metabolism and cytotoxicity of aflatoxin b1 in cytochrome ... - Toxicology

Journal of Toxicology and Environmental Health, Part A, 65:853–867, 2002 Copyright© 2002 Taylor & Francis 1528-7394 /02 $12.00 + .00 DOI: 10.1080/0098410029007121 6

METABOLISM AND CYTOTOXICITY OF AFLATOXIN B1 IN CYTOCHROME P-450-EXPRESSING HUMAN LUNG CELLS Terry R. Van Vleet, Patrick J. Klein, Roger A. Coulombe, Jr. Graduate Program in Toxicology, and Department of Veterinary Sciences, Utah State University, Logan, Utah, USA The mycotoxin aflatoxin B1 ( AFB 1) is a hepatocarcinogen in many animal models and probably a human carcinogen. Besides being a dietary carcinogen, AFB1 has been detected in dusts generated in the processing and transportation of AFB1-contaminated products. Inhalation of grain dusts contaminated with AFB1 may be a risk factor in human lung cancer. Aflatoxin B1 requires cytochrome P-450 ( CYP) -mediated activation to form cytotoxic and DNA-reactive intermediates, and this activation in human liver is mediated by the CYP 1A2 and 3A4 isoforms. Which isoforms are important in AFB1 activation in human lung is not well understood. To investigate whether these CYPs can activate AFB1 at low, environmentally relevant concentrations in human lung cells, SV40 immortalized human bronchial epithelial cells ( BEAS-2B) that were transfected with cDNA for CYPs 3A4 ( B3A4) or 1A2 ( B-CMV1A2) were used. B-CMV1A2 cultured in 15 nM AFB1 produced the AFB1–glutathione conjugate (AFB1–GSH) and aflatoxin M1 ( AFM1) , while B3A4 cells produced only aflatoxin Q1 ( AFQ1) at 0.15 µM AFB1. Nontransfected BEAS-2B cells produced no metabolites, even at 1.5 mM AFB 1. Microsomes prepared from B-CMV1A2 and B3A4 cells activated AFB1 to AFB1 8,9-epoxide ( AFBO) , while those from BEAS-2B cells did not produce AFBO. Cytosol from all three cell types was ineffective at glutathione S-transferase ( GST) -mediated trapping of enzymatically generated AFB1 8,9-epoxide. B-CMV1A2 cells were 100-fold more sensitive to AFB1 compared to B3A4 cells, and were 6000fold more sensitive than control BEAS-2B cells. Western immunoblots confirmed that only B-CMV1A2 cells expressed CYP 1A2 protein, while CYP 3A4 was only in B3A4 cells. B-CMV1A2 cells were the most sensitive to AFB1, followed by B3A4 cells. CYP 3A4, which has been predicted to activate AFB1 primarily at higher AFB1 concentrations, was also responsible for significant AFB1 toxicity at low concentrations. These data indicate that human lung cells expressing these CYP isoforms are capable of activating AFB1, even at environmentally relevant concentrations.

Aflatoxin B1 (AFB 1), a product of the molds Aspergillus flavus and A. parasiticus, is immunotoxic and carcinogenic in many animal models and is strongly suspected to be a human carcinogen (Bondy & Pestka, 2000; Wogan et al., 1974; Wong & Hsieh, 1976). AFB 1 contamination of grains is found in areas of high heat and humidity, or under inadequate storage Received 20 June 2001; sent for revision 26 July 2001; accepted 25 September 2001. The authors thank Dr. Katherine Macé of the Nestle Research Centre, Lausanne, Switzerland, for generously supplying the cells for this work, and Dr. Jeffrey O. Hall for supplying the MTT assay protocol for this study. This work was supported in part by NIH grant ES04813, USDA-NRI grants 970-3081 and 980-3754, and the Utah State Agricultural Experiment Station, where this paper is designated number 7415. Portions of this report were presented at the 37th annual meeting of the Society of Toxicology, Seattle, WA, March 1998 (abstr. 1970). Address correspondence to Roger A. Coulombe, Jr., Toxicology Program, Utah State University, 4620 Old Main Hill, Logan, UT 84322, USA. E-mail: [email protected] 853

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conditions. AFB 1 has been detected in respirable grain dusts at levels as high as 52 ppm (Burg et al., 1981), and some epidemiological data have linked pulmonary exposure to AFB 1-laden grain dusts with an increase in lung tumor incidence and pneumonia (Oyelami et al., 1997; Hayes et al., 1984). The immunotoxicity of AFB1 may be mediated by a reduction of nitric oxide production via inhibition of nitric oxide synthetase (Moon et al., 1998). In order to form cytotoxic and DNA-alkylating intermediates, AFB 1 requires activation by human cytochromes P-450 (CYP) to the electrophilic AFB1 8,9-epoxide (AFBO). While there are conflicting reports as to which isoform is most important for AFB 1 activation (Forrester et al., 1990; Mace et al., 1997; Ramsdell et al., 1991; Raney et al., 1992b; Shimada & Guengerich, 1989; Ueng et al., 1995), CYP 1A2 and 3A4 appear to be the most significant isoforms for AFB1 activation in human liver (Mace et al., 1996), while other isoforms, such as 2A6 and 2B7, play a minor role (Aoyama et al., 1990). Numerous studies have shown that AFB1 is activated in animal and human lung tissues (Ball & Coulombe, 1991; Ball et al., 1995; Coulombe et al., 1986; Donnelly et al., 1996; Kelly et al., 1997). However, many of these studies used high AFB1 concentrations (15–128 µM) that may not be relevant to the low level of exposure normally encountered by those occupationally exposed to AFB1-laden grain dusts (Burg et al., 1981). In human lung microsomes, AFB1 activation has been shown to be mediated by both CYP 3A4 (Kelly et al., 1997) and lipoxygenase (Donnelly et al., 1996). A factor that may increase risk associated with AFB1 inhalation is that the CYP 1A family is induced by polycyclic aromatic hydrocarbons (PAHs), to which many people may be exposed. Exposure to inducers may influence metabolism in vitro and in vivo in the lung (Langouet et al., 1995). Very recently, our laboratory has demonstrated that normal human bronchial epithelial cells induced by the polycyclic aromatic hydrocarbon (PAH) 3methylcholanthrene (3-MC) activate AFB1 at low, environmentally relevant (0.15–1.5 µM) concentrations (Van Vleet et al., 2001). Detoxification of AFBO in mammals is primarily by glutathione S-transferases (GSTs) (Hayes et al., 1991; Ketterer et al., 1993). GST M1-1 is the most important isoform in humans, although the M3-3, P1-1, A1-1, and A22 isoforms may also play a role (Johnson et al., 1997; Ketterer et al., 1993). The expression of GSTs can also be increased by chemical inducers, such as 3-MC (Donnelly et al., 1996; Yonamine et al., 1996). The bronchiolar epithelium in human lung is the major site of tumor formation (Mace et al., 1994). There are many cell types recognized in the bronchiolar epithelium, but there is significant variation in the cell types present between species and between the same cell types of different species (McDowell et al., 1978; Plopper, 1983; Plopper et al., 1983; St. George et al., 1988). The BEAS-2B cell line is an SV-40 immortalized cell line originating from normal human bronchiolar epithelium (NHBE)

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progenitor cells (Reddel et al., 1988). BEAS-2B cells remain nontumorigenic through numerous passages (Mace et al., 1994; Pfeifer et al., 1991; Reddel et al., 1988; Ura et al., 1989) and represent a good model for studying pulmonary carcinogenesis because they originate from cells targeted by chemical carcinogens in the lung (Mace et al., 1994; Reddel et al., 1988). BEAS-2B cells have also been transfected with cDNA for CYPs 1A2 and 3A4 isoforms, to stably express these CYPs (Mace et al., 1994, 1996). A possible advantage of using this cell line to study AFB1 activation, versus other CYP-transfected cell lines not originating from the lung, is that these cells retain some of the biochemistry of normal bronchial epithelial cells (Mace et al., 1994). This may provide some insight into AFB1 metabolism and cytotoxicity, particular to human lung, under conditions where appropriate CYPs are expressed (Van Vleet et al., 2001). Our laboratory has extensively studied AFB1 metabolism and adduct formation in the airways using human and animal airway and lung tissues (Ball & Coulombe, 1991; Ball et al., 1990, 1995; Coulombe et al., 1986; Kelly et al., 1997; Wilson et al., 1990). The use of animal models is limited for studying the human condition because of variability between species, and human tissues are extremely variable in CYP expression and activitie s (Kelly et al., 1997). In this study, the cytotoxicity and metabolism of low concentrations of AFB1 was compared in the B-CMV1A2 and B3A4 cells, which stably express CYP 1A2 and 3A4, two isoforms known to activate this mycotoxin in human liver. MATERIALS AND METHODS Chemicals and Reagents [3H]-AFB 1 (30 Ci/mmol; >98% radiochemical purity) was purchased

from Moravek Biochemicals, Inc. (Brea, CA), and diluted with unlabeled AFB1 to achieve desired concentrations. Spectral-grade dimethyl sulfoxide (DMSO) and unlabeled AFB1, AFM 1, AFQ1, and AFG1 standards were purchased from Sigma (St. Louis, MO). Exo-AFBO standard was a generous gift from Dr. Thomas Harris (Vanderbilt University). LHC-8 (Lechner and LaVeck media), epinephrine, and retinoic acid were obtained from Biofluids (Rockville, MD) and used to make LHC-9. LHC basal and bovine serum albumin (BSA) stock were also purchased from Biofluids. Fetal bovine serum (FBS) was from Hyclone (Logan, UT). Bovine fibronectin, 0.25% trypsin–ethylenediamine tetraacetic acid (EDTA), and trypsin inhibitor were purchased from Sigma (St. Louis, MO), and collagen was a product of Collagen Corp. (Fremont, CA). Molecular-weight markers and an enhanced chemiluminescence (ECL) detection kit were from Amersham International (Arlington Heights, IL). Western immunoblotting antibodies were rabbit anti-human CYP 3A4 from Gentest (Woburn, MA) and polyclonal rabbit anti-human CYP 1A2 from Oxford Biomedical (Oxford, MI). CYP 3A4 and 1A2 standards were also

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purchased from Oxford Biomedical (Oxford, MI). Secondary antibody was goat anti-rabbit horseradish peroxidase (Biorad, Hercules, CA). Sep-Pak C18 cartridges were purchased from Waters (Milford, MA). 3-[4,5-Dimethylthiazol-2-yl ]-2,5-diphenyltetrazolium bromide thiazolyl blue (MTT), butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and phenymethylsulfonyl fluoride (PMSF) were purchased from Sigma (St. Louis, MO). Culture of BEAS-2B Cells BEAS-2B cells were cultured with LHC-9 (LHC-8 with 50 ml 3.3 mM retinoic acid and 250 ml 0.1% epinephrine) in Corning T75 tissue culture flasks (Corning, Corning, NY) at 37°C and 5% CO2. Cells were passed and harvested using 0.25% trypsin–EDTA and trypsin inhibitor solutions and Hanks balanced saline solution (HBSS: 0.9% NaCl, 30 mM HEPES). The freeze medium was 10% FBS, 10% DMSO, and 80% LHC-9. Flasks were coated with a plate coat consisting of 5 mg bovine fibronectin, 5 ml Vitrogen 100, 50 ml BSA stock, and 500 ml LHC Basal, which was added to flasks 15 min prior to seeding. AFB1 Cytotoxicity Assay The 3-[4,5-dimethylthiazol-2-yl ]-2,5-diphenyltetrazolium bromide thiazolyl blue (MTT) cytotoxicity assay was used to measure the relative toxic effects of AFB1 to each cell type (Dombrink-Kurtzman et al., 1994). Microwell (96-well) plates were seeded at a density of 2.5 × 10 4 cells/well (in 0.18 ml medium/well). After 24 h of plating and growth, cells were incubated with AFB1. Each 96-well plate had a control (no AFB1), and 4 rows were used as blanks (no MTT) for each treatment column. Cells were incubated in concentration medium (LHC-9 with AFB1) for 24 h, at which time MTT was added (20 µl of 8.33 mg/ml; final concentration = 0.83 µg/ml) for 4 h. MTT was then aspirated and 100 µl DMSO was added to dissolve dye crystals formed by mitochondria. Plates were then agitated for 10 min and the absorbance was read (l = 540 nm) on a plate reader (model BT 2000, Fisher Biotech, Dallas, TX). Absorbance of blank wells (with no MTT added; l = 540 nm) was subtracted from treatment groups and controls (no AFB 1 added). The percent inhibition of MTT conversion by the cells was calculated as follows (Dombrink-Kurtzman et al., 1994): percent inhibition = [1 – (A540test – A540blank/A540control – A540blank) ] × 100. A540test is the spectral absorbance at 540 nm of a test (AFB1) concentration group, and A540blank is the spectral absorbance of the same test group with no MTT added. A540control is the 540-nm absorbance of the control (no AFB1) wells. Cellular AFB1 Metabolism T75 tissue culture flasks were seeded at a density of 6.3 × 105 cells/ flask in 10 ml LHC-9 and cultured for 3 d before incubating with 3H-AFB1 (0.015, 0.15, or 1.5 µM; 422 µCi/µg AFB1, as determined by liquid scintillation and spectrophotometry; l = 360 nm; e = 21,800) for 48 h. The growth


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medium was then collected and metabolites were removed using Sep-Pak C18 cartridges with 0.05 M acetic acid as previously reported (Walsh et al., 1992). AFB1 metabolites were eluted with 100% methanol and concentrated to 0.1 ml under N2 for analysis by reverse-phase high-performance liquid chromatography (HPLC). Standards and metabolites were detected by counting of fractions using a binary gradient program of two mobile phases as described (Klein et al., 2000). Mobile phases used were A, 0.1% ammonium phosphate, pH 3.6, and B, 95% methanol:5% tetrahydrofuran, at a flow rate of 1 ml min–1. Starting conditions were 90% A and 10% B, which were maintained for 2 min. Between 2 and 13 min, B was increased to 38%, and then to 60% between 13 and 16 min. Finally B was increased to 90% by 17 min and returned to starting conditions by 22 min. Fractions were collected and counted (Beckman LS 3801 scintillation counter; Beckman, Fullerton, CA). Recovery efficiency was greater than 95% as determined by recovery of the AFG1 internal standard. Controls without 3H-AFB1 and without cells were also run under identical conditions. The chromatographic system (Beckman System Gold Nuveau) consisted of a model 126 pump, a model 166 ultraviolet–visible (UV-VIS) detector, and an Econosphere C18 column (15 × 0.46 cm; Alltech, Deerfield, IL), which was maintained at 40°C. Preparation of Cellular Fractions and Homogenates Cells were harvested via trypsinization, then suspended in cold homogenizing buffer (0.05 M Tris, 1 mM EDTA, 0.25 M sucrose, 0.15 M KCl, 20 µM B HT, and 2 00 µM PMSF, pH 7.4), and ho mogenized. C rud e c ell homogenates for Western blotting were homogenized in storage buffer and then stored at –80°C. Homogenates were then centrifuged at 600 × g and at 10,000 × g for 10 min each. The supernatant was again centrifuged at 16,000 × g for 10 min, the soluble fraction from which was then spun at 105,000 × g for 1 h. The resulting supernatant (cytosol) was saved for use in GST activity assays. The pellet from the last spin was resuspended in cold wash buffer (0.1 M sodium phosphate, 1 mM EDTA, 20 µM BHT, and 200 µM PMSF, pH 7.4), then centrifuged at 105,000 × g for 1 h. The pellet was resuspended and stored at –80°C in cold storage buffer (0.05 M Tris, 1 mM EDTA, 0.25 M sucrose, 200 µM PMSF, and 20% glycerol, pH 7.4). BHAinduced mouse liver cytosol was isolated from Swiss Webster mice (male, 25–30 g), following treatment with a previously described dosing regimen (Klein et al., 2000). Turkey liver microsomes were isolated as described previously (Klein et al., 2000), from 21-d-old male Orlopp turkey livers. Microsomal AFB1 Activation and Cytosolic AFB 1 Conjugation Activation of AFB1 to the exo-AFBO was assayed by incubating microsomes (70 µg protein) in 0.1 mM Tris buffer (pH 7.6) and AFB1 (128 mM— a concentration determined in preliminary studies to be saturating) at 37°C for 15 min. The 250-µl reaction mixture also contained 2 mM NADPH, 5 mM glutathione (GSH), and 26 µl BHA-induced mouse cytosol (Ramsdell & Eaton, 1990). Incubations were stopped by adding 250 µl of cold 100%

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methanol with 12 µM AFG 1 (internal standard) and stored at –20°C overnight to precipitate protein. Samples were centrifuged at 13,000 × g for 10 min to generate a pellet, and 100 µl of supernatant was analyzed by HPLC. Controls were also run without microsomes but were otherwise identical to the reaction mixtures of treatment groups. AFB1–GSH was quantified as an indirect measurement of AFBO production. Cytosolic GST-mediated trapping of AFBO was analyzed by a variation of the AFB1 activation assay (Ramsdell & Eaton, 1990). Cytosol prepared from the BHT-induced mouse liver, BEAS-2B, B-CMV1A2, or B3A4 cells was added to an incubation consisting of turkey liver microsomes (source of AFB1 activating CYPs), 128 µM AFB1, 2 mM NADPH, and 5 mM GSH, for 15 min at 37°C. Proteins were then precipitated with cold MeOH (1 volume) and removed by centrifugation. Turkey liver microsomes were used because they were previously shown to be efficient at activating AFB 1 (Klein et al., 2000). Trapped AFBO, as the AFB 1–GSH adduct, was quantified by reverse-phase HPLC as described previously (Klein et al., 2000). Immunodetection of CYPs 1A2 and 3A4 Cell homogenates were loaded onto a 10% acrylamide sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel, and electrophoresed at 125 V for 8 h. Each gel was run with duplicate lanes and included positive control protein standards and negative control molecular-weight markers. One half of the gel was transferred to a nitrocellulose membrane using a semidry blotter (Buchler, Kansas City, MO). The duplicate half was stained with Coomassie blue for molecular weight comparison. Nitrocellulose membranes were immunostained using rabbit anti-human CYP 3A4 or polyclonal rabbit anti-human CYP 1A2, in high-salt Tween (HST) blocking buffer (10 mM Tris, 1 M NaCl 0.5% Tween 20, pH 7.4) for 1 h. Membranes were washed with HST, TBS (10 mM Tris and 140 mM NaCl, pH 7.4), and TBS-Tween (TBS/0.1% Tween 20). Blots were incubated in secondary antibody and HST for 1 h. Protein detection was by chemiluminescence using an ECL kit. Concentration-Response Modeling and Statistical Analysis Cytotoxicity plots (percent inhibition versus µM AFB1) were fitted using an empirical three-parameter Hill equation model (Melnick et al., 1998) using Sigma Plot software (SPSS, Chicago): Rmax dose n ____________ R= n K 0.5 + dosen where R is the measured response (% inhibition), K0.5 is the AFB1 concentration yielding half of the maximal response (Rmax), and n is the Hill exponent, which is a measure of cooperativity. A value of n > 1 indicates positive cooperativity. The IC50 values were calculated as the dose where R =


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50% inhibition. The Hill mathematical model was used because it allows for comparison of cooperativity between CYP-expressing cell lines that validates the observations of previous studies and shows that cytotoxicity is dependent on the activity of the transfected CYP isoforms. Groups were compared using t-test or one-way analysis of variance (ANOVA) (Sigma Stat, SPSS, Chicago). A value of p < .05 was judged significant. An experiment (n = 4) consisted of four separate repetitions (T75 flasks or 96-well plates) from separate frozen cell stock performed at a different time (n = 1). One flask or plate from each cell type was assayed together during each repetition (n = 1) using the same reagents for comparison. RESULTS AFB1 Cytotoxicity As can be seen in Figure 1, AFB1 was cytotoxic to all cell types in a concentration-dependent fashion. The data clearly show that there were profound differences in sensitivity to AFB1 between cell types resulting from the stable expression of CYPs 1A2 and 3A4. By far, the B-CMV1A2 cells were the most susceptible cell type to the cytotoxic effects of AFB1—they were approximately 100 times more sensitive to AFB1 than expressing B3A4 cells, and 6000 times more so than the nontransfected BEAS-2B cells under these experimental conditions (Table 1). B3A4 cells were approximately 60 times more sensitive to AFB1 than were BEAS-2B cells. Regression lines from the nonlinear Hill three-parameter model transformations of percent inhibition versus AFB1 concentration curves had good fit as judged by high r2 values. The Hill coefficient (n) for B3A4-mediated cytotoxicity (1.4) was indicative of positive cooperativity, as was that for BEAS-2B cells (n = 3.293). Cellular AFB1 Metabolism Metabolite profile can provide an indication of which CYPs may be involved in AFB1 metabolism. For example, in human liver, in addition to AFBO, AFM1 and AFQ1 are produced by CYP 1A2 and 3A4 isozymes, respectively (Gallagher et al., 1994, 1996). As in human liver, whole B-CMV1A2 cells produced both AFBO, measured indirectly as the stable, trapped AFB1–GSH conjugate, and the detoxified metabolite, AFM1. In contrast, B3A4 cells produced only AFQ1; no other metabolites were detected. Control, nontransfected BEAS-2B cells produced no metabolites, even when cultured with a higher AFB1 (i.e., 1.5 mM) concentration (Table 2). Because the AFB1 concentration of 0.15 µM was cytotoxic to B-CMV1A2 cells, metabolites were measured only at the lowest (i.e., 0.015 µM) concentration . Microsomal AFB1 Activation/Cytosolic Detoxification Microsomes prepared from B-CMV1A2 and B3A4 cells both activated AFB1 to AFBO at an approximately equal rate. B3A4 cell microsomes activated at a specific activity of 7.9 ± 2.1 nmol AFB1–GSH mg–1 min–1, while B-

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FIGURE 1. Comparative cytotoxicity of AFB1 in three human lung cell lines, (A) BEAS-2B, (B) B3A4, and (C) B-CMV1A2, as determined by the MTT assay. IC50 values were calculated using the Hill three-parameter mathematical model to estimate the concentration causing 50% inhibition of MTT dye conversion as described in Materials and Methods. Regression lines had good fit as judged by high r 2 values. Data points are the means ± SD (n = 4).


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TABLE 1. Comparative Cytotoxicity of AFB1 in BEAS2B, B3A4, and B-CMV1A2 Cells Cell type

IC50a

BEAS-2B B3A4 B-CMV1A2

348 ± 42b 6.4 ± 2.4c 0.06 ± 0.02d

a Values are µM, means ± SD (n = 4). Values were calculated as described in Materials and Methods. See Figure 1 for a graphical presentation of the data. IC50 values were calculated using the Hill three-parameter mathematical model to estimate the concentration causing 50% inhibition of MTT dye conversion. b,c,d Different letters indicate groups are statistically different.

CMV1A2 cell microsomes did so at 6.4 ± 2.7 nmol AFB1–GSH mg–1 min–1. In contrast, cytosolic fractions from both cell types were deficient in trapping enzymatically generated AFB1 8,9-epoxide, a specific measure of AFB1-relevant GST activity (Figure 2). AFB 1-relevant conjugation was not present in any of the cell types as determined by the AFBO epoxidetrapping assay. The positive standard, BHA-induced mouse liver cytosol, was clearly effective in trapping enzymatically generated AFB1 8,9-epoxide (Figure 2A). CYP Expression Western immunoblots probing for CYPs 1A2 and 3A4 in each cell type showed cell-specific CYP expression in that only B-CMV1A2 cells expressed CYP 1A2 protein and B3A4 cells exclusively expressed 3A4 protein (Figure 3). BEAS-2B cells did not express either CYP isoform. DISCUSSION Our data clearly show that both CYP 1A2 and 3A4 are capable of bioactivating AFB1, as determined by the ability of CYP-transfected cells to produce cytotoxic intermediates compared to the nontranfected BEAS-2B cell TABLE 2. AFB 1 Metabolism in Cultures of BEAS-2B, B3A4, and B-CMV1A2 Cells Cell type

[AFB1 ], µM

AFBOa

AFQ1b

AFM 1 a

BEAS-2B B3A4 B-CMV1A2

1.5 0.15 0.015

ND ND 0.73 ± 0.1

ND 10.5 ± 0.1 ND

ND ND 0.51 ± 0.1

Note. Metabolites produced in cell cultures exposed to 1.5, 0.15, and 0.015 µM 3 H-AFB 1 for 48 h. All numeric values shown are significantly different from controls. ND, not detected. a Values are mean nmol/flask ± SD (n = 4).

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FIGURE 2. Representative HPLC chromatograms showing lack of cytosolic GST-mediated conjugation of enzymatically generated AFBO. (A) Included as a positive control, AFBO conjugation by BHAinduced mouse cytosol; (B) negative control (no cytosol); lack of AFBO conjugation (above control levels) in cytosols prepared from (C) BEAS-2B, (D) B-CMV1A2 cytosol, and (E) B3A4 cells. These chromatograms were run at different times, which explains the slight differences in peak elution times. In any event, metabolite standards were run with each analysis to locate the elution time of each peak.


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FIGURE 3. Western immunoblots showing the expression of CYPs 3A4 and 1A2 in BEAS-2B, BCMV1A2, and B3A4 cells. (A) CYP 3A4 expression and (B) CYP 1A2 expression. Each line represents a separate crude cell homogenate from BEAS-2B cells (lanes 1–3), B3A4 cells (lanes 4–6), and BCMV1A2 cells (lanes 7–9). Cell homogenates (30 µg homogenate protein/well) were loaded into a 10% acrylamide SDS-PAGE gel and electrophoresed at 125 V for 8 h. The gel was transferred to a nitrocellulose membrane, then immunostained using rabbit anti-human CYP 3A4 or polyclonal rabbit anti-human 1A2, in high-salt Tween (HST) blocking buffer for 1 h as described in the Materials and Methods. Blots were then incubated in secondary antibody (goat anti-rabbit) and HST for 1 h. Protein detection was by chemiluminescence using an ECL kit.

line. Based on the relative cytotoxicity of AFB1, CYP 1A2-expressing cells appear to be more efficient at AFB1 activation, especially at low substrate concentrations. A similar observation was made by Gallagher et al., (1994), who demonstrated that CYP 1A2-expressing microsomes predominated in activation of AFB 1 concentrations, which are more relevant to occupational exposures compared to those expressing CYP 3A4. Previous literature (Shimada et al., 1992; Kelly et al., 1997; Mace et al., 1998) indicates that there is significant variability in whether CYP 1A2 is detected in human lung tissues. Although the 1A2 and 3A4 CYP isoforms have recently been detected in human lung tissue and cultured cells (Mace et al., 1998; Van Vleet et al., 2001), CYP 1A2 has not been detected in human lung in other studies (Kelly et al., 1997; Shimada et al., 1992). This may indicate that the 1A2 isoform is rarely expressed, or that this isoform is not particularly stable to isolation. Previous work from this laboratory has shown that human lung cells exposed to environmental CYP inducers such as 3MC express increased amounts of CYPs critical to AFB1 activation (Van Vleet et al., 2001). People are commonly exposed to similar inducers in components of cigarette smoke and automobile exhaust. Therefore, these CYPs may also be expressed transiently in response to such compounds. Cytotoxicity is believed to be primarily dependent on AFB1 activation, and the vast toxicity differences between BEAS-2B and CYP expressing cells strongly supports this. In all cell types, AFB 1 cytotoxicity curves reached

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plateau as would be expected of an enzyme-mediated activation (Figure 1), although this is not proof of enzyme saturation. Because toxicity is primarily due to the production of exo-AFBO, it appears that B-CMV1A2 cells are more efficient at producing exo-AFBO than B3A4 cells. In support of this finding, it is reported that human liver CYP 1A2 has a lower Km for AFBO production (32–47 µM) compared to CYP 3A4 (140–180 µM) (Gallagher et al., 1996). Interestingl y, the Hill constant generated from the AFB1-mediated cytotoxicity curve in B3A4 cells indicated positive cooperativity. If cytotoxicity in these cells is the result of CYP-mediated activation of AFB1 as we suspect, this observation seems to support previous evidence of two cooperative AFB1-binding sites in recombinant human liver 3A4 (Offord et al., 1995; Gallagher et al., 1996). Although there is significant evidence that CYPs 1A2 and 3A4 are key to AFB1 activation in their transfected cell lines, BEAS-2B cells were also susceptible to high concentrations of AFB1. This indicates the possibility of other modes of cytotoxicity for AFB1 or activation via other enzyme systems with a lower AFB1 affinity. It is not presently known, however, whether other enzyme systems effectively activate AFB1 at concentrations as low as 1.5 µM. Microsomes from both B-CMV1A2 and B3A4, but not BEAS-2B, cells were effective at activating AFB1, but at high concentrations. Using identical amounts of protein, activation was not significantly different between microsomal fractions from the CYP expressing cell types. Because mouse liver cytosol, used in the AFB1 activation assay, conjugates exo-AFBO almost exclusively (Raney et al., 1992a), microsomes from both cell types appear to have produced approximately equal amounts of the toxic exo-epoxide at a high (128 µM) AFB1 concentration. The production of CYP-specific AFB1 metabolites only in transfected cells demonstrates the critical role these CYPs play in AFB1 metabolism. For example, in human liver, the primary AFB1 metabolite of CYP 1A2 is AFBO, while that of CYP 3A4 is AFQ1 (Gallagher et al., 1996). Metabolite profiles observed in transfected cells reflected this specificity (Table 2). A key detoxification pathway for AFB1 in the livers of animal models is via GST-mediated conjugation. Cytosolic GST activity that conjugated enzymatically generated AFBO, a specific measurement of AFB1-relevant GST activity, was absent in all cell types. Thus, the lack of specific GST-mediated detoxification of AFB1 seems at odds with our detection of an AFB1–GSH peak in the metabolism studies. However, this observation is consistent with previous results using intact NHBE cells and NHBE cytosol to conjugate AFBO (Van Vleet et al., 2001). Differences in the phase II metabolic potential between cytosolic fractions and intact cells may be due to the loss of the low GST activity during the harvesting of cytosol. It is also possible that microsomal and not cytosolic GSTs are responsible for AFB1–GSH conjugation in human lung cells. While human lung cells are much less effective at activating AFB1 than human liver cells, the absence of key GSTs in


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human lung cells may make them susceptible to the toxic effects of AFB1 (Ryberg et al., 1997). This study demonstrates that both CYP 1A2-expressing and 3A4-expressing human lung cells appear to metabolize low, environmentally relevant concentrations of AFB1; however, any assessment of risk posed by inhaled AFB1 should take into account the relative expression of these isoforms in human lung. Although induction of these CYP isoforms has been demonstrated in primary cultures of bronchial epithelial cells after exposure to 3MC (Van Vleet et al., 2001), it is still possible that induction doesn’t occur in vivo. Under conditions where appropriate CYPs are expressed in the lung, however, it is possible that inhalation of AFB1 may result in an increased risk of lung cancer in exposed persons. REFERENCES Aoyama, T., Yamano, S., Guzelian, P. S., Gelboin, H. V., and Gonzalez, F. J. 1990. Five of 12 forms of vaccinia virus-expressed human hepatic cytochrome P450 metabolically activate aflatoxin B1. Proc. Natl. Acad. Sci. USA 87:4790–4783. Ball, R. W., and Coulombe, R. A., Jr. 1991. Comparative biotransformation of aflatoxin B1 in mammalian airway epithelium. Carcinogenesis 12:305–310. Ball, R. W., Wilson, D. W., and Coulombe, R. A., Jr. 1990. Comparative formation and removal of aflatoxin B1–DNA adducts in cultured mammalian tracheal epithelium. Cancer Res. 50:4918–4922. Ball, R. W., Huie, J. M., and Coulombe, R. A., Jr. 1995. Comparative activation of aflatoxin B1 by mammalian pulmonary tissues. Toxicol. Lett. 75:119–125. Bondy, G. S., and Pestka, J. J. 2000. Immunomodulation by fungal toxins. J. Toxicol. Environ. Health B 3:109–143. Burg, W. A., Shotwell, O. L., and Saltzman, B. E. 1981. Measurements of airborne aflatoxins during the handling of contaminated corn. Am. Ind. Hyg. Assoc. J. 42:1–11. Coulombe, R. A., Jr., Wilson, D. W., Hsieh, D. P., Plopper, C. G., and Serabjit-Singh, C. J. 1986. Metabolism of aflatoxin B1 in the upper airways of the rabbit: Role of the nonciliated tracheal epithelial cell. Cancer Res. 46:4091–4096. Dombrink-Kurtzman, M. A., Bennett, G. A., and Richard, J. L. 1994. An optimized MTT bioassay for determination of cytotoxicity of fumonisins in turkey lymphocytes. J. AOAC Int. 77:512–516. Donnelly, P. J., Stewart, R. K., Ali, S. L., Conlan, A. A., Reid, K. R., Petsikas, D., and Massey, T. E. 1996. Biotransformation of aflatoxin B1 in human lung. Carcinogenesis 17:2487–2494. Forrester, L. M., Neal, G. E., Judah, D. J., Glancey, M. J., and Wolf, C. R. 1990. Evidence for involvement of multiple forms of cytochrome P-450 in aflatoxin B1 metabolism in human liver. Proc. Natl. Acad. Sci. USA 87:8306–8310. Gallagher, E. P., Wienkers, L. C., Stapleton, P. L., Kunze, K. L., and Eaton, D. L. 1994. Role of human microsomal and human complementary DNA-expressed cytochromes P4501A2 and P4503A4 in the bioactivation of aflatoxin B1. Cancer Res. 54:101–108. Gallagher, E. P., Kunze, K. L., Stapleton, P. L., and Eaton, D. L. 1996. The kinetics of aflatoxin B1 oxidation by human cDNA-expressed and human liver microsomal cytochromes P450 1A2 and 3A4. Toxicol. Appl. Pharmacol. 141:595–606. Hayes, J. D., Judah, D. J., McLellan, L. I., and Neal, G. E. 1991. Contribution of the glutathione Stransferases to the mechanisms of resistance to aflatoxin B1. Pharmacol. Ther. 50:443–472. Hayes, R. B., van Nieuwenhuize, J. P., Raatgever, J. W., and Ten Kate, F. J. 1984. Aflatoxin exposures in the industrial setting: An epidemiological study of mortality. Food Chem. Toxicol. 22: 39–43. Johnson, W. W., Ueng, Y. F., Widersten, M., Mannervik, B., Hayes, J. D., Sherratt, P. J., Ketterer, B.,

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