| During the last decades, a variety of synthetic chemicals and their by-products have been released to the environment as a consequence of efforts to increase agricultural productivity or modern manufacturing, which caused severe harm for ecosystem, animal and human health. Some of these chemicals are similar to endogenous hormone and termed as the endocrine disrupting chemicals (EDCs). It induced reproductive abnormalities (reduction of sperm number, induction of testicular cancer and breast cancer), neural disorder (cognitive deficits and motor dysfunction) and immune disorder (increase of the allergic disease). Such wide toxicity of EDCs may impose great threat to the health of animal and human populations. Since the oxidative damage was the pivotal route of EDCs toxicity, any antioxidants, especially those plant compounds from the dietary sources, may prevent negative effects induced by the oxidative EDCs. Flavonoids are important phytochemicals ubiquitous in the plant kingdom and exhibit multiple biological activities on human health. Especially the low toxicity of flavonoids on human health has been given rise to much attention. Flavonoids possess the antioxidative potential such as scavenging free radicals, metal chelation. However, the attenuating mechanism of antioxidant flavonoids on reproductive and developmental toxicity induced by oxidative EDCs remains unclear at the cellular and molecular levels. Therefore, the present study adopted testicular cells of chicken embryo to evaluate the antioxidant effects of flavonoids and provided the firm evidence for the preventive effect of food-derived flavonoids on the reproductive toxicity induced by oxidative EDCs.1. Establishment and application of cell culture modelThe serum-free germ-somatic cell coculture model was established to evaluate the effect of EDCs. Testicular cells of 18-day-old chicken embryos were dispersed and cultured in different media. Results showed that somatic cells formed a monolayer to which germ cells were round in shape and adhered in the medium supplemented with insulin (Ins), transferrin (Tf) and selenite (Se) (ITS medium). Many growth factors such Ins, Tf could support cell survival in culture. FCS is a source of mixed growth factors that are capable of maintaining cell survival but only a few large germ cell masses can be observed in FCS-supplemented medium. However, it is clear to observe the morphological changes of germ cells in ITS medium as the consequence of the monolayer formed by somatic cells. Germ cells were characterized by expression of a specific antibody for stem cell factor receptor c-kit and follicle-stimulating hormone (FSH) increased c-kit expression and promoted germ cell proliferation, compared with the control. Three well-known EDCs were selected to test the feasibility of this model. Aroclor 1254 (A 1254, 10μg/ml), a mixture of PCB from the industry, induced condensed nuclei and vacuolated cytoplasm in germ cells, which was further confirmed by a cell proliferation assay. However after culture for 48h, the number of germ cells displayed a significant augment stimulated by A1254 (0.1-10μg/ml) (P<0.05). Similarly, the herbicide 2, 4-dichlorophenoxyacetic acid (2,4-D) from the agriculture and the antineoplastic drug busulfan from the medicine displayed notable toxic effects on germ cells, and the germ cell number and cell viability were significantly decreased in a dose-dependent manner (P<0.05). The above results indicate that the serum-free germ-somatic cell coculture model is a simple, rapid and veracious in vitro tool for studies such as the effects of the endogenous hormones and exogenous materials such as EDCs on the cultured testicular cells. Furthermore, the model is also used to evaluate the mechanism of reproductive toxicity and reproductive nutrition.2. The effects of gonadotropin and sex steroids on proliferation of testicular germ cellsThe proliferation and differentiation of germ cells require complex regulation of endogenous hormones in the auto/paracrine pattern. FSH from the adenohypophysis and sex sterids from testicular Leydig cells plays crucial roles in gonadal development. Therefore, a germ-somatic cell coculture model was used to study effects of FSH, testosterone (T) and 17β-estradiol (E2) on testicular germ cell proliferation. FSH (0.25-1.0 IU/ml) significantly increased the number of germ cells and proliferating cell nuclear antigen (PCNA)-labeling index (P<0.05). In the FSH-treated groups, germ cells were manifested distinct knob-like form. Similar stimulating effect was found in the germ cell number by both T (10-7-10-6 mol/L) and E2 (100 ng/mL) treatment. T (10-7-10-6 mol/L) also significantly increased PCNA-labeling index. Furthermore, FSH (0.5IU/mL) combined with T significantly promoted germ cell proliferation (P<0.05), compared with either FSH or T alone, which implied that interaction of FSH and T might be additive. The above results indicated that the serum-free germ-somatic cell coculture model allowed evaluating hormonal regulation of testicular germ cell proliferation. FSH and sex steroids promoted testicular germ cell proliferation probably through indirect effects on somatic cells.3. Protective effect of daidzein on PCB-induced oxidative damage in cultured chicken testicular germ cellsPolychlorinated biphenyls (PCBs) are members of the halogenated hydrocarbon class of EDCs and imposed adverse effects on human and animal. Though the manufacture and use of PCBs had been strictly prohibited in most industrial countries they are still common in the environment as a result of the large quantity of previous use, stable property and biomagnification along food chains. The isoflavonoid daidzein (DAI) is found at high levels in soy products and possess the antioxidant and estrogenic activities. So the aim of this study was to evaluate the protective effects of DAI on oxidative damage induced by PCBs in cultured testicular germ cells, with emphasis on the mechanisms. Results showed that A1254 (0.1-1μg/mL) manifested obvious hormonal effect to induce germ cell proliferation, while A1254 at 10μg/mL displayed remarked toxic and hormonal effects on germ and somatic cells after 48h culture. The antioxidantα-Tocopherol attenuated the toxic effect of A1254. Blockade of either androgen receptor by flutamide or estrogen receptor by tamoxifen inhibited hormonal effect of A1254 on germ cell proliferation and reinforced the cytotoxicity. As the consequence of the cytotoxicity of A1254, The mechanism of A1254 toxicity was further explored by assessment of the membrane integrity (loctate dehydrogenase leakage, LDH leakage), production of thiobarbituric acid reactive substances (TBARS, an indicator of lipid peroxidation), activity of superoxide dismutase (SOD, a scavenger of superoxide) and glutathione (GSH, an intracellular antioxidant). Exposure to A1254 reduced SOD activity and GSH content, but increased LDH leakage and the levels of TBARS significantly. Therefore, A1254 induced the cytotoxicity by oxidative damage. However, simultaneous supplementation with DAI restored these parameters to the control levels. Thus, DAI displayed protective effects on testicular cells from A1254-induced toxicity through preventing oxidative damage. Additionally, DAI increased germ cell number, which was inhibited by co-treatment with tamoxifen. So DAI exerted the estrogenic activity by inducing germ cell proliferation. Therefore, the hormonal and toxic effects of A1254 interrupt the development of germ cells. However, the phytoantioxidant DAI with the weak estrogenic activity attenuated the oxidative damage. Therefore, dietary-derived DAI would play critical role in the chemprotection of abnormal development of gonads induced by the oxidative EDCs and be beneficial for health of human and animals.4. Protective effect of quercetin on EDCs-induced oxidative damage in cultured chicken testicular germ cellsQuercetin (QUE), a dietary-derived falvonol-type flavonoid, is ubiquitous in fruits and vegetables and plays important roles in human health due to its antioxidant function. The present study was performed to investigate effects of QUE on oxidative damage induced by EDCs (A1254 and 2,4-D) with the germ-somatic cell coculture model. Results showed that exposure to A1254 (10μg/mL) and 2,4-D (50μg/mL) induced an elevation of LDH leakage. The present studies revealed that A1254 and 2,4-D caused an elevation of TBARS and induced lipid peroxidation but a decrease in SOD activity and GSH content and damaged the antioxidant defense system. However, QUE had no deleterious effect on germ cells at 0.01-1μg/mL. And compared with A1254 or 2,4-D treatment alone, simultaneous supplementation with QUE decreased LDH leakage to maintain the cell integrity, decreased the levels TBARS to quench the free radicals, increased SOD activity and GSH content to restore endogenous antioxidant defense system. Thus, QUE displayed protective effects on germ cells from A1254 or 2,4-D-induced oxidative damage through antioxidant action. Consequently, the antioxidant from plant food or feed may be a safety, innoxious and excellent method to attenuate negative effects of oxidative EDCs.Conclusion:The germ-somatic cell coculture model of embryonic chicken testes was established for endocrine and toxicological studies. The results showed testicular germ cells were c-kit positive and thus c-kit represented a molecular marker for germ cells. FSH increased c-kit expression and stimulated germ cell proliferation. It also combined with T to significantly promote higher PCNA expression. Furthermore, E2 also promoted germ cell proliferation. The industrial EDCs A1254 displayed obvious toxic and hormonal effects on germ cells while the agricultural and medical EDCs 2,4-D and busulfan displayed notable toxic effects. In addition, A1254 and 2,4-D damaged the membrane integrity, induced the lipid peroxidation, damaged antioxidant defense system, and thus induced the oxidative damage. However, flavonoids DAI and QUE inhibited the oxidative damage induced by A1254 and 2,4-D. Therefore, the plant-derived flavonoids could attenuate the oxidative damage induced by EDCs and inhibit the reproductive toxicity caused by EDCs. These results elucidated the antioxidative mechanism of natural dietary-derived flavonoids and provided potential application of flavonoids in preventing the reproductive toxicity induced by oxidative EDCs.
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