Reproductive Toxicity Of 4-Nitrophenol-Treated Male Rats And Investigation On Attenuating Effects Of Phytosterins

It is well known that the decline in human and wildlife endocrine health, which is mainly caused by environmental pollution, has received increasing attention from the public and the scientific communities. Meanwhile, many chemicals, such as environmental endocrine disruptors (EEDs), have been experimentally demonstrated to affect endocrine processes. EEDs are nonsteroidal, man-made chemicals that can enter the body by ingestion or absorption and mimic the actions of endogenous hormones. Exposure to EEDs, specifically during developing stages, may cause dysfunctions or abnormalities of reproductive organs later in adults, which may lead to infertility. As an EEDs,4-nitrophenol (PNP) is commonly found in water and soil environments and is typically used for agriculture and industry manufacturing. PNP has been isolated from diesel exhaust particles (DEP). It is also a degradation product of the insecticide parathion.Expression of most antioxidant enzymes is controlled by the antioxidant response element (ARE) and activated by nuclear factor erythroid 2-related factor 2 (Nrf2). Phytosterins (PS) have attracted much attention in recent decades because of their health benefits for humans. It is well established that certain PS reduce plasma cholesterol levels, ostensibly by inhibiting enterocytic cholesterol uptake through competition with dietary and biliary cholesterol for absorption. In addition, PS are recognized to exert antioxidative actions.In the present study, we focused on PNP-induced reproductive injuries. We investigated the PNP-induced testicular oxidative damage in rats using a variety of assays. The attenuating effects of PS on PNP-induced testicular toxicity were also studied. The main results are as follows:1. Effects of low dosage PNP on hormonal balance and ER, AR expression in male rat testesPNP is generally regarded as an environmental endocrine disruptor capable of estrogenic and anti-androgenic activities. To investigate PNP-induced reproductive effects, immature male rats were injected subcutaneously with PNP (0.1,1,10 mg/kg body weight or vehicle) daily for 4 weeks. We assessed reproductive tract alterations, sex hormone balance in the serum and estrogen receptor (ER) a, ERβ and androgen receptor (AR) expression in testes. Although no significant difference was observed in body weight or testes weights of PNP-treated rats compared with the controls, the serum concentrations of testosterone in the 10 mg/kg PNP-treated group were significantly elevated. This effect was accompanied by Leydig cells hyperplasia in the testes. Conversely, there was a significant decrease in estradiol concentration and aromatase expression in the testes of the 10 mg/kg PNP-treated group. Furthermore, we observed a significant increase in ERa expression in the testes of the 10 mg/kg PNP-treated group compared with the control group. Conversely, ERβ expression displayed a significant reduction. Moreover, AR expression was significantly increased in the 10 mg/kg PNP-treated group compared with the control group. The existence of AR, ERa and ERβ in the testes suggests that estradiol and testosterone directly affect germ cells and that differential modulation of AR, ERa and ERβ in the testis may be involved in the direct effects of PNP or either the indirect effects of PNP-induced disruption of the estradiol-to-testosterone balance or the Leydig cells hyperplasia. Thus, the measurement of many endpoints is necessary for good risk assessment.2. Effects of low dosage PNP on the endocrine Function of Adrenal cortex in male ratsPNP is generally regarded as an EEDs that accumulates in various organs. Studies of PNP have revealed that this chemical accumulates in adrenal glands in vivo. However, the impacts of exposure to this compound on adrenal endocrine disruption and steroidogenesis have not been investigated. The present study investigates the effect of PNP on the adrenocortical functions of male rats in vivo and in vitro. Immature male rats were injected subcutaneously with PNP (0.1,1,10 mg/kg body weight or vehicle) daily for 2 weeks.24 h after the final injection, the rats were weighed and decapitated. Serum was collected for radioimmunoassay of progesterone, testosterone and estradiol levels. Adrenal pathology was detected by HE staining. Steroid synthesis enzymes expression was measured by RT-PCR. To investigate the direct effects of PNP on the hormone secretion of adrenal cortex, we exposed cultured primary adrenal cells to PNP (10-6,10-5, or 10-4 M) for 24 h. Cell viability was detected by trypan blue under microscop. Supernatant was collected to determining the progesterone levels. In vivo experiment, body, organs and organ weight index of rats in PNP-treated groups showed no significant difference compared with the control group (P> 0.05);serum testosterone levels were significantly increased (P< 0.05) in 10 mg/kg PNP-treated rats, whereas progesterone levels were significantly decreased (P < 0.05), and estradiol levels were no significant difference (P> 0.05);histological examination did not found any abnormality of the adrenal cortex in PNP-treated groups; the results of RT-PCR revealed that the expression of StAR and P450c17 mRNA was significantly increased, however,3/3-HSD mRNA expression was significantly decreased (P < 0.05). In vitro experiment, the cells survival rate was not affected by PNP, but the progesterone levels in culture medium was significantly decreased (P< 0.05) in 10-4M PNP group compared with the control group. These results demonstrated that PNP disrupted hormone secretion of adrenal cortex mainly via affecting the expression of steroid synthase.3. Supplemental dietary PS attenuated high dosage of PNP induced abnormal spermatogenesis and germ cells apoptpsis in rat testesThis study examined PNP-induced testicular damage and PS-mediated protection against that damage. Twenty male rats were randomly assigned into four experimental groups (Control, PS, PNP, and PNP+PS). PS were added to the basal diet supplied by the commercial supplier (50 mg/kg) and pre-fed for one week. Rats were injected subcutaneously with PNP (100 mg/kg body weight) daily for 4 weeks.24 h after the final injection, rats were weighed, anesthetized with ethylic ether and sacrificed. PNP significantly reduced the caudal epididymal sperm counts and serum testosterone levels (P < 0.05). Typical morphological changes were also observed in the testes of PNP-treated animals by Transmission electron microscopy, including vacuoles between Sertoli cells and spermatogonia, the typical morphological changes of germ cells apoptosis (including pyknosis and apoptotic bodies), mitochondrial sheath deletion of microscler spermatids and lipid droplet deposition in spermatocytes. Immunostaining for caspase3, mainly distributed in spermatocytes and Leydig cells, was stronger in the testes of rats in the PNP-treated group than in the control group (P< 0.05). However, in combination with PS, these effects were mitigated compared with PNP treatment alone. These results demonstrated that high dosage of PNP affected the spermatogenesis and induced germ cells apoptpsis in male rats, moreover, supplemental dietary PS protect against PNP-induced damage.4. Protective effects of PS on high dosage of PNP induced oxidative stress in male rat testesThis study examined PNP-induced testicular oxidative damage and PS-mediated protection against that damage. The generation of MDA and H2O2 upon PNP and PS treatment was milder than that upontreatment with PNP alone. This mitigation was accompanied by partially reversed changes in SOD, CAT, GSH and GSH-Px, though their levels did not return to normal. PNP reduced the transcriptional level of Nrf2, as evaluated by RT-PCR, but it promoted the dissociation from the Nrf2 complex, stabilization and translocation into the nucleus, as evaluated by immunohistochemistry and western blotting. Moreover, there was also an increase in nuclear translocation upon PS treatment alone. In addition, PNP enhanced the Nrf2-dependent gene expression of heme oxygenase-1 (HO-1) and glutamate-cysteine ligase catalytic subunit (GCLC); however, simultaneous supplementation with PS restored these parameters to near the control levels. These results suggest that the Nrf2 pathway plays an important role in PNP-induced oxidative damage and that PS possesses modulatory effects on PNP-induced oxidative damage in rat testes.5. Reproductive toxicity of intratesticular injection of PNP on rats and investagation on attenuating effects of phytosterinsThe potential of PNP to affect testes function in the rat was assessed by intratesticular injection (IT). Experimental rats testes were injected with PNP (0.1 M,50 μl) in sterile vehicle (PBS,0.1M, pH 7.4, containing 0.05% Tween 80). The control testes were injected with vehicle. Rats were sacrificed on days 1,3 and 7 after IT of PNP. The seminiferous tubules from all control groups were similar at all time points observed, however, IT of PNP affected testes morphological appearance at current concentration, including haemorrhage in the intertubular areas, denudation and disorganization of the germinal epithelium and vacuolization of the germinal cells. All of these might adequately elucidate the increased expression of caspase-3 and sperm abnormalities (P< 0.05). In addition, the concentration of testosterone in serum fluctuated after PNP injection. PNP induced testes oxidative stress, which manifested increased SOD, CAT, GSH-Px activities and MDA, GSH, H2O2 concentrations (P< 0.05). Moreover, Nrf2 antioxidant pathway was activated, accompanyed with increased expression of Nrf2, HO-1, GCLC mRNA (P< 0.05). These results suggested that IT of PNP resulted in activation of Nrf2 antioxidant pathway and apotosis of the spermatogenic cell. Nrf2 displayed nuclear accumulation and protective activity in the process of PNP-induced oxidative stress in rat testes.