DNA-damaging carcinogen 3-amino-1,4-dimethyl-5H-pyrido [4,3-b]indole (Trp-P-1) induces apoptosis via caspase-9 in primary cultured rat hepatocytes
Bunsyo Shiotani,
Yuji Nonaka,
Takashi Hashimoto,
Kaori Kihara,
Kazuki Kanazawa,
Gen-ichi Danno1, and
Hitoshi Ashida1,2,
Division of Life Science, Graduate School of Science and Technology and
1 Department of Biofunctional Chemistry, Faculty of Agriculture, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan
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Abstract
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The mechanism of cytotoxicity induced by the DNA-damaging carcinogen 3-amino-1,4-dimethyl-5H-pyrido[4,3-b] indole (Trp-P-1) was investigated in primary cultured rat hepatocytes. Cytotoxicity was caused by intact Trp-P-1 and not by metabolically activated derivatives prepared using a recombinant yeast strain AH22/pAMR2 expressing rat cytochrome P450 1A1, and not by metabolically activated derivatives. We also found internucleosomal DNA fragmentation 6 h after treatment with 30 µM Trp-P-1, indicating that the cytotoxicity was due to the induction of apoptosis. After treatment with Trp-P-1, c-Myc protein level increased in a time-dependent manner and p53 protein also increased transiently with a subsequent increase in Bax protein level. This apoptotic pathway required the activation of caspase-9 as an initiator after leakage of cytochrome c into the cytosol from mitochondria and the activation of caspase-3 and -7 as executioners, but not caspase-1, -6 or -8 as measured using the corresponding peptide inhibitors and substrates or western blotting. The activated caspases in turn cleaved poly(ADP-ribose) polymerase as an intracellular substrate. Furthermore, we detected NUC18-like endonuclease activity during apoptosis induced by Trp-P-1. These findings suggest that this apoptosis may have a role against heterocyclic amine-type carcinogens in normal cells.
Abbreviations: AMC, 7-amino-4-methyl-coumarin; CYP, cytochrome P 450; DMSO, dimethyl sulfoxide; DTT, dithiothreitol; ECD, electrochemical detector; GDC, glycodeoxycholic acid; HRP, horseradish peroxidase; PARP, poly(ADP-ribose) polymerase; PMSF, phenylmethylsulfonylfluoride; TNF-
, tumor necrosis factor-
; Trp-P-1, 3-amino-1,4-dimethyl-5H-pyrido [4,3-b]indole.
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Introduction
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The heterocyclic amine 3-amino-1,4-dimethyl-5H-pyrido [4,3-b]indole (Trp-P-1) is a potent carcinogen (1,2). The metabolic activation of heterocyclic amines, including Trp-P-1, to reactive metabolites is a critical step in the mechanism of its carcinogenic effects (24). The primary step of metabolic activation, N-hydroxylation, is catalyzed by members of the cytochrome P450 (CYP) 1A subfamily (58). The activated metabolite was shown to induce sister-chromatid exchange in a human lymphoblastoid cell line (9), and the ingestion of Trp-P-1 was reported to be associated with a high incidence of hepatocellular carcinoma in F-344 rats (10). On the other hand, intact Trp-P-1 without activation interacts with DNA (11) and induces chromosomal aberrations in CHO cells (12), suggesting that Trp-P-1 causes DNA damage in mammalian cells without metabolic activation to the N-hydroxyl form. DNA damage triggers not only mutation and carcinogenesis but also apoptosis. Indeed, 2-acetamidofluorane, a chemical carcinogen with a Trp-P-1-related structure, shows a cytotoxic effect on hepatocytes (13) and induces apoptosis in the liver of rats in vivo (14). We previously reported that Trp-P-1 was cytotoxic to primary cultured rat hepatocytes, and that this cytotoxicity was due to induction of apoptosis (15). However, it is not clear which form, intact Trp-P-1 or metabolite, induces apoptosis of hepatocytes.
Apoptosis is essential for development, maintenance of tissue homeostasis and elimination of harmful cells in metazoan organisms (16,17). Malfunctions of apoptosis have been implicated in many forms of human disease such as cancer, neurodegenerative disease, AIDS and ischemic stroke (18). Unnecessary or damaged cells, for example pre-cancer cells carrying DNA mutations or immune cells directed against self-antigens, are eliminated through apoptosis. Thus apoptosis is part of the host defense mechanism.
The apoptotic signal pathway is not simple and is dependent on both the stimuli and target cells or organs. Non-physiological signals such as X-rays, UV irradiation and anti-cancer drugs primarily induce DNA damage resulting in tumor suppressor gene and proto-oncogene expression, which regulate apoptosis. Changes in expression of these genes leads to mitochondrial cytochrome c release, which triggers the caspase cascade. On the other hand, most physiological apoptotic signals, such as tumor necrosis factor-
(TNF-
) and Fas ligand, are transmitted to the cell by mediating specific receptors on the cell membrane. These receptors can activate death caspases within seconds of ligand binding, causing an apoptotic demise of the cell within hours. The most advanced studies analyzed the apoptotic effects of anti-cancer drugs on carcinoma cells (19) and Fas-mediated apoptosis in immune cells involved in the immune defense system or tissue development (20). However, little is known about carcinogen-induced apoptosis in normal cells, especially in hepatocytes. Therefore, it is of interest to clarify the mechanism of apoptosis in primary cultured hepatocytes induced by carcinogens such as Trp-P-1, because the seemingly contradictory outcome of drug treatment, i.e. mutation or death, is a critical issue in understanding carcinogenesis.
In this study, we showed that intact Trp-P-1, and not a metabolically activated derivative, induces apoptosis. The apoptotic pathway induced by Trp-P-1 may be c-Myc- and p53-dependent accompanied with a subsequent increase in Bax protein. This pathway requires the activation of caspase-9 following the leakage of cytochrome c from mitochondria into the cytosol, and this leads to initiation of a caspase cascade involving activation of the downstream executioner caspase-3 and -7 and then the cleavage of poly(ADP-ribose) polymerase (PARP). Furthermore, we also observed the activation of NUC18-like endonuclease in apoptosis.
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Materials and methods
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Materials
Trp-P-1 (acetate form), actinomycin D and murine TNF-
were purchased from Wako Pure Chemical Industries (Osaka, Japan). For culture of hepatocytes, fetal bovine serum and William's medium E were purchased from Life Technologies Oriental (Tokyo, Japan). For extraction and analysis of DNA, DNase-free proteinase K and RNase A were obtained from Sigma Chemical (St Louis, MO, USA). Substrates and inhibitors for caspase were purchased from Peptide Institute (Osaka, Japan). For western blotting analysis, the following antibodies were used: mouse monoclonal antibodies to c-Myc and p53 and a monoclonal rabbit antibody to Bax (Oncogene Research Products, Cambridge, MA, USA); mouse monoclonal antibody to cytochrome c (Pharmingen, San Diego, CA, USA); goat polyclonal antibody to caspase-3 and rabbit polyclonal antibodies to PARP and caspase-9 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); secondary antibodies to rabbit IgG and mouse IgG (Amersham Pharmacia Biotech, Tokyo, Japan) and secondary antibody to goat IgG (Wako Pure Chemical Industries). All other chemicals were of the highest quality commercially available.
Animals and primary culture of hepatocytes
All animal treatments in this study conformed to the `Guidelines for the care and use of experimental animals', Rokkodai Campus, Kobe University. Six to 10-week-old male Wistar rats (Japan SLC, Shizuoka, Japan) were housed in suspended steel cages, provided with commercial chow and water ad libitum, and maintained under a controlled environment (temperature 25°C, humidity 60% and 12 h light/dark cycle). Parenchymal hepatocytes were isolated from rats by in situ perfusion of the liver with collagenase solution by the method of Tanaka et al. (21). Isolated hepatocytes were suspended at a concentration of 5x105 cells/ml in William's medium E with 1 nM insulin, 1 nM dexamethasone, 100 mg/l kanamycin, 10 KIU/ml aprotinin and 5% fetal bovine serum. The cells were seeded on plastic multi-well plates or dishes (Becton Dickinson, Franklin Lakes, NJ) pre-coated with collagen type I, then cultured under an atmosphere of 95% air5% CO2 at 37°C for 2 h. Hepatocytes were treated with various concentrations of Trp-P-1 in dimethyl sulfoxide (DMSO) for various times as indicated in each figure. Parallel dishes were treated with vehicle alone to obtain control samples (maximum concentration of DMSO in the medium was 0.1% v/v). In some samples, cells were treated with peptide inhibitors of caspases, Ac-YVAD-CHO or Ac-DEVD-CHO, 30 min before treatment with Trp-P-1. As a positive control, apoptosis was induced by TNF-
in the presence of actinomycin D (22) as follows. After 30 min pretreatment with 1 µM actinomycin D, 100 ng/ml TNF-
was added to the cells followed by incubation for a further 6 h. These cells were used for the following experiments.
Metabolic activation of Trp-P-1
The recombinant yeast strain Saccharomyces cerevisiae AH22/pAMR2 expressing both rat CYP 1A1 and yeast reductase was a gift from Prof. Ohkawa (23), and its microsomal fraction was obtained as previously described (24). As a control assay, rat liver microsomal fraction (control microsomes) was used. Microsomes (0.15 nmol as CYP 1A1) were mixed with 225 pmol Trp-P-1 in 1.5 ml William's medium E containing 1.5 mg ß-NADPH. After 1 h incubation at 37°C, the enzymic reaction was stopped by heating at 100°C for 3 min and kept on ice for 10 min. The mixture was centrifuged at 1000 g for 10 min and 1 ml of the resultant supernatant was diluted with 4 ml William's medium E (final Trp-P-1 concentration was adjusted to 30 µM) and was used for measurement of cell viability (15) and analysis of DNA fragmentation.
Detection of Trp-P-1 and its metabolites by HPLC
Detection of metabolites derived from Trp-P-1 during incubation with microsomes from AH 22/pAMR2 cells was performed according to the method of Minamoto and Kanazawa (8) with some modifications. After incubation of Trp-P-1 with microsomes, an aliquot of the mixture (0.5 ml) was immediately added to cold acetonitrile (0.5 ml) and centrifuged at 3000 r.p.m. for 3 min. The resulting supernatant (20 µl) was analyzed. The metabolites were detected using a Hitachi HPLC (L-7100) equipped with a UV detector (Hitachi L-7420) and electrochemical detector (ECD; IRICA
875) connected in series. An Inertsil column, ODS (i.d. 4.6x150 mm), was maintained at 35°C. The mobile phase consisted of 40 mM potassium phosphate monobasic (pH 4.6)/acetonitrile (80/20, v/v) containing 0.1 mM EDTA and 0.05% acetic acid and the flow rate was 1.0 ml/min. The UV detector was set at 267 nm,
max of Trp-P-1. ECD with a glassy carbon electrode was set at +600 mV versus Ag/AgCl, because +600 mV of ECD was the optimal voltage for detecting metabolites under conditions in which various interfering substances were present.
Mutagenicity test
Salmonella typhimurium TA98, which is highly susceptible to the mutagenic effects of all heterocyclic amines, was used, and the previously described method (25) was modified by pre-incubation with heterocyclic amines and microsomes. Briefly, the bacteria were cultured in nutrient broth overnight. Trp-P-1 in DMSO was diluted to 150 µM with 0.1 M sodium phosphate buffer (pH 7.4) just before use, and pre-incubated for 60 min with the microsomes in 3 ml phosphate buffer containing 0.5 mM ß-NADPH at 37°C. The enzymatic reaction was stopped by heating at 100°C for 20 s. After cooling, the solution was diluted to various concentrations of Trp-P-1 (0.37, 1.85, 3.7, 30 and 150 µM) with 0.1 M sodium phosphate buffer (pH 7.4), and the solutions were mixed with 0.1 ml bacterial suspension and incubated at 37°C for a further 10 min. Suspensions were added to 2 ml molten top agar and poured onto minimal glucose agar medium. After a 2 day culture, the number of His+-revertant colonies was counted. The increase in the number of revertants as a result of exposure to each mutagen was calculated by subtracting the number of spontaneous revertants (14 ± 4, n = 3) without mutagen from the total. These assays were performed independently in triplicate with three plates each time.
Analysis of DNA fragmentation
Hepatocytes (2x105 cells on 35-mm dishes) were treated with Trp-P-1 and then lysed with 200 µl TE buffer (10 mM TrisHCl, pH 7.4 and 10 mM EDTA) containing 0.5% SDS. The resultant lysate was incubated with 500 µg/ml RNase A at 50°C for 30 min and then with 500 µg/ml proteinase K at 50°C for 60 min. After addition of 0.5 M NaCl and 1 mM EGTA (final concentrations), DNA was precipitated in 50% isopropanol at 20°C overnight. DNA precipitate was obtained by centrifugation at 17 000 g for 20 min, washed with 70% ethanol and resuspended in TE buffer. DNA was resolved by 2% agarose gel electrophoresis in TBE buffer (89 mM TrisHCl, 89 mM borate and 2 mM EDTA). After electrophoresis, the gel was stained with ethidium bromide and visualized on a UV transilluminator.
Preparation of subcellular fractions and whole cell lysate
Cultured hepatocytes on 100-mm dishes were washed twice with ice-cold buffer E [20 mM HEPESKOH, pH 7.5, 250 mM sucrose, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol (DTT) and 0.1 mM phenylmethylsulfonylfluoride (PMSF)] and harvested with 100 µl of the same buffer. The cells were homogenized with 10 strokes of a Teflon homogenizer, and the homogenate was centrifuged at 750 g for 10 min at 4°C. The supernatant was centrifuged at 10 000 g for 15 min at 4°C, and the resultant pellet was resuspended in buffer E and referred to as the mitochondorial fraction. The supernatant of the 10 000 g centrifugation step was further centrifuged at 100 000 g for 1 h at 4°C, and the resultant supernatant was used as the cytosolic fraction. Pellets of the 750 g centrifugation step were washed twice with buffer A (10 mM TrisHCl, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.1% Nonident P-40, 5 µg/ml aprotinin, 20 µg/ml leupeptin, 0.5 mM DTT and 1 mM PMSF) containing 1% Triton X-100 and once with buffer A without Triton X-100 under the same centrifugation conditions. The pellet was then resuspended in 30 µl extraction buffer (10 mM TrisHCl, pH 7.9, 25% glycerol, 0.42 M NaCl, 5 µg/ml aprotinin, 20 µg/ml leupeptin, 0.5 mM DTT and 1 mM PMSF) and homogenized. The homogenates were rotated at 4°C for 1 h, and the nuclear protein fraction was obtained as the supernatant by centrifugation at 17 000 g at 4°C for 20 min. The subcellular fractions were frozen at 80°C and used for the experiments within 2 weeks.
Another series of cultures on 60-mm dishes were used to prepare whole cell lysates. After treatment with Trp-P-1, the medium was aspirated off, and hepatocytes were washed twice with ice-cold Tris-buffered saline (25 mM TrisHCl, pH 7.6 and 120 mM NaCl). Then the cells were lysed with 1.25 ml RIPA buffer (50 mM TrisHCl, pH 8.0, 120 mM NaCl, 0.5% Nonident P-40, 20 µg/ml leupeptin, 5 µg/ml pepstatin, 5 µg/ml aprotinin, 2 mM PMSF and 2 mM EDTA) at 4°C for 15 min. The lysate was spun at 10 000 g for 15 min at 4°C, and the supernatant was collected and stored at 80°C. The protein content of the subcellular fractions and the whole cell lysate were measured by Bradford's method (26).
Fluorimetric assay of caspase proteolytic activities
Proteolytic activities of caspases were measured spectrofluorometrically (27) using the following synthetic fluorogenic peptide substrates: Ac-YVAD-MCA for caspase-1-like protease; Ac-DEVD-MCA for caspase-3-like protease; Ac-IETD-MCA for caspase-8, -6 and granzyme B; Ac-DMQD-MCA for caspase-3; Ac-VEID-MCA for caspase-6 and Ac-DQTD-MCA for caspase-7. Aliquots of cell lysate (0.1 ml containing 100150 µg protein) in 0.7 ml incubation buffer (100 mM HEPES, pH 7.5, 10% sucrose, 0.1% Nonident P-40, 1 mM EDTA, 1 mM PMSF and 10 µM leupeptin) were incubated at 37°C. The reaction was started by adding corresponding substrate (50 µM at final), and the 7-amino-4-methyl-coumarin (AMC) liberated from the substrate was continuously monitored for 4 min with excitation and emission wavelengths of 380 and 460 nm, respectively. The protease activities were calculated from the slope of the recording, calibrated with standard concentrations of AMC and expressed as percentage of activity obtained from the 0 h cells.
Western blotting analysis
Proteins related to apoptosis were detected by western blotting analysis using suitable subcellular fractions. For detection of c-Myc and p53, aliquots of 30 µg nuclear protein were separated by 10% SDSPAGE. For analysis of Bax, aliquots of 150 µg protein from the whole cell lysate were separated by 15% SDSPAGE. To determine the leakage of cytochrome c from mitochondria into the cytosol, 10 and 30 µg protein in the respective fractions were separated by 15% SDSPAGE. For determination of the cleavage of PARP and caspase-3, aliquots of 50 µg nuclear protein were separated by 7.5 and 15% SDSPAGE, respectively. For analysis of cleavage of caspase-9, aliquots of 50 µg cytosolic protein were separated by 15% SDSPAGE. Each protein sample was solubilized in SDS-treatment buffer (62.5 mM TrisHCl, pH 6.8, 1% SDS, 11% glycerol, 0.004% bromophenol blue and 5% 2-mercaptoethanol) and boiled at 100°C for 5 min. After SDSPAGE, the proteins were transferred onto polyvinylidene difluoride membranes followed by blocking of non-specific binding sites with 5% non-fat dried milk for Bax or 10% FBS for others in TBST buffer (10 mM TrisHCl, pH 8.0, 150 mM NaCl and 0.06% Tween 20) at 4°C overnight. The membranes were washed with TBST buffer four times for 5 min each time and incubated with respective primary antibodies for 1 h. Washing with TBST buffer under the same conditions, the membranes were incubated with the secondary antibodies conjugated with horseradish peroxidase for 30 min. Specific immune complexes were visualized with the ECL Detection System (Amersham Pharmacia Biotech).
Nuclease activity assay
To identify the nuclease activated during apoptosis, we used an active gel assay as described previously (28) with modifications. Briefly, cultured hepatocytes on 100-mm dishes were washed twice with ice-cold buffer B (0.25 mM sucrose, 15 mM TrisHCl pH 7.4, 60 mM KCl, 15 mM NaCl, 2 mM EDTA, 0.5 mM EGTA, 5 mM DTT and 0.2 mM PMSF), harvested and homogenized with 250 µl buffer B containing 0.3% Triton X-100. The homogenate was centrifuged at 600 g for 5 min at 4°C. The pellet obtained was washed with buffer B containing Triton X-100 under the same centrifugation conditions, resuspended in TK buffer (10 mM TrisHCl and 60 mM KCl) and stored at 80°C. To make substrate-containing gels, calf thymus DNA sodium salt (30 µg/ml at final) was incorporated directly into the 15% separating gel matrix during polymerization. Protein samples were solubilized in treatment buffer (0.63 M TrisHCl, pH 8.5, 10% glycerol, 2.3% SDS and 5 mM DTT) without heating, loaded onto the gel and electrophoresed at 4°C. The gel was washed in TMC buffer (50 mM TrisHCl, pH 7.5, 5 mM MgCl2 and 5 mM CaCl2) containing 20% isopropanol to remove SDS, thereby allowing protein renaturation, and incubated with 50 ml TMC buffer for 3 h at 37°C to activate nucleases. After incubation, the gel was stained with 1 µg/ml ethidium bromide in TMC buffer for 5 min and checked under UV to localize the area and the extent of apparent DNA loss. The data were visualized and analyzed by the IS-1000 Digital Imaging System (Alpha Innotech, San Leandro, CA, USA).
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Results
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Trp-P-1 but not its metabolites induces apoptosis in primary cultured rat hepatocytes
Among the 11 heterocyclic amines examined, Trp-P-1 showed the strongest cytotoxicity to primary cultured rat hepatocytes (15). Exposure of hepatocytes to Trp-P-1 resulted in DNA fragmentation and nucleosomal ladder formation, characteristic of apoptotic cells in the late stage as described previously (15,29). To determine whether intact Trp-P-1 or the metabolically activated form induces apoptosis, we designed a system in which Trp-P-1 was activated to the metabolic form by microsomes of AH 22/pAMR2 cells which express rat CYP 1A1 (24). Intact Trp-P-1 incubated without microsomes of AH 22/pAMR2 cells and ß-NADPH showed strong cytotoxicity in a time-dependent manner but metabolically activated Trp-P-1 did not (Figure 1A
). Similarly, intact Trp-P-1 caused DNA fragmentation but metabolically activated Trp-P-1 did not (Figure 1B
). ß-NADPH did not affect cell viability or DNA fragmentation.

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Fig. 1. Trp-P-1, and not its metabolite, induced apoptosis. (A) Hepatocytes treated with Trp-P-1 ( ) or metabolite () (30 µM each) for the indicated times. Cell viability was measured by MTT test. (B) Effects of Trp-P-1 or metabolite on DNA fragmentation. Hepatocytes were treated with 0.1% DMSO (vehicle alone) or Trp-P-1 directly without incubation for 6 h (lanes 1 and 2). Trp-P-1 or DMSO was incubated with or without the microsomes (1.5 mg as protein and 0.15 nmol as CYP 1A1) in the absence (lanes 3, 4 and 7, 8) or presence of 1.5 mg ß-NADPH (lanes 5, 6 and 9, 10) for 6 h. DNA fragmentation was analyzed by 2% agarose gel electrophoresis.
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Detection with UV absorption of metabolites such as N-hydroxyl-Trp-P-1 in the microsomal mixture has been difficult because of interference by proteins, NADPH and Trp-P-1. On the other hand, ECD specifically responds to electroactive compounds such as hydroxyl compounds that have a potential of oxidation or reduction. Therefore, an electrochemical detector (ECD) was used to detect metabolites in this study. The production of metabolite from Trp-P-1 by microsomes from AH 22/pAMR2 cells was analyzed after incubation with 150 µM Trp-P-1. The amounts of metabolite were below the limits of detection for UV. In contrast, the ECD at +600 mV detected the metabolite at a retention time of 4.8 min in the 4 min incubation mixture (Figure 2A
). Using the HPLCECD method, the production of metabolite in the 150 µM Trp-P-1 incubation mixture was measured at various time points (Figure 2B
, left). The amount of metabolite increased within the 60 min incubation period. In mixtures of various concentrations of Trp-P-1 between 0.05 and 150 µM after 4 min incubation, the metabolite increased dose-dependently and reached a maximum level of 15 µM (Figure 2B
, right). The production of metabolite after 60 min incubation with 150 µM Trp-P-1 was >4-fold higher than that incubated with control microsomes (data not shown). In addition, for comparison of mutagenicity, these mixtures were analyzed using the Salmonella test. On incubation with Trp-P-1, the number of revertants induced by microsomes from AH 22/pAMR2 cells was ~10-fold that in cultures incubated with normal rat microsomes (Figure 3
). Moreover, after incubation with microsomes of AH 22/pAMR2 cells, Trp-P-1 concentration was decreased to ~16 µM in the culture medium, which was about half the concentration used to induce apoptosis in hepatocytes (Figure 2C
). Trp-P-1 concentration in cultures incubated with normal rat microsomes was almost the same as that found in cultures incubated without microsomes. These results suggest not only that Trp-P-1 induced apoptosis without being metabolized by CYP 1A1, because Trp-P-1 level was decreased and the level of its metabolites were increased by incubation with microsomes from AH 22/pAMR2 cells in this system, but also the metabolite might be the N-hydroxyl form. In the following experiments, we applied intact Trp-P-1 to hepatocytes and investigated the mechanism of apoptosis.

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Fig. 2. Increase in metabolite derived from Trp-P-1 detected with ECD and decrease in Trp-P-1 during incubation with microsomes of AH 22/pAMR2 cells. (A) Trp-P-1 (150 µM) was incubated with microsomes from AH 22/pAMR2 cells (expressing rat CYP 1A1 gene) at concentrations of 0.15 nmol CYP 1A1 for 60 min. Metabolite derived from Trp-P-1 was extracted and analyzed by HPLC with ECD (+600 mV) as described in Materials and methods. (B) Metabolite derived from Trp-P-1 was determined by HPLC with ECD under the same conditions as in (A). Trp-P-1 (150 µM) incubated for 0, 1, 2, 4, 10, 30 or 60 min (left panel), and various concentrations of Trp-P-1 (0.05, 0.15, 0.5, 1.5, 5, 15, 50 and 150 µM) added for 4 min (right panel). (C) Trp-P-1 (150 µM) incubated for 60 min with microsomes from AH 22/pAMR2 cells and NADPH or with microsomes from normal rat liver and NADPH. The remaining Trp-P-1 was subjected to HPLC analysis and determined with a UV-detector. The values show the concentration diluted for culture.
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Expression of c-Myc, p53 and Bax proteins in Trp-P-1-treated hepatocytes
The c-Myc gene, which is well known for its proliferative action, has been implicated in the regulation of apoptosis (30,31). The p53 gene and its protein have been extensively tested in various tumors, particularly in hepatocarcinomas (32). Although a large number of studies have indicated transient induction of p53 protein in apoptosis, the significance of this effect in primary cultured hepatocytes remains unclear. To clarify the role of c-Myc and p53 during apoptosis in hepatocytes, changes in the levels of both proteins were measured in nuclear protein extracts from Trp-P-1-treated cells by western blotting analysis (Figure 4
). An increase in c-Myc protein was observed after treatment with 30 µM Trp-P-1 at all time points examined. The levels of p53 protein increased time-dependently until 4 h and decreased 6 h after treatment. These results suggested that Trp-P-1-induced apoptosis might be dependent on both c-Myc and p53 proteins.

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Fig. 4. Western blotting analysis of the time dependent-changes in c-Myc, p53 and Bax. Nuclear protein extracts or whole cell extracts were prepared from cells treated with or without 30 µM Trp-P-1 for the indicated times. Equal amounts of nuclear proteins (30 µg each) were separated by 10% SDSPAGE (for c-Myc and p53) or whole cell lysate (150 µg each) by 15% SDSPAGE (for Bax) followed by transfer onto PVDF membranes. The membranes were probed with primary antibodies to corresponding proteins following horseradish peroxidase (HRP)-conjugated secondary antibodies. The blots were developed with an ECL Chemiluminescence Detection kit (Amersham Pharmacia Biotech). 6C, control cells after 6 h in culture.
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The bcl-2 proto-oncogene discovered in malignant B lymphomas has been shown to prolong cell survival by inhibiting apoptosis in various cell types in culture (33). In contrast to Bcl-2, overexpression of Bax protein promotes apoptosis (34). The levels of Bax protein in whole cell lysates increased time-dependently until 2 h and decreased thereafter (Figure 4
). Trp-P-1 caused up-regulation of c-Myc protein and transient up-regulation of p53 and Bax protein. Thus, Trp-P-1 may stimulate the nuclear transcription factors and be involved in commitment to cell death.
Release of cytochrome c from mitochondria into cytosol
To determine whether mitochondorial proteins participate in this apoptotic pathway, we analyzed the release of cytochrome c into the cytoplasm from mitochondria. After 2 h treatment of hepatocytes with Trp-P-1, cytochrome c in the mitochondrial fraction decreased as that in the cytosolic fraction increased (Figure 5A
). The release of cytochrome c into the cytosolic fraction was detected at 1 h and increased in a time-dependent manner until 4 h (Figure 5B
).

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Fig. 5. Western blotting analysis of the release of cytochrome c from mitochondria into cytosol in hepatocytes treated with Trp-P-1. (A) Mitochondrial and cytosolic fractions prepared from cells treated with or without 30 µM Trp-P-1 for 2 h or (B) at the indicated time points. The resultant mitochondrial and cytosolic proteins (10 and 30 µg, respectively) were separated by 15% SDSPAGE followed by transfer onto PVDF membranes. The membranes were probed with a primary antibody to denatured cytochrome c followed by incubation with HRP-conjugated secondary antibody. The blots were developed with an ECL Chemiluminescence Detection kit.
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Involvement of caspase in Trp-P-1-induced apoptosis
Caspases that are believed to be the downstream mediators of apoptosis have been sub-classified into initiators (e.g. caspase-8, -9) and executioners (e.g. caspase-3, -6, -7). An increase in the cleavage activity has been suggested to be the most likely explanation for the processing/activation of one or more caspases (35). Since caspase-9 and caspase-3 are the most likely candidates, we performed immunoblotting to determine whether these caspases are activated during Trp-P-1-induced apoptosis. The level of p10-subunit of caspase-9 increased time-dependently until 2 h, and then inactive procaspase-3 was cleaved to p20-subunit in a time-dependent manner after treatment with Trp-P-1 (Figure 6
).

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Fig. 6. Detection of active caspase-9 and caspase-3 in hepatocytes after induction of apoptosis by Trp-P-1. Cytosolic and nuclear protein extracts were prepared from cells treated with or without 30 µM Trp-P-1 at the indicated time points. Equal amounts of cytosolic (for caspase-9) and nuclear (for caspase-3) proteins (50 µg each) were separated by 15% SDSPAGE followed by transfer onto PVDF membranes. The membranes were probed with primary antibody to p10-subunit of caspase-9 or p20-subunit of caspase-3, and then treated with suitable secondary antibodies. The blots were detected with an ECL Chemiluminescence Detection kit. The arrows indicate p10-subunit of caspase-9, procaspase-3 and p20-subunit of caspase-3. 6C, control cells after 6 h in culture.
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Next, we measured the caspase activities with six fluorogenic substrates. The caspase-3-like protease and caspase-7 activities in Trp-P-1-treated cells gradually increased in a time-dependent manner and reached 7.9- and 8.2-fold higher than those in the 0 h cells as controls (Figure 7B and E
). The other protease activities, caspase-1-like, -3, -6 and -8, did not show any changes within the period examined (Figure 7A
, C, D and F). Caspase-3-like activity increased in a dose-dependent manner but caspase-1-like protease activity did not (Figure 8A
). In addition, Trp-P-1-induced DNA fragmentation was inhibited by Ac-DEVD-CHO, an inhibitor of caspase-3-like protease, in a dose-dependent manner, while Ac-YVAD-CHO, an inhibitor of caspase-1-like protease, showed no inhibitory effect (Figure 8B and C
). Similar to the DNA fragmentation induced by Trp-P-1, that induced by glycodeoxycholic acid (GDC), a bile salt that is also an inducer of apoptosis in hepatocytes (36), was inhibited by Ac-DEVD-CHO but not by Ac-YVAD-CHO. On the other hand, not only Ac-DEVD-CHO but also Ac-YVAD-CHO inhibited DNA fragmentation by TNF-
with actinomycin D (Figure 8B
). Taken together these results indicate that Trp-P-1 activates caspase-9 as an initiator and mainly caspase-3 and -7 as an executioner. The apoptotic pathway of Trp-P-1 is similar to that mediated by GDC but is distinct from the TNF-
-mediated pathway.

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Fig. 7. Activation of caspase-3-like proteases and caspase-7 during Trp-P-1-mediated apoptosis. Whole cell lysates were prepared from hepatocytes cultured with or without 30 µM Trp-P-1 for the indicated times. The activities of (A) caspase-1-like protease, (B) caspase-3-like protease, (C) caspase-3, (D) caspase-6, (E) caspase-7 and (F) caspase-8 in the lysates were determined using the corresponding peptidyl substrates Ac-YVAD-MCA, Ac-DEVD-MCA, Ac-DMQD-MCA, Ac-VEID-MCA, Ac-DQTD-MCA and Ac-IETD-MCA. The protease activities were calculated from the slope of the recording, calibrated with AMC as a standard. Data are expressed as percentages of values in 0 h cells.
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Cleavage of PARP as a cellular substrate for caspases
PARP, a DNA repair enzyme, is one of the best-examined targets of activated caspases. PARP is cleaved from a 115 kDa active form polypeptide into an inactive 85 kDa polypeptide in the cells undergoing apoptosis (37). Our results clearly showed that PARP was cleaved from 115 into 85 kDa, and furthermore was cleaved into species of 54 and 42 kDa in a time-dependent manner by Trp-P-1 (Figure 9
). These observations suggested that in the pathway mediated by Trp-P-1, activated caspases cleave intracellular substrates during culture.

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Fig. 9. Cleavage of PARP in hepatocytes treated with Trp-P-1. Nuclear protein extracts were prepared from cells treated with or without 30 µM Trp-P-1 at the indicated time points. Equal amounts of nuclear proteins (50 µg each) were separated by 7.5% SDSPAGE followed by transfer onto PVDF membranes. Membranes were probed with primary antibody to active form (115 kDa) and inactive fragments (85, 54 and 42 kDa) of PARP indicated by the arrows followed by incubation with HRP-conjugated secondary antibody. Blots were developed with an ECL Chemiluminescence Detection kit. 6C, control cells after 6 h in culture.
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Activated nuclease during apoptosis induced by Trp-P-1
Internucleosomal DNA fragmentation, one of the hallmarks of apoptosis, is catalyzed by nucleases. Recent reports suggest that many inducers activate specific nucleases. For example, in the rat thymus glucocorticoid activates NUC18 (38), and in HL-60 cells ionomycin activates DNase II (39). DNase
and Mg2+-dependent endonuclease activities were reported in hepatocytes (40). To elucidate what type of endonuclease is activated during apoptosis induced by Trp-P-1, we performed nuclease activity assays. The nuclear extracts from hepatocytes treated with 30 µM Trp-P-1 for 6 h demonstrated the loss of DNA substrate at a position corresponding to 18 kDa in a dose-dependent manner (Figure 10
). This result suggested that NUC18-like endonuclease is activated during apoptosis induced by Trp-P-1.

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Fig. 10. Detection of NUC18-like-endonuclease during apoptosis induced by Trp-P-1. Nuclear proteins were prepared from cells treated with or without 30 µM Trp-P-1 for 6 h. Nuclease activity analysis is described in detail in Materials and methods. Nuclease activity is shown as a black hole on a white DNA background, as indicated by the arrow.
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Discussion
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We confirmed that the DNA-damaging carcinogen Trp-P-1 induces apoptosis in primary cultured rat hepatocytes without being metabolically activated by CYP 1A1. At the initial stage of apoptosis, when hepatocytes were exposed to Trp-P-1,c-Myc and p53 genes were activated directly or indirectly, followed by activation of Bax gene expression. Bax protein might be translocated to mitochondria, leading to mitochondrial matrix swelling, outer membrane disruption (41,42) and the release of cytochrome c into the cytosol. Released cytochrome c might form a protein complex with procaspase-9 and convert it into an active protease. The recruited initiator caspase-9 activates the downstream caspase cascade including the executioners caspase-3 and -7. The activated executioner caspases cleave PARP and subsequently activate NUC18-like endonuclease, thereby setting in motion the events that lead to DNA fragmentation and cell death.
Heterocyclic amines are the typical carcinogen and are considered to be one of the major causes of human cancer. They are pyrolysates formed during the cooking process, and the average intake has been estimated to be 0.416 µg per day per person (43). The distribution analysis of Trp-P-1 in the mouse by autoradiography showed that Trp-P-1 was detected in many tissues including liver and with high levels in the bile and feces. In liver, Trp-P-1 was detectable about a week after administration, in contrast to other tissues (44). These studies suggest that Trp-P-1 is accumulated in the liver and excluded from the body, and the concentration in the liver is higher than those in other tissues and organs. Therefore, apoptosis induced by Trp-P-1 may occur partially in the liver, especially near the bile ducts.
Trp-P-1 is metabolized to the N-hydroxyl form by the CYP 1A subfamily, and this metabolite shows mutagenicity and carcinogenicity (45). Both Trp-P-1 and the N-hydroxyl form damage DNA (9,12). In the present study, cytotoxicity and DNA-ladder formation were observed following treatment with only intact Trp-P-1 (Figure 1
). By incubation with microsomes from AH22/pAMR2 cells expressing rat CYP 1A1, Trp-P-1 would be metabolized to the N-hydroxyl-derivative with mutagenic activity. As a consequence, the concentration of Trp-P-1 was decreased to non-cytotoxic levels. In fact, at low concentrations (<20 µM) Trp-P-1 did not show cytotoxicity in hepatocytes (15) and Trp-P-1 increased the placental type glutatione S-transferase level, which is well known as an acute marker for the promotion stage of carcinogenesis, in the normal rat hepatocyte cell line RL-34 cells (our unpublished observations). This suggested that at the low concentrations Trp-P-1 may be metabolized to the N-hydroxyl form by endogenous CYP 1A subfamily, but at high concentrations Trp-P-1 would severely damage DNA leading to apoptosis because it could not be metabolized. Together, these results indicate that cells undergo apoptosis to protect the body when the cells cannot successfully rid themselves of Trp-P-1, although Trp-P-1 is first metabolized to be discharged out of the cells by the system that abolishes xenobiotics mediated by the CYP 1A subfamily.
Our findings, i.e. the accumulation of c-Myc, p53 and Bax proteins by Trp-P-1 (Figure 4
), are consistent with the apoptotic pathway induced by DNA-damaging agents. Upon DNA damage, p53 protein accumulates rapidly through a transcriptional mechanism mediated by c-Myc that has been demonstrated to trans-activate the p53 promoter and induce expression of p53 (46), or a post-transcriptional mechanism by which p53 is phosphorylated at serine 15, which in turn induces a conformational change that prevents MDM2 from binding to p53 (47). Transcriptionally activated p53 induced expression of the pro-apoptotic molecule Bax. Bax protein, which appears to be localized primarily in the cytosol but upon induction of apoptosis rapidly undergoes translocation to the mitochondria, promotes release of cytochrome c from the mitochondria into the cytosol (41,42).
Following exposure of hepatocytes to Trp-P-1, the recruitment of caspase-9 as an initiator caspase was observed accompanied by cytochrome c release from mitochondria into the cytosol (Figures 5 and 6
). Once in the cytosol, cytochrome c interacted with other factors to form a caspase-9-activating complex (48). Activated caspase-9 by Trp-P-1 in turn activated downstream caspase-3, and -7 as executioners (Figures 7 and 8
), and the activation of caspases resulted in the cleavage of PARP as an intracellular substrate (Figure 9
). In the above caspase cascade Trp-P-1 treatment did not cause cleavage of the caspase-3 specific substrate Ac-DMQD-MCA, although Trp-P-1 clearly cleaved Ac-DEVD-MCA (Figure 7
). DMQD and DEVD substrates were deduced from the cleavage site of protein kinase C
(49) and that of PARP (37), respectively. These results suggest that even if caspase-3 is activated, not all of its target proteins are cleaved. In contrast to caspase-9, another initiator, caspase-8, is recruited to ligand-bound death receptors such as Fas and TNFR1. Our results demonstrate that Trp-P-1 does not activate caspase-8, although all the caspases tested, including initiator caspase-8, were fully activated during apoptosis induced by TNF-
in the presence of actinomycin D (data not shown). Moreover, the inhibitory actions of peptidyl inhibitors of caspases in Trp-P-1-treated cells were distinct from those in TNF-
- and actinomycin D-treated cells (Figure 8B
). These observations indicate that the Trp-P-1-mediated apoptotic pathway is different from that mediated by death receptors.
Nuclease activation plays an essential role in nucleosomal DNA fragmentation during apoptosis. It has been suggested that the caspase cascade is involved in the activation of nucleases. Our experimental results show that a NUC18-like endonuclease is activated during apoptosis induced by Trp-P-1 in hepatocytes (Figure 10
). Activation of this endonuclease is Mg2+/Ca2+-dependent and inhibited by Zn2+ (data not shown). NUC18 was first identified in glucocorticoid-treated rat thymocytes (38). However, no information is available about the distribution of this endonuclease in other organs. This is the first report of the existence of a NUC18-like endonuclease in rat hepatocytes, but further experiments are required for the detailed characterization of this endonuclease activated by Trp-P-1.
In the present study, we demonstrated that the DNA-damaging carcinogen intact Trp-P-1, but not its activated metabolites, induced apoptosis, and investigated the mechanism of apoptosis induced by Trp-P-1 in rat hepatocytes in primary culture. The signal transduction pathway of Trp-P-1 was mediated by caspase-9. These findings are physiologically significant as they indicate a protective mechanism against carcinogens in the living body mediated by a cell death program that is highly conserved during evolution, since our findings indicated that normal cells have the latent ability to avoid malignant alterations when exposed to mutagens or carcinogens.
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Notes
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2 To whom correspondence should be addressedEmail: ashida{at}kobe-u.ac.jp 
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Acknowledgments
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The author wishes to thank Prof. Ohkawa (Kobe, Japan) for providing the recombinant yeast strain. This work was supported by a Grant-in-Aid for Scientific Research(c) (No. 11660126) from the Ministry of Education, Science, Sports and Culture of Japan to H.A.
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Received May 5, 2000;
revised December 14, 2000;
accepted December 22, 2000.