- Email: [email protected]
FasL pathway
Life Sciences 77 (2005) 3306 – 3320 www.elsevier.com/locate/lifescie
Nonylphenol-induced thymocyte apoptosis is related to Fas/FasL pathway Genhong Yao a, Yali Hu b, Junfeng Liang c, Yayi Hou a,T a
Immunology and Reproductive Biology Lab, Medical School & State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR China b The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing 210008, PR China c Department of Chemistry and Chemical Biology, Stevens Institute of Technology, Castle Point on Hudson, Hoboken, NJ 07030, USA Received 29 November 2004; accepted 2 May 2005
Abstract Nonylphenol (NP) is the final biodegradation product of nonylphenol polyethoxylates, which are widely used as surfactants in domestic and industrial products. NP has been reported to have estrogenic activity and shown to have potential reproductive toxicity. However, its influence on immune system function remains unclear. In this study, to determine the immunological effects of NP, the effects of NP on apoptosis and Fas/FasL gene expression in rat thymocyte in vitro were investigated. Thymocytes were treated with NP 0.1, 1, and 10 ppm, respectively. Viable cell numbers were determined by MTT assay. Apoptotic cells were identified by DNA fragment analysis. A semi-quantitative reverse transcriptase-polymerase chain reaction method was used to analyze Fas and FasL mRNA levels. Fas and FasL protein expression was evaluated by flow cytometry. The results showed that NP decreased the cellularity; induced apoptotic death and enhanced the expression of Fas and FasL mRNA as well as proteins in thymocytes. These findings suggest that NP may induce apoptosis by altering the expression of Fas and FasL in thymocytes so as to affect the immune system function. D 2005 Elsevier Inc. All rights reserved. Keywords: Nonylphenol; Thymocyte; Apoptosis; Fas/FasL
T Corresponding author. Tel.: +86 25 83686441; fax: +86 25 83686441. E-mail address: [email protected] (Y. Hou). 0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2005.05.035
G. Yao et al. / Life Sciences 77 (2005) 3306–3320
3307
Introduction Nonylphenol polyethoxylates are non-ionic surfactant widely used as components of detergents, paints, herbicides, wetting agents, cosmetics, pesticides and many other industrial and agricultural products (Arukwe et al., 2002; Bragadin et al., 1999; Burkhardt-Holm et al., 2000; Ferguson et al., 2002; You et al., 2002). They are discharged to aquatic ecosystems through sewage treatment, pulp mill, and industrial effluent, urban and agricultural runoff and so on (Ying et al., 2002). In the aquatic ecosystems, nonylphenol polyethoxylates are degraded by the actions of bacteria and finally form nonylphenol (NP) (Kinnberg et al., 2000). Studies showed that the concentration of NP in the aquatic environment, particularly in sediment, could reach up to 300 ppb (Nagao et al., 2001). Then NP is accumulated in our bodies through food chain. A convincing body of evidence indicates that NP is one of the potential endocrine disruptors and possesses estrogen-like properties (Folmar et al., 2002; Fujimoto et al., 2002; Hughes et al., 2000; Iguchi et al., 2001; Legler et al., 2002; Schwaiger et al., 2002; Yadetie and Male, 2002). Several studies also have shown that nonylphenol bound to estrogen receptor and activated the transcription genes, which are regulated by some estrogen-like compounds (Beato et al., 1995; Sakazaki et al., 2002; Yoon et al., 2001; Safe et al., 2001). Aoki reported that NP enhanced apoptosis induced by serum deprivation in PC12 cells (Aoki et al., 2004). Recent review suggests that the immune system also may serve as a potential target for NP (Ansar Ahmed, 2000). Natural estrogens, such as 17hestradiol, modulate immune responses and contributed to the etiology of immune-mediated diseases (Zajchowski and Hoffman-Goetz, 2000; Karpuzoglu-Sahin et al., 2001a,b; Geenen et al., 2003; Gaillard and Spinedi, 1998; Lindberg et al., 2002). Studies of other groups showed that the addition of E2 to thymic organ culture could induce apoptosis (Barnden et al., 1997). Our previous studies also demonstrated that in vivo administration of 17h-estradiol to rats triggered apoptosis in thymocyte through Fas/FasL pathway (Yao and Hou, 2004b). If natural estrogens have strong immunomodulatory properties and induce apoptosis, it may be reasonable to postulate that NP also modulates the immune system. However, until recently most research has focused on the toxicity of NP on development process and reproductive system, especially in aquatic ecosystem (Kinnberg et al., 2000; Sweeney, 2002; Yokota et al., 2002). We also found that NP disrupted the reproductive system by dramatically increasing Sertoli cells apoptosis in rats, which was mediated by Fas/FasL pathway (Wang et al., 2003). Therefore, to evaluate estrogenic effect of NP on the immune system, the apoptotic cell death and the expression of Fas and FasL in the thymocyte was detected with treatment of NP in the present study. Materials and methods Reagents Nonylphenol (NP) was purchased from Tokyo Kasei Kogyo Co. (Tokyo, Japan). Trizol regent was purchased from BioBasic Inc (Scarborough Ontario,Canada). Taq polymerase enzyme, M-MLV Reverse Transcriptase and rRNasin (Ribonuclease Inhibitor) were from Promega (Madison, WI, USA). Monoclonal anti-Fas and anti-FasL antibody and a FITC-labeled (fluorescein-isothiocyanate) secondary
3308
G. Yao et al. / Life Sciences 77 (2005) 3306–3320
antibody were from Boster-Biotechnology (Wuhan, China). All other reagents were of the highest grade commercially available. Tissue collection and single cell preparation The thymus was obtained from normal 3-week-old rats. The rats were killed by cervical dislocation. The thymus was removed immediately. To prepare a single-cell suspension, the cells were teased out in chilled phosphate-buffed saline (PBS) and filtered through a sterile wire mesh. The cells were centrifuged at 400 g for 5 min, and pelleted thymus cells were resuspended in Tris-bufered 0.83% NH4Cl solution to lyse erythrocytes. The resulting thymus cells were washed in ice-cold PBS three times and cells density was adjusted to 1 106 cells/mL. The viability of such cells was determined by trypan blue dye exclusion. It was more than 95% in each specimen routinely. A total of 1 105 thymus cells were dispersed into plastic 96-well round-bottomed microtiter plates and incubated with different concentrations of NP at 37 8C in humidified 5% CO2 atmosphere (final volume 100 AL/ well). Determination of viable cell numbers Cell viability was determined by an MTT assay using the cell proliferation Kit (Sigma USA). One hundred microtiter of thymocyte at a concentration of 1 105 cells/mL was plated into each well of a 96well round-bottom tissue-culture plate. Then different concentrations of NP were added to wells. After different time of incubation at 37 8C in humidified 5% CO2, 20 AL of MTT dissolved in PBS was added to cells for 4 h to give a final concentration of 0.1 mg/mL. The blue crystals were dissolved by the addition of SDS in HCl. An automated microplate reader (Bio-Rad Model 550) was used to measure the light absorbance value at 570 nm. DNA laddering detection For examination of apoptosis by electrophoresis of nucleosomal fragments, we used a standard procedure precipitating cytosolic nucleic acid (Jenkins et al., 2001). Briefly, 1 106 NP-treated thymocytes were pelleted (200 g, 5 min) and lysed for 15 min (250 Al, 0.4% Triton-X, 20 mM Tris, 0.4 mM EDTA) at 4 8C. Nuclei were then pelleted (13 000 g, 5 min, 4 8C) and the supernatant was transferred to a clean microfuge tube. Nucleosomal fragments were precipitated overnight with an equal volume of isopropanol after adjustment to 0.5 M NaCl. The pellet was washed twice in 70% ethanol, dried briefly, and resuspended in 40 Al TE (10 mM Tris–HCl, 0.1 mM EDTA, pH 8.8). The extracted DNA was mixed with the loading buffer. The DNA was then separated on 1.5% agarose gel containing 0.5 Ag/mL ethidium bromide. The gel was put on an UVtransilluminator and photographed. Fragmented DNA, shown as DNA ladder in the gel, represents the existence of apoptotic cell death. RNA isolation and reverse transcription-polymerase chain reaction Total RNAs were isolated from thymocytes by the Trizol method. RNA concentrations were quantified by spectrophotometer at 260 nm. A 2 Ag total RNA was reverse-transcribed using reverse
G. Yao et al. / Life Sciences 77 (2005) 3306–3320
3309
transcription-polymerase chain reaction (Perkin-Elmer Cetus DNA Thermal Cycler, Perkin-Elmer, CT, USA). Then the first strand complementary was gained in the presence of M-MLV reverse transcriptase and oligo-dT primers. After RT reaction, 2 AL of the incubation mixture was used as the template for the following PCR. The following components were added to the mixture: 5 AL 10 PCR buffer, 1 AL (10 pmol) of both sense and antisense primers (for Fas and h-actin, or for FasL and h-actin), 1 AL 10 mM mixture of all four deoxynucleotide triphosphates, 1 AL Taq DNA polymerase, and 12 AL nuclease-free water to adjust the final volume to 25 AL. The primer sequences used for PCR are shown below. After an initial incubation at 94 8C for 1 min, temperature cycling was initiated with each cycle as following: denaturation at 94 8C for 30 s, annealing at 65 8C for 1 min and elongation at 72 8C for 1 min. Then the reaction followed by a second round of 35 cycles of denaturation at 94 8C for 30 s, annealing at 65 8C for 1 min and elongation at 72 8C for 1 min. And the reactions were terminated by incubation at 72 8C for 5 min. The PCR conditions and the number of cycles were carefully chosen and have been described in previous reports (Lee et al., 1997; Lee et al., 1999). The PCR products were then separated on 1.5% agarose gel containing 0.5 `ıg/mL ethidium bromide. The gel was put on an UV-transilluminator and photographed. The Fas and FasL signal was measured by a densitometer and standardized against the hactin signal using a digital imaging and analysis system. Oligonucleotide primers for PCR reactions Based on the cDNA sequences referred to Lee et al. [Lee et al., 1997, 1999], primers were synthesized by Sangon Co (Shanghai, China). cDNA sequences and sizes of the amplified fragments are listed as follows: Fas, 5V-ATGCACACTCTGCGATGAAG -3V and V-ATGCACACTCTGCGATGAAG-3V (PCR product: 240 bp); FasL, 5V-GGAATGGGAAGACACATATGGAACTGC-3V and V-CATATCTGGCCAGTAGTGCAGTAATTC-3V (PCR product: 238 bp); h-actin, 5V-AGGCATCCTGACCCTGAA GTAC-3V and 5V-TCTTCATGAGGTAGTCTGTCAG-3V (PCR product: 389 bp). Flow cytometry analysis Expressions of Fas and FasL proteins were detected by anti-Fas and anti-FasL antibody and a FITC-labeled (fluorescein-isothiocyanate) secondary antibody (Boster-Biotechnology, Wuhan, China). Briefly, the prepared cell suspension was incubated with unconjugated monoclonal rabbit anti-mouse antibodies for 30 min on ice. The cells were then washed, followed by secondary antibody, FITCconjugated goat anti-rabbit antibody for 30 min on ice and washed twice. As a negative control, an aliquot of the cells from each case was stained with an irrelevant antibody of the same phenotype and a secondary antibody. Flow cytometric analysis was performed on a FACScan instrument (Becton Dickinson, San Jose, CA, USA) and data were processed by using the CellQuest program (Becton Dickinson). Forward scatter threshold was set to exclude debris from viable nucleated cells and the data were collected on 10 000 cells per sample. Statistical analysis Results were shown on the mean F S.E.M. Statistical significance between groups was analyzed by one-way ANOVA followed by the Student–Newman–Keuls Multiple Comparisons tests. A P value of b 0.05 was considered significant.
3310
G. Yao et al. / Life Sciences 77 (2005) 3306–3320
Results NP decreases the cellularity of thymocytes To determine the role of the NP on cellularity of thymocytes, thymocytes were treated with different doses of NP for 2, 4 and 6 h, respectively. The cell viability was determined by an MTT assay. Fig. 1 showed that cell survival did not cause significantly alteration in 2 h, in all the doses we used. However, the decrease of cell viability was observed in all the doses after treatment for 4 and 6 h. Furthermore, as shown in Fig. 2, the effect of NP on cellularity of thymocytes was time dependent. NP induces apoptosis of thymocytes According to Hughes (Hughes et al., 2000), DNA laddering is commonly used to establish if a decrease in cell viability is due to apoptosis rather than necrosis. Fig. 3B and C showed that exposure of thymocytes to 10, 1, and 0.1 ppm NP provoked DNA laddering in 4 and 6 h, suggesting that NP induced cell death by apoptosis. The results are consistent with observations of decreased cellularity of thymocytes by NP treatment. NP stimulates expression of Fas and FasL mRNA Semi-quantitative RT-PCR was performed on total RNA from control and NP-treated thymocytes to evaluate the expression levels of Fas and FasL mRNA. As shown in Fig. 4B, our results indicated that
Fig. 1. NP decreased the viable cells number of thymocytes. The cell viability was determined by an MTT assay. The results are presented as mean F S.E. with triplicate measurement. NP1, NP2, NP3 represent NP at levels of 0.1, 1, and 10 ppm respectively. *P b 0.05 vs. control. #P b 0.05 vs. NP1.
G. Yao et al. / Life Sciences 77 (2005) 3306–3320
3311
Fig. 2. Time dependent characteristics of NP on viabilities of thymocytes. The cell viability was determined by an MTT assay. The results are presented as mean F S.E. with triplicate measurement. NP1, NP2, NP3 represent NP at levels of 0.1, 1, and 10 ppm respectively. *P b 0.05 vs. 2 h. #P b 0.05 vs. 4 h.
Fig. 3. Agarose gel electrophoresis of DNA fragments in thymocytes cultured for 2, 4 and 6 h. Thymocytes were pretreated with different concentrations of NP. Fragmented DNA was collected and assessed by agarose gel electrophoresis containing ethidium bromide. Data shown are representative of three separate experiments. M stands for DNA marker. C stands for control. 1, 2, 3 represent NP at the concentrations of 0.1, 1, and 10 ppm respectively.
3312
G. Yao et al. / Life Sciences 77 (2005) 3306–3320
Fig. 4. Effects of NP on Fas mRNA transcripts in thymus Fas mRNA (lower lane) was determined by RT-PCR. PCR amplication of the housekeeping gene, h-actin (upper lane), was performed for each sample. (A) The representative agarose gel electrophoresis of three independent experiments is shown. PCR products of cDNA derived from vehicle control (1), NP (2, 3, 4 represent NP at levels of 0.1, 1, and 10 ppm respectively). (B) Densitometric analysis. The intensity of the band was scanned. The quotient of Fas/h-actin gene was calculated. The data shown are the average of three independent experiments including cDNA synthesis and PCR analysis. The results are shown as mean F S.E. from three representative independent experiments. *The difference from rats treated with the vehicle alone was statistically significant P b 0.05.
the expression of Fas mRNA was increased in NP 10, 1, and 0.1 ppm treated groups at 4 and 6 h. However, relative shorter time of 2 h NP treatment appeared no effect on Fas mRNA compared with control. The Fig. 4A showed the representative gel pattern. As for the expression of FasL, no significant alteration of FasL mRNA by NP treatment was shown in 2 h compared with vehicle, while up-regulation of FasL mRNA was observed in thymocytes exposed to NP for 4 and 6 h. Moreover the expressions of FasL mRNA were clearly enhanced in dose-dependent for 6 h (Fig. 5B). The Fig. 5A showed the representative gel pattern. NP increases expression of Fas and FasL proteins It is important to underline the fact that the susceptibility to Fas-induced apoptosis is usually related to the Fas and FasL proteins and not to their mRNA. Therefore, the expressions of Fas and FasL proteins in thymocytes treated with or without NP were evaluated by flow cytometry. As shown in Figs. 6 and 7, our results showed that relative shorter time of 2 h NP treatment appeared no effect on Fas and FasL proteins compared with control ( p N 0.5). However, when NP was added to culture for 4 h, the levels of Fas and FasL proteins were moderately increased compared with control at any concentration (Fas: control 0.58%, NP 0.1 ppm 2.17%, NP 1 ppm 2.32%, NP 10 ppm
G. Yao et al. / Life Sciences 77 (2005) 3306–3320
3313
Fig. 5. Effects of NP on FasL mRNA transcripts in thymus FasL mRNA(lower lane) was determined by RT-PCR. PCR amplication of the housekeeping gene, h-actin (upper lane), was performed for each sample. (A) The representative agarose gel electrophoresis of three independent experiments is shown. PCR products of cDNA derived from vehicle control (1), NP (2, 3, 4 represent NP at levels of 0.1, 1, and 10 ppm respectively). (B) Densitometric analysis. The intensity of the band was scanned. The quotient of FasL/h-actin gene was calculated. The data shown are the average of three independent experiments including cDNA synthesis and PCR analysis. The results are shown as mean F S.E. from three representative independent experiments. *The difference from rats treated with the vehicle alone was statistically significant P b 0.05. #The difference from rats treated with 0.1 ppm was statistically significant P b 0.05.
3.17%; FasL: control 0.66%, NP 0.1 ppm 2.97%, NP 1 ppm 3.16%, NP 10 ppm 4.14%;). Figs. 6 and 7 also illustrated that at the doses tested, NP significantly increase the Fas and FasL proteins in 6 h (Fas: control 0.70%, NP 0.1 ppm 5.87%, NP 1 ppm 6.93%, NP 10 ppm 7.09%; FasL: control 0.73%, NP 0.1 ppm 4.76%, NP 1 ppm 5.32%, NP 10 ppm 5.80%).
Discussion NP is one of biodegradation products of nonylphenol polyethoxylates and widely presents in the natural world. So it is practically unavoidable to contaminate our living environment. Moreover, NP can mimic natural hormones and disrupt the functions of endocrine, immune and reproductive system of living beings (Ying et al., 2002). Concern has been increased in recent years about the NP in recent years, but little is known about the toxicological effect of NP on immunocompetent cells. To address NP to cause animal and human immune disorders, the present in vitro study focuses on the effects of NP on the thymocytes, a type of important immunocompetent cells. Furthermore, there have been many evidences to suggest that NP may induce thymocyte apoptosis via regulating the expression of Fas and
3314
G. Yao et al. / Life Sciences 77 (2005) 3306–3320
NP(0.1ppm)
NP(1ppm)
NP(10ppm)
2h
4h
6h
Fig. 6. Flow cytometric analysis of Fas expression on thymocytes after NP treatment. Thymocytes were treated with different concentrations of NP for 2, 4 and 6 h respectively. Then thymocytes were labeled with Abs as described in Materials and methods. Control (Fas expression on untreated thymocytes) is shown as a thin line, and NP treatment is shown as a bold line. The results shown were representative of a series of at least three experiments.
FasL by mimicking estrogen. (1) NP has been found to act like estrogen; (2) studies showed estrogens influence the development and function of immune system, particularly the thymocytes (Okasha et al., 2001); (3) the substantial evidence indicated that estrogen receptors (de Fougerolles Nunn et al., 1999) and Fas/FasL (Mor et al., 2001; Sharva et al., 2001) were present in the thymocytes and their expression are regulated by estrogens. Therefore in the study we investigated the effects of NP on the apoptosis, expression of the Fas and FasL mRNA in thymocytes for exploring the immunological consequences of exposure to NP. NP decreased viable cell numbers of thymocytes. After exposure to all doses of NP we used in the study for 4 and 6 h, the cell viability significantly was decreased. The decrease was independent of doses of NP, but showed in a time dependent pattern. NP treatment of up to 4 h caused apoptotic cell death of thymocytes in vitro. To explore whether the decreased numbers of viable thymocytes were due to the induction of apoptosis or not, we determined the apoptosis of thymocytes by DNA ladder. The results were that NP treatment (10, 1,
G. Yao et al. / Life Sciences 77 (2005) 3306–3320
NP(0.1ppm)
NP(1ppm)
3315
NP(10ppm)
2h
4h
6h
Fig. 7. Flow cytometric analysis of FasL expression on thymocytes after NP treatment. Thymocytes were treated with different concentrations of NP for 2, 4 and 6 h respectively. Then thymocytes were labeled with Abs as described in Materials and methods. Control (FasL expression on untreated thymocytes) is shown as a thin line, and NP treatment is shown as a bold line. The results shown were representative of a series of at least three experiments.
and 0.1 ppm) for 4 and 6 h all induced cell damage compared to control. These findings are in agreement with our previous work, which showed that NP in vivo induced thymocytes apoptosis (Yao and Hou, 2004a). In addition, Nair-Menon groups demonstrated that exposure of splenic lymphocytes from rats and mice to octylphenol, an endocrine disrupter with estrogenic activity like NP, induced apoptosis (Nair-Menon et al., 1996). These observations apparently support our hypothesis that the decreased thymocyte viability by NP treatment may result from activation of apoptosis. The apoptosis-inducing cell death of NP has gained experimental support from studies of other environmental estrogens, including bisphenol A. Bisphenol A, known to be another a xenoestrogen, is widely used in industry. Studies shown that bisphenol A could induce apoptosis in a variety of cell types, such as stertoli cells, ovarian granulose cells and some specific nervous system cells (Hughes et al., 2000; Iida et al., 2003; Oka et al., 2003).
3316
G. Yao et al. / Life Sciences 77 (2005) 3306–3320
The expressions of Fas and FasL mRNA in the thymocytes were increased after NP treatment. According to Hengartner group, apoptosis in thymocytes is triggered mainly through two pathways: the death receptor pathway and the mitochondrial pathway (Hengartner, 2000). Fas is a death receptor expressed on immune cells. Ligation of this receptor by Fas ligand (FasL), a member of the tumor necrosis factor family, triggers the induction of apoptosis in cells (Do et al., 2002). Studies by independent groups show that Fas/FasL (CD95/CD95L) interaction is directly responsible for this kind of activation, which induced cell death along with the fact that activated lymphocytes express the receptor protein Fas and its ligand FasL (Sepiashvili et al., 2001; Sharma et al., 2000). The thymocytes surface receptor Fas and its ligand (FasL) are mediators of apoptosis. These mediators have been shown to act in the immune system: (1) to limit over-expression of an immune response by deleting unwanted lymphocytes; (2) to eliminate auto reactive T cells during thymus selection. Other studies also demonstrated that Fas/FasL interaction played an important role in chemicals or chemotherapeutic drug induced apoptosis (Eichhorst et al., 2001; Kamath et al., 1999). The results we obtained illustrated that NP at the level of 0.1; 1, and 10 ppm increased the Fas and FasL mRNA expression in the thymocytes after exposure for 4 and 6 h. It should be pointed out that the upregulations of Fas and FasL mRNA after NP treatment might not reflect the alterations of their proteins. Our results demonstrated that the expression of Fas and FasL mRNA changed in the same manner as those of the proteins. It is assumed that NP resulted in greatly increased apoptosis of thymocytes as well as the decreased surviving thymocytes. The up-regulations of Fas and FasL occurred after 4 h in concomitant with the onset of apoptosis. These data suggested that NP might induce thymocytes apoptosis by activating Fas/FasL pathway. The studies by French groups are in agreement with ours although they treated the thymocytes with other apoptosis-induced agents (French et al., 1997). However, the present results could not exclude the possibility that NP-induced apoptosis were mediated by other pathways other than Fas/FasL pathway. There is increasing evidence that apoptosis can also be induced by a variety of signal transudation events leading to stimulation of calcium flux, cAMP production, PLC activation, inositol phosphate generation and mitochondrial membrane transition pore permeability. The extent to which each pathway contributes to the thymocyte apoptosis induced by NP remains to be explored (Bjomstrom and Sjoberg, 2002; Dos Santos et al., 2002; Staples et al., 1999). There is substantial evidence indicating that high levels of FasL can result in both increased Fasmediated apoptosis and the development of T cells that express Fas (Cheng et al., 1997; Krammer, 2000; Weih et al., 1996). It is known that apoptosis has an essential role in the immune system. Aberrations in genes encoding pro- or anti-apoptotic proteins or defects in apoptotic process in thymocytes have been implicated in the initiation of conditions such as immunodeficiency, autoimmunity and cancer (Opferman and Korsmeyer, 2003; Dulos and Bagchus, 2001). Therefore, it is likely that an alteration of the immune system by environmental estrogens, including NP, could change the individual’s ability of regulating immune homeostasis and host resistance to infection and tumor growth. It is known that NP has a weak estrogenic effect. This result here is consistent with other studies, which have shown that the addition of E2 to thymic organ culture could induce apoptosis (Barnden et al., 1997). The findings also are in agreement with our previous work in vivo, which demonstrated that administration of 17h-estradiol and NP to rats triggered apoptosis in thymocyte through Fas/FasL pathway (Yao and Hou, 2004a,b). Therefore, we postulate that NP and estrogen may induce thymocyte apoptosis in the same manner in vitro.
G. Yao et al. / Life Sciences 77 (2005) 3306–3320
3317
Several studies have shown that nonylphenol bonded to estrogen receptor and activated the transcription genes, which are regulated by some estrogen-like compounds. (Beato et al., 1995; Sakazaki et al., 2002; Yoon et al., 2001) The estrogenic effects of nonylphenol appear to be blocked by an estrogen receptor antagonist (Odum et al., 1997). Despite the results demonstrating that NP induced thymocyte apoptosis similar to estrogen, the question whether NP would act directly on thymocytes through estrogen receptor remained to be answered. Taken together, in the present study, we found for the first time that NP induced thymocyte apoptosis and up-regulated expression of Fas receptor and level of FasL. Our findings may provide new insight on the toxicity of NP. However, whether NP directly or indirectly participate transcriptional regulation of other genes and whether the immunological effect of NP are mediated through binding to estrogen receptors in thymocytes require further investigation.
Acknowledgement This work was supported by Nanjing University b985Q Project and the key medical project foundation of Nanjing City (2KG 0002). The authors wish to thank Zhigang Tu for technical assistance and critical discussion.
References Ansar Ahmed, S., 2000. The immune system as a potential target for environmental estrogens (endocrine disrupters): a new emerging field. Toxicology 150 (1–3), 191 – 206. Aoki, M., Kurasaki, M., Saito, T., Seki, S., Hosokawa, T., Takahashi, Y., Fujita, H., Iwakuma, T., 2004. Nonylphenol enhances apoptosis induced by serum deprivation in PC12 cells. Life Sciences 74 (18), 2301 – 2312. Arukwe, A., Kullman, S.W., Berg, K., Goksoyr, A., Hinton, D.E., 2002. Molecular cloning of rainbow trout (Oncorhynchus mykiss) eggshell zona radiata protein complementary DNA: mRNA expression in 17-h-estradiol- and nonylphenol-treated fish. Comparative Biochemistry and Physiology. B. Comparative Biochemistry 132 (2), 315 – 326. Barnden, M.J., Heath, W.R., Carbone, F.R., 1997. Down-modulation of CD8 beta-chain in response to an altered peptide ligand enables developing thymocytes to escape negative selection. Cellular Immunology 175 (2), 111 – 119. Beato, M., Herrich, P., Schutz, G., 1995. Steroid hormones receptors: many actors in search of a plot. Cell 83 (6), 851 – 857. Bjomstrom, L., Sjoberg, M., 2002. Signal transducers and activators of transcription as downstream targets of nongenomic estrogen receptor action. Molecular Endocrinology 16 (10), 2202 – 2214. Bragadin, M., Perin, G., Iero, A., Manente, S., Rizzoli, V., Scutari, G., 1999. An in vitro study on the toxic effects of nonylphenol(NP) in mitochondria. Chemosphere 38 (9), 1997 – 2001. Burkhardt-Holm, P., Wahli, T., Meier, W., 2000. Nonylphenol affects the granulation pattern of epidermal mucous cells in rainbow trout Oncorhynchus mykiss. Ecotoxicology and Environmental Safety 46 (1), 34 – 40. Cheng, J., Liu, C., Yang, P., Zhou, T., Mountz, J.D., 1997. Increased lymphocyte apoptosis in Fas ligand transgenic mice. Journal of Immunology 159 (2), 674 – 684. de Fougerolles Nunn, E., Greenstein, B., Khamashta, M., Hughes, G.R., 1999. Evidence for sexual dimorphism of estrogen receptor in hypothalamus and thymus of neonatal and immature Wistar rats. International Journal of Immunopharmacology 21 (12), 869 – 877. Do, Y., Ryu, S., Nagarkatti, M., Nagarkatti, P.S., 2002. Role of death receptor pathway in estradiol-induced T-cells apoptosis in vivo. Toxicological Sciences 70 (1), 63 – 72. Dos Santos, E.G., Dieudonne, M.N., Pecquery, R., Moal, V.L., Giudicelli, Y., Lacasa, D., 2002. Rapid nongenomic E2 effects on p42/p44 MAPK, activator protein-1, and cAMP response element binding protein in rat white adipocytes. Endocrinology 143 (3), 930 – 940.
3318
G. Yao et al. / Life Sciences 77 (2005) 3306–3320
Dulos, G.J., Bagchus, W.M., 2001. Androgens indirectly accerate thymocyte apoptosis. International Immunopharmacology 1 (2), 321 – 328. Eichhorst, S.T., Muerkoster, S., Weigand, M.A., Krammer, P.H., 2001. The chemotherapeutic drug 5-flurouacil induces apoptosis in mouse thymocytes in vivo via activation of the CD95 (APO-1/Fas) system. Cancer Research 61 (1), 243 – 248. Ferguson, S.A., Flynn, K.M., Delclos, K.B., Newbold, R.R., Gough, B.J., 2002. Effects of lifelong dietary exposure to genistein or nonylphenol on amphetamine-stimulated striatal dopamine release in male and female rats. Neurotoxicology and Teratology 24 (1), 37 – 45. Folmar, L.C., Hemmer, M.J., Denslow, N.D., Kroll, K., Chen, J., Cheek, A., Richman, H., Meredith, H., Grau, E.G., 2002. A comparison of the estrogenic potencies of estradiol, ethynylestradiol, diethylstilbestrol, nonylphenol and methoxychlor in vivo and in vitro. Aquatic Toxicology 60 (1–2), 101 – 110. French, L.E., Wilson, A., Hahne, M., Viard, I., Tschopp, J., Macdonald, H.R., 1997. Fas ligand expression is restricted to nonlymphoid thymic components in situ. Journal of Immunology 159 (5), 2196 – 2202. Fujimoto, Y., Sakuma, S., Inoue, T., Uno, E., Fujita, T., 2002. The endocrine disruptor nonylphenol preferentially blocks cyclooxygenase-1. Life Sciences 70 (19), 2209 – 2214. Gaillard, R.C., Spinedi, E., 1998. Sex- and stress-steroids interactions and the immune system: evidence for a neuroendocrineimmunological sexual dimorphism. Domestic Animal Endocrinology 15 (5), 345 – 352. Geenen, V., Poulin, J.F., Dion, M.L., Martens, H., Castermans, E., Hansenne, I., Moutschen, M., Sekaly, R.P., Cheynier, R., 2003. Quantification of T cell receptor rearrangement excision circles to estimate thymic function: an important new tool for endocrine-immune physiology. Journal of Endocrinology 176 (3), 305 – 311. Hengartner, M.O., 2000. The biochemistry of apoptosis. Nature 407 (6805), 770 – 776. Hughes, P.J., McLellan, H., Lowes, D.A., Khan, 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 Ca2+ pumps. Biochemical and Biophysical Research Communications 277 (3), 568 – 574. Iguchi, T., Watanabe, H., Katsu, Y., 2001. Developmental effects of estrogenic agents on mice, fish, and frogs: a mini-review. Hormones and Behavior 40 (2), 248 – 251. Iida, H., Maehara, K., Doiguchi, M., Mori, T., Yamada, F., 2003. Bisphenol A-induced apoptosis of cultured rats sertoli cells. Reproductive Toxicology 17 (4), 457 – 464. Jenkins, J.K., Suwannaroj, S., Elbourne, K.B., Ndebele, K., McMurray, R.W., 2001. 17-h-estradiol alter Jurkat lymphocyte cell cycling and induces apoptosis through suppression of Bcl-2 and cyclin A. International Immunopharmacology 1 (11), 1897 – 1911. Kamath, A.B., Camacho, I., Nagarkatti, P.S., Nagarkatti, M., 1999. Role of Fas–Fas ligand interactions in 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD)-induced immunotoxicity: increased resistance of thymocytes from Fas-deficient (lpr) and Fas ligand-defective (gld) mice to TCDD-induced toxicity. Toxicology and Applied Pharmacology 160 (2), 141 – 155. Karpuzoglu-Sahin, E., Hissong, B.D., Ansar Ahmed, S., 2001a. Interferon-g levels are upregulated by 17-h-estradiol and diethylstilbestrol. Journal of Reproductive Immunology 52 (1-2), 113 – 127. Karpuzoglu-Sahin, E., Zhi-Jun, Y., Lengi, A., Sriranganathan, N., Ansar Ahmed, S., 2001b. Effects of long-term estrogen treatment on IFN-g, IL-2 and IL-4 gene expression and protein synthesis in spleen and thymus of normal C57BL/6 mice. Cytokine 14 (4), 208 – 217. Kinnberg, K., Korsgaard, B., Bjerregaard, P., 2000. Concentration-dependent effects of nonylphenol on testis structure in adult platyfish. Marine Environmental Research 50 (1–5), 169 – 173. Krammer, P.H., 2000. CD95’s deadly mission in the immune system. Nature 407 (6805), 789 – 795. Lee, J., Richburg, J.H., Younkin, S.C., Boekelheide, K., 1997. The Fas system is a key regulator of germ cell apoptosis in the testis. Endocrinology 138 (5), 2081 – 2088. Lee, J., Richburg, J.H., Shipp, E.B., Meistrich, M.L., Boekelheide, K., 1999. The Fas system, a regulator of testicular germ cell apoptosis, is differentially up-regulated in sertoli cell versus germ cell injury of the testis. Endocrinology 140 (2), 852 – 858. Legler, J., Dennekamp, M., Vethaak, A.D., Brouwer, A., Koeman, J.H., van der Burg, B., Murk, A.J., 2002. Detection of estrogenic activity in sediment-associated compounds using in vitro reporter gene assays. Science of the Total Environment 293 (1–3), 69 – 83. Lindberg, M.K., Weihua, Z., Andersson, N., Moverare, S., Gao, H., Vidal, O., Erlandsson, M., Windahl, S., Andersson, G., Lubahn, D.B., Carlsten, H., Dahlman-Wright, K., Gustafsson, J.A., Ohlsson, C., 2002. Estrogen receptor specificity for the effects of estrogen in ovariectomized mice. Journal of Endocrinology 174 (2), 167 – 178.
G. Yao et al. / Life Sciences 77 (2005) 3306–3320
3319
Mor, G., Munoz, A., Redlinger Jr, R., Silva, I., Song, J., Lim, C., Kohen, F., 2001. The role of the Fas/Fas ligand system in estrogen-induced thymic alteration. American Journal of Reproductive Immunology 46 (4), 298 – 307. Nagao, T., Wada, K., Marumo, H., Yoshimura, S., Ono, H., 2001. Reproductive effects of nonylphenol in rats after gavage administration: a two-generation study. Reproductive Toxicology 15 (3), 293 – 315. Nair-Menon, J.U., Campbell, G.T., Blake, C.A., 1996. Toxic effects of octylphenol on cultured rat and murine splenocytes. Toxicology and Applied Pharmacology 139 (2), 437 – 444. Odum, J., Lefevre, P.A., Tittensor, S., Paton, D., Routledge, E.J., Bresford, N.A., Sumpter, J.R., Ashby, J., 1997. The rodent uterotrophic assay: critical protocol features, studies with nonylphenols and comparison with a yeast estrogenicity assay. Regulatory Toxicology and Pharmacology 25 (2), 176 – 188. Oka, T., Adati, N., Shinkai, T., Sakuma, K., Nishimura, T., Kurose, K., 2003. Bisphenol A induces apoptosis in central neural cells during early development of Xenopus laevis. Biochemical and Biophysical Research Communications 312 (4), 877 – 882. Okasha, S.A., Ryu, S., Do, Y., McKallip, R.J., Nagarkatti, M., Nagarkatti, P.S., 2001. Evidence for estradiol-induced apoptosis and dysregulated T cell maturation in the thymus. Toxicology 163 (1), 49 – 62. Opferman, J.T., Korsmeyer, S.J., 2003. Apoptosis in the development and maintenance of the immune system. Nature Immunology 4 (5), 410 – 415. Safe, S.H., Pallaroni, L., Yoon, K., Gaido, K., Ross, S., Saville, B., McDonnell, D., 2001. Toxicology of environmental estrogens. Reproduction, Fertility and Development 13 (4), 307 – 315. Sakazaki, H., Ueno, H., Nakamuro, K., 2002. Estrogen receptor a´ in mouse splenic lymphocytes: possible involvement in immunity. Toxicology Letters 133 (2–3), 221 – 229. Schwaiger, J., Mallow, U., Ferling, H., Knoerr, S., Braunbeck, T., Kalbfus, W., Negele, R.D., 2002. How estrogenic is nonylphenol? A transgenerational study using rainbow trout (Oncorhynchus mykiss) as a test organism. Aquatic Toxicology 59 (3–4), 177 – 189. Sepiashvili, R.I., Shubich, M.G., Kolesnikova, N.V., Slavyanskaya, T.A., Lomtatidze, L.V., 2001. Apoptosis in immunological processes. Clinical and Applied Immunology Reviews 1 (3–4), 163 – 172. Sharma, K., Wang, R.X., Zhang, L.Y., Yin, D.L., Luo, X.Y., Solomon, J.C., Jiang, R.F., Markos, K., Davidson, W., Scott, D.W., Shi, Y.F., 2000. Death the Fas way: regulation and pathophysiology of CD95 and its ligand. Pharmacology & Therapeutics 88 (3), 333 – 347. Sharva, N.I., Dzutsev, A.K., Litvina, M.M., Pleskovskaya, G.N., Kharechenko, T.Y., Yarilin, A.A., 2001. Thymic epithelial cells induce Fas-independent activation apoptosis of thymocytes. Immunology Letters 78, 201 – 207. Staples, J.E., Gasiewicz, T.A., Fiore, N.C., Lubahn, D.B., Korach, K.S., Siversterone, A.E., 1999. Estrogen receptor-a` is necessary in thymic development and estradiol-induced thymic alterations. Journal of Immunology 163 (8), 4168 – 4174. Sweeney, T., 2002. Is exposure to endocrine disrupting compounds during fetal/post-natal development affecting the reproductive potential of farm animals? Domestic Animal Endocrinology 23 (1–2), 203 – 209. Wang, X., Han, X., Yao, G., Wang, Y., 2003. Effect of nonylphenol on apoptosis of sertoli cell in vitro. Bulletin of Environmental Contamination and Toxicology 70 (5), 898 – 904. Weih, F., Ryseck, R., Chen, L., Bravo, R., 1996. Apoptosis of nur77/N10-transgenic thymocytes involves the Fas/Fas ligand pathway. Proceedings of the National Academy of Sciences of the United States of America 93 (11), 5533 – 5538. Yadetie, F., Male, R., 2002. Effects of 4-nonylphenol on gene expression of pituitary hormones in juvenile Atlantic salmon (Salmo salar). Aquatic Toxicology 58 (1–2), 113 – 129. Yao, G., Hou, Y., 2004a. Nonylphenol induces thymocyte apoptosis through Fas/FasL pathway by mimicking estrogen in vivo. Environmental Toxicology and Pharmacology 17 (1), 19 – 27. Yao, G., Hou, Y., 2004b. Thymic atrophy via estrogen-induced apoptosis is related to Fas/FasL pathway. International Immunopharmacology 4 (2), 213 – 221. Ying, G.G., Williams, B., Kookana, R., 2002. Environmental fate of alkylphenols and alkylphenol ethoxylates—a review. Environment International 28 (3), 215 – 226. Yokota, H., Miyashita, N., Yuasa, A., 2002. High glucuronidation activity of environmental estrogens in the carp (Cyprinus carpino) intestine. Life Sciences 71 (8), 887 – 898. Yoon, K., Pallaoni, L., Stoner, M., Gaido, K., Safe, S., 2001. Differential activation of wild-type and variant forms of estrogen receptor a by synthetic and natural estrogenic compounds using a promoter containing three estrogen-responsive elements. Journal of Steroid Biochemistry 78 (1), 25 – 32.
3320
G. Yao et al. / Life Sciences 77 (2005) 3306–3320
You, H.J., Choi, C.Y., Jeon, Y.J., Chung, Y.C., Kang, S.K., Hahm, K.S., Jeong, H.G., 2002. Suppression of inducible nitric oxide synthase and tumor necrosis factor-a expression by 4-nonylphenol in macrophages. Biochemical and Biophysical Research Communications 294 (4), 753 – 759. Zajchowski, S., Hoffman-Goetz, L., 2000. Supraphysiological level of estrogen exposure in vivo increases lymphoid cell death in mice. Life Sciences 66 (15), 1451 – 1459.
Nonylphenol-induced thymocyte apoptosis is related to Fas/FasL pathway Genhong Yao a, Yali Hu b, Junfeng Liang c, Yayi Hou a,T a
Immunology and Reproductive Biology Lab, Medical School & State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR China b The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing 210008, PR China c Department of Chemistry and Chemical Biology, Stevens Institute of Technology, Castle Point on Hudson, Hoboken, NJ 07030, USA Received 29 November 2004; accepted 2 May 2005
Abstract Nonylphenol (NP) is the final biodegradation product of nonylphenol polyethoxylates, which are widely used as surfactants in domestic and industrial products. NP has been reported to have estrogenic activity and shown to have potential reproductive toxicity. However, its influence on immune system function remains unclear. In this study, to determine the immunological effects of NP, the effects of NP on apoptosis and Fas/FasL gene expression in rat thymocyte in vitro were investigated. Thymocytes were treated with NP 0.1, 1, and 10 ppm, respectively. Viable cell numbers were determined by MTT assay. Apoptotic cells were identified by DNA fragment analysis. A semi-quantitative reverse transcriptase-polymerase chain reaction method was used to analyze Fas and FasL mRNA levels. Fas and FasL protein expression was evaluated by flow cytometry. The results showed that NP decreased the cellularity; induced apoptotic death and enhanced the expression of Fas and FasL mRNA as well as proteins in thymocytes. These findings suggest that NP may induce apoptosis by altering the expression of Fas and FasL in thymocytes so as to affect the immune system function. D 2005 Elsevier Inc. All rights reserved. Keywords: Nonylphenol; Thymocyte; Apoptosis; Fas/FasL
T Corresponding author. Tel.: +86 25 83686441; fax: +86 25 83686441. E-mail address: [email protected] (Y. Hou). 0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2005.05.035
G. Yao et al. / Life Sciences 77 (2005) 3306–3320
3307
Introduction Nonylphenol polyethoxylates are non-ionic surfactant widely used as components of detergents, paints, herbicides, wetting agents, cosmetics, pesticides and many other industrial and agricultural products (Arukwe et al., 2002; Bragadin et al., 1999; Burkhardt-Holm et al., 2000; Ferguson et al., 2002; You et al., 2002). They are discharged to aquatic ecosystems through sewage treatment, pulp mill, and industrial effluent, urban and agricultural runoff and so on (Ying et al., 2002). In the aquatic ecosystems, nonylphenol polyethoxylates are degraded by the actions of bacteria and finally form nonylphenol (NP) (Kinnberg et al., 2000). Studies showed that the concentration of NP in the aquatic environment, particularly in sediment, could reach up to 300 ppb (Nagao et al., 2001). Then NP is accumulated in our bodies through food chain. A convincing body of evidence indicates that NP is one of the potential endocrine disruptors and possesses estrogen-like properties (Folmar et al., 2002; Fujimoto et al., 2002; Hughes et al., 2000; Iguchi et al., 2001; Legler et al., 2002; Schwaiger et al., 2002; Yadetie and Male, 2002). Several studies also have shown that nonylphenol bound to estrogen receptor and activated the transcription genes, which are regulated by some estrogen-like compounds (Beato et al., 1995; Sakazaki et al., 2002; Yoon et al., 2001; Safe et al., 2001). Aoki reported that NP enhanced apoptosis induced by serum deprivation in PC12 cells (Aoki et al., 2004). Recent review suggests that the immune system also may serve as a potential target for NP (Ansar Ahmed, 2000). Natural estrogens, such as 17hestradiol, modulate immune responses and contributed to the etiology of immune-mediated diseases (Zajchowski and Hoffman-Goetz, 2000; Karpuzoglu-Sahin et al., 2001a,b; Geenen et al., 2003; Gaillard and Spinedi, 1998; Lindberg et al., 2002). Studies of other groups showed that the addition of E2 to thymic organ culture could induce apoptosis (Barnden et al., 1997). Our previous studies also demonstrated that in vivo administration of 17h-estradiol to rats triggered apoptosis in thymocyte through Fas/FasL pathway (Yao and Hou, 2004b). If natural estrogens have strong immunomodulatory properties and induce apoptosis, it may be reasonable to postulate that NP also modulates the immune system. However, until recently most research has focused on the toxicity of NP on development process and reproductive system, especially in aquatic ecosystem (Kinnberg et al., 2000; Sweeney, 2002; Yokota et al., 2002). We also found that NP disrupted the reproductive system by dramatically increasing Sertoli cells apoptosis in rats, which was mediated by Fas/FasL pathway (Wang et al., 2003). Therefore, to evaluate estrogenic effect of NP on the immune system, the apoptotic cell death and the expression of Fas and FasL in the thymocyte was detected with treatment of NP in the present study. Materials and methods Reagents Nonylphenol (NP) was purchased from Tokyo Kasei Kogyo Co. (Tokyo, Japan). Trizol regent was purchased from BioBasic Inc (Scarborough Ontario,Canada). Taq polymerase enzyme, M-MLV Reverse Transcriptase and rRNasin (Ribonuclease Inhibitor) were from Promega (Madison, WI, USA). Monoclonal anti-Fas and anti-FasL antibody and a FITC-labeled (fluorescein-isothiocyanate) secondary
3308
G. Yao et al. / Life Sciences 77 (2005) 3306–3320
antibody were from Boster-Biotechnology (Wuhan, China). All other reagents were of the highest grade commercially available. Tissue collection and single cell preparation The thymus was obtained from normal 3-week-old rats. The rats were killed by cervical dislocation. The thymus was removed immediately. To prepare a single-cell suspension, the cells were teased out in chilled phosphate-buffed saline (PBS) and filtered through a sterile wire mesh. The cells were centrifuged at 400 g for 5 min, and pelleted thymus cells were resuspended in Tris-bufered 0.83% NH4Cl solution to lyse erythrocytes. The resulting thymus cells were washed in ice-cold PBS three times and cells density was adjusted to 1 106 cells/mL. The viability of such cells was determined by trypan blue dye exclusion. It was more than 95% in each specimen routinely. A total of 1 105 thymus cells were dispersed into plastic 96-well round-bottomed microtiter plates and incubated with different concentrations of NP at 37 8C in humidified 5% CO2 atmosphere (final volume 100 AL/ well). Determination of viable cell numbers Cell viability was determined by an MTT assay using the cell proliferation Kit (Sigma USA). One hundred microtiter of thymocyte at a concentration of 1 105 cells/mL was plated into each well of a 96well round-bottom tissue-culture plate. Then different concentrations of NP were added to wells. After different time of incubation at 37 8C in humidified 5% CO2, 20 AL of MTT dissolved in PBS was added to cells for 4 h to give a final concentration of 0.1 mg/mL. The blue crystals were dissolved by the addition of SDS in HCl. An automated microplate reader (Bio-Rad Model 550) was used to measure the light absorbance value at 570 nm. DNA laddering detection For examination of apoptosis by electrophoresis of nucleosomal fragments, we used a standard procedure precipitating cytosolic nucleic acid (Jenkins et al., 2001). Briefly, 1 106 NP-treated thymocytes were pelleted (200 g, 5 min) and lysed for 15 min (250 Al, 0.4% Triton-X, 20 mM Tris, 0.4 mM EDTA) at 4 8C. Nuclei were then pelleted (13 000 g, 5 min, 4 8C) and the supernatant was transferred to a clean microfuge tube. Nucleosomal fragments were precipitated overnight with an equal volume of isopropanol after adjustment to 0.5 M NaCl. The pellet was washed twice in 70% ethanol, dried briefly, and resuspended in 40 Al TE (10 mM Tris–HCl, 0.1 mM EDTA, pH 8.8). The extracted DNA was mixed with the loading buffer. The DNA was then separated on 1.5% agarose gel containing 0.5 Ag/mL ethidium bromide. The gel was put on an UVtransilluminator and photographed. Fragmented DNA, shown as DNA ladder in the gel, represents the existence of apoptotic cell death. RNA isolation and reverse transcription-polymerase chain reaction Total RNAs were isolated from thymocytes by the Trizol method. RNA concentrations were quantified by spectrophotometer at 260 nm. A 2 Ag total RNA was reverse-transcribed using reverse
G. Yao et al. / Life Sciences 77 (2005) 3306–3320
3309
transcription-polymerase chain reaction (Perkin-Elmer Cetus DNA Thermal Cycler, Perkin-Elmer, CT, USA). Then the first strand complementary was gained in the presence of M-MLV reverse transcriptase and oligo-dT primers. After RT reaction, 2 AL of the incubation mixture was used as the template for the following PCR. The following components were added to the mixture: 5 AL 10 PCR buffer, 1 AL (10 pmol) of both sense and antisense primers (for Fas and h-actin, or for FasL and h-actin), 1 AL 10 mM mixture of all four deoxynucleotide triphosphates, 1 AL Taq DNA polymerase, and 12 AL nuclease-free water to adjust the final volume to 25 AL. The primer sequences used for PCR are shown below. After an initial incubation at 94 8C for 1 min, temperature cycling was initiated with each cycle as following: denaturation at 94 8C for 30 s, annealing at 65 8C for 1 min and elongation at 72 8C for 1 min. Then the reaction followed by a second round of 35 cycles of denaturation at 94 8C for 30 s, annealing at 65 8C for 1 min and elongation at 72 8C for 1 min. And the reactions were terminated by incubation at 72 8C for 5 min. The PCR conditions and the number of cycles were carefully chosen and have been described in previous reports (Lee et al., 1997; Lee et al., 1999). The PCR products were then separated on 1.5% agarose gel containing 0.5 `ıg/mL ethidium bromide. The gel was put on an UV-transilluminator and photographed. The Fas and FasL signal was measured by a densitometer and standardized against the hactin signal using a digital imaging and analysis system. Oligonucleotide primers for PCR reactions Based on the cDNA sequences referred to Lee et al. [Lee et al., 1997, 1999], primers were synthesized by Sangon Co (Shanghai, China). cDNA sequences and sizes of the amplified fragments are listed as follows: Fas, 5V-ATGCACACTCTGCGATGAAG -3V and V-ATGCACACTCTGCGATGAAG-3V (PCR product: 240 bp); FasL, 5V-GGAATGGGAAGACACATATGGAACTGC-3V and V-CATATCTGGCCAGTAGTGCAGTAATTC-3V (PCR product: 238 bp); h-actin, 5V-AGGCATCCTGACCCTGAA GTAC-3V and 5V-TCTTCATGAGGTAGTCTGTCAG-3V (PCR product: 389 bp). Flow cytometry analysis Expressions of Fas and FasL proteins were detected by anti-Fas and anti-FasL antibody and a FITC-labeled (fluorescein-isothiocyanate) secondary antibody (Boster-Biotechnology, Wuhan, China). Briefly, the prepared cell suspension was incubated with unconjugated monoclonal rabbit anti-mouse antibodies for 30 min on ice. The cells were then washed, followed by secondary antibody, FITCconjugated goat anti-rabbit antibody for 30 min on ice and washed twice. As a negative control, an aliquot of the cells from each case was stained with an irrelevant antibody of the same phenotype and a secondary antibody. Flow cytometric analysis was performed on a FACScan instrument (Becton Dickinson, San Jose, CA, USA) and data were processed by using the CellQuest program (Becton Dickinson). Forward scatter threshold was set to exclude debris from viable nucleated cells and the data were collected on 10 000 cells per sample. Statistical analysis Results were shown on the mean F S.E.M. Statistical significance between groups was analyzed by one-way ANOVA followed by the Student–Newman–Keuls Multiple Comparisons tests. A P value of b 0.05 was considered significant.
3310
G. Yao et al. / Life Sciences 77 (2005) 3306–3320
Results NP decreases the cellularity of thymocytes To determine the role of the NP on cellularity of thymocytes, thymocytes were treated with different doses of NP for 2, 4 and 6 h, respectively. The cell viability was determined by an MTT assay. Fig. 1 showed that cell survival did not cause significantly alteration in 2 h, in all the doses we used. However, the decrease of cell viability was observed in all the doses after treatment for 4 and 6 h. Furthermore, as shown in Fig. 2, the effect of NP on cellularity of thymocytes was time dependent. NP induces apoptosis of thymocytes According to Hughes (Hughes et al., 2000), DNA laddering is commonly used to establish if a decrease in cell viability is due to apoptosis rather than necrosis. Fig. 3B and C showed that exposure of thymocytes to 10, 1, and 0.1 ppm NP provoked DNA laddering in 4 and 6 h, suggesting that NP induced cell death by apoptosis. The results are consistent with observations of decreased cellularity of thymocytes by NP treatment. NP stimulates expression of Fas and FasL mRNA Semi-quantitative RT-PCR was performed on total RNA from control and NP-treated thymocytes to evaluate the expression levels of Fas and FasL mRNA. As shown in Fig. 4B, our results indicated that
Fig. 1. NP decreased the viable cells number of thymocytes. The cell viability was determined by an MTT assay. The results are presented as mean F S.E. with triplicate measurement. NP1, NP2, NP3 represent NP at levels of 0.1, 1, and 10 ppm respectively. *P b 0.05 vs. control. #P b 0.05 vs. NP1.
G. Yao et al. / Life Sciences 77 (2005) 3306–3320
3311
Fig. 2. Time dependent characteristics of NP on viabilities of thymocytes. The cell viability was determined by an MTT assay. The results are presented as mean F S.E. with triplicate measurement. NP1, NP2, NP3 represent NP at levels of 0.1, 1, and 10 ppm respectively. *P b 0.05 vs. 2 h. #P b 0.05 vs. 4 h.
Fig. 3. Agarose gel electrophoresis of DNA fragments in thymocytes cultured for 2, 4 and 6 h. Thymocytes were pretreated with different concentrations of NP. Fragmented DNA was collected and assessed by agarose gel electrophoresis containing ethidium bromide. Data shown are representative of three separate experiments. M stands for DNA marker. C stands for control. 1, 2, 3 represent NP at the concentrations of 0.1, 1, and 10 ppm respectively.
3312
G. Yao et al. / Life Sciences 77 (2005) 3306–3320
Fig. 4. Effects of NP on Fas mRNA transcripts in thymus Fas mRNA (lower lane) was determined by RT-PCR. PCR amplication of the housekeeping gene, h-actin (upper lane), was performed for each sample. (A) The representative agarose gel electrophoresis of three independent experiments is shown. PCR products of cDNA derived from vehicle control (1), NP (2, 3, 4 represent NP at levels of 0.1, 1, and 10 ppm respectively). (B) Densitometric analysis. The intensity of the band was scanned. The quotient of Fas/h-actin gene was calculated. The data shown are the average of three independent experiments including cDNA synthesis and PCR analysis. The results are shown as mean F S.E. from three representative independent experiments. *The difference from rats treated with the vehicle alone was statistically significant P b 0.05.
the expression of Fas mRNA was increased in NP 10, 1, and 0.1 ppm treated groups at 4 and 6 h. However, relative shorter time of 2 h NP treatment appeared no effect on Fas mRNA compared with control. The Fig. 4A showed the representative gel pattern. As for the expression of FasL, no significant alteration of FasL mRNA by NP treatment was shown in 2 h compared with vehicle, while up-regulation of FasL mRNA was observed in thymocytes exposed to NP for 4 and 6 h. Moreover the expressions of FasL mRNA were clearly enhanced in dose-dependent for 6 h (Fig. 5B). The Fig. 5A showed the representative gel pattern. NP increases expression of Fas and FasL proteins It is important to underline the fact that the susceptibility to Fas-induced apoptosis is usually related to the Fas and FasL proteins and not to their mRNA. Therefore, the expressions of Fas and FasL proteins in thymocytes treated with or without NP were evaluated by flow cytometry. As shown in Figs. 6 and 7, our results showed that relative shorter time of 2 h NP treatment appeared no effect on Fas and FasL proteins compared with control ( p N 0.5). However, when NP was added to culture for 4 h, the levels of Fas and FasL proteins were moderately increased compared with control at any concentration (Fas: control 0.58%, NP 0.1 ppm 2.17%, NP 1 ppm 2.32%, NP 10 ppm
G. Yao et al. / Life Sciences 77 (2005) 3306–3320
3313
Fig. 5. Effects of NP on FasL mRNA transcripts in thymus FasL mRNA(lower lane) was determined by RT-PCR. PCR amplication of the housekeeping gene, h-actin (upper lane), was performed for each sample. (A) The representative agarose gel electrophoresis of three independent experiments is shown. PCR products of cDNA derived from vehicle control (1), NP (2, 3, 4 represent NP at levels of 0.1, 1, and 10 ppm respectively). (B) Densitometric analysis. The intensity of the band was scanned. The quotient of FasL/h-actin gene was calculated. The data shown are the average of three independent experiments including cDNA synthesis and PCR analysis. The results are shown as mean F S.E. from three representative independent experiments. *The difference from rats treated with the vehicle alone was statistically significant P b 0.05. #The difference from rats treated with 0.1 ppm was statistically significant P b 0.05.
3.17%; FasL: control 0.66%, NP 0.1 ppm 2.97%, NP 1 ppm 3.16%, NP 10 ppm 4.14%;). Figs. 6 and 7 also illustrated that at the doses tested, NP significantly increase the Fas and FasL proteins in 6 h (Fas: control 0.70%, NP 0.1 ppm 5.87%, NP 1 ppm 6.93%, NP 10 ppm 7.09%; FasL: control 0.73%, NP 0.1 ppm 4.76%, NP 1 ppm 5.32%, NP 10 ppm 5.80%).
Discussion NP is one of biodegradation products of nonylphenol polyethoxylates and widely presents in the natural world. So it is practically unavoidable to contaminate our living environment. Moreover, NP can mimic natural hormones and disrupt the functions of endocrine, immune and reproductive system of living beings (Ying et al., 2002). Concern has been increased in recent years about the NP in recent years, but little is known about the toxicological effect of NP on immunocompetent cells. To address NP to cause animal and human immune disorders, the present in vitro study focuses on the effects of NP on the thymocytes, a type of important immunocompetent cells. Furthermore, there have been many evidences to suggest that NP may induce thymocyte apoptosis via regulating the expression of Fas and
3314
G. Yao et al. / Life Sciences 77 (2005) 3306–3320
NP(0.1ppm)
NP(1ppm)
NP(10ppm)
2h
4h
6h
Fig. 6. Flow cytometric analysis of Fas expression on thymocytes after NP treatment. Thymocytes were treated with different concentrations of NP for 2, 4 and 6 h respectively. Then thymocytes were labeled with Abs as described in Materials and methods. Control (Fas expression on untreated thymocytes) is shown as a thin line, and NP treatment is shown as a bold line. The results shown were representative of a series of at least three experiments.
FasL by mimicking estrogen. (1) NP has been found to act like estrogen; (2) studies showed estrogens influence the development and function of immune system, particularly the thymocytes (Okasha et al., 2001); (3) the substantial evidence indicated that estrogen receptors (de Fougerolles Nunn et al., 1999) and Fas/FasL (Mor et al., 2001; Sharva et al., 2001) were present in the thymocytes and their expression are regulated by estrogens. Therefore in the study we investigated the effects of NP on the apoptosis, expression of the Fas and FasL mRNA in thymocytes for exploring the immunological consequences of exposure to NP. NP decreased viable cell numbers of thymocytes. After exposure to all doses of NP we used in the study for 4 and 6 h, the cell viability significantly was decreased. The decrease was independent of doses of NP, but showed in a time dependent pattern. NP treatment of up to 4 h caused apoptotic cell death of thymocytes in vitro. To explore whether the decreased numbers of viable thymocytes were due to the induction of apoptosis or not, we determined the apoptosis of thymocytes by DNA ladder. The results were that NP treatment (10, 1,
G. Yao et al. / Life Sciences 77 (2005) 3306–3320
NP(0.1ppm)
NP(1ppm)
3315
NP(10ppm)
2h
4h
6h
Fig. 7. Flow cytometric analysis of FasL expression on thymocytes after NP treatment. Thymocytes were treated with different concentrations of NP for 2, 4 and 6 h respectively. Then thymocytes were labeled with Abs as described in Materials and methods. Control (FasL expression on untreated thymocytes) is shown as a thin line, and NP treatment is shown as a bold line. The results shown were representative of a series of at least three experiments.
and 0.1 ppm) for 4 and 6 h all induced cell damage compared to control. These findings are in agreement with our previous work, which showed that NP in vivo induced thymocytes apoptosis (Yao and Hou, 2004a). In addition, Nair-Menon groups demonstrated that exposure of splenic lymphocytes from rats and mice to octylphenol, an endocrine disrupter with estrogenic activity like NP, induced apoptosis (Nair-Menon et al., 1996). These observations apparently support our hypothesis that the decreased thymocyte viability by NP treatment may result from activation of apoptosis. The apoptosis-inducing cell death of NP has gained experimental support from studies of other environmental estrogens, including bisphenol A. Bisphenol A, known to be another a xenoestrogen, is widely used in industry. Studies shown that bisphenol A could induce apoptosis in a variety of cell types, such as stertoli cells, ovarian granulose cells and some specific nervous system cells (Hughes et al., 2000; Iida et al., 2003; Oka et al., 2003).
3316
G. Yao et al. / Life Sciences 77 (2005) 3306–3320
The expressions of Fas and FasL mRNA in the thymocytes were increased after NP treatment. According to Hengartner group, apoptosis in thymocytes is triggered mainly through two pathways: the death receptor pathway and the mitochondrial pathway (Hengartner, 2000). Fas is a death receptor expressed on immune cells. Ligation of this receptor by Fas ligand (FasL), a member of the tumor necrosis factor family, triggers the induction of apoptosis in cells (Do et al., 2002). Studies by independent groups show that Fas/FasL (CD95/CD95L) interaction is directly responsible for this kind of activation, which induced cell death along with the fact that activated lymphocytes express the receptor protein Fas and its ligand FasL (Sepiashvili et al., 2001; Sharma et al., 2000). The thymocytes surface receptor Fas and its ligand (FasL) are mediators of apoptosis. These mediators have been shown to act in the immune system: (1) to limit over-expression of an immune response by deleting unwanted lymphocytes; (2) to eliminate auto reactive T cells during thymus selection. Other studies also demonstrated that Fas/FasL interaction played an important role in chemicals or chemotherapeutic drug induced apoptosis (Eichhorst et al., 2001; Kamath et al., 1999). The results we obtained illustrated that NP at the level of 0.1; 1, and 10 ppm increased the Fas and FasL mRNA expression in the thymocytes after exposure for 4 and 6 h. It should be pointed out that the upregulations of Fas and FasL mRNA after NP treatment might not reflect the alterations of their proteins. Our results demonstrated that the expression of Fas and FasL mRNA changed in the same manner as those of the proteins. It is assumed that NP resulted in greatly increased apoptosis of thymocytes as well as the decreased surviving thymocytes. The up-regulations of Fas and FasL occurred after 4 h in concomitant with the onset of apoptosis. These data suggested that NP might induce thymocytes apoptosis by activating Fas/FasL pathway. The studies by French groups are in agreement with ours although they treated the thymocytes with other apoptosis-induced agents (French et al., 1997). However, the present results could not exclude the possibility that NP-induced apoptosis were mediated by other pathways other than Fas/FasL pathway. There is increasing evidence that apoptosis can also be induced by a variety of signal transudation events leading to stimulation of calcium flux, cAMP production, PLC activation, inositol phosphate generation and mitochondrial membrane transition pore permeability. The extent to which each pathway contributes to the thymocyte apoptosis induced by NP remains to be explored (Bjomstrom and Sjoberg, 2002; Dos Santos et al., 2002; Staples et al., 1999). There is substantial evidence indicating that high levels of FasL can result in both increased Fasmediated apoptosis and the development of T cells that express Fas (Cheng et al., 1997; Krammer, 2000; Weih et al., 1996). It is known that apoptosis has an essential role in the immune system. Aberrations in genes encoding pro- or anti-apoptotic proteins or defects in apoptotic process in thymocytes have been implicated in the initiation of conditions such as immunodeficiency, autoimmunity and cancer (Opferman and Korsmeyer, 2003; Dulos and Bagchus, 2001). Therefore, it is likely that an alteration of the immune system by environmental estrogens, including NP, could change the individual’s ability of regulating immune homeostasis and host resistance to infection and tumor growth. It is known that NP has a weak estrogenic effect. This result here is consistent with other studies, which have shown that the addition of E2 to thymic organ culture could induce apoptosis (Barnden et al., 1997). The findings also are in agreement with our previous work in vivo, which demonstrated that administration of 17h-estradiol and NP to rats triggered apoptosis in thymocyte through Fas/FasL pathway (Yao and Hou, 2004a,b). Therefore, we postulate that NP and estrogen may induce thymocyte apoptosis in the same manner in vitro.
G. Yao et al. / Life Sciences 77 (2005) 3306–3320
3317
Several studies have shown that nonylphenol bonded to estrogen receptor and activated the transcription genes, which are regulated by some estrogen-like compounds. (Beato et al., 1995; Sakazaki et al., 2002; Yoon et al., 2001) The estrogenic effects of nonylphenol appear to be blocked by an estrogen receptor antagonist (Odum et al., 1997). Despite the results demonstrating that NP induced thymocyte apoptosis similar to estrogen, the question whether NP would act directly on thymocytes through estrogen receptor remained to be answered. Taken together, in the present study, we found for the first time that NP induced thymocyte apoptosis and up-regulated expression of Fas receptor and level of FasL. Our findings may provide new insight on the toxicity of NP. However, whether NP directly or indirectly participate transcriptional regulation of other genes and whether the immunological effect of NP are mediated through binding to estrogen receptors in thymocytes require further investigation.
Acknowledgement This work was supported by Nanjing University b985Q Project and the key medical project foundation of Nanjing City (2KG 0002). The authors wish to thank Zhigang Tu for technical assistance and critical discussion.
References Ansar Ahmed, S., 2000. The immune system as a potential target for environmental estrogens (endocrine disrupters): a new emerging field. Toxicology 150 (1–3), 191 – 206. Aoki, M., Kurasaki, M., Saito, T., Seki, S., Hosokawa, T., Takahashi, Y., Fujita, H., Iwakuma, T., 2004. Nonylphenol enhances apoptosis induced by serum deprivation in PC12 cells. Life Sciences 74 (18), 2301 – 2312. Arukwe, A., Kullman, S.W., Berg, K., Goksoyr, A., Hinton, D.E., 2002. Molecular cloning of rainbow trout (Oncorhynchus mykiss) eggshell zona radiata protein complementary DNA: mRNA expression in 17-h-estradiol- and nonylphenol-treated fish. Comparative Biochemistry and Physiology. B. Comparative Biochemistry 132 (2), 315 – 326. Barnden, M.J., Heath, W.R., Carbone, F.R., 1997. Down-modulation of CD8 beta-chain in response to an altered peptide ligand enables developing thymocytes to escape negative selection. Cellular Immunology 175 (2), 111 – 119. Beato, M., Herrich, P., Schutz, G., 1995. Steroid hormones receptors: many actors in search of a plot. Cell 83 (6), 851 – 857. Bjomstrom, L., Sjoberg, M., 2002. Signal transducers and activators of transcription as downstream targets of nongenomic estrogen receptor action. Molecular Endocrinology 16 (10), 2202 – 2214. Bragadin, M., Perin, G., Iero, A., Manente, S., Rizzoli, V., Scutari, G., 1999. An in vitro study on the toxic effects of nonylphenol(NP) in mitochondria. Chemosphere 38 (9), 1997 – 2001. Burkhardt-Holm, P., Wahli, T., Meier, W., 2000. Nonylphenol affects the granulation pattern of epidermal mucous cells in rainbow trout Oncorhynchus mykiss. Ecotoxicology and Environmental Safety 46 (1), 34 – 40. Cheng, J., Liu, C., Yang, P., Zhou, T., Mountz, J.D., 1997. Increased lymphocyte apoptosis in Fas ligand transgenic mice. Journal of Immunology 159 (2), 674 – 684. de Fougerolles Nunn, E., Greenstein, B., Khamashta, M., Hughes, G.R., 1999. Evidence for sexual dimorphism of estrogen receptor in hypothalamus and thymus of neonatal and immature Wistar rats. International Journal of Immunopharmacology 21 (12), 869 – 877. Do, Y., Ryu, S., Nagarkatti, M., Nagarkatti, P.S., 2002. Role of death receptor pathway in estradiol-induced T-cells apoptosis in vivo. Toxicological Sciences 70 (1), 63 – 72. Dos Santos, E.G., Dieudonne, M.N., Pecquery, R., Moal, V.L., Giudicelli, Y., Lacasa, D., 2002. Rapid nongenomic E2 effects on p42/p44 MAPK, activator protein-1, and cAMP response element binding protein in rat white adipocytes. Endocrinology 143 (3), 930 – 940.
3318
G. Yao et al. / Life Sciences 77 (2005) 3306–3320
Dulos, G.J., Bagchus, W.M., 2001. Androgens indirectly accerate thymocyte apoptosis. International Immunopharmacology 1 (2), 321 – 328. Eichhorst, S.T., Muerkoster, S., Weigand, M.A., Krammer, P.H., 2001. The chemotherapeutic drug 5-flurouacil induces apoptosis in mouse thymocytes in vivo via activation of the CD95 (APO-1/Fas) system. Cancer Research 61 (1), 243 – 248. Ferguson, S.A., Flynn, K.M., Delclos, K.B., Newbold, R.R., Gough, B.J., 2002. Effects of lifelong dietary exposure to genistein or nonylphenol on amphetamine-stimulated striatal dopamine release in male and female rats. Neurotoxicology and Teratology 24 (1), 37 – 45. Folmar, L.C., Hemmer, M.J., Denslow, N.D., Kroll, K., Chen, J., Cheek, A., Richman, H., Meredith, H., Grau, E.G., 2002. A comparison of the estrogenic potencies of estradiol, ethynylestradiol, diethylstilbestrol, nonylphenol and methoxychlor in vivo and in vitro. Aquatic Toxicology 60 (1–2), 101 – 110. French, L.E., Wilson, A., Hahne, M., Viard, I., Tschopp, J., Macdonald, H.R., 1997. Fas ligand expression is restricted to nonlymphoid thymic components in situ. Journal of Immunology 159 (5), 2196 – 2202. Fujimoto, Y., Sakuma, S., Inoue, T., Uno, E., Fujita, T., 2002. The endocrine disruptor nonylphenol preferentially blocks cyclooxygenase-1. Life Sciences 70 (19), 2209 – 2214. Gaillard, R.C., Spinedi, E., 1998. Sex- and stress-steroids interactions and the immune system: evidence for a neuroendocrineimmunological sexual dimorphism. Domestic Animal Endocrinology 15 (5), 345 – 352. Geenen, V., Poulin, J.F., Dion, M.L., Martens, H., Castermans, E., Hansenne, I., Moutschen, M., Sekaly, R.P., Cheynier, R., 2003. Quantification of T cell receptor rearrangement excision circles to estimate thymic function: an important new tool for endocrine-immune physiology. Journal of Endocrinology 176 (3), 305 – 311. Hengartner, M.O., 2000. The biochemistry of apoptosis. Nature 407 (6805), 770 – 776. Hughes, P.J., McLellan, H., Lowes, D.A., Khan, 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 Ca2+ pumps. Biochemical and Biophysical Research Communications 277 (3), 568 – 574. Iguchi, T., Watanabe, H., Katsu, Y., 2001. Developmental effects of estrogenic agents on mice, fish, and frogs: a mini-review. Hormones and Behavior 40 (2), 248 – 251. Iida, H., Maehara, K., Doiguchi, M., Mori, T., Yamada, F., 2003. Bisphenol A-induced apoptosis of cultured rats sertoli cells. Reproductive Toxicology 17 (4), 457 – 464. Jenkins, J.K., Suwannaroj, S., Elbourne, K.B., Ndebele, K., McMurray, R.W., 2001. 17-h-estradiol alter Jurkat lymphocyte cell cycling and induces apoptosis through suppression of Bcl-2 and cyclin A. International Immunopharmacology 1 (11), 1897 – 1911. Kamath, A.B., Camacho, I., Nagarkatti, P.S., Nagarkatti, M., 1999. Role of Fas–Fas ligand interactions in 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD)-induced immunotoxicity: increased resistance of thymocytes from Fas-deficient (lpr) and Fas ligand-defective (gld) mice to TCDD-induced toxicity. Toxicology and Applied Pharmacology 160 (2), 141 – 155. Karpuzoglu-Sahin, E., Hissong, B.D., Ansar Ahmed, S., 2001a. Interferon-g levels are upregulated by 17-h-estradiol and diethylstilbestrol. Journal of Reproductive Immunology 52 (1-2), 113 – 127. Karpuzoglu-Sahin, E., Zhi-Jun, Y., Lengi, A., Sriranganathan, N., Ansar Ahmed, S., 2001b. Effects of long-term estrogen treatment on IFN-g, IL-2 and IL-4 gene expression and protein synthesis in spleen and thymus of normal C57BL/6 mice. Cytokine 14 (4), 208 – 217. Kinnberg, K., Korsgaard, B., Bjerregaard, P., 2000. Concentration-dependent effects of nonylphenol on testis structure in adult platyfish. Marine Environmental Research 50 (1–5), 169 – 173. Krammer, P.H., 2000. CD95’s deadly mission in the immune system. Nature 407 (6805), 789 – 795. Lee, J., Richburg, J.H., Younkin, S.C., Boekelheide, K., 1997. The Fas system is a key regulator of germ cell apoptosis in the testis. Endocrinology 138 (5), 2081 – 2088. Lee, J., Richburg, J.H., Shipp, E.B., Meistrich, M.L., Boekelheide, K., 1999. The Fas system, a regulator of testicular germ cell apoptosis, is differentially up-regulated in sertoli cell versus germ cell injury of the testis. Endocrinology 140 (2), 852 – 858. Legler, J., Dennekamp, M., Vethaak, A.D., Brouwer, A., Koeman, J.H., van der Burg, B., Murk, A.J., 2002. Detection of estrogenic activity in sediment-associated compounds using in vitro reporter gene assays. Science of the Total Environment 293 (1–3), 69 – 83. Lindberg, M.K., Weihua, Z., Andersson, N., Moverare, S., Gao, H., Vidal, O., Erlandsson, M., Windahl, S., Andersson, G., Lubahn, D.B., Carlsten, H., Dahlman-Wright, K., Gustafsson, J.A., Ohlsson, C., 2002. Estrogen receptor specificity for the effects of estrogen in ovariectomized mice. Journal of Endocrinology 174 (2), 167 – 178.
G. Yao et al. / Life Sciences 77 (2005) 3306–3320
3319
Mor, G., Munoz, A., Redlinger Jr, R., Silva, I., Song, J., Lim, C., Kohen, F., 2001. The role of the Fas/Fas ligand system in estrogen-induced thymic alteration. American Journal of Reproductive Immunology 46 (4), 298 – 307. Nagao, T., Wada, K., Marumo, H., Yoshimura, S., Ono, H., 2001. Reproductive effects of nonylphenol in rats after gavage administration: a two-generation study. Reproductive Toxicology 15 (3), 293 – 315. Nair-Menon, J.U., Campbell, G.T., Blake, C.A., 1996. Toxic effects of octylphenol on cultured rat and murine splenocytes. Toxicology and Applied Pharmacology 139 (2), 437 – 444. Odum, J., Lefevre, P.A., Tittensor, S., Paton, D., Routledge, E.J., Bresford, N.A., Sumpter, J.R., Ashby, J., 1997. The rodent uterotrophic assay: critical protocol features, studies with nonylphenols and comparison with a yeast estrogenicity assay. Regulatory Toxicology and Pharmacology 25 (2), 176 – 188. Oka, T., Adati, N., Shinkai, T., Sakuma, K., Nishimura, T., Kurose, K., 2003. Bisphenol A induces apoptosis in central neural cells during early development of Xenopus laevis. Biochemical and Biophysical Research Communications 312 (4), 877 – 882. Okasha, S.A., Ryu, S., Do, Y., McKallip, R.J., Nagarkatti, M., Nagarkatti, P.S., 2001. Evidence for estradiol-induced apoptosis and dysregulated T cell maturation in the thymus. Toxicology 163 (1), 49 – 62. Opferman, J.T., Korsmeyer, S.J., 2003. Apoptosis in the development and maintenance of the immune system. Nature Immunology 4 (5), 410 – 415. Safe, S.H., Pallaroni, L., Yoon, K., Gaido, K., Ross, S., Saville, B., McDonnell, D., 2001. Toxicology of environmental estrogens. Reproduction, Fertility and Development 13 (4), 307 – 315. Sakazaki, H., Ueno, H., Nakamuro, K., 2002. Estrogen receptor a´ in mouse splenic lymphocytes: possible involvement in immunity. Toxicology Letters 133 (2–3), 221 – 229. Schwaiger, J., Mallow, U., Ferling, H., Knoerr, S., Braunbeck, T., Kalbfus, W., Negele, R.D., 2002. How estrogenic is nonylphenol? A transgenerational study using rainbow trout (Oncorhynchus mykiss) as a test organism. Aquatic Toxicology 59 (3–4), 177 – 189. Sepiashvili, R.I., Shubich, M.G., Kolesnikova, N.V., Slavyanskaya, T.A., Lomtatidze, L.V., 2001. Apoptosis in immunological processes. Clinical and Applied Immunology Reviews 1 (3–4), 163 – 172. Sharma, K., Wang, R.X., Zhang, L.Y., Yin, D.L., Luo, X.Y., Solomon, J.C., Jiang, R.F., Markos, K., Davidson, W., Scott, D.W., Shi, Y.F., 2000. Death the Fas way: regulation and pathophysiology of CD95 and its ligand. Pharmacology & Therapeutics 88 (3), 333 – 347. Sharva, N.I., Dzutsev, A.K., Litvina, M.M., Pleskovskaya, G.N., Kharechenko, T.Y., Yarilin, A.A., 2001. Thymic epithelial cells induce Fas-independent activation apoptosis of thymocytes. Immunology Letters 78, 201 – 207. Staples, J.E., Gasiewicz, T.A., Fiore, N.C., Lubahn, D.B., Korach, K.S., Siversterone, A.E., 1999. Estrogen receptor-a` is necessary in thymic development and estradiol-induced thymic alterations. Journal of Immunology 163 (8), 4168 – 4174. Sweeney, T., 2002. Is exposure to endocrine disrupting compounds during fetal/post-natal development affecting the reproductive potential of farm animals? Domestic Animal Endocrinology 23 (1–2), 203 – 209. Wang, X., Han, X., Yao, G., Wang, Y., 2003. Effect of nonylphenol on apoptosis of sertoli cell in vitro. Bulletin of Environmental Contamination and Toxicology 70 (5), 898 – 904. Weih, F., Ryseck, R., Chen, L., Bravo, R., 1996. Apoptosis of nur77/N10-transgenic thymocytes involves the Fas/Fas ligand pathway. Proceedings of the National Academy of Sciences of the United States of America 93 (11), 5533 – 5538. Yadetie, F., Male, R., 2002. Effects of 4-nonylphenol on gene expression of pituitary hormones in juvenile Atlantic salmon (Salmo salar). Aquatic Toxicology 58 (1–2), 113 – 129. Yao, G., Hou, Y., 2004a. Nonylphenol induces thymocyte apoptosis through Fas/FasL pathway by mimicking estrogen in vivo. Environmental Toxicology and Pharmacology 17 (1), 19 – 27. Yao, G., Hou, Y., 2004b. Thymic atrophy via estrogen-induced apoptosis is related to Fas/FasL pathway. International Immunopharmacology 4 (2), 213 – 221. Ying, G.G., Williams, B., Kookana, R., 2002. Environmental fate of alkylphenols and alkylphenol ethoxylates—a review. Environment International 28 (3), 215 – 226. Yokota, H., Miyashita, N., Yuasa, A., 2002. High glucuronidation activity of environmental estrogens in the carp (Cyprinus carpino) intestine. Life Sciences 71 (8), 887 – 898. Yoon, K., Pallaoni, L., Stoner, M., Gaido, K., Safe, S., 2001. Differential activation of wild-type and variant forms of estrogen receptor a by synthetic and natural estrogenic compounds using a promoter containing three estrogen-responsive elements. Journal of Steroid Biochemistry 78 (1), 25 – 32.
3320
G. Yao et al. / Life Sciences 77 (2005) 3306–3320
You, H.J., Choi, C.Y., Jeon, Y.J., Chung, Y.C., Kang, S.K., Hahm, K.S., Jeong, H.G., 2002. Suppression of inducible nitric oxide synthase and tumor necrosis factor-a expression by 4-nonylphenol in macrophages. Biochemical and Biophysical Research Communications 294 (4), 753 – 759. Zajchowski, S., Hoffman-Goetz, L., 2000. Supraphysiological level of estrogen exposure in vivo increases lymphoid cell death in mice. Life Sciences 66 (15), 1451 – 1459.