Mitogen-activated protein kinase and Akt pathways are involved in 4-n-nonyphenol induced apoptosis in mouse Sertoli TM4 cells

https://doi.org/10.1016/j.etap.2015.02.004Get rights and content

Abstract

Nonylphenol (NP) is considered an important environmental toxicant, which may disrupt male reproductive system. The aim of this study was to investigate 4-n-nonylphenol (4-n-NP) induced apoptosis and its related mechanism in mouse Sertoli cell line, TM4 cells. Our results showed that NP treatment (0.1, 1, 10, 20 and 30 μM) decreased cell viability and induced apoptosis in the cells, accompanied by alteration of Bcl-2 family mRNA expression, activation of caspases-3, release of Ca2+, and increase of reactive oxygen species (ROS) generation. Subsequently, it was found that the levels of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-PX) in the cells were markedly decreased, and maleic dialdehyde (MDA) content was increased by NP treatment. Then activation of the mitogen-activated protein kinases (MAPKs) pathways and inhibition of Akt pathway were simultaneously detected in NP challenged TM4 cells. Taken together, it was concluded that NP induced cytotoxicity and apoptosis in TM4 cells, and the apoptosis may be mediated via MAPKs and Akt pathways in addition to Ca2+ release and ROS generation.

Introduction

Nonylphenol (NP), the major biodegradation product of nonylphenol polyethoxylate, has been considered as one of the most common environmental endocrine-disrupting compounds (EDCs). Studies have demonstrated that NP could result in reproductive problems, and that the impacts of NP in the environment include decrease in male fertility, feminization of aquatic organisms and survival of juveniles (Soares et al., 2008). Today, NP has been used as emulsifiers and modifiers in paints, pesticides, textiles and some personal care products, as well as plasticizers and antioxidants in plastics and resins. Due to its wide usage, a large amount of NP was discharged into ecosystem. In the area of Chongqing, the concentrations of 4-NP in river water, drinking water and fish tissues, were measured up to 7.3 μg/L, 2.7 μg/L and 1.9 μg/g, respectively (Shao et al., 2005). In a diet survey in Germany NPs was found in all the investigated food samples with concentrations ranging from 0.1 up to 19.4 μg/kg (Guenther et al., 2002), and NP concentrations from 5.8 to 235.8 μg/kg in Taiwan foodstuff samples have been reported (Lu et al., 2007). It is noteworthy that levels of NP in samples of human milk were as high as 56.3 ng/ml (equivalent to 0.26 μM) (Ademollo et al., 2008) and as high as 268 ng/g (equivalent to 1.2 μM) in human blood plasma samples (Chen et al., 2008). Studies have been shown that pollutants such as NP and bisphenol-A were highly toxic to testicular cells in the μM concentration range. In order to better assess the effect of NP on human health, it is necessary to consider the toxic effects of NP from an environmental pollution levels viewpoint.

Testis is the target organ for most male reproductive toxicants. Sertoli cells, located in the seminiferous tubules of the testis, provide nutrition as well as morphogenetic support for germ cells during spermatogenesis (Guttenbach et al., 2001). Thus, Sertoli cell is a potential target for environmental toxicant-induced reproductive dysfunction in male mice. Any agent that impairs the viability and/or function of Sertoli cells may have profound effects on spermatogenesis. At present, various Sertoli cell lines from mice and rats have been established, in order to study the regulation of Sertoli cell function and the influence of toxicants in vitro (Guttenbach et al., 2001). In the present study, mouse Sertoli cell line TM4 was used to determine the cytotoxicity and apoptosis induced by NP to evaluate the potential mechanism of male reproductive dysfunction caused by NP.

Apoptosis, the process of programmed cell death, is the most common form of cell death. However, deregulation of apoptotic process leading to either increased or reduced cell death can contribute to various pathologic conditions. Recently, it has been reported that some EDCs act as chemical substances that cause apoptosis in cells (Hallegue et al., 2002, Qian et al., 2006). Meanwhile it has been demonstrated that apoptosis is one of the mechanisms of reproductive injury. NP can exert the toxicity on development process and reproductive system. However, the effects of NP on cell cytotoxicity and the related underlying mechanisms are not fully understood. In current study, we investigated NP induced apoptosis and the related mechanism on Sertoli TM4 cells.

Interestingly, Choi et al. (2013) have been confirmed that 4-NP reduced TM4 cell viability and induced apoptotic cell death, which was mediated via ROS generation and activation of the ERK signaling pathway in TM4 cells. Apparently, in their study, the concentrations of 4-NP (0, 5, 10, 15, 20 or 50 μg/mL), equivalent to 0, 22.70, 45.40, 68.10, 90.81 and 227.03 μM, were much higher than those in the environment and diet exposure as mentioned above. High levels of specific EDCs are known to cause endocrine, reproductive, or neurological problems. Studies found that cells treated with 200 μM NP exhibited a one hundred percent mortality or apoptosis response (Choi et al., 2013, Kuo et al., 2010). Health impacts of low-level exposure to EDCs are not as well known. Reports of EDCs effects in laboratory animals at low doses are highly controversial and it is still the subject of intense research. At present no firm conclusions can be drawn about low-dose effects. In the present study, low-dose effects of NP (0, 0.1, 1, 10, 20 or 30 μM) on Sertoli TM4 cells were evaluated.

Section snippets

Reagents and chemicals

4-n-NP (99.9% purity, CAS: 104-40-5), penicillin, streptomycin sulfate, and 5(6)-carboxy-2′-7′-dichlorofluorescein diacetate (DCFH-DA) were purchased from Sigma–Aldrich Inc., Dulbecco's modified Eagle's medium nutrient mixture F-12 HAM (DMEM/F-12 medium), fetal bovine serum and horse serum were purchased from Hyclone (Waltham, MA, USA). Annexin V Apoptosis Kit and Caspase-3 Colorimetric Assay Kit were obtained from KeyGen Biotech (Nanjing, Jiangsu, China). Fulo-3/AM was purchased from Beyotime

Effect of NP on the viability of TM4 cells

To evaluate cytotoxicity of NP on the TM4 cells, viability test was performed using MTT method. As shown in Fig. 1, no significant difference was observed in the cell viability between 0.1 and 1 μM treatment groups and the control group. Compared with control group, however, cell viability significantly decreased in high concentrations of NP-treated groups (10, 20 and 30 μM), (P < 0.05).

NP induced apoptosis in TM4 cells

NP induced apoptosis in Sertoli TM4 cells was analyzed by flow cytometry (Fig. 2). After treatment with NP at

Discussion

Alkylphenol polyethoxylates (APEs), known as environmental hormone by the whole world, are used as plasticizers in plastics and nonionic surfactants (Inoue et al., 2001, Liu et al., 2008). NP is a main biological degradation product of APEs. The presence of NP in the environment has been shown to be persistent and toxic, which means that NP will persist in the environment for a very long time, and can enter the food chain. NP can mimic natural hormones, and the level present in the environment

Conclusion

In this study, we found that exposure to NP (0.1, 1, 10, 20, 30 μM) can exert toxic effects on mouse Sertoli TM4 cells, resulting in the decrease of cell viability. The cytotoxicity of NP may be due to its ability to induce apoptosis. Mitochondrial apoptosis pathway may, in part, contribute to NP induced apoptosis of mouse Sertoli TM4 cells, as evidenced by increased expression of Bax mRNA and decreased expression of Bcl-2 mRNA, activation of caspases-3, release of Ca2+ and generation of ROS.

Conflict of interest

The authors declare that there are no conflict of interest.

Transparency document

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Acknowledgements

The financial support for this study by the National Basic Research Program of China (973 program) (No. 2012CB720805), the Joint Sino-German Research Project of National Natural Science Foundation of China (No. GZ 731), International Science & Technology Cooperation Program of China (No. 2010DFA31780), and the Program for New Century Excellent Talents in University (No. NCET-12-0749), is gratefully acknowledged.

References (37)

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