| Zn and Bisphenol-A (BPA) are the typical environmental pollutants of lower toxity, and there are lots of evidences showing that they threats aquatic life and human reproductive systems. Zn is an important biological essential element, however, excessive accumulation of Zn in organisms would endanger the health of individual organisms, and the mechanisms of related toxicological effects were complicated. BPA, a typical class of environmental endocrine disruptor, affect biological gonadal development and gender features, thereby affecting the sex ratio of the populated life, then endangering ecosystem health. Currently, the molecular mechanisms of reproduction toxicity resulted from them still keep unknown. The phenomenon of hermaphrodite and sex reversal can be found in oyster, which is one of the organisms that have the best performance in the bioaccumulation of Zn. Meanwhile, their gonads are rich and their gonadal developmental cycle is short. Thus, oysters are often chosen as the model organism to study the reproductive toxic effects of environmental pollutants.In this paper, oyster was also chosen as the model organism to study the toxic effects of chronic exposure of oyster gonad to ZnCl2and BPA. ICP-MS and GC-MS/MS were used to measure the bioaccumulation of Zn and BPA in oyster gonads. Next, different proteome of oyster gonads under the Zn2+or BPA stress were analyzed by the LC-MS/MS to reveal the mechanism of the related reproductive toxic effects.Firstly, a toxicoproteomic study was carried out in the Zn2+-exposed experiment. With the combined application of histological section, the measurement of SOD and CAT activities and the ICP-MS technology, the present study identified that the Zn bioaccumulation in oyster gonads still increased while the oxidative stress exerted a more strong inhibiting effects on the gonadal development. All these above suggested that the level of Zn bioaccumulation in oyster gonads was inversely proportional to the degree of gonadal development, which was similar to previous reports. It was speculated that Zn bioaccumulation in oyster gonads was closely related to the regulation of reproduction cycle. At the same time, according to the current proteomics study, we certified that the Zip family, ZnT family and the metallothionein/thionein systems were in charge of cellular zinc homeostasis in oyster gonads. Additionally, cavortin may also play a key role in the cellular zinc homeostasis. Other differentail proteins indentified in this experiment were used for the establishment of the STITCH (chemical-protein interactions) network. The whole STITCH network could be divided into some small functional networks concerning energy metabolism, oxidative stress and regulation of gene expression, respectively. STITCH networks analysis indicated that Zn2+stress in male oyster gonads resulted in gonads glucose metabolism dysfunction, i.e. the main energy supply of the gonads was switched from saccharometabolism to lipid mobilization, while the main energy supply of female gonads under Zn2+stress was still from saccharometabolism; Zn2+stress induced gene activation in male gonads while it caused gene silencing in female gonads; additionally, enzymatic activity analysis suggested in the male gonads, antioxidant enzyme activity were strongly and persistently inhibited by Zn2+stress, whereas in the female gonads, they recovered gradually after the inhibition. According to these results, we speculated that the responses to Zn2+stress were different between the male and female gonads. By the way, we selected some candidate biomarkers of Zn2+pollution from the differental proteins in the male and female groups, respectively. More specifically, in the male groups,14-3-3epsilon, galactosyltransferase and serine/threonine protein kinase can be regarded as the candidate biomarkers, while in the female groups, hemicentin, cyclohexadienyl dehydrogenase and ABC transporter permease also has the potential to be the candidate biomarkers.Next, the toxicoproteomic study was also performed in the BPA-exposed experiment. The toxicological effects of BPA were different from those of Zn2+exposure. The combination of morphological observation and determination of gonadosomatic index (GSI) showed that the chronic stress of lppm BPA accelerated the growth of female gonads while the effects of2ppm BPA to oyster ovaries were prohibitive. However, both of the lppm and2ppm BPA showed prohibitive effects to the development of male gonads. All this indicated that the toxicological effects of BPA showed closely related to its exposed concentration and the gender of the objects. In fact, the main mechanism of toxicological effects is closed to BPA’s estrogen-like effects. BPA has the ability to combine with the estrogen receptor. Once it finished combining with the estrogen receptor, it will affect the gene expression and disturb the physical function of estrogens. Thus, the toxicological effects of BPA varied by genders. Our current toxicoproteomic study further indicated that, in terms of female gonads, BPA restrained the various translation elongation factors, which may result in the disturbance of gene translation regulation; it also restrained the tubulins, possibly preventing the formation of follicles; moreover it stimulated the estrogen receptor, potentially activating the expression of vitellogenin to show its estrogen-like action; in addition, it can also inhibit the expression of nuclear autoantigenic sperm protein, probably preventing the germ cells entering the S phase, maybe resulting in losing a large number of oocytes. However, for the male oysters, the mechanism was different. Specifically, BPA inhibited the formation of periostin, resulting in the insufficient synthesis of collagen, finally maybe contributing to the inhibition of spermaducts in testis, thus the testis showed to be atrophic; it also regulated the voltage-dependent anion-selective channel protein2and the periostin, potentially disturbing the apoptosis in the testis. Furthermore, BPA stress upregulated the sperm surface protein Sp17possibly disturbing the sexuality differentiation and the formation of spermatic flagellum and cilia. In addition, upregulation of calmodulin induced by BPA possibly affected the formation of sperms and their mobility. Furthermore, these proteins were regarded as the potential candidate biomarkers for BPA pollution. Besides, the STITCH network also was successfully applied in the BPA-exposed experiment and the whole network also could be divided into some small functional networks including energy metabolism, oxidative stress, regulation of gene transcription and the vitellogenins interaction network. STITCH networks analysis suggested that the glycolysis and tricarboxylic acid cycle of the male gonads under BPA-stress were increased which enhanced their energy supply, while the female gonads only increased ATP synthetase expression level; moreover, enzymatic activity analysis suggested under the BPA stress, the activities of the antioxidant enzymes were inhibited befroe a gradual increase, and the speed of upregulation in female gonads faster than that of the males; BPA stress boosted the transcription elongation of the female gonads but not that of the males; with the stimulation of BPA’ estrogen-like effects, vitellogenins were remarkably upregulated in the male and female gonads, with greater level of upregulation observed in the female gonads. It was speculated that the BPA’estrogen-like effects worked obviously in the female oysters, however, these estrogen-like effects have little influences on the males while the oxidative stress effects might be more apparent in the males. |
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