Introduction

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The lung represents one of the major targets for exposure to xenobiotics not only because it is the primary site for the entrance of airborne agents but also because it receives 100% of the cardiac output. As one of the extrahepatic organs involved in the biotransformation, the lung contains several enzymatic pathways capable of xenobiotic metabolism (Devereux et al., 1993). These include the cytochrome P450 (CYP¹) superfamily of enzymes, which is the main system catalyzing the oxidative metabolism and metabolic activation of toxic compounds and procarcinogens, as well as phase II enzymes such as glutathione S-transferase, DT-diaphorase, and UDP-glucuronyltransferase, which also play a role in the early cellular defense against toxicity and tumorogenesis (Bogdanffy and Keller, 1999). The susceptibility of the lung to xenobiotics depends on the metabolic balance between toxication and detoxication pathways (Hayashi et al., 1992).

In the lung, CYP is localized in Clara cells, alveolar type II and type I cells, endothelial cells, macrophages, and ciliated bronchiolar cells (Devereux et al., 1993; Lee and Dinsdale, 1995). The nonuniform distribution of xenobiotic bioactivation and detoxication enzymes has been suggested as a basis for cell-specific toxicity observed with many lung-damaging chemicals (Minchin and Boyd, 1983). The use of isolated pulmonary cells constitutes a valuable system for studying mechanisms involved in xenobiotic-induced toxicity (Nemery and Hoet, 1993; Schwarze et al., 1996) and seems to be a good model system to examine the cellular regulation of the CYP monooxygenase pathways in the lung (Devereux et al., 1993; Låg et al., 1996).

Rodent type II pneumocytes and alveolar macrophages have been extensively used for in vitro toxicological studies. The type II epithelial cells play a critical role in toxicant-induced damage, can metabolize many xenobiotic compounds, and represent possible targets in lung carcinogenesis (Schwarze et al., 1996). Alveolar macrophages play an important role in lung defense and in inflammatory responses. Activated alveolar macrophages and their secretory products may also contribute to a variety of lung disorders induced by xenobiotics (Laskin and Laskin, 2001).

Type II pneumocytes are known to alter their phenotype within the first days in culture and to change toward characteristics that are more type I cell-like (Dobbs, 1990). However, most of the studies have focused on morphological changes, production of surfactant phospholipids and apoproteins, and antioxidant enzyme activities (Kalina and Riklis, 1988; Dobbs, 1990; Kinnula et al., 1992). Although xenobiotic-metabolizing enzymes have been extensively characterized in freshly isolated type II pneumocytes and alveolar macrophages (Domin et al., 1986; Devereux et al., 1993), little is known concerning the effect of time in culture on the maintenance of these enzymes. Several studies carried out on hepatocytes have shown the rapid loss of CYP and phase II-catalyzing enzymes after cell isolation and plating (Rogiers and Vercruysse, 1993; Kern et al., 1997). In cultures of rat type II pneumocytes and Clara cells, Låg et al. (1996) have found a decrease in CYP2B1 apoenzyme expression with time in culture.

The aim of the present study was to improve our knowledge of the activity of some xenobiotic-metabolizing enzymes of rat type II pneumocytes and alveolar macrophages in primary cultures.

Experimental Procedures

Chemicals and Materials. Trypsin type I (EC 3.4.214, catalog no. T-8003), reduced glutathione (GSH), NADH, NADPH, metaphosphoric acid, bovine serum albumin, Percoll, EDTA, o-phthaldialdehyde, 3,3'-methylene-bis(4-hydroxycoumarin) (dicumarol), cytochrome c, 1 chloro-2,4-dinitrobenzene, 2,6-dichlorophenol-indophenol, 7-ethoxyresorufin, 7-pentoxyresorufin, 7-benzyloxyresorufin, and a -glutamyl transferase diagnostic kit were purchased from Sigma-Aldrich NV/SA (Bornem, Belgium). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin solution (10,000 U/ml and 10,000 µg/ml, respectively), amphotericin B (250 µg/ml), and L-glutamine (200 mM) were obtained from Invitrogen (Merelbeke, Belgium). Deoxyribonuclease (DNase) I (EC 3.1.21.1) was purchased from Roche Molecular Biochemicals (Mannheim, Germany). Protein assay dye solution was obtained from Bio-Rad (Brussels, Belgium). All other chemicals were purchased from U.C.B. (Brussels, Belgium). Tissue culture plates (24- and 6-well plates) were purchased from Iwaki Glass (International Medical, Brussels, Belgium).

Animals. Male Wistar rats with a mean weight of 191 g (range, 174-215 g) were used. The animals were obtained from an in-house strain and were maintained in a conventional animal house with 12-h dark/light cycles in metal cages with a wired bottom. Animals were allowed free access to standard laboratory diet and tap water.

Cell Isolation and Plating. A population of enriched type II pneumocytes was isolated from rat lung, according to the method of Hoet et al. (1995), which includes lung perfusion, trypsin digestion, Percoll gradient centrifugation, and differential adherence. Briefly, the rats were anesthetized with pentobarbital (90-mg/kg i.p.) and euthanized by exsanguination. The trachea was cannulated, and the lungs were perfused with 0.9% NaCl via the pulmonary artery and ventilated 5 times with 5 to 8 ml of air. The lungs were excised and lavaged via the trachea with 0.9% NaCl (5 times with 3.5 ml/100 g of body weight). The lungs were trypsinized (250 mg of trypsin in 100 ml of phosphate-buffered saline with calcium and magnesium; PBS⁺, 130 mM NaCl, 5.2 mM KCl, 10 mM glucose, 10.6 mM Hepes, 2.6 mM Na₂HPO₄, 1.9 mM CaCl₂, 1.29 mM MgSO₄, pH 7.4, per rat lung) during 30 min at 37°C. The lungs were chopped, and 5 ml of FBS and 3 mg of DNase I were added. After shaking and filtering, the cell suspension was layered onto a discontinuous Percoll gradient (density, 1.089 and 1.040 g/ml) and centrifuged at 250g for 20 min (10°C). The cells of the creamy layer above the heavy gradient were plated in a 60-mm diameter culture dish and incubated for 1 h (5% CO₂, 37°C) to let the macrophages attach. The unattached cells were suspended in DMEM supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 1.25 µg/ml Fungizone, and 2 mM glutamine. An average of 5.5 × 10⁶ cells were isolated per animal (range, 4-8 × 10⁶), and the cell viability, as assessed by trypan blue exclusion, was 98% (range, 96-99). Identification of type II pneumocytes was carried out with the alkaline phosphatase stain. The proportions of alkaline phosphatase-stained cells in freshly isolated cell preparations and after 24 and 48 h in culture were 74 ± 8 (n = 6), 83 ± 1 (n = 4), and 96 ± 2% (n = 4), respectively.

Enzyme Assays. For the determination of enzyme activities, the cells were scraped on ice with a rubber policeman in buffer containing 10 mM Tris, 150 mM KCl, and 1 mM EDTA, pH 7.4. Aliquots of the freshly isolated cells were suspended in the same buffer. All samples were immediately frozen at 80°C. Before the assays, the cell samples were homogenized by sonication on ice with a probe sonicator (Instruments Scientifiques Analis, Namur, Belgium) at an amplitude of 18 µm peak-to-peak for 15 s. A 50-µl aliquot was taken for protein determination. The O-dealkylation of 7-ethoxyresorufin (EROD), PROD, and BROD were determined by the method of Lubet et al. (1985). The initial linear rate (5 min) of resorufin formation was measured spectrofluorometrically (excitation, 550 nm; emission, 586 nm) at 37°C using a Shimadzu RF-5001PC fluorescence spectrophotometer (Shimadzu, Benelux, Antwerpen, Belgium). The reaction mixture (1 ml) consisted of 50 mM Tris, 25 mM MgCl₂ buffer, pH 7.5, 100 µM NADPH, substrate (1 µM 7-ethoxyresorufin, 5 µM 7-pentoxyresorufin, or 5 µM 7-benzyloxyresorufin), and sonicated cell suspension (200-500 µg of protein). Dicumarol (20 µM final concentration) was added to the assay medium to prevent the biotransformation of resorufin by cytosolic diaphorase. At the end of the incubation, 10 pmol of resorufin was added as an internal standard. The results were expressed as picomoles of resorufin formed per minute per milligram of cell protein.

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GSH Determination. The cells were deproteinized in 200 µl of metaphosphoric acid (5% in 0.1 M phosphate, 0.005 M EDTA buffer, pH 8.0), samples were centrifuged at 14,000g for 15 min, and the supernatant was analyzed by the fluorometric method of Hissin and Hilf (1976) as previously described (Dimova et al., 2000). A standard curve using GSH was prepared for each run. GSH content was expressed as nanomoles per milligram of cell protein.

Protein Determination. The sonicated cell suspensions and precipitated proteins were dissolved in 1 N NaOH. The cell protein content was determined using the method of Bradford (1976) after neutralization with 0.333 N HCl. Bovine serum albumin was used as the standard.

Statistical Analysis. The results are presented as means ± S.D. of three to six independent experiments. The data were statistically analyzed by repeated measures ANOVA followed by Bonferroni's multiple comparison test or by Student's t test (GraphPad Prism package; GraphPad Software, San Diego, CA). The minimum level of significance was considered to be p < 0.05. Statistical analysis was performed before the data were transformed to percentages.

	Results

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Xenobiotic-metabolizing enzyme activities, intracellular GSH, and -GT activity were studied in freshly isolated rat type II pneumocytes (initial values, 0 h) and after 24 and 48 h in culture and in freshly isolated and 3-h-attached rat alveolar macrophages. The absolute values of the parameters studied in freshly isolated type II pneumocytes and alveolar macrophages are shown in Table 1. Freshly isolated type II pneumocytes possessed higher BROD, NADPH- and NADH-cytochrome c reductases, DT-diaphorase, and GST activities compared with freshly isolated alveolar macrophages. No difference was observed in intracellular GSH content, calculated per milligram of cell protein. At the protein concentrations used, EROD activity was undetectable in both type II pneumocytes and alveolar macrophages. In macrophages, PROD and -GT activities were also below the detection limits.

The CYP-dependent alkoxyresorufin-O-dealkylation in type II pneumocytes was considerably lost with time in culture (Fig. 1A). At 24 h, the BROD and PROD activities decreased by 84 and 82%, respectively, and they continued to decline over the next 24 h, with no measurable PROD activity at 48 h. The activity of NADPH-cytochrome c reductase, considered to be mainly due to the presence of CYP reductase, was relatively stable at 24 h but decreased by 31% at 48 h compared with freshly isolated type II pneumocytes (Fig. 1B). NADH-cytochrome c reductase activity decreased with time in culture by 42 and 51% at 24 and 48 h, respectively.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Effect of time in culture on xenobiotic-related enzyme activities in rat type II pneumocytes. A, BROD () and PROD (); B, NADPH ()- and NADH ()- cytochrome c reductase; C, GST; D, DT-diaphorase. All enzyme activities were measured in sonicated cells. The results are expressed as a percentage of the corresponding activity in freshly isolated cells (initial time value, 0). Data are the mean ± S.D.; n = 3 to 6; *p < 0.05, **p < 0.01, ***p < 0.001 significant versus freshly isolated type II pneumocytes; °p < 0.05 significant versus 24 h; repeated measures ANOVA followed by Bonferroni's multiple comparison test. ND, not detectable (see Table 1).

In contrast to CYP-related activities, the activity of cytosolic DT-diaphorase was increased at 24 h (by 50%) and then decreased to approximately the initial level at 48 h (Fig. 1D). The activity of GST decreased continuously as a function of time (Fig. 1C), but more slowly than did BROD and PROD activities. After 24 h and 48 h in culture, GST activity in type II pneumocytes significantly decreased by 25 and 42%, respectively. At the same time, the intracellular GSH content increased, and at 48 h, it was elevated by 159% compared with freshly isolated type II pneumocytes (Fig. 2A). The increase in intracellular GSH level was associated with an increase in -GT activity by 45 and 95% at 24 and 48 h, respectively (Fig. 2B).

View larger version (7K): [in this window] [in a new window]   Fig. 2.   Effect of time in culture on intracellular GSH level (A) and -GT activity (B) in rat type II pneumocytes. The results are expressed as a percentage of the corresponding value in freshly isolated cells (initial time value, 0). Data are the mean ± S.D.; n = 5 to 6; *p < 0.05, **p < 0.01 significant versus freshly isolated type II pneumocytes; °p < 0.05 significant versus 24 h; repeated measures ANOVA followed by Bonferroni's multiple comparison test.

In 3-h-attached alveolar macrophages, the BROD activity decreased by 38% compared with freshly isolated cells (Fig. 3A). There were no changes in NADPH- and NADH-cytochrome c reductase, GST, and DT-diaphorase activities in 3-h-attached alveolar macrophages (Fig. 3, A and B). An increase of intracellular GSH (by 24%) was observed in the cultured macrophages.

View larger version (12K): [in this window] [in a new window]   Fig. 3.   BROD, NADPH- and NADH-cytochrome c reductase (A) and DT-diaphorase, GST, and GSH (B) in freshly isolated and 3-h-attached rat alveolar macrophages. All enzyme activities were measured in sonicated cells. The results are expressed as a percentage of the corresponding activity in freshly isolated cells. Data are the mean ± S.D.; n = 4 to 6; *p < 0.05, **p < 0.01 significant versus freshly isolated alveolar macrophages; paired Student's t test.

	Discussion

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The present study demonstrates that rat type II pneumocyte cultures undergo a decrease in the activities of BROD, PROD, NADPH- and NADH-cytochrome c-reductase, and GST and an increase of DT-diaphorase, intracellular GSH, and -GT. In alveolar macrophages, a decrease in BROD activity and increase in GSH occur after 3 h in culture. The time points (0, 24, and 48 h in type II pneumocytes; 0 and 3 h in alveolar macrophages) were chosen because of their common use in studying xenobiotic toxicity in these cells.

We determined the activities, rather than apoprotein or mRNA levels of the xenobiotic-metabolizing enzymes, because activities represent a true measure of the corresponding enzyme performance. The activities found in freshly isolated rat type II pneumocytes and alveolar macrophages were in the range of those previously reported (Lacy et al., 1992; Devereux et al., 1993). The most important CYP isoenzymes identified in rat lung are CYP2B1, 3A, 4B, 2F, 1A1, and 2E1 (Bogdanffy and Keller, 1999). Benzyloxyresorufin is a substrate for several isoenzymes, including CYP1A1, 2B1 and 3A1 (Wolf et al., 1986). Pentoxyresorufin shows high specificity for CYP2B1, and it was established that it is also a substrate for CYP2F1 (Nhamburo et al., 1990). Because CYP2B1 is the main constitutive CYP present in rat lung (Guengerich, 1990), we suggest that the greatest part of PROD and BROD is related to CYP2B1, but CYP2F1 and 3A1 can also contribute to these activities. We found 37 times lower BROD activity and undetectable PROD activity in alveolar macrophages compared with type II pneumocytes, which is in agreement with the levels of expression of CYP2B1 mRNA in these cells, as reported by Låg et al. (1996). CYP1A1-associated EROD activity was undetectable in freshly isolated type II pneumocytes and alveolar macrophages. This may be due to a combination of low activity and limitation of the detection assay. In uninduced rat lung, CYP1A1 is generally present at very low levels (Marcus et al., 1990). EROD activity has been measured in microsomes isolated from Clara and type II cells (Domin et al., 1986) and macrophages (Germolec et al., 1995) but not in sonicated cell suspensions of rat type II pneumocytes and alveolar macrophages (Lacy et al., 1992).

Freshly isolated type II pneumocytes exhibited 6-fold greater DT-diaphorase activity than alveolar macrophages. This is in line with the results of Siegel et al. (1988), who found a minimal DT-diaphorase activity in mouse alveolar macrophages and showed that type II pneumocytes are the site of DT-diaphorase activity in rodent lung. In rat lung, the apical plasma membrane enzyme -GT is localized dominantly in Clara cells and type II pneumocytes and is a recognized marker of these cell types (Dinsdale et al., 1992). In alveolar macrophages, -GT activity is 10 times lower than in type II pneumocytes and has not been detected histochemically (Van Klaveren et al., 1997). Accordingly, we did not detect -GT activity in alveolar macrophages.

Similar to hepatocytes, the culturing of rat type II pneumocytes and alveolar macrophages results in changes in xenobiotic-metabolizing enzyme activities and intracellular GSH content. Our findings show that the CYP-related activities do not survive well in type II pneumocytes and alveolar macrophages in our simple but common culture conditions since most of the activity was lost at 24 and 3 h, respectively. The marked reduction in BROD and PROD activities in type II pneumocytes is consistent with previous studies, showing an approximate 50% decrease in CYP2B1 apoenzyme expression after 24 h in culture (Låg et al., 1996). Similar decreases in BROD and EROD activities were found in hamster lung slices after 24 h of incubation (Hoet et al., 1997). NADPH-cytochrome c reductase activity was more stable than CYP-related activities.

In general, the phase II-related activities were better kept than those of phase I enzymes. This is in line with the data obtained on hepatocytes (Rogiers and Vercruysse, 1993). In 3-h-attached alveolar macrophages, there were no changes in GST and DT-diaphorase activity. In type II pneumocytes, GST activity decreased to a lesser extent compared with BROD and PROD activities, and at 48 h in culture, it was 58% of the initial value. In cultured hepatocytes, Niemann et al. (1991) described a similar (40%) decrease in GST activity.

The activity of DT-diaphorase in culture has not been extensively studied. In human fibroblasts, the DT-diaphorase activity increases as the cells reach confluence and become density growth arrested (Schlager et al., 1993). Siegel et al. (1988) have found a 10% increase in DT-diaphorase activity 24 h after plating of rat type II pneumocytes. The observed transient increase in DT-diaphorase activity in type II pneumocytes at 24 h by 50% and return to the baseline level at 48 h is difficult to explain.

We observed an increase in intracellular GSH in cultured type II pneumocytes and alveolar macrophages compared with freshly isolated cells. These results confirmed our previous observation that there is a higher GSH content in type II pneumocytes cultured for 24 h rather than in freshly isolated cells (Dimova et al., 2000) and are in agreement with the results of Reynolds et al. (1999). The increase GSH content with time in culture has been shown in primary cultures of rodent hepatocytes (Ruch et al., 1989).

In cultured type II pneumocytes, the increase in GSH was associated with an increase in -GT activity. -GT plays an important role in the regulation of intracellular GSH levels via the -glutamyl cycle, and in type II pneumocytes, it has a major role in the use of extracellular GSH as a source for intracellular GSH (Dinsdale et al., 1992). -GT increases as a part of the adaptation of the cells to oxidative stress (Kugelman et al., 1994). Kinnula et al. (1992) have found that antioxidant enzyme activities (catalase, glutathione reductase, and glutathione peroxidase) but not reactive oxygen species generation by rat type II pneumocytes decrease rapidly in culture. The increase in -GT activity and intracellular GSH may represent an adaptation mechanism of the cells to the changed antioxidant status in type II pneumocytes in culture.

The balance of metabolic activation and detoxification of xenobiotics is an important factor for their toxicity and carcinogenesis. The results of this study have shown that in conventional cultures of type II pneumocytes and alveolar macrophages, phase II enzyme activities (GST and quinone oxidoreductase) were better kept than those of phase I (CYP-related enzymes) and an increase in intracellular GSH occurs. These shifts in the balance between toxication and detoxication pathways in cultured rat type II pneumocytes and alveolar macrophages make the cells in culture more resistant to the toxic action of xenobiotics than freshly isolated cells. This is apparent with agents such as acetaminophen in type II pneumocytes (Dimova et al., 2000).

In conclusion, type II pneumocytes and to a lesser extent alveolar macrophages in primary cultures undergo changes in biotransformation-related enzyme activities and intracellular GSH levels that may affect xenobiotic toxicity at different times in culture. These biochemical changes make the results obtained not necessarily comparable with the in vivo situation and suggest the need for the development of a culture model (mainly for type II pneumocytes) in which the xenobiotic-metabolizing activities are better kept.