Jiancao Gao

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.

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).

Effects of bisphenol A on lipid metabolism in rare minnow Gobiocypris rarus.

As one of the most abundant endocrine disrupting compounds (EDCs), bisphenol A (BPA) exists ubiquitously in an aquatic environment. Many studies on fish have focused on the reproductive toxicity effects of BPA. However, few has involved the effects of BPA on lipid metabolism. To evaluate the effects of BPA on lipid metabolism, we determine the hepato-somatic index, triglyceride contents in the liver and serum, the activities of acetyl-CoA carboxylase (ACC), fatty acid synthase (FASN), carnitine palmitoyltransferase (CPT1), and glycerol-3-phosphate acyltransferase (GPAT) enzymes and the mRNA expression of acaca, acacb, fasn, gpat1 and cpt1α in Gobiocypris rarus after exposure to BPA for 28days. BPA induced increasing tendency of triglyceride contents in male fish, possibly due to up-regulated lipid synthesis. Although in this process, fatty acid β-oxidation was up-regulated, it might be compensated by increasing lipogenesis. Our result also revealed that the GPAT enzyme might play a key role in lipid metabolism disturbance by BPA in females. Besides, the effect of BPA on the fatty acid β-oxidation pathway might be gender-dependent in G. rarus. Further studies are needed to investigate BPAs effects on the signaling pathway of lipid metabolism.

Effects of 17 α-methyltestosterone on transcriptome, gonadal histology and sex steroid hormones in rare minnow Gobiocypris rarus

The 17α-methyltestosterone (MT), a synthetic androgen, is known for its interference effects on the endocrine system. Aiming to investigate the transcriptome profiling of gonads induced by MT and to understand the molecular mechanism by which MT causes adverse effects in fish, transcriptome profiling of gonads, gonadal histology and the sex steroid hormones in response to MT were analyzed in Gobiocypris rarus. Eight libraries, 4 from the ovary and 4 from the testis, were constructed and sequenced and then a total number of clean reads per sample ranging from 7.03 to 9.99 million were obtained. In females, a total of 191 transcripts were differentially regulated by MT, consisting of 102 up-regulated transcripts and 89 down-regulated transcripts. In males, 268 differentially expressed genes with 108 up-regulated and 160 down-regulated were detected upon MT exposure. Testosterone serves as the major sex steroid hormone content in G. rarus of both sexes. The concentrations of 17β-estradiol, testosterone and 11-ketotestosterone were significantly increased in females and decreased in males after MT exposure. Interestingly, MT caused a decreased number of vitellogenic oocytes in the ovary and spermatozoa in the testis. After MT exposure, four differentially expressed genes (ndufa4, slc1a3a, caskin-2 and rpt3) were found in G. rarus of both sexes. Overall, we suggest that MT seemed to affect genes involved in pathways related to physiological processes in the gonads of G. rarus. These processes include the electron transfer of Complex IV, endothelial cell activation, axon growth and guidance, and proteasome assembly and glutamate transport metabolic.

Hepatic expression patterns of aryl hydrocarbon receptor, pregnane X receptor, two cytochrome P450s and five phase II metabolism genes responsive to 17alpha-methyltestosterone in rare minnow Gobiocypris rarus

17Alpha-methyltestosterone (MT), a synthetic androgen, is widely used in aquaculture. Aquatic organisms can receive continuous exposure to residual MT throughout their lives. Aiming to evaluate the effects of MT on genes involved in biotransformation pathway, meanwhile attempting to unravel the MT metabolic pathway at the transcriptional level in fish, here we isolated the cDNAs of previously unreported AHR2, Sult1 st1, Ugt2a1 and Ugt2b6 in rare minnow, and predominantly investigated the hepatic transcriptional patterns of AHR2, PXR and five biotransformation genes after MT exposure in both genders adult rare minnow Gobiocypris rarus. The present findings suggest that AHR2 and PXR should play important roles in regulating biotransformation enzymes related to MT catabolism, moreover, CYP1A, CYP3A, SULT1 ST4, SULT1 ST6 and UGT2A1 may play certain roles in catabolism of MT in adult G. rarus. Additionally, UGT2A1 may make greater contribution than SULT1 ST4 and SULT1 ST6 in MT catabolism in males.

Molecular mechanism of endocrine system impairment by 17α-methyltestosterone in gynogenic Pengze crucian carp offspring.

The effects of synthetic androgen 17α-methyltestosterone (MT) on endocrine impairment were examined in crucian carp. Immature 7-month old mono-female Pengze crucian carp (Pcc) F2 offspring were exposed to 50 and 100 μg/L of MT (week 2, 4, and 8). Gonadosomatic index, hepatosomatic index and intestine weight altered considerably and oocyte development was repressed. In the treatment groups, ovarian 11-ketotestosterone decreased, whereas 17β-estradiol and testosterone increased, and ovarian aromatase activities increased at week 4. However, in the brain tissue, those values significantly decreased. Quantitative RT-PCR analysis demonstrated changes in steroid receptor genes and upregulation of steroidogenic genes (Pcc-3bhsd, Pcc-11bhsd2 Pcc-cyp11a1), while the other three steroidogenic genes (Pcc-cyp17a1, Pcc-cyp19a1a and Pcc-star) decreased from week 4 to week 8. Ovarian, hepatic Pcc-vtg B and vitellogenin concentration increased in both 50 and 100 μg/L of MT exposure groups. This study adds further information regarding the effects of androgens on the development of previtellogenic oocytes, which suggests that MT could directly target estrogen signaling pathway, or indirectly affect steroidogenesis and vitellogenesis.

Accumulation and detoxification dynamics of microcystin-LR and antioxidant responses in male red swamp crayfish Procambarus clarkii.

MC-LR is one of major microcystin isoforms with potent hepatotoxicity. In the present study, we aim to: 1) explore the dynamics of MC-LR accumulation and elimination in different tissues of male red swamp crayfish Procambarus clarkii; 2) reveal the mechanisms underlying hepatic antioxidation and detoxification. In the semi-static toxicity tests under the water temperature of 25±2°C, P. clarkii were exposed to 0.1, 1, 10 and 100μg/L MC-LR for 7days for accumulation and subsequently relocated to freshwater for another 7days to depurate MC-LR. MC-LR was measured in the hepatopancreas, intestine, abdominal muscle and gill by HPLC. The enzyme activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione S-transferase (GST), content of glutathione (GSH), and transcripts of Mn-sod, cat, gpx1, Mu-gst, heat shock protein90 (hsp90), hsp70 and hsp60 in hepatopancreas were detected. The results showed that P. clarkii accumulated more MC-LR in intestine, and less in abdominal muscle and gill during accumulation period and eliminated the toxin more quickly in gill and abdominal muscle, and comparatively slowly in intestine during depuration period. The fast increase of SOD and CAT activities at early stage, subsequent decrease at later stage of accumulation period and then fast increase during depuration period were partially consistent with the transcriptional changes of their respective genes. GPx was activated by longer MC-LR exposure and gpx1 mRNA expression showed uncoordinated regulation pattern compared with its enzyme. Hsp genes were up-regulated when P. clarkii was exposed to MC-LR.

Responsiveness of four gender-specific genes, figla, foxl2, scp3 and sox9a to 17α-ethinylestradiol in adult rare minnow Gobiocypris rarus.

Proteins encoded by figla, foxl2, scp3 and sox9a play important roles in gonad differentiation and reproduction. In the present study, we aimed to determine the responsiveness of figla, foxl2, scp3 and sox9a to 17α-ethinylestradiol (EE2) in the gonads of adult Gobiocypris rarus. Full-length cDNAs of figla, scp3 and sox9a were cloned and characterized by RT-PCR and RACE methods. Expression patterns in adult tissues were investigated. Results indicated that figla was predominantly expressed in adult ovaries and scp3 was restrictively expressed in the male testes and sox9a was principally expressed in the brains of both genders and the testes of males. Gene expression profiles of figla, foxl2, scp3 and sox9a were analyzed in the gonads of adult G. rarus exposed to EE2 at 1, 5, 25, and 125ng/L for 3 and 6days. Three-day EE2 treatment at 1-125ng/L all caused a significant increase of figla transcript in testes and foxl2 transcript in ovaries. However, six-day EE2 exposure at 1-125ng/L repressed figla and scp3 transcript in testes and foxl2 transcript in ovaries. The present study indicates that the testicular transcripts of figla and scp3 in males and the ovarian foxl2 transcript in females have high responsiveness to EE2 and they can be used as sensitive molecular biomarkers for early warning to monitor the environmental estrogenic chemicals in fresh water environment. The present study also suggests that the effective EE2 dosage for feminization in male G. rarus might be at least 25ng/L.