Mitochondrial DNA Stress-Mediated Health Risk to Dibutyl Phthalate Contamination on Zebrafish (Danio rerio) at Early Life Stage.

Plastics contaminations are found globally and fit the exposure profile of the planetary boundary threat. The plasticizer of dibutyl phthalate (DBP) leaching has occurred and poses a great threat to human health and the ecosystem for decades, and its toxic mechanism needs further comprehensive elucidation. In this study, environmentally relevant levels of DBP were used for exposure, and the developmental process, oxidative stress, mitochondrial ultrastructure and function, mitochondrial DNA (mtDNA) instability and release, and mtDNA-cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) signaling pathway with inflammatory responses were measured in zebrafish at early life stage. Results showed that DBP exposure caused developmental impairments of heart rate, hatching rate, body length, and mortality in zebrafish embryo. Additionally, the elevated oxidative stress damaged mitochondrial ultrastructure and function and induced oxidative damage to the mtDNA with mutations and instability of replication, transcription, and DNA methylation. The stressed mtDNA leaked into the cytosol and activated the cGAS-STING signaling pathway and inflammation, which were ameliorated by co-treatment with DBP and mitochondrial reactive oxygen species (ROS) scavenger, inhibitors of cGAS or STING. Furthermore, the larval results suggest that DBP-induced mitochondrial toxicity of energy disorder and inflammation were involved in the developmental defects of impaired swimming capability. These results enhance the interpretation of mtDNA stress-mediated health risk to environmental contaminants and contribute to the scrutiny of mitochondrial toxicants.

Antibiotic-Induced Recruitment of Specific Algae-Associated Microbiome Enhances the Adaptability of Chlorella vulgaris to Antibiotic Stress and Incidence of Antibiotic Resistance.

Insights into the symbiotic relation between eukaryotic hosts and their microbiome lift the curtain on the crucial roles of microbes in host fitness, behavior, and ecology. However, it remains unclear whether and how abiotic stress shapes the microbiome and further affects host adaptability. This study first investigated the effect of antibiotic exposure on behavior across varying algae taxa at the community level. Chlorophyta, in particular Chlorella vulgaris, exhibited remarkable adaptability to antibiotic stress, leading to their dominance in phytoplankton communities. Accordingly, we isolated C. vulgaris strains and compared the growth of axenic and nonaxenic ones under antibiotic conditions. The positive roles of antibiotics in algal growth were apparent only in the presence of bacteria. Results of 16S rRNA sequencing further revealed that antibiotic challenges resulted in the recruitment of specific bacterial consortia in the phycosphere, whose functions were tightly linked to the host growth promotion and adaptability enhancement. In addition, the algal phycosphere was characterized with 47-fold higher enrichment capability of antibiotic resistance genes (ARGs) than the surrounding water. Under antibiotic stress, specific ARG profiles were recruited in C. vulgaris phycosphere, presumably driven by the specific assembly of bacterial consortia and mobile genetic elements induced by antibiotics. Moreover, the antibiotics even enhanced the dissemination potential of the bacteria carrying ARGs from the algal phycosphere to broader environmental niches. Overall, this study provides an in-depth understanding into the potential functional significance of antibiotic-mediated recruitment of specific algae-associated bacteria for algae adaptability and ARG proliferation in antibiotic-polluted waters.

Metagenomic insights into comparative study of nitrogen metabolic potential and microbial community between primitive and urban river sediments.

As a result of anthropogenic pollution, the nitrogen nutrients load in urban rivers has increased, potentially raising the risk of river eutrophication. Here, we studied how anthropogenic impacts alter nitrogen metabolism in river sediments by comparing the metagenomic function of microbial communities between relatively primitive and human-disturbed sediments. The contents of organic matter (OM), total nitrogen (TN), NO3--N and NO2--N were higher in primitive site than in polluted sites, which might be due to vegetation density, sediment type, hydrology, etc. Whereas, NH4+-N content was higher in midstream and downstream, indicating that nitrogen loading increased in the anthropogenic regions and subsequently leading higher NH4+-N. Hierarchical cluster analyses revealed significant changes in the community structure and functional potential between the primitive and human-affected sites. Metagenomic analysis demonstrated that Demequina, Streptomyces, Rubrobacter and Dechloromonas were the predominant denitrifiers. Ardenticatena and Dechloromonas species were the most important contributors to dissimilatory nitrate reduction. Furthermore, anthropogenic pollution significantly increased their abundance, and resulting in a decrease in NO3-, NO2--N and an increase in NH4+-N contents. Additionally, the SOX metabolism of Dechloromonas and Sulfuritalea may involve in the sulfur-dependent autotrophic denitrification process by coupling the conversion of thiosulfate to sulfate with the reduction of NO3--N to N2. From pristine to anthropogenic pollution sediments, the major nitrifying bacteria harboring Hao transitioned from Nitrospira to Nitrosomonas. This study sheds light on the consequences of anthropogenic activities on nitrogen metabolism in river sediments, allowing for better management of nitrogen pollution and eutrophication in river.

Biofilm characteristics and transcriptomic profiling of Acinetobacter johnsonii defines signatures for planktonic and biofilm cells.

Most bacteria in the natural environment have a biofilm mode of life, which is intrinsically tolerant to antibiotics. While until now, the knowledge of biofilm formation by Acinetobacter johnsonii is not well understood. In this study, the characteristics and the effect of a sub-inhibitory concentration of antibiotic on A. johnsonii biofilm and planktonic cells were determined. We discovered a positive relationship between biofilm formation and tetracycline resistance, and biofilms rapidly evolve resistance to tetracycline they are treated with. Persister cells commonly exist in both planktonic and biofilm cells, with a higher frequency in the latter. Further transcriptomic analysis speculates that the overexpression of multidrug resistance genes and stress genes were mainly answered to sub lethal concentration of tetracycline in planktonic cells, and the lower metabolic levels after biofilm formation result in high resistance level of biofilm cells to tetracycline. Altogether, these data suggest that A. johnsonii can adjust its phenotype when grown as biofilm and change its metabolism under antibiotic stress, and provide implications for subsequent biofilm control.

Metagenomic assembly and binning analyses the prevalence and spread of antibiotic resistome in water and fish gut microbiomes along an environmental gradient.

The pristine river and urban river show an environmental gradient caused by anthropogenic impacts such as wastewater treatment plants and domestic wastewater discharges. Here, metagenomic and binning analyses unveiled antibiotic resistance genes (ARGs) profiles, their co-occurrence with metal resistance genes (MRGs) and mobile genetic elements (MGEs), and their host bacteria in water and Hemiculter leucisculus samples of the river. Results showed that the decrease of ARG abundances from pristine to anthropogenic regions was attributed to the reduction of the relative abundance of multidrug resistance genes in water microbiomes along the environmental gradient. Whereas anthropogenic impact contributed to the enrichment of ARGs in fish gut microbiomes. From pristine to anthropogenic water samples, the dominant host bacteria shifted from Pseudomonas to Actinobacteria. Potential pathogens Vibrio parahaemolyticus, Enterobacter kobei, Aeromonas veronii and Microcystis aeruginosa_C with multiple ARGs were retrieved from fish gut microbes in lower reach of Ba River. The increasing trends in the proportion of the contigs carrying ARGs (ARCs) concomitant with plasmids along environmental gradient indicated that plasmids act as efficient mobility vehicles to enhance the spread of ARGs under anthropogenic pressures. Moreover, the higher co-occurrence of ARGs and MRGs on plasmids revealed that anthropogenic impacts accelerated the co-transfer potential of ARGs and MRGs and the enrichment of ARGs. Partial least squares path modeling revealed anthropogenic contamination could shape fish gut antibiotic resistome mainly via affecting ARG host bacteria in water microbiomes, following by ARGs co-occurrence with MGEs and MRGs in gut microbiomes. This study enhanced our understanding of the mechanism of the anthropogenic activities on the transmission of antibiotic resistome in river ecosystem and emphasized the risk of ARGs and pathogens transferring from an aquatic environment to fish guts.

Mitochondrial changes in fish cells in vitro in response to serum deprivation.

Mitochondria are critical to cellular activity that implicated in expansive networks to maintain organismal homeostasis under external stimuli of nutrient variability, a common and severe stress to fish performance during the intensive culture conditions. In the present study, zebrafish embryonic fibroblast cells were used to investigate the fish mitochondrial changes upon serum deprivation. Results showed that mitochondrial content and membrane potential were significantly reduced with increased intracellular ROS level in the serum deprivation treated fish cells. And the impaired mitochondria were characterized by rough and fracted outer membrane, and more fused mitochondria were frequently observed with the upregulated mRNA expressions of mitochondrial fusion genes (mfn1b, mfn2, and opa1). Besides, the mitochondrial DNA (mtDNA) copy numbers of mtatp6, mtcox1, mtcytb, mtnd4, and mtnd6 were overall showing the highly significant reduction, together with the mRNA expressions of these genes significantly increased, exhibiting the compensatory effects in mitochondria. Furthermore, the methyl-cytosine of whole mtDNA was compared and the methyl-reads numbers were distinctly increased in the treatment group, reflecting the instability of fish mtDNA with mitochondrial dysfunction under nutrient fluctuations. Collectively, current findings could facilitate the integrated research between fish mitochondrial response and external variables that indicates the potentially profound and durative deficits in fish health during the aquaculture processes.

Phenotype profiles and adaptive preference of Acinetobacter johnsonii isolated from Ba River with different environmental backgrounds.

Acinetobacter johnsonii is a potentially opportunistic pathogen widely distributed in nosocomial and natural environments, but little attention has been paid to this bacillus. Here A. johnsonii strains from Ba River with different pollution levels were isolated. In this study, we found that the increasing anthropogenic contaminants accounted for the emergence of multidrug-resistant (MDR) A. johnsonii strains. Correlation analysis results showed that the resistance phenotype of strains could be generated by co-selection of heavy metals or non-corresponding antibiotics. The whole genome sequence analysis showed that the relative heavy pollution of water selects strains containing more survival-relevant genes. We found that only some genes like blaOXA-24 were responsible for its corresponding resistance profile. Additionally, the tolerance profiles toward heavy metals also attribute to the expression of efflux pumps rather than corresponding resistance genes. In summary, our finding revealed that the resistance profiles of A. johnsonii could be generated by cross or co-selection of anthropogenic contaminants and mediated by efflux pumps instead of corresponding resistance determinants. Our study also has deep-sight into the adaptive preference of bacteria in natural environments, and contributes to surveillance studies and MDR- A. johnsonii monitoring worldwide.

Shifts in bacterial communities and antibiotic resistance genes in surface water and gut microbiota of guppies (Poecilia reticulata) in the upper Rio Uberabinha, Brazil.

Anthropogenic activities especially water pollution can affect the diversity and composition of microbial communities and promote the spread of antibiotic resistance genes (ARGs). In this study, water samples and guppies (Poecilia reticulata) were sampled from six sampling sites along the Uberabinha River in southeastern Brazil, both microbial communities and ARGs of surface waters and intestinal microbiota of guppies (Poecilia reticulata) were detected. According to the results of 16S rRNA amplicon sequencing, Proteobacteria, Bacteroidetes, Firmicutes and Actinobacteria were dominant phyla in both water and intestinal microbiota, but the abundance of putative pathogens was higher at heavily polluted sites. Up to 83% of bacteria in intestinal microbiota originated from water microbiota; this proportion was relatively higher in less polluted compared to polluted environments. ARGs providing resistance of tetracyclines and quinolones were dominant in both water and gut microbiota. The relative abundances of class I integrons and ARGs were as high as 1.74 × 10-1/16S rRNA copies and 3.61 × 10-1/16S rRNA copies, respectively, at heavily polluted sites. Correlation analysis suggests that integrons and bacteria play key roles in explaining the widespread occurrence of ARGs in the surface, but not in intestinal microbiota. We could rule out the class I integrons a potential intermediary bridge for ARGs between both types of microbiomes. Our results highlight the tight link in microbial communities and ARGs between ambient microbiota of stream ecosystems and intestinal microbiota of fish. Our study could have far-reaching consequences for fisheries and consumer safety and calls for investigations of gut microbiota of target species of both commercial fisheries and recreational (hobby) angling.

Characterization of tetracycline effects on microbial community, antibiotic resistance genes and antibiotic resistance of Aeromonas spp. in gut of goldfish Carassius auratus Linnaeus.

The gut of aquatic animals was a significant niche for dissemination of antibiotic resistance genes (ARGs) and direct response of living conditions. In this study, the gut microbiota of goldfish Carassius auratus Linnaeus was sampled at 7 days and 21 days after treatment with tetracycline at 0.285 and 2.85 µg L-1 to investigate the influences on the microbial structure and antibiotic resistance. The proportion of tetracycline resistance bacteria was 1.02% in the control group, while increased to 23.00%, 38.43%, 62.05% in groups of high concentration for 7 days (H7), low concentration for 21 days (L21) and high concentration for 21 days (H21), respectively. Compared to the control group, the diversity of isolated Aeromonas spp. was decreased in the treatment groups and the minimal inhibitory concentration (MIC) of resistant isolates was enhanced from 32 to 256 µg mL-1 with the treatment of tetracycline in time- and dose-dependent manners. Furthermore, the abundance of most genes was increased in treatment groups and efflux genes mainly responded to the stress of tetracycline with an average level of 1.0 × 10-2. After treatment with tetracycline, the predominant species were changed both at phylum and genus levels. The present study explored the impact of tetracycline on gut microbiota of goldfish at environmentally realistic concentrations for the first time and our findings will provide a reference for characterizing the microbiome of fish in the natural environment.

N-acetylcysteine alleviated bisphenol A-induced testicular DNA hypermethylation of rare minnow (Gobiocypris rarus) by increasing cysteine contents.

Ubiquitous BPA exposure resulted in DNA methylation errors and oxidative stress. Numerous studies have demonstrated that oxidative stress can lead to changes in DNA methylation levels and supplementation with antioxidants, including N-acetylcysteine (NAC), was able to restore these changes. Our previous study supposed that BPA-induced de novo synthesis of glutathione (GSH) promoted DNA methylation process in Gobiocypris rarus testes. To validate this conjecture and explore the protective effects of NAC on BPA toxicity, the present study was carried out. Adult male G. rarus was treated with 225 µg L-1 BPA and/or NAC for 7 days. The sperm motility and DNA integrity of G. rarus were determined. Meanwhile, the levels of 5-methylcytosine (5mC), GSH, hydrogen peroxide (H2O2), DNA methyltransferase proteins (DNMTs), γ-glutamyl cysteine synthetase (GCS), S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), homocysteine (HCY), nicotinamide adenine dinucleotide phosphate (NADPH) and cysteine in the testes were detected. Furthermore, the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) were measured. Results indicated that NAC addition resulted in increase of cysteine contents and partially inhibited the BPA-induced DNA hypermethylation of G. rarus testes. In addition, the changes in DNA methylation levels in the testes after BPA and/or NAC treatment might be controlled by DNA methylation process that mediated by DNMTs. Moreover, BPA exposure caused oxidative stress in the testes and the elimination of H2O2 might be mainly accomplished by CAT while it changed to mainly through GPx after NAC supplement. Finally, the positive response of testicular antioxidant enzyme system and the antioxidant activity of NAC itself protected sperm motility and DNA integrity from oxidative damage in each group.

Adverse effects of bisphenol A on Sertoli cell blood-testis barrier in rare minnow Gobiocypris rarus.

Bisphenol A (BPA), an environmental contaminant, has been shown to disturb the dynamics of Sertoli cell blood-testis barrier (BTB) in mammal testis. However, the effects of BPA on Sertoli cell barrier (SC barrier) were little known in fish to date. To evaluate the potential mechanism of reproductive toxicity of BPA, we studied the damage of SC barrier using in vivo models. In this study, male adult rare minnow Gobiocypris rarus were exposed to 15 µg/L BPA for 7-35 days. Gonadal histology and the integrity of SC barrier were analyzed. Meanwhile, the expressions of SC barrier -associated proteins, tumor necrosis factor (TNFα) content, and the mRNA expressions of genes in the mitogen activated protein kinase (MAPK) pathway were detected. Histological analysis demonstrated 15 µg/L BPA promoted the infiltration of inflammatory cells in fish testes after 7-days exposure. The biotin tracer assay showed that 7-days BPA exposure increased permeability for spermatid cysts. In addition, the BPA treatment caused increased TNFα in testis, which was reportedly related to SC barrier impairment. The expressions of Occludin and ß-Catenin protein were significantly decreased in the testes after 7- and 21-days exposure. BPA also altered the mRNA expressions of occludin, ß-catenin, p38 MAPK and JNK. Therefore, the detrimental effects of BPA on reproduction of male fish may attribute to the disturbed expressions of SC junction proteins.

Bisphenol A induced abnormal DNA methylation of ovarian steroidogenic genes in rare minnow Gobiocypris rarus.

Bisphenol A (BPA), an ubiquitous environmental endocrine disruptor chemical, disturbs the mRNA expressions of steroidogenic genes and subsequently steroid hormone synthesis in mammals and aquatic species. However, the underlying regulation mechanisms are barely understood, especially in fish. To explore the regulation mechanism, we exposed female rare minnow Gobiocypris rarus (G. rarus) to BPA at a nominal concentration of 15 µg/L for 7 and 14 days in the present study. Results showed significant increase of gonad somatic index (GSI) and serum estradiol (E2) levels in response to BPA at day 14. The 7-day BPA exposure notably repressed the expression of two ovarian steroidogenic genes (star and hsd11b2) and suppressed their capacity of estrogen response elements (ERE) to recruit estrogen receptor (ER), while the 14-day BPA treatment remarkably induced transcript of hsd3b and enhanced the capacity of ERE to recruitment ER in ovaries. Furthermore, the 7-day BPA exposure caused DNA hypermethylation of star (CpGs: -742 bp and -719 bp) and hsd11b2 (CpG: -1788 bp). However, 14-day BPA exposure resulted in DNA hypomethylation of hsd3b (CpG: -181 bp). Correlation analysis revealed that the DNA methylation levels at specific CpGs in star, hsd3b and hsd11b2 were significantly correlated to their mRNA levels and ER-EREs interactions. These findings suggest that the disturbed steroidogenesis and the transcripts of ovarian steroidogenic genes might attribute to the altered DNA methylation status of these ovarian steroidogenic genes in response to BPA.

Bisphenol A regulates rare minnow testicular vitellogenin expression via reducing its promoter Er recruitment.

Vitellogenins (Vtgs) are major precursor of the egg-yolk proteins. They are synthesized in liver of adult female ovipara, but normally silent in males. For their sensitive response to estrogen, Vtgs are usually used as biomarkers for environmental estrogenic compounds. In the present study, three vtg subtypes (vtg1, vtg2 and vtg3) were proved to present in the testis of rare minnow Gobiocypris rarus for the first time. Immunohistochemistry result showed that Vtg proteins mainly deposit in spermatogonium and spermatocytes. Following 225µg/L bisphenol A (BPA) exposure 1, 3 and 9 weeks, testicular vtg mRNAs were mostly significantly decreased. The further chromatin immunoprecipitation showed that BPA could decrease estrogen receptor (Er) recruitment in vtg promoter, which possibly reduced Er's transcription activation effect on vtgs. However, different from the continuously decreased vtg mRNA levels, testicular Vtg protein levels were recovered at week 9. Considering the induced hepatic Vtg expression, testicular Vtgs may be replenished by the induced hepatic Vtgs under BPA exposure.

Maternal Bisphenol A exposure impaired endochondral ossification in craniofacial cartilage of rare minnow (Gobiocypris rarus) offspring.

Bisphenol A (BPA), an endocrine disrupting compound, is present in the aquatic environment. BPA can mimic estrogen and cause adverse effects on development and reproduction in different organisms. As epigenetic modifications due to BPA exposure have been reported, the interest on the effects of this chemical has increased. To assess the potential effects of maternal BPA exposure on offspring bone development, adult Gobiocypris rarus (G. rarus) females were exposed to 15 µg L-1 and 225 µg L-1 BPA for 21 days. Eggs were collected after artificial spawning and fertilized with the fresh milt of non-exposed male fish. The offspring were raised in clean water and randomly selected for examination at different development stages. Our results showed that specific effects including poor quality of the embryos, increased malformation (bent spine and tail), and delayed craniofacial cartilage ossification of the larvae. Additionally, the transcripts of ossification related genes were significantly downregulated in offspring, and the lysyloxidase activity decreased. The present study demonstrated the maternal-mediated skeleton toxicity of BPA and its adverse effects on G. rarus.

Responses of gonadal transcriptome and physiological analysis following exposure to 17α-ethynylestradiol in adult rare minnow Gobiocypris rarus.

17α-ethynylestradiol (EE2), a synthetic estrogen commonly used in the oral contraceptive pills, disrupts the sexual differentiation, gonadal development and reproduction in aquatic species. Nowadays aquatic species and even humans still have the potential risks of exposure to EE2. However, the mechanism of EE2 endocrine disruption is still unclear. Aiming to elucidate molecular mechanisms, we analyzed transcriptome profiling of gonads, gonadal histology and the sex steroid hormones in response to EE2 in G. rarus. Through this study, we obtained eight RNA-Seq libraries upon EE2 exposure, and found some key genes and pathways in correlation with the disruption effects of EE2. We found EE2 could disrupt oocyte development and spermatogenesis in adult G. rarus, and EE2 has more obvious disruption effects on male G. rarus than females. Interestingly, EE2 was indicated to be an exogenous DPC-inducing agent and ppp2r3b was suggested to be a spermatogenesis candidate gene in rare minnow. The differential gene expressions of rps30, samp9, ppp2r3b and spartan upon EE2 exposure suggest EE2's disruption effects on gonads could attribute to altered pathways of translation, ribosome biogenesis and cell division.

Molecular identification of Kiss/GPR54 and function analysis with mRNA expression profiles exposure to 17α-ethinylestradiol in rare minnow Gobiocypris rarus.

17α-ethinylestradiol (EE2) is a widely existed endocrine disrupting chemical in water environment. Kisspeptins act as indispensable regulators through GPR54 in the hypothalamic-pituitary-gonadal (HPG) axis. This study aimed to provide further understanding of the effect of EE2 on HPG axis. Molecular cloning and tissue distribution of kiss genes and GPR54s were performed in Gobiocypris rarus. The mRNA expression profiles of kiss1, kiss2, GPR54s and GnRHs were detected in G. rarus brain and/or gonad following 3- and 6-days EE2 (1, 5, 25 and 125 ng/L) exposure. Results showed that kiss genes and GPR54s were highly expressed in brain and gonad. Both kiss1 and kiss2 were increased in female brain and suppressed in male brain following EE2 exposure. GnRHs were inhibited in a concentration-dependent manner in male brain following 3-days EE2 exposure. In gonad, GPR54b was almost suppressed in all of EE2 concentrations. The present findings suggest that EE2 impacts the genes expression of Kiss/GPR54-GnRH system in G. rarus, thereby probably disturbing the neuroendocrine homeostasis.

Effect of low-dose malathion on the gonadal development of adult rare minnow Gobiocypris rarus.

Malathion is an organophosphorus pesticide that extensively used in agriculture and veterinary practices. To investigate the effects of low dose malathion on rare minnow Gobiocypris rarus gonadal development, we exposed adult rare minnow to environmentally relevant concentration malathion (2 and 20µg/L) for 21 days. Gonadal histology, sex hormone levels and mRNA expressions of steroidogenic genes were investigated. Malathion at both 2 and 20µg/L significantly up-regulated rare minnow testicular weight and promoted the progression of spermatogenesis. Neither ovarian weight nor process of ovary development was markedly changed. In testis, 2µg/L malathion significantly down-regulated testosterone and 11-ketotestosterone levels, and up-regulated mRNA expression of steroidogenic genes. In ovaries, 2 and 20µg/L malathion significantly inhibited estradiol17ß levels and induced testosterone levels, both in concentration dependent manners; mRNA expressions of almost all the detected ovarian steroidogenic genes were up-regulated. The present result suggested that malathion even at low dose could pose a potential threat to adult rare minnow gonadal development.

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).

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 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 17ß-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.