* Department of Pharmacology and Toxicology, University of Utah, Salt Lake City, Utah 841125820;
Nestlé Research Centre, 1000 Lausanne 26, Switzerland; and
College of Pharmacy, Toxicology Program, University of New Mexico, Albuquerque, New Mexico 87131-5691
Received August 21, 2002; accepted October 29, 2002
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ABSTRACT |
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Key Words: 3-methylindole (3MI); human bronchial epithelial cells; BEAS-2B; cytochrome P4502F1 (CYP2F1); apoptosis.
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INTRODUCTION |
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Isolated rabbit nonciliated bronchiolar epithelial cells (Clara) and type-II alveolar epithelial cells, which contain the highest concentrations of cytochrome P450 enzymes in the lung, were susceptible to cytotoxicity induced by 3MI (Nichols et al., 1990). Treatment with 1-aminobenzotriazole (ABT), a suicide inhibitor of CYP, protected these cells from 3MI-mediated toxicity, indicating a requirement for cytochrome P450 enzyme bioactivation. ABT has also been shown to inhibit 3MI turnover and covalent binding in human lung microsomes (Ruangyuttikarn et al., 1991
). Isolated rabbit Clara cells efficiently bioactivated 3MI to form 3-methyleneindolenine, which was detoxified by glutathione (GSH) (Thornton-Manning et al., 1993
). These studies showed that the relatively low susceptibility of rabbits to the pneumotoxic effects of 3MI may be explained by the highly effective detoxication by nucleophilic trapping of the reactive 3-methyleneindolenine with GSH, and is not due to less efficient formation of the reactive intermediate by P450 enzymes in rabbit Clara cells. One of the objectives in the current studies was to evaluate the hypothesis that human lung cells might also efficiently detoxify the reactive intermediate of 3MI though GSH conjugation.
Several studies have investigated which human P450 enzymes might be capable of bioactivating 3MI to an intermediate that covalently binds to cellular macromolecules. Although cytochrome P450s expressed in lung tissue are less well characterized than hepatic P450s, a cDNA encoding CYP2F1 was isolated from a human lung library (Nhamburo et al., 1990), and mRNA corresponding to this form was found in low abundance in several human lung samples. Human CYP2F1 bioactivated 3MI to a covalent-binding intermediate at a rate that was the highest among all human P450s tested by Thornton-Manning et al.(1991)
. In addition, CYP2F1 produced the highest rates of formation of 3-methyleneindolenine (Thornton-Manning et al., 1996
), the putative reactive intermediate, and CYP2F1 expressed in a lymphoblast cell line catalyzed only the dehydrogenation of 3MI without detectable formation of oxygenation products (Lanza et al., 1999
; Lanza and Yost, 2001
).
Some established human cell lines with indefinite life spans may serve as in vitro models to assess potential human toxicity. However, most of these cell lines have low or no metabolic capacity, primarily due to the loss of P450 enzyme expression. To overcome this limitation, metabolically competent human cell lines have been developed though cDNA overexpression techniques in order to restore specific catalytic activities (Macé et al., 1996). The human bronchial epithelial BEAS-2B cell line, immortalized by the SV-40 T-antigen gene (Reddel et al., 1988
), retained the expression of several phase-II enzymes including glutathione S-transferase (GST), but expressed low levels of endogenous P450 (Macé et al., 1994
). This cell line has been used to express recombinant CYP1A2 and was used for cytotoxic and genotoxic testing of aflatoxin B1 (Macé et al., 1994
). Other human P450 enzymes that have been stably expressed in BEAS-2B cells include CYP2A6, 2B6, 2D6, 2E1, 3A4, and 3A5 (Macé et al., 1997
).
In this study, BEAS-2B cells transfected with human cytochrome P450 cDNAs for 2A6, 3A4, 2E1, and 2F1 were used to evaluate the bioactivation of 3MI. Normal BEAS-2B cells were utilized to assess the unresolved molecular mechanisms by which electrophilic metabolites of 3MI induce cell death. The ability of 3MI to induce apoptosis was assessed in BEAS-2B cells by measuring the externalization of phosphatidylserine and by measuring DNA fragmentation. Preliminary studies using fluorescent microscopy after incubation of these cells with high concentrations of 3MI demonstrated apoptotic body formation as well as cell necrosis (Nichols et al, 2000). The current studies confirmed that P450-mediated bioactivation of 3MI induced DNA fragmentation and apoptosis in a human bronchial epithelial cell line and demonstrated that apoptosis occurred at low concentrations of 3MI. Overexpression of CYP2F1 increased the susceptibility of this human lung cell line to 3MI, as assessed by the extent of cytotoxicity and apoptosis.
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MATERIALS AND METHODS |
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Human bronchial cell line.
BEAS-2B cells (American Type Culture Collection, Rockville, MD) were obtained by infection of normal bronchial epithelial cells with an adenovirus 12simian virus 40 hybrid preparation (Reddel et al., 1988). To generate the CYP450-expressing vector, the 1.8-kb 2F1 (kindly provided by Frank Gonzalez, NIH, Bethesda, MD) cDNA was individually inserted by blunt-ended cloning into the BamH1 site of the pCVMneo vector (kindly provided by B. Vogelstein, Johns Hopkins University, Baltimore, MD). The DNA construct was introduced into the BEAS-2B cells by liposome-mediated transfection and selected as previously described (Macé et al., 1994
). These immortalized clones are nontumorigenic cells up to a very high passage number (Pfeifer et al., 1989
). Control cells containing only the pCMVneo vector are designated B-CMVneo cells (Macé et al., 1997
). The cells were cultured in a serum-free medium called LHC-9 (Biofluids, Rockville, MD). For subculturing, cells were trypsin-dissociated and plated into fibronectin/collagen-coated culture plates. The CYP450 expressing BEAS-2B cells utilized in the present study included three cell lines (B-CMV2A6, B-CMV3A4, B-CMV2E1) in which P450 expression has been previously characterized by western blots and measurement of catalytic activities using specific fluorescent substrates such as 7-alkoxycoumarins, coumarins, and 7-alkoxyresorufins (Macé et al., 1997
). A non-toxic substrate for CYP2F1 is not available for quantitation of this enzyme's activity.
Western blot analysis of CYP2F1 in B-CMV2F1 cells.
Western blot analysis was done as described by Wang et al. (1998), using rabbit antipeptide polyclonal antibodies to CYP2F1 that were produced by Genemed Synthesis, Inc. (San Francisco, CA). These antibodies were raised to a keyhole limpet hemocyanin (KLH) conjugate of a cyclic amino acid peptide (KGCCSVHDHQASLDPRSPRDFIQC). This peptide was chosen to mimic the strategy employed by Schulz-Utermoehl et al. (2000). In this work, effective and selective antibodies were made to a hydrophilic loop region between helices G and I of CYP2D6. The peptide corresponding to residues 248267 of CYP2F1 contained three extraneous amino acid N-terminal residues (KGC) designed to provide an N-terminal lysine to couple with KLH, a spacer glycine, and a cysteine that was oxidized with the other cysteine to form a disulfide to cyclize the peptide. These antibodies detected a 55 kDa protein band in the B-CMV2F1 cells that was not detected in the BEAS-2B cell line.
Detection of CYP2F1 transcripts in B-CMV2F1 cells.
Total cellular RNA was isolated from confluent 75cm2 flasks using an RNeasy Kit and QIAshredder microspin homogenizer (Qiagen, Valencia, CA). RNA samples were stored at 70°C. First strand cDNA synthesis for RT/PCR was performed using Superscript II RNase H-reverse transcriptase (Gibco BRL, Rockville, MD); 5 mg total RNA was used in each synthesis. The cDNA was stored at 20°C for PCR amplification.
PCR primers were designed to amplify a cytochrome P450 2F1 product, a small fragment of 427 bp that contained the entire CYP2F1 open reading frame. The small-fragment primers were 5'-GCTGCGGAAAACTGAAGG-3' (sense) and 5'-GCCAAAGAGCAGGTTATGTGT-3' (antisense). The PCR reactions were performed using 1 ml of cDNA (out of a 20-ml total from the reverse transcriptase step), 2.5 U Taq DNA polymerase (Gibco BRL, MD), 5 ml 10x PCR buffer, 1.5 ml 50-mM MgCl2, 1 ml 10-mM dNTP mix (at a final concentration of 200 mM), 0.2 mM of each primer, and water to a final concentration of 50 mM. PCR reaction conditions were to denature at 94°C for 3 min, followed by 30 cycles of melting at 94°C for 1 min, annealing at 55°C for 1 min, extending at 72°C for 2 min, and adding a 10-min final extension. PCR product was visualized by electrophoresis on a 1% v/v agarose gel stained with ethidium bromide. The small fragment was cut from the gel and subcloned into pCR2.1-TOPO using the TOPO TA-cloning kit (Invitrogen, Carlsbad, CA) for sequencing.
Lactate dehydrogenase leakage.
Cytotoxicity was measured by detection of the release of lactic dehydrogenase (LDH) into the media. Cultured cells were grown to confluence in 24-well plates coated with fibronectin and collagen in a humidified incubator with 95% air and 5% CO2 in 0.5 ml of LHC-9 medium. 3MI and DEM were dissolved in DMSO, which was diluted to a final concentration of 0.5%. LDH leakage was determined by removing the supernatant from each well and centrifuging at 2000 x g for 10 min at 4°C. Total LDH was determined by adding 1% Triton X-100 to the replicate wells. LDH leakage and total LDH were determined using SIGMA Procedure No. 340-UV. The change in absorption/time was monitored every 11 s for a total of 6 min at 340 nm, using Molecular Devices SpectraMax 250 Microplate Reader (Molecular Devices, Sunnyvale, CA). Each experiment was carried out in triplicate and cytotoxicity was expressed in terms of cell viability determined by formula: 100 minus the percentage of total cellular LDH released into the medium. Cytotoxicity results were expressed as the percent of DMSO-treated control cell viability (86 ± 2%).
Dojindo colorimetric cytotoxicity assay.
The cytotoxic effect of 3MI was assessed utilizing a 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8) (Dojindo Molecular Technologies, Inc., Gaithersburg, MD) bioassay, according to the manufacturer's recommendation for this cell-counting kit. This bioassay utilizes Dojindo's highly water-soluble tetrazolium salt, WST-8, which produces a water-soluble formazan dye upon reduction in the presence of an electron carrier. The amount of the formazan dye generated by the activity of dehydrogenases in cells is directly proportional to the number of living cells. Briefly, cells were subcultured into 96-well, collagen/fibronectin-coated plates and grown to 5080% confluence. A range of concentrations of 3MI in 0.5% v/v DMSO containing LH-9 serum-free medium, with or without 0.5 mM ABT, was incubated for 624 h. Data are reported as percentages of cell viability, compared to untreated control cells.
Analysis of cellular glutathione content.
B-CMV2F1 cells were grown to confluence in 6-well plates on a collagen/fibronectin matrix in a humidified incubator with 95% air and 5% CO2 in LHC-9 medium. 3MI and DEM were dissolved in DMSO, which was diluted to a final concentration of 0.5% during the incubations. GSH was measured by HPLC, following monobromobimane (mBBr) (Molecular Probes, Eugene, OR) derivatization by the method described by Chichester et al.(1994). The bimane derivative of GSH was separated by Beckman System Gold HPLC (Beckman, San Ramon, CA) on a reversed-phase Ultremex 5 mm IP column (250 x 4.6 mm, Phenomenex, Torrance, CA) using the ion-pair reagent tetrabutylammonium phosphate (TBAP). A Shimadzu RF-535 Fluorescence HPLC Monitor (Shimadzu, Inc, Kyoto, Japan) with excitation and emission wavelengths set at 360 and 460 nm, respectively, attached to a Beckman Recorder (Beckman, Scientific Instruments Division, Irvine, CA), was used for the detection. A linear standard curve with derivatized GSH was run with each batch of samples analyzed by this HPLC assay.
Glutathione S-Transferase (GST) activity.
B-CMV2F1 cells were grown to confluence in Corning 150 cm2 flasks on a collagen/fibronectin matrix. After removing media, cells were trypsin-dissociated and centrifuged at 2000 x g for 7 min at 4°C to remove trypsin. The pellet (cells) was resuspended in a cold TrisHCl buffer, pH 7.4 containing 10 mM TrisHCl, 0.15 M KCl, 10 mM EDTA, and 1 mM dithiothreitol. Protease inhibitor, phenylmethysufonyl fluoride (PMSF), was added to the 0.25 mM final concentration and cells were sonicated. Microsomes were prepared by centrifugation at a low speed (3000 x g for 5 min), followed by 10,000 x g for 10 min to remove the cell debris. Supernatant (S9 fraction) was then centrifuged at 105,000 x g for 30 min, and cytosol was separated. The pellet (microsomes) was washed three times with TrisHCl buffer, resuspended, and recentrifuged at 105,000 x g for 30 min. Finally, the microsomal pellet was resuspended in 0.25 M sucrose, 0.05 M Tris, and 1 mM EDTA, pH 7.4, and stored at 80°C. Protein content of the cytosolic fraction was determined using the BCA protein assay.
GST activity was determined using a modification of the method by Habig et al.(1974). Briefly, 15 mM GSH and 2.5 mM 1-chloro-2,4-dinitrobenzene (CDNB) were combined in a 1-ml cuvette in 0.1 M potassium phosphate buffer (pH 6.5) at room temperature.
Annexin-V binding.
BEAS-2B cells were plated in LHC-9 media on FNC (FNC Coating Mix; Biological Research Faculty and Facility, Inc.; Ijamsville, MD)-coated 6-well plates at 2 x 104 cells per well, and the medium was replaced 48 h later. Five days after plating cells that had grown to subconfluence, the cells were treated for the times indicated and harvested with trypsin-EDTA and soybean trypsin inhibitor (SBTI; BioFluids Division, BSI Rockville, MD).
The detached cells were collected and added to the appropriate supernatant sample. Cells were centrifuged and then washed twice with cold DPBS+ (with calcium and magnesium). After the last wash, the supernatant was aspirated and binding buffer (Annexin-V-FITC Apoptosis Detection Kit II, PharMingen; San Jose, CA) was added to controls, Annexin-V-FITC only, propidium iodide only, and block. The block control was incubated for 15 min at room temperature in the dark, then Annexin-V-FITC and propidium iodide were added and the sample was incubated an additional 15 min. After all samples had been incubated for a total of 30 min, binding buffer was added and each sample was analyzed by flow cytometry on a FACScan flow cytometer (Becton Dickinson Company, Franklin Lakes, NJ).
For DMSO (dimethylsulfoxide) control cultures, forward-angle light scatter and side-angle light scatter were used to establish a gate of viable cells. The DMSO control samples were also used to set the regions of positive staining for Annexin-V-FITC and propidium iodide. The Annexin-V-FITConly controls and propidium iodide-only controls were used to identify nonspecific binding. A four-quadrant dot plot was set using these controls, and the percentage of cells reflect the number of events recorded in each quadrant.
DNA fragmentation assay (cell-death assay for quantitative in vitro determination of cytoplasmic histone-associated DNA fragments).
Cells were incubated in 48-well plates (104 cells per well) coated as described for Annexin-V studies. Experiments were initiated 24 h after plating cells that were at subconfluence when the test compounds were added. The adherent cells were gently lysed to release nucleosomes from the cytoplasm of apoptotic cells, using the buffer solutions provided in the commercially available Cell Death Detection ELISA kit (Boehringer-Mannheim, Indianapolis, IN) for the quantitative determination of in vitro cytoplasmic histone-associated DNA fragments (mono- and oligonucleosomes). The enrichment factor of nucleosomes in the cytoplasm of cells treated with 3MI compared to untreated cells was calculated using the formula: (absorbance at 405 nm using a reference absorbance at 490 nm for treated cells) divided by (corresponding absorbance for untreated control cells).
Statistical analysis.
All the experiments were repeated, at least in triplicate, with three different passage numbers of cells. For a given set of experiments, each parameter, including controls, were evaluated with between 2 and 6 replicates, depending on the type of experiment. All the data were reported as mean ± SEM. The difference between (3MI) and (3MI + DEM) was tested using two-way analysis of variance (ANOVA). The individual groups were compared using Student's t-test. The difference was considered significant with a probability of p 0.05.
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RESULTS |
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The CMV transfected cell lines were incubated with 1 mM 3MI for 48 h in the presence and absence of 5 mM ABT, a suicide substrate inhibitor of cytochrome P450. Cytotoxicity was measured by the amount of LDH released into the medium compared to that by control untreated cell cultures (Fig. 2). The B-CMV2F1 cells were the most susceptible to 3MI-mediated cytotoxicity after 48 h, followed by B-CMV3A4 (34 and 45% viability of control, respectively). BEAS-2B and B-CMVneo were only slightly affected (80% viability of control) by 3MI under the same conditions. The responses of B-CMV2A6 and B-CMV2E1 cells were not significantly different from control cells. The presence of ABT protected the susceptible cells from 3MI-mediated cytotoxicity, indicating a requirement for P450-mediated bioactivation of 3MI.
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A role for GST in the detoxification of 3MI was suspected, based on the increased susceptibility of cells treated with DEM, which decreased GSH levels without causing cytotoxicity. Therefore, the GST activity in the cytosol of B-CMV2F1 cells was measured at different protein concentrations. The GST activity was linear in the range of 0.10.4 mg protein with the correlation coefficient of 0.96. The GST activity in the cell cytosol was 101 ± 21 nmol/min/mg protein with 15 mM GSH and 2.5 mM CDNB. The experiment was carried out at room temperature, and the value is a mean of 3 different batches of cell cytosol, each with a different passage number of cells. The activity was measured in duplicate for each individual batch of cell cytosol. The reported activity is the mean ± SEM. The GST activity in microsomes was undetectable with these conditions.
Increased susceptibility of B-CMV2F1 cell line to low concentrations of 3MI.
The relative susceptibility of the B-CMV2F1 and BEAS-2B cell lines to 3MI-induced cytotoxicity was assessed over a range of concentrations from 5 to 100 µM by measuring the metabolic activity of the cells, using the Dojindo bioassay for cell viability. The results shown in Figure 4 clearly demonstrated that B-CMV2F1 cells were more susceptible to 3MI-induced cytotoxicity after a 24-h incubation at 10 µM 3MI than were the BEAS-2B control cells (10% vs. 50%, respectively). The increased susceptibility of the 2F1 overexpressing cells compared to BEAS-2B cells was also statistically significant at 50 and 100 µM 3MI (5% vs. 20%, respectively).
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DISCUSSION |
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Cytotoxicity of bioactivated compounds depends upon the balance between activation and the ability of the cells to detoxify the resulting electrophilic metabolites. In general, metabolites generated by cytochrome P450 enzymes cause cytotoxicity by a combination of processes that can involve direct interaction with critical macromolecules in target cells and/or depletion of cofactors important in detoxification processes such as GSH, which may indirectly result in cell injury. Studies by Nocerini et al. (1983), exemplifying these processes, have demonstrated that 3MI depletes GSH significantly in lung, liver, and kidney tissues when administered to goats. Depletion of pulmonary GSH by administration of DEM prior to 3MI administration significantly enhanced pneumotoxicity in goats. Conversely, administration of a GSH precursor, cysteine, a rate-limiting compound in cellular GSH biosynthesis, prolonged survival times, decreased covalent binding of 3MI metabolites, and reduced the severity of 3MI-induced lung injury when given prior to 3MI administration (Nocerini et al., 1983
).
Studies with several animal species have been reported which document that 3MI induces severe pulmonary damage to epithelial cells in the bronchioles and alveoli after systemic administration of this toxicant (Adams et al., 1988; Yost, 1999
). In order to study the involvement of GSH in a human lung cell model, B-CMV2F1 cells were incubated with 3MI in the presence of DEM, and 3MI toxicity was apparent after a shorter time of incubation (18 vs. 48 h, Fig. 3
). However, the GSH levels were not decreased by 3MI alone at any of the times studied, from 5 min to 48 h. The human B-CMV2F1 cells were more susceptible to 3MI if the availability of glutathione was reduced by DEM, but depletion of total cellular GSH only increased susceptibility by about 50%. These results can be contrasted with previous studies with isolated rabbit Clara cells (Thornton-Manning et al., 1993
), which showed that GSH adduct formation efficiently detoxified 3MI reactive intermediates to protect rabbits from 3MI-mediated pneumotoxicity. The cytosolic fraction of the B-CMV2F1 cells showed a GST activity (101 ± 21 nmol/min/mg protein) that is in close agreement with the published value of GST activity reported for BEAS-2B cells (91.9 nmol/min/mg protein) by Macé et al.(1997)
and for human lung cells (77 nmol/min/mg protein) by Tateoka et al., 1987
. In the current study, B-CMV2F1 cells were shown to possess relatively normal levels of GSH and active GST enzymes, but detoxification via this pathway did not efficiently protect these human lung cells from 3MI-mediated damage.
High concentrations of 3MI clearly induced cell necrosis based on the measurement of leakage of lactate dehydrogenase into the medium in these human lung-cell lines. However, it was also evident that bioactivation of 3MI by BEAS-2B cells can lead to a dose-dependent induction of apoptosis when low concentrations (10100 µM) of this selective pneumotoxicant are present. Inhibition of the P450 enzymes in the BEAS-2B cells protected cells from both necrosis and apoptosis at early time periods (1224 h). The increased extent of cytotoxicity and apoptosis observed in the lung-cell-line overexpression of CYP2F1 demonstrated a clear role for this pulmonary-expressed enzyme in the bioactivation of 3MI to a cytotoxic intermediate.
The ability of CYP2F1 to metabolize 3MI to a metabolite that can alkylate DNA through the covalent bond formation of 3-methyleneindolenine with the exocyclic amine nitrogens of deoxyguanosine and deoxycytidine has recently been demonstrated (Regal et al., 2001). These adducts were formed from in vitro microsomal incubations with calf thymus DNA, as well as with intact DNA from incubations of 3MI with rat hepatocytes. Therefore, apoptosis caused by 3MI could occur though the direct alkylation of nuclear DNA, which signals cellular apoptotic mechanisms in BEAS-2B cells.
The ability of a low concentration of 3MI to induce apoptotic changes in BEAS-2B cells as early as 6 h was clearly demonstrated using Annexin-V binding, which occurred in cells that were not necrotic. The earliest evidence of necrosis, measured in 2F1 overexpressing cells treated with DEM to deplete glutathione, was at 18 h when cells were incubated with a high concentration of 3MI. Consequently, the apoptotic pathway was initiated at a much lower concentration of 3MI than the concentration required to induce cellular necrosis. A conclusion consistent with these results is that 3MI causes cell death though an apoptotic mechanism, even at relatively low concentrations and at shorter time periods than observed with studies that measured leakage of LDH into the media. However, when higher concentrations and/or longer time periods are evaluated, the cells become "leaky" and secondary necrosis can be observed.
In conclusion, BEAS-2B cells transfected with CYP2F1 and CYP3A4 were susceptible to 3MI-mediated cytotoxicity, which was moderately enhanced if cells overexpressing CYP2F1 were treated with DEM, a chemical known to deplete cellular GSH. Studies of 3MI metabolism by recombinant CYP3A4 showed that only 3-methyloxindole was produced. However, reactive intermediates (the 2,3-epoxide and 3-hydroxy-3-methylindolenine) may be formed from the ring oxidation of 3MI, as reported in earlier studies by our laboratory (Skordos et al, 1998a,b
). Recombinant CYP2F1 only produced the dehydrogenation product of 3MI (Lanza and Yost, 2001
), which covalently bound to cellular macromolecules (Thornton-Manning et al, 1991
). The dehydrogenated metabolite, 3-methyleneindolenine, was the only 3MI product detected from human HepG2 cells infected with vaccinia virus containing CYP2F1 cDNA (Thornton-Manning et al, 1991
). Production of the dehydrogenated metabolite by various P450 enzymes in this cellular model was precisely correlated to the relative levels of covalent binding to cellular proteins. Therefore, the current result with B-CMV2F1 cells correlated very well with previous studies from our laboratory that CYP2F1 efficiently produces 3-methyleneindolenine, which covalently binds to critical cellular proteins or DNA (Regal et al., 2001
) and results in cell death. It was apparent from the current studies that human P450 enzymes participate in the bioactivation of 3MI to reactive intermediates that initiate apoptosis in human lung cell lines. Since the BEAS-2B cell line was derived from normal human bronchial epithelial cells, these results strongly suggest that human lung epithelial cells that express higher levels of this enzyme may be susceptible to damage by 3MI, which may induce DNA fragmentation and apoptosis.
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ACKNOWLEDGMENTS |
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NOTES |
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2 Present address: Department of Clinical Pharmacology and Discovery Medicine, GlaxoSmithKline, Research Triangle Park, NC 27709.
3 Present address: Department of Pharmaceutics, College of Pharmacy, Rutgers University, The State University of New Jersey, Piscataway, NJ 08854-8020.
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