Alveolar macrophage cytotoxic activity is inhibited by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a carcinogenic component of cigarette smoke

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a carcinogenic compound of cigarette smoke that generates electrophilic intermediates capable of damaging DNA. Recently, we have shown that NNK can modulate mediator production by alveolar macrophages (AM) and bronchial and alveolar epithelial cells, suggesting that cigarette smoke can alter lung immune response. Thus, we investigated the effect of NNK and cigarette smoke extract (CSE) on AM capacity to eliminate tumoral cells. Rat AM cell line, NR8383, was treated with NNK (500 μM) or CSE (3%) and stimulated with lipopolysaccharide (10 ng/ml). The release of cytotoxic mediators, tumor necrosis factor (TNF) and reactive oxygen species (ROS), was measured in cell-free supernatants using ELISA and superoxide anion production. TNF- and ROS-dependent cytotoxicity were studied using a ⁵¹Chromium-release assay and WEHI-164 and P-815 cell lines. Treatment of AM with NNK and CSE for 18 h significantly inhibited AM TNF release. CSE exposure resulted in a significant increase of ROS production, whereas NNK did not. TNF-dependent cytotoxic activity of NR8383 and freshly isolated rat AM was significantly inhibited after treatment with NNK and CSE. Interestingly, although ROS production was stimulated by CSE and not affected by NNK, CSE inhibited AM ROS-dependent cytotoxicity. These results suggest that NNK may be one of the cigarette smoke components responsible for the reduction of pulmonary cytotoxicity. Thus, NNK may have a double pro-carcinogenic effect by contributing to DNA adduct formation and inhibiting AM cytotoxicity against tumoral cells.     

Acute in vivo treatment with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone does not alter base excision repair activities in murine lung and liver

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent pulmonary carcinogen found in unburned tobacco and tobacco smoke, and is believed to play an important role in human tobacco-induced cancers. In previous studies, NNK has been reported to induce oxidative DNA damage, and to alter DNA repair processes, effects that could contribute to pulmonary tumorigenesis in rodent models. The goal of this study was to determine the effects of NNK on levels of 8-hydroxydeoxyguanosine (8-OHdG), a biomarker of DNA oxidation, and activity of base excision repair (BER), which repairs oxidative DNA damage. Female A/J mice were treated with a tumorigenic dose of NNK (10 μmol) i.p. At 1, 2 and 24 h post treatment, there were no statistically significant differences in lung or liver 8-OHdG levels between control and NNK-treated mice (P > 0.05). Furthermore, NNK did not alter lung or liver BER activity compared to control at any time point (P > 0.05). In summary, acute treatment with a tumorigenic dose of NNK did not stimulate oxidative DNA damage or significantly alter BER activity, and these effects may not be major mechanisms of action of NNK in mouse models.     

Increased susceptibility of mice lacking Clara cell 10-kDa protein to lung tumorigenesis by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, a potent carcinogen in cigarette smoke

Ninety percent of all human lung cancers are related to cigarette smoking. Both tobacco smoke and lung tumorigenesis are associated with drastically reduced levels of Clara cell 10-kDa protein (CC10), a multifunctional secreted protein, naturally produced by the airway epithelia of virtually all mammals. We previously reported that the expression of CC10 is markedly reduced in animals exposed to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, NNK, a potent carcinogen in tobacco smoke. Furthermore, it has been reported that CC10 expression, induced in certain tumor cells, reverses the transformed phenotype. We demonstrate here that NNK exposure of CC10-knock-out (CC10-KO) mice causes a significantly higher incidence of airway epithelial hyperplasia and lung adenomas compared with wild type (WT) littermates (30% CC10-KO versus 5% WT, p = 0.041). We also found that compared with NNK-treated WT mice, CC10-KO mice manifest increased frequency of K-ras mutation, elevated level of Fas ligand (FasL) expression, and increased MAPK/Erk phosphorylation, all of which are considered predisposing events in NNK-induced lung tumorigenesis. We propose that CC10 has a protective role against NNK-induced lung tumorigenesis mediated via down-regulation of the above-mentioned predisposing events.     

Modulation of reduction of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone by vitamin C-palmitate

An in vitro study of effects of vitamin C-palmitate on the metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in rat microsomes was performed. A sensitive assay method has been developed for the detection of metabolites of NNK in microsomes. Only the reduced metabolite of NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-butanol (NNAL), was detected and measured in a time-course study. Vitamin C-palmitate enhanced the reduction of NNK in a concentration-dependent manner. The results indicate a significant increase in V_max and K_m in the presence of vitamin C. However, the rate of formation of NNAL at low substrate concentration varied. The ratio of V_max to K_m decreases. The results suggest that the kinetics are accounted for best by an uncompetitive activator binding model at low concentration of vitamin C. The uncompetitive binding model becomes sketchy at higher concentration of vitamin C. These observations infer that vitamin C loosely binds to the substrate-enzyme complex. Furthermore, the nature of the binding would facilitate the modulation of NNK biotransformation leading to the formation of NNAL. The results also show that vitamin C-palmitate is a potent activator of NNK reduction in rat liver microsomes. Thus, vitamin C-palmitate would mediate the metabolism of NNK through reduction. The resulting NNAL-glucuronide is more readily eliminated in urine.     

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)     

A high-level expression of CYP2A in the lung of the suncus (Suncus murinus) and its role in the activation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

Northern blot analysis of mRNA prepared from the lung of Suncus murinus (suncus), which was classified as an ancestor of primates, revealed that the expression level of cytochrome P450 2A (CYP2A) mRNA was about 100-fold higher than in the lung from rats and mice. To confirm that the pulmonary CYP2A of the suncus had a catalytic activity, the metabolism of a specific substrate for CYP2A6, (+)-cis-3,5-dimethyl-2-(3-pyridyl) thiazolidin-4-one hydrochloride (SM-12502), was determined. The intrinsic clearance for SM-12502 S-oxidation by the suncus lung microsomes was calculated to be 99-fold higher than that by rat liver microsomes. The mutagen-producing activity of a 9,000 g supernatant fraction prepared from suncus lung was examined using 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) as a promutagen. The results showed that the suncus lung possessed 82-fold higher mutagen-producing activity than the rat lung, indicating that NNK was efficiently activated by the CYP2A isoform expressed in the suncus lung and that the suncus was a sensitive animal species to the genotoxicity of NNK contained in tobacco smoke.     

The effect of a 2-h exposure to cigarette smoke on the metabolic activation of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in A/J mice.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific nitrosamine, induces lung adenomas in A/J mice following a single intraperitoneal (i.p.) injection. However, inhalation of mainstream cigarette smoke does not induce or promote NNK-induced lung tumors in this mouse strain purported to be sensitive to chemically-induced lung tumorigenesis. The critical events for NNK-induced lung tumorigenesis in A/J mice is thought to involve O(6)-methylguanine (O(6)MeG) adduct formation, GC-->AT transitional mispairing, and activation of the K-ras proto-oncogene. The objective of this study was to test the hypothesis that a smoke-induced shift in NNK metabolism led to the observed decrease in O(6)MeG adducts in the lung and liver of A/J mice co-administered NNK with a concomitant 2-h exposure to cigarette smoke as observed in previous studies. Following 2 h nose-only exposure to mainstream cigarette smoke (600 mg total suspended particulates/m(3) of air), mice (n=12) were administered 7.5 micromol NNK (10 microCi [5-3H]NNK) by i.p. injection. A control group of 12 mice was sham-exposed to HEPA-filtered air for 2 h prior to i.p. administration of 7.5 micromol NNK (10 microCi [5-3H]NNK). Exposure to mainstream cigarette smoke had no effect on total excretion of NNK metabolites in 24 h urine; however, the metabolite pattern was significantly changed. Mice exposed to mainstream cigarette smoke excreted 25% more 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) than control mice, a statistically significant increase (P<0.0001). Cigarette smoke exposure significantly reduced alpha-hydroxylation of NNK to potential methylating species; this is based on the 15% reduction in excretion of the 4-(3-pyridyl)-4-hydroxybutanoic acid and 42% reduction in excretion of 4-(3-pyridyl)-4-oxobutanoic acid versus control. Detoxication of NNK and NNAL by pyridine-N-oxidation, and glucuronidation of NNAL were not significantly different in the two groups of mice. The observed reduction in alpha-hydroxylation of NNK to potential methylating species in mainstream cigarette smoke-exposed A/J mice provides further mechanistic support for earlier studies demonstrating that concurrent inhalation of mainstream cigarette smoke results in a significant reduction of NNK-induced O(6)MeG adduct formation in lung and liver of A/J mice compared to mice treated only with NNK.     

Biomonitoring of environmental tobacco smoke (ETS)-related exposure to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

The exposure of non-smokers to the tobacco-specific N-nitrosamine 4-(N-methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a rodent lung carcinogen, was determined in the air of various indoor environments as well as by biomonitoring of non-smokers exposed to environmental tobacco smoke (ETS) under real-life conditions using the urinary NNK metabolites 4-(N-methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and [4-(N-methylnitrosamino)-1-(3-pyridyl)but-1-yl]-beta-O-D-glucosiduronic acid (NNAL-Gluc). NNK was not detectable (&lt;0.5 ng m^-3) in 11 rooms in which smoking did not occur. The mean NNK concentration in 19 rooms in which smoking took place was 17.5 (2.4-50.0) ng m^-3. The NNK levels significantly correlated with the nicotine levels (r=0.856; p&lt; 0.0001). Of the 29 non-smokers investigated, 12 exhibited no detectable NNAL and NNAL-Gluc excretion (&lt;3 pmol day) in their urine. The mean urinary excretion of NNAL and NNAL-Gluc of the 17 remaining non-smokers was 20.3 (&lt;3-63.2) and 22.9 (&lt;3-90.0) pmol day^-1, respectively. Total NNAL excretion (NNAL+NNAL-Gluc) in all non-smokers investigated significantly correlated with the amount of nicotine on personal samplers worn during the week prior to urine collection (r=0.88; &lt;0.0001) and with the urinary cotinine levels (r=0.40; p=0.038). No correlation was found between NNAL excretion and the reported extent of ETS exposure. Average total NNAL excretion in the non-smokers with detectable NNAL levels was 74 times less than in 20 smokers who were also investigated. The cotinine/total NNAL ratios in urine of smokers (9900) and non-smokers (9300) were similar. This appears to be at variance with the ratios of the corresponding precursors (nicotine/NNK) in mainstream smoke (16400) and ETS (1000). Possible reasons for this discrepancy are discussed. The possible role of NNK as a lung carcinogen in non-smokers is unclear, especially since NNK exposure in non-smokers is several orders of magnitude lower than the ordinary exposure to exogenous and endogenous N-nitrosamines and the role of NNK as a human lung carcinogen is not fully understood.     

The profile of urinary metabolites of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in rats is determined by its pulmonary metabolism.

Metabolism of the tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in rats was compared to metabolism in primary lung and liver cells. Untreated rats and rats pretreated with phenobarbital, acetone or phenethyl isothiocyanate (PEITC) were used for all experiments. Also the influence of [-]-1-methyl-2-[3-pyridyl]-pyrrolidine (nicotine) administered concomitantly with NNK, or incubated with isolated cells, upon NNK metabolism was investigated and found to be only marginal upon alpha-hydroxylation and pyridine N-oxidation in vivo. In hepatocytes nicotine inhibited NNK pyridine N-oxidation, alpha-hydroxylation and glucuronidation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), whereas in lung cells the influence of nicotine was not as pronounced. In vivo phenobarbital induced alpha-hydroxylation and pyridine N-oxidation. In vitro the effects of the modulators were most pronounced upon hepatocytes, where phenobarbital greatly induced pyridine N-oxidation and PEITC inhibited alpha-hydroxylation. NNAL was conjugated to its beta-glucuronide in lung cells at four times higher rates than in hepatocytes. The ratios of the sum of N-oxides to the sum of alpha-hydroxylation products in vivo were similar to those in lung cells, especially at low NNK concentrations (1 microM), while in hepatocytes alpha-hydroxylation was more pronounced. The same correlation of metabolism in isolated lung cells with whole rats was observed if oxidative NNAL metabolism was related to oxidative NNK metabolism. Here hepatocytes showed a much higher formation of NNAL oxidation products than either lung cells formed, or rats excreted in urine. This was true despite a lower rate of metabolism in the lung than in liver if based on cell number, the rate based on mg protein was four times higher in lung than liver. Only after phenobarbital treatment was the contribution of hepatic metabolism to excreted metabolites important. In conclusion the lung which is also the target of NNK carcinogenesis, and not the liver, is the organ with the most important contribution to NNK and NNAL metabolism at concentrations relevant to human exposure.     

Mutations induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in the lacZ and cII genes of Muta™ Mouse

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) found in chewing tobacco, snuff, cigarettes, and cigars is a tobacco-specific nitrosamine and classified as a possible human carcinogen (Class 2B) by the International Agency for Research on Cancer (IARC). NNK given intraperitoneally was seen to induce lung and liver adenomas.To evaluate the genotoxicity of NNK in vivo, NNK was intraperitoneally administered to Muta™ Mouse at two concentrations (125 and 250 mg/kg, once a week for 4 weeks) followed by the measurement of mutant frequencies in the lacZ and cII genes from lung and liver in the same mice. Characterization of the types of the mutation was determined by sequencing the cII genes from mutant plaques. The mutant frequencies in both target genes from both organs dose-dependently increased up to 10 times compared to those of the control group. For the types of mutations, the ratio of the G:C to A:T mutation in the total number of mutants was less than the ratio of A:T to T:A and A:T to C:G transversion, contrary to a previous report [Cancer Res, 49 (1989) 5305]. The A:T to T:A transversion was the most highly induced mutation both in the lung and liver cII genes. The increasing rate of mutant frequencies in lung and liver over the vehicle control was 55 and 56 times, respectively, while the increasing rate of G:C to A:T transition was only 1.9 and 2.8 times, respectively.These observations show that NNK predominantly induces DNA adducts leading to A:T to T:A and/or A:T to C:G mutations in the transgene.     

Characterization of enzymes participating in carbonyl reduction of 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) in human placenta

4-Methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) has been identified as one of the strongest nitrosamine carcinogens in tobacco products in all species tested. Carbonyl reduction to 4-methylnitrosamino-1-(3-pyridyl)-1-butanol (NNAL) followed by glucuronosylation is considered to be the main detoxification pathway in humans. In previous investigations, we have identified a microsomal NNK carbonyl reductase as being identical to 11ß-hydroxysteroid dehydrogenase 1, a member of the short-chain dehydrogenase/reductase (SDR) superfamily. Recently, we provided evidence that carbonyl reduction of NNK does also take place in cytosol from mouse and human liver and lung. In human liver cytosol, carbonyl reductase, a SDR enzyme, and AKR1C1, AKR1C2 and AKR1C4 from the aldo-keto reductase (AKR) superfamily were demonstrated to be responsible for NNK reduction. Since NNK and/or its metabolites can diffuse through the placenta and reach fetal tissues, we now investigated NNK carbonyl reduction in the cytosolic fraction of human placenta in addition to that in microsomes. Concluding from the sensitivity to menadione, ethacrynic acid, rutin and quercitrin as specific inhibitors, mainly carbonyl reductase (EC 1.1.1.184) seems to perform this reaction in human placenta cytosol. The presence of carbonyl reductase was confirmed by RT-PCR. This is the first report to provide evidence that NNAL formation in placenta is mediated by carbonyl reductase.     

Characterization of enzymes participating in carbonyl reduction of 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) in human placenta

4-Methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) has been identified as one of the strongest nitrosamine carcinogens in tobacco products in all species tested. Carbonyl reduction to 4-methylnitrosamino-1-(3-pyridyl)-1-butanol (NNAL) followed by glucuronosylation is considered to be the main detoxification pathway in humans. In previous investigations, we have identified a microsomal NNK carbonyl reductase as being identical to 11beta-hydroxysteroid dehydrogenase 1, a member of the short-chain dehydrogenase/reductase (SDR) superfamily. Recently, we provided evidence that carbonyl reduction of NNK does also take place in cytosol from mouse and human liver and lung. In human liver cytosol, carbonyl reductase, a SDR enzyme, and AKR1C1, AKR1C2 and AKR1C4 from the aldo-keto reductase (AKR) superfamily were demonstrated to be responsible for NNK reduction. Since NNK and/or its metabolites can diffuse through the placenta and reach fetal tissues, we now investigated NNK carbonyl reduction in the cytosolic fraction of human placenta in addition to that in microsomes. Concluding from the sensitivity to menadione, ethacrynic acid, rutin and quercitrin as specific inhibitors, mainly carbonyl reductase (EC 1.1.1.184) seems to perform this reaction in human placenta cytosol. The presence of carbonyl reductase was confirmed by RT-PCR. This is the first report to provide evidence that NNAL formation in placenta is mediated by carbonyl reductase.     

Effects of vitamins and common drugs on reduction of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in rat microsomes.

4-(Methylnitrosamino)-1-(3-pyridyl)-butanone (NNK) is a tobacco-specific nitrosamino that requires metabolic activation by cytochrome P450 enzymes. The activation of NNK by cytochrome P450 enzymes leads to the formation of different metabolites. Detoxification of NNK usually occurs via carbonyl reduction to its hydroxyl product, 4-(methylnitrosamino)-1-(3-pyridyl)-butanol (NNAL). In the present study, the influences of common vitamins and P450 modulators on the reduction of NNK by rat microsomes were studied. The formation of NNAL but not other metabolites was detected by the described HPLC method. Among the vitamins tested, vitamins E, A (retinol), B6 and B5 were found to be marginal effective upon reduction of NNK while vitamins A (cis-acid), A (trans-acid), D2, D3, K1, K3, B1 and A (crocetin) increased the formation of NNAL from 3 to 21%. The effect of vitamin C-palmitate (<10 microM) was most pronounced followed by crocetin upon reduction of NNK. Clonidine, tolbutamide and atropine slightly increased the reduction of NNK while cimetidine showed no effects. The modulation of NNK reduction could reduce the carcinogenic potential of NNK, since the main detoxification pathway of NNK involves carbonyl reduction.     

Characterization of cytochrome P450 2A4 and 2A5-catalyzed 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) metabolism

The tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is a potent lung carcinogen in the A/J mouse, and is believed to be a causative agent for human lung cancer. NNK requires metabolic activation by alpha-hydroxylation to exert its carcinogenic potential. The human P450, 2A6 is a catalyst of this reaction. There are two closely related enzymes in the mouse, P450 2A4 and 2A5, which differ from each other by only 11 amino acids. In the present study these two mouse P450s were expressed in Spodoptera frugiperda (Sf9) cells using recombinant baculovirus. The catalysis of NNK metabolism by Sf9 microsomal fractions containing either P450 2A4 or 2A5 was determined. Both enzymes catalyzed the alpha-hydroxylation of NNK but with strikingly different efficiencies and specificities. P450 2A5 preferentially catalyzed NNK methyl hydroxylation, while P450 2A4 preferentially catalyzed methylene hydroxylation. The KM and Vmax for the former were 1.5 microM and 4.0 nmol/min/nmol P450, respectively, and for the latter 3.9 mM and 190 nmol/min/nmol P450. The mouse coumarin 7-hydroxylase, P450 2A5 is a significantly better catalyst of NNK alpha-hydroxylation than is the closely related human enzyme, P450 2A6.     

4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, a nicotine derivative, induces apoptosis of endothelial cells

Smoking causes endothelial cell (EC) injury; however, neither the components of cigarette smoke nor the mechanisms responsible for this injury are understood. The nitrosated derivative of nicotine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), has been implicated in the carcinogenic effects of tobacco; however, the effects of NNK on the cardiovascular system are largely unknown. NNK binds to beta1- and beta2-adrenergic receptors. Because beta-adrenergic receptor activation causes arachidonic acid (AA) release and cellular injury, we postulated that NNK causes EC injury by a mechanism that involves beta-adrenergic-mediated release of AA. NNK stimulated [3H]AA release from ECs, and this effect was mediated by both beta1- and beta2-adrenergic receptors because pretreatment with atenolol or ICI 118,551 inhibited the response. NNK also induced EC apoptosis, as measured by terminal deoxyribonucleotide transferase-mediated dUTP nick-end labeling and annexin V staining. NNK-mediated apoptosis was attenuated by pretreatment with atenolol or ICI 118,551. Furthermore, depletion of cellular AA by incubation with eicosapentaenoic acid abolished the apoptotic effect of NNK. These data suggest that NNK causes EC apoptosis by a mechanism that involves beta1- and beta2-adrenergic receptor-mediated release of AA.     

Tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone promotes functional cooperation of Bcl2 and c-Myc through phosphorylation in regulating cell survival and proliferation

Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is formed by nitrosation of nicotine and has been identified as the most potent carcinogen contained in cigarette smoke. NNK significantly contributes to smoking-related lung cancer, but the molecular mechanism remains enigmatic. Bcl2 and c-Myc are two major oncogenic proteins that cooperatively promote tumor development. We report here that NNK simultaneously stimulates Bcl2 phosphorylation exclusively at Ser(70) and c-Myc at Thr(58) and Ser(62) through activation of both ERK1/2 and PKCalpha, which is required for NNK-induced survival and proliferation of human lung cancer cells. Treatment of cells with staurosporine or PD98059 blocks both Bcl2 and c-Myc phosphorylation and results in suppression of NNK-induced proliferation. Specific depletion of c-Myc expression by RNA interference retards G(1)/S cell cycle transition and blocks NNK-induced cell proliferation. Phosphorylation of Bcl2 at Ser(70) promotes a direct interaction between Bcl2 and c-Myc in the nucleus and on the outer mitochondrial membrane that significantly enhances the half-life of the c-Myc protein. Thus, NNK-induced functional cooperation of Bcl2 and c-Myc in promoting cell survival and proliferation may occur in a novel mechanism involving their phosphorylation, which may lead to development of human lung cancer and/or chemoresistance.     

Mutations induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in the lacZ and cII genes of Muta Mouse

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) found in chewing tobacco, snuff, cigarettes, and cigars is a tobacco-specific nitrosamine and classified as a possible human carcinogen (Class 2B) by the International Agency for Research on Cancer (IARC). NNK given intraperitoneally was seen to induce lung and liver adenomas. To evaluate the genotoxicity of NNK in vivo, NNK was intraperitoneally administered to Muta Mouse at two concentrations (125 and 250 mg/kg, once a week for 4 weeks) followed by the measurement of mutant frequencies in the lacZ and cII genes from lung and liver in the same mice. Characterization of the types of the mutation was determined by sequencing the cII genes from mutant plaques. The mutant frequencies in both target genes from both organs dose-dependently increased up to 10 times compared to those of the control group. For the types of mutations, the ratio of the G:C to A:T mutation in the total number of mutants was less than the ratio of A:T to T:A and A:T to C:G transversion, contrary to a previous report. The A:T to T:A transversion was the most highly induced mutation both in the lung and liver cII genes. The increasing rate of mutant frequencies in lung and liver over the vehicle control was 55 and 56 times, respectively, while the increasing rate of G:C to A:T transition was only 1.9 and 2.8 times, respectively. These observations show that NNK predominantly induces DNA adducts leading to A:T to T:A and/or A:T to C:G mutations in the transgene.     

In vitro and in vivo modulation of the bioactivation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in hamster lung tissues

The metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) by hamster lung explants was studied. The three major metabolic pathways were alpha-C-hydroxylation (activation), pyridine N-oxidation (deactivation) and carbonyl reduction. alpha-C-Hydroxylation and pyridine N-oxidation were linear with time (0.5-5 h) and number of explants per dish (3-10). Addition of [2-(diethylamino)ethyl 2,2-diphenylpentenoate] hydrochloride (SKF-525A) to the culture medium reduced alpha-C-hydroxylation and pyridine N-oxidation. alpha-C-Hydroxylation was enhanced by treatment of the hamsters with the two cytochrome P-450 inducers, phenobarbital and 3-methylcholanthrene. These results suggest that cytochrome P-450 monooxygenases are involved in the activation of NNK by alpha-C-hydroxylation. Three groups of hamsters were fed a control diet or diet supplemented with 2% 2(3)-tert-butyl 4-hydroxyanisole (2(3)-BHA) or given a 0.002% solution of (S)-nicotine to drink for two weeks. Lung explants were then cultured with NNK in vitro. Treatment with 2(3)-BHA and (S)-nicotine induced the alpha-C-hydroxylation pathways. Pyridine N-oxidation was increased by (S)-nicotine treatment. These results indicate that dietary factors and tobacco smoke components can modulate the metabolism of NNK.     

Gene expression profiles in HPV-immortalized human cervical cells treated with the nicotine-derived carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

Human papillomavirus (HPV) infection is an established etiological factor for cervical cancer. Epidemiological studies suggest that smoking in combination with HPV infection plays a significant role in the etiology of this disease. We have previously shown that the tobacco carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is present in human cervical mucus. Here, we hypothesized that treatment of HPV-16-immortalized human ectocervical cells (Ecto1/E6E7) with NNK would alter the expression of genes involved in cellular transformation. Ecto1/E6E7 cells were treated with water (vehicle control) alone or with 1 μM, 10 μM, and 100 μM of NNK in water for 12 weeks. The colony-forming efficiency increased following NNK treatment; the maximum effect was observed after 12 weeks with 100 μM NNK. Microarray analysis revealed that, independent of the dose of NNK, expression of 30 genes was significantly altered; 22 of these genes showed a dose-response pattern. Genes identified are categorized as immune response (LTB4R), RNA surveillance pathway (SMG1), metabolism (ALDH7A1), genes frequently expressed in later stages of neoplastic development (MT1F), DNA binding (HIST3H3 and CHD1L), and protein biosynthesis (UBA52). Selected genes were confirmed by qRT-PCR. Western blot analysis indicates that phosphorylation of histone 3 at serine 10, a marker of cellular transformation, was up-regulated in cells treated with NNK. This is the first study showing that NNK can alter gene expression that may, in part, account for transformation of HPV-immortalized human cervical cells. The results support previous epidemiological observations that, in addition to HPV, tobacco smoking also plays an important role in the development of cervical cancer.     

Tobacco carcinogen induces microglial activation and subsequent neuronal damage

4-Methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) is a tobacco-specific procarcinogen. We have investigated whether NNK causes inflammatory upheaval in the brain by activation of resident microglia and astrocyte and result in bystander neuronal damage. We have carried out the work in both in vitro and in vivo models. We have found that treatment with NNK causes significant activation of mouse microglial (BV2) cell line as evident by increase in reactive oxygen species and nitric oxide level. Western blot analysis has showed increase in proinflammatory signaling proteins, proinflammatory effector proteins, and other stress-related proteins. Interestingly, increased levels of proinflammatory cytokines like interleukin (IL)-6, tumor necrosis factor-α, monocyte chemoattractant protein 1 (MCP1), and IL-12p70 are also detected. Work from our in vivo studies has demonstrated similar increase in proinflammatory signaling and effector molecules along with the proinflammatory cytokine levels, following NNK treatment. Immunohistochemical staining of the brain sections of NNK-treated mice reveals massive microglial and astrocyte activation along with distinct foci of neuronal damage. Both in vitro and in vivo results provide strong indication that NNK causes significant upheaval of the inflammatory condition of brain and inflicts subsequent neuronal damage.