Metabolic Detoxication Pathways for ... - ACS Publications

Chem. Res. Toxicol. 2010, 23, 1673–1681

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Metabolic Detoxication Pathways for Sterigmatocystin in Primary Tracheal Epithelial Cells Odile Cabaret,†,‡ Olivier Puel,§ Françoise Botterel,†,‡ Michel Pean,| Khaled Khoufache,† Jean-Marc Costa,‡,⊥ Marcel Delaforge,# and Ste´phane Bretagne*,†,‡ UMR BIPAR, U-PEC, AFSSA, ENVA, Faculte´ de Me´decine, Cre´teil Cedex F-94010, France, AP-HP, Groupe hospitalier Henri Mondor-Albert CheneVier, Laboratoire de Parasitologie-Mycologie, Cre´teil Cedex F-94010, France, INRA, UR 66, Laboratoire de Pharmacologie-Toxicologie, Toulouse F-31027, France, CEA, DSV, IBEB, Group Rech Appl Phytotechnol, Saint-Paul-lez-Durance F-13108, France, CNRS, UMR Biol Veget & Microbiol EnViron, Saint-Paul-lez-Durance F-13108, France, Aix-Marseille UniVersite´, Saint-Paul-lez-Durance F-13108, France, Laboratoire Pasteur Cerba, Saint Ouen l’Aumone F-95310, France, and CEA Saclay, iBiTec-S, SB2SM, and URA CNRS 2096, Gif sur YVette Cedex F-91191, France ReceiVed April 6, 2010

Human health effects of inhaled mycotoxins remain poorly documented, despite the large amounts present in bioaerosols. Among these mycotoxins, sterigmatocystin is one of the most prevalent. Our aim was to study the metabolism and cellular consequences of sterigmatocystin once it is in contact with the airway epithelium. Metabolites were analyzed first in vitro, using recombinant P450 1A1, 1A2, 2A6, 2A13, and 3A4 enzymes, and subsequently in porcine tracheal epithelial cell (PTEC) primary cultures at an air-liquid interface. Expressed enzymes and PTECs were exposed to sterigmatocystin, uniformly enriched with 13C to confirm the relationship between sterigmatocystin and metabolites. Induction of the expression of xenobiotic-metabolizing enzymes upon sterigmatocystin exposure was examined by realtime quantitative real-time polymerase chain reaction. Incubation of 50 µM sterigmatocystin with recombinant P450 1A1 led to the formation of three metabolites: monohydroxy-sterigmatocystin (M1), dihydroxy-sterigmatocystin (M2), and one glutathione adduct (M3), the latter after the formation of a transient epoxide. Recombinant P450 1A2 also led to M1 and M3. P450 3A4 led to only M3. In PTEC, 1 µM sterigmatocystin metabolism resulted in a glucuro conjugate (M4) mainly excreted at the basal side of cells. If PTEC were treated with β-naphthoflavone prior to sterigmatocystin incubation, two other products were detected, i.e., a sulfo conjugate (M5) and a glucoro conjugate (M6) of hydroxysterigmatocystin. Exposure of PTEC for 24 h to 1 µM sterigmatocystin induced an 18-fold increase in the mRNA levels of P450 1A1, without significantly induced 7-ethoxyresorufin O-deethylation activity. These data suggest that sterigmatocystin is mainly detoxified and is unable to produce significant amounts of reactive epoxide metabolites in respiratory cells. However, sterigmatocystin increases the P450 1A1 mRNA levels with unknown long-term consequences. These in vitro results obtained in the porcine pulmonary tract need to be confirmed in human epithelial cells. Introduction Mycotoxins are secondary metabolites of filamentous fungi that can cause a wide range of acute and chronic systemic effects. Most publications deal with hazards due to toxic effects after gastrointestinal uptake (1). Fewer data with regard to health effects after inhalation are available, although mycotoxins are detected in bioaerosols and dust at workplaces and homes (2-4) and are associated with inhaled spores (5-7). The more studied mycotoxin is aflatoxin B1. Some epidemiological data link pulmonary exposure to aflatoxin B1 with an increase in lung cancer incidence in Dutch workers (8). In addition, aflatoxin B1 can be activated in human and animal tissues or in human bronchial cells (9-15). * To whom correspondence should be addressed. Telephone: 33 (0)1 49 81 36 41. Fax: 33 (0)1 49 81 36 01. E-mail: [email protected]. † UMR BIPAR, U-PEC, AFSSA, ENVA. ‡ AP-HP, Groupe hospitalier Henri Mondor-Albert Chenevier. § INRA, UR 66. | CEA, DSV, IBEB, Group Rech Appl Phytotechnol, CNRS, UMR Biol Veget & Microbiol Environ, and Aix-Marseille Universite´. ⊥ Laboratoire Pasteur Cerba. # CEA Saclay, iBiTec-S, SB2SM, and URA CNRS 2096.

Among mycotoxins, sterigmatocystin (STG),1 a biochemical precursor in the biosynthesis of aflatoxins (16), is one of the most prevalent in the environment. Engelhardt and co-workers determined STG concentrations between 2 and 4 ng/g in carpet dust from damp dwellings (17). STG was also detected in dwellings after water flooding (18, 19). The production of mycotoxins is highly dependent on the fungal species and on environmental conditions. STG is produced by certain fungal species in the Aspergillus, Bipolaris, and Chaetomium genera and by Penicillium luteum (20-22). Emericella nidulans (syn. Aspergillus nidulans) and Aspergillus Versicolor are among the most efficient producers of STG. This latter species is also very prevalent in the environment (23-25), and STG can represent up to 1% of the biomass if A. Versicolor grows at an optimal water activity (23). After ingestion, STG is carcinogenic in animal models (26-28) and classified as possibly carcinogenic 1 Abbreviations: STG, sterigmatocystin; PTEC, porcine tracheal epithelium cells; qPCR, quantitative real-time polymerase chain reaction; UG, Ultroser G; GSTs, glutathione S-transferases; UGTs, UDP-glucuronosyltransferases; MRP1, multidrug resistance-associated protein 1; AhR, aryl hydrocarbon receptor; SULT, sulfo-transferase; EROD, 7-ethoxyresorufin O-deethylation.

10.1021/tx100127b  2010 American Chemical Society Published on Web 10/07/2010

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in humans (20). STG is activated by P450 to a liver carcinogen (29). To date, no metabolite has been reported in airway epithelial cells. The aim of this study was to elucidate the metabolism of STG after it makes contact with airway epithelium and to assess any possible cellular consequences. For this purpose, we used primary cultures of porcine tracheal epithelium cells (PTEC) at an air-liquid interface (30). Preliminary studies consisted of identifying STG metabolites in recombinant P450 enzymes. On the basis of the structural homology of STG with aflatoxins, we considered P450 isoforms 1A, 2A, and 3A that are reported to metabolize aflatoxin B1 (14, 15, 31, 32). We analyzed formed metabolites following exposure of PTEC to nonlethal concentrations of STG, with or without prior P450 1A induction. These metabolic studies were performed using uniformly 13C-enriched STG to confirm the relationship between the metabolite and STG. Finally, we evaluated the effect of STG on mRNA expression for a number of xenobiotic-metabolizing enzymes by quantitative real-time polymerase chain reaction (qPCR).

Materials and Methods Chemicals. Ham’s F-12 nutrient medium, Dulbecco’s modified Eagle’s medium, penicillin/streptomycin, amphotericin B, fetal calf serum, 0.05% trypsin-EDTA (1×), and PBS were purchased from Invitrogen Life-Technologies (Cergy-Pontoise, France). Gentamicin, collagen IV, commercial [12C]STG with a purity of 98%, acetonitrile, NADPH, GSH, β-naphthoflavone, R-naphthoflavone, 7-ethoxyresorufin, resorufin, β-glucuronidase type HP-2 from Helix pomatia, and ammonium acetate were obtained from Sigma (Saint Quentin Fallavier, France). Ultroser G (UG) was purchased from BioSepra SA (Cergy-Saint-Christophe, France) and Me2SO from Prolabo (VWR International, Fontenay sous Bois, France). Production of uniformly 13C-enriched STG was performed using autoclaved 10% 13C wheat grains (Triticum aestiVum cv. Ardente) as the exclusive nutrient source for the fungal culture, as previously described (33, 34). The grains were moistened to a water activity value of 0.98 before sterilization. Approximately 30 g of this plant material was inoculated with a suspension of 2 × 105 Emericella nidulans conidia cells. STG was extracted from 14-day-old cultures with chloroform. The raw extract was fractioned by an automated flash chromatography instrument (Teledyne Isco, Lincoln, NE). The fractionation was performed on a normal phase RediSep 12 g column (Teledyne Isco) at a flow rate of 25 mL/min with linear gradient. The solvent system was composed of 10% acetic acid in toluene as solvent A and 10% acetic acid in ethyl acetate as solvent B. The initial solvent B part was from 0 to 45.5% over 10 min. The fraction volume was set to 5 mL. The presence of STG in fractions was checked by HPLC-DAD (Kontron Instruments, Neufahru, Germany) with a 250 mm × 4.6 mm Modul-cart Strategy 5 µm C18-2 column (Interchim, Montluçon, France). Gradient chromatography (run of 46 min) was performed with 0.2% acetic acid in water (eluent A) and acetonitrile (eluent B) as the mobile phase at a flow rate of 1.5 mL/min. The compounds were eluted by starting from 31% solvent B for 30 min followed by a linear gradient to 90% B over 5 min. After an isocratic elution for 5 min, the gradient was decreased to an initial value within 5 min and remained at this value for the last 5 min. The [13C]STG was purified from positive fractions by semiquantitative HPLC. The HPLC unit was fitted with a 250 mm × 7.8 mm Modul-cart Strategy 5 µm C18-2 column (Interchim) and a diode array detector. The gradient program was the same as described above, except the flow rate was increased to 4.5 mL/min. Metabolite Studies in Recombinant Human P450. To identify oxidized metabolites to be investigated in cellular cultures, we first analyzed STG metabolism in Bactosome-expressed human P450 1A1, 1A2, 2A6, 2A13, and 3A4 obtained from Cypex (Tebu-bio, Le Perray en Yvelines, France). Metabolism of 50 µM commercial [12C]STG or [13C]STG was studied at 37 °C in 0.1 M potassium

Cabaret et al. phosphate buffer (pH 7.4) with 0.2 µM human isoforms, using 0.5 mM NADPH for a 0.2 mL incubation. Metabolism was studied after incubation for 30 min or 10, 20, 30, or 60 min for time course studies. To test for the GSH adduct, we conducted the assay using 5 mM GSH. R-Naphthoflavone (20 µM) was used to test the inhibition of STG P450 1A1 metabolism. Controls contained all the reaction components except NAPDH or STG. A control with STG and GSH in the absence of P450 was also performed. Incubations were arrested via addition of the same volume of cold acetonitrile and then stored at -20 °C for 20 min to precipitate proteins. The mixture was centrifuged at 10000 rpm for 5 min, and the supernatant was analyzed by HPLC-MS/MS. Porcine Tracheal Epithelial Cells (PTEC). Preparation of primary cultures was performed as previously described (30). Briefly, trachea were sampled in euthanized pigs. After dissociation, 106 cells were plated in 12 mm insert wells (Transwell, Costar, Cambridge, MA) with microporous membranes coated with type IV collagen and maintained in a humidified incubator at 37 °C in the presence of 5% CO2. After 24 h, the culture medium was removed from the apical compartment to produce an air-liquid interface. The basal compartment was filled with 1 mL of the PTEC culture medium (UG medium), consisting of Dulbecco’s modified Eagle’s medium/Ham’s F-12 nutrient medium with 2% UG, antibiotics (100 units/mL penicillin, 100 µg/mL streptomycin, and 100 µg/mL gentamicin), and 2.5 µg/mL amphotericin B. The culture medium was changed daily, and the electrophysiological properties of the cultures were checked once a week using a microvoltmeter (EVOM World Precision Instruments, Aston-Stevenage, U.K.). For 14-day-old cultures, the usual ranges of transepithelial resistances and potential differences are 800-1200 Ω/cm2 and -20 to -30 mV, respectively. Transepithelial resistances and potential differences measure intercellular junction permeability and transcellular ionic transport, respectively. Biological Activity of STG on PTEC. Stock solutions (5 × 10-3 M) of STG were prepared in Me2SO. Dilutions from 0.5 to 10.0 µM were made from this stock containing a maximal amount of 0.2% Me2SO. The control solution was a 0.2% Me2SO solution in UG medium. All experiments were performed on 14-day-old PTEC cultures, such that stable cell differentiation had been established. PTEC were exposed to STG at the apical side for 3 h. Electophysiological effects of STG on PTEC were evaluated using bioelectrical properties of PTEC cultures: transepithelial resistances and potential differences were measured after exposure for 3 h, as previously described (35). The culture medium containing STG was removed, and the electrophysiological values were again measured after 24 h. These electrophysiological tests allowed us to determine the noncytotoxic concentration to be used for metabolic studies. STG Metabolism in PTEC. PTEC were exposed for 48 h to 1 mL of 1 µM [13C]STG through the basal side of cultures under air-liquid conditions (without any medium on the apical side of cells). Then, the basal medium was collected for extraction. For STG time course metabolism, cells were exposed with 750 µL of 1 µM [12C]STG to the apical side of cultures and 1 mL of UG medium to the basal side (liquid-liquid conditions). The media in apical and basal sides were collected for extraction after 3, 6, and 24 h. When the effect of P450 1A1 induction was examined, cells were pretreated for 24 h with 25 µM β-naphthoflavone or with 0.2% Me2SO in UG medium. This medium was then removed and the STG incubated for 24 or 48 h. P450 1A1 mRNA levels were checked under such conditions. Controls were obtained after incubation for 24 or 48 h with [12C]- or [13C]STG in coated wells without cells and with 0.2% Me2SO in UG medium. Before being extracted, proteins were precipitated via addition of 500 µL of cold methanol. After 30 min at -20 °C, the mixture was centrifuged at 10000 rpm for 5 min. Then, 3 mL of water was added to the supernatant, and the resulting solution was purified on a SepPack C18 cartridge (Waters Corp., Milford, MA). Samples were eluted with 1 mL of acetonitrile. This extract was reduced to dryness by speed-vac concentration (Savant SC-200, Savant Instruments, Farmingdale, NY) and then dissolved in the HPLC starting solvent

Pulmonary Metabolism of Sterigmatocystin

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Figure 1. Isotopic cluster of uniformly 13C-enriched sterigmatocystin after 10% 13C enrichment (A) and natural [12C]sterigmatocystin (B) recovered by mass spectrometry analysis.

[50% ammonium acetate in water (10 mM) and 50% acetonitrile]. For glucuronide hydrolysis, conjugates were enzymatically digested using 500 units/mL H. pomatia β-glucuronidase in 0.05 M acetate buffer (pH 5) overnight at 37 °C and therefore extracted. HPLC-MS/MS Analysis. HPLC-MS/MS analyses were performed on an Esquire Ion Trap (Bruker Daltonick, GmbH, Bremen, Germany) with an electrospray ionization source operating in alternative positive/negative ionization mode, coupled with a 150 mm × 2.1 mm C18 Kromasil column (Interchim). The flow rate was 0.2 mL/min for a 25 min linear gradient ranging from 10 to 80% B (90% acetonitrile in water) into A [90% ammonium acetate in water (10 mM) and 10% acetonitrile]. Electrospray ionization was performed at 4.5 kV, and the capillary temperature was set to 310 °C using a gas flow of 40 mL/min and an auxiliary gas flow of 10 mL/min. The collision energy was 70%. MS and MSn optimizations were conducted using infusion of commercial STG. Injections were performed in a 50/50 acetonitrile/water mixture containing 10 mM ammonium acetate. mRNA Expression Studies. After being cultured for 14 days, cells were treated with 1 µM STG or 25 µM β-naphthoflavone to the apical side. Cells were collected for RNA extraction after being exposed for 3, 6, and 24 h using the MagNA Pure Compact RNA isolation kit (Roche Diagnostics, Mannheim, Germany). Determination of nucleic acid concentrations and RNA quality control were performed using a spectrophotometer (Nanodrop ND 1000, Thermo Scientific, Wilmington, DE). RNA integrity was assessed using a microfluidic-based electrophoresis system (2100 Bioanalyzer, Agilent Technologies, Santa Clara, CA). Reverse transcription was performed as previously described (36). For each reaction, negative controls without RNA and without reverse transcriptase were added. cDNAs were diluted in sterile water (1:20) and stored at -80 °C until amplification. The primers for amplifying porcine mRNA are listed in Table S1 of the Supporting Information. qPCRs were conducted on a LightCycler 480 (Roche). Each sample was normalized on the basis of its mRNA content for the two reference genes cyclophilin A (GenBank accession number NM214353) and TATA box binding protein (GenBank accession number DQ845178), as described previously (36). The results, expressed as the N-fold difference in target gene expression relative to the cyclophilin A or TATA box binding protein

genes (termed Ntarget), were determined by the following formula: Ntarget ) 2∆Cq sample, where the ∆Cq sample was determined by subtracting the average Cq value of the target gene from the average Cq value of the TATA box binding protein (or cyclophilin A) gene. The Ntarget values of the samples were subsequently normalized so that the median Ntarget values of the control samples without STG were 1, and the values are presented according to the standardization of Willems et al. (37). Statistical significance was tested by the Wilcoxon matched-pairs signed-ranks test for untreated and treated cells, using a p < 0.05 significance level. 7-Ethoxyresorufin O-Deethylation (EROD) Assay. PTEC cultures were incubated with 10 µM 7-ethoxyresorufin and dicumarol each. Aliquots of the supernatant medium were withdrawn after incubation for 1 h. Samples were stored frozen at -20 °C until they were analyzed. After thawing, resorufin conjugates were cleaved using β-glucuronidase in 0.05 M acetate buffer (pH 5.0) at 37 °C. After 2 h, the reaction was stopped with 1.6 M glycine buffer (pH 10.3). Afterward, the formation of resorufin was quantified by fluorometry with an excitation wavelength of 530 nm and an emission wavelength of 580 nm. The spectrofluorometer was calibrated using resorufin standards. Statistical significance was tested by the Wilcoxon matched-pairs signed-ranks test for untreated and treated cells, using a p < 0.05 significance level.

Results Uniform 13C Enrichment in STG. Before use in metabolic studies, we checked that 13C-enriched STG presented an isotopic cluster characteristic of 10% 13C enrichment. The MS spectrum contained the molecular ion (nominal ion at m/z 325) and also M + 1 (m/z 326), M + 2 (m/z 327), M + 3 (m/z 328), M + 4 (m/z 329), and M + 5 (m/z 330) (Figure 1). These peaks are in a 0.50/1.00/0.93/0.57/0.23/0.02 ratio relative to the most intense ion at m/z 326. These isotopic ratios are close to those expected for 10% 13C enrichment (38). This isotopic cluster shape is present for all metabolites, confirming them as STG metabolites. Metabolite Studies in Recombinant Human P450. A 30 min incubation of either commercial [12C]STG or 10% [13C]STG with 0.2 µM recombinant human P450 1A1 in the presence of

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Figure 2. Representative HPLC-MS chromatograms showing formation of sterigmatocystin metabolites after incubation for 30 min in vitro (0.2 µM cytochrome P450 1A1 in the presence of 50 µM sterigmatocystin and 5 mM GSH). Chromatogram of the ion at (A) m/z 325 in positive ionization mode (sterigmatocystin), (B) m/z 341 in positive ionization mode (M + 16, metabolite M1), (C) m/z 355 in negative ionization mode (M + 32, metabolite M2), and (D) m/z 644 in negative ionization mode (M + 321, metabolite M3).

NADPH led to significant formation of two metabolites M1 and M2. These metabolites, having tR values of 19.5 min (M1) and 15.5 min (M2), were more polar than STG (tR ) 24.8 min) and had molecular weight differences of M + 16 and M + 32, respectively. According to their mass, their polarity, and the monooxygenase activity of P450 1A1, M1 was identified as monohydroxy-sterigmatocystin and M2 as dihydroxy-sterigmatocystin. After 30 min, 5 nmol of STG was metabolized (∼125 nmol per 30 min per nanomole of P450). Between 30 and 60 min, no significant decrease in STG concentration was observed. Incubations in the presence of GSH led to the formation of a third metabolite (M3) having a tR of 11 min and an m/z ratio of M + 321 in comparison to that of STG (Figure 2). M3 was identified as a GSH adduct of an oxidized metabolite. M1, M2, and M3 were not detected in the presence of 20 µM R-naphthoflavone, a P450 1A1 inhibitor (Figure S1 of the Supporting Information). Incubations of STG with human P450 1A2 led to the formation of M1 but not M2. Incubations of STG with human P450 2A6, 2A13, and 3A4 did not lead to the formation of any significantly detectable metabolite. Incubations of STG with human P450 1A2 or 3A4 in the presence of glutathione led to the formation of M3. No demethylated metabolite was detectable in STG incubations with human P450 1A1, 1A2, 2A6, 2A13, or 3A4. The isotopic cluster profile was present for all the metabolites of STG, which confirms their relationship to STG. No metabolite was observed in the control incubations performed in the absence of NADPH, GSH, or enzymatic preparations. No metabolite was observed in the control incubation performed with STG in the presence of GSH but without P450. Determination of Nontoxic Concentrations of STG on PTEC. These studies were performed to determine the noncytotoxic concentrations to use for cellular metabolite studies. After

incubation for 3 h, 5 and 10 µM STG induced a significant decrease in transepithelial resistances and potential differences compared to controls. After 24 h at these concentrations, STG induced a cell disruption resulting in decreases in the electrophysiological parameters (data not shown). In contrast, 1 µM STG did not significantly alter transepithelial resistance after incubation for 3 or 24 h, so this concentration was chosen for further cellular incubations. STG Metabolism in PTEC. After incubation for 48 h with PTEC at the air-liquid interface, STG applied on the basal side led to the formation of only one metabolite, M4, identified as a glucuro conjugate. M4 was more polar than STG (tR ) 11.5 min) and had an m/z ratio of M + 176 in comparison to STG. Its MS fragmentation led to the formation of the MS ion of STG, and after incubation with β-glucuronidase, the M4 peak was partially converted to a peak with the same retention time as that of STG (Figure S2 of the Supporting Information). Only glucuronide conjugation on C8 is compatible with the mass of M4. When STG was added to the apical side (liquid-liquid conditions), M4 appeared on the basal side after 3 and 6 h and was predominantly present on the basal side after 24 h (Figure 3). After exposure of PTEC to 25 µM β-naphthoflavone for 24 h, 24 or 48 h STG incubations led to the formation of two new polar metabolites, M5 and M6. These metabolites had tR values of 12.5 and 5.5 min and m/z ratios of M + 96 and M + 192, respectively, as compared to STG (Figure 4). Their MS fragmentation led to the STG +16 ion. M5 was identified as a sulfo conjugate (M + 16 + 80) and M6 as a glucuro conjugate (M + 16 + 176) of monohydroxy-sterigmatocystin. mRNA Expression Levels. When PTEC were exposed to 1 µM STG for 24 h, a significant (p < 0.05) 18-fold increase in P450 1A1 mRNA levels was observed using qPCR, with either



Figure 3. Representative HPLC-MS chromatograms of the ion at m/z 499 in negative mode, showing formation of sterigmatocystin glucuro conjugate (M4) on the apical (A) and basal (B) side of cells after exposure of PTEC to 1 µM sterigmatocystin for 3, 6, and 24 h and exposure to 0.2% Me2SO for 24 h (controls) through the apical side of cells.

TATA box binding protein (Figure 5) or cyclophilin A (data not shown) as the reference gene. After exposure for 3 and 6 h, no significant increase in P450 1A1 mRNA levels was observed. After STG exposure for 3, 6, and 24 h, no significant increase in mRNA levels for P450 1A2, glutathione S-transferases A (GSTAs), UDP-glucuronosyltransferases (UGTs), multidrug resistance-associated protein 1 (MRP1), or aryl hydrocarbon receptor (AhR) was observed. When PTEC were exposed to a nontoxic concentration of β-naphthoflavone (25 µM) for 24 h, a 160-fold increase in P450 1A1 mRNA levels was observed, a 14-fold induction in 1A2 mRNA levels, a 3-fold induction in UGTs mRNA levels, and a 4-fold induction in GSTAs mRNA levels (Figure S3 of the Supporting Information). On the basis of Cq values, we estimated P450 1A1 was expressed 1000-fold more strongly than P450 1A2 in PTEC and 10000-fold more strongly after 25 µM β-naphthoflavone induction. EROD Assay. When PTEC were exposed to 1 µM STG for 24 h, no EROD activity was detected, as in the control cells. After exposure for 24 h to 25 µM β-naphthoflavone, EROD activity was significantly increased [71 ( 11 pmol of resorufin min-1 (mg of protein)-1].

Discussion Our study shows that STG is metabolized in porcine epithelial airway cells mainly through glucuronidation (Scheme 1). This

is the first study to report metabolite formation and detoxification pathways in a pulmonary stage using primary cells at an air-liquid interface. However, several technical choices can limit our conclusions. First, the chosen concentration of 10-6 M can seem high with regard to those previously reported in indoor environments, ∼10-9 mol/m3 in aerial dust from Aspergillus-contaminated buildings (17, 18, 39). This concentration is only 10-fold lower than the lytic doses but was required for the detection of metabolites by MS. Second, we performed most of our experiments using the basal compartment of the cultured cells because the prolonged immersion of the cells leads to a loss of differentiation. Third, our experiments were performed on pig cells instead of human primary cells. However, pigs present obvious advantages and constitute a validated alternative to traditional nonrodent species in pharmacological and toxicological studies, mainly because pigs share essential physiological characteristics with humans (40). Great similarities have been reported for P450 between human and pig liver (41-43). There are several arguments suggesting that porcine hepatocytes are more relevant than rodent hepatocytes for in vitro P450 metabolism studies (44). In addition, we show here that EROD activity was detected after β-naphthoflavone induction in PTEC. Therefore, it means that porcine P450 mRNA efficiently translates to the active enzyme in our model. On the other hand, we do agree that some catalytic differences and different substrate specificities between porcine and human P450 1A1 may exist (45). As a consequence, our results warrant being

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Figure 4. Representative HPLC-MS/MS chromatograms showing formation of sterigmatocystin metabolites in PTEC treated with 25 µM β-naphthoflavone prior to 1 µM sterigmatocystin incubation through the basal side of cultures. (A) m/z 419 in negative ionization mode (M5) and MS2 spectrum of the M5 metabolite. (B) m/z 515 in negative ionization mode (M6) and MS2 spectrum of the M6 metabolite.

and GST, we also observed a glutathione adduct (M3). These three metabolites have never been described with P450 1A1. The detection of a glutathione adduct after in vitro metabolism through P450 1A and 3A4 activities strongly suggests the formation of a transient reactive epoxide of STG, as described using either human hepatic microsomes or the Ames test (46, 47). Such metabolic pathways have been described for aflatoxin B1 hepatotoxicity (48). These mutagenic properties could be related to the pulmonary carcinomas described in mice after oral STG administration (20, 49). Figure 5. Standardized mRNA expression of the genes encoding P450 1A1 after exposure of PTEC to 1 µM sterigmatocystin for 3, 6, and 24 h, calculated with the ∆∆Ct method with porcine TATA box binding protein as the reference gene and presented using the system described by Willems et al. (37). Significant overexpression at 24 h was analyzed by a Wilcoxon paired t test. *p < 0.05 compared with control. Data are averages (histograms) and 95% confidence intervals (error bars) after the sequential standardization steps from two separate experiments, each performed in duplicate.

validated on a human model. Nevertheless, human tracheal primary cells are difficult to obtain, and in contrast with PTEC, a wide variability of responses is expected because of the genetic and therapeutic background of each patient (30). Our preliminary in vitro experiments using human recombinant P450 enzymes were designed to identify the metabolites resulting from P450 action. Only P450 1A and 3A4 formed metabolites. P450 1A1 led to the formation of two oxidized metabolites, monohydroxy-sterigmatocystin (M1) and dihydroxy-sterigmatocystin (M2). In the presence of cytosolic GSH

The two hydroxylated STG metabolites (M1 and M2) and the glutathione adduct (M3) identified using expressed P450 were not detected in PTEC. Rather than oxidation, studies with PTEC exhibited significant formation of a sterigmatocystinglucuro conjugate (M4). This metabolic pathway is generally involved in drug elimination and detoxification. Because our qPCR studies did not show any increase in UGTs mRNA levels after exposure to STG, basal UGTs activities seem sufficient for conjugating STG under our experimental conditions. This conjugation pathway participates in elimination of the STG in the urine of rat or monkey (50, 51). Glucuronidation has already been identified as an important pathway in the respiratory detoxification of environmental carcinogens such polycyclic aromatic hydrocarbons (52). The sterigmatocystin-glucuro conjugate (M4) was preferentially excreted at the basal side of the cells. Such a mechanism must involve at least one transporter in basal sterigmatocystin-glucuro conjugate excretion. MRP1 (ATP-binding cassette, subfamily C, member 1) may be involved in this excretion. Our qPCR results showed that MRP1



Scheme 1. Putative Pulmonary Sterigmatocystin Metabolisma

a Pathway A (top) indicates the metabolism of sterigmatocystin in “normal” pulmonary epithelial cell cultures. Pathway B (meddle) indicates metabolic pathways of sterigmatocystin in “P450 1A1-induced” pulmonary cultures. P450 1A1 was inducted by being treated for 24 h with 25 µM β-naphthoflavone. For high levels of P450 1A1 and in the presence of glutathione S-transferases and GSH, transient epoxide formation led to the glutathione conjugate or to dihydrodiol (pathway C, bottom).

mRNAs were detected in PTEC, although STG exposure did not increase their level of expression. MRP1 is strongly expressed in lung epithelium and localized at the basolateral side of human bronchial epithelial cells (53). MRP1 is known to be involved in transport of aflatoxin B1-glutathione-S conjugates (54). The low basal expression level of P450 1A1 in our model, as already reported in the lung (55, 56), could explain the absence of detection of two hydroxylated STG metabolites (M1 and M2) and the glutathione adduct (M3) formed with recombinant P450 1A1. Therefore, we investigated the metabolites produced when PTEC were exposed to β-naphthoflavone, a P450 1A inducer, before STG challenge. One sulfo conjugate (M5) and one glucuro conjugate (M6) were detected. The mass fragmentations of M5 and M6 show the presence of an M + 16 ion, which could correspond to a monohydroxy-sterigmatocystin product (M1 or hydroxylation at another location). Therefore, P450 1A1 metabolism may result in the formation of M1 rapidly transformed into M5 and M6. Evidence of epoxide formation and the subsequent formation of dihydroxysterigmatocystin (M2) or the glutathione conjugate (M3) were not found in porcine primary cells.

Because incubations of STG with recombinant P450 1A2 led to the formation of M1, the intervention of P450 1A2 might also contribute to M1 formation in PTEC. However, P450 1A1 is 1000-fold more strongly expressed in PTEC and 10-fold more strongly induced after β-naphthoflavone challenge than P450 1A2. Similarly, Chirulli and co-workers found that the mRNA level of P450 1A1 was markedly increased in lungs of β-naphthoflavone-treated pigs, whereas the mRNA level of P450 1A2 was barely increased (45). In humans, P450 1A2 mRNA were not detected in bronchial epithelium (57), and no P450 1A2 was found in lung microsomes (58). Although we cannot exclude the possible contribution of human P450 1A2 in STG pulmonary metabolism after P450 1A induction, this seems to us unlikely. Finally, we investigated whether ST can increase the level of expression of metabolizing enzymes. Our results show that STG increased the level of expression of the P450 1A1 messenger, though only weakly (18-fold induction) as compared to that of β-naphthoflavone results (160-fold induction). Because STG is a planar polycyclic compound, STG may be a ligand of the nuclear receptor Ahr to explain the increase in the level of the P450 1A1 messenger. However, despite this increase in

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mRNA levels, no significant increase in P450 1A1 activity was detected after STG challenge in PTEC. These data suggest that airway cells, which express under normal conditions both UGTs and MRP1, are able to conjugate and to transport STG. We also suggest that the metabolism of STG in the porcine airway tract in vitro is unable to produce significant amounts of reactive epoxide metabolites. On the other hand, STG increases the P450 1A1 mRNA levels with unknown long-term consequences. Because differences in xenobioticmetabolizing enzymes have been demonstrated between human and pig, these results need to be confirmed on primary human epithelial cells. Acknowledgment. We thank Sandrine Blondel and Anaı¨s Roussel for technical assistance. We thank Je´roˆme Bouligand for attentive rereading of the manuscript. These studies were supported in part by AFSSET grants (EST-2007-63 and EST2008-27). Supporting Information Available: Primer sequences used in the study for the relative quantification of porcine gene expression (Table S1), additional representative HPLC-MS chromatograms (Figures S1 and S2), and standardized mRNA expression of genes (Figure S3). This material is available free of charge via the Internet at http://pubs.acs.org.

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