Carbamazepine at environmentally relevant concentrations caused DNA damage and apoptosis in the liver of Chinese rare minnows (Gobiocypris rarus) by the Ras/Raf/ERK/p53 signaling pathway.

To assess genetoxicity and the underlying mechanisms of carbamazepine (CBZ) toxicity in fish, adult Chinese rare minnows (Gobiocypris rarus) were exposed to 1, 10, and 100 µg/L CBZ for 28 d. Comet assays indicated that hepatic DNA damage was significantly increased in groups of minnows exposed to CBZ at all concentrations in a dose-dependent manner compared to those of the control groups (p < 0.05). Liver levels of 8-hydroxydeoxyguanosine (8-OHdG) were significantly increased at 10 and 100 µg/L CBZ (p < 0.05). TUNEL assays indicated that the average apoptotic rates of the livers of female and male minnows were significantly increased following exposure to CBZ at all concentrations for 28 d (p < 0.05). Significant increases in caspase 3 and 9 activities after CBZ exposure at all concentrations and caspase 8 at 10 and 100 µg/L CBZ exposure reflected the presence of mitochondrial apoptosis (p < 0.05). The mRNA levels of gadd45a, mdm2, casp3 and casp9 in female and male minnows exposed to CBZ at all concentrations were significantly increased compared with those in the control groups (p < 0.05). Significant increases in the levels of p21 in female minnows exposed to 1 and 100 µg/L CBZ, p53 in female minnows at all CBZ treatments and bcl2 in male minnows exposed to 1 and 100 µg/L CBZ were observed, indicating p53 pathway activation. The inhibition of ras levels in females and males exposed to CBZ at all concentrations and increased levels of raf1 in males exposed to CBZ at all concentrations indicated Ras/Raf1/MAPK (ERK) activation. Therefore, the present study demonstrates that CBZ at environmentally relevant levels induces DNA damage and apoptosis in Chinese rare minnows by the Ras/Raf/ERK/p53 signaling pathway.

Evaluation and mechanistic study of chlordecone-induced thyroid disruption: Based on in vivo, in vitro and in silico assays.

The present study aimed to evaluate the thyroid-disrupting potency of chlordecone, and reveal the underlying mechanism. In the in vivo assays, rare minnow embryos were exposed to 0, 0.01, 0.1, 1 and 10 µg·L-1 chlordecone until sexually mature. The results showed decreased T4 but increased T3 concentrations in plasma, upregulated mRNA levels of thyrotropin-releasing hormone receptor (trhr) and sodium-iodide symporter (nis) in the brain, and transthyretin (ttr), thyroid hormone receptor α (trα) and deiodinase enzymes (dio1 and dio2) in the liver of adult fish. In the in vitro assays, single chlordecone treatments promoted growth hormone (GH) and prolactin (PRL) secretion in GH3 cells. Transcription of thyroid receptor (trß) was inhibited, but this is not likely responsible for chlordecone-induced GH secretion and altered transcription. When co-treated with T3, chlordecone acted independently of the effect of T3 on GH secretion; chlordecone-induced GH/PRL secretion and mRNA expression were further promoted when co-treated with E2, but inhibited when co-treated with ICI, indicating an important role for estrogen receptors (ERs) in chlordecone-induced changes in GH3 cells. Furthermore, in silico prediction suggested no stable interactions between chlordecone and thyroid hormone-related proteins, as well as a regulatory role for ERs in thyroid systems. Overall, our results indicated that chlordecone may have adverse effects on thyroid systems upon long-term exposure. However, rather of TRs, ERs may be responsible for thyroid disruption following chlordecone exposure.

Reproductive toxicity and estrogen activity in Japanese medaka (Oryzias latipes) exposed to environmentally relevant concentrations of octocrylene.

The growing use of octocrylene (OC) in sunscreens has posed a great threat to aquatic organisms. In the present study, to assess its reproductive toxicity and mechanism, paired Japanese medaka (Oryzias latipes) (F0) were exposed to OC at nominal concentrations of 5, 50, and 500 µg/L for 28 d. Significant increases were observed in the gonadosomatic index (GSI) and hepatosomatic index (HSI) of F0 medaka at 500 µg/L OC (p < 0.05) without significant differences in fecundity. The fertility was significantly decreased at all treatments (p < 0.05). Significant increases in the percent of mature oocytes were observed at 5 and 500 µg/L OC, in which contrary to the percent of spermatozoa (p < 0.05). The plasma sex hormones and vitellogenin levels significantly increased in males at all treatments and in females at 50 and 500 µg/L OC (p < 0.05). In addition, the levels of fshß and lhß in the brains and the levels of fshr, lhr and cyp17α in the gonads were significantly upregulated in males at all treatments (p < 0.05), in line with those of ar, erα, erß and cyp19ß in the brains of male and female. The upregulation of vtg in male and female livers was observed only at 500 µg/L OC and upregulation of star and hsd3ß was observed in testis at all treatments (p < 0.05). Continued exposure to OC significantly induced increases in the time to hatching, morphological abnormality rates, and cumulative death rates of F1 embryos, inconsistent with body length of F1 larvae (p < 0.05). Therefore, the responses of the exposed fish at the biochemical and molecular levels indicated reproductive toxicity and estrogenic activity of OC, providing insights into the mechanism of OC.

Subchronic effects of dietary selenium yeast and selenite on growth performance and the immune and antioxidant systems in Nile tilapia Oreochromis niloticus.

Selenium is an essential element but toxic at high levels in animals. The effects of Se on growth performance and the immune system in Nile tilapia remain inconclusive. In this study, Nile tilapia Oreochromis niloticus was fed on selenium yeast (Se(Y))- and selenite (Se(IV))-enriched feed at 0, 3, 6, and 12 µg/g (dry wt) for 45 and 90 d. The growth, bioaccumulation, biochemical markers related to antioxidant, immunological, nervous and digestive systems were evaluated in various fish tissues (liver, intestine, kidney, muscle, brain, spleen, gills). The results showed that the accumulation of Se(Y) was 1.3-2 folds of Se(IV) in most tissues. The growth of tilapia was enhanced by both Se(Y) and Se(IV) at 3 µg/g after 90 d, with Se(Y) better than Se(IV) in tilapia feed. After 45 d, the levels of lipid peroxidation, the activity of the antioxidant enzymes, and the transcriptional levels of the immune related genes (IL-1ß, IFN-γ and TNF-α) and stress proteins (HSP70 and MT) were enhanced in all treatments, except that of MT in the 12 µg/g Se(Y) group. In addition, both Se species inhibited the activity of acetylcholinesterase (AChE) in the brain and one digestive enzyme α-glucosidase (α-Glu) in the intestine at 12 µg/g. However, after 90 d, the effects on most biochemical markers were less pronounced, implying a possible acclimation after prolonged duration. The results demonstrate Se is beneficial to O. niloticus at low levels and toxic at elevated levels. The immunostimulation by Se might be greatly weakened after long term feeding Se-enriched feed. This study helps to better understand the effects of Se on the antioxidant and immune systems and to establish the optimal Se levels in the feed and duration for O. niloticus.

Diastereoisomer-specific neurotoxicity of hexabromocyclododecane in human SH-SY5Y neuroblastoma cells.

Hexabromocyclododecane (HBCD) is a widely applied brominated flame retardant (BFR) and is regarded as a persistent organic pollutant. It has been found in human tissues and has the potential to cause neurological disorders. However, our understanding of HBCD neurotoxicity at the diastereoisomer level remains lacking. Here, we investigated the neurotoxicity of three HBCD diastereoisomers, i.e., α-, ß-, and γ-HBCD, in SH-SY5Y human neuroblastoma cells. Results showed that the HBCD diastereoisomers decreased cell viability, increased lactate dehydrogenase (LDH) release, and impaired cytoskeleton development. Typical morphological features and apoptosis rates showed that the HBCD diastereoisomers induced SH-SY5Y cell apoptosis. The expression levels of several cell apoptosis-related genes and proteins, including Bax, caspase-3, caspase-9, cytochrome c, Bcl-2, and X-linked inhibitor of apoptosis (XIAP), as well as the cell cycle arrest, DNA damage, adenosine triphosphate (ATP) consumption, reactive oxygen species (ROS) levels, and intracellular calcium ion (Ca2+) levels, were examined. Results showed that the HBCD diastereoisomer neurotoxicity was ranked ß-HBCD > γ-HBCD > α-HBCD. The cell apoptosis and caspase expression levels of the three HBCD diastereoisomers followed the same order, suggesting that caspase-dependent apoptosis may be one mechanism responsible for the structure-selective HBCD diastereoisomer neurotoxicity. The levels of intracellular Ca2+ and ROS increased significantly. The ROS levels were ordered ß-HBCD > γ-HBCD > α-HBCD, whereas those of intracellular Ca2+ were γ-HBCD > ß-HBCD > α-HBCD. Thus, ROS may be a key factor regulating the neurotoxicity of HBCD diastereoisomers. To the best of our knowledge, this is the first study to report on the diastereoisomer-specific toxicity of HBCD in human neural cells and on the possible mechanisms responsible for the selective neurotoxicity of HBCD diastereoisomers.

Environmentally relevant concentrations of carbamazepine induce liver histopathological changes and a gender-specific response in hepatic proteome of Chinese rare minnows (Gobiocypris rarus).

To assess hepatotoxicity and to determine the underlying mechanisms of carbamazepine (CBZ) toxicity in fish, histopathology and the liver proteome were examined after Chinese rare minnow (Gobiocypris rarus) were exposed to 1, 10, and 100 µg/L CBZ for 28 days. Histopathological changes included disruption of spatial structure, pyknotic nuclei, cellular vacuolization and deformation of cell nuclei, in addition to marked swelling of hepatocytes in all treatment groups. Protein analysis revealed that there were gender-specific responses in rare minnow following exposure, and there were 47 proteins in females and 22 proteins in males identified as differentially abundant following CBZ treatments. Pathway analysis revealed that cellular processes affected by CBZ included apoptosis, cell differentiation, cell proliferation, and the respiratory chain, indicating impaired energy homeostasis. Noteworthy was that 15 proteins identified as different in abundance were associated with carcinogenicity. Relative mRNA levels for select transcripts were consistent with the changes of proteins N-myc downstream regulated gene (NDRG), Tropomyosin 2-Beta (TPM2) and annexin A4 (ANXA4). Protein pyruvate kinase, liver and RBC (PKLR) were increased at 1 and 100 µg/L CBZ without significant difference in transcript levels. These findings characterize molecular responses and histological changes in the liver that generate new insights into CBZ hepatotoxicity in Chinese rare minnow.

Changes of hematological and biochemical parameters revealed genotoxicity and immunotoxicity of neonicotinoids on Chinese rare minnows (Gobiocypris rarus).

Adverse impacts of immunity in terrestrial non-target organisms exposed to neonicotinoid insecticides have been reported, but the causal link between insecticide exposure and possible immune alterations in fish remains limited. In the present study, the potential genotoxicity and immunotoxicity of three neonicotinoids (imidacloprid, nitenpyram, and dinotefuran) were assessed in Chinese rare minnows by using a 60-day chronic toxicity test. The hematological and biochemical parameters of juvenile Chinese rare minnows and changes in the transcription of six inflammation-related genes were determined after exposure to neonicotinoids at 0.1, 0.5, or 2.0 mg/L. A clear difference in the frequency of erythrocytes with micronuclei (MN) was observed after treatment with 2.0 mg/L imidacloprid (p < .05). Additionally, exposure to 0.5 or 2.0 mg/L imidacloprid significantly increased the binucleated (BN) erythrocytes and those with notched nuclei (NT) (p < .05). A serum protein electrophoresis (SPE) assay showed significant alterations in the serum protein in all treatments (p < .05), and further analysis indicated decreases in immunoglobulin (Ig) in treatments with 0.5 or 2.0 mg/L imidacloprid or dinotefuran or with 0.1 mg/L nitenpyram (p < .05). Moreover, a biochemical assay confirmed that immunoglobulin M (IgM) levels were indeed significantly decreased upon treatment with imidacloprid or dinotefuran at 0.5 or 2.0 mg/L (p < .05). In addition, the transcriptional levels of the inflammatory cytokines IL-6, INF-α, TNF-α, and IL-1ß were markedly down-regulated after all imidacloprid treatments (p < .05), whereas the expression levels of only TNF-α and IL-1ß were significantly down-regulated following the 0.5 and 2.0 mg/L dinotefuran treatments (p < .05). Taken together, our results clearly demonstrate that imidacloprid, rather than nitenpyram and dinotefuran, can induce genotoxicity. The responsiveness of these immune indicators provides new insight into and evidence of the adverse effects of neonicotinoids on aquatic non-target organisms.

Benzo[a]pyrene induced p53-mediated cell cycle arrest, DNA repair, and apoptosis pathways in Chinese rare minnow (Gobiocypris rarus).

The p53 pathways play an important role in carcinogenesis. In mammals, p53 and p53 target genes have been extensively studied, but little is known about their functions and regulation in fish. In this study, the cDNA fragments of p53 network genes, including p53, p21, mdm2, gadd45α, gadd45ß, igfbp-3, and bax, were cloned from Chinese rare minnow (Gobiocypris rarus). These genes displayed high amino acid sequence identities with their zebrafish orthologs. The mRNA levels of p53 network genes and pathological changes in the liver were determined after adult rare minnow were exposed to 0.4, 2, and 10 µg/L of benzo[a]pyrene (BaP) for 28 days. The results showed that p53, p21, mdm2, gadd45α, and bax mRNA expressions in the livers from males and females were significantly upregulated compared with those of the controls (p < 0.05), but gadd45ß and igfbp-3 expression was not significantly changed. Microphotographs revealed enlargement of the cell nuclei and cellular degeneration in males, while atrophy and vacuolization of hepatocytes were observed in females (10 µg/L). These results suggested that BaP induced liver DNA repair and apoptosis pathways and caused adverse pathological changes in rare minnow. The strongly responsive p53 network genes in the livers suggest that rare minnow is suitable as an experimental fish to screen environmental carcinogens. In addition, the p53 network genes in rare minnow could feasibly be used to identify the mechanism of environmental carcinogenesis. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 979-988, 2017.

Mechanistic study of chlordecone-induced endocrine disruption: Based on an adverse outcome pathway network.

The adverse outcome pathway (AOP) framework could be helpful for chemical risk assessment and mechanistic research. The aim of the present study was to unravel the mechanism of chlordecone-induced endocrine disruption by illustrating the main molecular initiating event (MIE)/perturbations responsible for the observed effects. In silico simulations were performed to predict the MIE(s), and the results pointed to agonistic interaction with estrogen receptors (ERα, ERß), androgen receptor (AR), cytochrome P450 (CYP19A) by chlordecone. In vivo endocrine disruptions were evaluated in rare minnow (Gobiocypris rarus) exposed to 0.01, 0.1, 1 and 10 µg L(-1) chlordecone from 2 h post-fertilization until sexually mature. In the females, increases of vitellogenin (vtg) mRNA levels in liver and gonad, plasma estradiol (E2), testosterone (T) and E2/T, and renalsomatic index confirmed the role of agonism of ER and CYP19A as MIEs, but the decreased gonadosomatic index, degenerated ovaries as well as the feed-forward response pointed to other potential but important MIEs and corresponding AOPs. In the males, increased E2/T ratio, increased testis vtg mRNA levels and occurrence of intersex confirmed the roles of agonism of ERα and CYP19A as main MIEs in chlordecone-induced endocrine disruptions. Our results also fetches out the limit of AOPs in predicting the adverse outcomes and explaining the mechanism of chemicals at present, thus reflected a critical need for expanding AOPs and AOP network before using it in chemical risk assessment.

2, 4-Dichloro-6-nitrophenol, a photonitration product of 2, 4-dichlorophenol, caused anti-androgenic potency in Chinese rare minnows (Gobiocypris rarus).

2,4-Dichloro-6-nitrophenol (DCNP) is an environmental transformation product of 2,4-dichlorophenol that has been identified as widespread in effluent wastewater, but little is known about its toxicity because this compound is not regulated. Therefore, to investigate the endocrine disruption potency of DCNP in Chinese rare minnows (Gobiocypris rarus), adult and juvenile fish were exposed to various concentrations of DCNP (2, 20, and 200 µg/L) for 28 d. After 28 d exposure, the plasma vitellogenin (VTG) levels were reduced in females while increased in males and juvenile fish considerably, as compared with the control. These results suggested that DCNP affects the HPG-axis in a sex-dependent way. Testosterone (T) levels in the plasma were significantly lower in adult and juvenile fish and were accompanied by an increased estradiol (E2)/T ratio. Histopathological observation revealed hypertrophy of the hepatocytes and nuclear pyknosis in the liver, the inhibition of spermatogenesis in the testes, and the degeneration of oocytes in the ovaries after DCNP exposure. The expression pattern of selected genes indicated that the nuclear receptor, steroidogenesis and gonadotropin regulation pathways were perturbed after DCNP exposure. Above all, our results demonstrated that DCNP clearly had anti-androgenic activity in both adult and juvenile fish and can therefore be considered as an endocrine-disrupting chemical.

Metabolic pathways of decabromodiphenyl ether (BDE209) in rainbow trout (Oncorhynchus mykiss) via intraperitoneal injection.

Decabromodiphenyl ether (BDE209) was of great concern due to its biotransformation in different organisms. However, most studies devoted to the metabolic intermediates of BDE209, less has been done on the metabolic pathways in vivo, especially on the relationships among debrominated-BDEs, OH-BDEs and MeO-BDEs. In this study, the metabolic pathways and intermediates of BDE209 in rainbow trout (Oncorhynchus mykiss) were investigated, and the time-dependent transformations of the metabolites were also examined. The primary debrominated metabolites were BDE47, 49, 99, 197, 207; the main MeO-BDEs were MeO-BDE47, MeO-BDE68 and MeO-BDE100; OH-BDEs were primarily composed of OH-BDE28 and OH-BDE42. From the time-dependent and dose-effect relationships, the debromination should be followed by hydroxylation, and then by methoxylation. The increasing in body burden of MeO-BDEs corresponded to the decreasing of OH-BDEs, which could indirectly prove the inter-conversion between OH-BDEs and MeO-BDEs. This study would motivate the future research of toxicological mechanisms of BDEs.

Endocrine disrupting effects of benzotriazole in rare minnow (Gobiocypris rarus) in a sex-dependent manner.

Benzotriazole (BT), an anticorrosive agent, is widely used in industrial applications and household dishwashing agents. Despite its reported toxicity to aquatic organisms, little is known about its endocrine disrupting effects. In this study, adult Chinese rare minnows (Gobiocypris rarus) were exposed to 0.05, 0.5, and 5 mg L(-1) BT for 28 d. The pathological damage in liver was associated with hypertrophy of the hepatocytes, nuclei pyknosis and vacuolization at 5 mg L(-1) groups. Additionally, the degeneration of the ovary and the stimulation of spermatogenesis were observed at 5 mg L(-1) groups. The plasma 17ß-estradiol level was significantly increased in the males but decreased in the females at 5 mg L(-1) (p<0.05). In the brain, the up-regulation of CYP19B, GnRHs, and LHß mRNA was detected across all doses (p<0.05). In the gonad, the transcriptional levels of StAR, CYP11A, 3ßHSD, CYP17, 17ßHSD, and CYP19A were generally decreased in the males at 5 mg L(-1) (p<0.05), whereas these genes, except for 3ßHSD, were significantly increased in females at all concentrations (p<0.05). Moreover, the expression level of VTG in the livers from all exposure groups was significantly increased compared with controls (p<0.05). Taken together, our results indicate that BT could adversely affect the rare minnows in a sex-dependent manner.

Pyruvate carboxylase as a sensitive protein biomarker for exogenous steroid chemicals.

Assessing protein responses to endocrine disrupting chemicals is critical for understanding the mechanisms of chemical action and for the assessment of hazards. In this study, the response of the liver proteome of male rare minnows (Gobiocypris rarus) treated with 17ß-estradiol (E2) and females treated with 17α-methyltestosterone (MT) were analyzed. A total of 23 and 24 proteins were identified with differential expression in response to E2 and MT, respectively. Pyruvate carboxylase (PC) was the only common differentially expressed protein in both males and females after E2- and MT-treatments. The mRNA as well as the protein levels of PC were significantly down-regulated compared with that of the controls (p < 0.05). Our results suggest that endocrine disruptors interfere with genes and proteins of the TCA cycle and PC may be a sensitive biomarker of exposure to exogenous steroid chemicals in the liver of fish.

Long-term exposure investigating the estrogenic potency of estriol in Japanese medaka (Oryzias latipes).

The growth, development, and ERα and Vtg-I gene expressions of Japanese ricefish (Oryzias latipes; medaka) exposed to different concentrations of estriol (E3), including one environmentally relevant concentration, during embryo-adult life stages were evaluated. At the early life stage, fertilized eggs were exposed to 5, 50, 500, 5000ng/L E3 for 15days, and the hatched fry were exposed continuously to the same concentrations for an additional 15days. Exposure to 500 and 5000ng/L E3 resulted in adverse effects on hatchability and time to hatching. At 5000ng/L, the gross abnormality rate was increased and the number of females that hatched was twice that of males. When the fish were exposed to 5-5000ng/L E3 for further 60days, the male hepatosomatic index (HSI) was increased at 5000ng/L. The female gonadosomatic index (GSI) was decreased at 500 and 5000ng/L E3, while the male GSI at 5000ng/L E3 was increased and sex reversal was also found at this concentration. Quantitative RT-PCR showed that the hepatic vitellogenin-I (Vtg-I) genes were up-regulated in females at 500 and 5000ng/L E3 and in males at all E3 concentrations, whereas E3 did not affect estrogen receptor α (ERα) mRNA transcription. These results showed that E3 at environmental concentration of 5ng/L has no adverse effects on growth and development of the Japanese medaka. However, in this study, if we only focused on Vtg gene change in males, E3 had strong estrogenic effects on male medaka under the conditions of these experiments.

Effects of decabromodiphenyl ether (BDE-209) on mRNA transcription of thyroid hormone pathway and spermatogenesis associated genes in Chinese rare minnow (Gobiocypris rarus).

Polybrominated diphenyl ethers (PBDEs) are widely used as flame retardants, which are ubiquitous environmental contaminant found in both abiotic and biotic environmental samples. Deca-BDE (BDE-209) is the principal component, which is currently used worldwide. In this study, the effect of BDE-209 on the mRNA levels of thyroid hormone (TH) related genes and spermatogenesis associated genes were determined from larvae and adult rare minnow (Gobiocypris rarus) exposed to concentrations 0.01, 0.1, 1, and 10 µg/L for 21 days. The results showed that the type II deiodinase (dio2) and sodium iodide symporter (nis) mRNA levels were significantly up-regulated in the larvae at 10 µg/L treatment. In adult, histopathological observations showed that liver of female fish were degenerated at 10 µg/L treatment, and inhibition of spermatogenesis were observed in testis of male fish. In addition, the thyroid hormone receptor α (trα), dio2, and nis mRNA levels in the liver of male and female fish were significantly up-regulated, whereas dio2 and nis mRNA levels were significantly down-regulated in the brain. These results indicate that exposure to BDE-209 could result in tissue-specific alternations of TH-related genes expression in adults. Moreover, the mRNA levels of the testis-specific apoptosis genes, the spermatogenesis-associated 4 (spata4) and spermatogenesis-associated 17 (spata17), were down-regulated at 10 µg/L treatment in testis of male fish. Our results suggest that BDE-209 may pose threat to normal thyroid and reproductive function in fish.

Effects of estrone on the early life stages and expression of vitellogenin and estrogen receptor genes of Japanese medaka (Oryzias latipes).

The fertilized eggs of Japanese medaka (Oryzias latipes) were exposed to estrone (E1) at 5-5000 ng L(-1) for 15 d, and the hatched fry were exposed continuously to the same concentrations for the additional 15 d. Adverse effects on hatchability, time to hatching, and gross abnormalities occurred at 50 ng L(-1) or above. Then the fry were divided into a continual exposure group, and a water recovery group. When the fry were exposed to E1 for another 60 d, there was a decrease in the hepatosomatic index (HSI) of males and the influence disappeared in the water recovery group. The gonadosonatic index (GSI) of females at 500 ng L(-1) decreased significantly in another 60 d exposure. While the fry were maintained in dechlorinated tap water for 60 d, a significant decrease in female GSI was observed at 50 ng L(-1) or above. An increased GSI was found in males in both continual exposure and water recovery groups at all E1 treatments. Quantitative RT-PCR showed that vitellogenin-I (Vtg-I) gene expressions in the female liver were significantly down-regulated at 50 ng L(-1) in the continual exposure group, and at 500 ng L(-1) in the water recovery group, while male Vtg-I genes were significantly up-regulated for all E1 treatments. In addition, all E1 treatments caused sex reversal of males. These results suggest that E1 at 5 ng L(-1) or above have unrecoverable impacts on the gonadal growth and development of medaka, even if only early life stages were exposed to E1.

New cytochrome P450 1B1, 1C1, 2Aa, 2Y3, and 2K genes from Chinese rare minnow (Gobiocypris rarus): Molecular characterization, basal expression and response of rare minnow CYP1s and CYP2s mRNA exposed to the AHR agonist benzo[a]pyrene.

Cytochrome P450 (CYP450) genes play an important role in catalyzing oxidative metabolism of toxicants. Recently, CYP1 subfamily were discovered and reported in fish, however, little is known regarding the CYP2 isoforms in fish. In the present study, the cDNA fragments of CYP 1B1 and 1C1 and CYP2Aa, 2Y3, and 2K of rare minnow were cloned and exhibited a high amino acid sequence identity compared with their zebrafish orthologs. Basal expression showed CYP1C1 and CYP 2Aa expression were observed in all eight tissues analyzed (liver, gill, intestine, kidney, spleen, brain, skin, and muscle). CYP 1A, and 1B1 expression was found in all tissues except for muscle and skin. However, CYP 2Y3 was expressed in liver, spleen, intestine and muscle whereas CYP 2K in liver, kidney and intestine. 4 and 100µgL(-1) Benzo[a]pyrene (BaP) induced patterns showed that CYP 1A, 1B1 and 1C1 expression in liver, gill, and intestine was strongly up-regulated (p<0.05). Furthermore, CYP 2Y3 was strongly induced in liver from BaP treatments (p<0.05). The high induction on mRNA level of CYP1s and CYP 2Y3 by BaP could be associated with catalyzing detoxification and indicated that CYP2s may also be potential biomarker to screen AHR agonist. The high responsiveness of CYP1 and 2 genes suggested Chinese rare minnow is feasible to screen and assess pollution with AHR agonist.

ß-estradiol 17-valerate affects embryonic development and sexual differentiation in Japanese medaka (Oryzias latipes).

ß-estradiol 17-valerate (EV) is a synthetic estrogen widely used in combination with other steroid hormones in hormone replacement therapy drugs and is detected in natural waters. Although EV is known as an estrogenic chemical, there is still a lack of data on its developmental and reproductive toxicities in fish following exposure to EV during embryo-larval-, juvenile- and adult-life stages in Japanese medaka (Oryzias latipes). At the early life stage, the fertilized eggs of medaka were exposed to 1, 10, 100 and 1000 ng/L EV for 15 days, and hatched larval fish were continually exposed to the same concentration range for an additional 15 days. The results showed that exposure to 10 ng/L or above resulted in adverse effects on hatchability and time to hatching, and the number of hatched females was twice that of males at 10 ng/L or above. When the hatched fish were continually exposed to 1, 10 and 100 ng/L of EV for another 40 days, the hepatosomatic index (HSI) was increased in both males and females, and the gonadosomatic index (GSI) was decreased in females, and increased in males. Sex reversal was found in fish exposed to 1 ng/L and above. Quantitative real-time RT-PCR showed that mRNA levels of estrogen receptor α (ER-α) and vitellogenin-I (VTG-I) in the liver of females were significantly down-regulated, while those of vitellogenin-I (VTG-I) in the liver of males were significantly up-regulated at all concentrations. These findings suggest that EV is a reproductive toxicant and estrogenic chemical in both male and female fish.

Occurrences of six steroid estrogens from different effluents in Beijing, China.

Concentration levels of six natural and anthropogenic origin steroid estrogens, namely, diethylstilbestrol (DES), estrone (E1), estradiol (E2), estriol (E3), ethinylestradiol (EE2), and estradiol-17-valerate (Ev), from different effluents in Beijing were assessed. Sampling sites include two wastewater treatment plants (WWTPs), a chemical plant, a hospital, a pharmaceutical factory, a hennery, and a fish pool. In general, concentrations of estrogens in the effluents varied from no detection (nd) to 11.1 ng/l, 0.7 to 1.2 × 10(3) ng/l, nd to 67.4 ng/l, nd to 4.1 × 10(3) ng/l, nd to 1.2 × 10(3) ng/l, and nd to 11.2 ng/l for DES, E1, E2, EE2, E3, and Ev, respectively. The concentration levels of steroid estrogens from different effluents decreased in the order of pharmaceutical factory and WWTP inlets > hospital > hennery > chemical factory > fish pool. This study indicated that natural estrogens E1, E2, and E3 and synthetic estrogen EE2 are the dominant steroid estrogens found in the different Beijing effluents. For source identification, an indicator (hE = E3/(E1 + E2 + E3)) was used to trace human estrogen excretion. Accordingly, hE in effluents from the hospital and WWTP inlets exceeded 0.4, while much smaller values were obtained for the other effluents. Human excretions were the major contributor of natural estrogens in municipal wastewater. Estimation results demonstrated that direct discharge was the major contributor of steroid estrogen pollution in receiving waters.

Occurrence and fate of steroid estrogens in the largest wastewater treatment plant in Beijing, China.

Concern over steroid estrogens has increased rapidly in recent years due to their adverse health effects. Effluent discharge from wastewater treatment plants (WWTPs) is the main pollutant source for environmental water. To understand the pollutant level and fate of steroid estrogens in WWTPs, the occurrence of estrone (E1), 17-ß-estradiol (E2), estriol (E3), and 17-ß-ethinylestradiol (EE2) was investigated in the Gaobeidian WWTP in Beijing, China. Water samples from influent as well as effluent from second sedimentation tanks and advanced treatment processes were taken monthly during 2006 to 2007. In influent, steroid estrogen concentrations varied from 11.6 to 1.1 × 10(2) ng/l, 3.7 to 1.4 × 10(2) ng/l, no detection (nd) to 7.6×10(2) ng/l and nd to 3.3 × 10(2) ng/l for E1, E2, E3, and EE2, respectively. Compared with documented values, the higher steroid estrogen concentrations in the WWTP influent may be due to higher population density, higher birthrate, less dilution, and different sampling time. Results revealed that a municipal WWTP with an activated sludge system incorporating anaerobic, anoxic, and aerobic processes could eliminate natural and synthetic estrogens effectively. The mean elimination efficiencies were 83.2%, 96.4%, 98.8%, and 93.0% for E1, E2, E3, and EE2, respectively. The major removal mechanism for natural estrogens and synthetic estrogen EE2 were biodegradation and sorption on the basis of mass balance in water, suspension particles, and sludge. In the WWTP effluent, however, the highest concentrations of E1, E2, E3, and EE2 attained were 74.2, 3.9, 5.1, and 4.6 ng/l, respectively. This is concerning as residual steroid estrogens in WWTP effluent could lead to pollution of the receiving water. Advanced flocculation treatment was applied in the WWTP and transformed the residual estrogen conjugates to free species, which were reduced further by filtration with removal shifting from 32% to 57% for natural estrogen, although no EE2 was removed.