1 Department of Pharmacology and Toxicology,
2 Department of Surgery and
3 Department of Medicine, Queen's University, Kingston, ON K7L 3N6, Canada and
4 Laboratory of Cancer Etiology and Chemoprevention, Faculty of Pharmacy, Laval University, Quebec City, PQ G1K 7P4, Canada
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abbreviations: 11ß-HSD, 11ß-hydroxysteroid dehydrogenase; ANOVA, analysis of variance; d.p.m., disintegrations per minute; diol, 4-hydroxy-1-(3-pyridyl)-1-butanol; HPLC, high-performance liquid chromatography; LOX, lipoxygenase; NNAL, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol; NNAL-N-oxide, 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; PHS, prostaglandin H synthase.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bioactivation to reactive intermediates is required for both NNK and its keto reduced form 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) to exert carcinogenicity (2). Hepatic and extrahepatic biotransformation of NNK have been relatively well characterized in rodents, and to a lesser extent in humans (Figure 1). Reversible carbonyl reduction of NNK results in NNAL. Both NNK and NNAL can undergo similar pathways of biotransformation. Pyridine N-oxidation of NNK and NNAL results in 4-(methylnitrosamino)-1-(3-pyridyl N-oxide)-1-butanone (NNK-N-oxide) and 4-(methylnitrosamino)-1-(3-pyridyl N-oxide)-1-butanol (NNAL-N-oxide), respectively. These N-oxidation pathways are termed detoxification pathways, and the polarity of the resulting metabolites allows for ready excretion (1). The two carbons
to the nitrosamino groups of NNK and NNAL can be hydroxylated, yielding unstable intermediates which spontaneously decompose to produce alkylating agents.
-Methylene hydroxylation of NNK and NNAL results in the formation of methyl diazohydroxides which can methylate DNA.
-Methyl hydroxylation of NNK and NNAL results in the formation of oxobutyl and hydroxybutyl diazohydroxides, respectively, which can bind to DNA via processes termed pyridyloxobutylation and pyridylhyroxybutylation. These
-hydroxylation pathways are considered metabolic activation pathways, and are believed to be entirely responsible for DNA alkylation and resulting carcinogenesis (3 and references therein).
|
Human pulmonary NNK biotransformation was first demonstrated by Castonguay et al. (9) in cultured bronchus and whole peripheral lung explants over a 45 day culture period. In subsequent studies, Smith et al. (10,11) reported that peripheral human lung microsomes catalyzed NNK metabolism by cytochrome P450-dependent polysubstrate monooxygenase systems (P450s), lipoxygenases (LOXs) and other peroxidases.
The lung is a complex tissue, and all human lung NNK biotransformation studies to date have involved only lung explant incubates or whole peripheral lung as a source of homogenate, and do not provide information regarding the cellular sites of NNK metabolism. Pulmonary P450 activities are, in general, much lower than hepatic activities, reflecting the six to 20 times lower concentrations of monooxygenase enzymes in lung (12). Overall NNK metabolic activation capacity may be low in total human lung, but high in a certain cell type representing a small percentage of total lung cells. Human alveolar type II cells are believed to be relatively high in P450 content due to their endoplasmic reticulum content (13,14), while human alveolar macrophages possess P450 mRNAs (15,16), in addition to prostaglandin H synthase (PHS) (17) and LOX activities (18). Thus, type II cells and macrophages might be important cell types for NNK bioactivation.
The identification, cellular localization and inter-individual variabilities in activities of NNK biotransformation systems in human lung have yet to be established. In the present study, lung cell types potentially involved in the biotransformation of NNK were characterized using isolated human peripheral lung cell preparations, including isolated unseparated cells (cell digest), preparations enriched in alveolar type II cells, and preparations enriched in alveolar macrophages. The isozyme non-selective P450 inhibitor SKF-525A was used to assess the contributions of P450s to NNK metabolism in these cell preparations.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tissue procurement
Human lung tissue, devoid of macroscopically visible tumours, was obtained from Kingston General Hospital, in accordance with procedures approved by the Queen's University Ethics Review Committee for Research on Human Subjects. Following informed consent, sections of peripheral lung (10100 g) were removed during clinically indicated lobectomy. Immediately after removal, the tissue was placed in 0.9% NaCl solution and kept on ice. Elapsed time between surgical resection and initiation of cell isolation was ~15 min. Prior to lung cell isolation, a 1.5 cm3 section was removed from the tissue and placed in 10% buffered formalin. The fixed tissue was dehydrated, embedded in paraffin, and 5 mm sections were stained with haematoxylin and eosin (20). They were then examined by light microscopy to confirm the absence of microscopic tumours. Patients were characterized with respect to age, gender, surgical diagnosis, possible occupational exposure to carcinogens, drug treatment one month prior to surgery and smoking history. Patients were classed as former smokers if smoking cessation was >2 months before surgery. This time interval was chosen to eliminate the inductive effects of cigarette smoke on biotransformation enzymes (21).
Lung cell isolation
Human lung cells were isolated as described previously (22) with minor modifications that resulted in increased cell yield and improved purity. Following protease digestion, a 2 ml aliquot of cell digest was saved, and the remaining 10 ml underwent centrifugal elutriation using a Beckman J221M/E centrifuge with a JE-6B elutriation rotor. Three elutriation fractions, each having a different profile of cell types, were collected. The first fraction (150 ml, 2200 r.p.m., 13 ml/min) contained primarily red blood cells and cellular debris and was not used in experiments. For cells from poorly perfused tissue specimens, which contained large amounts of red blood cells, up to 250 ml were collected in the first fraction. The second fraction (100 ml, 2200 r.p.m., 21 ml/min) was comprised of 2060% alveolar type II cells and was saved for further enrichment. The third fraction (100 ml, 1200 r.p.m., 18 ml/min) consisting of 6080% macrophages, was saved for use in experiments.
Alveolar type II cells were further enriched from the second elutriation fraction by Percoll density gradient centrifugation. Cells were layered on a 38% (v/v) solution of Percoll in RPMI-1640 medium and centrifuged at 1000 g for 15 min. The layer of cells remaining on top of the Percoll contained 6585% type II cells and was saved for use in experiments.
Cell viability and yield were estimated by 0.5% trypan blue dye exclusion on a haemocytometer (23). Using the modified Papanicolaou stain (24), alveolar type II cells were identified by the presence of stained lamellar bodies in the cytoplasm, while macrophages were identified by the absence of lamellar bodies, large size, round shape and nucleus and the absence of stained cytoplasmic granules.
Incubations with NNK
Immediately following isolation, lung cells were incubated in sterile 24-well Nunclon polystyrene multidish plates with 4.2 mM (10 mCi) [5-3H]NNK (>98% purity) in 1 ml RPMI-1640 medium, supplemented with antibiotic-antimycotic (1 ml/l), for 24 h at 37°C under 95% air5% CO2. Incubates were performed in duplicate, and all available cells were used in incubates to ensure low level detection of the various NNK metabolites. Incubations were terminated by removal of cells from incubation medium by centrifugation (800 g for 15 min). Supernatants and cell pellets were transferred to separate cryovials, frozen in liquid N2, and stored at 80°C until analysis. Inhibition experiments involved the inclusion of 1.0 mM SKF-525A in the incubation medium. For each patient, control incubates containing no cells were subjected to the same incubation conditions as the cell-containing incubates.
NNK purification
Prior to use, the purity of the [5-3H]NNK was tested using the metabolite analysis protocol (see below). When [5-3H]NNK purity was <98%, the compound was re-purified by a normal phase gradient high-performance liquid chromatography (HPLC) protocol generously provided by Dr Neil Trushin, American Health Foundation, Valhalla, NY. Briefly, 100ml of contaminated [5-3H]NNK was injected onto a Zorbax 5 micron silica column (4.6x250 mm). The elution program was: solvent A (70:30 hexane: chloroform) for 5 min, followed by a linear gradient to 7% solvent B (methanol) in 28 min. Solvent delivery was at 1.0 ml/min. Absorbance was monitored at 254 nm, and [5-3H]NNK eluted from 36 to 41 min. The mobile phase was then removed using N2 gas and the remaining [5-3H]NNK was re-suspended in HPLC grade H2O.
Measurement of NNK biotransformation
NNK metabolites were analysed by reverse phase HPLC, essentially as described by Castonguay et al. (19). Briefly, 500 ml of filtered (0.45 mm polypropylene, Millipore) incubation medium plus 5 ml of NNK metabolite reference standards (UV markers) were co-injected onto a Waters µBondapak C18 column. The elution program was: solvent A (0.06 M sodium acetate pH 6.0) for 10 min, followed by a linear gradient to 60% solvent B (CH3OH:H2O, 1:1, v/v) in 60 min. Solvent delivery was at 1.0 ml/min. UV marker peaks were detected by absorbance at 254 nm and quantitation of corresponding [5-3H]NNK precursor and metabolite peaks was carried out by liquid scintillation spectroscopy. For each NNK metabolite, amount produced was expressed as a percentage of the total radioactivity recovered from [5-3H]NNK plus metabolites per 106 cells per 24 h. This expression of metabolite formation accounted for loss of radioactivity during removal of media from cell pellets, and during analysis. NNK metabolite values from control incubates were subtracted from each individual patient's cellular metabolite profile prior to normalizing to the number of cells used per incubate.
Data analysis
Data are presented as means ± SD, and as individual patient's results. Statistically significant differences between cell preparations were determined by repeated measures analysis of variance (ANOVA) followed by the TukeyKramer multiple comparisons test. When Bartlett's test revealed heterogeneity of variance, a positive integer (k = 1) was added to each datum because some points were equal to zero (non-detectable), and either a log or reciprocal transformation was performed on the resultant values prior to conducting the ANOVA (25). If the transformed data did not achieve homogeneity of variance, the Friedman non-parametric repeated measures test was used (25). Statistically significant differences following SKF-525A treatments were determined by paired t-test. If the F-statistic revealed heterogeneity of variance, data were transformed as described above. If the transformed data did not meet the criteria for homogeneity of variance, the non-parametric Wilcoxon signed rank test was used. P < 0.05 was considered statistically significant in all cases.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
[5-3H]NNK biotransformation in isolated lung cells
Recovery of radioactivity in filtered media ranged from 3662% of that added to cell incubates, of which 7590% represented NNK and its recovered metabolites. Analysis of duplicate cell digest preparations from two patients revealed variability in metabolite quantities of <5%. Subsequently, metabolite analysis was routinely conducted in a single sample for each cell type and treatment.
NNAL was the major metabolite in all cell preparations (Table III), and its formation ranged from 0.50 to 13%/106 cells/24 h (representing ~5155 pmol NNAL/106 cells/24 h), reflecting considerable variability between patients and individual cell types. Both
-hydroxylation and N-oxidation metabolites were detected. However, levels of certain metabolites were not detectable in cells from certain individuals (Table III
). Both total NNK bioactivation (sum of all four
-hydroxylation end-point metabolites) and total NNK detoxification (sum of two N-oxidation metabolites) were highly variable among the 12 patients and three cell fractions examined (Figures 2 and 3
). Formation of
-hydroxylation end-point metabolites ranged from non-detectable to ~0.60%/106 cells/24 h, representing up to 10 pmol
-hydroxylation metabolites/106 cells/24 h, whereas formation of N-oxidation metabolites ranged from non-detectable to ~1.5%/106 cells/24 h (up to 20 pmol/106 cells) (Table III
). For most patients' cell preparations, the balance between
-hydroxylation and N-oxidation weighed heavily towards
-hydroxylation.
|
|
|
There was no apparent effect of smoking status on NNK biotransformation. The NNK metabolite pattern in cells from patient 115, the only reported lifetime non-smoker, was typical of the pattern seen in cells from smokers (Tables I and III; Figures 2 and 3
). Similarly, there were no differences in formation of any NNK metabolites in cells from smokers (n = 6) versus non/former smokers (n = 6) (Student's t-test, P > 0.05).
There were no observable differences in NNK metabolism between cells from four female and eight male subjects (Student's t-test, P > 0.05). Cells from patient 117, an individual who was taking the non-steroidal anti-inflammatory drug sulindac (200 mg twice daily prior to surgery), demonstrated the lowest activity for total NNK biotransformation. All three cell fractions from this patient formed the lowest amounts of NNAL (major metabolite) relative to cell fractions from all other patients examined (Table III). Similarly, this patient's cells formed very low levels of
-hydroxylation metabolites relative to cells from other patients (Figure 2
).
[5-3H]NNK biotransformation: grouped results
When results from all 12 patients were combined, NNK keto reduction to NNAL and subsequent -methyl hydroxylation to the end-point metabolite 4-hydroxy-1-(3-pyridyl)-1-butanol (diol) were 3- to 5-fold higher in fractions enriched in type II cells than in cell digest and macrophage preparations (Figure 4
). No significant differences in the formation of other specific metabolites or in formation of total
-hydroxylation or N-oxidation metabolites occurred between the different cell preparations (P > 0.05).
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Rat and hamster studies have demonstrated that non-ciliated bronchial epithelial (Clara) cells are major sites of NNK activation and toxicity (2628), whereas a role for alveolar type II cells in NNK activation and tumorigenesis has been demonstrated in the A/J mouse (27,29). However, extrapolation of these results to humans is problematic because of significant species differences in morphological and functional characteristics of lung cells (3032). Notably, Clara cells are rare in humans and are decreased in number by cigarette smoke (33). Additionally, human Clara cells do not contain the large amounts of endoplasmic reticulum found in rodent Clara cells (31). Consequently, human Clara cells are not likely to play as large a role in xenobiotic biotransformation as do their rodent counterparts. Human alveolar type II cells do contain endoplasmic reticulum, and have been demonstrated to have high P450 activities relative to other pulmonary cell types (13,14).
Smith et al. (11) reported that 549% of human lung microsomal NNK metabolism may be related to one or more of the various P450 isozymes expressed in human lung. We found bioactivation (reflected by the sum of four -hydroxylation end-point metabolites) to be decreased in some patients' cell preparations treated with the P450 inhibitor SKF-525A, while in some cases it had no effect or increased metabolite formation. In contrast, addition of SKF-525A to type II cell incubates significantly decreased total
-hydroxylation, and diol formation specifically. This could conceivably be an indirect effect, since SKF-525A decreased production of NNAL, the diol precursor. However, since SKF-525A did not decrease formation of other NNAL-derived metabolites (NNAL-N-oxide and hydroxy acid), a role for P450s in NNAL bioactivation by the diol pathway, is implicated.
The type II cell results are consistent with the observation that many peripherally arising human lung adenocarcinomas have ultrastructural and biochemical features characteristic of progenitor cells of the peripheral lung, including type II cells (34), and that persistent DNA pyridyloxobutylation adducts have been demonstrated in rat lung tumours apparently derived from type II cells (2 and references therein). Furthermore, rats treated with phenethyl isothiocyanate, an inhibitor of NNK tumourigenesis, showed a reduction of pyridyloxobutylation in type II cells and in NNK-induced lung tumours (35).
P450-independent (i.e. arachidonic acid) pathways have been suggested for NNK metabolism in human lung (11). However, Rioux and Castonguay (36) did not observe arachidonic acid-supported NNK oxidation in murine pulmonary microsomes, and purified LOX-mediated NNK activation was completely inhibited by exogenous arachidonic acid. Human lung LOX- and PHS-mediated NNK biotransformation have not been fully investigated. However, PHS and LOXs are found at high levels relative to P450s in the lung, and studies from this laboratory have demonstrated cooxidative metabolism of the carcinogen aflatoxin B1 to be concentrated in human alveolar macrophages (22).
It may be that NNK activation is mediated by either PHS or LOX in human pulmonary macrophages as well as in other human lung cell types making up the cell digest preparations, since SKF-525A had no significant effect on mean -hydroxylation in these cells. Furthermore, SKF-525A tended to increase detoxification (reflected by pyridine N-oxidation metabolites), particularly in cells producing detectable N-oxide metabolites in the absence of inhibitor. It has been proposed that SKF-525A inhibits P450-arachidonic acid epoxygenases, making more arachidonic acid available for cooxidative metabolism (22). This could increase the availability of arachidonic acid in these cells and potentiate NNK metabolism.
Consistent with previous reports regarding pulmonary xenobiotic biotransformation activities in general (37,38), and NNK in particular (911), we found NNK metabolism to be highly variable between individuals. Contributing factors to variability include smoking history, diet, alcohol intake, age, race, gender, previous exposure to carcinogens, medications and genetic polymorphisms (4,39,40). Most human lung NNK metabolism studies have involved the use of samples almost entirely from long-term smokers, whose capacity to metabolize NNK may have been affected by years of mainstream smoke exposure (1). Our observation of no apparent difference in NNK metabolism in cells between smokers and non-smokers is consistent with the findings of Castonguay et al. (9) in cultured human bronchus and whole lung explants. It should also be noted that, since four of five former smokers in our study reported not having smoked for at least five years, potential inductive effects of smoking on NNK biotransformation are unlikely to have persisted (21).
In agreement with a previous study of pulmonary microsomal NNK metabolism (10), gender had no apparent effect on NNK metabolism in isolated lung cells, suggesting that the reported higher risk of adenocarcinoma per given number of cigarettes smoked in women (41), is not a result of differences in NNK activation/detoxification within lung cells.
Medications and lifestyle may have affected NNK metabolism in some patients' cells. For example, cells from patient 117 demonstrated very low abilities to metabolize NNK. Interestingly, this patient was taking sulindac, a non-steroidal anti-inflammatory drug, 200 mg twice daily, prior to surgery. Sulindac and its metabolite sulindac sulfone, inhibit pulmonary NNK carcinogenesis in the A/J mouse (42,43). It is possible that administration of sulindac could account for the relatively low levels of NNK metabolites observed in this patient's lung cell fractions. Conversely, the high total biotransformation activity of lung cells from patient 132 might be explained by induction of CYP2E1 due to this patient's alcohol intake of two drinks/day for the last 25 years. Cell digest, but not macrophages or type II cells from patient 112, an asphalt worker, demonstrated abnormally high levels of NNAL and NNAL oxidation metabolites. Thus, chronic exposure to asphalt fumes, coupled with cigarette smoking, may have induced biotransformation enzymes in (a) cell type(s) other than alveolar type II cells or macrophages.
Maser et al. (44) identified 11ß-hydroxysteroid dehydrogenase (11ß-HSD), a microsomal enzyme, as the carbonyl reductase responsible for conversion of NNK to NNAL in mouse lung microsomes. Whether this is the major enzyme responsible for NNK keto reduction in humans is not known (45,46). We found no significant differences between cell types in conversion of NNK to NNAL, suggesting no cellular differences in lung tissue expression of NNK carbonyl reductase. Our observation that three patients (130, 132 and 135) formed high amounts of NNAL, and one patient (117) formed very low amounts of NNAL, is consistent with the suggestion that 11ß-HSD genetic polymorphisms may exist and play a functional role in the conversion of NNK to NNAL (47). In all freshly isolated human lung cell preparations examined, carbonyl reduction of NNK to NNAL was significantly inhibited by SKF-525A. In contrast, this agent had no effect on conversion of NNK to NNAL in rat, hamster and pig lung microsomes (48). Thus, a species variation apparently exists in reduction of NNK to NNAL.
In conclusion, both type II cells and macrophages have the ability to biotransform NNK, and may be potential target cells for NNK toxicity based on -hydroxylation activities. In particular, NNAL bioactivation via
-methyl hydroxylation to hydroxybutyl diazohydroxides, as suggested by diol formation, was concentrated in alveolar type II cells. Thus, type II cells might be target cells for NNK-mediated DNA pyridylhydroxybutylation via the diol formation pathway. Inhibition of total
-hydroxylation formation by SKF-525A suggested a role for P450s in alveolar type II cell NNK bioactivation, but not in alveolar macrophages. NNK bioactivation in different lung cell types showed a high degree of inter-individual variation, which may be attributed in part to environmental factors. Finally, carbonyl reduction of NNK to NNAL is SKF-525A sensitive in human lung cells.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|