Reproductive Toxicity Of Di-(2-ethylhexyl)Phthalate On Male Immature SD Rats And The Mechanism Involved

BackgroundThere is an increasing concern focusing on the potential of environmental toxicants to impair human health as more and more environmental chemicals have been identified as EDCs. Exposure to endocrine disruptors results in multiple health problems including neurodegenerative disorders, cancer and especially infertility. MFI (male factor infertility) is a major problem worldwide, and is also a common reproductive dysfunction. There are several potential causes of male infertility, such as genetic, environmental, and hormonal factors. A number of studies suggest that male infertility and environmental exposure to endocrine disruptors is closely related. Some researchers found that compounds with endocrine disrupters effects were associated with idiopathic male infertility and semen parameters. Phthalates are a class of industrial chemicals that are dialkyl-or alkylarylesters of 1,2-benzenedicarboxylic acid and have been used for a variety of purposes. Among the phthalates, Di-(2-ethylhexyl)phthalate (DEHP) is one of the most widely used phthalates, which are widely used as plastic softeners and plasticizers. DEHP is a ubiquitously used EDCs that has raised substantial public concern due to potential detrimental health effects in the general popolation. DEHP has a constelltion of adverse effects, including reproductive toxicity. The productive toxicity of DEHP in males is well established, but a unifying mechanism explaining the male reproductive toxicity is lacking. Additional experiments are therefore necessory to elucidate the toxicological and pathogenic pathways affected by DEHP.ObjectivesThrough studies of the weanling rats, rat whole embryo culture and cell culture in vitro,, we systematically measured the effects of DEHP on male reproduction and the detailed mocular mechanism invloved. In vitro cells culture, DNA replication, flow cytometry and western blotting were performed to explore the exact apoptotic pahtways of testicular toxicity and roles of mitochondrial dysfunction, SIRT1 activity and ATP levels in male infertility.1. To check the antiandrogenic effect of DEHP. Both extrinsic and mitrochondrial-associated intrinsic apoptotic pahtways in testicular and liver was systematically investigated.2. To assess developmental toxic properties of DEHP with rat postimplantation whole embryo culture.3. To investigate the potential role of mitochondrial in DEHP-induced testicular toxicity. DNA replication, cellular/mitochondrial ROS and ATP levels were explored to determine whether DEHP treatment lead to DNA damage.4. To investigate the role of NAD+levels, SIRT1 activity and mitochondrial malfunction in DEHP-induced testicular toxicity.Methods1. The weanling rat assay was performed, effects of DEHP on rat testis, epididymides, anogenital distance and androgen dependent organs such as seminal vesicles, ventral prostate, levator plus bulbocavemosus muscles (LABC) were investigated.2. Testis and liver sections of DEHP-fed rats were stained with H&E for histophthology examination. In situ detection of cell death in testicular and liver tissues using TUNEL assay was applied for detecting DNA fragmentation.3. Rat postimplantation whole embryo culture was performed to assess developmental toxic properious of DEHP.4. By western blotting, We systematically investigated expression levels of designated members in both extrinsic and mitrochondrial-associated intrinsic apoptotic pathways in testicular and liver tissues, such as caspase-3、 caspase-9、caspase-8、PARP-1, Bcl-2 family of Bcl-XL, Bid, Bak, Bax and so on.5. DNA fiber experiment was performed to detect whether DEHP inhibit DNA replication.6. Flow cytometry was using to measure intracellular/mitochondiral ROS levels in a list of testicular cell lines and NIH 3T3 cells.7. ATPlite luminescence assay was performed to detect cellular ATP levels and NAD+levels was measured with ELISA assay in control and DEHP-treated groups.8. Immunoblot for OXPHOS complex, SIRT1, PGC-1a, p53 and acetylated p53 as well as PARP expression in rat testes of designated groups were performed.ResultsDEHP elicted antiandrogenic effects, the weight of rat testis, epididymides, anogenital distance and androgen-dependent accessory reproductive glands (seminal vesicles, ventral prostate, levator ani plus bulbocavernosus muscles/LABC) were decreased in DEHP treated group in dose-dependent manner. The testicular histopathology of DEHP-fed rats with H&E showed a significant decrease of spermatocytes (C) and spermatids (T) in a dose-dependent manner in DEHP-treated rats. An increased number of TUNEL-positive staining cells (indicating DEHP induced more fragmented DNA) was noted in DEHP-exposed rat tissues in a dose-dependent way.By immunoblotting, we found that an increased cleaved form of caspase-8 (p43, C-caspase-8) and processed form p37 of caspase-9 pro-form (C-caspase-9) in both liver and testes in DEHP-exposed animals, respectively. DEHP upregulated the protein levels of Bax, Bak and Bid. Accordingly, we detected a decreased expression of Bcl-xL in the DEHP-exposed group. In conclusion, under DEHP exposure, the testicular apoptosis appeared to be caused by a mitochondrion-associated apoptotic signaling.The results of the rat postimplantation whole embryo culture (WEC) showed that a dose-dependent decrease in total morphology score (TMS), growth (head length, crown-rump length), yolk sac diameter and final somite number for DEHP exposed group. Embryos showed concentration-dependent inhibition on embryonic growth and morphological development. The results of Malformation of embryos showed that embryos exposed to DEHP showed malformations including cardiomegaly, pericardial effusion and metencephalon enlargement.DNA fiber tract analysis showed that DEHP exposure strongly inhibited DNA replication in both NIH3T3 cells and CRL-2714 cells. DEHP inhibited replication leading to activation of a DNA damage response. We postulate that DEHP induces apoptosis at least partially by stalling of replication forks leading to DNA strand breaks.In vitro assays showed that ROS in both intracellular and mitochondrial levels were increased as determined by flow cytometry in DEHP-treated cells. The results of NAD+assays showed that NAD+levels were decreased in DEHP-treated groups. Since NAD+consumption by hyperPARylation may impair SIRT1 activity, we further asked whether DEHP treatment could also reduce SIRT1 activity. Intriguingly, DEHP decreased the SIRT1 and PGC-1a protein level, and also the deacetylation activity of SIRT1 was blunted, which was evident from increased acetylated p53 levels in DEHP-treated testes. Results of ATPlite luminescence assay showed that ATP levels were decreased in DEHP-treated groups. We next examined the changes in the ATP production machinery, oxidative phosphorylation (OXPHOS) complexes in testicular tissues. Immunoblotting showed a substantial decrease in protein levels of subunits of OXPHOS complex II, III, IV and V.Thus, the mitochondrial dysfunction in DEHP-treated cells may be ascribed to impairment of the SIRT1 deacetylation activity via overconsumption of NAD+by hyperPARylation. Our findings unveil the role of SIRT1, mitochondrial function and DNA replication in DEHP-induced testicular toxicity. The testicular toxicity of DEHP may be caused by direct inhibition of DNA replication leading to activation of the DNA damage response enzyme PARP1. This in turn lead to SIRT1 attenuation, apoptosis and mitochondrial dysfunction.The liver is also DEHP-sensitive and included in this study. The liver histopathology of DEHP-fed rats shows that intercellular space diameter was significantly greater than the control group. Hepatic steatosis, cell necrosis, and expanded hepatocyte were appeared in DEHP-treated group. An increase number of TUNEL-positive staining cells were noted in DEHP-exposed rat tissues. By immunoblotting, caspase-8, caspase-9, caspase-3 and PARP-1 were activated in DEHP-treated rat liver tissues. However, there was a small increase in pro-apoptotic Bcl-2 members, the liver was less sensitive to intrinsic apoptotic cell death. Unexpectedly, we detected an elevated Bcl-XL in DEHP-exposed liver which may be due to a secondary protective cellular response. Consistent with the abovementioned data showing a decreased intrinsic (mitochondrial) apoptosis pathway in DEHP-exposed rat liver tissues, we did not detect significant changes in the OXPHOS complex in liver tissues from the same groups.Conclusion1. DEHP showed antiandrogenic effects and caused reproductive toxicity including testicular toxicity in male reproduction.2. DEHP showed developmental toxicity in a concerntration-dependent manner.3. DEHP induces apoptosis at least partially by stalling of replication forks leading to DNA strand breaks.4. DEHP induced activation of apoptosis of testes in intrinsic/mitochondrial apoptotic pathways in testes, partially be attributed to its induction of mitochondrial damage and increased ROS production..5. The mitochondrial dysfunction in DEHP-treated cells may be ascribed to impairment of the SIRT1 deacetylation activity via overconsumption of NAD+by hyperPARylation.6. SIRT1 may play an important role in male fertility.