Toxicology in Vitro 28 (2014) 812–821

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Isolation and cultivation of metabolically competent alveolar epithelial cells from A/J mice Tanja Hansen a, Anil Chougule b, Jürgen Borlak b,⇑ a b

Fraunhofer Institute of Toxicology and Experimental Medicine, Nikolai-Fuchs-Str. 1, 30625 Hannover, Germany Centre for Pharmacology and Toxicology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany

a r t i c l e

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Article history: Received 19 September 2013 Accepted 18 March 2014 Available online 27 March 2014 Keywords: A/J mice AECs isolation and cultivation Characterization of AECs Cytochrome P450 monooxygenases Gene expression EROD and testosterone metabolism

a b s t r a c t The A/J mouse strain is used in lung cancer studies. To enable mechanistic investigations the isolation and cultivation of alveolar epithelial cells (AECs) is desirable. Based on four different protocols dispase digestion of lung tissue was best and yielded 9.3 ± 1.5  106 AECs. Of these 61 ± 13% and 43 ± 5% were positive for AP and NBT staining, respectively. Purification by discontinuous Percoll gradient centrifugation did not change this ratio; however, reduced the total cell yield to 4.4 ± 1.1  106 AECs. Flow cytometry of lectin bound AECs determined 91 ± 7% and 87 ± 5% as positive for Helix pomatia and Maclura pomifera to evidence type II pneumocytes. On day 3 in culture the ethoxyresorufin-O-demethylase activity was 251 ± 80 pmol/4 h  1.5  106 and the production of androstenedione proceed at 243.5 ± 344.4 pmol/ 24 h  1.5  106 AECs. However, 6-a, 6-b and 16-b-hydroxytestosterone were produced about 20-fold less as compared to androstenedione and the production of metabolites depended on the culture media supplemented with 2% mouse serum or 10% FCS. Finally, by RT-PCR expression of CYP genes was confirmed in lung tissue and AECs; a link between testosterone metabolism and CYP2A12, 3A16 and 2B9/10 expression was established. Taken collectively, AECs can be successfully isolated and cultured for six days while retaining metabolic competence. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction The physiological role of the lung is gas exchange and oxygen supply to the body. Because of its large surface area and extensive vascularization administration of drugs via inhalation opens new possibilities for drug entry into systemic circulation particularly for drugs with pharmacokinetics defects, i.e. those that suffer from presystemic metabolic inactivation at the intestinal barriers or due to large hepatic extraction that will result in minimized drug delivery into systemic circulation. In the past, we demonstrated that

Abbreviations: AECs, alveolar epithelial cells; AP, alkaline phosphatase; B[a]P, benzo[a]pyrene; CYP, cytochrome P; DMEM, Dulbecco’s modified Eagle Medium; EROD, ethoxyresorufin-O-deethylase; FCS, Fetal Calf Serum; FITC, fluorescein isothiocyanate; FMO, flavine monooxygenases; GSH, glutathione S-transferase; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HPLC, high-performance liquid chromatography; i.p., intraperitoneal; NBT, Nitro Blue Tetrazolium; NNK, 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone; PAH, polycyclic aromatic hydrocarbon; SD, Sprague Dawley. ⇑ Corresponding author. Tel.: +49 511 532 7250. E-mail addresses: [email protected] (T. Hansen), Chougule. [email protected] (A. Chougule), [email protected] (J. Borlak). http://dx.doi.org/10.1016/j.tiv.2014.03.009 0887-2333/Ó 2014 Elsevier Ltd. All rights reserved.

pulmonary delivery of verapamil abrogated extensive first pass metabolism (Borlak et al., 2005, 2003; Walles et al., 2003, 2002a, 2002b; Koch et al., 2001) with liquid chromatography–tandem mass spectrometry identifying 25 phase I and 14 phase II metabolites of verapamil in cultures of rat hepatocytes, respectively (Walles et al., 2003). Notably, for medications to be delivered via inhalation drug induced pulmonary toxicity need to be considered. The lung is also a major organ exposed to airborne chemicals to possibly cause organ specific toxicity. Entry into systemic circulation requires the passage of inhaled drugs and/or chemicals across the alveolar barrier. In the case of human lung about 500 million alveoli are present with the alveolar space being composed of type I squamous and type II cuboidal alveolar epithelial cells to account for 95 and 5% of the surface area, respectively (Crapo et al., 1982). Next to alveolar macrophages the tracheobronchial airway is lined with Club (formerly called Clara cells), goblet and serous cells. Alveolar epithelial type I and II cells are morphologically and functionally distinct in structural integrity (cellular thickness, composition, absorptive volume) as well in biochemical function and exhibit different behavior (Steimer et al., 2006) with alveolar type II cells to synthesize and secrete

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pulmonary surfactant as to regulate surface tension, alveolar fluid balance and repair. Furthermore, there is evidence for alveolar epithelial stem and progenitor cells to replace injured cells thereby contributing to regeneration of the lung (Barkauskas et al., 2013). To enable mechanistic studies metabolically competent alveolar epithelial cells (AECs) are required. Therefore, researchers will benefit from an isolation and cultivation protocol to investigate mechanistic endpoints in toxicology. Furthermore, an assessment of organ specific toxicity in organotypic cultures derived from animals is complex and hurdled by cell heterogeneity and inter/ intraspecies variation (Bhogal et al., 2005). Owing to the 3R principle (Reduce, Replace and Refine) in the use of animals for toxicity testing, alternative testing approaches are needed. An establishment of a cell-culture system consisting of a population of target cell enables researchers to design superior mechanistic toxicological assays; for example, AECs are predominantly involved in B[a]P metabolism (Kuriharal et al., 1993). Moreover, the distribution of xenobiotic-metabolizing CYP enzymes in lungs is cell type specific. The pulmonary expression of CYP monooxygenases and of other biotransformation enzymes was reported for laboratory animals and humans and was shown to encompass glutathione Stransferases (GST), esterases, peptidases, cyclooxygenases and flavine monooxygenases (FMO). Moreover, it was shown that CYP enzymes are mostly expressed in Club cells (Patton et al., 2004), type II pneumocytes and macrophages (Hukkanen et al., 2002). We previously characterized drug metabolism enzymes of AECs isolated from rats and demonstrated AECs to express and to retain a range of CYP monooxygenases (1A1, 1A2, 3A2, 1B1, 2B1, 2E1 and 2J3) when cultured under optimal conditions (Hansen et al., 2006). Most protocols for an isolation of murine AECs stem from the earlier work of Corti et al. (1996) and Harrison et al. (1995). The principle differences among the applied methods are types and concentration of enzymes used for lung tissue digestion during cell isolation and includes enzyme preparations based on non-specific protease type I, dispase, elastase, trypsin and collagenase. An elastase based digestion of lung tissue and panning of cell suspension on plates coated with IgG was reported by Dobbs et al. (1986) and was found to provide high yield and purity of AECs. Except for rat the method is suitable for many species. While methods developed for a particular species can be employed as guidance these need to be adopted when used across species and require the lung anatomy to be considered. Specifically, the airway immediately proximal to the bronchiole–alveolar duct junctions in the mouse differs from other species with fewer respiratory bronchioles and airway generations (Irvin and Bates, 2003). Because of these anatomical features the mouse lung has a smaller alveolar surface than rat and human; an absence of submucosal glands is noted but the proportion of Club cells is higher as compared to other rodents. Thus, enzymatic digests of lung tissue contain mostly Club cells. To enrich for AECs flow cytometry based sorting of CD31 and CD45 was reported (Driscoll et al., 2012). However, cell sorting is a significant stress to cells. IgG depletion, magnetic bead isolation and density gradient as well as counterflow centrifugation elutriation techniques enabled separation and enrichment of alveolar epithelial and Club cell fractions isolated from lung tissue (Reddy et al., 2004). In the present study, we report a protocol for an efficient isolation and cultivation of differentiating AECs from the A/J mice suitable for studies over 6 days. This strain of mice has been used successfully in carcinogenesis studies and was shown to develop lung adenomas within 6–8 month following treatment with PAHs or tobacco carcinogens, such as NNK or B[a]P (Witschi, 1998).

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2. Materials and methods 2.1. Chemicals and reagents Dulbecco’s modified Eagle Medium (DMEM), Fetal Calf Serum (FCS) and phosphate buffered saline solution (PBS) were purchased from Biochrom (Berlin, Germany). The medium to culture primary alveolar epithelial was supplemented with 6.3 lg/ml insulin (InsumanÒ Rapid, Hoechst Marion Roussel, Frankfurt/Main, Germany), 0.67 lg/ml Prednisolone, 0.016 lg/ml glucagon (Novo, Mainz, Germany), 200 U/ml penicillin and 200 lg/ml streptomycin (Biochrom, Berlin, Germany). Trypsin–EDTA solution (0.25%) was obtained from Sigma (Deisenhofen, Germany). Dispase grade II was obtained from Roche Diagnostics (Berlin, Germany). Mouse serum was prepared from A/J mice using standard procedure. Collagen was prepared from A/J mouse tails according to the method of Elsdale and Bard (1972). Testosterone, 11-a-hydroxyprogesterone, 2-a-hydroxytestosterone, 6-a-hydroxy-testosterone, 6-a-hydroxytestosterone, 16a-hydroxytestosterone, androstendione, ethoxyresorufin and resorufin were obtained from Sigma (Deisenhofen, Germany). 2.2. Animals A/J mice and SD rats (n = 5) were purchased from Charles Rivers Laboratory (Sulzfeld, Germany). All animal work followed strictly the Public Health Service (PHS) policy on Human Care and Use of Laboratory Animals of the National Institutes of Health, USA. Formal approval to carry out animal studies was granted by the animal welfare ethics committee of the State of Lower Saxony, Germany (‘Lower Saxony State office for Consumer Production and Food Safety’, LAVES). The approval ID is Az: 33.9-42502-0406/1087. 2.3. AECs isolation and culture A total of n = 4 different protocols (A–D) were tested and initially explorative studies were done with n = 10 animals. Subsequently, and for each protocol n = 5 A/J mice were used for AECs isolation and cultivation. In the case of rat AECs, a total n = 5 SD rats were used. Male A/J mice weighting approximately 30 g were anesthetized by i.p. injection of KetaminÒ and RompunÒ. After tracheotomy the trachea was cannulated with a bulb head cannula. Following the midline incision and removal of the rib cage, a Luer cannula was inserted into the right ventricle. The pulmonary circulation was perfused with ice cold PBS using a peristaltic pump at a flow rate of 4 ml/min. Then, the perfusate was removed by incision of the left atrium and perfusion with the buffer was continued for 2–3 min until the lungs were completely free of blood. During the perfusion, the lungs were manually ventilated via the tracheal cannula. Then the lungs were explanted and transferred to a sterile flow cabinet. Lungs were lavaged three times with 1 ml PBS with a 22 G needle and 10-cc syringe followed by a 1 ml Trypsin–EDTA solution that was instilled via the trachea and incubated in a shaking water bath at 37 °C for 15 min. Lungs were treated separately with trypsin at two different concentrations of 0.25% and 0.025%, respectively, as depicted in Fig. 1 for protocol A and B. The incubation was stopped by instillation of 10 ml ice cold trypsin-inhibitor solution and the lungs were stored on ice for tissue dissection. For protocol C and D the lungs were initially filled with 1.5 ml warm dispase solution (>2.4 U/ml) without previous bronchoalveolar lavage at room temperature for 45 min followed by instillation of 0.55 ml of 45 °C warm agarose (1% w/v, low melting). Immediately afterwards, the lungs were covered with crushed ice and

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Fig. 1. A schematic overview of the main stages of mouse alveolar epithelial cell isolation protocol A, B, C and D.

stored on ice for 2 min to gelate the agarose. For manual dissection the lungs were transferred to a 100 mm Petri dish containing 7 ml buffer, and after removal of the trachea and main bronchi the parenchymal lung tissue was dissected into 1–2 mm pieces on ice. A comparison of the different protocols (A–D) employed for the isolation of AECs is given in Fig. 1. The lung tissue suspension was incubated with 10 ml DNAase solution for 10 min (250 lg/ml, 37 °C, for protocol A, B and 100 lg/ml, room temperature for protocol C and D, respectively) and subsequently, filtered through 100 lm and 40 lm cell strainer (Becton, Dickinson, Germany) and a nylon mesh with 25 lm pore size. According to protocol D, the crude cell suspension was purified by discontinuous Percoll gradient centrifugation (heavy density 1.089 and low density 1.040). Following centrifugation for 30 min at 250g and 4 °C the cell fraction at the interface between the heavy and low density gradient was removed and mixed with DNAase solution containing 50 lg/ml DNAase. The cell suspension was again centrifuged at 140g at 4 °C for 6 min (for further details see Hansen et al., 2006). AECs isolated from protocol A, B, C and D were cultured on mouse tail collagen-coated 24-well tissue culture plates. Approximately 1.5  106 cells were seeded per well. To assess the effect of extracellular matrix component cells were either seeded on a collagen monolayer or plated onto plastic culture plates. Cells were cultured for a maximum of 6 days in DMEM medium supplemented with either 2% mouse serum (MS) or 10% Fetal Calf Serum

(FCS). For comparison, AECs were isolated from SD rats and isolated AECs were cultured according to our previously published method (Hansen et al., 2006). 2.4. Histochemistry Cytospots were prepared from freshly isolated AECs and frozen at 80 °C. Staining of alveolar type II cells for alkaline phosphatase (AP) was done with 2 mg naphthol-AS-MX-phosphate (Sigma, Deisenhofen, Germany), 200 ll N,N-dimethylformamide (DMF, Merck, Darmstadt, Germany), 9.8 ml of 0.1 M Tris HCl-buffer pH 8.2 and 10 mg fast-red salt (Sigma, Deisenhofen, Germany). Naphthol-AS-MX-phosphate was dissolved in DMF and mixed with Tris HCl to obtain a total volume of 10 ml. Fast-red was dissolved in naphthol-Tris HCl immediately before use. Club-cells were identified by staining for NADPH-dependent Nitro Blue Tetrazolium (NBT) reductase activity. Cytospin preparations were fixed with 10% buffered formalin for 40 s, washed in 25 mM HEPES buffer and incubated with 0.1% NBT and 0.1% NADPH in PBS at 37 °C for 10 min. Methylene green was used as a counter stain. 2.5. Flow cytrometry of lectin binding Approximately 1  106 AECs isolated either by the use of protocol C or D were stained with different fluorescein isothiocyanate (FITC) labeled lectins immediately after isolation and on day 3 in

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culture. Cells were incubated with either 10 lg/ml Helix pomatia lectin, 20 lg/ml Maclura pomifera lectin or 20 lg/ml Lycopersicon esculentum lectin for 30 min at room temperature in the dark. In order to estimate non-specific binding, control experiments were done with all three lectins in the presence of their specific inhibiting sugars that is 0.1 M D-(+) galactose and 0.1 M N-acetyl-galactosamin for M. pomifera and H. pomatia and 0.2 M N-acetylgalactosamin and 0.2 M N-acetyl-glucosamin in the case of L. esculentum, respectively. Cells were washed three times with PBS and used for flow cytometry using a Facscan cytometer (Becton, Dickinson, Germany) equipped with a 488 nm argon laser. 2.6. Preparation of lung microsomes from Aroclor 1254 treated mice Aroclor 1254 is a pleiotropic agent that induces a variety of CYP monooxygenases in a tissue specific manner (Thum and Borlak, 2008; Borlak and Thum, 2001; Thum et al., 2000). To induce CYP monooxygenase expression, male A/J mice were given a single i.p. dose of 100 mg/kg body weight of Aroclor1254 dissolved in vegetable oil. Control animals received vehicle only. Animals were sacrificed after 48 h by i.p. injection of KetaminÒ and RompunÒ. The lung microsomes of Aroclor 1254 treated mice were prepared according to Hansen et al. (2006). The protein content of lung microsomes was determined according to Smith et al. (1985). Microsomal suspensions were shock frozen in liquid nitrogen and stored at 80 °C until further use. 2.7. CYP monooxygenase induction studies in cultured AECs AECs were isolated according to protocol C or D. Cells were cultured in a medium supplemented with either 2% mouse serum or 10% FCS. On day 3 in culture cells were treated with 10 lm Aroclor 1254 dissolved in DMSO. The final DMSO concentration in culture medium was 0.5 vol.%. The testosterone and ethoxyresorufin assays were performed as described below. 2.8. Testosterone assay Testosterone is a substrate to probe for CYP monooxygenase activity in lung tissue and cultures of AECs. Lung microsomes isolated from Aroclor 1254 and vehicle treated mice were incubated with 50 lM testosterone and 1 mM NADPH for 60 min in a 1 ml reaction mixture containing 50 lg total protein. Cultured AECs isolated according to protocol C or D were exposed to culture medium containing 50 lM testosterone for 48 h. The cell culture supernatant was collected and testosterone and its metabolites were analyzed by HPLC using 11–hydroxyprogesterone as internal standard as previously described (Hansen et al., 2006). 2.9. EROD activity Fluorimetric determination of 7-ethoxyresorufin-O-deethylation (EROD) was done according to the method of Burke and Mayer (1983). Enzyme activities were assayed in cultures of AECs by adding the substrate at 2 lM to the culture medium. Dicumarol (10 lm) was added to the culture medium to prevent further biotransformation of resorufin by cytosolic diaphorase. The incubation was stopped by removal and immediate storage of the cell culture supernatant at 20 °C. Lung microsomal preparations were incubated with 10 lm ethoxyresorufin and 1 mM NADPH for 120 min in 1 ml reaction mixture containing 100 lg protein. Formation of resorufin was measured using a VersaFluor fluorimeter (Biorad) with an excitation wavelength of 515 nm and an emission wavelength of 610 nm. Fluorescence was converted to pmol metabolite formation with a calibration curve for resorufin (range: 5–100 nmol/l). The formation of glucuronides was assessed by

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overnight treatment of the cell culture supernatants with b-glucuronidase at 37 °C. 2.10. RT-PCR Freshly explanted lung tissue was homogenized in ice-cold Tri Reagent (Lucerna Chem AG, Luzern, Switzerland) and total RNA was extracted using the Nucleospin RNA II Kit (Macherey–Nagel, including DNAase digest) according to the manufacturer’s instruction, with the exception that all extraction steps were performed on ice. Likewise, total RNA was isolated from cultured AECs and stored at 80 °C in DEPC-treated water. The concentration and purity of RNA were determined by measuring the absorbance at 260 nm and by determining the A260/A280 ratio. For cDNA synthesis, 2 lg of total RNA from each sample was used. RNA was preheated for 5 min at 65 °C. Reverse transcription was done in a final volume of 20 ll containing 1 RT-buffer (Qiagen), 0.5 mM of each dNTP (Qiagen), 0.5 lM random hexamers (Promega), 10 U RNase inhibitor (Promega), 4 U Omniscript Reverse Transcriptase (Qiagen) and DEPC-treated water. Reverse transcription was carried out for 60 min at 37 °C and stopped by heating to 95 °C for 5 min. The resulting cDNA was frozen at 20 °C. For PCR amplification of cDNA, a 20 ll reaction mixture was prepared with cDNA equivalent to 25–100 ng of total RNA (lung tissue) or 2 ng total RNA (cultured cells) and HotStarTaq MasterMix Kit (Qiagen) containing HotStar Taq DNA polymerase, PCR buffer (with 1.5 mM MgCl2) and 400 lM each of dNTPs. Primers were designed by the Primer3 free online tool. The oligos were synthesized by Life Technologies (Darmstadt, Germany) and sequences are given in Supplementary Table S1. A final concentration of 500 nM of each primer was used in the PCR reaction. PCR amplification was done at 95 °C for 15 min (HotStar activation), 94 °C for 45 s, at 57–58 °C for 45 s and 72 °C for 45 s. Final extension was at 72 °C for 10 min and cooled down at 4 °C. Additionally, PCR reaction product were visualized as follow: The amplification products were mixed with 6 loading buffer (40% glycerol, 0.001% xylencyanol blue, 0.002% bromphenol blue in ddH2O) and separated on a 1.5% agarose gel. Gels were stained with ethidium bromide (2 ll of 10 mg/ml EtBr solution per each 100 ml agarose gel) and photographed on a transilluminator (Kodak Image Station 440 CF). Quantification of bands and serial dilution of PCR product was performed by using the Li-COR image studio Lite software (http://www.licor.com/bio/products/software/image_studio_lite/index.jsp). 2.11. Statistical analysis At least three independent experiments were performed. To determine significant differences amongst treatment groups the Student’s t-test was applied and considered significant at p-values of

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The A/J mouse strain is used in lung cancer studies. To enable mechanistic investigations the isolation and cultivation of alveolar epithelial cells (...
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