NP was reported to cause intracellular accumulation of ROS in multiple cell types (Qi et al. 2013; Okai et al. 2004; Gong and Han 2006 ), and induce oxidative stress within testis of rats (Chitra and Mathur 2004). Our interesting findings implied that NP65 treatment caused ROS over generation and oxidative stress in TM4 cells, as proven by rapid increase of MDA content and the marked decrease of SOD activity in NP65 stimulated cells. Oxidative stress was thought to be the underlying mechanism for the death of apoptotic cells (Kitazawa et al. 2001). The current results demonstrated that NP in TM4 cells could induce oxidative stress, which might lead to cellular apoptotic damages potentially. Apoptosis accompanied by alteration in the mRNA expression of Bcl-2 family, suggesting that in TM4 cells, mitochondrial pathway should be contributed to NP65 induced apoptosis.
Ca2+, as the widely employed intracellular messenger, can encode a variety of cellular information based on the regulation of signals of Ca2+. Signals of Ca2+ have pivotal roles within various cellular behaviours including differentiation, proliferation as well as transcription of genes (Bootman et al. 2012). In response to different stimuli, increase of the Ca2+ concentration within the cytosol induces plenty of types of events, which plays substantial role in cell apoptosis. Wang et al. (2005) have found that Ca2+ elevation and Ca2+-independent cell death could be induced by NP in MG63 human osteosarcoma cells. Studies have found that Ca2+ release and homeostasis disorder were involved in the apoptosis induced by alkylphenol in TM4 cells (Michelangeli et al. 2008), and NP caused Ca2+-dependent apoptosis in SCM1 human gastric cancer cells (Kuo et al. 2010). Analysis of flow cytometry illustrated that NP65 dramatically rised intracellular Ca2+ level in TM4 cells through a manner of dose dependence, and ROS scavenging contributed to weakening NP-induced Ca2+ overloading. The results suggested that ROS generation was involved in Ca2+ release in NP65 challenged TM4 cells, and Ca2+ homeostasis disorder might act an important role in apoptosis of cells induced by NP65.
SERCA has always been accepted as playing a central role in the mechanism of Ca2+ transport across the membrane, from the cell cytosol into the endoplasmic reticulum, and this is the reason why low levels of free cytosolic Ca2+ are maintained in the cells. Under stimulation, [Ca2+]i becomes higher and remains high if SERCA is blocked. Nonylphenol was reported to influence Ca2+ signaling mechanisms within cells by influencing transporters of Ca2+ such as Ca2+ pumps of SERCA (Hughes et al. 2000). Many estrogenic alkylphenols such as NP have been tested to inhibit the sarcoplasmic reticulum Ca2+-ATPase of skeletal muscle, which functions in a similar manner to the endoplasmic reticulum Ca2+-ATPase (Mason et al. 1993). Therefore, we hypothesized that the evaluation of Ca2+ induced by NP65 may be related to alteration of SERCA Ca2+ pumps function. The inhibition of Ca2+-ATPase activity and SERCA-type Ca2+ pumps expression in this study suggested that NP65 might act as an inhibitor of SERCA. We proposed that NP65 could release Ca2+ from intracellular stores by inhibiting the activity of SERCA Ca2+ pumps. Ca2+ channels were regulated by different kinases, calmodulin-dependent kinase, protein tyrosine kinase, as well as G protein subunits, etc. (Keef et al. 2001). Since cAMP and cGMP are important for regulation of protein kinase C, cGMP-dependent protein kinase and cAMP-dependent protein kinase to exert vital functions of cell (e.g. differentiation, proliferation, transcription of genes as well as cellular apoptosis,) within all organisms, we concentrated our researches upon the effects of cAMP as well as cGMP in TM4 cells. The decrease of cAMP concentration could be one of the factors that were involved in the elevation of Ca2+ level in TM4 cells, although the mechanism was still unclear. Interestingly, the Ca2+ disorder was moderately inhibited by NAC, no matter the inhibition of the activity of Ca2+-ATPase or the decrease of cAMP content was weakened by NAC pretreatment. We concluded that ROS pathway was involved in NP65 disturbed Ca2+ signaling in TM4 cells.
Apoptosis and Ca2+ overload were related to the formation of ROS, several antioxidants can block cell apoptosis. NAC has been reported to alleviate damage of cells induced by toxic chemicals (Spagnuolo et al. 2006). In the present study, NAC was employed for investigation of the putative role of ROS production in apoptosis and loss of Ca2+ homeostasis induced by NP65. NP65-induced apoptosis and loss of Ca2+ homeostasis were blocked by NAC, suggesting that ROS generation was involved in NP65 induced apoptosis as well as loss of Ca2+ homeostasis. Thus, the current study built a direct linkage between NP65 induced generation of ROS and apoptosis as well as Ca2+ disorder in TM4 cells.
NPs exerted effects not only through binding to estrogen receptors but also through cell signal pathways. It is known that the the structure of the side chain may influence the estrogenic effect of a single NP isomer to a large extent. The estrogenic potency of NP65 was 6.1 × 10− 6, and 4-n-NP showed no estrogen effect (Preuss et al. 2006). Michelangeli et al. (2008) reported that the SERCA inhibition potency of alkylphenols was relevant to length of chain for linear chain alkylphenols, and branched chain alkylpenols in general had higher potencies compared with their counterparts of linear chain. There were different effects between NP65 and 4-n-NP on the apoptosis and Ca2+ disorder in TM4 cells, which further proved that the effects of NPs to organism were related to their structures or their potential xenoestrogenic activity.
In summary, this study has shown that the NP isomer, NP65, could trigger oxidative stress as well as mitochondrial pathway apoptosis in mouse Sertoli TM4 cells. In addition, NP65 was capable of elevating intracellular [Ca2+]i levels in Sertoli TM4 cells. Our study found that ROS was related to both the apoptosis as well as Ca2+ disorder induced by NP65 in TM4 cells. However, whether NP65 evoked Ca2+ disorder is related to NP65-induced apoptosis is an important issue, which will be investigated in our further study. Anyway, results in this study offered an alternative vision that NP isomer might disrupt the development of male reproduction by inducing apoptosis and disturbing relative signaling pathways in the cells, but not require to be mediated solely by direct interaction with the estrogen receptor.