Cell selective alkylation of DNA in rat lung following low dose exposure to the tobacco specific carcinogen 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone

The molecular dosimetry of O6-methylguanine (O6MG) in DNA from lung and specific cell populations isolated from lung was determined during multiple administrations of the tobacco specific carcinogen 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) to Fischer 344 rats. O6MG accumulated with doses of NNK ranging from 0.1 to 100 mg/kg/day. The dose response for NNK was nonlinear; the O6MG to dose ratio, an index of alkylation efficiency, increased dramatically as the dose of carcinogen decreased. These data suggest that low and high Km pathways may exist for activation of NNK to a methylating agent. Marked differences in O6MG concentration were observed in specific lung cell populations. The Clara cell, one of the suggested progenitor cells for nitrosamine-induced neoplasia, was found to possess the greatest concentration of O6MG. Moreover, as the dose of NNK was decreased from 100 to 0.3 mg/kg, the alkylation efficiency in this cell population increased 38-fold. The high level of DNA adduct formation in Clara cells following low dose exposure to NNK was supported by autoradiographic studies. Four h after treatment with 1 mg/kg [3H]NNK, silver grains were more heavily concentrated over Clara cells than over other cell types in the lung. Comparative studies on dimethylnitrosamine, a weak carcinogen in the rat lung, did not demonstrate this cell specificity for DNA alkylation. Thus, the presence of a high affinity pathway in the Clara cell for activation of NNK may contribute to the carcinogenicity of this tobacco specific carcinogen.     

Metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in human lung and liver microsomes and cytochromes P-450 expressed in hepatoma cells.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a potent tobacco-specific carcinogen in animals, has been linked to tobacco-related cancers in humans. The cytochrome(s) P-450 (P-450) responsible for the metabolic activation of NNK in humans has not been identified. The present work investigated the ability of human lung and liver microsomes and 12 forms of human P-450, expressed in Hep G2 (hepatoma) cells, to metabolize NNK. Of the 12 P-450 forms, P-450 1A2 had the highest activity in catalyzing the conversion of NNK to the keto alcohol, 4-hydroxy-1-(3-pyridyl)-1-butanone. P-450s 2A6, 2B7, 2E1, 2F1, and 3A5 also had measurable activities in the formation of keto alcohol. The apparent Km and Vmax for the formation of keto alcohol in the P-450 1A2-expressed Hep G2 cell lysate were 309 microM and 55 pmol/min/mg protein, respectively. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol, a reductive product, was the major metabolite formed, whereas the formation of keto alcohol and its aldehyde and acid derivatives (all alpha-hydroxylation products) constituted approximately 1% of the initial amount of NNK in P450-expressed Hep G2 cell lysate. A similar metabolite pattern was observed with human lung or liver microsomes. In human lung microsomes, the apparent Kms for the formation of 4-hydroxy-4-(3-pyridyl)butyric acid, 4-oxo-1-(3-pyridyl)-1-butanone, NNK-N-oxide, and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol were 526, 653, 531, and 573 microM, respectively; the formation of keto alcohol was not observed. For human lung microsomes, there was no sex-related difference in NNK metabolism. Carbon monoxide (90% atmosphere) significantly inhibited the metabolism of NNK in human lung and liver microsomes. 7,8-Benzoflavone, an inhibitor of P-450s 1A1 and 1A2, had no effect on NNK metabolism in human lung microsomes but decreased the formation of keto alcohol by 47% in human liver microsomes. Similarly, antibodies against human P-450s 1A2 and 2E1 decreased keto alcohol formation by 42% and 53%, respectively, in human liver microsomes but did not affect NNK metabolism in lung microsomes. Inhibitory antibodies against P-450s 2A1, 2C8, 2D1, or 3A4 had little or no effect on the metabolism of NNK in human liver or lung microsomes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Dose-response relationship between O6-methylguanine formation in Clara cells and induction of pulmonary neoplasia in the rat by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

The relationship between the formation of O6-methylguanine (O6MG) and the induction of lung, liver, and nasal tumors in the Fisher 344 rat by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was examined in a dose-response study. Animals were treated for 20 wk (3 times/wk) with concentrations of NNK ranging from 0.03 to 50 mg/kg to induce tumors. Steady-state concentrations of O6MG were quantitated, and cytotoxicity was assessed in target cells and tissues after 4 wk of treatment with NNK. No cytotoxicity was detected in the lung during treatment with NNK. The formation of O6MG was greatest in Clara cells compared with macrophages, type II cells, small cells, and whole lung at all doses examined. The difference in adduct concentration between the Clara cell and other pulmonary cell types was most pronounced with low doses of carcinogen. The O6MG:dose ratio, an index of alkylation efficiency, increased 29-fold as the dose of NNK was decreased from 50 to 1 mg/kg of carcinogen. In contrast, only a small increase in alkylation efficiency was observed in type II cells and whole lung. A significant number of tumors were induced in the lung at doses of 0.1 to 50 mg/kg with incidences ranging from 10% at the lowest dose up to 87% in the group of animals which received 50 mg/kg of NNK. A linear relationship was observed when the concentration of O6MG in Clara cells as a function of dose was plotted against the corresponding tumor incidence. This relationship was not observed using DNA adduct concentrations in type II cells or whole lung. The development of pulmonary tumors appeared to involve the formation of alveolar hyperplasias which progressed to adenomas and finally to carcinomas. The majority of adenomas were solid, whereas carcinomas were mainly papillary. Examination of the ultrastructure of the hyperplasias, adenomas, and carcinomas revealed morphological structures (e.g., lamellar bodies, tubular myelin) which are associated with type II cells. Thus, these data suggest that the majority of neoplasms in the lung begin as type II cell proliferations with progression to adenomas and carcinomas within the areas of hyperplasia. The lack of agreement between biochemical and morphological findings makes it difficult to hypothesize a cell of origin for the pulmonary neoplasms. In contrast to the lung, tumors were induced in the liver and nasal passages only after exposure to high doses of NNK. Moreover, both the formation of DNA adducts and cytotoxicity appear obligatory for the generation of tumors in these tissues.(ABSTRACT TRUNCATED AT 400 WORDS)     

Molecular dosimetry of O6-methylguanine formation and cell toxicity in lung and nasal mucosa by 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone

The molecular dosimetry of O6-methylguanine (O6-meG) in DNA from lung and nasal mucosa was determined during administration of 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK) to Fischer 344 rats. O6-MeG accumulated in lung during 12 days of treatment with doses of NNK ranging from 0.1 to 100 mg/kg per day. The dose-response to NNK was nonlinear; the ratio of O6-meG to dose, an index of alkylation efficiency, increased dramatically as the dose of carcinogen decreased. These data suggest that high and low Km pathways may exist for activation of NNK to a methylating agent. Clara cells, when compared to Type-II cells, macrophages and alveolar small cells, were found to possess the greatest concentration of O6-meG. Moreover, as the dose of NNK was decreased, a marked increase in the alkylation efficiency of Clara cells was observed. Thus, the presence of a high-affinity, low-Km pathway in Clara cells for activation of NNK may be a significant factor in the carcinogenicity of this tobacco-specific carcinogen. The dose-response for O6-meG differed considerably between respiratory and olfactory mucosa. The dose-response to NNK was nonlinear in respiratory mucosa and linear in the olfactory mucosa, and the concentration of O6-meG was five times greater in respiratory than in olfactory mucosa after treatment with 1 mg/kg NNK. As the dose of NNK was increased, alkylation in the two regions of the nose became similar. Histological examination of the nasal passages following treatment with NNK indicated that the olfactory region was more sensitive than the respiratory region to toxicity induced by NNK.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

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

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

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

Metabolic activation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone as measured by DNA alkylation in vitro and its inhibition by isothiocyanates

The bioactivation of the tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), by microsomes from target organs was studied with an in vitro microsome-mediated DNA alkylation system. Mouse lung, rat lung, and rat nasal microsomes catalyzed a time- and protein-dependent DNA methylation by [methyl-3H]NNK with activities of 4.11, 0.95, and 137.4 pmol/mg DNA/mg protein/h, respectively. The DNA methylation of NNK catalyzed by all three microsomal systems was inhibited by cytochrome P-450 inhibitors, such as carbon monoxide and metyrapone, but not by the cyclooxygenase inhibitor, aspirin, or by prolonged preincubation in the absence of NADPH. The possible involvement of specific P450 isozymes was assessed by specific inhibitory antibodies. An anti-P450IIB1&2 antibody significantly inhibited the DNA methylation by 45 and 32% in mouse lung and rat lung, respectively, whereas anti-P450IA1 and anti-P450IIE1 antibodies failed to show significant inhibition. All antibodies showed no inhibition in rat nasal microsomes. Glutathione inhibited the DNA methylation in a concentration-dependent manner in all three microsomal systems. Phenethyl isothiocyanate (PEITC), at doses of 0.25 and 1.00 mmol/kg body weight, was given intragastrically 2 h before sacrifice to mice and 24 h before sacrifice to rats, respectively; both mouse and rat lung microsomal activities were inhibited by about 40 and 90% by the low- and high-dose PEITC treatments, respectively. The rat nasal microsomes were only inhibited by the high-dose PEITC treatment by about 40%. PEITC, 4-phenylbutyl isothiocyanate, and 6-phenylhexyl isothiocyanate all inhibited the microsome-mediated DNA methylation of NNK in vitro, with 4-phenylbutyl isothiocyanate and 6-phenylhexyl isothiocyanate being more potent than PEITC and the mouse lung microsomes more sensitive than the rat lung and nasal microsomes. All three microsomal systems were shown to catalyze the in vitro DNA pyridyloxobutylation by [5-3H]NNK. On an equal protein basis, the rat nasal microsomes were much more active in catalyzing the DNA pyridyloxobutylation.     

Comparative tumorigenicity and DNA methylation in F344 rats by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N-nitrosodimethylamine

The tumorigenic activities and DNA methylating abilities in F344 rats of the tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and the structurally related nitrosamine N-nitrosodimethylamine (NDMA) were compared. Groups of 30 male rats were given 60 s.c. injections of 0.0055 mmol/kg of either NNK or NDMA over a 20-week period (total dose, 0.33 mmol/kg). The experiment was terminated after 104 weeks. The numbers of rats with tumors were as follows for NNK and NDMA, respectively: liver, 10 and 6; lung 13 and 0; and nasal cavity, 6 and 1. NNK was significantly more tumorigenic than was NDMA toward the lung (P less than 0.01) and nasal cavity (P less than 0.05). Groups of rats were treated with a single s.c. injection of 0.39 mmol/kg or 0.055 mmol/kg of NNK or NDMA and the levels of 7-methylguanine and O6-methylguanine were measured in liver, lung, and nasal mucosa 1-48 h after treatment. In liver and lung, levels of 7-methylguanine and O6-methylguanine in DNA were 3-22 times (P less than 0.001) greater in NDMA treated rats than in NNK treated rats. Levels of methylation induced by NDMA and NNK in the nasal mucosa were similar. The results of this study demonstrate that NNK is a more potent tumorigen than NDMA in the F344 rat and suggest that DNA methylation alone does not account for its strong tumorigenicity in rat lung and nasal mucosa.     

Effects of indole-3-carbinol on lung tumorigenesis and DNA methylation induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and on the metabolism and disposition of NNK in A/J mice

The effects of indole-3-carbinol (I3C) on lung neoplasia induced by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were assessed in an A/J mouse pulmonary adenoma bioassay. Mice were administered corn oil or I3C (25 or 125 mumol/mouse/day) by gavage for 4 consecutive days. Two h after the final pretreatment, mice were administered a single dose of NNK (10 mumol/mouse) i.p. Pulmonary adenomas were quantitated 16 wk after NNK dosing. Mice pretreated with corn oil developed 10.7 tumors/mouse; I3C pretreatment at either dose level inhibited tumor multiplicity by approximately 40%. The effects of I3C on NNK-induced DNA methylation in the lungs and livers of A/J mice were assessed using the same dosing regimen as in the bioassay. Both dose levels of I3C inhibited pulmonary O6-methylguanine formation by at least 50%, but enhanced hepatic DNA methylation at 2 or at 6 h after NNK administration. The effects of I3C pretreatment on NNK metabolism were also investigated. Hepatic microsomes of I3C-pretreated mice showed increased formation of alpha-hydroxylation products, while no significant effect of I3C pretreatment was observed in pulmonary microsomes. The effects of I3C on [5-3H]NNK disposition were also evaluated. I3C pretreatment produced lower levels of total radioactivity in the lung when compared with controls. Additionally, lower proportions of NNK and its carcinogenic metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol were found in the lungs of I3C-pretreated mice. These results demonstrate that I3C inhibits NNK-induced lung neoplasia in A/J mice and suggest that the basis of this inhibition is the decrease in O6-methylguanine formation in A/J lung caused by I3C pretreatment. This decrease in lung DNA methylation appears to be due to the decreased bioavailability of NNK and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in the lungs of I3C-treated mice which, in turn, may be a result of increased metabolic alpha-hydroxylation of NNK by the liver.     

Kinetics of DNA methylation by the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in F344 rats

The carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was injected intravenously (0.41 mmol/kg) into F344 rats. DNA from target organs (lung, liver) and a non-target organ (kidney) was extracted hydrolysed and analysed for methylated guanines by cation-exchange high-performance liquid chromotography-fluorimetry. Levels of O6-methylguanine, a promutagenic lesion, and 7-methylguanine were three to eight times higher in the liver than in the lung. Neither base could be detected in the kidneys. The extent of methylation of hepatic DNA by NNK was 35 times lower than observed with an equimolar dose of NDMA by Swann et al. (1983). The levels of the two methylated guanines in liver and lung DNA increased between 4 and 24 h following NNK injection. NNK is metabolized rapidly in F344 rats to 4-(methylnitrosamino)-1(3-pyridyl)-butan-1-ol (NNA1). The relatively slow methylation of hepatic DNA after injection of NNK could be due to a slow release of methylating species from the major circulating metabolite NNA1. This low but sustained level of O6-methylguanine induced by NNK could, in part, explain its carcinogenic potency.     

Absence of metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) by flavin-containing monooxygenase (FMO)

The N-nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent lung carcinogen present in tobacco and tobacco smoke. Carbonyl reduction, alpha-carbon hydroxylation (activation) and N- oxidation of the pyridyl ring (detoxification) are the three main pathways of metabolism of NNK. In this study, metabolism of NNK was studied with lung and liver microsomes from F344 rats, Syrian golden hamsters and pigs and cloned flavin-containing monooxygenases (FMOs) from human and rabbit liver. Thermal inactivation at 45 degrees C for 2 min reduced FMO S-oxygenating activity but did not affect N-oxidation of NNK, leading to the conclusion that FMOs are not implicated in the detoxification of NNK. Detoxification of NNK was not increased by n- octylamine or by incubation at pH 8.4, supporting the conclusion that FMOs are not involved in the metabolism of NNK. SKF-525A (1 mM) significantly reduced N-oxidation and alpha-carbon hydroxylation, suggesting that these two pathways were catalyzed by cytochromes P450. Metabolism of NNK was lower with lung microsomes than with liver microsomes. Inhibition of metabolism of NNK by SKF-525A was also observed with rat lung microsomes, leading to the conclusion that cytochromes P450 are involved in pulmonary metabolism of NNK. Cloned FMOs did not metabolize NNK. In conclusion, cytochromes P450 rather than FMOs are involved in N-oxidation of NNK. The high capacity of hamster liver microsomes to activate NNK does not correlate with the resistance of this tissue to NNK-induced hepatocarcinogenesis.      

Cell specificity for the pulmonary metabolism of tobacco-specific nitrosamines in the Fischer rat

The activity and distribution of the metabolic pathways of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), its major metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and the structurally related nitrosamine, N'-nitrosonornicotine (NNN) were examined in pulmonary cells from F344 rats in order to investigate the mechanisms by which NNK and NNAL, but not NNN, cause lung tumors. The tritium labeled nitrosamines were incubated with Clara cells, alveolar macrophages, alveolar type II cells, or small cells and metabolites were analyzed by HPLC. O6-Methyl-guanine (O6MG) formation was also quantified in the cells incubated with NNK. Clara cells metabolized all compounds more extensively than the other cell types. Total alpha-hydroxylation, carbonyl reduction to NNAL, and pyridine N-oxidation in cells incubated with NNK, as well as concentrations of O6MG in DNA were higher in Clara cells than in other cell types. Carbonyl reduction of NNK predominated over the other metabolic pathways in all cell types. The high activity for alpha-hydroxylation of NNK in Clara cells is consistent with previous studies which proposed that the cell specificity for O6MG formation and the accumulation of this adduct during low-dose exposure to NNK may stem from the presence of a high affinity pathway in Clara cells for NNK activation. Metabolism of NNAL by alpha-hydroxylation, and by reconversion to NNK followed by alpha-hydroxylation were observed. Total alpha-hydroxylation of NNAL was less extensive than alpha-hydroxylation of NNK. NNN was metabolized by both the 2'- and 5'-alpha-hydroxylation pathways. 2'-Hydroxylation of NNN produces the same DNA pyridyloxobutylating agent as does methyl hydroxylation of NNK. However, NNN is not a methylating agent and does not induce lung tumors in rats. Metabolism of NNN by 2'-hydroxylation was, depending on cell type, 41-85% as extensive as total alpha-hydroxylation of NNK, indicating that the rates of formation of the DNA pyridyloxobutylating agent were similar from NNN and NNK. The results of this study demonstrate that Clara cells have a high capacity to metabolically activate NNK, NNAL and NNN and provide further support for the hypothesis that DNA methylation of pulmonary cells is important in NNK carcinogenesis.     

Quantitation of microsomal alpha-hydroxylation of the tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is activated to DNA alkylating species via two different alpha-hydroxylation pathways. Methylene hydroxylation leads to DNA methylation, whereas methyl hydroxylation yields DNA pyridyloxobutylation. We have developed a high-pressure liquid chromatography assay utilizing radiochemical detection that permits the determination of the extent of metabolism through each pathway in microsomal preparations. Levels of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) were used to measure the extent of methyl hydroxylation, whereas levels of the aldehyde, 4-oxo-1-(3-pyridyl)-1-butanone (OPB), were used to quantify the extent of methylene hydroxylation. Incubations of [5-3H]NNK with microsomes and cofactors were conducted in the presence of 5 mM sodium bisulfite to trap the reactive OPB. The inclusion of bisulfite did not affect the rate of NNK metabolism. Trapping the aldehyde also inhibited its further oxidation to the corresponding acid or reduction to HPB. Furthermore, the conversion of HPB to OPB made only a minor contribution to the OPB levels under our incubation conditions. Analysis of incubation mixtures containing [5-3H]NNK, cofactors, and either A/J mouse liver or lung microsomes demonstrated that OPB was a significant metabolite of NNK. The OPB:HPB ratio was greater in liver (1.5) than in lung (0.2-1) microsomal preparations. Apparent Km values for OPB and HPB formation in lung microsomes were 23.7 and 3.6 microM, respectively, whereas the corresponding values for liver microsomes were 19.1 and 73.8 microM, respectively. These data are consistent with the involvement of more than one cytochrome P-450 isozyme in the activation of NNK to DNA reactive species.     

Kinetics and enzyme involvement in the metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in microsomes of rat lung and nasal mucosa.

The rat lung and nasal cavity are two target organs for carcinogenesis by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). In order to characterize further the enzymes involved in the bioactivation of NNK, detailed kinetic and inhibitory studies were conducted with rat lung and nasal mucosa microsomes, and the results were compared with previous studies. The enzymes in rat lung microsomes catalyzed the alpha-hydroxylation, pyridine N-oxidation and carbonyl reduction of NNK. The apparent Km for the formation of the NNK-derived keto aldehyde, NNK-N-oxide, the NNK-derived keto alcohol and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol were 28.8, 10.4, 7.0 and 178.1 microM respectively. In rat nasal microsomes, alpha-hydroxylation was the predominant pathway and the rate was approximately 200 times higher than that in lung microsomes. The apparent Kms for keto aldehyde and keto alcohol formation in rat nasal microsomes were 9.6 and 10.1 microM respectively. The cytochrome P450 inhibitors metyrapone and carbon monoxide markedly inhibited the metabolism of NNK in both rat lung and nasal microsomes. In rat lung microsomes, alpha-naphthoflavone and monospecific antibodies against P450s 1A2, 2A1 and 2B1 inhibited the formation of keto aldehyde by 39, 46, 64 and 23% respectively. In rat nasal microsomes, alpha-naphthoflavone and antibodies against P450s 1A2, 2A1 and 3A inhibited the metabolism of NNK by 80, 35, 20 and 14% respectively. The results indicate that cytochromes P450 play a major role in the metabolic activation of NNK in rat lung and nasal microsomes, and that there are tissue-related differences in NNK metabolism.     

Metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) by cytochrome P450IIB1 in a reconstituted system.

Several previous studies have suggested that cytochrome P450IIB1 is involved in the bioactivation of the tobacco-specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), in rats as well as in mouse lung microsomes. The present investigation was undertaken to study the metabolism of NNK by purified cytochrome P450IIB1 in a reconstituted system. The metabolites 4-hydroxy-4-(3-pyridyl) butyric acid (hydroxy acid), 4-oxo-4-(3-pyridyl) butyric acid (keto acid), 4-oxo-4-(3-pyridyl) butanol (keto aldehyde), 4-(methylnitrosamino)-1-(3-pyridyl-N-oxide)-1-butanone (NNK-N-oxide) and 4-oxo-4-(3-pyridyl)-1-butanol (keto alcohol) were quantitated by HPLC. The results showed that, in addition to alpha-hydroxylations, cytochrome P450IIB1 also catalyzed the formation of NNK-N-oxide efficiently, and to a certain extent, the conversion of NNK primary hydroxylation metabolites (keto aldehyde and keto alcohol) to secondary metabolites (keto acid and hydroxy acid). Cytochrome b5 at a ratio of 1:1 or 2:1 to P450IIB1 had no significant effect on the metabolic activities and profiles of NNK. The apparent Km values for the formation of keto aldehyde, NNK-N-oxide and keto alcohol were respectively 191.2, 131.4 and 318.0 microM with corresponding apparent Vmax values of 89.7, 295.5 and 333.3 pmol/min/nmol P450, indicating that hydroxylation at the alpha-methyl position is preferred over the alpha-methylene position. Measurement of formaldehyde, a product derived from the alpha-methyl hydroxylation, was developed as a convenient method to study NNK metabolism. Thiourea activated cytochrome P450IIB1-catalyzed NNK metabolism significantly. Phenethyl isothiocyanate, an inhibitor of NNK-induced lung carcinogenesis, inhibited P450IIB1-catalyzed NNK demethylation in a concentration-dependent manner. This work demonstrates that purified P450IIB1 can catalyze the conversion of NNK to most of its oxidative metabolites.     

Inhibitory effects of diallyl sulfide on the metabolism and tumorigenicity of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in A/J mouse lung.

Diallyl sulfide (DAS), a component of garlic oil, has been shown to inhibit tumorigenesis by several chemical carcinogens. Our previous work demonstrated that DAS inhibited the metabolic activation of carcinogenic nitrosamines, including the tobacco-specific 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), in rat lung and nasal mucosa microsomes. In the present study, the effects of DAS on the tumorigenicity and the metabolism of NNK in A/J mouse lung were examined. Female A/J mice at 7 weeks of age were pretreated with DAS (200 mg/kg body wt in corn oil, p.o) daily for 3 days. Two hours after the final DAS treatment, the mice were either given a single dose of NNK (2 mg/mouse, i.p.) and kept for an additional 16 weeks for determining the production of pulmonary tumors, or were killed immediately so as to measure the microsomal activity in metabolizing NNK. In comparison to the vehicle control group, DAS pretreatment significantly decreased the incidence of NNK-induced lung tumors (37.9 versus 100%) and the tumor multiplicity (0.6 versus 7.2 tumors/mouse). In pulmonary metabolism of NNK, DAS pretreatment reduced the rates of formation of keto aldehyde, keto alcohol, NNAL-N-oxide, and NNK-N-oxide by 70-90%. In addition, the formation of NNK oxidative metabolites from NNK in the liver microsomes from DAS-pretreated mice was remarkably reduced. DAS also inhibited the metabolism of NNK in mouse lung microsomes in vitro. These results demonstrate that DAS is an effective chemopreventive agent against NNK-induced lung tumorigenesis, probably by inhibiting the metabolic activation of NNK.     

Role of ras protooncogene activation in the formation of spontaneous and nitrosamine-induced lung tumors in the resistant C3H mouse

The role of ras activation in the formation of spontaneous and chemically induced tumors was evaluated in the C3H mouse, a strain that has a low incidence of spontaneous lung tumors. Lung tumors were induced in C3H mice by treatment with 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 50 mg/kg, or nitrosodimethylamine (NDMA), 3 mg/kg for 7 weeks (3 times/week, i.p.). Eleven tumors from each treatment group were evaluated for activated ras genes by direct sequencing and oligonucleotide hybridization to slot blots of amplified DNA from these tumors. An activated K-ras gene was detected in 100% of NDMA- and NNK-induced lung tumors, and the activating mutation detected in all samples was a GC to AT transition (GGT to GAT) in codon 12. In contrast, only 40% of the seven spontaneous lung tumors analyzed contained an activated K-ras gene and the mutations identified were not localized to either a specific base or codon. Both NNK and NDMA can be activated via alpha-hydroxylation to methylating agents. The GC to AT mutation observed in codon 12 in the nitrosamine-induced tumors is consistent with the formation of an O6-methylguanine (O6MG) adduct. Similar concentrations (13-15 pmoles/mumol deoxyguanosine) of this promutagenic adduct were detected in lungs during treatment with either NNK or NDMA. Thus, both these nitrosamines appear to activate the K-ras gene in lung through a direct genotoxic mechanism involving the formation of the O6MG adduct. The frequency of K-ras activation was similar in chemically induced lung tumors from the sensitive A/J strain and the C3H mouse, indicating that susceptibility for neoplasia in these stains is not related to the ability to activate this gene. Although tumors were induced in lung from 100% of C3H mice following chronic carcinogen exposure, both the size and the multiplicity was significantly less, while latency was longer than that observed in the A/J mouse. These differences could not be attributed to an altered propensity for DNA damage, but rather suggest that genetic loci which regulate clonal expansion and growth of initiated cells play a major role in the susceptibility of pulmonary neoplasia.     

Prostaglandin synthetase and cytochrome P-450-dependent metabolism of (+/-)benzo(a)pyrene 7,8-dihydrodiol by enriched populations of rat Clara cells and alveolar type II cells

The metabolism of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene (BP-7,8-diol) by prostaglandin synthetase and cytochrome P-450-dependent monooxygenases was studied using enriched fractions of Clara cells and alveolar type II cells from rat lung. Arachidonic acid-fortified fractions enriched in Clara cells and alveolar type II cells metabolized BP-7,8-diol to the 7,10/8,9-tetrol of benzo(a)pyrene and the 7/8,9,10-tetrol of benzo(a)pyrene. These tetrols are formed upon solvolysis of (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha- epoxy-7,8,9,10-tetrahydrobenzo(a)-pyrene (BP diol-epoxide I). Arachidonic acid-dependent metabolism of BP-7,8-diol to BP diol-epoxide I in enriched Clara cells and alveolar type II cells was completely inhibited by indomethacin, a classical inhibitor of prostaglandin synthetase. Enriched Clara cells and alveolar type II cells also metabolized BP-7,8-diol to BP diol-epoxide I in the presence of NADPH. Amounts of BP diol-epoxide I-derived tetrols formed from BP-7,8-diol by the prostaglandin synthetase-dependent and the cytochrome P-450-dependent pathways varied significantly between the two pulmonary cell fractions examined. In fractions enriched in Clara cells, cytochrome P-450-dependent BP-7,8-diol oxidation was higher than was prostaglandin synthetase-dependent BP-7,8-diol oxidation; while in fractions of alveolar type II cells, prostaglandin synthetase-dependent BP-7,8-diol oxidation to BP diol-epoxide I predominated. Pretreatment of rats with beta-naphthoflavone resulted in a 2- to 3-fold increase in BP diol-epoxide I formation by prostaglandin synthetase and cytochrome P-450-dependent monooxygenases in both enriched Clara cells and alveolar type II cells. These increases in BP-7,8-diol oxidation to BP diol-epoxide I appear to be due to induction of the two enzymatic pathways in both pulmonary cell types. No qualitative changes in the pattern of BP-7,8-diol metabolism by either enzymatic pathway in enriched Clara cells or alveolar type II cells were observed following beta-naphthoflavone treatment. The results suggest that pulmonary prostaglandin synthetase may serve as either an additional or an alternative bioactivating enzyme to the cytochrome P-450-dependent monooxygenases for the formation of reactive chemical carcinogens in the lung.     

In vitro metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone by lung cells isolated from Syrian golden hamster

The nicotine derived N-nitrosamine, 4-(methylnitro-samino)-1-(3-pyridyl)-1-butanone (NNK), is a potent respiratory carcinogen in the Syrian golden hamster. The in vitro metabolism of NNK by three enriched lung cell populations were compared. Clara cells had more than ten times higher capacity to activate NNK by alpha-carbon hydroxylation than alveolar macrophages and fibroblasts. In the former cell types, levels of deactivation of NNK by pyridine N-oxidation were ten times lower than those of alpha-carbon hydroxylation. In alveolar macrophages, carbonyl reduction was the major metabolic pathway. Our results suggest that DNA damages induced by NNK in hamster lung is more likely to occur in Clara cells than in macrophages or fibroblasts.     

SHORT COMMUNICATION: Inhibition of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone mouse lung tumorigenesis by arylalkynes, mechanism-based inactivators of cytochrome P450

Arylalkynes such as 4-phenyl-1-butyne (PBY), 5-phenyl-1-pentyne(PPY) and 2-ethynylnaphthalene (2-EN) are suicide inhibitorsof cytochrome P450 enzymes. Arylalkyl isothiocyanates such as6-phenylhexyl isothiocyanate (PHITC) are structurally relatedto arylalkynes and are known to inhibit the cytochrome P450mediated metabolic activation and tumorigenicity of a tobacco-specificlung carcinogen, 4-(methylnitrosamino)-1-(3-pyridyI)-1-butanone(NNK). In this study, we compared the ability of PBY, PPY, 2-ENand PHTTC to inhibit A/J mouse lung tumorigenesis by NNK. Groupsof 20 female mice were gavaged with 5µmol of arylalkyneor PHTTC in corn oil. Two hours later they were given a singlei.p. injection of 10 µmol NNK. The mice were killed 16weeks later. PPY and PHlTC were both potent inhibitors of tumorigenesisby NNK, reducing lung tumor multiplicity from 8.35 tumors permouse to 0.40 and 0.35 respectively. PBY and 2-EN also significantlyinhibited tumor multiplicity. The results of this study demonstratethat arylalkynes and PHITC are potent inhibitors of NNK inducedlung tumorigenesis in A/J mice, consistent with the hypothesisthat inhibition of specific cytochrome P450 enzymes is involvedin inhibition of tumorigenesis.