Polycyclic aromatic hydrocarbons induce both apoptotic and anti-apoptotic signals in Hepa1c1c7 cells

Anita Solhaug, Magne Refsnes, Marit Låg, Per E. Schwarze, Trine Husøy and Jørn A. Holme1

Division of Environmental Medicine, Norwegian Institute of Public Health, PO Box 4404 Nydalen, N-0403 Oslo, Norway

1 To whom correspondence should be addressed. Tel: +47 22 04 22 47; Fax: +47 22 04 26 86; Email: jorn.holme{at}fhi.no


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this study we show that benzo[a]pyrene (B[a]P) and the cyclopenta polycyclic aromatic hydrocarbons (CP-PAH) cyclopenta[c,d]pyrene (CPP), benz[j]aceanthrylene (B[j]A) and benz[l]aceanthrylene (B[l]A) induce apoptosis in Hepa1c1c7 cells, as measured by fluorescence microscopy and flow cytometry. The compounds induced formation of the active form of caspase-3, cleavage of its intracellular substrate, poly(ADP-ribose)polymerase (PARP), and DNA fragmentation. B[j]A was found to be the most potent in inducing apoptosis, followed by B[a]P, CPP and B[l]A. All compounds increased expression of CYP1A1 with relative potencies B[j]A > B[a]P >> CPP > B[l]A, corresponding well with their relative apoptotic responses. {alpha}-Naphthoflavone ({alpha}NF), an inhibitor of CYP1A1, reduced the induced apoptosis. B[a]P and CP-PAH exposure also resulted in an accumulation of the tumour suppressor protein p53. No changes were observed in the protein levels of Bax and Bcl-2, whereas the anti-apoptotic Bcl-xl protein was down-regulated, as judged by western blot analysis. Fluorescence microscopic analysis revealed a translocation of p53 to the nucleus and of Bax to the mitochondria. Furthermore, caspase-8 was activated and Bid cleaved. Interestingly, the levels of anti-apoptotic phospho-Bad (Ser155 and Ser112) had a biphasic increase after B[a]P or CPP treatment. Whereas {alpha}NF markedly reduced the activation of B[a]P to reactive metabolites, as measured by covalent binding to macromolecules, it did not inhibit the up-regulation of phospho-Bad. Neither of the compounds triggered apoptosis in primary cultures of rat lung cells (Clara cells, type 2 cells and lung alveolar macrophages), possibly due to a lack of CYP1A1 induction. In conclusion, B[a]P and the CP-PAH induced apoptotic as well as anti-apoptotic signals in Hepa1c1c7 cells.

Abbreviations: AhR, aryl hydrocarbon receptor; B[j]A, benz[j]aceanthrylene; B[l]A, benz[l]aceanthrylene; B[a]P, benzo[a]pyrene; BPDE, benzo[a]pyrene 7,8-diol-9,10-epoxide; CPP, cyclopenta[c,d]pyrene; CP-PAH, cyclopenta polycyclic aromatic hydrocarbons; DMSO, dimethyl sulphoxide; EGF, epidermal growth factor; FBS, foetal bovine serum; 3-MC, 3-methylcholanthrene; {alpha}NF, {alpha}-naphthoflavone; PAH, polycyclic aromatic hydrocarbons; PARP, poly(ADP-ribose) polymerase; PBS, phosphate-buffered saline; PI, propidium iodide; PMSF, phenylmethylsulfonyl fluoride


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Polycyclic aromatic hydrocarbons (PAH) are major pollutants in the environment formed during incomplete combustion of organic materials such as gasoline, diesel fuel, coal and oil. The substances are therefore found in heavily polluted air, water, soil and smoked food (1,2). Their exposure and metabolism to DNA-reactive metabolites in the body are considered to contribute to the aetiology of human lung cancer (3).

Cyclopenta polycyclic aromatic hydrocarbons (CP-PAH), a group of environmental contaminants structurally related to PAH, have been detected in gasoline and diesel exhaust, carbon black, coal combustion emissions and cigarette smoke (46). CP-PAH contain an ethylene fragment fused to an unsubstituted PAH, forming an unsaturated 5-membered ring. Theoretical and experimental studies have indicated that the cyclopenta ring contributes to the genotoxic activities of CP-PAH and that some of these compounds might be even more reactive than the well known PAH, benzo[a]pyrene (B[a]P) (7,8). The cyclopenta ring serves as a major site for metabolism and metabolic activation by CYP enzymes (8,9). The reactive metabolites, epoxides, form covalent adducts with macromolecules and mediate the toxic effects and act as initiators of carcinogenesis. Cyclopenta[c,d]pyrene (CPP) (Figure 1) has been detected in diesel and automobile exhaust at levels 8–15 times higher than B[a]P (10). Two other CP-PAH, benz[j]aceanthrylene (B[j]A) and benz[l]aceanthrylene (B[l]A) (Figure 1), have been shown to initiate skin tumours in mice with approximately the same activity as B[a]P (11).



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Fig. 1. Chemical structures of the CP-PAH compounds and B[a]P.

 
Most of the biological effects of B[a]P and other PAH are considered to be mediated via the aryl hydrocarbon receptor (AhR)-dependent gene expression (12). AhR is a ligand-activated member of a family of transcription factors that regulates multiple genes, including members of the cytochrome P450 family, such as CYP1A1. CYP1A1 metabolizes several PAH to reactive electrophilic metabolites that form DNA adducts and induce oxidative DNA damage, thereby causing mutations and cancer initiation (13).

In addition to carcinogenic and mutagenic effects, B[a]P and other PAH compounds have been shown to induce apoptosis in vitro in Hepa1c1c7 hepatoma cells (14), Daudi human B cells, (15), human ectocervical cells (16) and A20.1 murine B cells (17). In combination with carbon black, B[a]P has also been shown to induce apoptosis in macrophages (18). Recently, it has also been reported that B[a]P coated onto hematite particles induces apoptotic events in rat lungs (19). Apoptosis is an active and physiological mode of cell death characterized by condensation of chromatin in the nucleus and DNA fragmentation. Mitochondria, Bcl-2 protein, cytochrome c and caspases, a family of intracellular cysteine proteases, are considered to be essential components of the intracellular apoptotic signalling pathways. It is well known that PAH may cause an accumulation of the tumour suppressor protein p53 (20) and more recent studies have indicated that the resulting accumulation of p53 may be important for the induction of apoptosis (21). In the present study we extend previous findings with B[a]P and examine possible apoptotic effects of B[j]A, B[l]A and CPP in Hepa1c1c7 cells and primary rat lung cells.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals and cell culture media
B[j]A and B[l]A were synthesized and purified according to published methods (22) and kindly provided by Dr S.Nesnow (US Environmental Protection Agency, Research Triangle Park, NC). CPP was obtained from the National Cancer Institute Midwest Research Institute (Kansas City, MO). [3H]B[a]P and leupeptin was purchased from Amersham Biosciences (Buckinghamshire, UK). B[a]P,{alpha}-naphthoflavone ({alpha}NF), Triton X-100, protease (type 1, crude P-4630), DNase I (type III), HEPES, insulin, hydrocortisone, transferrin, epidermal growth factor (EGF), ponceau S, dimethyl sulphoxide (DMSO), propidium iodide (PI), Nonidet P-40, RNase A (R5000), phenylmethylsulfonyl fluoride (PMSF), Hoechst 33258, Hoechst 33342, aprotinin, proteinase K (KP0390), sodium selenite, ascorbic acid and glutathione were obtained from Sigma-Aldrich Chemical Co. (St Louis, MO). Pepstatin A was from Calbiochem (La Jolla, CA). SeaKem GTG agarose and Gel star nucleic acid gel stain were obtained from FMC Bioproducts (Rockland, ME) and Bio-Rad DC protein assay from Bio-Rad Laboratories Inc. (Hercules, CA). MEM{alpha} medium with L-glutamine, without ribonucleosides and deoxyribonucleosides, foetal bovine serum (FBS) and gentamycin were from Gibco BRL (Paisley, UK). William's E medium without glutamine and penicillin/streptomycin were from BioWhittaker (Walkersville, MD). Ampicillin and fungizone were obtained from Bristol Myers Squibb (Bromma, Sweden) and MitoTracker Red CMX Ros from Molecular Probes (Leiden, The Netherlands). All other chemicals were purchased from commercial sources and were of analytical grade.

Antibodies
Antibodies against Bad, phospho-Bad(Ser112), phospho-Bad(Ser136), phospho-Bad(Ser155), Bcl-xl, Bid, poly(ADP-ribose) polymerase (PARP) and caspase-3 were obtained from Cell Signaling (Beverly, MA). Antibodies against Bax, Bcl-2, CYP1A1 and pro-caspase-8 were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA); p53 (CM5) from Novocastra Laboratories Ltd (Newcastle, UK) and ß-actin from Sigma-Aldrich Chemical Co. As secondary antibody horseradish peroxidase-conjugated goat anti-rabbit (Sigma-Aldrich Chemical Co., St Louis, MO) and horseradish peroxidase-conjugated anti-goat or anti-mouse IgG (Dako, Glostrup, Denmark) were applied.

Animals
Male rats (WKY/NHsd), weighing 200–250 g, were purchased from Harlan, UK. The animals were given Ewos R3 standard pelleted laboratory chow from Astra Ewos AB (Södertälje, Sweden) and water ad libitum.

Isolation of lung cells
Rat lung cells were isolated as previously described (23). Briefly, alveolar macrophages were collected by lavage and the epithelial cells (type 2 and Clara cells) isolated by enzymatic digestion of the lung followed by centrifugal elutriation of the cells.

Cell cultures and treatments
The mouse hepatoma cell line Hepa1c1c7 (purchased from the European Collection of Cell Culture) was maintained in MEM{alpha} medium with L-glutamine without ribonucleosides and deoxyribonucleosides and supplemented with 10% heat-inactivated FBS and 0.1 mg/ml gentamycin in 5% CO2 at 37°C. The cells were routinely kept in logarithmic growth phase at 1.0–9.0 x 106 cells/75 cm2 flask by splitting the cells twice a week. The cells were seeded near confluence the day before treatment. The medium was replaced with fresh medium before treating the cells with inhibitors and PAH/CP-PAH. For treatment with inhibitors, cells were preincubated with the inhibitor for 1 h, followed by PAH/CP-PAH treatment for the time indicated. The inhibitors and PAH/CP-PAH were dissolved in DMSO (final concentration of DMSO in cell culture was <=0.5%). Appropriate controls containing the same amount of solvent were included in each experiment.

Type 2 cells and Clara cells were suspended in William's E medium supplemented with insulin (5 µg/ml), hydrocortisone (0.087 µg/ml), transferrin (5 µg/ml), EGF (10 ng/ml), sodium selenite (6.2 ng/ml), ascorbic acid (5 µg/ml), glutathione (5 µg/ml), ampicillin (100 µg/ml), streptomycin (100 µg/ml), fungizone (0.25 µg/ml), HEPES (15 mM) and 5% heat- inactivated FBS. Type 2 cell and Clara cell populations were cultured on tissue culture dishes as described (23). The cells were incubated at 37°C in a humidified atmosphere of 5% CO2 in air and cultured for 2 days. The 2 day cultures were exposed to B[a]P or CPP (30 µM) for 25–30 h in fresh medium with 5% heat-inactivated FBS.

Microscopic characterization of cells
Plasma membrane damage and changes in nuclear morphology associated with necrosis and apoptosis were determined after staining cells (~0.5 x 106 cells) with PI (10 µg/ml) and Hoechst 33342 (5 µg/ml) for 30 min. Smears made from Hepa1c1c7 or rat lung cells suspended in FBS were air dried quickly. Cell morphology was evaluated using a Nikon Eclipse E 400 fluorescence microscope. Cells with distinct condensed nuclei, segregated nuclei and apoptotic bodies were counted as apoptotic and determined as a fraction of the total number of cells. Non-apoptotic cells, excluding PI, were categorized as viable cells. PI-stained cells with a round morphology and homogeneously stained nucleus were termed necrotic. A minimum of 300 cells was counted.

Flow cytometry
The percentage of apoptotic cells and cells in different phases of the cell cycle were determined by flow cytometry. After treatment, the cells were prepared for flow cytometry with Triton X-100 (0.1%) and Hoechst 33258 (1.0 µg/ml) for staining of cellular DNA. The histograms were recorded on a Skatron Argus 100 flow cytometer and analysed using the Multiplus Program (Phoenix Flow Systems, San Diego, CA). The different cell phases, as well as apoptotic cells/bodies and secondary necrotic cells, were distinguished on the basis of their DNA content (Hoechst fluorescence; channel number) and cell size (forward light scatter; channel number) (24,25). Apoptotic index was determined as the percentage of signals between the G1 peak and the channel positioned at 20% of the G1 peak.

DNA fragmentation assay
DNA fragmentation was assayed according to the method of Gorczyca et al. (24), with minor modifications (25). The results from one representative experiment are shown.

Cell lysis and western blotting
Total cellular protein was lysed in 20 mM Tris buffer, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerol phosphate, 1 mM Na3VO4, 1 mM NaF, 10 µg/ml leupeptin, 1 mM PMSF, 10 µg/ml aprotinin and 10 µg/ml pepstatin A, sonicated and clarified by centrifugation. Protein concentration was measured using a Bio-Rad DC protein assay kit. The samples were adjusted to an equal protein concentration with lysis buffer and diluted with 5x SDS sample buffer containing ß-mercaptoethanol (at a final concentration of 5%) and bromophenol blue and then boiled for 5 min. A sample of 12.5 µg protein in each well was subjected to 12% SDS–PAGE. The proteins were electrotransferred to nitrocellulose membranes and stained with ponceau S. Blots with equal protein loading were probed with a particular primary antibody in 1% fat-free dry milk or 5% bovine serum albumin, according to the recommendations of the manufacturer. The incubations with primary antibodies were overnight at 4°C or 2 h at room temperature. The blots were then incubated with horseradish peroxidase-conjugated antibody for 1 h at room temperature. The western blots were developed using the ECL chemiluminescence system according to the manufacturer's instructions (Amersham Pharmacia, Little Chalfont, UK). The results from one representative experiment are shown.

Covalent binding of [3H]B[a]P to macromolecules
The amount of B[a]P covalently bound to macromolecules was determined as previously described (26). In short, Hepa 1c1c7 cells were incubated with 5 µM [3H]B[a]P (~16 000 c.p.m./nmol) for various time intervals. The reactions were stopped by placing the dishes on ice. The cells were scraped off with a rubber policeman and protein precipitated with trichloroacetic acid. The precipitates were washed sequentially with trichloroacetic acid, methanol and ethanol:ether (1:1). The amount of radioactivity covalently bound to macromolecules was determined by scintillation counting. Protein concentration was measured using a Bio-Rad DC protein assay kit.

Immunofluorescence
The cells were fixed in cold absolute ethanol for 10 min and permeabilized in phosphate-buffered saline (PBS) with 0.75% Triton X-100 for 30 min. Avidin–biotin blocking kit (Vector Laboratories, Burlingame, CA) was used to quench the endogenous biotin–avidin. After 1 h in 5% dried milk, the cells were incubated for 30 min with primary rabbit antibody. The cells were then incubated with biotin-conjugated secondary antibody for 10 min followed by fluorescein–avidin DCS for 5 min. Between each step, the cells were washed in PBS. The cells were finally mounted in Vectashield mounting medium (Vector Laboratories). Mitochondria were labelled with mitotracker red (fluorescent stain) according to the producer's prescription.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Induction of apoptosis by B[a]P and CP-PAH
B[a]P and the CP-PAH compounds B[j]A, B[l]A and CPP all induced cell death in Hepa1c1c7 cells, measured by fluorescence microscopy after staining with Hoechst 33342 and PI (Figures 2 and 3). Treatment with B[a]P or CP-PAH for 20 h induced concentration-dependent apoptosis (Figure 3B). B[j]A had the greatest response at 3 µM, whereas higher concentrations produced only minor increases. In contrast, exposure to increased concentrations of both B[a]P and CPP caused a more linear increase in the number of apoptotic cells, with B[a]P more potent than CPP. B[l]A exposure resulted in only a slight induction of apoptosis at 20 h. Some of the apoptotic cells had taken up PI, indicating secondary development of plasma membrane damage (Figure 2). The relative effects on necrosis were similar to those observed on apoptosis (Figure 3C).



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Fig. 2. Effects of B[a]P and the CP-PAH compounds B[j]A, B[l]A and CPP on cell morphology. Hepa1c1c7 cells were incubated without or with B[a]P, B[j]A, B[l]A or CPP at 30 µM for 20 h, stained with PI and Hoechst 33342 and analysed by fluorescence microscopy. Arrows indicate apoptotic cells, arrowheads indicate necrotic cells and thin arrows indicate apoptotic/necrotic cells. Original magnification 1000x.

 


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Fig. 3. Concentration-dependent increase in B[a]P-, B[j]A-, B[l]A- or CPP-induced apoptosis and necrosis as judged by fluorescence microscopy. Hepa1c1c7 cells were exposed to the test substance for 20 h and analysed by fluorescence microscopy for (A) living (viable), (B) apoptotic and (C) necrotic cells. The data represent mean ± SE of three different experiments.

 
Flow cytometry analysis of Hepa1c1c7 cells exposed to B[a]P or CP-PAH (30 µM) for 20 h revealed apoptotic cells/bodies with lower DNA fluorescence signals than G1 cells (Figure 4). The flow cytometry and microscopic analyses results correspond well, as illustrated by comparing Figures 3B and 5A. Further, flow cytometry analyses were used in the following experiments (Figures 5 and 8), and confirmed by microscopic analysis (data not shown). Cells with normal DNA content within a DNA histogram were also analysed with respect to cell cycle. As shown in Figure 4, both B[l]A and CPP caused marked accumulation of cells in S phase, whereas only a minor accumulation could be seen for B[a]P and B[j]A at 30 µM. However, at lower concentrations of B[a]P and B[j]A similar accumulations in S phase and corresponding decreases, mainly in G1 cells, were seen (data not shown). An increase in the number of apoptotic cells was observed after 8–45 h exposure to B[a]P or CP-PAH (30 µM) (Figure 5B). In contrast, B[a]P and CP-PAH did not induce apoptosis in primary lung cells (alveolar macrophages, type 2 cells and Clara cells), as judged by microscopic analyses and flow cytometry (data not shown).



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Fig. 4. Effects of B[a]P and CP-PAH compounds on cell cycle parameters as measured by flow cytometry. Hepa1c1c7 cells were exposed to test substance (30 µM) for 20 h and analysed by flow cytometry. Computer drawn two-parameter histograms (cell size versus DNA content; channel number) representing light scatter versus Hoechst 33258 fluorescence are shown. In general, apoptotic cells (Ap) have a smaller cell size and less DNA than G1 cells, while necrotic cells are often of increased cell size.

 


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Fig. 5. Concentration- and time-dependent increase in B[a]P- and CP-PAH-induced apoptosis. (A) Hepa1c1c7 cells were incubated for 20 h with different PAH/CP-PAH concentrations and analysed by flow cytometry. The data represent mean ± SE of three different experiments. (B) Hepa1c1c7 cells were treated with 30 µM B[a]P or CP-PAH for 8–45 h and analysed by flow cytometry. The data are representative of two independent experiments and are expressed as mean ± SE of three parallel incubations.

 


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Fig. 8. Effect of {alpha}NF on B[a]P/CPP-induced apoptosis and the formation of reactive B[a]P metabolites. (A) Hepa1c1c7 cells were incubated with B[a]P (20 µM) or CPP (30 µM) for 25 h in the presence or absence of {alpha}NF (10, 25 or 50 µM) and analysed for apoptotic cells by flow cytometry. (B) Hepa1c1c7 cells were incubated with [3H]B[a]P (5 µM) in the presence or absence of {alpha}NF (25 µM) and analysed for the amount of [3H]B[a]P covalently bound to macromolecules. The data represent mean ± SE of three different experiments (A) or three different incubations from one representative experiment of three (B).

 
Cleaving of caspase-3 and PARP and DNA fragmentation
Caspase-3 is a central effector of apoptosis, activating the characteristic DNA endonucleases responsible for fragmentation of DNA (27). Exposure to B[a]P, B[j]A, CPP (Figure 6) or B[l]A (data not shown) resulted in the appearance of the pro-caspase-3 cleavage product (active caspase-3). Processing was first observed after a 20 h exposure period. Cleavage of PARP, a substrate for caspase-3 and caspase-7, is commonly used as an indicator of caspase-3 and caspase-7 activity. The 116 kDa proform of PARP was cleaved to give a 89 kDa fragment in cells treated with B[a]P, B[j]A, CPP (Figure 6A) or B[l]A (data not shown). Figure 6B illustrates that B[a]P and the CP-PAH compounds B[j]A, B[l]A and CPP all caused fragmentation of DNA in Hepa1c1c7 cells, as analysed by DNA gel electrophoresis.



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Fig. 6. B[a]P and CP-PAH induce cleavage of caspase-3 and PARP and DNA fragmentation in Hepa1c1c7 cells. (A) Hepa1c1c7 cells were incubated without or with 30 µM B[a]P, B[j]A or CPP for 8, 20 and 30 h. The cell lysates were subjected to SDS–PAGE, followed by western blotting. The caspase-3 blot was also reprobed with an anti-ß-actin antibody as a control for the amount of protein added in each lane. (B) Agarose gel electrophoresis of DNA from cells incubated with 30 µM B[a]P or CP-PAH for 25 h.

 
CYP1A1 induction and the importance of reactive metabolites
To determine whether the CP-PAH can bind to the AhR and increase the expression of CYP1A1, Hepa1c1c7 cells were exposed to different concentrations of B[a]P or CP-PAH for 25 h and protein extracts analysed by western blotting. As shown in Figure 7A, B[a]P and the CP-PAH compounds elicited concentration-dependent expression of CYP1A1. B[a]P and B[j]A induced a marked expression of CYP1A1 at 0.03 µM, whereas CPP and B[l]A first showed increased CYP1A1 expression at 0.1 and 1 µM, respectively. Expression of CYP1A1 was also investigated in primary lung cells. The different cell types were exposed to B[a]P or CPP for 25 h and protein extracts were analysed by western blotting. Untreated Clara cells have a higher expression of CYP1A1 compared with the other lung cells, but neither B[a]P nor CPP induced expression of CYP1A1 in primary lung cells (Figure 7B). {alpha}NF, an inhibitor of CYP1A1 activity (28), markedly reduced B[a]P- and CPP-induced apoptosis in Hepa1c1c7 cells, measured by flow cytometry (Figure 8A). Analysis of the metabolism of B[a]P in Hepa1c1c7 cells showed that {alpha}NF reduced removal of the parent compound, formation of the major metabolite 3-OH-B[a]P (Steinar Øvrebø et al., unpublished data) and formation of reactive metabolites, as judged by covalent binding of radiolabelled B[a]P to macromolecules (Figure 8B).



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Fig. 7. Expression of CYP1A1 following B[a]P or CP-PAH exposure. (A) Hepa1c1c7 cells were incubated without or with B[a]P, B[j]A, B[l]A or CPP at different concentrations for 25 h. The cell lysates were subjected to SDS–PAGE, followed by western blotting. (B) Macrophages, Clara cells and type 2 cells were incubated with B[a]P or CPP (30 µM) for 25 h. The cell lysates were subjected to SDS–PAGE, followed by western blotting.

 
Accumulation of the p53 gene product is considered to be an important component of the cellular response to DNA damage. Marked increases in p53 protein levels were observed after 20 h exposure to B[a]P, B[j]A and CPP, as analysed by western blotting (Figure 9A). The effect of B[l]A was less pronounced and a clear increase was only observed after 30 h exposure (data not shown). The increased levels of p53 following exposure to B[a]P and CPP were inhibited by the addition of {alpha}NF (Figure 9B). In contrast, no accumulation of p53 could be seen in the primary lung cells (Figure 9C).



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Fig. 9. Expression of p53 following B[a]P or CP-PAH exposure. (A) Hepa1c1c7 cells were incubated with B[a]P, B[j]A or CPP (30 µM) for 8, 20 and 30 h. The cell lysates were subjected to SDS–PAGE, followed by western blotting. (B) Hepa1c1c7 cells were incubated with B[a]P (20 µM) or CPP (30 µM) for 25 h in the presence or absence of the CYP1A1 inhibitor {alpha}NF (25 µM) and analysed by western blotting. (C) Macrophages, Clara cells and type 2 cells were incubated with B[a]P or CPP (30 µM) for 25 h. The cell lysates were subjected to SDS–PAGE, followed by western blotting. Hepa1c1c7 cells exposed to B[a]P (30 µM; 20 h) were used as a positive control (P.C.).

 
Expression of Bcl-2 family proteins
The Bcl-2 family proteins are important regulators of apoptotic pathways (29) and p53 has been suggested to cause apoptosis through changes in the level or localization of Bax, a member of the Bcl-2 family. To elucidate whether the levels of Bax or other proteins in this family were changed in B[a]P- or CP-PAH-induced apoptosis, we examined the expression of several Bcl-2-proteins by western blot analysis. As shown in Figure 10A, exposure to B[a]P, B[j]A or CPP (B[l]A not done) led to a down-regulation of Bcl-xl, which is anti- apoptotic, while no marked effects on the cellular levels of Bcl-2 (anti-apoptotic) or Bax (pro-apoptotic) could be seen. Surprisingly, the level of the pro-apoptotic Bad was also decreased, whereas phospho-Bad(Ser155) and phospho-Bad (Ser112), which are both anti-apoptotic, were increased after B[a]P and CP-PAH treatment. In order to investigate the role of reactive metabolites in B[a]P/CPP-induced up-regulation of phospho-Bad, we added {alpha}NF. As seen in Figure 10B, the inhibitor had no effect on the induced levels of these proteins. A more detailed time analysis revealed that phospho-Bad was increased after 30 min and 2 h exposure to B[a]P, and at 4 h the level had decreased back to control levels. After 8 h another increase in the level of phosho-Bad protein was observed (Figure 10C, Ser 112; data not shown, Ser 155).



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Fig. 10. Expression of different Bcl-2 proteins following B[a]P or CP-PAH exposure. (A) Hepa1c1c7 cells were incubated without or with B[a]P, B[j]A or CPP (30 µM) for 8, 20 and 30 h. The cell lysates were subjected to SDS–PAGE and analysed for expression of different Bcl-2 proteins by western blotting. (B) Hepa1c1c7 cells were incubated with B[a]P (20 µM) or CPP (30 µM) for 25 h in the presence or absence of {alpha}NF (25 µM) and analysed for the expression of phospho-Bad (p-Bad) by western blotting. (C) The time course of the increase in p-Bad following exposure to B[a]P (20 µM) or CPP (30 µM) as analysed by western blotting.

 
Immunocytochemistry
A translocation of p53 to the nucleus 15 h after exposure of Hepa1c1c7 cells to B[a]P was visualized by fluorescence microscopic analysis (Figure 11A). Translocation of p53 to the nucleus was inhibited by {alpha}NF.



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Fig. 11. Immunofluoresence of p53 and bax and subcellular distribution. Hepa1c1c7 cells were incubated without or with B[a]P (20 µM) for 15 h. Cells were immunostained for (A) p53 (green) and (B) bax (green) and mitochondria (red) were labelled with mitotracker red. Fluorescence microscopy showed translocalization of p53 into the nucleus, and merged pictures of bax and mitotracker red indicated translocalization of bax to the mitochondria (co-localization, yellow).

 
Western blots of total cell lysates revealed no changes in the level of Bax after exposure to B[a]P (Figure 10A). However, B[a]P-treated cells exhibited a punctate staining, and Bax co-localized extensively with a specific mitochondrial marker (mitotracker red), indicating translocation of Bax to the mitochondria (Figure 11B).

Possible involvement of the death receptor pathway in B[a]P/CP-PAH-induced apoptosis
To examine the possible involvement of the death receptor pathway in B[a]P/CP-PAH-induced apoptosis we looked at the activation of caspase-8 (degradation of pro-caspase-8) and cleavage of the pro-apoptotic protein Bid. As can be seen from the western blot results presented in Figure 12, treatment with B[a]P or B[j]A leads to a marked down-regulation of pro-caspase-8 and cleavage of Bid, whereas no effects were seen after exposure to CPP (B[l]A was not tested).



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Fig. 12. Expression of pro-caspase-8 and Bid following B[a]P or CP-PAH exposure. Hepa1c1c7 cells were incubated without or with B[a]P, B[j]A or CPP (30 µM) for 8, 20 and 30 h. The cell lysates were subjected to SDS–PAGE, followed by western blotting.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Epidemiological and experimental studies have revealed that PAH may cause mutation and cancer. More recent studies have indicated that some of these compounds, including B[a]P, 3-methylcholanthrene (3-MC) and dimethylbenz[a]anthracene (14,21,30), also induce apoptosis. In the present study we confirm the findings with B[a]P (14), but also find that CP-PAH compounds cause apoptosis in the Hepa1c1c7 cells, as judged by microscopic analysis, flow cytometry, cleavage of caspase-3 and PARP and DNA fragmentation. The compounds also induced necrosis, as analysed by fluorescence microscopy. This might be due to an incomplete apoptotic process and a switch of induced apoptosis to necrosis, most probably due to inhibition of mitochondrial respiration or inactivation of caspases (31,32). As is often seen with cells in culture that enter apoptosis asynchronously, some of the apoptotic cells had secondary plasma membrane damage.

The caspases, a family of intracellular cysteine proteases, appear to play an important role in the initiation and execution of apoptosis induced by various stimuli (33). Caspases are normally present in the cell in an inactive proform and are proteolytically processed to the active form during apoptosis, usually by other caspases. Caspase-3 is one of the key executioners of apoptosis, being responsible for cleavage of a variety of proteins, including PARP (34), DNA-dependent protein kinase (35), DNA fragmentation factor (36) and the Bcl-2 proteins Bcl-2 and Bcl-xl (37,38). We show here that upon treatment with B[a]P or CP-PAH a marked cleavage of pro-caspase-3 was observed, in accordance with previous findings with B[a]P (39). Furthermore, the cleavage of PARP was consistent with activation of caspase-3 and time-dependent induction of apoptosis by these compounds.

B[j]A was somewhat more potent than B[a]P at inducing apoptosis, followed by CPP and B[l]A. Interestingly, the ability of these compounds to induce CYP1A1 was B[j]A > B[a]P >> CPP > B[l]A, corresponding quite well with their apoptotic effects. These differences in potencies are, however, not directly correlated with the reported genotoxic and mutagenic potentials. In most genotoxicity tests (using isolated microsomes), their DNA-damaging potentials are reported to be: B[l]A > B[j]A > B[a]P > CPP (8,9,40). Thus it appears that in a cellular system such as Hepa1c1c7 cells, the ability to bind to the AhR and induce CYP1A1 determines the overall toxicity of these compounds, rather than their affinity for and activation by CYP1A1. However, B[l]A is considered to be a fjord region PAH, which are usually activated by CYP1B1 rather than CYP1A1 (41). This may also be part of the explanation for the low toxicity observed with B[l]A. {alpha}NF markedly reduced the covalent binding of B[a]P and apoptosis by B[a]P and CPP, indicating that the formation of reactive metabolites was important for the initiation of apoptosis.

Accumulation and activation of p53 occur in response to various cellular stresses, including DNA damage, and may lead to the activation of several genes whose products trigger cell cycle arrest, DNA repair or apoptosis (42). Several PAH metabolites are highly genotoxic and elicit an accumulation of p53. B[a]P treatment of murine 3T3 cells (43), as well as treatment of normal human fibroblasts with the reactive benzo [a]pyrene 7,8-diol-9,10-epoxide (BPDE) (44), have been shown to result in DNA damage associated with elevated levels of nuclear p53. p53 has also been shown to be necessary for the efficient repair of BPDE adducts in human cells (45) and for induction of the murine multidrug resistance mdr1 gene by B[a]P or 3-MC in Hepa1c1c7 cells (46) and is suggested to be required in 3-MC-induced apoptosis in HepG2-cells (21). In accordance with this, we found that CP-PAH, in addition to B[a]P, induced an accumulation of p53 and that B[a]P increased translocation of p53 into the nucleus. These findings further support a role for p53 in B[a]P- and CP-PAH-induced apoptosis in Hepa1c1c7 cells.

The commitment of a cell to apoptosis requires the integration of numerous inputs involving multiple signal pathways. It is well known that Bcl-2 family members play a pivotal role in the regulation of cell death and survival. The Bcl-2-related proteins, which may have either anti-apoptotic or pro-apoptotic functions, are capable of physically interacting with each other through a complex network of homo- and heterodimers and are regulated by cytokines and other death/survival signals at different levels (47). In this study, B[a]P/CP-PAH-induced apoptosis of Hepa1c1c7 cells was accompanied by down-regulation of Bcl-xl. Following a variety of stimuli, Bcl-xl has been shown to block apoptosis (48), and even to be a stronger protector against apoptosis than Bcl-2 under certain circumstances (49). Low levels of Bcl-xl expression correlate with a greater tendency to undergo apoptosis (29) and, based on these results, it is possible that the down-regulation of Bcl-xl is associated with B[a]P/CP-PAH-induced apoptosis. No changes in the level of Bcl-2 or Bax protein were observed in response to B[a]P or CP-PAH. However, double immunofluorescence staining indicated that B[a]P induced translocation of Bax to the mitochondria (none of the CP-PAH were tested). Such changes, possibly due to phosphorylation (50) or other conformational changes (51), appear to be important for cytochrome c release and subsequent apoptosis.

Bad is a pro-apoptotic member of the Bcl-2 family of proteins that exerts its death-promoting activity by heterodimerizing with Bcl-xl or Bcl-2 (52). In response to stimulation by survival factors, Bad has been shown to be phosphorylated at Ser112 and Ser136, upon which it dissociates from Bcl-xl/Bcl-2 and becomes sequestered by 14-3-3 proteins in the cytosol (53). Recently it was suggested that phosphorylation of Bad at Ser155 by protein kinase A also contributes to cell survival (54,55). Only active non-phosphorylated Bad can form heterodimers with Bcl-xl or Bcl-2 at membrane sites to promote apoptosis/inhibit survival (53,55), which eventually results in the release of free Bax and induction of apoptosis (52,56). In our study we found that the level of non-phosphorylated Bad is reduced while phosphorylation of Bad at Ser155 or Ser112 is increased after exposure to B[a]P, B[j]A or CPP (no effects on Ser136 were seen). Thus, exposure to these compounds appears to give anti-apoptotic as well as pro-apoptotic signals. Interestingly, the parent compounds appear to be able to cause anti-apoptotic signals, judged by an early up-regulation (30 min after start of exposure) of phospho-Bad and since {alpha}NF had no effect on Bad phosphorylation. This suggestion, however, needs further verification using cell knockouts for the CYP1A1 gene. No increase in phospho-Bad(Ser115) or phospho- Bad(Ser112) was seen in Clara cells (data not shown).

Taking into consideration the B[a]P/CP-PAH-induced phosphorylation of Bad, it is interesting to note that B[a]P has been shown to increase the growth of primary cultures of human mammary epithelial cells and to increase intracellular Ca2+, which appears to play an important second messenger role in growth factor control of cell proliferation (57,58). B[a]P has also been shown to activate insulin-like growth factor signalling pathways under insulin-deficient conditions (58). An anti-apoptotic signal in combination with DNA damage may increase the possibility of the cells surviving and thus the probability of producing mutations. Altered growth factor receptor activation may also cause tumour promotion and progression by increasing cell proliferation or reducing cell death (60). Anti-apoptotic signals of the parent PAH compounds may therefore be of great importance when explaining their carcinogenic potential.

Following activation, p53 may be translocated to the nucleus and up-regulate pro-apoptotic proteins, such as Bax, or ‘death receptors’, such as CD95 and DR5. Pro-caspase-8 is believed to cluster within the death-inducing signalling complex in the plasma membrane, undergo trans-catalytic activation and process and activate effector caspases and Bid (27). Bid and cleaved Bid have also been shown to facilitate a conformational change in and mitochondrial association of Bax after apoptotic stimuli (61). In the present study we observed a reduction in pro-caspase–8 and cleavage of Bid following exposure to B[a]P, B[j]A and CPP (B[l]A not tested). Thus, these compounds may induce apoptosis through p53-mediated activation of death receptors, cleavage of Bid and translocation of Bax to the mitochondria.

When evaluating potential lung carcinogens, important information may be obtained by using specific cells from the target organ. The lung is a heterogeneous tissue consisting of at least 40 different cell types (62). Alveolar macrophages, playing an important role in lung defence and inflammatory responses, have been shown to exhibit monooxygenase activity and be able to activate some lung carcinogens (63). Clara cells and type 2 cells are known to have relatively high levels of CYP isoforms compared with other pulmonary cell types (64,65) and have been suggested to be target cells in lung carcinogenesis (63). Untreated type 2 cells have practically no CYP1A1 activity, whereas Clara cells have high activity, compared with type 2 cells of the rat lung (66). Thus, after induction with {alpha}NF increased CYP1A1 activity could be seen in both Clara and type 2 cells (66). Diesel exhaust particles, which contain different types of PAH and CP-PAH (10), have also been shown to induce expression of CYP1A1 in human airway epithelial cells (67). However, in the present study no induction of CYP1A1 could be seen in rat lung cells after treatment with B[a]P or CPP. As earlier reported (66), Clara cells were found to have higher CYP1A1 expression compared with macrophages and type 2 cells. In the present study neither accumulation of p53 nor apoptosis could be seen in rat lung cells after exposure to B[a]P or CPP. This may be due to the experimental conditions used, which are optimized for a high proliferation rate (23) and not with regard to giving an optimal level of CYP1A1 or AhR response. Accordingly, the metabolism of B[a]P and CP-PAH has been reported to be rather low in these rat lung cells (7). In humans relatively high levels of CYP1A1 (68) and DNA adducts (69,70) resulting from PAH exposure have been found, thus sufficient levels of reactive PAH and CP-PAH to cause apoptosis might be reached in human lung.

In conclusion, we have shown that B[a]P and the CP-PAH result in apoptotic as well as anti-apoptotic signals. The final result may be an increased probability for the cells to survive with DNA damage. Such properties may be of importance when explaining the carcinogenic effects of PAH and CP-PAH compounds.


    Acknowledgments
 
The technical assistance of Leni Johanne Ekeren and Heidi Skjønhaug Hopen is very much appreciated. Anita Solhaug is a research fellow supported by the Norwegian Research Council.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received June 5, 2003; revised December 5, 2003; accepted December 18, 2003.