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PubMed1988

Effects of dietary sinigrin or indole-3-carbinol on O6-methylguanine-DNA-transmethylase activity and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced DNA methylation and tumorigenicity in F344 rats.

The effects of dietary sinigrin and indole-3-carbinol (I3C) on DNA methylation and O6-methylguanine--DNA-transmethylase activity, factors which may be of importance in the induction of tumorigenicity by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), were investigated. Additionally, the effects of dietary sinigrin on NNK tumorigenicity were assessed in a two-year bioassay in F344 rats. DNA methylation in target tissues of NNK tumorigenesis was examined in F344 rats administered [3H-CH3]NNK (0.6 mg/kg, four doses) s.c. and fed control or experimental diets for two weeks. Dietary sinigrin at a concentration of 3 mumol/g diet decreased 7-methylguanine formation in hepatic DNA, but had no effect on 7-methylguanine levels of lung or nasal mucosa DNA. Dietary I3C at a concentration of 30 mumol/g diet increased 7-methylguanine levels in hepatic DNA, but decreased DNA methylation in lung and nasal mucosa. No effects on O6-methylguanine--DNA-transmethylase activity were observed in tissue extracts derived from the livers, lungs and nasal mucosae of rats fed diets containing sinigrin or I3C. These results suggested that dietary sinigrin might reduce the incidence of NNK-induced hepatic tumors with no effect on NNK tumorigenesis of the lung and nasal cavity, whereas I3C might increase hepatic tumor incidence and reduce NNK tumorigenesis of the lung and nasal cavity. The bioassay results showed that dietary sinigrin had no effect on NNK tumorigenesis in these target tissues. However, dietary sinigrin plus NNK resulted in a significant incidence of pancreatic tumors, a rare occurrence in F344 rats. While the results from DNA methylation studies are in agreement with the bioassay data for lung and nasal cavity, the absence of any inhibitory effect of dietary sinigrin on NNK hepatic tumorigenesis indicates that factors other than DNA methylation and O6-methylguanine repair should be considered in assessing the effects of dietary compounds on NNK hepatic tumorigenesis. The contrary effects on NNK-induced hepatic DNA methylation by sinigrin and I3C, two major components of cruciferous vegetables, demonstrate the complexities of dietary modulation of carcinogenesis.

Animals↗
PubMed1988

In vitro genotoxicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone activated by hamster maternal and fetal tissues.

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was found to be a potent genotoxic agent, in the V79 cell line, when activated by liver and lung homogenates from polychlorinated biphenyl (Aroclor)-treated adult Syrian golden hamsters. The damage was observed in the metaphase as well as in the anaphase--telophase divisions. Liver and lung homogenates from non-induced adult hamster were less effective in activating NNK. Micronuclei were also induced by NNK activated by fetal liver and lung homogenates from fetuses on the 15th day of gestation. The observed aberrations: micronuclei, chromatid aberrations particularly chromatid-type exchanges, anaphase bridges with or without acentric fragments, were concentration (8-40 mM NNK)-dependent. A significant number of aberrations were observed. These results suggest that NNK induces chromosome damage when activated by adult and fetal tissues. This damage could play, at least in part, a role in the NNK-induced carcinogenesis observed in vivo and underlines the importance of this compound in the transplacental carcinogenesis induced by tobacco smoke.

Animals↗
PubMed2004

Cytokine production by alveolar macrophages is down regulated by the alpha-methylhydroxylation pathway of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK).

NNK, a nicotine-derived nitrosamine, is a potent lung carcinogen that generates electrophilic intermediates capable of damaging DNA. The effects of NNK on the immune response, which may facilitate lung carcinogenesis, are poorly understood. Alveolar macrophages (AM), a key cell in the maintenance of lung homeostasis, metabolize NNK via two major metabolic activation pathways: alpha-methylhydroxylation and alpha-methylenehydroxylation. We have shown previously that NNK inhibits the production of interleukin-12 (IL-12) and tumor necrosis factor (TNF), but stimulates the production of IL-10 and prostaglandin E(2) (PGE(2)) by AM. In the present study, we investigated the contribution of each activation pathway in the modulation of AM function. We used two precursors, 4-[(acetoxymethyl)-nitrosamino]-1-(3-pyridyl)-1-butanone (NNKOAc) and N-nitro(acetoxymethyl)methylamine (NDMAOAc), which generate the reactive electrophilic intermediates [4-(3-pyridyl)-4-oxo-butanediazohydroxide and methanediazohydroxide, respectively] in high yield and exclusively. Rat AM cell line, NR8383, was stimulated and treated with different concentrations of NNKOAc or NDMAOAc (12, 25 and 50 microM). Mediator release was measured in cell-free supernatants. NNKOAc significantly inhibited the production of IL-10, IL-12, TNF and nitric oxide but increased the release of PGE(2) and cyclooxygenase-2 expression suggesting that the alpha-methylhydroxylation pathway might be responsible for NNK modulation of AM cytokine release. In contrast, NDMAOAc did not modulate AM mediator production. However, none of these precursors, alone or in combination, could explain the stimulation of AM IL-10 production by NNK. Our results suggest that the alpha-methylhydroxylation of NNK leading to DNA pyridyloxobutylation also modulates cytokine production in NNK-treated AM.

Animals↗
PubMed2004

Stereoselective metabolism and tissue retention in rats of the individual enantiomers of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), metabolites of the tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK).

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is reduced to its main metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in a reaction that is both stereoselective and reversible. (S)-NNAL has been shown to be equivalent to NNK in carcinogenic potency, and significantly more potent than (R)-NNAL. It was hypothesized that stereoselective differences in metabolism or tissue distribution contributed to the difference in carcinogenicity between the enantiomers. The individual NNAL enantiomers were therefore administered to bile duct-cannulated rats. Male Fisher F344 rats received i.v. doses of either (R)-NNAL (n = 10) or (S)-NNAL (n = 9) and bile, urine, blood and tissue samples were collected over 24 h. (R)/(S)-NNAL and metabolites were quantified by HPLC and radioflow detection. NNAL was also collected from the HPLC and silylated, and the two NNAL enantiomers were separated by chiral GC-TEA. (S)-NNAL had a much larger tissue distribution (Vss = 1792 +/- 570 ml) than did (R)-NNAL (Vss = 645 +/- 230 ml). Overall, (R)-NNAL tended to enter detoxification pathways, particularly glucuronidation, while reversible metabolism of (S)-NNAL to NNK was favored. For example, after (R)-NNAL administration, approximately 50% of the dose was excreted as (R)-NNAL-Gluc in bile and urine, and <5% was excreted as NNK or NNK metabolites. In contrast, only 10% of an (S)-NNAL dose was excreted as a glucuronide, while almost 20% of the (S)-NNAL dose was excreted as NNK or NNK metabolites. In tissues, particularly the lung, (S)-NNAL appeared to be stereoselectively retained. These findings suggest that the difference in carcinogenicity between the NNAL enantiomers may be attributed to stereoselective differences in tissue distribution and excretion.

Animals↗
PubMed2005

Mechanisms of chemopreventive effects of 8-methoxypsoralen against 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced mouse lung adenomas.

Recently we reported that the occurrence of lung adenoma caused by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was completely prevented by pretreatment of female A/J mice with 8-methoxypsoralen, a potent inhibitor of cytochrome P450 (P450 or CYP) 2A [Takeuchi et al. (2003) Cancer Res., 63, 7581-7583]. Thus, the aim of this study was to confirm that 8-methoxypsoralen exhibits chemopreventive effects by inhibiting CYP2A in the mouse lung. The involvement of CYP2A in the metabolic activation of NNK in the lung was first evidenced by the fact that the mutagenic activation of NNK by mouse lung microsomes was inhibited by 8-methoxypsoralen, coumarin and antibodies to rat CYP2A1. Supporting this, the mutagenic activation of NNK was efficiently catalyzed by mouse CYP2A4 and CYP2A5 co-expressed with NADPH-P450 reductase in a genetically engineered Salmonella typhimurium YG7108. The expression of mRNA for CYP2A5, but not for CYP2A4 or CYP2A12, in the mouse lung was proven by reverse transcriptase-polymerase chain reaction, probably indicating that CYP2A5 present in the mouse lung was involved in the metabolic activation of NNK. In accordance with these in vitro data, treatment of gpt delta transgenic mice with 8-methoxypsoralen prior to NNK completely inhibited the mutation of the gpt delta gene. The in vivo chemopreventive effects of 8-methoxypsoralen towards NNK-induced adenoma was seen only when the agent was given to female A/J mice prior to, but not posterior to, NNK, lending support to the idea that NNK is activated by CYP2A5 in the mouse lung as an initial step to cause adenoma. The inhibition by 8-methoxypsoralen of NNK-induced adenoma was seen in a dose-dependent manner: the dose to show apparent 50% suppression was calculated to be 1.0 mg/kg. To our surprise, CYP2A protein(s) was expressed in the lesion of NNK-induced lung adenomas, probably suggesting that 8-methoxypsoralen could inhibit the possible occurrence of further mutation of the adenoma cells induced by NNK. Based on these lines of evidence, we propose that 8-methoxypsoralen inhibits the CYP2A5-mediated metabolic activation of NNK in the mouse lung, leading to the prevention of NNK-induced adenoma.

Adenoma↗
PubMed2005

Permeation and reservoir formation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and benzo[a]pyrene (B[a]P) across porcine esophageal tissue in the presence of ethanol and menthol.

Environmental influences may affect carcinogen absorption and residency in the tissues of the aero-digestive tract. We quantified the effect of ethanol and menthol on the rates of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and benzo[a]pyrene (B[a]P) absorption using a fully validated in vitro diffusion system, capable of accurately and precisely quantifying tobacco carcinogen permeation and reservoir formation in porcine esophageal mucosa. Confocal microscopy was employed to visualize the location of B[a]P in the exposed membranes. Markedly different extents of permeation and reservoir formation for the tobacco carcinogens were recorded in the presence of ethanol and menthol. The water-soluble NNK permeated the membrane rapidly, while the lipophilic B[a]P did not appreciably diffuse through the tissue. Significantly different extents of reservoir formation were observed for the different carcinogens and in the presence of the different penetration-enhancer solvents. Alcohol (at 5% concentration) did not influence the permeation or reservoir formation of NNK. A mentholated donor solution (0.08%) both decreased the flux of NNK and significantly increased the tissue reservoir formation. The magnitude of the reservoir formed by B[a]P was relatively extensive (even though membrane permeation rates were negligible), being greatest in the presence of both ethanol and menthol. This suggests synergy between the two penetration-enhancer species acting on this carcinogen. Confocal microscopy studies confirmed that there was an appreciable intra-cellular, and specifically nuclear, association of the B[a]P species during the reservoir formation process. The aqueous solubility of the diffusing species and the presence of penetration enhancers appeared to be key factors in the absorption and cellular binding processes. The results presented support the hypothesis that the use of mentholated cigarettes, or the concomitant consumption of alcohol while smoking, may have marked effects on the fate of tobacco chemicals. This finding may help to explain elevated rates of esophageal squamous cell carcinoma in African Americans.

Animals↗
PubMed1997

Recovery from 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced immunosuppression in A/J mice by treatment with nonsteroidal anti-inflammatory drugs.

BACKGROUND: We have previously reported that nonsteroidal anti-inflammatory drugs inhibit lung tumorigenesis induced by the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in mice. PURPOSE: The aims of this study were to determine if NNK suppresses humoral (i.e., antibody) and cellular immune responses in mice and if nonsteroidal anti-inflammatory drugs could attenuate these immune responses. METHODS: Female A/J mice (7-8 weeks old) were fed nonsteroidal anti-inflammatory drugs starting 2 weeks before the beginning of NNK treatment (9.1 mg per mouse in total) and continuing through the 7 weeks of NNK treatment. Eight groups (two control groups and six experimental groups) of 10 mice each were used per experiment. Animals in the two control groups received the same diet and water as animals in the six experimental groups; one control group received no nonsteroidal anti-inflammatory drugs or NNK and the other control group received only NNK. The primary humoral and cellular immune responses to the various treatments were assayed by the plaque-forming cell technique and by measurement of natural killer cell cytotoxic activity, respectively. At the end of each experiment, the animals were killed, blood was collected, plasma was prepared, and levels of the immune system modulator prostaglandin E2 were measured. RESULTS: NNK treatment inhibited the plaque-forming cell response by approximately 50%; this inhibition was attenuated by treatment with sulindac or acetylsalicylic acid (P = .0001 for both). In contrast, treatment with naproxen, which had no chemopreventive (i.e., tumor inhibitory) efficacy, further increased by 26% (P = .05) the immunosuppressive effect of NNK. The cytotoxic activity of splenic natural killer cells against YAC-1 cells was reduced by 60% (P = .002); treatment with acetylsalicylic acid (254 mg/kg of diet) reduced the NNK-induced natural killer cell cytotoxicity inhibition by 50% (P = .02), whereas the administration of the specific cyclooxygenase-2 inhibitor NS-398 (7 mg/kg of diet) resulted in an almost complete recovery (approximately 95%, P = .04) of natural killer cell activity. The prostaglandin E2 plasma concentration was approximately 100% greater in NNK-treated mice than in untreated mice. Treatment of the mice with nonsteroidal anti-inflammatory drugs attenuated this elevation (from approximately 25% to 100%), and NS-398 (7 mg/kg of diet) was the most effective (100%). CONCLUSIONS AND IMPLICATIONS: The ability of various nonsteroidal anti-inflammatory drugs to inhibit NNK-induced carcinogenesis appears to be directly related to the ability of these drugs to inhibit NNK-induced immunosuppression. Our results suggest that the chemopreventive effect of nonsteroidal anti-inflammatory drugs may be mediated through the modulation of prostaglandin E2 synthesis.

Animals↗
PubMed2002

Combined treatment with 4-(N-methyl-N-nitrosamino)-1- (3-pyridyl)-1-butanone and dibutyl phthalate enhances ozone-induced genotoxicity in B6C3F1 mice.

Potential toxicological interactions of 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and/or dibutyl phthalate (DBP) with ozone were investigated. Male and female B6C3F1 mice were exposed to ozone (0.5 p.p.m.), NNK (1.0 mg/kg), DBP (5000 p.p.m.) and different combinations of these toxicants 6 h/day for 16, 32 and 52 weeks. Two cytogenetic end-points, determined by the chromosomal aberration (CA) and supravital micronucleus (SMN) assays, were investigated in vivo. Our results show that all treated groups of both sexes showed genotoxic effects when compared with the control group. Additive and/or synergistic responses were observed in the CA assay for all test periods when mice of both sexes were exposed to ozone and NNK, ozone and DBP and the combination of ozone, NNK and DBP. In the SMN assay, additive interactions were noted for both sexes in the 16 and 32 week studies, similar to the results with the CA assay. All combination groups of both sexes showed synergistic interactions in the 52 week study. The results indicate that combined exposure to ozone, NNK and DBP in both sexes of mice has enhanced genotoxic effects compared with exposure to ozone alone.

Animals↗
PubMed1999

The effect of cotinine or cigarette smoke co-administration on the formation of O6-methylguanine adducts in the lung and liver of A/J mice treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

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 tobacco smoke has not induced or promoted tumors in these mice. NNK-induced lung tumorigenesis is thought to involve O6-methylguanine (O6MeG) formation, leading to GC-->AT transitional mispairing and an activation of the K-ras proto-oncogene in the A/J mouse. NNK can be metabolized by several different cytochromes P450, resulting in a number of metabolites. Formation of the promutagenic DNA adduct O6MeG is believed to require metabolic activation of NNK by cytochrome P450-mediated alpha-hydroxylation of the methylene group adjacent to the N-nitroso nitrogen to yield the unstable intermediate, methanediazohydroxide. Nicotine, cotinine (the major metabolite of nicotine), and aqueous cigarette tar extract (ACTE) have all been shown to effectively inhibit metabolic activation of NNK to its mutagenic form, most likely due to competitive inhibition of the cytochrome P450 enzymes involved in alpha-hydroxylation of NNK. The objective of the current study was to monitor the effects of cotinine and cigarette smoke (CS) on the formation of O6MeG in target tissues of mice during the acute phase of NNK treatment. To test the effect of cotinine, mature female A/J mice received a single intraperitoneal injection of NNK (0, 2.5, 5, 7.5, or 10 mumole/mouse) with cotinine administered at a total dose of 50 mumole/mouse in 3 separate i.p. injections, administered 30 min before, immediately after, and 30 min after NNK treatment. To test the effect of whole smoke exposure on NNK-related O6MeG formation, mice were exposed to smoke generated from Kentucky 1R4F reference cigarettes at 0, 0.4, 0.6, or 0.8 mg wet total particulate matter/liter (WTPM/L) for 2 h, with a single i.p. injection of NNK (0, 3.75, or 7.5 mumole/mouse) midway through the exposure. Cigarette smoke alone failed to yield detectable levels of O6MeG. The number of O6MeG adducts following i.p. injection of NNK was significantly (p < 0.05) reduced in both lung and liver by cotinine and by cigarette smoke exposure. Our results demonstrate that NNK-induced O6MeG DNA adducts in A/J mice are significantly reduced when NNK is administered together with either cotinine, the major metabolite of nicotine, or the parental complex mixture, cigarette smoke.

Animals↗
PubMed2001

Transcriptional regulation of 3,4-dihydroxy-2-butanone 4-phosphate synthase.

The filamentous hemiascomycete Ashbya gossypii is a strong riboflavin overproducer. A striking but as yet uninvestigated phenomenon is the fact that the overproduction of this vitamin starts when growth rate declines, which means that most of the riboflavin is produced in the stationary phase, the so-called production phase. The specific activity of 3,4-dihydroxy-2-butanone 4-phosphate (DHBP) synthase, the first enzyme in the biosynthetic pathway for riboflavin, was determined during cultivation and an increase during the production phase was found. Furthermore, an increase of RIB3 mRNA, encoding DHBP synthase, was observed by competitive RT-PCR in the production phase. The mRNAs of two housekeeping genes, ACT1 (encoding actin) and TEF (encoding translation elongation factor-1 alpha), served as standards in the RT-PCR. Reporter studies with a RIB3 promoter-lacZ fusion showed an increase of beta-galactosidase specific activity in the production phase. This investigation verified that the increase of RIB3 mRNA in the production phase is caused by an induction of promoter activity. These data suggest that the time course of riboflavin overproduction of A. gossypii is correlated with a transcriptional regulation of the DHBP synthase.

Gene Expression Regulation, Fungal↗
PubMed1996

Effects of K-ras gene mutations in the development of lung lesions induced by 4-(N-methyl-n-nitrosamino)-1-(3-pyridyl)-1-butanone in A/J mice.

The relationship between the development of peripheral lung lesions induced by tobacco-specific 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and K-ras gene mutation in A/J mice, and the correlations between histological alterations and the course of lung lesion development after NNK treatment and K-ras gene mutation were investigated. The acquisition of a selective growth advantage by the lung lesions with mutations was also examined using immunohistochemical labeling with bromodeoxyuridine. Thirty female 5 weeks old A/J mice were each injected intraperitoneally with a single dose of NNK (100 mg/kg body weight) and subdivided into 6 groups according to the time after NNK treatment. The lung lesions were characterized histologically as alveolar/bronchiolar hyperplasia, adenoma and adenocarcinoma, and point mutations in codons 12 and 61 of the K-ras gene were detected by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) and dideoxy sequencing methods. K-ras gene mutations were identified in 7 (58.3%) of 12 hyperplasias, 42(75.0%) of 56 adenomas and 3 (75.0%) of 4 adenocarcinomas. The most frequent K-ras gene mutation was a G-to-A transition at the second base of codon 12 and this accounted for 86.5% of all the mutations detected. Neither the frequency of activation of this gene nor the specific mutation was affected by the time after NNK treatment and there was no positive correlation between the proliferative activity of lung lesions and the presence of K-ras gene mutations. Thus, K-ras gene mutation is closely associated with the development of NNK-induced peripheral lung lesions in A/J mice, but it plays no role in the selective growth advantage of these lesions.

Animals↗
PubMed1996

The identification of 11 beta-hydroxysteroid dehydrogenase as carbonyl reductase of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NC), which is generated by nitrosation of nicotine, requires enzymatic activation by cytochrome-P450-mediated alpha-carbon hydroxylation to yield particularly powerful carcinogenic alkylating intermediates. Pyridine-N-oxidation and carbonyl reduction are detoxification pathways, the latter by providing the functional hydroxy moiety necessary for glucuronosylation and final excretion of NC. For more than a decade, the enzyme responsible for NC carbonyl reduction has awaited characterization. In the present study, we demonstrate that the NC carbonyl reductase is identical to 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD), the physiological function of which is the oxidoreduction of glucocorticoids. We conclude that the expression of 11 beta-HSD (together with glucuronosyl transferase) may have profound influence on the carcinogenic potency of NC and that many compounds of endogenous and exogenous origin, which are known to be substrates or inhibitors of 11 beta-HSD, may modulate NNK-induced carcinogenicity by competing for the same enzyme. In light of the known species and tissue differences in the expression of 11 beta-HSD isozymes, important aspects of NNK-induced tumorigenesis, such as metabolic activation versus inactivation or organospecificity, can now be re-evaluated.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed2004

Identification of critical amino acid residues of human CYP2A13 for the metabolic activation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, a tobacco-specific carcinogen.

Among all the known human cytochrome P450 enzymes, CYP2A13 has the highest efficiency in catalyzing the metabolic activation (keto aldehyde and keto alcohol formation) of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a potent lung carcinogen in animals and a suspected human lung carcinogen. As part of the structure-activity relationship (SAR) study, the present work was done to identify the key amino acid residues in CYP2A13 that are responsible for this high catalytic efficiency by using a series of mutants (Ala117Val, His164Gly, Ser208Ile, His372Arg, and Pro465Ser). In these CYP2A13 mutants, the amino acid residues were substituted by the residues at the corresponding positions of CYP2A6, which shares 93.5% amino acid sequence identity with CYP2A13 but is significantly less active (<5%) than CYP2A13 in NNK alpha-hydroxylation. We demonstrated that, except for the His164Gly mutant, all the CYP2A13 mutant proteins showed a significant decrease in the catalytic efficiency (Vmax/Km) for NNK alpha-hydroxylation. The His372 to Arg substitution resulted in a 20-fold increase in the Km value and a 7-fold decrease in the Vmax value for keto aldehyde formation as well as a total loss of detectable keto alcohol formation. The Ala117 to Val substitution, however, only caused a selective decrease in the Vmax value for keto aldehyde formation. The role of these amino acid residues in CYP2A13-catalyzed reactions is clearly substrate-dependent, since the same Ala117Val and His372Arg mutants showed a 9-fold increase in the catalytic efficiency for coumarin 7-hydroxylation. Together with the computational substrate docking, our study provides new SAR in formation of human CYP2A13.

Amino Acid Substitution↗
PubMed2005

Nicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-butanone metabolism by cytochrome P450 2B6.

Nicotine is the major addictive agent in tobacco; it is primarily metabolized by 5'-oxidation. 4-(Methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK), a potent lung carcinogen generated from nicotine during the curing of tobacco, is metabolically activated by P450 enzymes. P450 2A6 is the primary hepatic catalyst of nicotine metabolism and also catalyzes NNK alpha-hydroxylation, albeit less efficiently. It was previously reported that P450 2B6 catalyzed nicotine 5'-oxidation. The studies presented here investigate the relative importance of P450 2B6 as a catalyst of nicotine 5'-oxidation and NNK alpha-hydroxylation by human liver microsomes (HLMs). Radioflow high-performance liquid chromatography analysis and tritiated substrates were used to monitor the products of nicotine and NNK metabolism. The primary product of P450 2B6-catalyzed nicotine metabolism was the delta1'(5') iminium ion. The only other metabolite detected was nornicotine, the product of methyl oxidation, formed at about one-fourth the rate of the delta1' (5') iminium ion. We determined that P450 2B6 was a much less efficient catalyst of nicotine 5'-oxidation than previously reported, with an estimated K(m) of 820 microM. In contrast, the K(m) of NNK alpha-hydroxylation was 33 muM. Experiments with P450 2A6- and P450 2B6-selective inhibitory antibodies did not support P450 2B6 as a significant catalyst of nicotine 5'-oxidation by HLMs, and it is unlikely that this enzyme contributes to nicotine metabolism in smokers who express P450 2A6. However, P450 2B6 contributed significantly to NNK metabolism in HLMs expressing both P450 2B6 and P450 2A6, suggesting a possible role for P450 2B6 in NNK metabolic activation.

Aryl Hydrocarbon Hydroxylases↗
PubMed2003

Comparative metabolism of the tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol by rat cytochrome P450 2A3 and human cytochrome P450 2A13.

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its carbonyl-reduction product, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), are potent lung carcinogens in rats and are presumed human lung carcinogens. NNK and NNAL are bioactivated to DNA-binding intermediates via hydroxylation of the carbon atoms adjacent to the nitroso moiety (i.e., alpha-hydroxylation) by cytochrome p450s (p450s). Therefore, it is important to delineate which p450s are efficient catalysts of this metabolic transformation. In this study, the kinetic parameters for NNK and NNAL metabolism were determined for two extrahepatic p450s that are expressed in the lung: rat p450 2A3 and human p450 2A13. p450s 2A3 and 2A13 exhibited Vmax values for NNK 4-hydroxylation of 10.8 +/- 0.4 and 13.8 +/- 0.8 pmol min-1 pmol P450-1, respectively; the corresponding Km values were 4.6 +/- 0.5 and 3.6 +/- 0.7 microM. The respective Vmax values for p450 2A3- and 2A13-mediated N-methyl hydroxylation of NNK were 8.2 +/- 0.3 and 4.6 +/- 0.2 pmol min-1 pmol p450-1. These data indicate that p450s 2A3 and 2A13 are both efficient catalysts of the metabolic activation of NNK and are, along with mouse p450 2A5, the best catalysts of this reaction currently known. Both enzymes also catalyzed the alpha-hydroxylation and N-oxidation of NNAL, and its oxidation to NNK. In general, Vmax/Km values for NNAL metabolism were 1 to 2 orders of magnitude lower than those for NNK metabolism, and p450 2A3 was a slightly better catalyst of NNAL metabolism than was p450 2A13. Given the exquisite sensitivity of the rat lung to NNK-induced carcinogenesis, the efficient bioactivation of NNK by rat p450 2A3, and the similar catalytic efficiency of p450s 2A3 and 2A13, p450 2A13 may be an important contributor to NNK bioactivation in the human lung.

Animals↗
PubMed2004

The modulating role of nuclear factor-kappaB in the action of alpha7-nicotinic acetylcholine receptor and cross-talk between 5-lipoxygenase and cyclooxygenase-2 in colon cancer growth induced by 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone.

4-(N-Methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), the tobacco-specific nitrosamine, induces lung cancer in all animal species tested and is thought to contribute significantly to the high lung cancer burden associated with smoking. However, there is no report whether NNK could promote colon cancer growth. To address this hypothesis and the possible signaling pathways involved, we used SW1116 colon cancer cell line to study these biological events in vitro. Results showed that NNK, after 5-h treatment, stimulated cell proliferation, enhanced alpha7-nicotinic acetylcholine receptor (alpha7-nAChR) mRNA levels and nuclear factor-kappaB (NF-kappaB) DNA binding activity, as well as 5-lipoxygenase and cyclooxygenase-2 protein expressions. alpha-Bungarotoxin, the specific alpha7-nAChR antagonist, inhibited these biological effects. However, 5-lipoxygenase inhibition had no effect on alpha7-nAChR mRNA expression, but significantly inhibited cell proliferation and activation of NF-kappaB and cyclooxygenase-2, whereas NF-kappaB-specific inhibitor caffeic acid phenethyl ester reduced both cell proliferation and cyclooxygenase expression induced by NNK without affecting alpha7-nAChR mRNA level and 5-lipoxygenase expression. Together, the present study demonstrated that NNK promoted colon cancer growth in vitro. NF-kappaB not only conveys the biological effect of alpha7-nAChR activation but is also involved in the cross-talk between 5-lipoxygenase and cyclooxygenase-2 in response to NNK in colon cancer cell development.

Bungarotoxins↗
PubMed2001

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.

Animals↗
PubMed2005

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone from cigarette smoke stimulates colon cancer growth via beta-adrenoceptors.

Cigarette smoking is a risk factor for colorectal cancer. It is suggested that 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific nitrosamine, mediates the carcinogenic action of cigarette smoking by promoting cancer growth. In the present study, the proliferative response of a cultured colon cancer cell line HT-29 to NNK was determined. It was found that NNK dose-dependently stimulated HT-29 cell proliferation. In this regard, the stimulatory action of NNK was abolished by atenolol and ICI 118,551, a beta1- and beta2-selective antagonist, respectively. In addition, cell growth was stimulated by the nonselective adrenergic agonist, noradrenaline, and more effectively by the beta-selective agonist, isoproterenol. The second message cyclic AMP level for beta-adrenoceptor activation was elevated by isoproterenol and NNK treatment. These agents also up-regulated cyclooxygenase-2 expression, cytosolic phospholipase A2 expression, and prostaglandin E2 release. Beta2-adrenoceptor blockade with ICI 118,551, in contrast, significantly decreased cyclooxygenase-2 expression, cytosolic phospholipase A2 expression and prostaglandin E2 release induced by NNK and isoproterenol. To conclude, it is proposed that NNK stimulates HT-29 cell proliferation through beta-adrenoceptors, preferentially beta2 receptors. Activation of the beta-adrenoceptors, and the consequent cyclic AMP elevation coupled with the downstream arachidonic acid pathway, is perhaps an important mechanistic cascade in the promotion of colon cancer growth. These findings partly elucidate the carcinogenic actions of cigarette smoke and shed new light on the novel modulatory role of beta-adrenoceptors in the development of colon cancer.

Adrenergic beta-Agonists↗