Researches On The Roles Of Endoplasmic Reticulum Stress And Mitochondrial Damage In Dibutyl Phthalate-induced Male Reproductive Toxicity

Background:Phthalic acid esters(PAEs), namely phthalates, are a class of man-made organic chemicals, which are produced largely in industry. Most of PAEs are used as plasticizers in polyvinyl chloride(PVC) plastics, as well as used widely in lacquers, coatings, pesticide and personal care products such as perfumes and cosmetics. Thus, the opportunity is high for non-occupational human exposure. Because the phthalate is not covalently incorporated into plastic matrix and easily released into the environment under certain conditions, inevitable human exposure through ingestion, inhalation, and dermal contamination has caused public health concerns. The testis is the primary target of PAEs, and epidemiological and experimental researches have demonstrated a close connection between PAEs exposure and male reproductive toxicity. The recent studies indicated that spermatogenic cell apoptosis might account for the phthalate-induced testicular atrophy. However, the target organelles and molecular events that are triggered by the phthalate to initiate apoptosis remain unclear. The result from another research group that PAEs exposure can cause endoplasmic reticulum(ER) expanding and mitochondrial dropsy in testicular cells provided novel insights into our study. In the present study, we used dibutyl phthalate(DBP) as the model compound to investigate the roles of endoplasmic reticulum stress(ER stress), autophagy and mitochondrial damage in DBP-induced spermatogenic cell apoptosis in vitro and in vivo. Furthermore, we investigated the associations between PAEs exposure and mitochondrial membrane potential(MMP), mtDNA copy number and mt DNA integrity in human spermatozoa in the Male Reproductive Health in Chongqing College Students(MARHCS) cohort study, which was established in 2013. Content:1. Researches on the role of ER stress in DBP-induced germ cell apoptosis. We first established DBP-induced spermatocyte-derived GC-2 cell apoptosis model. To evaluate the cytotoxicity of DBP on GC-2 cells, the CCK-8 assay was applied in the present study. Parallel, apoptotic GC-2 cells were examined using Annexin V-FITC staining by flow cytometry analysis. We evaluated the protein levels of ER stress marker GRP78, ATF6, and p-e IF2α, as well as evaluate those of ER stress-related apoptosis pathways p-JNK/p-p38, CHOP and Caspase-12 using Western Blot. To evaluate the role of ER stress in DBP-induced GC-2 cell apoptosis, 4-PBA which is a chemical with chaperone-like activity, was used to inhibit ER stress in the present study. The expression levels of LC3 and P62 in DBP-treated CG-2 cells were measured, and the number of autophagosomes was detected by transmission electron microscopy(TEM). We further investigated the effects of ER stress inhibition by 4-PBA on DBP-induced autophagy. Then, 3-MA, a special autophagy inhibitor, was applied to evaluate the role of autophagy in DB-induced GC-2 cell apoptosis. Finally, we verified the in vitro results in DBP-treated male prepubertal rats.2. Researches on the molecular mechanism linking DBP-induced ER stress and autophagy. We used chloroquine(CQ) to monitor whether the cause of this increased autophagy was due to increased flux or a block in fusion or degradation. To investigate the molecular pathways linking ER stress and autophagy in DBP-treated GC-2 cells, we detected the protein levels of p-eIF2α/ATF4/Atg12, mTOR and IRE1α/JNK/Beclin 1 pathways. Furthermore, intracellular ROS was evaluated using DFCH-DA in DBP-treated GC-2 cells, and the antioxidant melatonin was applied to inhibit the increased level of ROS and investigate the role in DBP-induced ER stress and autophagy.3. Researches on the role of PERK/Nrf2/ARE pathway in DBP-induced mitochondrial damage and apoptosis. To evaluate whether DBP exposure can cause mitochondrial damage and mitochondrial pathway apoptosis, mitochondrial mass, ATP concentrations, mt DNA copy number and Caspase cascade were examined in DBP-treated GC-2 cells. Melatonin pretreatment was applied to explore the role of ROS in DBP-induced mitochondrial damage and mitochondrial pathway apoptosis. We further evaluated the activation of Nrf2-regulated antioxidant response element(ARE) by measuring the protein levels of Nrf2, HO-1 and NQO-1 in DBP-treated GC-2 cells. Then, the si RNA against Nrf2 was employed to investigate the effects of Nrf2/ARE inhibition on DBP-induced mitochondrial damage and mitochondrial pathway apoptosis. The special PERK inhibitor PERKi was used to investigate the association between the PERK pathway of ER stress and Nrf2/ARE, as well as to investigate the association between PERK inhibition and DBP-induced effects on GC-2 cells.4. The effects of PAEs exposure on the mitochondrial functionality and mt DNA structure in human spermatozoa. As a portion of the MARHCS cohort study, we measured mitochondrial membrane potential, mt DNA copy number, and mt DNA integrity in human spermatozoa, and evaluate the associations between these mitochondrial biomarkers and semen parameters. Furthermore, we investigated the effects of PAEs exposure on human semen parameters, as well as these mitochondrial biomarkers. Results:1. DBP-induced ER stress in male germ cells causes autophagy, which has a cytoprotective role against apoptosis in vivo and in vitro. We found that DBP induced marked apoptosis in GC-2 cells. The levels of the major ER stress markers GRP78, ATF6, and p-eIF2α were elevated by the exposure of GC-2 cells to 25 μM DBP and increased in a dose-dependent manner at higher concentrations. Furthermore, at a concentration that results in significant apoptosis(100 μM), CHOP that plays a convergent role in ER stress-mediated apoptosis and can be regulated by various upstream ER stress signals, was activated and contributed partially to DBP-induced apoptosis. However, the inhibition of ER stress by 4-PBA augmented the GC-2 cell apoptosis induced by DBP. Further experiments demonstrated that DBP-induced ER stress also had a protective role mediated through the activation of autophagy, which is distinguished by the increased number of autophagosomes and the increased conversion of LC3-I to LC3-II. These results were further confirmed in prepubertal rat testis germ cells. DBP treatment significantly induced testicular atrophy accompanied by spermatogenic cell apoptosis, ER stress, and autophagy. The inhibition of ER stress and autophagy significantly aggravated the DBP-induced damage of germ cells and the testes.2. The p-e IF2a/ATF4 pathway links endoplasmic reticulum stress to autophagy following the production of reactive oxygen species. We showed that exposure to DBP enhanced autophagic flux in GC-2 cells. Then, we found that the m TOR and IRE1α/JNK/Beclin 1 pathways were not activated in DBP-treated GC-2 cells. And the results indicated the p-e IF2α/ATF4 pathway mediated ER stress-related autophagy via regulating the expression of Atg12. Moreover, we demonstrated that DBP treatment led to the excessive generation of ROS which in turn can be attenuated the antioxidant melatonin. Furthermore, that the inhibition of ROS by melatonin abrogated both ER stress and autophagy in GC-2 treated GC-2 cells.3. DBP-activated PERK/Nrf2/ARE pathway protects GC-2 cells against mitochondrial damage and apoptosis. We found that exposure to DBP decreased mitochondrial mass, mt DNA copy number, COX IV protein level and ATP concentrations, and caused caspases cascade in GC-2 cells. And the antioxidant melatonin was capable of restoring significantly DBP-induced mitochondrial damage and mitochondrial pathway apoptosis. We also found that DBP exposure activated the Nrf2/ARE pathway against increased ROS in GC-2 cells, and the inhibition of Nrf2/ARE by si RNA augmented the GC-2 cell apoptosis and mitochondrial damage induced by DBP. That PERKi attenuated DBP-activated Nrf2/ARE pathway indicated the PERK pathway of ER stress can regulate the antioxidant response pathway. Furthermore, both the PERK inhibition by PERKi and the Nrf2/ARE inhibition by siRNA augmented DBP-induced mitochondrial damage observed under a confocal laser scanning microscope. In vivo animal experiments also demonstrated PERKi treatment inhibited Nrf2/ARE pathway and augmented DBP-induced mitochondrial pathway apoptosis in testicular cells.4. In the study of population, we found that mitochondrial membrane potential, mt DNA copy number and mt DNA integrity were associated with conventional semen parameters, which suggested that these mitochondrial biomarkers might be capable of serving as indicators of semen quality. The concentrations of urinary monoethyl phthalate(MEP), mono-n-butyl phthalate(MnBP) and mono(3-carboxypropyl) phthalate(MCPP) were associated negatively with sperm progressive motility, which is an important indicator of men fertility. Though there were not significant relationships between PAEs exposure and mtDNA copy number, we found that the urinary mono-(2-ethylhexyl) phthalate(MEHP) concentration was positively mitochondrial membrane potential in human spermatozoa. MtDNA integrity in those participators whose concentrations of urinary monocyclohexyl phthalate(MCHP), mono-n-octyl phthalate(Mn OP), monobenzyl phthalate(MBzP) and mono-isononyl phthalate(MiNP) were higher than relative limits of detection(LOD) was significantly poor. Conclusion:We first found that DBP-induced endoplasmic reticulum stress in male germ cells causes autophagy, which has a cytoprotective role against apoptosis in vitro and in vivo. And the p-e IF2α/ATF4/Atg12 pathway mediated ER stress-related autophagy independent of the mTOR and JNK/Beclin 1 pathways. Further study revealed that exposure to DBP can cause mitochondrial damage and mitochondrial pathway apoptosis, in which excessive ROS generation was involved. Besides causing autophagy, ER stress also activated the Nrf2/ARE via PERK pathway protecting male germ cells against DBP-induced cytotoxic effects. Finally, the population study revealed that some of urinary PAEs metabolites concentrations were associated negatively with sperm progressive motility, which might be explained by poor mt DNA integrity to some extent. The results provide novel insights into the molecular mechanism mediating DBP-induced male reproductive toxicity, as well as the self-defense mechanism of spermatogenic cell upon environment stress such as DBP exposure.