Effects Of 4-n-NP And Its Isomers On Mouse Sertoli TM4 Cells And The Underlying Molecular Mechanism

Nonylphenols(NPs) are considered as environmental toxicants and endocrine disrupting compounds which can disrupt male reproductive system. Actually, NPs consist mainly of a mixture of para-substituted mono-alkylphenols with various isomeric and branched nonyl groups, 4-nonylphenol(4-NP), in which the side chain is attached to the carbon directly opposite the hydroxyl group is the most common member of this family, 4-n-NP is the 4-NP with straight-chain. The activity of an individual NP isomer was heavily dependent upon the structure of the side chain, consequently, it is necessary to consider NPs from an isomer-specific viewpoint. Xenoestrogen can exhibit effects by two ways, one is that xenoestrogen activate the cell signaling pathays in cells and then induce cell proliferation or apoptosis; another one is that xenoestrogen binds to hormone receptor and then disturbs endocrine secretion. The present study is designed to investigate the effects of 4-n-NP and 4-NP isomers on Sertoli TM4 cells, and try to illustrate the possible underlying mechanisms from cell signaling pathway and endocrine disruption section, and then discuss the relativity between the activity and nonyl branches structure for nonylphenol isomers. The main research results obtained in this dissertation are concluded as follows:1. The toxic effects of 4-NP isomers on Sertoli TM4 cellsIn this study, MTT was used to detect the survival of Sertoli TM4 cells and flow cytometry was performed to detect apoptosis. The results showed that in low concentration, NP9, NP10 and NP37 promoted TM4 cells proliferation; 4-n-NP, NP65, NP70, NP111, NP112, NP143, NP153 and NP194 induced significant apoptosis in TM4 cells; the results suggested that the NP isomers whose structures are of longer branch chain, fewer branches in nonyl promote cells proliferation, while those NP isomers with shorter branch chain, bigger size and more complex in α-C substituent group showed strong apoptosis effects to TM4 cells(4-n-NP exclude). NPs induced apoptosis of Sertoli TM4 cells by oxidative stress and mitochondrial apoptosis pathway. In high concentration, all the NP isomers exhibit the direct toxic actions to kill TM4 cells.2. Effects of 4-NP isomers on the Ca2+ homeostasis in Sertoli TM4 cellsNP65, NP70, NP111, NP112, NP143, NP152, NP194 isomers could increase Ca2+ concentration in Sertoli TM4 cells, which have statistical significance in NP65, NP70, NP111 and NP152 treatment. The mechanism of NP isomers on Ca2+ homeostasis in TM4 cells are related to the Ca2+-ATPase activity, SERCA function and SERCA mRNA expression, Ca2+ channel kinase activity. The isomers with big branches in nonyl could result in Ca2+ disorder in Sertoli TM4 cells.3. Effects of 4-NP isomers on the MAPK and Akt pathways in Sertoli TM4 cellsThe effects of 4-NP isomers on MAPK and Akt pathways were detected by western bot. Results showed that 4-n-NP, NP41, NP42, NP65, NP111, NP112, NP143, NP152 activated JNK pathway; 4-n-NP, NP111, NP112, NP143, NP152, NP194 activated p38pathway; NP143, NP152 and NP194 showed no effects on ERK pathway, other isomers active or inhibited ERK pathway. 4-n-NP inhibited Akt pathway, all other isomers activated Akt pathway. The effects of 4-NP isomers on MAPKs pathways are related to their stuctures, and that depend on the chain length, α-C substituent group. The NP isomers whose structures are of shorter branch chain, bigger size and more complex in α-C substituent group could activate the JNK and p38 pathways, but showed no effects on ERK pathway; the NP isomers with shorter branch chain and fewer branches in nonyl inhibit JNK and p38 pathway(exclude 4-n-NP).4. Effects of 4-NP isomers on the hormone receptor and secretion functinon in Sertoli TM4 cells4-n-NP decreased the expression of estrogen receptor(ER)-β and BTB-associated elements, increased the secretion of ABP and decreased the secretion of inhibin B, 4-n-NP also increased the secretion of IL-1α, TNF-α, TGF-β1, and decreased NO secretion and iNOS expression in TM4 cells. All 4-NP isomers exposure have adverse effects on the expression of hormone receptor including ER-α, ER-β, PR and AR; the levels of BTB-associated elements including vimentin, vinculin, N-cadherin and occluding; 4-NPs had effects on endocrine secretion as evidenced by the changes in the levels of ABP and inhibin B.5. Effects of 4-NP isomers on hormone secretion in Sertoli TM4 cellsCells were treated with 4-NP isomers, the release of PGE2 was used to investigated the effects of 4-NP isomers on hormone secretion in Sertoli TM4 cells. The results showed that 4-n-NP, NP10, NP41, NP42 and NP70 induced COX-2 expression and PGE2 release; NP9, NP37 and NP65 showed no effects on COX-2 expression and PGE2 release in TM4 cells; NP111, NP112, NP143, NP152 and NP194 inhibited PGE2 release in TM4 cells. 4-n-NP regulates COX-2 expression and PGE2 release via ROS-activated NF-κB pathway in Sertoli TM4 cells. The effects of 4-NP isomers on COX-2 expression, PGE2 and IL-6 release are related to their structures. 4-NP isomers with long chain(6-7), a methyl in α-C and a substituent in β-C could induce the expression of COX-2 and secretion of PGE2 and IL-6, isomers with short chain(4-5) and big size substituent in α-C inhibited the secretion of PGE2 and IL-6. 4-n-NP isomers regulate COX-2 expression, PGE2 and IL-6 release via ROS-activated NF-κB pathway in Sertoli TM4 cells.Taken together, 4-NP isomers exposure affect the function of Sertoli cells can through all of these cell signaling pathways(including mitochondrial apoptosis pathway, Ca2+ signaling pathway and MAPKs pathway) and endocrine disrupting effects(including effects on hormone receptor, BTB-associated elements and endocrine secretion). The effects of 4-NP isomers to TM4 cells are individual, which may be related to their structures, including lenth and size of nonyl and the substituent in α-C and β-C.