Phototoxicity of benzo[a]pyrene by ultraviolet A irradiation: induction of apoptosis in Jurkat cells

Environmental Toxicology and Pharmacology 11 (2002) 101– 109 www.elsevier.com/locate/etap

Phototoxicity of benzo[a]pyrene by ultraviolet A irradiation: induction of apoptosis in Jurkat cells Yuko Ibuki *, Rensuke Goto Laboratory of Radiation Biology, Graduate School of Nutritional and En6ironmental Sciences, Uni6ersity of Shizuoka, 52 -1, Yada, Shizuoka-shi 422 -8526, Japan Received 20 August 2001; received in revised form 16 October 2001; accepted 17 October 2001

Abstract The toxicology of benzo[a]pyrene (BaP) has been mainly studied with regard to the carcinogenicity of its metabolites, but its phototoxicity is not well understood. Although some studies have indicated the lethal phototoxicity of BaP, there have been no reports regarding the pattern of cell death induced by this agent. In this study, we investigated the pattern and mechanism of cell death induced by coexposure to BaP plus ultraviolet A (UVA) in Jurkat cells. Coexposure to BaP plus UVA showed dose-dependent cytotoxicity. The pattern of cell death was apoptotic as determined by cell shrinkage, chromatin condensation, appearance of subdiploid apoptotic nuclei and translocation of phosphatidylserine to the outer membrane leaflet. Coexposure also strongly increased caspase-3/7 activity and slightly elevated those of caspase-8/6 and -9. The pan caspase inhibitor Z-VAD-CH2DCB partially inhibited the phototoxicity of BaP. Cytochrome c release was observed 6 h after coexposure, but not after 1 h. Furthermore, the phototoxicity was inhibited by NaN3 (quencher of singlet oxygen), but not by mannitol (quencher of hydroxy radicals). Chromatin condensation and translocation of phosphatidylserine were also inhibited by NaN3, suggesting that the induction of apoptosis by coexposure to BaP plus UVA was due to singlet oxygen production. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Phototoxicity; PAHs; Ultraviolet A; Apoptosis; Singlet oxygen

1. Introduction Benzo[a]pyrene (BaP) is one of the polycyclic aromatic hydrocarbons (PAHs), which are widely present in the environment. Traditionally, toxicological concern regarding BaP has focused on its metabolites such as

Abbre6iations: Ac-DEVD-MCA, acetyl-Asp-Glu-Val-Asp-a-(4methyl-coumaryl-7-amide); Ac-IETD-MCA, acetyl-Ile-Glu-Thr-Aspa-(4-methyl-coumaryl-7-amide); Ac-LEHD-MCA, acetyl-Leu-GluHis-Asp-a-(4-methyl-coumaryl-7-amide); BaP, benzo[a]pyrene; DTT, dithiothreitol; FBS, fetal bovine serum; FCM, flow cytometer; FDA, fluoresceine diacetate; Hoechst33342, bis-benzimide; PAHs, polycyclic aromatic hydrocarbons; PBS, phosphate-buffered saline; PDT, photodynamic therapy; PI, propidium iodide; PS, phosphatidylserine; ROS, reactive oxygen species; T-PBS, PBS containing 0.1% Tween 20; UVA, ultraviolet A; Z-VAD-CH2-DCB, carbobenzoxy-Val-AlaAsp-CH2-2,6-dichlorobenzolate. * Corresponding author. Tel.: +81-54-264-5795; fax: +81-54-2645799. E-mail address: [email protected] (Y. Ibuki).

(9 )-trans-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, because BaP becomes carcinogenic after metabolic activation (Weinstein et al., 1976; Sims et al., 1974; Albert et al., 1991). PAHs are generally not acutely toxic in the dark, whereas they show toxicity with exposure to ultraviolet (UV) in sunlight (Arfsten et al., 1996). Bowling et al. (1983) reported that coexposure to anthracene plus sunlight showed toxicity at concentrations of anthracene previously reported to have no effect, suggesting that this was due to photo-induced toxicity. Toxicological studies for defining the potential hazard of PAHs have been conducted in the absence of UV radiation. Therefore, comparative studies of the effects of PAHs in the presence and absence of UV irradiation are important. A number of studies have shown that toxicity was increased by coexposure to PAHs plus UV in bacteria, protozoa and cells of mammals and fish (Kagan and Kagan, 1986; Warshawsky et al., 1995; Kagan et al., 1989; Schirmer et al., 1998; Utesch et al., 1996).

1382-6689/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 2 - 6 6 8 9 ( 0 1 ) 0 0 1 1 0 - 7

102

Y. Ibuki, R. Goto / En6ironmental Toxicology and Pharmacology 11 (2002) 101–109

Schirmer et al. (1998) reported that BaP was found to be the most phototoxic as judged from the EC50 values among the 16 priority PAHs. Upon absorption of UV radiation, PAHs undergo photochemical reactions that involve the formation of singlet oxygen, free radicals and photomodified PAH products, which damage essential cellular components such as cell membranes or DNA, and induce cell death (Arfsten et al., 1996; Kagan et al., 1989, 1990). Two patterns of cell death have been defined, i.e. apoptosis and necrosis (Schwarzman and Cidlowski, 1993; Vermes and Haanen, 1994), which are distinguishable both morphologically and biochemically. Apoptosis is programmed cell death accompanied by cell shrinkage, nuclear condensation, endonuclease activation and DNA fragmentation. Necrosis is accidental cell death induced by lethal chemical, biological or physical events. There have been no studies of the pattern of cell death induced by coexposure to PAHs plus UV. In this study, we examined the pattern of cell death induced by coexposure to BaP plus UVA in Jurkat cells, a human lymphoma cell line. BaP showed definite photocytotoxicity, the death pattern of which was apoptotic as determined by the occurrence of cell shrinkage, chromatin condensation, appearance of subdiploid apoptotic nuclei, translocation of phosphatidylserine (PS) to the outer membrane leaflet, cytochrome c release and caspase activation. Furthermore, the induction of apoptotic cell death was attributed to singlet oxygen production.

2.2. Cells and culture conditions Jurkat cells were provided by RIKEN Cell Bank, Japan. They were cultured in RPMI1640 medium supplemented with 10% FBS and maintained at 37 °C in a humidified atmosphere containing 5% CO2 in air. For the experiments, logarithmic phase cells were used.

2.3. UVA irradiation A UV lamp (HP-30LM; Atto Co., Japan) which is characterized by a spectral output of 3% in UVB region (B 320 nm), 17% UVA2 region (320–340 nm) and 74% UVA1 region (340–400 nm) with an emission wavelength peak at 365 nm was used to irradiate the cells. The culture plates were placed on a table 2 cm below the UV lamp. During UV exposure, the fluence was simultaneously measured and integrated using a radiometer (ATV-3W; Atto Co., Japan) with a 365 nm detector placed at the same distance as the culture plates from the UV source. The UVA irradiance at the sample level was approximately 1.85 mW/cm2. The duration of UVA irradiation to obtain a fluence of 0.5 J/cm2 was typically 5 min.

2.4. Cytotoxicity assay Alamar Blue and FDA were used for detection of cytotoxicity. Alamar Blue can be used to monitor the activity of dehydrogenase in mitochondria of living cells, whereas FDA, a substrate of esterases, is hydrolyzed, charged, and then entrapped in living cells.

2. Materials and methods

2.1. Materials Alamar Blue™ and fluorescein diacetate (FDA) were purchased from Wako Pure Chemicals Ind., Ltd, Japan. Bis-benzimide (Hoechst33342) and propidium iodide (PI) were obtained from Sigma– Aldrich Co., USA. The caspase substrates acetyl-Asp-Glu-Val-Aspa-(4-methyl-coumaryl-7-amide) (Ac-DEVD-MCA), acetyl-Ile-Glu-Thr-Asp-a-(4-methyl-coumaryl-7-amide) (Ac-IETD-MCA) and acetyl-Leu-Glu-His-Asp-a-(4methyl-coumaryl-7-amide) (Ac-LEHD-MCA) were obtained from Peptide Inst. Inc., Japan. CarbobenzoxyVal-Ala-Asp-CH2-2,6-dichlorobenzolate (Z-VAD-CH2DCB) was obtained from Phoenix Pharmaceuticals, Inc. USA. Annexin V– FITC kit was purchased from Trevigen, Inc., USA. Anti-cytochrome c IgG, anti-actin IgG and peroxidase-conjugated anti-goat IgG were purchased from Santa Cruz Biotechnology, Inc., USA. RPMI1640 medium and fetal bovine serum (FBS) were purchased from Nissui Pharmaceutical Co., Japan, and JRH Biosciences, USA, respectively.

2.4.1. Assay by alamar Blue BaP (40 mMBaP stock solution in dimethyl sulfoxide) was diluted in RPMI1640 medium. Jurkat cells (2.5× 104 cells/ 100 ml/96 well) were treated with several doses of BaP for 1 h, then irradiated with several doses of UVA in the presence of BaP dissolved in RPMI1640 medium. After incubation for 24 h, alamar Blue (10 ml) was added. After incubation for 4 h, the fluorescence intensity was measured using a microplate fluorescence reader (FL500, BIO-TEC, Japan) (ex. 530 nm, em. 590 nm). 2.4.2. Assay by FDA Jurkat cells (5× 105 cells/1.4 ml/ 35 mm dish) were treated with BaP (250 or 500 nM) for 1 h, then irradiated with UVA (0.5 J/cm2) in the presence of BaP dissolved in RPMI1640 medium. They were incubated for predetermined times, suspended in phosphatebuffered saline (PBS) containing FDA (0.1 mg/ml) and incubated for 10 min at 37 °C. After addition of PI (50 mg/ml), the cells were further incubated for 10 min at

Y. Ibuki, R. Goto / En6ironmental Toxicology and Pharmacology 11 (2002) 101–109

room temperature. Viability was determined using a flow cytometer (FCM) (Epics XL; Coulter, USA).

2.5. Changes in nuclear morphology Changes in nuclear morphology were detected by staining with the DNA-binding fluorochome Hoechst -33342. Cells were washed with PBS and fixed in 2% glutaraldehyde at 4 °C for 4 h. Then, the cells were washed again and resuspended in 40 ml of PBS containing 1 mM Hoechst33342. Following incubation for 10 min at room temperature, aliquots (10 ml) were placed on glass slides, and the numbers of apoptotic nuclei per field of 200 cells were scored microscopically in triplicate.

2.6. Detection of subG1 fraction The percentage of subdiploid apoptotic nuclei was determined by staining with propidium iodide (PI). Cells were washed with PBS and fixed in 70% ethanol at −20 °C for 20 h. The cells were washed again and resuspended in PBS containing PI (50 mg/ml). Following incubation for 10 min at room temperature, the percentage of apoptotic nuclei, recognized by their subdiploid DNA content, was determined using the FCM.

2.7. Detection of Annexin V-binding to phosphatidylserine (PS) on the cell surface PS translocation to the outer leaflet of the plasma membrane was assessed by reaction with Annexin V– FITC. Cells were washed with PBS, suspended in 100 ml of binding buffer (10 mM HEPES, pH7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2) plus 1 ml of Annexin V– FITC solution, then incubated for 15 min at room temperature. Binding buffer (400 ml) containing PI (50 mg/ml) was added and Annexin V–FITC fluorescence in PI staining negative cells was detected using the FCM.

2.8. Determination of caspase acti6ity Caspase activity in lysates was measured using synthetic fluorogenic substrates (Ac-DEVD-MCA; substrate for caspase-3/7, Ac-IETD-MCA; substrate for caspase-8/6 and granzyme B, Ac-LEHD-MCA; substrate for caspase-9). Following incubation for several hours after coexposure to BaP plus UVA, cells were washed with PBS and lysed in 10 mM Tris– HCl, pH 7.5, 130 mM NaCl, 1% TritonX-100, 10 mM Na4P2O7 and 10 mM Na2HPO4 on ice. Aliquots of cell lysates were added to reaction buffer (20 mM HEPES, 10% glycerol, 2 mM dithiothreitol (DTT)

103

and 250 mM fluorogenic substrate), and the mixture was incubated for 1 h at 37 °C. Amounts of fluorogenic MCA moiety released were measured using a spectrofluorometer (FL4500, Hitachi, Japan) (ex. 380 nm, em. 460 nm). The fluorescence intensity was converted to micromoles of MCA released using the standard curve of 7-amino-4-methylcoumarin.

2.9. Immunoblotting analysis for cytochrome c To evaluate mitochondrial cytochrome c release, cytosolic protein extracts were obtained as described by Granville et al. (1998). Following incubation for predetermined times after coexposure to BaP and UVA irradiation, cells were washed with PBS and suspended in lysis buffer (20 mM HEPES, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT and 1 mM phenylmethylsulfonyl fluoride). Cells were disrupted using a Dounce homogenizer. Lysates were centrifuged at 10 000×g for 10 min and the supernatants were further centrifuged at 100 000× g for 30 min. The proteins in the supernatants were separated by 15% SDS-PAGE followed by electroblotting onto PVDF membrane (ImmobilonP, Millipore Co., USA). After blocking of nonspecific binding with 10% skimmed milk, the membrane was incubated with anti-cytochrome c IgG or anti-actin IgG at a dilution of 1:1000 for 1 h, and washed with PBS containing 0.1% Tween 20 (T-PBS). The membrane was then incubated with horseradish peroxidase-conjugated anti-goat IgG (1/1000 dilution) for 1 h. After washing with T-PBS, the secondary antibody was detected with the enhanced chemiluminescence detection system ECL (Amersham Pharmacia Biotech, UK).

3. Results

3.1. Cytotoxicity after coexposure to BaP plus UVA Jurkat cells are relatively sensitive to UVA irradiation. Fluorescence intensity of alamar Blue was reduced to half 24 h after UVA irradiation at a dose of 1 J/cm2 (data not shown). Therefore, doses below 0.5 J/cm2, which showed no cytotoxicity, were used in this study. BaP alone below 1 mM showed no cytotoxicity 24 h after treatment, but coexposure with UVA (0.5 J/cm2) markedly enhanced cytotoxicity in a BaP-dose dependent manner (Fig. 1A). The metabolic activity of alamar Blue 24 h after coexposure to BaP (250 nM) plus UVA (0.5 J/cm2) was about 10–20% of activity of unirradiated controls. Furthermore, the phototoxicity was dependent on UVA dose (Fig. 1B). The cytotoxicity was also examined using FDA (Fig.

104

Y. Ibuki, R. Goto / En6ironmental Toxicology and Pharmacology 11 (2002) 101–109

2). The right histograms in Fig. 2A show the results of double staining with FDA and PI. The percentage of FDA-negative region increased rapidly from 10 h after coexposure. Although coexposure to BaP (500 nM) plus UVA (0.5 J/cm2) completely inhibited the metabolism of alamar Blue (Fig. 1B), the percentage of cell death determined by FDA (FDA staining negative region) was about 30– 40% (Fig. 2B).

3.2. Induction of apoptosis by coexposure to BaP plus UVA As shown in the left histograms in Fig. 2A, coexposure to BaP plus UVA resulted in the appearance of a smaller cell size group (forward scattering was low), suggesting the induction of apoptosis. To further study the pattern of cell death, the morphological nuclear changes were observed by staining with Hoechst33342 24 h after coexposure to BaP (250 and 500 nM) plus UVA (0.5 J/cm2). As shown in Fig. 3A, the occurrence of chromatin condensation was detected and the percentage of chromatin-condensed cells was almost the same as that determined using FDA (Fig. 2B). Furthermore, the percentages of subdiploid apoptotic nuclei (subG1 fraction) were determined by staining with PI, 6 or 24 h after coexposure to BaP (250 nM) plus UVA (0.5 J/cm2) (Fig. 3B). The subG1 percentage was about 10% at 6 h and further increased at 24 h. Apoptotic cells were identified by selective binding of FITC– Annexin V to PS on the outer plasma membrane (Fig. 4). About 20 and 30% of the cells were labeled 6 and 18 h, respectively, after coexposure to BaP (250 nM) plus UVA (0.5 J/cm2).

3.3. Caspase acti6ation and cytochrome c release by coexposure to BaP plus UVA DEVD-ase activity (indicative of caspase-3/7 cleavage activity) was time-dependently enhanced after treatment (Fig. 5A). Furthermore, IETD-ase activity (indicative of caspase-8/6 and granzyme B cleavage activity) and LEHD-ase activity (indicative of caspase-9 cleavage activity) were also increased slightly (Fig. 5B and C). To confirm that caspases were involved in BaP-induced phototoxicity, cells were pretreated with the pan caspase inhibitor Z-VAD-CH2-DCB prior to UVA irradiation, and cytotoxicity was determined by alamar Blue assay (Fig. 5D). Z-VAD-CH2-DCB partially inhibited BaP-induced phototoxicity. Cytochrome c release from mitochondria was observed 6 h after coexposure, but not after 1 h (Fig. 6).

3.4. Participation of reacti6e oxygen species (ROS) To study the participation of ROS in BaP-induced phototoxicity, cells were treated with NaN3 and mannitol before UVA irradiation. NaN3 inhibited the phototoxicity completely, whereas mannitol showed no effect (Fig. 7A). Furthermore, NaN3 inhibited chromatin condensation (Fig. 7B) and selective binding of FITC–Annexin V to PS on the outer plasma membrane (Fig. 7C).

4. Discussion In this study, we showed that BaP can trigger apoptosis in a human lymphoma cell line irradiated with

Fig. 1. Determination of photocytotoxicity of BaP using alamar Blue. A. Cells were exposed to several doses of BaP plus UVA and incubated for 24 h. After further incubation for 4 h with addition of alamar Blue, the fluorescence intensity was determined: , 0 J/cm2; “, 0.5 J/cm2. B. Cells were exposed to BaP (250 or 500 nM) plus several doses of UVA and incubated for 24 h. After further incubation for 4 h with addition of alamar Blue, the fluorescence intensity was determined: , BaP 0 nM; “, 250 nM; , 500 nM. Values are means 9 S.D. (n =5).

Y. Ibuki, R. Goto / En6ironmental Toxicology and Pharmacology 11 (2002) 101–109

105

UVA. Apoptotic events such as cell shrinkage, chromatin condensation, appearance of subdiploid apoptotic nuclei, PS movement to the outer leaflet, cytochrome c release and caspase activation were observed. Apoptosis requires a proteolytic system involving a family of cysteine-dependent aspartate-specific proteases known as the caspases (Thornberry and Lazebnik, 1998; Wolf and Green, 1999). Activated caspase-8 and -9 (initiator caspases) are able to proteolytically activate downstream caspases such as caspase-3 (executor caspase), which can result in apoptotic cell death. Coexposure to BaP plus UVA was found to activate both initiator and executor caspases. There have been no previous reports regarding the induction of apoptosis by photosensitization of PAH, whereas many studies concerning photodynamic therapy (PDT) have demonstrated induction of apoptosis by many kinds of photosensitizers such as silicon phthacyanine Pc4, benzoporphyrin derivative monoacid ring A (Oleinik and Evans, 1998; Luo et al., 1996; Luo and Kessel, 1997; Granville et al., 1998; Ru¨ ck et al., 2000; Varnes et al., 1999). The activation of some caspases by PDT was reported previously (Granville et al., 1998). In apoptosis, cytochrome c released from permeability transition (PT) pores is known to form a complex with Apaf-1 and procaspase-9, and its proteolytically cleaved active form, caspase-9, also activated caspase-3 (Li et al., 1997). Some reports indicated the rapid release of cytochrome c (within 10 min) from mitochondria damaged by photooxidation, which induced acute activation of the caspase cascade (within 1 h) (Granville et al., 1998; Varnes et al., 1999). However, we found that cytochrome c was not released 1 h after treatment, and activation of caspase-9 and -3 occurred 3 h after treatment showing that the induction of apoptosis by coexposure to BaP plus UVA was not due to cytochrome c released from physically damaged mitochondria. Photosensitized oxidation is the basis of photodynamic actions subclassified as either type I or type II reactions (Foote, 1991). The exited state of sensitizers induced by absorption of light can either react with the substrate or solvent (Type I) or with oxygen (Type II),

Fig. 2.

Fig. 2. Determination of photocytotoxicity of BaP using FDA as a function of time after coexposure. A. Cells were exposed to BaP (500 nM) plus UVA (0.5 J/cm2). They were incubated for predetermined times (6, 10 and 24 h), suspended in PBS containing FDA for 10 min at 37 °C. After addition of PI, the viability was determined using the FCM. Left histogram: the horizontal axis shows forward scattering (reflecting cell size) and the vertical axis shows side scattering (reflecting density of a cell). Right histogram: the horizontal axis shows fluorescence intensity of FDA and the vertical axis shows fluorescence intensity of PI. FDA( + ) and PI( − ), living cells; FDA( − ) and PI(− ), early apoptotic cells; FDA( −) and PI(+ ), late apoptotic cells and necrotic cells. The numbers show the percentages of each fraction. B. Percentages of cell death were calculated from the numbers of FDA staining negative cells. , BaP 250 nM; “, 500 nM. Values are means 9 S.D. (n = 3).

106

Y. Ibuki, R. Goto / En6ironmental Toxicology and Pharmacology 11 (2002) 101–109

Fig. 3. Induction of chromatin condensation and DNA degradation. A. Cells were exposed to BaP (250 or 500 nM) plus UVA (0.5 J/cm2) and incubated for predetermined times. They were fixed in 2% glutaraldehyde and stained with Hoechst 33342. Photographs show cells with condensed chromatin (arrows). These cells were scored microscopically (200 cells per slides). , BaP 250 nM; “, 500 nM. Values are means 9S.D. (n= 3). B. Cells were exposed to BaP (250 nM) plus UVA (0.5 J/cm2) and incubated for 6 or 24 h. They were fixed in 70% ethanol and stained with PI. The percentage of apoptotic nuclei, recognized by their subdiploid DNA content, was determined using the FCM. Black columns, UVA 0 J/cm2; gray columns, UVA 0.5 J/cm2. Values are means 9 S.D. (n=3).

Fig. 4. Annexin V binding to PS on the cell surface. Cells were exposed to BaP (250 nM) plus UVA (0.5 J/cm2) and incubated for 6 (C, E) or 18 (A, B, D, F) h. They were suspended in buffer containing Annexin V – FITC for 15 min at room temperature. After addition of binding buffer containing PI, Annexin V –FITC fluorescence in PI staining negative cells was detected using the FCM. A, non-treated (in the absence of both BaP and UVA); B, BaP only; C and D, UVA only; E and F, BaP plus UVA. The horizontal axis shows fluorescence intensity of Annexin V –FITC on a logarithmic scale and the vertical axis shows cell number.

yielding radicals or radical ions (Type I) or singlet oxygen (Type II). Singlet oxygen accounts for a substantial portion of the damage produced during PDT (Oleinik and Evans, 1998). In the present study, the phototoxicity of BaP was inhibited by NaN3 (quencher of singlet oxygen (Pocci et al., 1999)) but not by mannitol (quencher of hydroxy radicals (Goldstein and

Czapski, 1984)), showing that BaP-induced phototoxicity was due to the production of singlet oxygen but not hydroxyl radicals. BaP, similar to photosensitizers for PDT, has been reported to enhance the formation of 8-hydroxy-2%-deoxyguanosine by UVA irradiation (Mauthe et al., 1995; Liu et al., 1998), which might be associated with photo-

Y. Ibuki, R. Goto / En6ironmental Toxicology and Pharmacology 11 (2002) 101–109

107

Fig. 5. Enhancement of caspase activity. A –C. Following incubation for 1, 3 or 6 h after coexposure to BaP (250 nM) plus UVA (0.5 J/cm2), cells were lysed. Aliquots of cell lysates were added to reaction buffer containing fluorogenic substrate (Ac-DEVD-MCA (A), Ac-IETD-MCA (B), Ac-LEHD-MCA (C)) and the mixtures were incubated for 1 h at 37 °C. Amounts of fluorogenic MCA moiety released were measured by spectrofluorometry. The fluorescence intensity was converted to micromoles of MCA released using the standard curve of 7-amino-4-methylcoumarin. Values are means 9S.D. (n= 3). (D) After pretreatment with Z-VAD-CH2-DCB (100 mM) for 1 h, cells were exposed to BaP (250 nM) plus UVA (0.5 J/cm2) and further incubated for 24 h. After incubation for 4 h with addition of alamar Blue, fluorescence intensity was determined. Black columns, absence of Z-VAD-CH2-DCB; gray columns, presence of Z-VAD-CH2-DCB. Values are means 9 S.D. (n =5). Student’s t-test was used to test the significance of differences between groups. *P B0.05, **PB 0.01.

Fig. 6. Cytochrome c release. Following incubation for 1 or 6 h after coexposure of BaP (500 nM) and UVA (0.5 J/cm2), cells were homogenized and centrifuged at 100 000 ×g. The supernatants were subjected to SDS-PAGE followed by Western blotting with anti-cytochrome c IgG (A) and anti-actin IgG (B), respectively.

carcinogenesis. Evans et al. (1997) reported that PDTinduced mutagenicity was not detected in cells in which it was easy to induce apoptosis, but detected in cells with a lower frequency of induction of apoptosis. As shown in this experiment, apoptosis induction might prevent photocarcinogenesis enhanced by environmental contaminants. In conclusion, coexposure to BaP plus UVA induced lethality in Jurkat cells. The pattern of cell death was apoptotic, showing caspase activation,

chromatin condensation and PS translocation to the outer membrane. We examined apoptosis mainly at doses of 250 and 500 nM BaP, and of 0.5 J/cm2 UVA. The photosensitizer, chloroaluminum phthalocyanine has been reported to induce apoptosis at low doses, and to shift from an apoptotic to a necrotic response at higher doses (Luo and Kessel, 1997). Therefore, some other combinations of both doses of BaP and UVA might change the pattern of cell death. Many kinds of PAHs enter the aquatic and atmospheric environments as the result of human activities. BaP was reported to be the most potent photocytotoxic PHA among the 16 priority PAHs (Schirmer et al., 1998). However, with respect to environmental relevant concentrations, other PAHs such as pyrene or fluoranthene appeared to be more potent. In this study, we examined only BaP-induced cell death in a human cell line. Higher aquatic organisms are continuously exposed to PAHs and direct sunlight. Further studies are necessary to clarify the association of other PAHs with cell death (apoptosis) and mutagenicity in other cell lines (e.g. fish cell lines). Acknowledgements This work was supported by Showa Shell Sekiyu Foundation for Promotion of Environmental Research.

108

Y. Ibuki, R. Goto / En6ironmental Toxicology and Pharmacology 11 (2002) 101–109

Fig. 7. Participation of ROS in BaP-induced phototoxicity. A. alamar Blue assay. After pretreatment with NaN3 (10 and 100 mM) or mannitol (10 and 100 mM) for 1 h, cells were exposed to BaP (250 nM) plus UVA (0.5 J/cm2) and further incubated for 24 h. After incubation for 4 h with addition of alamar Blue, fluorescence intensity was determined. Black columns, UVA 0 J/cm2; gray columns, UVA 0.5 J/cm2. Values are means9S.D. (n =5). B. Hoechst 33342 staining. After pretreatment with NaN3 (10 mM), cells were coexposed to BaP (250 or 500 nM) plus UVA (0.5 J/cm2) and incubated for 24 h. They were fixed in 2% glutaraldehyde and stained with Hoechst33342. Apoptotic cells were scored microscopically (200 cells per slides). Black columns, absence of NaN3; gray columns, presence of NaN3. Values are means 9S.D. (n= 3). C. Annexin V staining. After pretreatment with NaN3 (10 mM), cells were exposed to BaP (250 nM) plus UVA (0.5 J/cm2) and incubated for 18 h. They were stained by Annexin V –FITC and PI. Upper histogram: distribution of cells stained with Annexin V – FITC and PI. The horizontal axis shows fluorescence intensity of Annexin V –FITC and the vertical axis shows fluorescence intensity of PI. Lower histogram: fluorescence intensity of Annexin V – FITC in PI-negative region of upper histogram. The horizontal axis shows fluorescence intensity of Annexin V – FITC and the vertical axis shows cell number. (a) UVA +NaN3 (− BaP); (b) BaP + UVA (− NaN3); (c) BaP +UVA+NaN3.

Y. Ibuki, R. Goto / En6ironmental Toxicology and Pharmacology 11 (2002) 101–109

References Albert, R.E., Miller, M.L., Cody, T., Andringa, A., Shukla, R., Baxter, C.S., 1991. Benzo[a]pyrene-induced skin damage and tumor promotion in the mouse. Carcinogenesis 12, 1273. Arfsten, D.P., Schaeffer, D.J., Mulveny, D.C., 1996. The effect of near ultraviolet radiation on the toxic effects of polycyclic aromatic hydrocarbons in animals and plants: a review. Ecotoxicol. Environ. Saf. 33, 1. Bowling, J.W., Leversee, G.J., Landrum, P.F., Giesy, J.P., 1983. Acute mortality of anthracene-contaminated fish exposed to sunlight. Aquat. Toxicol. 3, 79. Evans, H.H., Horng, M.F., Ricanati, M., Deahl, J.T., Oleinick, N.L., 1997. Mutagenicity of photodynamic therapy as compared to UVC and ionizing radiation in human and murine lymphoblast cell lines. Photochem. Photobiol. 66, 690. Foote, C.S., 1991. Definition of type I and type II photosensitized oxidation. Photochem. Photobiol. 54, 659. Goldstein, S., Czapski, G., 1984. Mannitol as an OH’ scavenger in aqueous solutions and in biological systems. Int. J. Radiat. Biol. 46, 725. Granville, D.J., Carthy, C.M., Jiang, H., Shore, G.C., McManus, B.M., Hunt, D.W.C., 1998. Rapid cytochrome c release, activation of caspase 3, 6, 7 and 8 followed by Bap31 cleavage in Hela cells treated with photodynamic therapy. FEBS Lett. 437, 5. Kagan, J., Kagan, E.D., 1986. The toxicity of benzo[a]pyrene and pyrene in the mosquito Aedes aegypti, in the dark and in the presence of ultraviolet light. Chemosphere 15, 243. Kagan, J., Tuveson, R.W., Gong, H.H., 1989. The light-dependent cytotoxicity of benzo[a]pyrene: effect on human erythrocytes, Escherichia coli cells, and Haemophilus influenzae transforming DNA. Mutat. Res. 216, 231. Kagan, J., Wang, T.P., Benight, A.S., 1990. The phototoxicity of nitro polycyclic aromatic hydrocarbons of environmental importance. Chemosphere 20, 453. Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S.M., Ahmad, M., Alnemri, E.S., Wang, X., 1997. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91, 479. Liu, Z., Lu, Y., Rosenstein, B., Lebwohl, M., Wei, H., 1998. Benzo[a]pyrene enhances the formation of 8-hydroxy-2%-deoxyguanosine by ultraviolet A radiation in calf thymus DNA and human epidermoid carcinoma cells. Biochemistry 37, 10307. Luo, Y., Kessel, D., 1997. Initiation of apoptosis versus necrosis by photodynamic therapy with chloroaluminum phthalocyanine. Photochem. Photobiol. 66, 479.

109

Luo, Y., Chang, C.K., Kessel, D., 1996. Rapid initiation of apoptosis by photodynamic therapy. Photochem. Photobiol. 63, 528. Mauthe, R.J., Cook, V.M., Coffing, S.L., Baird, W.M., 1995. Exposure of mammalian cell cultures to benzo[a]pyrene and light results in oxidative DNA damage as measured by 8-hydroxydeoxyguanoside formation. Carcinogenesis 16, 133. Oleinik, N.L., Evans, H.H., 1998. The photobiology of photodynamic therapy: cellular targets and mechanisms. Radiat. Res. 150, S146. Pocci, L., Costa, M., Montefoschi, G., Antonucci, A., Cavallini, D., 1999. Oxidation of hypotaurine to taurine with photochemically generated singlet oxygen: the effect of azide. Biochem. Biophys. Res. Commun. 254, 661. Ru¨ ck, A., Heckelsmiller, K., Kaufmann, R., Grossman, N., Haseroth, E., Akgu¨ n, N., 2000. Light-induced apoptosis involves a defined sequence of cytoplasmic and nuclear calcium release in AIPcS4-photosensitized rat bladder RR 1022 epithelial cells. Photochem. Photobiol. 72, 210. Schirmer, K., Chan, A.G.J., Greenberg, B.M., Dixon, D.G., Bols, N.C., 1998. Ability of 16 priority PAHs to be photocytotoxic to a cell line from the rainbow trout gill. Toxicology 127, 143. Schwarzman, R.A., Cidlowski, J.A., 1993. Apoptosis: the biochemistry and molecular biology of programmed cell death. Endocr. Rev. 14, 133. Sims, P., Grover, P.L., Swaisland, A., Pal, K., Hewer, A., 1974. Metabolic activation of benzo(a)pyrene proceeds by a diol-epoxide. Nature 252, 326. Thornberry, N.A., Lazebnik, Y., 1998. Caspases: enemies within. Science 281, 1312. Utesch, D., Eray, K., Diehl, E., 1996. Phototoxicity testing of polycyclic aromatic hydrocarbons (PAH) in mammalian cells in vitro. Polycyclic Aromatic Compounds 10, 117. Vermes, I., Haanen, C., 1994. Apoptosis and programmed cell death in health and disease. Adv. Clin. Chem. 31, 177. Varnes, M.E., Chiu, S.M., Xue, L.Y., Oleinick, N.L., 1999. Photodynamic therapy-induced apoptosis in lymphoma cells: translocation of cytochrome c causes inhibition of respiration as well as caspase activation. Biochem. Biophys. Res. Commun. 255, 673. Warshawsky, D., Cody, T., Radike, M., Reilman, R., Schumann, B., LaDow, K., Schneider, J., 1995. Biotransformation of benzo[a]pyrene and other polycyclic aromatic hydrocarbons and heterocyclic analogs by several green algae and other algal species under gold and white light. Chem. Biol. Interact. 97, 131. Weinstein, I.B., Jeffrey, A.M., Jennette, K.W., Blobstein, S.H., 1976. Benzo[a]pyrene diol epoxides as intermediates in nucleic acid binding in vitro and in vivo. Science 193, 592. Wolf, B.B., Green, D.R., 1999. Suicidal tendencies: apoptotic cell death by caspase family proteinases. J. Biol. Chem. 274, 20049.