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Biotransformation of 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone in Lung Tissue from Mouse, Rat, Hamster, and Man Elmar Richter,*,† Johannes Engl,† Susanne Friesenegger,† and Anthony R. Tricker‡ Walther Straub Institute, Department of Toxicology, Ludwig-Maximilians UniVersity of Munich, Nussbaumstrasse 26, D-80336 Munich, Germany, and PMI Research & DeVelopment, Philip Morris Products S.A., Quai Jeanrenaud 56, CH-2000 Neuchaˆtel, Switzerland ReceiVed December 4, 2008
Exposure to the tobacco-specific N-nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is considered to be an important etiological risk factor for lung cancer in tobacco users. The metabolism of NNK via carbonyl reduction to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), R-hydroxylation to form both DNA methylating and pyridyloxobutylating intermediates, and detoxification by pyridyl N-oxidation and glucuronide formation are well-characterized in laboratory animals but less so in man. The in vitro kinetics of 0.03-250 µM [5-3H]NNK metabolism were determined under identical experimental conditions using female A/J mouse, male Fischer 344 rat, female Syrian golden hamster, and human lung tissue explants in tissue culture. The concentration-dependent percentage contribution of the three major pathways of NNK metabolism (carbonyl reduction, R-hydroxylation, and N-oxidation) showed large interspecies variation. Quantitatively, in mouse, carbonyl reduction to NNAL increased steadily with an increasing substrate concentration (10-74% total NNK metabolism), while concurrent decreases occurred in end products of R-hydroxylation (60 to 18%) and N-oxidation (42 to 5%). In rat lung, there were no apparent concentration-dependent trends (NNAL, 42 ( 4%; R-hydroxylation, 35 ( 2%; and N-oxidation, 24 ( 3%). In hamster lung, a clear concentration-dependent increase in the contribution of NNAL to total NNK metabolism (from 47 to 87%) was paralleled by a steady decline in end products of R-hydroxylation (31 to 11%) and N-oxidation (22 to 2%). Human lung metabolism showed no concentration-dependent tendencies (NNAL, 89 ( 1%; R-hydroxylation, 8.8 ( 1.1%; and N-oxidation, 2.1 ( 0.3%). The major R-hydroxylation product in human lung was 4-hydroxy-1-(3-pyridyl)1-butanone (keto alcohol), thus supporting the potential pyridyloxobutylation of lung DNA. Metabolism to 4-(3-pyridyl)-4-oxobutanoic acid (keto acid), which could result in lung DNA methylation, was only sporadically seen in human lung but present to a far greater extent in rodent lung. No evidence for glucuronidation was found in any species. Generally, the rate of formation of all NNK metabolites showed two different enzyme kinetics, resulting in large differences between apparent Km and Vmax values in the low (up to 2.8 µM) and high substrate concentration ranges. The metabolism of NNK by R-hydroxylation is considerably lower in human lung as compared to that observed in rodent species, suggesting that extrapolation of in vitro rodent data to man may result in invalid conclusions about the capacity of the human lung to activate NNK under realistic conditions of NNK exposure expected to occur in man. Introduction The tobacco-specific N-nitrosamine 4-(methylnitrosamino)1-(3-pyridyl)-1-butanone (NNK, 1) is classified as “carcinogenic to humans” (group 1) according to the International Agency for Research on Cancer (IARC)1 (1) and is considered to be an important etiological risk factor for lung cancer in tobacco users. NNK has a remarkable specificity for tumorigenesis of the lung, inducing predominantly adenocarcinomas in the lungs of mice, rats, and hamsters, independent of the route of administration (2). The major pathways of NNK metabolism in laboratory animals are illustrated in Figure 1. Metabolism of NNK is complex, and metabolic pathways include carbonyl reduction of NNK to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL, * To whom correspondence should be addressed. Tel: +49-89-218073802. Fax: +49-89-2180-73801. E-mail:
[email protected]. † Ludwig-Maximilians University of Munich. ‡ Philip Morris Products S.A.
2), glucuronidation of NNAL, R-hydroxylation of NNK and NNAL, and pyridine N-oxidation of NNK and NNAL (2). The major metabolic pathway for NNK in most tissues is carbonyl reduction to NNAL (2). NNK and NNAL are procarcinogens that require metabolic activation by cytochrome P450 enzymes to express their biological activity. R-Hydroxylation of the methylene carbon atoms adjacent to the N-nitroso group is believed to result in metabolic activation of NNK and NNAL 1 Abbreviations: diol, 4-(3-pyridyl)butane-1,4-diol; EBSS, Earle’s balanced salt solution; HPB, 4-hydroxy-1-(3-pyridyl)-1-butanone; hydroxy acid, 4-(3-pyridyl)-4-hydroxybutanoic acid; IARC, International Agency for Research on Cancer; keto acid, 4-(3-pyridyl)-4-oxobutanoic acid; keto alcohol, 4-hydroxy-1-(3-pyridyl)-1-butanone; KH, Krebs-Henseleit buffer; LDH, lactate dehydrogenase; NNAL, 4-(methylnitrosamino)-1-(3-pyridyl)1-butanol; NNAL-N-Gluc, 4-(methylnitrosamino)-1-(3-pyridyl-N-β-D-glucopyranuronosyl)-1-butanolonium inner salt; NNAL-O-Gluc, 4-(methylnitrosamino)-1-(3-pyridyl)-1-(O-β-D-glucopyranuronosyl)butane; NNAL-Noxide, 4-(methylnitrosamino)-1-(3-pyridyl-N-oxide)-1-butanol; NNK, 4(methylnitrosamino)-1-(3-pyridyl)-1-butanone; NNK-N-oxide, 4-(methylnitrosamino)-1-(3-pyridyl-N-oxide)-1-butanone; NSCLC, nonsmall cell lung carcinoma; SCC, squamous cell carcinoma.
10.1021/tx800461d CCC: $40.75 2009 American Chemical Society Published on Web 05/01/2009
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Figure 1. Metabolic scheme of NNK. Structures in brackets represent hypothetical intermediates (2, 4).
since the unstable intermediates produced decompose to methanediazohydroxide, which can methylate DNA and other cellular macromolecules, and to aldehydes, which are further hydrolyzed to the corresponding acids, 4-(3-pyridyl)-4-oxobutanoic acid (keto acid, 4) and 4-(3-pyridyl)-4-hydroxybutanoic acid (hydroxy acid, 5), respectively (2). By a similar process, R-hydroxylation of the methyl group of NNK and NNAL yields unstable intermediates that decompose to formaldehyde, which can react with DNA to yield various adducts in vitro and on administration of NNK to rats (3) and to either 4-(3-pyridyl)4-oxobutanediazohydroxide or 4-(3-pyridyl)-4-hydroxybutanediazohydroxide, respectively (2). The NNK-specific reactive intermediate, 4-(3-pyridyl)-4-oxobutanediazohydroxide, can pyridyloxobutylate cellular macromolecules, resulting in the formation of HPB-releasing adducts (4), or is hydrolyzed to 4-hydroxy-1-(3-pyridyl)-1-butanone (keto alcohol, 3; also referred to as HPB). HPB can also be further oxidized to keto acid. The specific reactive intermediate of R-methyl hydroxylation of NNAL gives rise to pyridylhydroxybutylation of DNA and the major end product of hydrolysis, 4-(3-pyridyl)butane1,4-diol (diol, 6), which is also further oxidized to hydroxy acid (5) (4). Detoxification of NNK occurs by glucuronidation of NNAL to yield 4-(methylnitrosamino)-1-(3-pyridyl)-1-(O-β-Dglucopyranuronosyl)butane (NNAL-O-Gluc, 9) (2) and, to a lesser extent, 4-(methylnitrosamino)-1-(3-pyridyl-N-β-D-glucopyranuronosyl)-1-butanolonium inner salt (NNAL-N-Gluc, 10) (5). Pyridine N-oxidation of NNK and NNAL to yield 4-(methylnitrosamino)-1-(3-pyridyl-N-oxide)-1-butanone (NNK-Noxide, 7) and 4-(methylnitrosamino)-1-(3-pyridyl-N-oxide)-1butanol (NNAL-N-oxide, 8), respectively, are considered to result in detoxification (2, 6, 7). Most of these metabolic pathways have been demonstrated to occur in various rodent species (2, 8-17) and nonhuman primates (2, 18-20). In man, the major pathway for NNK metabolism is reduction to NNAL (>95% of total metabolism) in most tissues (6, 21, 22) including both lung (6, 13) and freshly isolated lung cells (23).
Similar observations have been reported for NNK metabolism in microsome preparations from various human tissues (17, 24-29) including lung (17, 24, 30-33). Contrary to rodents (2, 12, 34-36) and nonhuman primates (2, 18, 19), NNAL and its conjugated detoxification products, NNAL-N-Gluc and NNAL-O-Gluc, are the major metabolites of NNK detected in human urine (5, 37-40). NNAL-N-oxide, another detoxification product of NNK, is also found, albeit at lower concentrations (41). Other NNK-derived metabolites observed in the urine of laboratory animals are not specific to NNK metabolism in man, since they are also formed by metabolism of nicotine present in both tobacco and tobacco smoke (2, 42). The extrapolation of animal data to humans has many uncertainties, which can lead to tenuous assessments of risk (2, 43). Perhaps the most critical parameters are the effects of dose, interspecies differences in metabolism, and human enzyme polymorphisms that result in interindividual differences in metabolism. The lowest total dose of NNK required to induce a significant incidence of lung tumors in experimental animals is at least 10 times higher than the hypothetical lifetime exposure of a smoker (44). In contrast to many experimental animal models, humans show large interindividual variation in the expression of 11β-hydroxysteroid dehydrogenase type 1 and carbonyl reductase, which convert NNK to 2 (45-47), and functional polymorphisms in P450 enzymes, in particular P450 2A6, 2A13, and 2E1, involved in the metabolic activation of NNK (31-33, 48-50). In addition, natural components present in the diet and tobacco smoke may also effect NNK bioavailability or modulate enzyme activities that are involved in NNK metabolism (13, 36, 51, 52). The metabolism of NNK has been studied in lung explants from mouse (11, 13, 53-56), rat (10, 20, 57), hamster (8, 14, 58-60), nonhuman primates (20), and man (6, 13). However, metabolic data have not been obtained under identical experimental conditions in different species to allow comparison of interspecies differences in pulmonary metabolism. Therefore,
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we investigated the in vitro kinetics of NNK metabolism under identical experimental conditions using A/J mouse, Fischer 344 rat, and human lung tissue explants in tissue culture. In addition, previously published results for Syrian golden hamster (14) obtained using identical experimental conditions are included to extend the comparison to another rodent species.
Materials and Methods Caution: NNK is a carcinogen and should be handled in a wellVentilated hood with extreme care and with personal protectiVe equipment. Materials. [5-3H]NNK with a specific activity of 30 Ci/mmol and radiochemical purity of >98% was obtained from SibTech (Tenafly, NJ). Unlabeled NNK was purchased from Chemsyn (Lenexa, KS). NNK metabolite standards were kindly donated by Dhimant H. Desai and Shantu Amin (Penn State University, Hershey, PA). Earle’s balanced salt solution without phenol red (EBSS) and Krebs-Henseleit buffer (KH) were purchased from Sigma (Deisenhofen, Germany). All other chemicals, which were either HPLC or analytical grade, were obtained from Merck (Darmstadt, Germany). Animals. Female A/J mice (7 weeks old; 18-20 g body weight), male Fischer 344 rats (9 weeks old; 180-210 g body weight), and female Syrian golden hamsters (9 weeks old; 110 g body weight) were obtained from Charles River (Sulzfeld, Germany) and allowed free access to ssniff H laboratory animal diet (ssniff, Soest, Germany) and drinking water. Animals were maintained on a 12 h light cycle at 20 ( 2 °C with a relative humidity of 50 ( 10% and allowed to acclimatize to these conditions for at least 7 days before use. All experimental studies were performed in accordance with German legislation. Preparation of Rodent Lung Tissue Samples. Animals were killed by cervical dislocation, and their lungs were excised through a midventral incision and immediately placed in ice-cold EBSS. Under aseptic conditions, the peripheral lungs were cut with a scalpel into pieces of approximately 2 mm3 (tissue weight, approximately 3.0 ( 0.5 mg). Human Lung Tissue Samples. Human lung tissue was obtained from peripheral lung removed during clinically indicated lobectomy. Immediately after removal, the tissue was placed in 0.9% NaCl solution and kept on ice. The elapsed time between surgical resection and initiation of tissue explant preparation was ∼1 h. All lung tissue samples were devoid of macroscopically visible tumors and obtained from different subjects. Patient records were used to characterize subjects with respect to age, gender, surgical diagnosis, and self-reported smoking history. The study was performed with approval from the Ethics Committee of the Medical Faculty of the Ludwig-Maximilians University of Munich (Germany). Culture of Lung Tissue. Incubation studies for the determination of kinetic parameters were performed using an identical optimized experimental protocol (14) according to the following conditions: Lung tissue explants were preincubated with 0.6 mL of KH buffer (pH 7.4) in 8 mL polypropylene tubes using a gyratory shaker (90 rpm) for 0.5 h at 37 °C under an atmosphere of 5% CO2/95% O2. The test substrate [5-3H]NNK (0.57-5.7 µCi/incubation plus appropriate amounts of unlabeled NNK at concentrations >0.03 µM) was dissolved in acetonitrile and added to the culture medium at a final concentration of 0.3% (v/v) to minimize inhibition of the in vitro activity of cytochrome P450 enzymes (61). A range of 13 different concentrations was used for the test substrate (0.03, 0.1, 0.19, 0.32, 1.15, 2.0, 2.8, 8.6, 16.8, 25, 84, 166, and 250 µmol/L), and incubation was performed for 6 h at 37 °C under an atmosphere of 5% CO2/95% O2. Each pharmacokinetic study was performed using a minimum of four concentration curve replicates per lung. Measurement of NNK Biotransformation. The culture medium was centrifuged (18000g, 15 min) to remove tissue debris and filtered through a 0.22 µm Millipore Ultrafree filter (Millipore, Ko¨nigstein, Germany), and NNK metabolites were determined by reversed-phase HPLC with online radioflow detection (62). Radioactive metabolites were identified by cochromatography with
Richter et al. Table 1. Patient Demographics patient
age (years)
sex
smoking history
1
53
F
smoker
2 3 4 5 6 7 8 9 10
77 58 57 53 48 52 50 67 69
M M M M M F M M M
smoker smoker smoker smoker smoker smoker smoker smoker ex-smoker (15 month cessation)
diagnosis leading to surgery nonsmall cell bronchial carcinoma SCC SCC SCC adenocarcinoma SCC SCC SCC SCC SCC
unlabeled reference compounds using UV detection at 234 and 254 nm. The distribution of radioactive metabolites was determined by integration of the individual peaks and calculation of the percentage of total radioactivity eluting from the column. Borderline amounts of radioactivity in a peak were verified by chromatogram addition using GINA Software Vs. 4.2 (Raytest, Straubenhardt, Germany), allowing a combination of up to four chromatograms from parallel incubations for enhanced sensitivity of detection. Viability of Lung Tissues and Biochemical Investigations. Separate tissue cultures from mouse and human lung were used to determine the viability of tissue samples by tissue lactate dehydrogenase (LDH) leakage into the culture medium and intracellular K+ content at times 0, 1, 2, 3, 4, 5, and 6 h (63). Aliquots of culture media were sampled for LDH activity by determining the UV spectra for a decrease of NADH during the transformation of pyruvate to lactose (64). Results for LDH leakage were expressed in terms of tissue protein content determined according to the method of Lowry et al. (65), employing bovine serum albumin as the standard. The intracellular K+ content (66) was determined as follows: Tissue samples were removed from the culture medium, blotted and weighed, and homogenized by sonication with a cell disrupter in 1.0 mL of deionized water containing 20 µL of perchloric acid (70%) to precipitate proteins. The homogenate was centrifuged (13500g, 5 min) to pellet precipitate materials. The supernatant fraction was assayed for K+ using a FCM6341 flame photometer (Eppendorf, Hamburg, Germany). Data Analysis. All statistical data analyses were performed using Prism 4 for Windows (GraphPad Software Inc., San Diego, CA). Data are expressed as means ( SEs. Statistical analyses of differences in groups were performed by the Student’s t test. The Michaelis-Menten kinetic parameters apparent Km and Vmax were derived by nonlinear least-squares regression analysis, which are reported as means ( SEs by Prism 4. Estimates of catalytic specificity were calculated as Vmax/Km.
Results Patient Demographics. Lung tissue explants were prepared from sections of peripheral lung obtained from 10 patients (eight males and two females) aged 58.9 ( 9.3 years (Table 1). On the basis of self-reported smoking histories, nine patients were current smokers, and one patient was classified as a former smoker (smoking cessation 15 months prior to surgery). Six of eight male patients had a diagnosis of squamous cell carcinoma (SCC), one had a nonsmall cell lung carcinoma (NSCLC), and one had an adenocarcinoma. One female patient had a SCC, and the other had a NSCLC. None of the patients had received chemotherapy prior to surgery. Viability of Tissue Culture Conditions. To optimize the conditions to study the metabolism of NNK, the time course for metabolite formation was studied over 6 h (data not presented), and an incubation time of 6 h was selected for all studies. Intracellular K+ and LDH leakages from mouse and human lung explants into culture medium were used as indicative measures of the functional cell viability. Over 6 h of
Biotransformation of NNK in Lung Tissue
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Figure 2. Viability of mouse and human lung tissue explants during 6 h of incubation. The intracellular K+ content was determined as described in the Materials and Methods. Results are means ( SEs of three samples per time interval.
incubation, intracellular K+ levels remained stable in lung tissue explants in both species. No time-dependent effect was observed for lung tissue samples incubated immediately after sacrifice of mouse or human lung tissue samples following collection and transport to the laboratory (Figure 2). Functional cell viability was confirmed since tissue LDH leakage into the culture medium was less than 20% following 6 h of incubation at the highest concentration of NNK. The storage of tissue samples in physiological saline at 4 °C up to 2 h following surgical removal did not affect metabolism of NNK and viability criteria. Species-Specific [5-3H]NNK Metabolism in Lung. Typical HPLC radiochromatograms obtained after incubation of lung tissue explants are shown in Figure 3. According to the proposed pathways of NNK metabolism (Figure 1), quantitative speciesspecific differences were observed for lung metabolism of NNK (Figure 4). Metabolism was evaluated based on the three major pathways of NNK metabolism: carbonyl reduction of NNK to NNAL (2), total NNK R-hydroxylation (sum of 3, 4, 5, and 6), and total NNK N-oxidation (sum of 7 and 8). Visual inspection of the data suggested that all three major pathways of NNK metabolism were highly variable in the 10 human subjects. Under the experimental conditions applied, the total metabolism of NNK in the lung at low substrate concentrations (0.03-1.15 µM [5-3H]NNK) was highest in hamster > rat > mouse ∼ human lung tissue (Figure 4). The carbonyl reduction of NNK to 2 was highest in hamster > human > mouse ∼ rat lung. End products of R-hydroxylation, which represent the most important metabolic activation pathways of NNK, were highest in hamster > rat > mouse > human lung. Pyridine N-oxidation of both NNK and 2, putative detoxification pathways, was higher in hamster > rat > mouse > human lung. At high substrate concentrations (2.8-250 µM [5-3H]NNK), the total metabolism of NNK was highest in hamster > human ∼ rat ∼ mouse lung. Carbonyl reduction to 2 was higher in hamster > human > rat ) mouse lung. R-Hydroxylation was higher in hamster and rat but relatively low in mouse and human lung. Major products
Figure 3. Radiochromatograms of culture medium incubated for 6 h with 2.0 µM [5-3H]NNK (control) and with rodent and human lung tissue explants. Individual peaks are identified as follows: NNK (1), NNAL (2), keto alcohol (3), keto acid (4), hydroxy acid (5), diol (6), NNK-N-oxide (7), and NNAL-N-oxide (8). See the Material and Methods for details.
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Richter et al.
Figure 4. Concentration-dependent metabolism of NNK (fmol/min/mg of protein) by lung tissue explants from A/J mice (4, n ) 6), Fischer 344 rats (], n ) 6), Syrian golden hamsters (O, n ) 5), and humans (0, n ) 10) after 6 h of incubation with 0.03-250 µM NNK. Each pharmacokinetic study was performed using a minimum of four concentration curve replicates per lung, and values represent means ( SEs. The major pathways of NNK metabolism are presented as follows: NNK reduction to NNAL (2), total R-hydroxylation [sum of keto alcohol (3), keto acid (4), hydroxy acid (5), and diol (6)], and total pyridine N-oxidation [sum of NNK-N-oxide (7) and NNAL-N-oxide (8)]. NNK metabolites were determined in the incubation medium by radioHPLC.
of NNK R-hydroxylation in rodents were 3 > 4 > 6 in A/J mouse and rat and 4 > 3 > 6 in hamsters. All three metabolites were found in roughly equal amounts in man (data not shown). Little difference was observed for pyridine N-oxidation between rodents and man. Over the whole substrate concentration range, hydroxy acid (5) was not observed as a metabolite of NNK in mouse and rat and only sporadically observed in hamster and human lung. Kinetics of [5-3H]NNK Metabolism in Lung. The Michaelis-Menten kinetic parameters Km and Vmax were calculated by regression from the linear regions of LineweaverBurke plots for all major NNK metabolites except 5 (Table 2). The rate of formation of all NNK metabolites showed two different enzyme kinetics, resulting in large differences between apparent Km and Vmax values in the low and high substrate concentration ranges. The catalytic specificity (Vmax/Km) of all pathways of NNK metabolism was generally higher in the low as compared to the high substrate concentration range in all species with the exception of carbonyl reduction to 2 and further N-oxidation to 8 in the A/J mouse. In the low and high substrate concentration ranges, human lung exhibited the lowest turnover to NNK metabolites with the exception of carbonyl reduction to 2. Concentration-Dependent Patterns of [5-3H]NNK Metabolism in Lung. The concentration-dependent percentage
contribution (means ( SE) of the three major pathways of NNK metabolism (carbonyl reduction, total R-hydroxylation, and total N-oxidation) showed large species-specific variations (Figure 5). In A/J mouse lung, NNK carbonyl reduction to 2 increased steadily with increasing NNK substrate concentrations, and this was accompanied by a concurrent decrease of R-hydroxylation and N-oxidation pathways. At high substrate concentrations, 2 was the major metabolite in mouse lung and accounted for up to 74% of total NNK metabolism. With increasing substrate concentrations, products of R-hydroxylation and N-oxidation decreased to a low of 18 and 5% of total NNK metabolism, respectively. Overall, the contribution of R-hydroxylation pathways was significantly higher (P < 0.05) than N-oxidation pathway of NNK metabolism in the mouse lung. In Fischer 344 rat lung, there were no apparent concentrationdependent trends in NNK metabolism. The formation of 2 and end products of R-hydroxylation and N-oxidation pathways contributed between 42 ( 4, 35 ( 2, and 24 ( 3% to total NNK metabolism, respectively. At low substrate concentrations, the only significant trend was a lower contribution of total N-oxidation as compared to R-hydroxylation (P < 0.05) to total
a
0.92 ( 1.31 93 ( 35 0.57 ( 0.32 58 ( 16 0.50 ( 0.26 160 ( 42 1.6 ( 0.9 218 ( 67 0.55 ( 0.25 88 ( 30 4.2 ( 15 82 ( 35
lowb highc low high low high low high low high low high
Vmax/Km 60 139 105 20 49 13 20 9 124 19 7 11
Vmax 56 ( 64 12900 ( 1990 60 ( 18 1170 ( 152 27 ( 7 2010 ( 315 32 ( 12 2000 ( 408 68 ( 16 1690 ( 265 31 ( 79 911 ( 161
A/J mouse (n ) 6)
Km
range (µM) 387 ( 322 15900 ( 3550 129 ( 26 22200 ( 16700 42 ( 10 4020 ( 1360 2.4 ( 0.9 951 ( 1290 138 ( 37 8780 ( 3390 19 ( 9 113 ( 117
Vmax
Fischer 344 rat (n ) 6) 3.6 ( 4.3 194 ( 82 1.1 ( 0.5 308 ( 371 0.67 ( 0.41 70 ( 67 0.08 ( 0.07 83 ( 142 1.0 ( 0.7 81 ( 92 0.32 ( 0.83 14 ( 23
Km 108 82 116 72 63 58 30 11 135 108 66 8
Vmax/Km 1.5 ( 0.9 275 ( 102 0.72 ( 0.18 1950 ( 6560 1.0 ( 0.4 550 ( 670 1.4 ( 0.9 25 ( 25 0.79 ( 0.23 82 ( 36 1.6 ( 1.9 117 ( 125
1060 ( 325 125000 ( 31900 95 ( 10 35200 ( 110000 215 ( 36 28900 ( 26300 34 ( 13 533 ( 301 161 ( 20 4370 ( 790 44 ( 26 3170 ( 1450
Vmax 709 454 131 18 212 53 25 21 200 53 28 27
Vmax/Km
Syrian golden hamster (n ) 5) Km 8.4 ( 21.7 1310 ( 2510 2.3 ( 2.9 517 ( 239 2.5 ( 2.2 471 ( 469 2.7 ( 6.9 1080 ( 5810 0.69 ( 1.20 323 ( 290 1.5 ( 1.6 373 ( 554
Km
Km, µM (mean ( SE); Vmax, fmol/min/mg protein (means ( SE). b Calculated in the concentration range of 0.03-2.8 µM. c Calculated in the concentration range of 1.15-250 µM.
NNAL-N-oxide (8)
NNK-N-oxide (7)
diol (6)
keto acid (4)
keto alcohol (3)
NNAL (2)
metabolite
Table 2. Michaelis-Menten Kinetic Parameters for NNK Metabolism by Rodent and Human Lunga
1610 ( 3710 239000 ( 395000 28 ( 19 2600 ( 990 31 ( 15 2560 ( 1960 45 ( 86 10200 ( 51300 8.6 ( 6.6 2960 ( 1680 23 ( 11 2100 ( 2600
Vmax
man (n ) 10) 190 182 12 5 12 5 17 9 12 9 16 6
Vmax/Km
Biotransformation of NNK in Lung Tissue Chem. Res. Toxicol., Vol. 22, No. 6, 2009 1013
Figure 5. Concentration-dependent contribution of major NNK metabolism pathways in lung tissue explants of A/J mice (n ) 6), Fischer 344 rats (n ) 6), Syrian golden hamsters (n ) 5), and man (n ) 10) after 6 h of incubation with 0.03-250 µM NNK. Each pharmacokinetic study was performed using a minimum of four concentration curve replicates per lung, and values represent means ( SEs. The major pathways of NNK metabolism are presented as follows: NNK reduction to NNAL (4, 2), total R-hydroxylation [O, sum of keto alcohol (3), keto acid (4), hydroxy acid (5), and diol (6)], and total pyridine N-oxidation [0, sum of NNK-N-oxide (7) and NNAL-N-oxide (8)]. NNK metabolites were determined in the incubation medium by radioHPLC.
NNK metabolism. At high substrate concentrations, 2 was the major metabolite formed by rat lung, mostly at the expense of N-oxidation. In Syrian golden hamster lung, the major pathway of NNK metabolism was also carbonyl reduction to 2, and there was a concentration-dependent increase in the contribution of 2 (from
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47 to 87%) to total NNK metabolites, which was paralleled by a steady decline in the sum of R-hydroxylation products (31 to 11%) and N-oxidation products (22 to 2%), respectively. Overall, the formation of 2 was significantly higher (P < 0.05) than the formation of end products of N-oxidation at all substrate concentrations and significantly higher (P < 0.05) than the formation of end products of R-hydroxylation at all but the two lowest substrate concentrations. Products of R-hydroxylation contributed consistently more to NNK metabolism than products of N-oxidation. The contribution was significant at 7 of 11 substrate concentrations (P < 0.05). In human lung, 2 was consistently the major metabolite formed, accounting for 82-92% of all metabolites. End products of R-hydroxylation contributed slightly more to total NNK metabolism at low substrate concentrations (0.32 µM [5-3H]NNK; 7-10%). No concentrationdependent tendency was seen for the sum of N-oxides, which constituted
