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Nonylphenol enhances apoptosis induced by serum deprivation in PC12 cells
Life Sciences 74 (2004) 2301 – 2312 www.elsevier.com/locate/lifescie
Nonylphenol enhances apoptosis induced by serum deprivation in PC12 cells Miho Aoki a, Masaaki Kurasaki a,*, Takeshi Saito b, Sayaka Seki a, Toshiyuki Hosokawa c, Yasumitsu Takahashi b, Hiroyoshi Fujita b, Toshio Iwakuma a a
Department of Environmental Medicine and Informatics, Graduate School of Environmental Earth Science, Hokkaido University, Kita-10, Nishi-5, Kita, Sapporo 060-0810, Japan b Laboratory of Environmental Biology, Hokkaido University School of Medicine, Sapporo 060-8638, Japan c Research Division for Higher Education, Center for Research and Development in Higher Education, Hokkaido University, Sapporo 060-0817 Japan Received 27 June 2003; accepted 30 September 2003
Abstract Although nonylphenol is well known as an endocrine disrupting chemical, there is little information concerning biological effect of nonylphenol. In this study, we investigated effect of nonylphenol on apoptosis induced by serum deprivation in PC12 cells using TUNEL and DNA fragmentation assays. In addition, changes in contents of proapoptotic factors, Bad and Bax, and antiapoptotic factor, Bcl-2, and enzyme activity of caspase-3 were studied. Below 100 ng/ml of nonylphenol increased TUNEL signals, DNA fragmentation and content of proapoptotic factor, Bad as compared to those by serum deprivation without nonylphenol. Furthermore, addition of nonylphenol enhanced caspase-3 activity and Z-VAD, caspase-3 inhibitor, diminished such effect. These results indicated that below 100 ng/ml of nonylphenol enhanced apoptosis induced by serum deprivation via caspase-3 activation in PC12 cell. D 2004 Elsevier Inc. All rights reserved. Keywords: Apoptosis; Caspase; Endocrine-disrupter; Nonylphenol; PC12 cell; Serum deprivation; Z-VAD
Introduction Alkylphenol (AP) is used in the synthesis of alkylphenol polyethoxylate (APE) detergents and as antioxidant (Sonnenschein and Soto, 1998). APEs are a group of nonionic surfactants that have been used * Corresponding author. Tel.: +81-11-706-2243; fax: +81-11-706-4864. E-mail address: [email protected] (M. Kurasaki). 0024-3205/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2003.09.066
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for over 40 years, primarily in the manufacture of plastics, elastomers, agricultural chemicals, pulp, and paper (Kwak et al., 2001). Annual global production of APEs is over 500,000 tons, which consists of approximately 80% nonylphenol ethoxylates and 20% octylphenol ethoxylates (Hawrelak et al., 1999). It has been reported that APEs are degraded by microbes in sewage treatment plants (Staples et al., 1999). During this process, APE chains are converted to short chains such as APs. Nonylphenol in particular, has been identified as the most critical metabolite of APEs because of its enhanced resistance towards biodegradation, toxicity, and ability to bioaccumulate in aquatic organisms (Arukwe et al., 2000). Moreover, it was reported that the nonylphenol leached from centrifuge tubes showed estrogenic properties and its estrogenic activity induced both cell and progesterone receptors in human estrogensensitive MCF-7 breast tumor cells (Soto et al., 1991). Endocrine-disrupting chemicals include a wide variety of natural and man-made chemicals in the environment and have been reported to modify sexual development and reproductive function in wildlife (Sonnenschein and Soto, 1998; Harris et al., 2000). Most of these chemicals disrupt endocrine function by binding to hormone receptors (Arnold et al., 1996; Gronen et al., 1999). AP and 17h-estradiol have a common structural motif, and it has been reported that APEs show weak estrogenic activity (Harris et al., 2000; White et al., 1994; Routledge and Sumpter, 1997). On the other hand, it has been reported that some endocrine disrupters act as chemical substances that cause apoptosis in cells (Aw et al., 1990; Hughes et al., 2000; Yamanoshita et al., 2000, 2001). APs, in particular, have been shown to induce apoptosis in a wide variety of cells (Hughes et al., 2000; Raychoudhury et al., 1999). Apoptosis is a morphological and biochemical description of a physiological cell death mechanism that is commonly associated with programmed events necessary for the differentiation and development of individuals and organs (Kerr et al., 1972; Maroto and Perez-Polo, 1997). Apoptotic cell death is characterized by chromatin condensation, DNA fragmentation, cellular shrinkage, and membrane blebbing that result in the formation of apoptotic bodies (Kerr et al., 1972). It is mediated by members of the caspase family of proteases and eventually causes the degradation of chromosomal DNA (Enari et al., 1998). Phenochromocytoma PC12 cells undergo apoptosis when cultured in serum-deprived medium and the apoptotic PC12 cells exhibit DNA fragmentation (Batistatou and Greene, 1991, 1993). Therefore, the PC12 cells have proven a useful model for studying the mechanism of induction and inhibition of apoptosis (Maroto and Perez-Polo, 1997). It has been shown that caspase is involved in the death of trophic factor-deprived PC12 cells and that caspase-3 like activity is induced in these cells (Stefanis et al., 1996; Haviv et al., 1997; Troy et al., 1996). In this study, the influence of nonylphenol on apoptosis induced by serum deprivation was investigated using the PC12 cell system. Furthermore, assays of some factors related to apoptosis such as caspase-3 and the Bcl-2 family were performed using PC12 cells exposed to nonylphenol.
Materials and methods Materials PC12 cells, a cell line of rat pheochromocytoma cells, were purchased from the American Type Culture Collection (USA and Canada). Ribonuclease A, streptavidin-conjugated peroxidase and o-
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phenylendiamine dihydrochloride (OPD) were obtained from Sigma (St. Louis, MO). Fetal bovine serum (FBS) was bought from HyClone (Rockville, MD). AP ELISA kit was from Takeda (Osaka, Japan). Non-radioactive cytotoxicity assay and caspase assay kits and benzyloxycalbonyl-Val-Ala-AspCH2F (Z-VAD) were from Promega (Madison, WI). p-Nonylphenol was from Nacalai Tesque (Kyoto, Japan). Biotin-16-2V-deoxy-uridine-5V-triphosphate, proteinase K and the blocking reagent were from Roche Diagnostics (Mannheim, Germany). Terminal deoxynucleotidyl transferase (TdT) was from Toyobo (Osaka, Japan). Polyclonal antibodies against Bax, Bad and Bcl-2 were from Calbiochem (San Diego, CA) and Santa Cruz Biotechnology (Santa Cruz, USA). Biotinylated donkey anti-rabbit immunoglobulin was from Amersham Pharmacia Biotech (Buckinghamshire, UK). Cell culture PC12 cells were maintained in DMEM supplemented with 10% FBS in a humidified incubator at 37 jC and 5% CO2. The cells were preincubated in 25 cm2 flasks or 6-well plates overnight, and then the medium was replaced with serum/serum-free DMEM with or without nonylphenol. When the medium was changed to serum-deprived medium, cells in the flask were washed twice with serum-free DMEM. Quantification of nonylphenol contents in PC12 cells Nonylphenol contents in the PC12 cells were determined using an AP ELISA kit. The AP ELISA kit can specifically detect nonylphenol, and the detection range is 5 to 500 ng/ml. The nonylphenol contents in the PC12 cells were expressed as the value minus the control value per mg of protein. Cytotoxicity assay The cytotoxicities of nonylphenol were estimated using Non-radioactive cytotoxicity assay kit, based on the assay for lactate dehydrogenase (LDH) activity released from dead cells into the medium. As a result, LDH activity in the medium can be expressed as cytotoxicity. Electrophoresis of genomic DNA from PC12 cells After that, the PC12 cells were cultured in the serum/serum-free medium with 0 to 100 ng/ml of nonylphenol for 3 days and genomic DNA was isolated by the method of Yamanoshita et al. (2000). The ladder pattern of DNA was analyzed by agarose gel electrophoresis. Five micrograms of DNA was subjected to electrophoresis on 1.5% of agarose gel. DNA was visualized by staining with ethidium bromide under UV illumination. Quantification of DNA fragmentation in PC12 cells by the TdT-mediated dUTP-biotin nick end labeling (TUNEL) method Next the PC12 cells were cultured in serum-free medium with 0 to 100 ng/ml of nonylphenol for 3 days and nuclear DNA was isolated from PC12 cells. The obtained DNA was resuspended in 1xTBE buffer and equal amounts of DNA were put into wells of 96-well plates. Quantification of DNA fragmentation in PC12 cells was measured by the method of Kurasaki et al. (2001).
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Microscopic detection of apoptotic cells by TUNEL method Cells were cultured on sterilized coverglass in the 6 well plate with and without 10 ng/ml of nonylphenol in serum-free medium for 3 days. The glass were prewashed with 10 mM Tris-HCl buffer, pH 7.4 and incubated for 20 min at room temperature in the same buffer, containing 40–67 mg/ml proteinase K. The slides were washed 5 times with PBS with 0.01% Tween 20. TdT reaction was performed in 100 mM sodium cacodylate, pH 7.2, 1 mM cobalt chloride, 0.1 unit TdT/ml and 4 mM biotinylated-d UTP at 37 jC for 2 hr. Nonspecific binding sites were blocked with 2% BSA for 30 min at room temperature. After blocking the slides were incubated for 1 hr at room temperature with streptavidin-conjugated FITC (diluted 1:100 in PBS), and subsequently washed 5 times with PBS. In addition, nuclei of the cells were stained with 0.005% propidium iodide in glycerol. The fluorescent signals of FITC depending on DNA damage and of propidium iodide depending on nuclei in the cells were observed using a Bio Rad MRC-1024 confocal imaging system (Bio Rad Microscience, UK). Measurement of caspase-3-like activity The activity of caspase-3-like protease was detected using the caspase assay system kit according to the instruction manual. This kit uses DEVD-pNA as the substrate and this chemical is cleaved by DEVDases such as caspase-3 protease. The activity of caspase-3-like protease in the PC12 cells was indirectly measured as absorbance at 405 nm depending on released pNA from the cleaved substrate. PC12 cells were incubated in medium containing serum or serum-free medium with 0 to 100 ng/ml of nonylphenol for 72 hr. After the incubation, the cells were washed with phosphate-buffered saline, harvested, centrifuged and lysed. Each 30 Ag of protein in the cell lysate was divided into two tubes for the assay. After the caspase inhibitor, Z-VAD, was added to one tube at 400 AM, the both tubes were adjusted to equal volumes using distilled water. Then the assay buffer and DEVD-pNA substrate were added to all tubes, and the reaction mixtures were incubated overnight. A caspase-3-like activity was expressed as relative content against that in the cells incubated in the medium containing serum without nonylphenol. ELISA of apoptotic factors in PC12 cells The PC12 cells were incubated in medium containing serum or serum-free medium with 0 to 100 ng/ ml of nonylphenol for 3 hr. The 3 hr time point was chosen to determine the contents of apoptotic factors such as Bax, Bad and Bcl-2, which were expected to change in early stage in the apoptosis pathway described by Maroto and Perez-Polo (1997). The cells were washed with 40 mM Tris-HCl buffer, pH 7.4, containing 150 mM NaCl. The harvested cells were centrifuged at 1000 g for 5 min to remove the supernatants. Then cells were resuspended in the same buffer and disrupted by sonication for 20 sec with a Sonifier 250 (Branson). The cell debris and unbroken cells were removed by centrifugation. The contents of Bad, Bax and Bcl-2 in the cells were measured by the method of Yamanoshita et al. (2000). Statistical analysis Each value is expressed as mean F SEM obtained from three independent experiments. The number (n) refers to the number of independent experiments. Statistical analyses were performed by one-way
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analysis of variance (ANOVA), followed by Fisher’s test. P < 0.05 was considered statistically significant.
Results Accumulation of nonylphenol in PC12 cells To investigate whether nonylphenol accumulated in PC12 cells treated with nonylphenol, nonylphenol contents in the cytosol of the cells were measured using an immunoassay system. After exposure to 10 and 100 ng/ml of nonylphenol for 24 to 72 hr, as shown in Fig. 1, nonylphenol contents in PC12 cells increased depending on the exposure concentration and incubation time. Cell cytotoxicity To study whether nonylphenol showed cell toxicity, the LDH activity in the cultured medium was measured after PC12 cells were exposed to nonylphenol for 24 to 72 hr. There was no significant difference between LDH activities in the medium of control cells and nonylphenol-exposed cells in the range from 0.01 to 100 ng/ml (data not shown). Detection of DNA fragmentation by agarose gel electrophoresis To investigate whether nonylphenol affected apoptosis, DNA fragmentation of the PC12 cells incubated in the serum or serum-free medium containing nonylphenol was examined (Fig. 2). The morphological characteristics of apoptosis are frequently accompanied by multiple cleavage of DNA into 180–200 bp. The oligonucleosomal-sized fragments can be visualized as a characteristic DNA ladder following agarose gel electrophoresis (Woodgate et al., 1999). When apoptosis was induced by serum deprivation in the PC12 cells, DNA fragmentation of the cells incubated in the serum-free
Fig. 1. Nonylphenol contents in PC12 cells cultured in medium containing 10 and 100 ng/ml of nonylphenol for 24 to 72 hr. Nonylphenol contents are expressed as ng of nonylphenol per mg of protein. Error bar indicates SEM (n = 3).
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Fig. 2. Agarose gel electrophoresis of DNA isolated from PC12 cells incubated in medium containing serum (A) or serum-free medium (B) with 0 (lane 1), 0.01 (lane 2), 0.1 (lane 3), 1 (lane 4), 10 (lane 5) and 100 (lane 6) ng/ml of nonylphenol. DNA molecular weight markers are on the left (M). Lane M shows 23.1, 9.4, 6.6, 4.4, 2.3 and 2.0 kb.
medium containing nonylphenol was observed (Fig. 2B). It was noted that the DNA ladder was enhanced by addition of 0.01 to 100 ng/ml of nonylphenol (Fig. 2B). On the other hand, the DNA ladder pattern was hardly observed in the cells incubated in the medium containing serum with nonylphenol (Fig. 2A). Determination of DNA fragmentation by quantificational TUNEL method DNA fragmentation occurs via the action of endonucleases. The TUNEL method specifically labels DNA ends generated by endonuclease activity (Gollapudi and Oblinger, 1999). Quantification of DNA ladders was carried out by TUNEL methods to evaluate the degree of apoptosis in the cells treated with 0
Fig. 3. TUNEL assay for quantification of DNA fragmentation of PC12 cells incubated in serum-deprived medium containing 0 to 100 ng/ml of nonylphenol for 72 hr. The degree of TUNEL signals is expressed relative to that of the control cells ( = 1.0). Error bars indicate SEM (n = 3). Asterisks denote values significantly different (*P < 0.05) from the control value.
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to 100 ng/ml of nonylphenol. As shown in Fig. 3, TUNEL signals increased in the cells exposed to nonylphenol in the serum-free medium as shown in Fig. 2B. In the cells exposed to 1 and 100 ng/ml of nonylphenol in the serum-free medium TUNEL signals increased significantly (p < 0.05). Detection of apoptotic cells treated with nonylphenol by TUNEL method In situ TUNEL method was carried out to show the evidence that apoptosis was enhanced in the cells treated with 10 ng/ml of nonylphenol. As shown in Fig. 4A, TUNEL signals increased in the cells exposed to nonylphenol in comparison with those in the cell cultured with serum-free medium (Fig. 4C).
Fig. 4. TUNEL signals (A and C) and nuclei (B and D) signals using confocal microscopy. Fluorescent FITC signals depending on cellular DNA damage in PC12 cells cultured in the serum-free medium with (A) and without (C) 10 ng/ml of nonylphenol. In the same area, nuclei stained with propidium iodide are shown in the cells treated with nonylphenol (B) and without it (D).
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Measurement of caspase-3 like activity To investigate whether apoptotic-related factors under conditions of apoptosis induced by serum deprivation were changed by addition of nonylphenol, the activity of caspase-3 was measured in PC12 cells cultured in serum-free medium containing nonylphenol. The obtained results are shown in Fig. 5. Caspase-3-like activity was measured in the cells incubated in the serum-free medium containing 0 to 100 ng/ml nonylphenol for 72 hr (Fig. 5A). The activity was inhibited by addition of the caspase inhibitor Z-VAD. These results confirmed that the increased activity was due to caspase-3.
Fig. 5. Caspase-3-like activity of PC 12 cells incubated in serum-free medium containing nonylphenol for 72 hr with (closed column) and without Z-VAD (open column). In A, caspase-3-like activity is expressed relative to that of cells incubated in serum containing medium without nonylphenol ( = 1.0). Error bar indicates SEM (n = 3). *: Significantly different from the cells incubated in serum-free medium without nonylphenol (P < 0.05). In B, agarose gel electrophoresis of DNA isolated from the PC12 cells under the same condition as shown in Fig. 5A.
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Fig. 6. The contents of Bax, Bad and Bcl-2 in PC12 cells exposed to 0 (control), 0.1 and 100 ng/ml of nonylphenol in serumfree medium for 3 hr. The contents of each apoptosis factor are expressed relative to that of the control cells incubated in serumfree medium without nonylphenol ( = 100%). Error bar indicates SEM (n = 3). Asterisks denote values significantly different (**P < 0.01) from cells incubated in serum-free medium without nonylphenol.
To confirm that the apoptosis of the PC12 cells was mediated by the caspase pathway, Z-VAD, an inhibitor of the caspase family, was used for analysis of ladder formation caused by apoptosis. As shown in Fig. 5B, when nonylphenol was added to the serum-free medium, ladder formation of DNA from the PC12 cells was enhanced. However, caspase-inhibitor Z-VAD strongly inhibited the ladder formation of DNA from the cells incubated in the serum-free medium containing 0 to 100 ng/ml of nonylphenol. These results indicated that the enhancing effect on apoptosis by nonylphenol was mediated by caspase-3. Detection of apoptosis factors in PC12 cells To investigate whether the apoptosis enhanced by nonylphenol was regulated by the Bcl-2 family, the contents of Bax, Bad and Bcl-2 in cells cultured in the serum-free medium containing 0 to 100 ng/ml of nonylphenol were measured by ELISA. As shown in Fig. 6, the contents of Bax and Bcl-2 did not change significantly. However, the content of Bad in the cells exposed to 0.1 and 100 ng/ml of nonylphenol for 3 hr increased significantly as compared with that of the cells incubated in the serumfree medium without nonylphenol (p < 0.01).
Discussion In this study, it was successfully demonstrated that nonylphenol enhanced apoptosis induced by serum deprivation in PC12 cells (Figs. 2–5). Furthermore, it was indicated that the enhanced apoptosis caused by exposure to nonylphenol was mediated by the caspase pathway, from the results showing that caspase-3-like activity was significantly increased (p < 0.05) in the cells incubated in the serum-free medium with nonylphenol (Fig. 5). Recent studies reported that APEs and APs were detected in many countries. In the United States and Canada, nonylphenol in the river water was detected with concentrations of 0.11–0.64 Ag/l, and 0.8–
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15.1 Ag/l in final effluents from sewage treatment plants in Texas and Toronto, respectively (Gronen et al., 1999). Blackburn et al. (1999) reported concentrations of 15–76 Ag/l of total extractable APEs containing 2–22 Ag/ml of dissolved nonylphenol in river water in the United Kingdom. In Japan, it was reported that 0.11–3.08 ng/ml of 4-nonylphenols were detected in river water flowing into Lake Biwa (Tsuda et al., 2000). It is important that the concentration of nonylphenol detected in the environmental water, as mentioned above (Gronen et al., 1999; Blackburn et al., 1999; Tsuda et al., 2000), may affect apoptosis in aquatic wildlife. Previous studies on endocrine disrupting chemicals such as nonylphenol, have been focused on its estrogen-like function. For example, Soto et al. (1991) showed that nonylphenol had estrogenic activity measured by cell proliferation assay using human estrogen-sensitive MCF-7 breast tumor cells. Lech et al. (1996) described that expression of vitellogenin mRNA, which was evidence of the presence of estrogen, was induced by exposure of 10–150 ng/ml of nonylphenol in rainbow trout. Coldham et al. (1997) reported that nonylphenol was increased in the mouse uterus, and showed the weakest estrogen-like activity in the binding assay to the estrogen receptor. Recently, it has been reported that some endocrine disrupting-chemicals act as chemical substances that cause apoptosis in cells (Aw et al., 1990; Yamanoshita et al., 2001). Apoptosis is a fundamental process necessary for development of individuals and organs, as well as differentiation of the nervous system (Bredesen, 1995). Since apoptosis is essential for cells in development and in elimination of harmful cells, the adverse effects of endocrine-disrupting chemicals on apoptosis could cause serious damage to bioorganisms (Saito and Kurasaki, 2000). Nonylphenol has been reported to induce apoptosis (Kwak et al., 2001; Hughes et al., 2000). Kwak et al. (2001) reported that 4 to 100 ng/ml of nonylphenol increased the number of apoptotic cells in a dosedependent manner in the swordtail fish. Wang et al. (2003) also described that apoptosis was observed in rat Sertoli cell exposured with high concentration (3000–5000 ppb) of nonylphenol. In addition, in TM4 cells, a Sertoli cell line, incubated in serum-free medium with 30 AM (approximately 600 ng/ml) nonylphenol cell death was induced by apoptosis (Hughes et al., 2000). Bevan et al. (2003) reported that exposure to three environmental estrogens, nonylphenol, octylphenol, and methoxychlor, as well as to the natural estrogen 17h-estradiol, increased mortality, induced morphologic deformations, increased apoptosis, and altered the deposition and differentiation of neural crest-derived melanocytes in tailbud stage Xenopus laevis embryos ranging from 10 nM to 10 AM. In this study, DNA fragmentation in PC12 cells cultured in medium containing serum with nonylphenol was scarcely observed. Thus below 100 ng/ml of nonylphenol did not act as chemicals caused apoptosis in PC12 cells. However, under the apoptotic conditions, nonylphenol enhanced apoptosis in the PC12 cells at a lower concentration than the apoptosis-induced concentration of nonylphenol, as reported by Hughes and co-worker (Figs. 2–6). In a previous study, it was reported that cell survival measured by trypan blue staining dramatically decreased after exposure to 20 and 50 AM (approximately 400 and 1000 ng/ml) nonylphenol in TM4 and Sertoli cells (Hughes et al., 2000; Wang et al., 2003). However, in this study, when LDH activity released into the medium from the cells was measured as a marker of cytotoxicity, nonylphenol ranging from 0 to 100 ng/ml did not significantly change the LDH activity in the medium for the PC12 cells incubated under the serum/serum-free conditions. From these results, it was suggested that nonylphenol did not causes cytotoxicity at those concentrations, although it was accumulated in a dose-dependent manner in the PC12 cells (Fig. 1). The present study indicated that apoptosis enhanced by nonylphenol was mediated by the caspase-3 pathway (Fig. 5). The apoptotic cell death observed in this study was inhibited completely by addition of
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Z-VAD (Fig. 5). This was evidence that apoptosis enhanced by nonylphenol and induced by serum deprivation depended on the caspase-3 pathway. Moreover, in this study, the contents of Bad, a proapoptotic protein, increased in the cells incubated in serum-free medium for 3 hr (Fig. 6). It was reported that Bad translocated to the mitochondria to exert pro-apoptotic activity after dephosphorylation by phosphatase during apoptosis, although phosphorylated Bad mainly localized in the cytoplasm in living cells (Tsujimoto and Shimizu, 2000). Maroto and Perez-Polo (1997) reported that expression of Bad was decreased by serum deprivation for 3 hr in PC12 cells. It is necessary to elucidate the reason for these differences in future studies. However, it was considered that the enhanced apoptotic cell death was dependent on the pathway regulating release of cytochrome c from mitochondria. Acknowledgements The authors are grateful to Ms. Tomoe Miura and Ms. Kazuyo Maekawa for their technical assistance. This research was supported by Grant-in-Aids from Japan Society for the Promotion of Science (M.K.) and from Ministry of Education, Science, Sports, and Culture, Japan (T.S.). References Arnold, S.F., Collins, B.M., Robinson, M.K., Guillette Jr., L.J., McLachlan, L.A., 1996. Differential interaction of natural and synthetic estrogens with extracellular binding proteins in a yeast estrogen screen. Steroids 61 (11), 642 – 646. Arukwe, A., Thibaut, R., Ingebrigtsen, K., Celius, T., Goksoyr, A., Cravedi, J., 2000. In vivo and in vitro metabolism and organ distribution of nonylphenol in Atlantic salmon (Salmo salar). Aquatic Toxicology 49 (3), 289 – 304. Aw, T.Y., Nicotera, P., Manzo, L., Orrenius, S., 1990. Tributyltin stimulates apoptosis in rat thymocytes. Archives of Biochemistry and Biophysics 283 (1), 46 – 50. Batistatou, A., Greene, L.A., 1991. Aurintricarboxylic acid rescues PC12 cells and sympathetic neurons from cell death caused by nerve growth factor deprivation: correlation with suppression of endonuclease activity. Journal of Cell Biology 115 (2), 461 – 471. Batistatou, A., Greene, L.A., 1993. Internucleosomal DNA cleavage and neuronal cell survival/death. Journal of Cell Biology 122 (3), 523 – 532. Bevan, C.L., Porter, D.M., Prasad, A., Howard, M.J., Henderson, L.P., 2003. Environmental Estrogens Alter Early Development in Xenopus laevis. Environmental Health and Perspective 111 (4), 488 – 496. Blackburn, M.A., Kirby, S.J., Waldock, M.J., 1999. Concentrations of alkylphenol polyethoxylates entering UK. Marine Pollution Bulletin 38 (2), 109 – 118. Bredesen, D.E., 1995. Neural apoptosis. Annals of Neurology 38 (6), 839 – 851. Coldham, N.G., Dave, M., Sivapathasundaram, S., McDonnell, D.P., Connor, C., Sauer, M.J., 1997. Evaluation of a recombinant yeast cell estrogen screening assay. Environmental Health Perspective 105 (7), 734 – 742. Enari, M., Sakahira, H., Yokoyama, H., Okawa, K., Iwamatsu, A., Nagata, S., 1998. A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature 391 (6662), 43 – 50. Gollapudi, L., Oblinger, M.M., 1999. Stable transfection of PC12 cells with estrogen receptor (ERalpha): protective effects of estrogen on cell survival after serum deprivation. Journal of Neuroscience Research 56 (1), 99 – 108. Gronen, S., Denslow, N., Manning, S., Barnes, S., Barnes, D., Brouwer, M., 1999. Serum vitellogenin levels and reproductive impairment of male Japanese Medaka (Oryzias latipes) exposed to 4-tert-octylphenol. Environmental Health Perspective 107 (5), 385 – 390. Harris, R.M., Waring, R.H., Kirk, C.J., Hughes, P.J., 2000. Sulfation of ‘‘estrogenic’’ alkylphenols and 17beta-estradiol by human platelet phenol sulfotransferases. Journal of Biologycal Chemistry 275 (1), 159 – 166. Haviv, R., Lindenboim, L., Li, H., Yuan, J., Stein, R., 1997. Need for caspases in apoptosis of trophic factor-deprived PC12 cells. Journal of Neuroscience Research 50 (1), 69 – 80.
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Hawrelak, M., Bennett, E., Metcalfe, C., 1999. The environmental fate of the primary degradation products of alkylphenol ethoxylate surfactants in recycled paper sludge. Chemosphere 39 (5), 745 – 752. Hughes, P.J., McLellan, H., Lowes, D.A., Kahn, S.Z., Bilmen, J.G., Tovey, S.C., Godfrey, R.E., Michell, R.H., Kirk, C.J., Michelangeli, F., 2000. Estrogenic alkylphenols induce cell death by inhibiting testis endoplasmic reticulum Ca(2+) pumps. Biochemistry and Biophysics Research Communications 277 (3), 568 – 574. Kerr, J.F., Wyllie, A.H., Currie, A.R., 1972. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. British Journal of Cancer 26 (4), 239 – 257. Kurasaki, M., Aoki, M., Miura, T., Seki, S., Toriumi, S., Hosokawa, T., Okabe, M., Numata, T., Saito, S., Saito, T., 2001. A developed method of Terminal deoxynucleotidyl transferase system for quantification of DNA damage caused by apoptosis. Analytical Science 17, i1547 – i1550 (Supplement). Kwak, H.I., Bae, M.O., Lee, M.H., Lee, Y.S., Lee, B.J., Kang, K.S., Chae, C.H., Sung, H.J., Shin, J.S., Kim, J.H., Mar, W.C., Sheen, Y.Y., Cho, M.H., 2001. Effects of nonylphenol, bisphenol A, and their mixture on the viviparous swordtail fish (Xiphophorus helleri). Environmental Toxicology and Chemistry 20 (4), 787 – 795. Lech, J.J., Lewis, S.K., Ren, L., 1996. In vivo estrogenic activity of nonylphenol in rainbow trout. Fundamental Applied Toxicology 30 (2), 229 – 232. Maroto, R., Perez-Polo, J.R., 1997. BCL-2-related protein expression in apoptosis: oxidative stress versus serum deprivation in PC12 cells. Journal of Neurochemistry 69 (2), 514 – 523. Raychoudhury, S.S., Blake, C.A., Millette, C.F., 1999. Toxic effects of octylphenol on cultured rat spermatogenic cells and Sertoli cells. Toxicology and Applied Pharmacology 157 (3), 192 – 202. Routledge, E.J., Sumpter, J.P., 1997. Structural features of alkylphenolic chemicals associated with estrogenic activity. Journal of Biological Chemistry 272 (6), 3280 – 3288. Saito, T., Kurasaki, M., 2000. Apoptosis and endocrine disrupters. Biomedical. Research 21 (6), 353 – 359. Sonnenschein, C., Soto, A.M., 1998. An updated review of environmental estrogen and androgen mimics and antagonists. Journal of Steroid Biochemistry and Molecular Biology 65 (2), 143 – 150. Soto, A.M., Justicia, H., Wray, J.W., Sonnenschein, C., 1991. p-Nonyl-phenol: an estrogenic xenobiotic released from ‘‘modified’’ polystyrene. Environmental Health Perspective 92, 167 – 173. Stefanis, L., Park, D.S., Yan, C.Y., Farinelli, S.E., Troy, C.M., Shelanski, M.L., Greene, L.A., 1996. Induction of CPP32-like activity in PC12 cells by withdrawal of trophic support. Dissociation from apoptosis. Journal of Biological Chemistry 271 (48), 30663 – 30671. Staples, C.A., Williams, J.B., Blessing, R.L., Varineau, P.T., 1999. Measuring the biodegradability of nonylphenol ether carboxylates, octylphenol ether carboxylates, and nonylphenol. Chemosphere 38 (9), 2029 – 2039. Troy, C.M., Stefanis, L., Prochiantz, A., Greene, L.A., Shelanski, M.L., 1996. The contrasting roles of ICE family proteases and interleukin-1beta in apoptosis induced by trophic factor withdrawal and by copper/zinc superoxide dismutase down-regulation. Proceedings of the National Academy of Science of the United State of America 93 (11), 5635 – 5640. Tsuda, T., Takino, A., Kojima, M., Harada, H., Muraki, K., Tsuji, M., 2000. 4-Nonylphenols and 4-tert-octylphenol in water and fish from rivers flowing into Lake Biwa. Chemosphere 41 (5), 757 – 762. Tsujimoto, Y., Shimizu, S., 2000. Bcl-2 family: Life-or-death switch. FEBS Letters 466 (1), 6 – 10. Wang, X., Han, X., Hou, Y., Yao, G., Wang, Y., 2003. Effect of nonylphenol on apoptosis of Sertoli cslls in vitro. Bulletin of Environmental Contamination and Toxicology 70 (5), 898 – 904. White, R., Jobling, S., Hoare, S.A., Sumpter, J.P., Parker, M.G., 1994. Environmentally persistent alkylphenolic compounds are estrogenic. Endocrinology 135 (1), 175 – 182. Woodgate, A., MacGibbon, G., Walton, M., Dragunow, M., 1999. The toxicity of 6-hydroxydopamine on PC12 and P19 cells. Brain Research Molecular Brain Research 69 (1), 84 – 92. Yamanoshita, O., Kurasaki, M., Saito, T., Takahasi, K., Sasaki, H., Hosokawa, T., Okabe, M., Mochida, J., Iwakuma, T., 2000. Diverse effect of tributyltin on apoptosis in PC12 cells. Biochemistry and Biophysics Research Communications 272 (2), 557 – 562. Yamanoshita, O., Saito, T., Takahashi, K., Hosokawa, T., Okabe, M., Ito, K., Kurasaki, M., 2001. 2,4,5-Trichlorophenoxyacetic acid inhibits apoptosis in PC12 cells. Life Sciences 69 (4), 403 – 408.
Nonylphenol enhances apoptosis induced by serum deprivation in PC12 cells Miho Aoki a, Masaaki Kurasaki a,*, Takeshi Saito b, Sayaka Seki a, Toshiyuki Hosokawa c, Yasumitsu Takahashi b, Hiroyoshi Fujita b, Toshio Iwakuma a a
Department of Environmental Medicine and Informatics, Graduate School of Environmental Earth Science, Hokkaido University, Kita-10, Nishi-5, Kita, Sapporo 060-0810, Japan b Laboratory of Environmental Biology, Hokkaido University School of Medicine, Sapporo 060-8638, Japan c Research Division for Higher Education, Center for Research and Development in Higher Education, Hokkaido University, Sapporo 060-0817 Japan Received 27 June 2003; accepted 30 September 2003
Abstract Although nonylphenol is well known as an endocrine disrupting chemical, there is little information concerning biological effect of nonylphenol. In this study, we investigated effect of nonylphenol on apoptosis induced by serum deprivation in PC12 cells using TUNEL and DNA fragmentation assays. In addition, changes in contents of proapoptotic factors, Bad and Bax, and antiapoptotic factor, Bcl-2, and enzyme activity of caspase-3 were studied. Below 100 ng/ml of nonylphenol increased TUNEL signals, DNA fragmentation and content of proapoptotic factor, Bad as compared to those by serum deprivation without nonylphenol. Furthermore, addition of nonylphenol enhanced caspase-3 activity and Z-VAD, caspase-3 inhibitor, diminished such effect. These results indicated that below 100 ng/ml of nonylphenol enhanced apoptosis induced by serum deprivation via caspase-3 activation in PC12 cell. D 2004 Elsevier Inc. All rights reserved. Keywords: Apoptosis; Caspase; Endocrine-disrupter; Nonylphenol; PC12 cell; Serum deprivation; Z-VAD
Introduction Alkylphenol (AP) is used in the synthesis of alkylphenol polyethoxylate (APE) detergents and as antioxidant (Sonnenschein and Soto, 1998). APEs are a group of nonionic surfactants that have been used * Corresponding author. Tel.: +81-11-706-2243; fax: +81-11-706-4864. E-mail address: [email protected] (M. Kurasaki). 0024-3205/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2003.09.066
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for over 40 years, primarily in the manufacture of plastics, elastomers, agricultural chemicals, pulp, and paper (Kwak et al., 2001). Annual global production of APEs is over 500,000 tons, which consists of approximately 80% nonylphenol ethoxylates and 20% octylphenol ethoxylates (Hawrelak et al., 1999). It has been reported that APEs are degraded by microbes in sewage treatment plants (Staples et al., 1999). During this process, APE chains are converted to short chains such as APs. Nonylphenol in particular, has been identified as the most critical metabolite of APEs because of its enhanced resistance towards biodegradation, toxicity, and ability to bioaccumulate in aquatic organisms (Arukwe et al., 2000). Moreover, it was reported that the nonylphenol leached from centrifuge tubes showed estrogenic properties and its estrogenic activity induced both cell and progesterone receptors in human estrogensensitive MCF-7 breast tumor cells (Soto et al., 1991). Endocrine-disrupting chemicals include a wide variety of natural and man-made chemicals in the environment and have been reported to modify sexual development and reproductive function in wildlife (Sonnenschein and Soto, 1998; Harris et al., 2000). Most of these chemicals disrupt endocrine function by binding to hormone receptors (Arnold et al., 1996; Gronen et al., 1999). AP and 17h-estradiol have a common structural motif, and it has been reported that APEs show weak estrogenic activity (Harris et al., 2000; White et al., 1994; Routledge and Sumpter, 1997). On the other hand, it has been reported that some endocrine disrupters act as chemical substances that cause apoptosis in cells (Aw et al., 1990; Hughes et al., 2000; Yamanoshita et al., 2000, 2001). APs, in particular, have been shown to induce apoptosis in a wide variety of cells (Hughes et al., 2000; Raychoudhury et al., 1999). Apoptosis is a morphological and biochemical description of a physiological cell death mechanism that is commonly associated with programmed events necessary for the differentiation and development of individuals and organs (Kerr et al., 1972; Maroto and Perez-Polo, 1997). Apoptotic cell death is characterized by chromatin condensation, DNA fragmentation, cellular shrinkage, and membrane blebbing that result in the formation of apoptotic bodies (Kerr et al., 1972). It is mediated by members of the caspase family of proteases and eventually causes the degradation of chromosomal DNA (Enari et al., 1998). Phenochromocytoma PC12 cells undergo apoptosis when cultured in serum-deprived medium and the apoptotic PC12 cells exhibit DNA fragmentation (Batistatou and Greene, 1991, 1993). Therefore, the PC12 cells have proven a useful model for studying the mechanism of induction and inhibition of apoptosis (Maroto and Perez-Polo, 1997). It has been shown that caspase is involved in the death of trophic factor-deprived PC12 cells and that caspase-3 like activity is induced in these cells (Stefanis et al., 1996; Haviv et al., 1997; Troy et al., 1996). In this study, the influence of nonylphenol on apoptosis induced by serum deprivation was investigated using the PC12 cell system. Furthermore, assays of some factors related to apoptosis such as caspase-3 and the Bcl-2 family were performed using PC12 cells exposed to nonylphenol.
Materials and methods Materials PC12 cells, a cell line of rat pheochromocytoma cells, were purchased from the American Type Culture Collection (USA and Canada). Ribonuclease A, streptavidin-conjugated peroxidase and o-
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phenylendiamine dihydrochloride (OPD) were obtained from Sigma (St. Louis, MO). Fetal bovine serum (FBS) was bought from HyClone (Rockville, MD). AP ELISA kit was from Takeda (Osaka, Japan). Non-radioactive cytotoxicity assay and caspase assay kits and benzyloxycalbonyl-Val-Ala-AspCH2F (Z-VAD) were from Promega (Madison, WI). p-Nonylphenol was from Nacalai Tesque (Kyoto, Japan). Biotin-16-2V-deoxy-uridine-5V-triphosphate, proteinase K and the blocking reagent were from Roche Diagnostics (Mannheim, Germany). Terminal deoxynucleotidyl transferase (TdT) was from Toyobo (Osaka, Japan). Polyclonal antibodies against Bax, Bad and Bcl-2 were from Calbiochem (San Diego, CA) and Santa Cruz Biotechnology (Santa Cruz, USA). Biotinylated donkey anti-rabbit immunoglobulin was from Amersham Pharmacia Biotech (Buckinghamshire, UK). Cell culture PC12 cells were maintained in DMEM supplemented with 10% FBS in a humidified incubator at 37 jC and 5% CO2. The cells were preincubated in 25 cm2 flasks or 6-well plates overnight, and then the medium was replaced with serum/serum-free DMEM with or without nonylphenol. When the medium was changed to serum-deprived medium, cells in the flask were washed twice with serum-free DMEM. Quantification of nonylphenol contents in PC12 cells Nonylphenol contents in the PC12 cells were determined using an AP ELISA kit. The AP ELISA kit can specifically detect nonylphenol, and the detection range is 5 to 500 ng/ml. The nonylphenol contents in the PC12 cells were expressed as the value minus the control value per mg of protein. Cytotoxicity assay The cytotoxicities of nonylphenol were estimated using Non-radioactive cytotoxicity assay kit, based on the assay for lactate dehydrogenase (LDH) activity released from dead cells into the medium. As a result, LDH activity in the medium can be expressed as cytotoxicity. Electrophoresis of genomic DNA from PC12 cells After that, the PC12 cells were cultured in the serum/serum-free medium with 0 to 100 ng/ml of nonylphenol for 3 days and genomic DNA was isolated by the method of Yamanoshita et al. (2000). The ladder pattern of DNA was analyzed by agarose gel electrophoresis. Five micrograms of DNA was subjected to electrophoresis on 1.5% of agarose gel. DNA was visualized by staining with ethidium bromide under UV illumination. Quantification of DNA fragmentation in PC12 cells by the TdT-mediated dUTP-biotin nick end labeling (TUNEL) method Next the PC12 cells were cultured in serum-free medium with 0 to 100 ng/ml of nonylphenol for 3 days and nuclear DNA was isolated from PC12 cells. The obtained DNA was resuspended in 1xTBE buffer and equal amounts of DNA were put into wells of 96-well plates. Quantification of DNA fragmentation in PC12 cells was measured by the method of Kurasaki et al. (2001).
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Microscopic detection of apoptotic cells by TUNEL method Cells were cultured on sterilized coverglass in the 6 well plate with and without 10 ng/ml of nonylphenol in serum-free medium for 3 days. The glass were prewashed with 10 mM Tris-HCl buffer, pH 7.4 and incubated for 20 min at room temperature in the same buffer, containing 40–67 mg/ml proteinase K. The slides were washed 5 times with PBS with 0.01% Tween 20. TdT reaction was performed in 100 mM sodium cacodylate, pH 7.2, 1 mM cobalt chloride, 0.1 unit TdT/ml and 4 mM biotinylated-d UTP at 37 jC for 2 hr. Nonspecific binding sites were blocked with 2% BSA for 30 min at room temperature. After blocking the slides were incubated for 1 hr at room temperature with streptavidin-conjugated FITC (diluted 1:100 in PBS), and subsequently washed 5 times with PBS. In addition, nuclei of the cells were stained with 0.005% propidium iodide in glycerol. The fluorescent signals of FITC depending on DNA damage and of propidium iodide depending on nuclei in the cells were observed using a Bio Rad MRC-1024 confocal imaging system (Bio Rad Microscience, UK). Measurement of caspase-3-like activity The activity of caspase-3-like protease was detected using the caspase assay system kit according to the instruction manual. This kit uses DEVD-pNA as the substrate and this chemical is cleaved by DEVDases such as caspase-3 protease. The activity of caspase-3-like protease in the PC12 cells was indirectly measured as absorbance at 405 nm depending on released pNA from the cleaved substrate. PC12 cells were incubated in medium containing serum or serum-free medium with 0 to 100 ng/ml of nonylphenol for 72 hr. After the incubation, the cells were washed with phosphate-buffered saline, harvested, centrifuged and lysed. Each 30 Ag of protein in the cell lysate was divided into two tubes for the assay. After the caspase inhibitor, Z-VAD, was added to one tube at 400 AM, the both tubes were adjusted to equal volumes using distilled water. Then the assay buffer and DEVD-pNA substrate were added to all tubes, and the reaction mixtures were incubated overnight. A caspase-3-like activity was expressed as relative content against that in the cells incubated in the medium containing serum without nonylphenol. ELISA of apoptotic factors in PC12 cells The PC12 cells were incubated in medium containing serum or serum-free medium with 0 to 100 ng/ ml of nonylphenol for 3 hr. The 3 hr time point was chosen to determine the contents of apoptotic factors such as Bax, Bad and Bcl-2, which were expected to change in early stage in the apoptosis pathway described by Maroto and Perez-Polo (1997). The cells were washed with 40 mM Tris-HCl buffer, pH 7.4, containing 150 mM NaCl. The harvested cells were centrifuged at 1000 g for 5 min to remove the supernatants. Then cells were resuspended in the same buffer and disrupted by sonication for 20 sec with a Sonifier 250 (Branson). The cell debris and unbroken cells were removed by centrifugation. The contents of Bad, Bax and Bcl-2 in the cells were measured by the method of Yamanoshita et al. (2000). Statistical analysis Each value is expressed as mean F SEM obtained from three independent experiments. The number (n) refers to the number of independent experiments. Statistical analyses were performed by one-way
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analysis of variance (ANOVA), followed by Fisher’s test. P < 0.05 was considered statistically significant.
Results Accumulation of nonylphenol in PC12 cells To investigate whether nonylphenol accumulated in PC12 cells treated with nonylphenol, nonylphenol contents in the cytosol of the cells were measured using an immunoassay system. After exposure to 10 and 100 ng/ml of nonylphenol for 24 to 72 hr, as shown in Fig. 1, nonylphenol contents in PC12 cells increased depending on the exposure concentration and incubation time. Cell cytotoxicity To study whether nonylphenol showed cell toxicity, the LDH activity in the cultured medium was measured after PC12 cells were exposed to nonylphenol for 24 to 72 hr. There was no significant difference between LDH activities in the medium of control cells and nonylphenol-exposed cells in the range from 0.01 to 100 ng/ml (data not shown). Detection of DNA fragmentation by agarose gel electrophoresis To investigate whether nonylphenol affected apoptosis, DNA fragmentation of the PC12 cells incubated in the serum or serum-free medium containing nonylphenol was examined (Fig. 2). The morphological characteristics of apoptosis are frequently accompanied by multiple cleavage of DNA into 180–200 bp. The oligonucleosomal-sized fragments can be visualized as a characteristic DNA ladder following agarose gel electrophoresis (Woodgate et al., 1999). When apoptosis was induced by serum deprivation in the PC12 cells, DNA fragmentation of the cells incubated in the serum-free
Fig. 1. Nonylphenol contents in PC12 cells cultured in medium containing 10 and 100 ng/ml of nonylphenol for 24 to 72 hr. Nonylphenol contents are expressed as ng of nonylphenol per mg of protein. Error bar indicates SEM (n = 3).
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Fig. 2. Agarose gel electrophoresis of DNA isolated from PC12 cells incubated in medium containing serum (A) or serum-free medium (B) with 0 (lane 1), 0.01 (lane 2), 0.1 (lane 3), 1 (lane 4), 10 (lane 5) and 100 (lane 6) ng/ml of nonylphenol. DNA molecular weight markers are on the left (M). Lane M shows 23.1, 9.4, 6.6, 4.4, 2.3 and 2.0 kb.
medium containing nonylphenol was observed (Fig. 2B). It was noted that the DNA ladder was enhanced by addition of 0.01 to 100 ng/ml of nonylphenol (Fig. 2B). On the other hand, the DNA ladder pattern was hardly observed in the cells incubated in the medium containing serum with nonylphenol (Fig. 2A). Determination of DNA fragmentation by quantificational TUNEL method DNA fragmentation occurs via the action of endonucleases. The TUNEL method specifically labels DNA ends generated by endonuclease activity (Gollapudi and Oblinger, 1999). Quantification of DNA ladders was carried out by TUNEL methods to evaluate the degree of apoptosis in the cells treated with 0
Fig. 3. TUNEL assay for quantification of DNA fragmentation of PC12 cells incubated in serum-deprived medium containing 0 to 100 ng/ml of nonylphenol for 72 hr. The degree of TUNEL signals is expressed relative to that of the control cells ( = 1.0). Error bars indicate SEM (n = 3). Asterisks denote values significantly different (*P < 0.05) from the control value.
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to 100 ng/ml of nonylphenol. As shown in Fig. 3, TUNEL signals increased in the cells exposed to nonylphenol in the serum-free medium as shown in Fig. 2B. In the cells exposed to 1 and 100 ng/ml of nonylphenol in the serum-free medium TUNEL signals increased significantly (p < 0.05). Detection of apoptotic cells treated with nonylphenol by TUNEL method In situ TUNEL method was carried out to show the evidence that apoptosis was enhanced in the cells treated with 10 ng/ml of nonylphenol. As shown in Fig. 4A, TUNEL signals increased in the cells exposed to nonylphenol in comparison with those in the cell cultured with serum-free medium (Fig. 4C).
Fig. 4. TUNEL signals (A and C) and nuclei (B and D) signals using confocal microscopy. Fluorescent FITC signals depending on cellular DNA damage in PC12 cells cultured in the serum-free medium with (A) and without (C) 10 ng/ml of nonylphenol. In the same area, nuclei stained with propidium iodide are shown in the cells treated with nonylphenol (B) and without it (D).
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Measurement of caspase-3 like activity To investigate whether apoptotic-related factors under conditions of apoptosis induced by serum deprivation were changed by addition of nonylphenol, the activity of caspase-3 was measured in PC12 cells cultured in serum-free medium containing nonylphenol. The obtained results are shown in Fig. 5. Caspase-3-like activity was measured in the cells incubated in the serum-free medium containing 0 to 100 ng/ml nonylphenol for 72 hr (Fig. 5A). The activity was inhibited by addition of the caspase inhibitor Z-VAD. These results confirmed that the increased activity was due to caspase-3.
Fig. 5. Caspase-3-like activity of PC 12 cells incubated in serum-free medium containing nonylphenol for 72 hr with (closed column) and without Z-VAD (open column). In A, caspase-3-like activity is expressed relative to that of cells incubated in serum containing medium without nonylphenol ( = 1.0). Error bar indicates SEM (n = 3). *: Significantly different from the cells incubated in serum-free medium without nonylphenol (P < 0.05). In B, agarose gel electrophoresis of DNA isolated from the PC12 cells under the same condition as shown in Fig. 5A.
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Fig. 6. The contents of Bax, Bad and Bcl-2 in PC12 cells exposed to 0 (control), 0.1 and 100 ng/ml of nonylphenol in serumfree medium for 3 hr. The contents of each apoptosis factor are expressed relative to that of the control cells incubated in serumfree medium without nonylphenol ( = 100%). Error bar indicates SEM (n = 3). Asterisks denote values significantly different (**P < 0.01) from cells incubated in serum-free medium without nonylphenol.
To confirm that the apoptosis of the PC12 cells was mediated by the caspase pathway, Z-VAD, an inhibitor of the caspase family, was used for analysis of ladder formation caused by apoptosis. As shown in Fig. 5B, when nonylphenol was added to the serum-free medium, ladder formation of DNA from the PC12 cells was enhanced. However, caspase-inhibitor Z-VAD strongly inhibited the ladder formation of DNA from the cells incubated in the serum-free medium containing 0 to 100 ng/ml of nonylphenol. These results indicated that the enhancing effect on apoptosis by nonylphenol was mediated by caspase-3. Detection of apoptosis factors in PC12 cells To investigate whether the apoptosis enhanced by nonylphenol was regulated by the Bcl-2 family, the contents of Bax, Bad and Bcl-2 in cells cultured in the serum-free medium containing 0 to 100 ng/ml of nonylphenol were measured by ELISA. As shown in Fig. 6, the contents of Bax and Bcl-2 did not change significantly. However, the content of Bad in the cells exposed to 0.1 and 100 ng/ml of nonylphenol for 3 hr increased significantly as compared with that of the cells incubated in the serumfree medium without nonylphenol (p < 0.01).
Discussion In this study, it was successfully demonstrated that nonylphenol enhanced apoptosis induced by serum deprivation in PC12 cells (Figs. 2–5). Furthermore, it was indicated that the enhanced apoptosis caused by exposure to nonylphenol was mediated by the caspase pathway, from the results showing that caspase-3-like activity was significantly increased (p < 0.05) in the cells incubated in the serum-free medium with nonylphenol (Fig. 5). Recent studies reported that APEs and APs were detected in many countries. In the United States and Canada, nonylphenol in the river water was detected with concentrations of 0.11–0.64 Ag/l, and 0.8–
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15.1 Ag/l in final effluents from sewage treatment plants in Texas and Toronto, respectively (Gronen et al., 1999). Blackburn et al. (1999) reported concentrations of 15–76 Ag/l of total extractable APEs containing 2–22 Ag/ml of dissolved nonylphenol in river water in the United Kingdom. In Japan, it was reported that 0.11–3.08 ng/ml of 4-nonylphenols were detected in river water flowing into Lake Biwa (Tsuda et al., 2000). It is important that the concentration of nonylphenol detected in the environmental water, as mentioned above (Gronen et al., 1999; Blackburn et al., 1999; Tsuda et al., 2000), may affect apoptosis in aquatic wildlife. Previous studies on endocrine disrupting chemicals such as nonylphenol, have been focused on its estrogen-like function. For example, Soto et al. (1991) showed that nonylphenol had estrogenic activity measured by cell proliferation assay using human estrogen-sensitive MCF-7 breast tumor cells. Lech et al. (1996) described that expression of vitellogenin mRNA, which was evidence of the presence of estrogen, was induced by exposure of 10–150 ng/ml of nonylphenol in rainbow trout. Coldham et al. (1997) reported that nonylphenol was increased in the mouse uterus, and showed the weakest estrogen-like activity in the binding assay to the estrogen receptor. Recently, it has been reported that some endocrine disrupting-chemicals act as chemical substances that cause apoptosis in cells (Aw et al., 1990; Yamanoshita et al., 2001). Apoptosis is a fundamental process necessary for development of individuals and organs, as well as differentiation of the nervous system (Bredesen, 1995). Since apoptosis is essential for cells in development and in elimination of harmful cells, the adverse effects of endocrine-disrupting chemicals on apoptosis could cause serious damage to bioorganisms (Saito and Kurasaki, 2000). Nonylphenol has been reported to induce apoptosis (Kwak et al., 2001; Hughes et al., 2000). Kwak et al. (2001) reported that 4 to 100 ng/ml of nonylphenol increased the number of apoptotic cells in a dosedependent manner in the swordtail fish. Wang et al. (2003) also described that apoptosis was observed in rat Sertoli cell exposured with high concentration (3000–5000 ppb) of nonylphenol. In addition, in TM4 cells, a Sertoli cell line, incubated in serum-free medium with 30 AM (approximately 600 ng/ml) nonylphenol cell death was induced by apoptosis (Hughes et al., 2000). Bevan et al. (2003) reported that exposure to three environmental estrogens, nonylphenol, octylphenol, and methoxychlor, as well as to the natural estrogen 17h-estradiol, increased mortality, induced morphologic deformations, increased apoptosis, and altered the deposition and differentiation of neural crest-derived melanocytes in tailbud stage Xenopus laevis embryos ranging from 10 nM to 10 AM. In this study, DNA fragmentation in PC12 cells cultured in medium containing serum with nonylphenol was scarcely observed. Thus below 100 ng/ml of nonylphenol did not act as chemicals caused apoptosis in PC12 cells. However, under the apoptotic conditions, nonylphenol enhanced apoptosis in the PC12 cells at a lower concentration than the apoptosis-induced concentration of nonylphenol, as reported by Hughes and co-worker (Figs. 2–6). In a previous study, it was reported that cell survival measured by trypan blue staining dramatically decreased after exposure to 20 and 50 AM (approximately 400 and 1000 ng/ml) nonylphenol in TM4 and Sertoli cells (Hughes et al., 2000; Wang et al., 2003). However, in this study, when LDH activity released into the medium from the cells was measured as a marker of cytotoxicity, nonylphenol ranging from 0 to 100 ng/ml did not significantly change the LDH activity in the medium for the PC12 cells incubated under the serum/serum-free conditions. From these results, it was suggested that nonylphenol did not causes cytotoxicity at those concentrations, although it was accumulated in a dose-dependent manner in the PC12 cells (Fig. 1). The present study indicated that apoptosis enhanced by nonylphenol was mediated by the caspase-3 pathway (Fig. 5). The apoptotic cell death observed in this study was inhibited completely by addition of
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Z-VAD (Fig. 5). This was evidence that apoptosis enhanced by nonylphenol and induced by serum deprivation depended on the caspase-3 pathway. Moreover, in this study, the contents of Bad, a proapoptotic protein, increased in the cells incubated in serum-free medium for 3 hr (Fig. 6). It was reported that Bad translocated to the mitochondria to exert pro-apoptotic activity after dephosphorylation by phosphatase during apoptosis, although phosphorylated Bad mainly localized in the cytoplasm in living cells (Tsujimoto and Shimizu, 2000). Maroto and Perez-Polo (1997) reported that expression of Bad was decreased by serum deprivation for 3 hr in PC12 cells. It is necessary to elucidate the reason for these differences in future studies. However, it was considered that the enhanced apoptotic cell death was dependent on the pathway regulating release of cytochrome c from mitochondria. Acknowledgements The authors are grateful to Ms. Tomoe Miura and Ms. Kazuyo Maekawa for their technical assistance. This research was supported by Grant-in-Aids from Japan Society for the Promotion of Science (M.K.) and from Ministry of Education, Science, Sports, and Culture, Japan (T.S.). References Arnold, S.F., Collins, B.M., Robinson, M.K., Guillette Jr., L.J., McLachlan, L.A., 1996. Differential interaction of natural and synthetic estrogens with extracellular binding proteins in a yeast estrogen screen. Steroids 61 (11), 642 – 646. Arukwe, A., Thibaut, R., Ingebrigtsen, K., Celius, T., Goksoyr, A., Cravedi, J., 2000. In vivo and in vitro metabolism and organ distribution of nonylphenol in Atlantic salmon (Salmo salar). Aquatic Toxicology 49 (3), 289 – 304. Aw, T.Y., Nicotera, P., Manzo, L., Orrenius, S., 1990. Tributyltin stimulates apoptosis in rat thymocytes. Archives of Biochemistry and Biophysics 283 (1), 46 – 50. Batistatou, A., Greene, L.A., 1991. Aurintricarboxylic acid rescues PC12 cells and sympathetic neurons from cell death caused by nerve growth factor deprivation: correlation with suppression of endonuclease activity. Journal of Cell Biology 115 (2), 461 – 471. Batistatou, A., Greene, L.A., 1993. Internucleosomal DNA cleavage and neuronal cell survival/death. Journal of Cell Biology 122 (3), 523 – 532. Bevan, C.L., Porter, D.M., Prasad, A., Howard, M.J., Henderson, L.P., 2003. Environmental Estrogens Alter Early Development in Xenopus laevis. Environmental Health and Perspective 111 (4), 488 – 496. Blackburn, M.A., Kirby, S.J., Waldock, M.J., 1999. Concentrations of alkylphenol polyethoxylates entering UK. Marine Pollution Bulletin 38 (2), 109 – 118. Bredesen, D.E., 1995. Neural apoptosis. Annals of Neurology 38 (6), 839 – 851. Coldham, N.G., Dave, M., Sivapathasundaram, S., McDonnell, D.P., Connor, C., Sauer, M.J., 1997. Evaluation of a recombinant yeast cell estrogen screening assay. Environmental Health Perspective 105 (7), 734 – 742. Enari, M., Sakahira, H., Yokoyama, H., Okawa, K., Iwamatsu, A., Nagata, S., 1998. A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature 391 (6662), 43 – 50. Gollapudi, L., Oblinger, M.M., 1999. Stable transfection of PC12 cells with estrogen receptor (ERalpha): protective effects of estrogen on cell survival after serum deprivation. Journal of Neuroscience Research 56 (1), 99 – 108. Gronen, S., Denslow, N., Manning, S., Barnes, S., Barnes, D., Brouwer, M., 1999. Serum vitellogenin levels and reproductive impairment of male Japanese Medaka (Oryzias latipes) exposed to 4-tert-octylphenol. Environmental Health Perspective 107 (5), 385 – 390. Harris, R.M., Waring, R.H., Kirk, C.J., Hughes, P.J., 2000. Sulfation of ‘‘estrogenic’’ alkylphenols and 17beta-estradiol by human platelet phenol sulfotransferases. Journal of Biologycal Chemistry 275 (1), 159 – 166. Haviv, R., Lindenboim, L., Li, H., Yuan, J., Stein, R., 1997. Need for caspases in apoptosis of trophic factor-deprived PC12 cells. Journal of Neuroscience Research 50 (1), 69 – 80.
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