Experimental Procedures

Animals.

Rat AII cells were isolated from male Wistar rats (100–150 g) that had been given free access to laboratory chow and water and had been maintained on a 12-h light/dark cycle.

Materials.

Trypsin, trypsin inhibitor, insulin, deoxyribonuclease, hydrocortisone hemisuccinate, 3,3′,5′-triiodo-l-thyronine, Na-selenite, and HEPES were obtained from Sigma (Deisenhofen, Germany). Dulbecco's modified Eagle's medium and Ham's F-12 was purchased from Life Technologies (Berlin, Germany), fetal calf serum was obtained from PAA (Coelbe, Germany), and collagen type I was purchased from Becton Dickinson (Heidelberg, Germany). Cell culture dishes were obtained from Nunc (Wiesbaden, Germany). Trizol reagent was purchased from Life Technologies. T4 polynucleotide kinase and herring sperm DNA were purchased from Roche Molecular Biochemicals (Mannheim, Germany). BSA was obtained from Paesel & Lorei (Frankfurt, Germany). Polyvinylidene difluoride membranes were purchased from Millipore (Eschborn, Germany); Hybond N nylon membranes and the enhanced chemiluminescence system were purchased from Amersham (Braunschweig, Germany). [γ-³²P]ATP was purchased from DuPont-NEN (Bad Homburg, Germany). The CYP2B1 antibody was obtained from Gentest (Woburn, MA). The CYP2B1 specific oligonucleotide probe (Omiecinski et al., 1985) (5′-GGT TGG TAG CCG GTG TGA-3′, exon 7, Genbank L00318) was synthesized by MWG Biotech (Ebersberg, Germany). The oligonucleotide probe specific for mouse and rat β-actin (exon 3) was obtained from Oncogene Research Products (Cambridge, MA). The secondary enzyme-conjugated anti-goat IgG antibody was purchased from Sigma. [5-³H]NNK with a specific activity of 2.89 Ci/mmol was obtained from Campro Scientific (Emmerich, Germany). HPLC analysis confirmed >98% radiochemical purity. Unlabeled reference compounds for known metabolites of NNK were a generous gift from Dr. D. Hoffmann (American Health Foundation, Valhalla, NY). HPLC grade solvents were obtained from Merck (Darmstadt, Germany). Quickszint Flow was purchased from Zinser Analytic (Frankfurt, Germany).

AII Cell Isolation.

AII cells were isolated according to the procedure described byRichards et al. (1987). AII cell suspensions showed viabilities greater than 90% as determined by trypan blue exclusion. AII cells were suspended in medium (Dulbecco's modified Eagle's medium/Ham's F-12) supplemented with 3% fetal calf serum, 5 μg/ml insulin, 5 μg/ml human transferrin, 50 nM hydrocortisone hemisuccinate, 4.75 pM 3,3′,5′-triiodo-l-thyronine, and 50 nM Na-selenite. Cells were plated at a density of 0.28 to 0.46 × 10⁶ cells/cm² on collagen type I-coated (4.1 μg/cm²) culture dishes (58 mm diameter) and cultivated at 37°C in a humidified atmosphere of 5% CO₂ and 90% air. At the end of the culture period, AII cells were washed once with PBS, pH 7.4, frozen in liquid nitrogen, and stored at −80°C before isolation of total RNA or proteins. Staining of characteristic lamellar bodies of AII cells, rendering a proof of culture purity, was performed according to the procedure described by Mason et al. (1985).

Northern Blot Analysis.

Total cellular RNA was isolated according to a modification of the procedure described by Chomczynski and Sacchi (1987) using the Trizol reagent. For Northern blots, 20 μg of total RNA per lane was separated electrophoretically through 1% formaldehyde/agarose gels. RNA was subsequently blotted onto Hybond N nylon membranes by capillary transfer (Sambrook et al., 1989) using 20× standard saline citrate (3 M NaCl, 0.3 M trisodium citrate) as transfer buffer. RNA blots were hybridized to oligonucleotide probes specific for CYP2B1 (Sambrook et al., 1989) or β-actin mRNA, which had been 5′-end-labeled with T4 polynucleotide kinase using [γ-³²P]ATP (Sambrook et al., 1989). Membranes were prehybridized for 2 h and hybridized for 16 h at 38°C in hybridization buffer containing 1 M NaCl, 0.1 M trisodium citrate, 50% (v/v) formamide, 0.5% (w/v) SDS, 5% (v/v) Denhardt's solution, and 400 μg/ml herring sperm DNA. The blots were then washed up to a stringency of 0.1× standard saline citrate (15 mM NaCl, 1.5 mM trisodium citrate) and 0.1% SDS (w/v) at 38°C (CYP2B1) or 60°C (β-actin), respectively. The expression of specific mRNAs was quantified by a phosphorimaging system (BAS 1500 Bio-Imaging Analyzer; Raytest, Straubenhardt, Germany).

Immunoblot Analysis.

Total cellular protein was isolated by using the Trizol reagent. Ten micrograms of protein per lane, determined according to Lowry et al. (1951), were subjected to electrophoresis through 7.5% SDS-polyacrylamide gels (Laemmli, 1970). Proteins were subsequently transferred to polyvinylidene difluoride membranes by semidry-blotting (Kyhse-Andersen, 1984) using a continuous buffer system [48 mM Tris, 39 mM glycine, 0.038% (w/v) SDS, and 15% (v/v) methanol, pH 9.0]. CYP2B1 was detected by using a primary polyclonal antibody and a peroxidase-conjugated anti-rabbit IgG as secondary antibody. Protein bands were visualized by enhanced chemiluminescence using the ECL-system.

In Vitro Assay of NNK Metabolism.

Immediately after plating of the AII cells, [5-³H]NNK was added to the medium, leading to initial concentrations of 0.05 μM or 5 μM, respectively. Cells were incubated for 48 h without media changes. At the end of the culture period, the supernatant medium was removed and stored at −20°C. Analysis of NNK metabolites in medium was performed by HPLC according to a modified procedure developed by Carmella and Hecht (1985). After precipitation of proteins by trichloroacetic acid, 200 to 600 μl of the clear supernatant was adjusted to pH 7.4 with 2 M Tris buffer. Samples were chromatographed on a 4 × 250 mm LiChrospher 60 RP-selectB 5-μm column fitted with a 4 × 4 mm precolumn (Merck, Darmstadt, Germany) by elution with a gradient that was linear from 100% A to 85% A/15% B in 25 min and linear to 70% A/30% B in 5 min (A: 20 mM Tris buffer, pH 7.2; B: acetonitrile) at a flow rate of 0.7 ml/min. Radioactivity was monitored by liquid scintillation counting using a radioactivity monitor fitted with a flow cell (LB 506 C-1; Berthold, Wildbad, Germany). Radioactive metabolites were identified by cochromatography with unlabeled reference compounds detected by their UV absorption at 254 nm (Kontron, Ecking, Germany). The detection limit for a single peak was 740 dpm, corresponding to 1% of the total radioactivity in a sample.

Results

Several studies suggest that AII cells might be target cells of NNK-induced carcinogenesis, which underlines the importance of investigations in AII cell-specific NNK metabolism (Belinsky et al., 1990, 1991). In this study, primary cultures of AII cells were chosen as an experimental model offering the advantage of longer incubation times than are possible in investigations involving suspended cells. For determination of purity and demonstration of differentiation of the cultured cells, characteristic lamellar bodies of AII cells were stained with tannic acid (Mason et al., 1985), which revealed an enrichment of the AII cells to approximately 80%. As apparent from Fig. 2, cells cultured for 72 h before staining exhibit several dark inclusions (surfactant containing lamellar bodies), indicating differentiated AII cells.

• Download figure
• Open in new tab
• Download powerpoint

Figure 2

Primary culture of rat AII cells (72 h) after tannic acid/polychrome staining.

Several lamellar bodies can be identified as dark inclusions (↓).

Expression of CYP2B1 in Primary Culture of AII Cells.

CYP2B1 is one of the major CYP isoforms expressed in the lung (Bond, 1993). It is constitutively expressed in the lung and participates on the metabolic activation of NNK (Bond, 1993; Hecht, 1996). Down-regulation of xenobiotic-metabolizing enzymes is a common phenomenon in primary cell culture (Guguen-Guillouzo et al., 1988); therefore, before the investigation of NNK metabolism we examined the expression of CYP2B1 in the primary cultures of AII cells.

Northern blot analysis of CYP2B1 mRNA (Fig.3) revealed a substantial expression of CYP2B1 mRNA in freshly isolated AII cells, which was comparable to the expression in the whole lung. However, CYP2B1 mRNA content decreased in a time-dependent manner during primary AII cell culture. Nevertheless, even after 72 h of culture a marked signal for CYP2B1 mRNA expression was observed.

• Download figure
• Open in new tab
• Download powerpoint

Figure 3

CYP2B1 mRNA expression in the whole lung (Lu), in freshly isolated AII cells (AII), and in AII cells cultured for differing periods of time (24, 48, and 72 h).

The membrane was rehybridized to a β-actin-specific oligonucleotide. Equal loading of lanes was ensured by evaluation of ethidium bromide staining of the 18 and 28 rRNA, respectively.

To determine whether the results observed on the mRNA level correlated with CYP2B1 protein expression in primary cultured AII cells, Western blot analyses were performed using a polyclonal antibody that recognizes both closely related isoforms of rat CYP2B (2B1 and 2B2). However, because CYP2B2 is not expressed in the rat lung (Srivastava et al., 1989), signals observed in the Western blot analysis of protein obtained from lung tissue or AII cells can be attributed to CYP2B1. As apparent from Fig. 4, a substantial CYP2B protein content was observed in total protein of freshly isolated AII cells, which was slightly enriched in comparison to the CYP2B protein content in the whole lung tissue. In analogy to the kinetics of CYP2B1 mRNA expression, CYP2B1 protein expression decreased during the primary culture of AII cells. However, a marked CYP2B1 protein content was obtained in AII cells after 24 h of primary culture, suggesting that primary cultured AII cells are metabolically active for at least 24 h.

• Download figure
• Open in new tab
• Download powerpoint

Figure 4

Western blot analysis of CYP2B protein expression in total protein of the whole lung (Lu), in freshly isolated AII cells (AII), and in AII cells cultured for differing periods of time (24, 48, and 72 h).

Aliquots of 10 μg total protein were subjected to immunoblot analysis. Hepatic microsomes of phenobarbital-treated rats served as a CYP2B protein reference.

Factors known to stabilize expression or induction of CYP2B1 in primary cultured rat hepatocytes such as culture in the absence of serum or in the presence of metyrapone (Aubrecht et al., 1996) failed to maintain CYP 2B1 expression in AII cell culture (data not shown).

NNK Metabolism in Primary Cultured AII Cells.

Taking into consideration the time-dependent reduction of CYP2B1 expression in primary cultured AII cells, metabolism of NNK was investigated within the first 48 h of culture. To examine whether NNK metabolism in AII cells exhibits dose-dependent alterations, AII cells were incubated at different concentrations of NNK (0.05 and 5 μM). Analysis of NNK and its metabolites were performed by HPLC. The radioactively labeled metabolites in the supernatant of cell culture were identified by cochromatography with the unlabeled reference metabolites. Typical HPLC runs of an analysis of a standard cocktail containing the nonradiolabeled reference metabolites and a medium sample are shown in Fig. 5.

• Download figure
• Open in new tab
• Download powerpoint

Figure 5

HPLC analysis of nonradiolabeled metabolites (top); separation of radiolabeled metabolites of [5-³H]NNK in cell culture medium after 48 h of culture with 0.05 μM NNK (bottom). n.i.: not identified.

During the first 48 h of culture a remarkable activity to metabolize NNK was revealed in primary cultured AII cells, leading to the formation of a broad spectrum of metabolites whereas only minor amounts of unmetabolized NNK remained in the medium. When AII cells were incubated with 0.05 μM NNK, N-oxidation of NNK, which is thought to be a detoxification pathway, was predominant, accounting for 42% of total NNK metabolism (Fig.6A). Nevertheless, substantial amounts of metabolites resulting from α-hydroxylation (representing the pathway of metabolic activation of NNK) were obtained in the cell culture media. In summary, metabolites resulting from metabolic activation (4-oxo-4-(3-pyridyl)-butyric acid, 4-oxo-(3-pyridyl)-butanol, and diol) contributed to 35% of all detected metabolites.

• Download figure
• Open in new tab
• Download powerpoint

Figure 6

NNK metabolite concentrations in cell culture media of AII cells after 48 h of culture.

Mean ± S.D. of three incubations. Filled columns represent the proportions of metabolites resulting from metabolic activation, hatched columns represent metabolites from detoxification of NNK and NNAL. n.i.: not identified. A, culture in the presence of 0.05 μM NNK. B, culture in the presence of 5 μM NNK.

However, when AII cells were incubated with a 100-fold higher concentration of NNK (5 μM), carbonyl reduction of NNK leading to the formation of NNAL became predominant (Fig. 6B). NNAL amounted to 32% of all detected metabolites, whereas the proportion of NNK-N-oxide decreased from 42% at 0.05 μM NNK to 22% at 5 μM NNK. Simultaneously, the proportion of metabolites from metabolic activation of NNK decreased to 17% when the concentration of NNK was increased. At both concentrations marked amounts of diol, a metabolite resulting from α-hydroxylation of NNAL, were detected (Fig. 6, A and B), indicating that AII cells are also capable of activating NNAL.

Interestingly, in all incubations a NNK metabolite that did not correspond to the used reference metabolites was observed (Figs. 5 and6). Incubations of the medium with β-glucuronidase to examine whether this metabolic product might be a glucuronidated metabolite (e.g., NNAL-Glu) (Schrader et al., 1998) revealed no enzymatic transformation of the substance (data not shown).

Discussion

As other nitrosamines, NNK requires metabolic activation to elicit its tumorigenic effects. Metabolic activation is suggested to proceed via α-hydroxylation of NNK and NNAL, resulting in the formation of reactive intermediates that bind to cellular proteins and DNA (Hecht, 1996). Several studies have demonstrated that α-hydroxylation as well as N-oxidation of NNK and NNAL are predominantly catalyzed by enzymes of the CYP superfamily (for review see Hecht, 1996). Furthermore, recent studies revealed evidence that other enzymes like cyclooxygenases and lipoxygenases might also be involved in metabolic activation of NNK (Smith et al., 1997; Rioux and Castonguay, 1998). On the other hand, an enzyme responsible for steroid metabolism, namely 11β-hydroxysteroid dehydrogenase, is suggested to catalyze the carbonyl reduction of NNK, resulting in the formation of NNAL (Maser, 1997, 1998).

Enzymes capable of metabolizing NNK are localized in several mammalian organs and tissues. However, numerous carcinogenicity studies demonstrated that NNK primarily induces pulmonary adenoma and adenocarcinoma in mice, rats and hamsters, independent of the route of administration (for review see Hoffmann et al., 1994). Within the lung AII cells and nonciliated bronchiolar epithelial (Clara) cells seem to be the most sensitive cell populations in NNK-induced carcinogenesis (Belinsky et al., 1990; Yang et al., 1997). Sensitivity of AII cells toward the carcinogenic effects of NNK is underlined by the occurrence of the characteristic lamellar bodies in NNK-induced hyperplasia. Furthermore, early proliferative lesions in mice and rats after NNK treatment occurred preferentially in the alveolar area of the lung (Belinsky et al., 1991).

Belinsky et al. (1989) demonstrated that NNAL is the main metabolite in suspensions of AII cells incubated with NNK at a concentration of 150 μM. However, this concentration of NNK is several magnitudes higher than the expected NNK plasma levels in human smokers who are exposed to 0.1 to 1.5 of nmol NNK per cigarette (Adams et al., 1985; Hoffmann et al., 1994).

In this study primary cultures of AII cells were investigated according to their ability to metabolize NNK as well as to their expression of CYP2B1, which is involved in the metabolic activation of NNK. Primary cell culture offers the advantage of longer incubation times than are possible in experiments with suspended cells. Furthermore, primary culture of AII cells enables investigation whether NNK affects cellular functions, e.g., gene expression or proliferation of the target cells of NNK-induced carcinogenesis. On the other hand, culture of cells is often accompanied by a decrease in the capacity to metabolize xenobiotics (Guguen-Guillouzo et al., 1988) which was also observed in the present study. Primary cultures of AII cells exhibited a time-dependent decrease of the expression of CYP 2B1. However, because the investigation of CYP2B1 mRNA and CYP2B protein levels in the cultured AII cells revealed a retained expression up to 24 to 48 h, metabolism of NNK in AII cells was studied within the first 48 h of culture. Besides CYP2B1, other CYP isoforms have been shown to also be involved in the metabolism of NNK in the lung, e.g., CYP1A or CYP2A subfamily members (Smith et al., 1992a; Hecht, 1994). It remains to be resolved in which manner expression/induction of other CYP enzymes in AII cells might be affected during cell culture.

During the first 48 h, the cultured AII cells demonstrated a remarkable activity to metabolize NNK at two concentrations (5 and 0.05 μM), leading to the formation of a broad spectrum of metabolites. This indicates the usefulness of primary cultures of AII cells, which due to the relatively long incubation time (48 h) allow the investigation of an extensive metabolism of NNK also at low NNK concentrations. Nevertheless, because the half-life of NNK in rats is in the range of hours (Adams et al., 1985), examination of NNK metabolism at shorter periods of AII cell culture would be of interest in future studies.

Formation of NNAL was the predominant pathway in the metabolism of 5 μM NNK. However, NNK-N-oxide was the main metabolite in the medium of cells incubated with 0.05 μM NNK. The proportion of metabolites resulting from metabolic activation was substantially lower in incubations of AII cells at 5 μM NNK compared with incubations performed at 0.05 μM NNK. These data suggest that formation of NNAL preferentially takes place at high concentrations, whereas CYP-mediated biotransformations, including the metabolic activation of NNK, occur already at low NNK concentrations. Taken together, the present data indicate that metabolic activation of NNK in AII cells proceeds more effectively than suggested from previous studies (Belinsky et al., 1989).

The different fractions of hydroxylated metabolites and NNAL on the total metabolism of NNK at low and high NNK concentrations may be explained by the kinetic data of the underlying biotransformations. Whereas K_m values in the micromolar range were demonstrated for the CYP-catalyzed α-hydroxylation of NNK (Peterson et al., 1991; Smith et al., 1992b), aK_m value in the millimolar range was obtained for the carbonyl reduction of NNK catalyzed by 11β-hydroxysteroid dehydrogenase 1, which is one of the enzymes capable of forming NNAL (Maser, 1997, 1998).

Interestingly, the observed pattern of NNK metabolites in cultured AII cells is in agreement with the recently described metabolic pathways of NNK in the whole rat lung (Schrader et al., 1998;Schulze et al., 1998). This might indicate that besides Clara cells AII cells may contribute to a main part to the overall metabolism of NNK in the whole lung.

Apart from the dose-dependent differences in NNK metabolism in primary cultured AII cells, a NNK metabolite that did not correspond to the used common reference metabolites was observed in the culture medium of AII cells after 48 h of culture. Considering the fact that AII cells appear to exhibit a high sensitivity to the carcinogenic effects of NNK (Belinsky et al., 1991), additional examination of this metabolite might be helpful to clarify the reasons for the susceptibility of AII cells.

In summary, the present study indicates the usefulness of primary cultures of AII in the investigation of AII cell-specific biotransformation of NNK. Cultured AII cells exhibited a remarkable capacity to metabolize NNK, leading to a substantial metabolic activation of NNK, which was more pronounced in incubations at low NNK concentrations. Because human exposure toward cigarette smoke leads to low plasma concentrations of NNK ranging in the picomolar scale (Adams et al., 1985; Hoffmann et al., 1994), these data suggest that metabolic activation of NNK in human smokers might be more effective than expected from previous studies mainly performed at the micromolar range.