (clearance 6.9 ± 1.6 ml/min, half-life 14.6 ± 1.2 min) than by lung (clearance 2.1 ± 0.5 ml/min, half-life 47.9 ± 7.4 min). When the clearance is calculated for a gram of organ or for metabolically active cell forms, the risk with respect to carcinogenic mechanisms was higher in lung than in liver. The metabolism of NNK in liver yielded the two products of NNK α-hydroxylation, the 4-oxo-4-(3-pyridyl)-butyric acid (keto acid) and 4-hydroxy-4-(3-pyridyl)-butyric acid (hydroxy acid). In lung, the major metabolite of NNK was 4-(methylnitrosamino)-1-(3-pyridyl-N-oxide)-1-butanone (NNK-N-oxide). Substantial amounts of metabolites formed from methyl hydroxylation of NNK, which is one of the two possible pathways of α-hydroxylation, were detected in lung but not in liver perfusion. Formation of these metabolites (4-oxo-4-(3-pyridyl)-butanol (keto alcohol), and 4-hydroxy-4-(3-pyridyl)-butanol (diol) can give rise to pyridyloxobutylating of DNA. When isolated rat livers were perfused with 150 μM NNK, equal to a dosage which is sufficient to induce liver tumors in rat, glucuronidation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) was increased when compared to the concentration of 35 nM NNK. Nevertheless, the main part of NNK was also transformed via α-hydroxylation for this high concentration of NNK. Received: 13 March 1997 / Accepted: 21 November 1997     

Biotransformation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in peripheral human lung microsomes.

The contributions of different enzymes to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) biotransformation were assessed in human lung microsomes prepared from peripheral lung specimens obtained from seven subjects. Metabolite formation was expressed as a percentage of total recovered radioactivity from [5-3H]NNK and its metabolites per milligram of protein per minute. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol was the major metabolite formed in the presence of an NADPH-generating system, with production ranging from 0.5186 to 1.268%/mg of protein/min, and total NNK bioactivation (represented by the sum of the four alpha-carbon hydroxylation endpoint metabolites) ranged from 0.002100 to 0.005685% alpha-hydroxylation/mg of protein/min. Overall, production of bioactivation metabolites was greater than that of detoxication (i.e., N-oxidation) products. Based on total bioactivation, subjects could be classified as high or low NNK bioactivators. In the presence of an NADPH-generating system, microsomal formation of the endpoint metabolite 1-(3-pyridyl)-1-butanone-4-carboxylic acid (keto acid) was consistently higher than that of all other alpha-carbon hydroxylation endpoint metabolites. Contributions of cytochrome p450 (p450) enzymes to NNK oxidation were demonstrated by NADPH dependence, inhibition by carbon monoxide, and inhibition by the nonselective p450 inhibitors proadifen hydrochloride (SKF-525A) and 1-aminobenzotriazole (ABT), particularly in lung microsomes from high bioactivators. At 5.0 mM, ABT inhibited total NNK bioactivation by 54 to 100%, demonstrating the importance of ABT-sensitive enzyme(s) in human pulmonary NNK bioactivation. Contributions of CYP2A6 and/or CYP2A13, as well as CYP2B6, to NNK bioactivation were also suggested by selective chemical and antibody inhibition in lung microsomes from some subjects. It is likely that multiple p450 enzymes contribute to human pulmonary microsomal NNK bioactivation, and that these contributions vary between individuals.     

Evidence that a hemoglobin adduct of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone is a 4-(3-pyridyl)-4-oxobutyl carboxylic acid ester.

Hemoglobin adducts of the carcinogenic tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) release 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) upon mild base or acid hydrolysis. HPB has been detected in hydrolysates of human hemoglobin and has been proposed as a dosimeter of exposure to and metabolic activation of NNK in people exposed to tobacco products. In this study, labeling experiments were carried out with Na18OH which provide strong evidence that the globin adduct which releases HPB upon base hydrolysis is a carboxylic acid ester. Globin was isolated from rats treated with NNK. This globin was reacted with NaCNBH3, followed by hydrolysis at room temperature with 0.2 N NaOH. Analysis of the products demonstrated the presence of 4-hydroxy-1-(3-pyridyl)-1-butanol (7), but not HPB. These results demonstrate that the adduct in globin has a free carbonyl group and is not a Schiff base. This sequence of reactions was then carried out with Na18OH, under conditions which would have resulted in incorporation of 18O into 7 if nucleophilic displacement at carbon 4 of the adduct had occurred. Analysis of the products by GC-MS showed no detectable incorporation of 18O into 7. These results demonstrate that the globin adduct which releases HPB upon base hydrolysis is a 4-(3-pyridyl)-4-oxobutyl carboxylic ester. Consistent with this conclusion, a model ester, alpha-methyl beta-[4-(3-pyridyl)-4-oxobutyl] N-(carbobenzyloxy)-L-aspartate (13), hydrolyzed in base and acid in a manner similar to that observed with globin from NNK-treated rats.     

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.     

Evidence for phosphate adducts in DNA from mice treated with 4-(N-Methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

The weakly alkylating capacity of phosphotriesters (PTE) has been used for the determination of adducts to phosphate groups in DNA by specific transfer to the strongly nucleophilic compound cob(I)alamin [Cbl(I)]. When enzymatically degraded liver DNA from mice treated with 1-(N-methyl-N-nitrosamino)-4-(3-[3H]pyridyl)-4-oxobutane ([3H]NNK) was added to Cbl(I), a 4-(3-[3H]pyridyl)-4-hydroxy-1-butyl-cobalamin ([3H]PHB-Cbl) complex was formed and determined by HPLC and liquid scintillation counting. The PHB-Cbl formed was compared with a synthetic standard verified by LC/MS and 1H NMR and corresponds to phosphate adducts formed from the pyridyloxobutylating species from NNK and from the pyridylhydroxybutylating species from NNAL, NNK being to a large extent converted to NNAL in vivo. It was concluded that about 22% of the total level of pyridyl (oxo or hydroxy) butyl adducts to DNA was bound to phosphate groups.     

K-ras gene sequence effects on the formation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-DNA adducts

The tobacco specific pulmonary carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is metabolically activated to electrophilic species that form methyl and pyridyloxobutyl adducts with genomic DNA, including O(6)-methylguanine, N7-methylguanine, and O(6)-[4-oxo-4-(3-pyridyl)butyl]guanine. If not repaired, these lesions could lead to mutations and the initiation of cancer. Previous studies used ligation-mediated polymerase chain reaction (LMPCR) in combination with PAGE to examine the distribution of NNK-induced strand breaks and alkali labile lesions (e.g., N7-methylguanine) within gene sequences. However, LMPCR cannot be used to establish the distribution patterns of highly promutagenic O(6)-methylguanine and O(6)-[4-oxo-4-(3-pyridyl)butyl]guanine adducts of NNK. We have developed methods based on stable isotope labeling HPLC-electrospray ionization tandem mass spectrometry (HPLC-ESI MS/MS) that enable us to accurately quantify NNK-induced adducts at defined sites within DNA sequences. In the present study, the formation of N7-methylguanine, O(6)-methylguanine, and O(6)-[4-oxo-4-(3-pyridyl)butyl]guanine adducts at specific positions within a K-ras gene-derived double-stranded DNA sequence (5'-G(1)G(2)AG(3)CTG(4)G(5)TG(6)G(7)CG(8)TA G(9)G(10)C-3') was investigated following treatment with activated NNK metabolites. All three lesions preferentially formed at the second position of codon 12 (GGT), the major mutational hotspot for G-->A and G-->T base substitutions observed in smoking-induced lung tumors. Therefore, our data support the involvement of NNK and other tobacco specific nitrosamines in mutagenesis and carcinogenesis.     

Bayesian derivation of an oral cancer slope factor distribution for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

The tobacco-specific nitrosamine (TSNA) 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is classified by the International Agency for Research on Cancer as a Group 1 carcinogen. Cancer risk assessment in humans exposed to TSNAs largely relies on potency values estimated from animal studies, but available cancer potency values for NNK derived from such studies are conflicting. In this analysis, oral cancer slope factors (CSFo) for NNK were derived according to U.S. Environmental Protection Agency guidelines. An animal study in which rats were exposed to NNK in drinking water was selected as the key study. The multistage-cancer model was fit to the tumor incidence data to determine a point of departure for low dose linear extrapolation, using a benchmark response of 10%. CSFo distributions were then computed using Bayesian methods and Monte Carlo simulation. The resultant CSFo point estimate (BMR/BMDL₁₀) was 19.2 (mg/kg day)⁻¹ based on lung tumor data and 12.2 (mg/kg day)⁻¹ based on pancreatic tumors. The 95th percentiles of the CSFo distributions were 27.3 and 19.3 (mg/kg day)⁻¹ based on lung and pancreatic tumors, respectively. The approach using Bayesian methods better accounts for the uncertainty inherent in the values generated using input assumptions and provides for a more robust probabilistic dose–response assessment.     

Metabolism of 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in primary cultures of rat alveolar type II cells.

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induces primarily lung tumors, which are assumed to derive from malignant transformation of alveolar type II (AII) cells within the lung. To elicit its carcinogenic effects, NNK requires metabolic activation by cytochrome P-450 (CYP)-mediated alpha-hydroxylation. Therefore, in this study the metabolism of NNK and expression of the NNK-activating CYP isoform CYP2B1 were investigated in primary cultures of rat AII cells. Although basal expression of CYP2B1 decreased in a time-dependent manner during culture of AII cells, substantial CYP2B1 protein expression was observed in AII cell cultures after the first 24 h. When AII cells were incubated with 0. 05 microM [5-(3)H]NNK, N-oxidation of NNK, which is thought to represent a detoxification pathway, was predominant (42%). alpha-Hydroxylated metabolites resulting from metabolic activation of NNK amounted to 35% of all detected metabolites. However, the proportion of alpha-hydroxylated metabolites decreased to 17% of all detected metabolites when AII cells were incubated with a 100-fold higher concentration of NNK (5 microM). In summary, this study indicates a remarkable activity of cultured AII cells to metabolize NNK, leading to substantial metabolic activation of NNK, which was more pronounced in incubations at low NNK concentration. Because exposure to NNK via cigarette smoking is thought to lead to very low plasma NNK concentrations (1-15 pM), these data suggest that metabolic activation of NNK in cigarette smokers might occur to a larger extent than would be expected according to previous metabolic studies performed with high (micromolar) NNK concentrations.     

Arsenite promotes centrosome abnormalities under a p53 compromised status induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

Epidemiological evidence indicated that residents, especially cigarette smokers, in arseniasis areas had significantly higher lung cancer risk than those living in non-arseniasis areas. Thus an interaction between arsenite and cigarette smoking in lung carcinogenesis was suspected. In the present study, we investigated the interactions of a tobacco-specific carcinogen 4- (methylnitrosamino)-1-(3-pyridyl)-1-butanone (nicotine-derived nitrosamine ketone, NNK) and arsenite on lung cell transformation. BEAS-2B, an immortalized human lung epithelial cell line, was selected to test the centrosomal abnormalities and colony formation by NNK and arsenite. We found that NNK, alone, could enhance BEAS-2B cell growth at 1-5 μM. Under NNK exposure, arsenite was able to increase centrosomal abnormality as compared with NNK or arsenite treatment alone. NNK treatment could also reduce arsenite-induced G2/M cell cycle arrest and apoptosis, these cellular effects were found to be correlated with p53 dysfunction. Increased anchorage-independent growth (colony formation) of BEAS-2B cells cotreated with NNK and arsenite was also observed in soft agar. Our present investigation demonstrated that NNK could provide a p53 compromised status. Arsenite would act specifically on this p53 compromised status to induce centrosomal abnormality and colony formation. These findings provided strong evidence on the carcinogenic promotional role of arsenite under tobacco-specific carcinogen co-exposure.     

Investigation of the role of lipoxygenase in bioactivation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in human lung

4-Methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) is a potent tobacco-specific carcinogen believed to play a role in human lung cancer. Bioactivation of NNK involves alpha-carbon hydroxylation that could be catalyzed by cytochrome P450, hemoglobin, and lipoxygenases (LOX). In the present study, the role of LOX in NNK bioactivation was investigated. Formation of keto acid, the endpoint metabolite of alpha-methylene NNK hydroxylation, was observed in human lung cytosols incubated with 4.2 microM [5-(3)H]NNK (N = 6). Following concanavalin A affinity chromatography to enrich human lung lipoxygenase (HLLO), the fraction containing cytosolic components less LOX (fraction 1) retained the ability to bioactivate NNK. Although enriched HLLO exhibited the characteristic dioxygenase and hydroperoxidase activities, it did not bioactivate NNK. The LOX inhibitor nordihydroguaiaretic acid inhibited dioxygenase activity of HLLO by 83 +/- 19% (P < 0.05, N = 6), but did not inhibit keto acid formation in the crude cytosols (N = 6, P > 0.05). Failure of soybean LOX to catalyze NNK bioactivation supported the results observed in human lung cytosols, and failure of chemically generated alkylperoxyl radicals to bioactivate NNK further suggested that the dioxygenase activity of LOX is not likely to be involved in NNK bioactivation. Horseradish peroxidase and myeloperoxidase catalyzed NNK bioactivation were also nondetectable. Our results demonstrate that, although human lung cytosols can bioactivate NNK to form keto acid, LOX is not involved. We have attributed the ability of crude human lung cytosols to bioactivate NNK to hemoglobin. The inhibitory effect of 1-aminobenzotriazole and arachidonic acid on keto acid formation in the crude cytosols and in fraction 1, respectively (P < 0.05, N = 6), is consistent with hemoglobin-catalyzed NNK bioactivation.