Zaizhao Wang

Effects of 17α-ethinylestradiol and bisphenol A on steroidogenic messenger ribonucleic acid levels in the rare minnow gonads

Previous studies showed that the endocrine disrupting chemicals (EDCs) affect reproductive physiology in teleosts. How the EDCs regulate gonadal steroidogenesis remains to be determined. The gonadal transcript changes of steroidogenic enzyme genes in adult rare minnow Gobiocypris rarus exposed to 17α-ethinylestradiol (EE2) and bisphenol A (BPA) were detected in the present study. The full-length cDNAs encoding steroidogenic enzymes, including steroidogenic acute regulatory protein (StAR), cytochrome P450-mediated side-chain cleavage enzyme (CYP11A1), 3β-hydroxysteroid dehydrogenase (3β-HSD), and cytochrome P450 17 A1 (CYP17A1) were isolated and characterized by RT-PCR and RACE methods. The homology and phylogenetic analyses of the amino acid sequences confirmed that the nucleotide sequences of these steroidogenic genes were correct. The mRNA tissue distribution results indicated that StAR, cyp11a1, and cyp17a1 mRNAs were mainly expressed in the gonads and 3β-HSD was mainly expressed in both the gonads and the brains. The 233 dpf adult G. rarus were exposed to EE2 (25ng/L) and BPA (5, 15, and 50 μg/L) dissolved in dimethyl sulfoxide (DMSO) or control for 7 days. The gonadal mRNA levels of StAR, cyp11a1, 3β-HSD, cyp17a1 and ovarian cytochrome P450 aromatase (cyp19a1a) were quantified by qRT-PCR. Our data indicated that 25 ng/L EE2 had different degrees of inhibitory effects on the expression of steroidogenic genes in the gonads. BPA at different levels caused concentration-specific effects on the mRNA expression of the steroidogenic genes. The transcripts of several ovarian steroidogenic genes were more sensitive to 15 μg/L BPA than that at other two levels. These findings suggest that EE2 could impair gonadal steroidogenesis by suppressing mRNA expression of steroidogenic genes and BPA could cause variations in gonadal steroidogenesis modulation with a potential consequence of compensation for the disturbance.

Molecular cloning and characterization of cat, gpx1 and Cu/Zn-sod genes in pengze crucian carp (Carassius auratus var. Pengze) and antioxidant enzyme modulation induced by hexavalent chromium in juveniles

Hexavalent chromium (Cr(6+)) is a common pollutant transient metal with high toxicity in the environment. The toxicological effects partly result from oxidative damage due to the production of excessive reactive oxygen species (ROS) in the reductive process of Cr(6+). To explore the influence of ROS induced directly by Cr(6+) on the oxidative stress generation and antioxidant system, the full length cDNAs of antioxidant-related genes cat, gpx1 and Cu/Zn-sod were successfully acquired from pengze crucian carp first and analyzed. Furthermore, the mRNA expression of the antioxidant genes encompassing catalase (cat), copper/zinc superoxide dismutase (Cu/Zn-sod) and glutathione peroxidase (gpx1), antioxidant enzyme activities of CAT, SOD, and GPx and total protein content were further studied in the gill, intestine and liver of pengze crucian carp (Carassius auratus var. Pengze) juveniles upon acute exposure to Cr(6+) at concentrations of 0.1, 1.0, 10 and 100 mg/L for 4 days. Differential significant changes of the antioxidant enzymes and gene expression were observed in different tissues. The findings contribute to better understanding the antioxidant mechanisms induced by Cr(6+) and selecting the organic-specific sensitive biomarkers to monitor the safety of the aquatic ecosystem.

Bisphenol A affects gene expression of gonadotropin-releasing hormones and type I GnRH receptors in brains of adult rare minnow Gobiocypris rarus.

Recent studies support the notion that endocrine disrupting chemicals (EDCs) could affect the reproductive regulations of the neuroendocrine system. The objectives of the present study were to determine whether the weak estrogenic chemical, bisphenol A (BPA), disrupts gonadotropin-releasing hormone (GnRH) system by altering the transcription of GnRHs and GnRH receptor (GnRHR) genes in adult rare minnow Gobiocypris rarus. In the present study, the histological examination of the ovary after 35-day BPA exposure at 15 μg/L demonstrated the perturbing effects of environmentally relevant BPA on the ovarian development in G. rarus. In addition mRNA expression of ovarian P450 aromatase in both ovaries and testes were significantly down-regulated by 15 μg/L BPA. GnRH2, GnRH3, GnRHR1A and GnRHR1B gene were identified in G. rarus. The expression patterns of GnRHs and GnRHR1s were analyzed in various tissues of G. rarus by quantitative real-time PCR. GnRHs and GnRHR1s were all predominantly expressed in the brains. Both GnRH3 and GnRHR1A were significantly upregulated in the brains of female exposed to 15 μg/L BPA for 35 days. It would suggest a potential negative feedback in the GnRH system in response to the disturbance of downstream of the brain-pituitary-gonadal axis. Collectively, the present findings suggest that the transcripts of some key genes in the neuroendocrine system can be used as critical biomarkers in endocrine disruption assays of teleost fish.

Induction of oxidative stress and the transcription of genes related to apoptosis in rare minnow (Gobiocypris rarus) larvae with Aroclor 1254 exposure.

Polychlorinated biphenyls (PCBs) are a group of environmental contaminants widely dispersed in aquatic system. Recent data have shown that Aroclor 1254 (a highly chlorinated PCB mixture) has the potential to induce oxidative stress. The antioxidant genes are usually up-regulated in response to the oxidative stress. However, the mechanisms underlying the modulation are little known. We hypothesized that nuclear factors erythroid 2-related factor 2 (Nrf2) and nuclear factor-kappa B (NF-κB) might be involved in the regulation. In this study, rare minnow (Gobiocypris rarus) larvae were exposed to the Aroclor 1254 at four concentrations (5, 50, 500 and 5000μg/L) for 7 days. We found that the mRNA expressions of antioxidant genes (Cu/Zn-sod, Mn-sod, Cat, Gpx1 and Gclc) were strongly enhanced by Aroclor 1254 at high concentrations. H2O2 was significantly induced by 500 and 5000μg/L Aroclor 1254 exposure and protein thiol significantly decreased with 5000μg/L Aroclor 1254 exposure. The expression of Nrf2 and NF-κB were significantly up-regulated. Taken together, we proposed that the activation of Nrf2 and NF-κB by ROS might be a potential mechanism underlying the antioxidant gene expression induction in G. rarus larvae by Aroclor 1254. Furthermore, we investigated that the expression of genes related with apoptosis. The gene expression patterns reveal that waterborne Aroclor 1254-induced apoptosis is probably through DNA damage (p53 and p53 upregulated modulator of apoptosis (Puma)), disrupting mitochondrial membrane integrity (B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X (Bax)) and c-jin N-terminal kinase (JNK)-mediated apoptotic pathway (thioredoxin 1 (Trx1) and c-jun N-terminal kinase (JNK)). Aroclor 1254 at 5μg/L did not cause any changes in G. rarus. The findings will help us to understand the toxicological mechanism of Aroclor 1254 in fish and properly assess the risk of the environmental contaminant.

Oxidative stress and immunotoxic effects of bisphenol A on the larvae of rare minnow Gobiocypris rarus.

Bisphenol A (BPA), a known endocrine disrupting chemical, is ubiquitous in the aquatic environment and can pose risk to the health of aquatic organisms. Studies on immunotoxicity of BPA in aquatic organisms are limited. In this study, rare minnow (Gobiocypris rarus) larvae were exposed to 1, 225 and 1000μg/L BPA for 7 days. Inflammatory effects of BPA exposure were assessed from the increased production of nitric oxide (NO) and reactive oxygen species (ROS), the change of iNOS mRNA and other TLRs-associated immune gene expression. Our findings provide evidences that different concentrations of BPA can induce a toxic response in fish to produce reactive free radicals which can affect the function of T lymphocytes and decrease the transcription levels of cytokine genes. The excess production of H2O2, induced oxidative stress and suppressed TLR4/NF-κB signaling, leading to immunosuppressive effects in fish larvae. The present results suggest that BPA has the potential to induce oxidative stress accompanied by immunosuppression in rare minnow larvae.

Molecular characterization of five steroid receptors from pengze crucian carp and their expression profiles of juveniles in response to 17α-ethinylestradiol and 17α-methyltestosterone.

Pengze crucian carp (Carassius auratus var. pengze, Pcc), a triploid gynogenetic fish, was used in this study to investigate the cross-talk between EDCs and steroid receptors. The full-length cDNAs of five steroid receptors (esr1, er alpha2, esr2a, esr2b, ar) and partial cDNA of vtg B were isolated. The tissue distributions of these genes were analyzed in adult fish by qRT-PCR. Then the expression profiles of five steroid receptors (esrs and ar) and vtg B were detected in the juveniles exposed to 17α-ethinylestradiol (EE2, 0.1, 1 and 10ng/L) and 17α-methyltestosterone (MT, 50μg/L) for 4weeks. The results demonstrated that esrs, ar, and vtg B were predominantly expressed in liver of adult fish. However, among these detected genes, esr1 and er alpha2 mRNAs are sensitive biomarkers in response to EE2 at 0.1, 1, and 10ng/L for 1 and 2weeks compared to esr2a, esr2b, ar, and vtg B in the juveniles of mono-female gynogenetic fish. Totally, the subtypes of esrs show biphasic responses to EE2 exposures for 4weeks, and most of the EE2 exposures at 0.1, 1, and 10ng/L for 1, 2, 3 and 4weeks did not induce the mRNA expressions of vtg B. However, 1-, 2-, and 4-week 50μg/L MT all significantly stimulated vtg B transcripts. Further investigations are needed to elucidate the mechanism underlying the insensitivity or down-regulation of vtg B mRNA in response to EE2 in juvenile Pcc.

Cloning, sequencing and expression analysis of cDNA encoding a constitutive heat shock protein 70 (HSC70) in Fenneropenaeus chinensis

The cDNA encodinghsc70 of Chinese shrimpFenneropenaeus chinensis was cloned from hepatopancreas by RT-PCR based on its EST sequence. The full length cDNA of 2090 bp contained an open reading frame of 1956 nucleotides and partial 5′- and 3′-untranslated region(5′- and 3′-UTR). PCR amplification and sequencing analysis showed the existence of introns in the region of 1–547 bp, but they did not exist in the region of 548–2090 bp ofhsc70 cDNA. When the deduced 652 amino acid sequence of HSC70 was compared with the members of HSP70 family from other organisms, the results showed 85.9% similarity with HSC71 fromOncorhynchus mykiss and HSC70 fromHomo sapiens. It also exhibited 85.8% similarity with HSP70 fromMus musculu and 85.4% with HSC70 fromManduca sexta. Expression analysis showed that hsc70 mRNA was espressed constitutively in hepatopancreas, muscle, eyestalks, haemocytes, heart, ovary, intestine and gills inFenneropenaeus chinensis. No difference could be detected onhsc70 mRNA level in muscle between heat-shocked and control animals.

Global DNA methylation in gonads of adult zebrafish Danio rerio under bisphenol A exposure.

Altered DNA methylation is pervasively associated with changes in gene expression and signal transduction after exposure to a wide range of endocrine disrupting chemicals. As a weak estrogenic chemical, bisphenol A (BPA) has been extensively studied for reproductive toxicity. In order to explore the effects of BPA on epigenetic modification in gonads of zebrafish Danio rerio, we measured the global DNA methylation together with the gene expression of DNA methyltransferase (dnmts), glycine N-methyltransferase (gnmt), and ten-eleven translocation (tets) in gonads of D. rerio under BPA exposure by ELISA and quantitative real-time PCR method, respectively. The global level of DNA methylation was significantly decreased in ovaries after exposed to BPA for 7 days, and testes following 35-day exposure. Moreover, the global level of DNA methylation was also significantly reduced in testes after exposed to 15μg/L BPA for 7 days. Besides the alteration of the global level of DNA methylation, varying degrees of transcriptional changes of dnmts, gnmt and tets were detected in gonads of D. rerio under BPA exposure. The present study suggested that BPA might cause the global DNA demethylation in gonads of zebrafish by regulating the transcriptional changes of the DNA methylation/demethylation-associated genes (dnmts, gnmt, and tets).

Non-monotonic dose-response effect of bisphenol A on rare minnow Gobiocypris rarus ovarian development.

Bisphenol A (BPA) is widely spread in the environment, and can cause various reproductive disrupting effects on different organisms, including fish. Our previous published study showed that BPA has non-monotonic (inverted U-shaped) dose-response effect on rare minnow Gobiocypris rarus ovarian weight at different concentrations. To investigate the potential mechanism, we exposed female rare minnow to 1, 15 and 225 µg L(-1) BPA for 7 days in the present study. The levels of vitellogenin (Vtg), sex hormones, hydrogen peroxide (H2O2), glutathione (GSH) and triglyceride (TG) were measured. RNA-seq of ovary tissues was also performed. Result showed that Vtg, sex hormone and TG levels showed an inverted U-shaped increased response, while H2O2 and GSH levels showed a U-shaped inhibited response. RNA-seq data showed that many genes involved in lipid metabolism, oxidative stress, and proteolysis processes were altered. The change of Vtg, H2O2, GSH and TG levels was possibly related to the altered sex hormone levels. Sex hormones direct effect, Vtg accumulation, TG accumulation and oxidative stress induced proteolysis may contribute to the change of ovary weight.

Characterization of four nr5a genes and gene expression profiling for testicular steroidogenesis-related genes and their regulatory factors in response to bisphenol A in rare minnow Gobiocypris rarus

Bisphenol A (BPA) widely used in the manufacture of numerous products is ubiquitous in aquatic environment. To explore the mechanisms of BPA-mediated actions, male rare minnow Gobiocypris rarus were exposed to BPA at concentrations of 5, 15, and 50 μg/L for 14 and 35 days in the present study. Four subtypes of nr5a gene encoding important transcription factors for steroidogenesis were characterized, and tissue distribution analysis demonstrated distinct expression profiling of the four genes in G. rarus. BPA at environmentally relevant concentration (5 μg/L) caused increase of gonadosomatic index (GSI) of male fish. In response to BPA, no obvious changes on the testis development were observed. Modulation of vtg mRNA expression by BPA suggests estrogenic and/or anti-estrogenic effects of BPA were dependent on exposed duration (14 or 35 days). Gene expression profiling for testicular steroidogenesis-related genes, sexual steroid receptors, gonadotropin receptors, and transcription factors indicates differential regulation was dependent on exposure duration and dose of BPA. The correlation analysis at mRNA level demonstrates that the BPA-mediated actions on testicular steroidogenesis might involve sex steroid hormone receptor signaling, gonadotropin/gonadotropin receptor pathway, and transcription factors such as nuclear receptor subfamily 5, group A (Nr5a), fork head box protein L2 (Foxl2).