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.     

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.     

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.     

The effect of tobacco smoke, nicotine, and cotinine on the mutagenicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL).

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) is a rodent carcinogen that is metabolically derived from carbonyl reduction of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). NNAL can be pyridine N-oxidized to form NNAL-N-oxide, or conjugated to form NNAL-glucuronide - non-genotoxic metabolites that can be excreted in urine. Alternatively, NNAL can be alpha-hydroxylated at the methyl and methylene carbons adjacent to the nitroso group to generate electrophiles that can react with biological macromolecules, such as DNA and proteins. Our laboratory has previously demonstrated that the mutagenicity of NNK was significantly inhibited by the aqueous extract of tobacco smoke, as well as pyridine alkaloids in cigarette smoke, such as nicotine, cotinine and nornicotine. Given the structural similarity between NNK and NNAL, and the metabolic activation of both by cytochromes P450, we hypothesized that there may be a similar inhibition of NNAL metabolism, and consequently, inhibition of the mutagenic activity of NNAL by tobacco smoke and its pyridine alkaloid constituents. In the present study, we evaluated the ability of two pyridine alkaloids (nicotine and cotinine) and aqueous cigarette smoke condensate extract (ACTE) to inhibit the mutagenicity of NNAL in Salmonella typhimurium strain TA1535 in the presence of a metabolic activation system (S9). Both pyridine alkaloids tested, as well as ACTE, inhibited the mutagenicity of NNAL in a concentration-dependent manner. The observed reductions in mutagenicity were not the result of cell killing due to cytotoxicity. These results demonstrate that tobacco smoke contains pyridine alkaloids, as well as other unidentified constituents that inhibit the mutagenicity of NNAL, a major metabolite of NNK.     

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.     

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.     

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.     

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.     

DNA adduct formation from tobacco-specific N-nitrosamines

Tobacco-specific N-nitrosamines are a group of carcinogens derived from the tobacco alkaloids. They are likely causative factors for cancers of the lung, esophagus, pancreas, and oral cavity in people who use tobacco products. The most carcinogenic tobacco-specific nitrosamines in laboratory animals are 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and N'-nitrosonornicotine (NNN). DNA adduct formation from NNK and NNN has been studied extensively and is reviewed here. NNK is metabolically activated by cytochromes P450 to intermediates which methylate and pyridyloxobutylate DNA. The resulting adducts have been detected in cells and tissues susceptible to NNK carcinogenesis in rodents. The methylation and pyridyloxobutylation pathways are both important in carcinogenesis by NNK. NNK also induces single strand breaks and increases levels of 8-oxodeoxyguanosine in DNA of treated animals. NNAL, which like NNK is a potent pulmonary carcinogen, is also metabolically activated to methylating and pyridyloxobutylating intermediates. NNN pyridyloxobutylates DNA in its rat target tissues, esophagus and nasal mucosa. Methyl and pyridyloxobutyl DNA adducts are detected in human tissues. The methyl adducts most likely result in part from exposure of smokers to NNK, but these adducts are also detected in non-smokers. Some of the methyl adducts detected in non-smokers may be due to environmental tobacco smoke exposure. There are also potential dietary and endogenous sources of these adducts. Pyridyloxobutyl DNA adducts in human tissues result mainly from exposure to tobacco-specific N-nitrosamines. In laboratory animals, DNA adduct formation and carcinogenicity of tobacco-specific N-nitrosamines are closely correlated in many instances, and it is likely that similar relationships will hold in humans.     

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.     

Cytotoxicity, sister-chromatid exchanges and DNA single-strand breaks induced by 4-oxo-4-(3-pyridyl)butanal, a metabolite of a tobacco-specific N-nitrosamine

The tobacco-specific N-nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is metabolized by alpha-carbon hydroxylation to reactive diazohydroxides and aldehydes. The aim of this study was to determine the relative ability of one NNK-derived aldehyde, 4-oxo-4-(3-pyridyl)butanal, to induce cytotoxicity, sister-chromatid exchanges (SCEs) and DNA single-strand breaks (SSBs) in V79 cells. Our data demonstrate that this aldehyde is cytotoxic for V79 cells (IC50 = 0.4 mM) and induces SCEs at concentrations ranging from 0.01 to 0.5 mM. DNA SSBs were observed at concentrations ranging from 0.05 to 1 mM and were repaired within 8 h. When V79 cells were cultured with primary hepatocytes, there was a reduction in the frequency of SCEs induced by the aldehyde. This suggests that hepatocytes can partially deactivate the aldehyde. Our results suggest that this aldehyde is one of the reactive intermediates generated during NNK metabolism.     

Cytotoxicity, genotoxicity and transforming activity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in rat tracheal epithelial cells.

The cytotoxicity, genotoxicity and transforming activity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were studied by the assays of colony-forming efficiency (CFE), micronucleus formation (MN), and cell transformation in rat tracheal epithelial (RTE) cells both in vitro and in vivo. Liver S9, primary hepatocytes and RTE cells from normal and Aroclor-1254 induced rats were compared for bioactivation of NNK using Salmonella mutagenesis as the endpoint. Results from the in vitro experiments indicated that low concentrations of NNK (0.01-25 micrograms/ml) caused from 15% to greater than 100% increases in CFE of RTE cells. At high concentrations (100-200 micrograms/ml), NNK was significantly toxic to RTE cells. NNK treatment in vitro (50-200 micrograms/ml) increased MN frequency as much as 3-fold above background and significantly increased the transformation frequency (TF) in 4/5 (50 micrograms/ml) and 6/8 (100 micrograms/ml) experiments. The in vivo exposure of rats to NNK (150-450 mg/kg, given i.p.) resulted in a 60-85% reduction in CFE and a 3-5-fold increase in MN formation in RTE cells. In vivo treatment with cumulative doses of 150 and 300 mg/kg of NNK produced significant increases in TF of tracheal cells from 3/3 and 2/3 rats, respectively. Without activation, NNK was not mutagenic in Salmonella TA1535. The bioactivation of NNK to a mutagenic metabolite was achieved by incubation of NNK with liver S9 fraction from Aroclor-1254 induced rats or primary hepatocytes from both untreated and Aroclor-1254 pretreated rats. RTE cells did not produce sufficient quantities of mutagenic NNK metabolites to be detected by the Salmonella assay.     

Transport of the beta -O-glucuronide conjugate of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) by the multidrug resistance protein 1 (MRP1). Requirement for glutathione or a non-sulfur-containing analog

Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) play a crucial role in the induction of lung cancer, and NNAL-O-glucuronide formation and elimination are important steps in detoxification of these compounds. In the present study, we investigated the ATP-binding cassette (ABC) protein, MRP1 (ABCC1), as a candidate transporter responsible for NNAL-O-glucuronide export. MRP1 mediates the active transport of numerous GSH-, sulfate-, and glucuronide-conjugated organic anions and can transport certain xenobiotics by a mechanism that may involve co-transport with GSH. Using membrane vesicles prepared from transfected cells, we found that MRP1 transports [3H]NNAL-O-glucuronide but is dependent on the presence of GSH (Km 39 microm, Vmax 48 pmol x mg(-1) x min(-1)). We also found that the sulfur atom in GSH was dispensable because transport was supported by the GSH analog, gamma-glutamyl-alpha-aminobutyryl-glycine. Despite stimulation of NNAL-O-glucuronide transport by GSH, there was no detectable reciprocal stimulation of [3H]GSH transport. Moreover, whereas the MRP1 substrates leukotriene C4 (LTC4) and 17beta-estradiol 17beta-(d-glucuronide) (E(2)17betaG) inhibited GSH-dependent uptake of [3H]NNAL-O-glucuronide, only [3H]LTC4 transport was inhibited by NNAL-O-glucuronide (+GSH) and the kinetics of inhibition were complex. A mutant form of MRP1, which transports LTC4 but not E(2)17betaG, also did not transport NNAL-O-glucuronide suggesting a commonality in the binding elements for these two glucuronidated substrates, despite their lack of reciprocal transport inhibition. Finally, the related MRP2 transported NNAL-O-glucuronide with higher efficiency than MRP1 and unexpectedly, GSH inhibited rather than stimulated uptake. These studies provide further insight into the complex interactions of the MRP-related proteins with GSH and their conjugated organic anion substrates, and extend the range of xenotoxins transported by MRP1 and MRP2 to include metabolites of known carcinogens involved in the etiology of lung and other cancers.     

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone modulation of cytokine release in U937 human macrophages

 The nicotine-derived N-nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is one of the most abundant and potent carcinogens found in tobacco smoke. NNK induces lung tumors in rodents and is most likely involved in lung carcinogenesis in humans. Studies on the metabolism and carcinogenicity of NNK have been extensive. However, its effects on the immune system have not been investigated thoroughly. Considering that tobacco smoking partially suppresses the immune response in humans, and that immune surveillance plays a critical role in cancer development, we examined the effects of NNK on the production of selected cytokines. In a previous study, we observed an inhibition of NK cell activity and IgM secretory cell number in NNK-treated A/J mice [Rioux and Castonguay (1997) J Natl Cancer Inst 89: 874]. In this study, we demonstrate that U937 human macrophages activate NNK to alkylating intermediates by α-carbon hydroxylation and detoxify NNK by N-oxidation. We observed that NNK, following activation, induces the release of soluble tumor necrosis factor (TNF), but inhibits interleukin(IL)-10 synthesis. We also report that 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone, and nitroso(acetoxymethyl)methylamine, which generate the same alkylating intermediates as NNK, have similar effects on TNF and IL-10. This suggests that pyridyloxobutylating and methylating intermediates generated from NNK are potent modulators of the immune response. The levels of IL-6, granulocyte/macrophage-colony-stimulating factor and macrophage chemotactic protein 1 were also decreased in supernatants of NNK-treated U937 macrophages. In contrast, IL-2 synthesis in Jurkat cells was inhibited by NNK treatment. This is the first study demonstrating that NNK, via its alkylating intermediates, alters the cytokine synthesis profile in human cells. Modulation of cytokine synthesis by NNK might partially explain the immunosuppresion observed in smokers. Inhibition of immune functions, resulting from NNK activation to alkylating agents, may facilitate lung tumor development. Received: 3 February 2000 / Accepted: 15 September 2000     

Tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in smokers in the united states: NHANES 2007–2008

The tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), a metabolite of the tobacco-specific nitrosamine (TSNA) 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), has been measured in urine samples from all participants aged 6 years and older from the National Health and Nutrition Examination Survey 2007–2008. Participants with a serum cotinine concentration of ≥10?ng/mL were identified as tobacco users, primarily cigarette smokers. Regression models were developed to calculate geometric mean NNAL concentrations adjusted for serum cotinine, urinary creatinine, cigarettes per day, and Federal Trade Commission tar values of the cigarettes smoked. Significant differences were found by gender (p?=?0.003) and race/ethnicity (p?=?0.022 for non-Hispanic white versus non-Hispanic black smokers), but not by menthol type of the cigarettes. Females and non-Hispanic white smokers had the highest adjusted means for urinary NNAL (353 and 336 pg/mL, respectively). The results from this study demonstrated significant relationships between NNAL concentrations and serum cotinine (p?<?0.001) and urine creatinine (p?<?0.001). The joint effect of linear and quadratic terms for number of cigarettes smoked per day was also statistically significant (p?=?0.001). In addition to addressing current NNK exposure levels, these results will form a baseline for future estimates of tobacco users’ exposure to this carcinogen.     

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.     

Simultaneous determination of four tobacco-specific N-nitrosamines (TSNA) in human urine

Tobacco-specific N-nitrosamines (TSNA) include 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosonornicotine (NNN), N-nitrosoanabasine (NAB) and N-nitrosoanatabine (NAT). TSNA are suggested to play an important role in tobacco smoke carcinogenesis. We have developed and validated an LC–MS/MS method for the determination of total (free and conjugated) TSNA in human urine. The limits of detection (LOD) were 2.0, 0.8, 1.1 and 0.7 pg/ml for NNAL, NNN, NAB and NAT, respectively. Smokers were found to have significantly higher levels of TSNA in their urine than nonsmokers. In conclusion, the newly developed method is suitable for assessing the tobacco use-related exposure to NNK, NNN, NAB and NAT.     

High-performance liquid chromatographic analysis of metabolites of the nicotine-derived nitrosamines, N'-nitrosonornicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

An improved high-performance liquid chromatographic system was developed for separation of 11 metabolites of the nicotine-derived nitrosamines N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). The new system employed a 5-microns octadecylsilane bonded column eluted with aqueous sodium acetate-methanol gradients of varying pH. Analysis times were typically 30 min for NNN metabolites and 50 min for NNK metabolites, compared to 80 and 90 min, respectively, when 10-microns columns were used. The E and Z isomers of all nitrosamine-containing metabolites of NNK were separated. The chromatographic behavior of the 11 metabolites as well as NNN and NNK was studied between pH 4.0 and 7.5. The retention times of several metabolites were altered significantly as a function of pH. The results of the pH study provide valuable additional criteria for metabolite identification as well as optimized conditions for their separation. Applications of the system to the metabolism of [2'-14C]NNN in cultured rat esophagus and [carbonyl-14C]NNK in rat liver slices are presented.