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Tris (2-ethylhexyl) phosphate (TEHP) is one of the most commonly used organophosphorus flame retardant (OPFR) analogues and is commonly detected in surface water and sediments. Limited information is available about the metabolic pathway or metabolite formation related to TEHP in fish. In this study, rare minnows (Gobiocyprisrarus) were exposed to TEHP in static water for 30 d to investigate the bioaccumulation and metabolite distribution in the fish muscle, liver, kidney, gill, GI-tract, ovary and testis. Based on the estimated k up , parent and k d,parent values, the bioconcentration factors (BCF parent ) of TEHP in fish tissues were calculated in the order of kidney > ovary ≈ liver ≈ testis > gill ≈ GI-tract > muscle; this finding was consistent with the results of our previous study on other alkyl-substituted OPFRs. In addition, this study identified the metabolic profiles of TEHP in the liver. TEHP was oxidatively metabolized by the fish to a dealkylated metabolite (di 2-ethylhexyl phosphate; DEHP) and hydroxylated TEHP (OH-TEHP). OH-TEHP further underwent extensive phase II metabolism to yield glucuronic acid conjugates. DEHP was mainly distributed in rare minnow in the following order: liver > GI-tract > kidney ≫ other tissues. However, the metabolite showed lower accumulation potential in fish tissues than TEHP, with metabolite parent concentration factors (MPCFs) for DEHP of less than 0.1 in all the investigated tissues. The BCF parent values of TEHP in various fish tissues were only 9.0 × 10 -3 -7.2 × 10 -4 times its estimated tissue-water partition coefficient (K tissue-water ) values based on tissue lipid, protein and water contents, which indicated the significance of biotransformation in reducing the bioaccumulation potential of TEHP in fish. The toxicokinetic data in the present study help in understanding the tissue-specific bioaccumulation and metabolism pathways of TEHP in fish and highlight the importance of toxicology research on TEHP metabolites in aquatic organisms., Competing Interests: Declaration of competing interest The authors declared that they have no conflicts of interest to this work entitled “Tissue-specific bioaccumulation, metabolism and excretion of tris(2-ethylhexyl) phosphate (TEHP) in rare minnow (Gobiocypris rarus)” by Rui Hou, Yiping Xu, Kaifeng Rao, Chenglian Feng, and Zijian Wang for publication in the Environmental Pollution., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Thiamethoxam has emerged as an environmental contaminant detected in aqueous environments, and its endocrine-disrupting effect at chronic exposure in teleosts remains unknown. In the present study, a docking experiment and an in vivo test were integrated to systematically explore the toxic mechanisms of thiamethoxam in fish. Histological analysis, plasma VTG and hormone level (E2, 11-KT, T3 and T4) determinations, and HPG and HPT gene expression quantification were performed after Chinese rare minnow (Gobiocypris rarus) was exposed to thiamethoxam (0, 0.5, 5, and 50 μg/L) for 90 days. According to the docking study, thiamethoxam had different interactions with ERα, AR and TRα via hydrogen bonding. A decrease in body length and plasma T4 was observed in both genders. The histological damage in liver and delayed gonadal development were observed in both genders at 50 μg/L thiamethoxam treatment. In males, the following HPG axis genes were upregulated: gnrh and cyp19b in the brain; vtg and cyp19a in the liver; and cyp17 and cyp19a in the gonad. In females, erɑ in the liver was significantly upregulated with 0.5 μg/L thiamethoxam treatment, and cyp17 in the gonad was upregulated with all treatment. The suppression of cyp19a, gnrh, cyp11a, and ttr was observed at the concentration of 5 μg/L in the female liver. Taken together, the endocrine system of Chinese rare minnow might be disrupted after chronic exposure to thiamethoxam., (Copyright © 2019. Published by Elsevier Inc.)

Alkylated organophosphate esters (alkyl-OPEs) are widely used and extensively detected in aquatic organisms. This work investigated the tissue-specific toxicokinetics of two common alkyl-OPEs, tri(2‑butoxyethyl) phosphate (TBOEP) and tri‑n‑butyl phosphate (TNBP) in Chinese rare minnow (Gobiocypris rarus) through a 50 day uptake and depuration experiment. The tissue-specific bioconcentration factor (BCF) values for the two alkyl-OPEs ranged from 1 to 30 L/kg wet weight (ww), with the kidney and ovary as the tissues with the highest accumulation. The tissue BCFs only exhibited a significant correlation with lipid contents only in storage tissues (i.e., muscle, brain, ovary and testis), indicating that lipids might not be the major contributor to tissue distribution of TBOEP and TNBP. However, the contribution of blood perfusion and active transport to tissue-specific OPE accumulation needs to be further investigated. Lower accumulation of metabolites than parent chemicals was observed, with metabolite parent concentration factors (MPCFs) <1. Di-alkyl phosphate (DAP), bis(2‑butoxyethyl) phosphate (BBOEP) and di(n-butyl) phosphate (DNBP) were the most abundantly formed metabolites of TBOEP and TNBP in various tissues, followed by the monohydroxylated OPEs (OH-OPEs). However, bis(2‑butoxyethyl) hydroxyethyl phosphate (BBOEHEP), was detected at much lower levels in the tissues. All the investigated metabolites showed high production rates (k prod,metabolites ) in the fish liver, followed by the GI tract and the kidney, indicating the importance of the hepatobiliary and urinary systems in eliminating the metabolites. Our study suggested that metabolism plays an important role in eliminating these two alkyl-OPEs in rare minnow and results in different tissue distribution mechanisms for metabolites and their compounds., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

In the present study, Chinese rare minnows (Gobiocypris rarus) were exposed to 1, 10, and 100 μg/L of carbamazepine (CBZ) under flow-through conditions for 28 d. A hepatic-specific custom microarray identified 111 and 71 differentially expressed genes in the livers of females and males, respectively, exposed to 100 μg/L of CBZ (ratio ≥ 2, p ≤ 0.05). The levels of five differentially expressed genes associated with the hypothalamic-pituitary-gonadal (HPG) axis were quantified by qPCR, and the results indicated the feasibility of screening endocrine-disrupting chemicals using a custom microarray. The mRNA levels of genes related to the HPG axis differed significantly in different organs of Chinese rare minnows (p < 0.05). Significant differences were observed in the 11-ketotestosterone and plasma vitellogenin levels in all treatments and in the 17β-estradiol (E2) levels in the 100 μg/L CBZ treatment. In contrast, the gonadosomatic index was significantly higher in females and slightly higher in males without significant differences. A pathological analysis determined that 10 and 100 μg/L of CBZ could lead to ova-testis in males and significantly promoted ovum maturation in females. Therefore, our results demonstrate that environmentally relevant concentrations of CBZ have homologous estrogenic activity and induce reproductive toxicity in Chinese rare minnows.

To examine the effects of BaP on tissue apoptosis, laboratory studies were conducted using juvenile Chinese rare minnows (Gobiocypris rarus) exposed to 1, 5, 20, and 80 μg/L of BaP for 28 days. The post-treatment pathological findings in the liver were associated with hepatocyte swelling, karyopyknosis, and karyorrhexis. Moreover, an increase in the goblet cells in the intestine, epithelial hyperplasia of the gills and fusion of gill lamellae were observed. Significant increases in hepatocyte apoptosis using the TUNEL stain were observed in the liver tissue but not in the intestine and gills. In addition, BaP exposure significantly up-regulated the mRNA levels of cyp1a1, p53, bax, bcl-2, and caspase-9 in the liver following the 5, 20, and 80 μg/L treatments, whereas the apaf-1 was significantly down-regulated following all treatments. Moreover, the activities of caspase 3 and caspase 8 were markedly elevated, whereas the protein expression levels of Apaf-1 were down-regulated following the 20 and 80 μg/L treatments. Taken together, our results suggested that BaP strongly induces tissue-specific apoptosis in vivo, leading to significant pathological changes. The responsiveness of apoptotic-related genes demonstrates that BaP induced apoptosis in the liver may be through a mitochondria-independent pathway., (Copyright © 2017. Published by Elsevier Ltd.)

Benzotriazole (BT) and its associated derivatives are used ubiquitously in industrial processes, and can be detected in indoor temperature coolants and in chemicals designed to inhibit corrosion. This chemical has been widely detected in aquatic environments and shows some degree of environmental persistence. Evidence has shown that BT exposure can negatively affect endocrine systems and can result in neurotoxicity in fish. However, no study has examined whether this chemical exhibits hepatotoxicity in fish, and if so, what are the underlying mechanism associated with the damage. To address this knowledge gap, we measured the liver proteome of adult male Chinese rare minnow (Gobiocypris rarus) exposed to either 0.05, 0.5, or 5 mg/L BT for 28 days. Overall, 17 proteins were induced and 9 were reduced in abundance following BT treatment (ratio > 1.5, p < 0.05). Pathway analysis revealed that cellular processes affected by BT included xenobiotic clearance, oxidative stress response, apoptosis, and translation. Moreover, transcripts related to these toxic pathways were also significantly affected by BT. In addition, rare minnows exposed to BT showed signs of hypertrophy of hepatocytes, nuclei pyknosis, and higher levels of cellular vacuolization compared to the controls, thus these early proteomic responses in the liver may be related to pathology (i.e. adverse outcome pathway). Our data demonstrate that BT dysregulates molecular responses in the liver and tissue pathology indicative of damage. This study provides new insight into BT hepatotoxicity in Chinese rare minnow., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

In the present study, to evaluate embryonic toxicity and the thyroid-disrupting effects of 2,4-dichloro-6-nitrophenol (DCNP), embryos and adults of Chinese rare minnow (Gobiocypris rarus) were exposed to 2, 20, and 200μg/L DCNP. In the embryo-larval assay, increased percentages of mortality and occurrence of malformations, decreased percentage of hatching, and decreased body length and body weight were observed after DCNP treatment. Moreover, the whole-body T3 levels were significantly increased at 20 and 200μg/L treatments, whereas the T4 levels were markedly decreased significantly (p<0.05) for all DCNP concentrations. In the adult fish assay, plasma T3 levels were significantly increased whereas plasma T4 levels were significantly reduced in the fish treated with 20 and 200μg/L (p<0.05). In addition, DCNP exposure significantly changed the transcription levels of thyroid system related genes, including dio1, dio2, me, nis, tr, and ttr. The increased responsiveness of thyroid hormone and mRNA expression levels of thyroid system related genes suggested that DCNP could disrupt the thyroid hormone synthesis and transport pathways. Therefore, our findings provide new insights of DCNP as a thyroid hormone-disrupting chemical., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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, gadd45β, 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 gadd45β 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., (© 2016 Wiley Periodicals, Inc.)

Benzotriazole (BT) is a high-production volume chemical which has been ubiquitously detected in aquatic environments. Although adverse effects from acute and chronic exposure to BT have been reported, the neurotoxic effect of BT and the mechanisms of toxicity are not well documented. In this study, adult female Chinese rare minnow (Gobiocypris rarus) were exposed to 0.05, 0.5, and 5mg/L BT for 28days. The brain proteome showed that BT exposure mainly involved in metabolic process, signal transduction, stress response, cytoskeleton, and transport. Pathway analysis revealed that cellular processes affected by BT included cellular respiration, G-protein signal cascades, Ca 2+ -dependent signaling, cell cycle and apoptosis. Moreover, data on relative mRNA levels demonstrated that genes related to these toxic pathways were also significantly affected by BT. Furthermore, proteins affected by BT such as CKBB, GS, HPCA, VDAC1, and FLOT1A are associated with neurological disorders. Therefore, our finding suggested that BT induced molecular responses in the brain and could provide new insight into BT neurotoxicity in Chinese rare minnow., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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, ERβ), 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., (Copyright © 2016. Published by Elsevier Ltd.)

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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: MicroRNAs (miRNAs), which comprise a large family of endogenous small non-coding RNA molecules, play important roles in the regulation of gene expression in various biological processes. The Chinese rare minnow (Gobiocypris rarus) is a Chinese native fish species and is used extensively as an experimental fish in China; however, relevant biological data, especially miRNA transcriptome data, have not been well documented. To discover conserved and potential novel miRNAs in Chinese rare minnows, a pool of equal amounts of RNA obtained from 6 different adult rare minnow tissues (brain, eye, gill, liver, muscle and heart) was sequenced using illumina deep sequencing technology., Results: In the present study, 26,930,553 raw reads, representing 2,118,439 unique high-quality reads, were obtained from the pooled small RNA library. Using bioinformatics analysis, 352 conserved and 112 novel Chinese rare minnow miRNAs were first discovered and characterized in this study. Moreover, we found extensive sequence variations (isomiRs) in rare minnow miRNAs, including internal miRNA isomiRs and terminal isomiRs at both the 5' and 3' ends and nucleotide variants. Six conserved and 4 novel miRNAs were selected and validated in 6 different adult rare minnow tissues using quantitative real-time PCR (qPCR). The results showed that miR-30a, miR-30b, and Novel-37 are ubiquitously expressed in a variety of tissues. miR-16a, miR-9, miR-125b, miR-34a, and Novel-69 were predominantly expressed in the brain. Novel-115 and Novel-7 were highly expressed in gills, but were relatively weakly expressed in other tissues. These results provided the expression patterns of miRNA genes in Chinese rare minnow. Finally, based on bioinformatics predictions, we mainly found that Novel-94 and Novel-1b-5p were simultaneously targeted to the 3'UTR of Dmrt1, which controls sex determination and/or sexual differentiation in a variety of metazoans at different sites. Novel-29b targeted the 3'UTR of Foxl2, which is involved in the maintenance of ovarian function and the transcriptional regulation of gonadal differentiation-related genes. Novel-62 and Novel-53 targeted the 3'UTR of ERbeta1 and ERbeta2 (which regulate the transcription of target genes), respectively., Conclusions: Rare minnow is a widely used model for assessing the risk of environmental pollution in China. Identifying and characterizing rare minnow miRNA genes is necessary to discover the biological function of miRNAs and to screen for new molecule biomarkers to assess the risk of environmental pollution in the future.

Organophosphate flame retardants (OPFRs) have been detected at high concentrations in various environmental and biotic samples, but little is known about their toxicity. In this study, the potential neurotoxicity of three OPFRs (TCEP, TDCPP, and TPP) and Chlorpyrifos (CPF, an organophosphate pesticide) were compared in Chinese rare minnow using an acute toxicity test and a 21-day fish assay. The acute test demonstrated significant inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) by CPF. Although significant AChE inhibition at high concentration of TPP was also observed, none of the OPFRs had effects similar to CPF on these enzymes, indicating that their acute toxicities to Chinese rare minnow may be unrelated to cholinesterase inhibition. In addition, the 21-day fish assay with TDCPP demonstrated no significant effects on cholinesterase activities or neurotransmitter levels. Nonetheless, this OPFR exhibited widespread effects on the neurotrophic factors and their receptors (e.g., ntf3, ntrk1, ntrk2, ngfr, and fgf2, fgf11, fgf22, fgfr4), indicating that TDCPP or other OPFRs may elicit neurological effects by targeting neurotrophic factors and their receptors in Chinese rare minnow., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

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 17β-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 LHβ mRNA was detected across all doses (p<0.05). In the gonad, the transcriptional levels of StAR, CYP11A, 3βHSD, CYP17, 17βHSD, and CYP19A were generally decreased in the males at 5 mg L(-1) (p<0.05), whereas these genes, except for 3βHSD, 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., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Butachlor is a chloroacetamide herbicide widely used in Asia, and may enter the aquatic environment through agricultural application. In this study, plasma VTG and hormone levels (E2, 11-KT, T3 and T4) were determined after the female rare minnow (Gobiocypris rarus) was exposed to butachlor at environmental relevant concentrations (0, 0.1, 1, and 10μg/L) for 40days. The mRNA levels of the HPG axis-related genes (gnrh, erα, vtg, star, lhr, 3β-hsd, cyp11a, cyp17, cyp19a and cyp19b), and the HPT axis-related genes (trα, dio1, dio2, and dio3) were quantified after 20 and 40days exposure to butachlor. For the HPG axis, the plasma 11-KT was increased at exposure concentration of 10μg/L, and VTG was significantly decreased at 1μg/L. Functional genes like gnrh and cyp19b in the brains, star, lhr, cyp11a, 3β-hsd, and cyp19a in the ovaries, and erα and vtg in livers were up-regulated. For the HPT axis, the results showed that plasma T4 levels were significantly increased, the gene expression of dio1 was up-regulated, dio2 showed no significant variation, and dio3 was down-regulated in the livers. These results indicated that butachlor may promote the accumulation of T4 in fish through inactive deiodinase type 3. The transcription of HPG axis-related genes could serve as an auto-regulation of hormone levels after exposure to butachlor. Furthermore, the activation of gnrh may play an important role as a feed-back mechanism in the regulation of hormone levels and crosstalk of endocrine axes., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

ATP-binding cassette (ABC) transporters together with phase I and II detoxification enzymes have been considered as included in a cellular detoxification system. Previous studies have highlighted the involvement of aryl hydrocarbon receptor (AHR) and Cyp1a in PAH-induced embryo toxicity. However, the response of other xenobiotic enzymes/transporters in PAH-mediated embryo toxicity is not fully characterized. In the present study, rare minnow embryos were exposed to 10 and 100µg/L BaP within 4h post-fertilization (hpf) up to 168 hpf. RNA was extracted at 24, 48, 96, and 168 hpf. The basal and BaP-induced expression of phase I enzyme genes (cyp1a, 1b1, and 1c1), phase II enzyme gene (gstm and ugt1a), and ABC transporter genes (abcb1, abcc1, abcc2, and abcg2) mRNA was determined using real-time PCR. Severe developmental defects (e.g., spinal deformities, pericardial and yolk-sac edema) were observed in the BaP treated groups. The basal expression showed that gstm was most strongly expressed, followed by abcb1, ugt1a, and abcc2, whereas cyp1a, 1b1, 1c1, and abcg2 showed weak expression. BaP significantly induced the mRNA expression of three CYP1s (cyp1a, 1b1, and 1c1) (p<0.05) and the ABC transporters (abcc1, abcc2, and abcg2) in a dose-dependent manner. However, the mRNA expression of Phase II enzymes (gstm, ugt1a) for the BaP treatments showed no significant difference with that of the controls. Furthermore, distinct induced patterns of these genes were observed during different exposure periods. Simultaneous up-regulation of the cyp and ABC transporter gene transcripts suggests that a possible involvement and cooperation in the detoxification process could provide protection against the BaP toxicity of rare minnows at the early life stage., (Copyright © 2014 Elsevier Inc. All rights reserved.)

In the present study, the cDNA fragments of five ABC transporter genes (ABCB1, ABCB11, ABCC1, ABCC2, and ABCG2) in the rare minnow were cloned, and their tissue-specific expression patterns were evaluated across eight rare minnow tissues (liver, gill, intestine, kidney, spleen, brain, skin, and muscle). Furthermore, the transcriptional effects on these ABC transporter genes and five xenobiotic-metabolizing enzyme genes (CYP1A, GSTm, GSTp1, GCLC, and UGT1a) were determined in the rare minnow liver after 12 days of pyrene exposure. Basal expression analysis showed that the tissues with high expression of the ABC transporters included the liver, kidney, and intestine. Moreover, the most highly expressed of the ABC genes were ABCB1 and ABCC2 in all eight of the tissues tested. The ABCB11 gene was almost exclusively expressed in the liver of the rare minnow, whereas ABCC1 and ABCG2 showed weak expression in all eight tissues compared to ABCB1 and ABCC2. Our results provide the first thorough examination of the expression patterns of toxicologically relevant ABC transporters in the rare minnow and serve as a necessary basis for further studies of these ABC transporters in fish. Furthermore, synergistic up-regulation of CYP1A, GSTp1, GCLC, UGT1a, and ABCC2 was observed in the rare minnow liver following pyrene exposure, while GSTm, ABCB1, ABCB11, ABCC1, and ABCG2 were not significantly affected (p < 0.05). The synergistic up-regulation of the xenobiotic-metabolizing enzymes and ABC transporters by pyrene suggests a possible involvement and cooperation of these genes in the detoxification process in rare minnows., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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., (Copyright © 2011 Wiley Periodicals, Inc.)

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., (Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.)

In this study, the expression time and profiles of thyroid hormone receptor alpha (trα), type I and II deiodinase enzymes (d1 and d2), transthyretin (ttr), sodium iodide symporter (nis), and thyrotropin-releasing hormone receptor (trhr) genes in Chinese rare minnow (Gobiocypris rarus) were determined using real-time PCR. Meanwhile, the changes of these genes were investigated by exogenous T(3) (3.8 nM) in larvae and adult fish. The retardation of swim bladder development and growth inhibition were observed for larvae, and the transcription of trα, d1, d2, nis, and trhr was significantly down-regulated at the end of exposure (21 d). In adults, a down-regulation of trα, d1, nis, and trhr mRNA levels occurred at 7th or 14th day of exposure, but returned back to their normal levels similar to control at the end of exposure. The down-regulation of gene mRNA expression could serve as a compensatory mechanism for the activation of thyroid system., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The chaperon role of heat shock proteins (HSPs) throughout the life cycle of steroid receptors have been demonstrated in vitro. However, the actions of HSPs in steroid pathways in animals especially in fish were unclear. In this study, sexually mature rare minnows (Gobiocypris rarus) were exposed to typical endocrine disruptors (atrazine and p,p'-DDE). Hypertrophy of hepatocytes at the 333 microg/l atrazine treatment and atrophy of hepatocytes in all p,p'-DDE treatments were observed. The expression of liver hsp70 and hsp90 in atrazine treatments were significantly up-regulated. Moreover, remarkable increases in the expression of androgen receptor (ar) and estrogen receptor (er) were observed, while alterations of the glucoticorcoid receptor (gr) expression was not significant in atrazine exposed fish. The expression of ar, er, gr, hsp70 and hsp90 were significantly suppressed following p,p'-DDE exposure. These results demonstrate that the expression of hsp70 and hsp90 is altered along with changes of steroid receptors in vivo. Therefore, the results are consistent with the possibility that in fish heat shock proteins (HSPs) play chaperon roles for the steroid receptors in vivo, which also concurs with previous in vitro mammalian studies., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

Atrazine, one of the most widely used herbicides, has been proved to interfere with sexual hormones. However few studies have considered the effects of atrazine on adrenal hormones (AH). In this study, rare minnow (Gobiocypris rarus) was exposed to 0, 3, 10, 33, 100 and 333microg/l atrazine for 28 days. The histopathology of kidney and gill was examined and the expressions of AHs-related genes including Na(+),K(+)-ATPase, glucocorticoid receptor (gr), heat shock protein 70 (hsp70), and heat shock protein 90 (hsp90) in kidney and gill were quantitatively determined. Histopathological observation revealed obvious lesions in gill including hyperplasia, necrosis in epithelium region, aneurysm and lamellar fusion at concentrations as low as 10microg/l. The observed lesions in kidney included extensive expansion in the lumen, degenerative and necrotic changes of the tubular epithelia, shrinkage of the glomerulus as well as increase of the Bowman's space at concentrations as low as 10microg/l. The expressions of Na(+),K(+)-ATPase, gr, hsp70 and hsp90 in the kidney of females were significantly decreased at all concentrations. For males, the expressions of hsp90 in the kidney of all treated groups were significantly down-regulated, while gr at all concentrations and hsp70 at 10, 33, 100microg/l were significantly up-regulated. However in the gill, the expressions of these genes were not significantly different from the control. These results indicated that exposure to atrazine caused impairments of kidney and gill of fish at environmental related concentrations. Histopathological lesions could partly attribute to the changes of the expressions of AHs-related genes in kidney. We concluded also that atrazine is a potential AHs-disruptor and AHs-related genes in kidney of fish could be used as sensitive molecular biomarkers.

Acetochlor is a commonly used herbicide in agricultural environments, which can accelerate T(3)-induced metamorphosis in amphibians. However, effects of acetochlor on the thyroid system in fish are still unclear at environmentally relevant concentrations. In this study, expression profiles of thyroid hormone receptor alpha (tralpha), deiodinase (d1 and d2), malic enzyme (me), and sodium iodide symporter (nis) genes were determined in larval and adult rare minnow (Gobiocypris rarus) after exposure to different levels of acetochlor (20, 200, and 2000ng/l) for 21 days, respectively. Furthermore, plasma thyroid hormones (THs) levels in adults were evaluated simultaneously. The results showed that d1, d2, me, and nis mRNA levels were significantly down-regulated in the larvae. Similar expression changes of these genes in female brains were found. However, in adult livers, these gene expressions had no significantly effects, except the tralpha mRNA level significantly up-regulated. These results indicated that exposure to acetochlor could result in tissue-specific alternative expression of TH-related genes in adults. Moreover, the expression of d2 and me showed a positive correlation with plasma T(4) levels in female brains. Therefore, larval development and adult brain of rare minnow could be affected by acetochlor at environmentally relevant concentrations.

Thyroid hormones (THs) play an important role in the development and metabolism of fish through their influences on genetic transcription and are targets for endocrine disruptive agents in the aquatic environment. Amitrole is a pesticide potentially interfering with thyroid hormone regulation. In this study, the rare minnow (Gobiocypris rarus) was exposed to different levels of 3-amino-1,2,4-triazole (amitrole) and allowed to recover in clean water. Plasma TH levels and the expression of TH-related genes, including transthyretin (ttr), deiodinases (d1 and d2), and the thyroid hormone receptor (tralpha) from the livers and brains were evaluated. After exposure, the plasma TH levels did not change. Histopathological observations showed that livers were degenerated at 10,000 ng/l and these damages could be recovered by the withdrawal of amitrole. However, the ttr, d1, and d2 mRNA levels in the livers of males were significantly up-regulated in all exposure groups (p<0.05). The ttr and d2 mRNA levels were significantly up-regulated at 10,000 ng/l and 10, 100, and 1000 ng/l in the livers of females, respectively (p<0.05). In the brains of males, a twofold increase of d2 mRNA levels at > or = 100 ng/l and a fivefold decrease of tralpha mRNA levels at > or = 10 ng/l were observed (p<0.05), whereas no significant differences were observed in the expression of d2 and tralpha in the brains of females. After a recovery period, the ttr, d1, and d2 mRNA levels in the livers of males returned to control levels, but the tralpha mRNA levels were irreversibly decreased at all treatments (p<0.05). In addition, the d2 mRNA levels in the livers of females were significantly induced at > or = 100 ng/l. Moreover, the d2 mRNA levels in the brains of males and females were up-regulated at 10,000 ng/l. These results indicated that amitrole exposure could result in alternations of ttr, d1, d2, and tralpha gene expression in different tissues of the rare minnow. The expression of these TH-related genes in males was more sensitive to amitrole than those of females. Recovery in clean water was associated with the selective regulation of TH-related gene transcription in the rare minnow. Therefore, these TH-related genes can serve as biomarkers to screen the effects of thyroid disruption chemicals in rare minnow.

2,4-Dichlorophenol (2,4-DCP) is known as a toxicant, but unknown as an environmental endocrine disruptor. In the present work, Chinese rare minnow (Gobiocypris rarus) was exposed to 0.03, 0.3, and 3 mg/L 2,4-DCP for 3 d and 0.1, 0.3, and 1 mg/L 2,4-DCP for 21 d, respectively. Endpoints including somatic index, vitellogenin (VTG) mRNA and protein level, estrogen receptor (ER) mRNA and histopathology were measured. In the 3-d exposure experiment, the effect concentrations were 0.3 mg/L and above, and in 21-d exposure, the effect concentrations were 0.1 mg/L and above. When exposed to the effect concentrations, GSI was significantly reduced for both male and female, ER mRNA was upregulated in male and downregulated in female. There were no significant variations of VTG mRNA in both male and female in 3-d exposure and in male in 21-d exposure. However, VTG mRNA in female in 21-d exposure was upregulated, corresponding to an increase of VTG protein in serum. Histopathological observation showed that ovaries were degenerated in the effect concentrations, where follicular atresias were more frequently observed. Because the sex hormones related genes and toxicological endpoints were affected in the dose-dependent manner, the results suggest that 2,4-DCP could be a potential endocrine disruptor and might cause adverse effects in female sex organs through interruption of ER-mediated processes., (2008 Wiley Periodicals, Inc.)

Breeding pairs of rare minnows were exposed to 5, 10, 20 microg/L 4-nonylphenol (NP) or 4 ng/L 17alpha-ethinylestradiol (EE(2)) for 21 days. Among reproductive parameters investigated, fecundity, fertility, and laying interval were significantly affected by 4 ng/L EE(2), but were not affected at any exposure level of NP. These effects coincided with male feminization, ova-testis, increased plasma VTG in both males and females as well as the accumulation of eosinophilic material in kidneys and livers. Histopathologic evaluation revealed severe kidney lesions in both EE(2) and NP-exposed fish. The bioassay succeeded in demonstrating that EE(2) was several orders of magnitude more potent than the industrial chemical NP. The rare minnow was more sensitive to EE(2) than medaka, but comparable to zebrafish in terms of sensitivity. The results of the present investigation indicated that a short-term reproductive bioassay is feasible using the Chinese rare minnow as the test species.

17alpha-Ethinylestradiol (EE(2)) is a synthetic estrogen used primarily in birth control pills and in hormone replacement therapy. Owing to its occurrence in surface waters at concentrations frequently greater than 1 ng/l and its projected future use, EE(2) is expected to pose a significant risk to aquatic organisms. This study was conducted to obtain long-term exposure data necessary for the establishment of water quality criteria and to investigate mechanisms associated with toxic effects. In a multigeneration experiment, Chinese rare minnows (Gobiocypris rarus) were constantly exposed to environmentally relevant concentrations of the synthetic estrogen EE(2). Mortality, deformities, reproductive parameters, plasma vitellogenin and histopathology were assessed. The results showed that, in the F(0) generation, all endpoints were significantly affected at concentrations higher than 0.2 ng/l EE(2). No F(1) phenotypic males developed to maturity at 0.2 ng/l and, when adult females of this exposure group were crossed with unexposed males, no F(2) fertile eggs were produced. Kidney histopathology and ultrastructure suggest anomalies possibly associated with increased vitellogenin accumulation. We concluded that the reproduction of the F(1) minnows was completely inhibited at the lowest concentration tested, 0.2 ng/l EE(2), a concentration frequently detected in surface waters. Growth effects may be related to increased energy requirements including the energy used in VTG synthesis. Reproductive effects are presumably associated with male feminization and the occurrence of testis-ova in males; however, ovarian degeneration observed in females may also have contributed to reproductive failure.

The mab-3 of Caenorhabditis elegans and doublesex of Drosophila are two sex-determining genes that are found to have only a common DNA binding motif called a doublesex and mab-3 (DM) domain among phyla. The DM domain regulates some aspects of sexual development such as yolk protein synthesis. In the present study, by using DM degenerate primers' 13 sequences of doublesex and mab-3-related transcription factor (dmrt), genes were cloned from genomic DNA of rare minnow (Gobiocypris rarus) and expression of dmrt1 was detected only in testis of adult males. In addition, partial sequences of vitellogenin (vtg) and beta-actin genes as comparative expression of genes were cloned from the female liver by degenerate primers based on conserved regions. The vtg mRNA in male liver was induced significantly in concentrations higher than 2 microg/L treatments, although adult rare minnow were exposed to nominal concentrations of 2, 10, 50, 250, and 1,250 microg/L 4-nonylphenol (4-NP) for 21 d. The dmrt1 mRNA level was significantly downregulated in a dose-dependent manner in all exposure groups of males. Moreover, other toxic endpoints, including growth, gonadosomatic index, hepatosomatic index, and histopathology, were determined. However, significant effects were observed only at higher exposure concentrations. Therefore, sex-specific genes such as dmrt1 can be used as a more sensitive biomarker for 4-NP and for discovering molecular mechanisms underlying toxic response.

Perfluorooctanoic acid (PFOA) is an important perfluorinated compound (PFC) with various applications and has been widely disseminated in the environment, wildlife, and humans. The present study investigated the effects of waterborne PFOA on the expression of hepatic estrogen-responsive genes, vitellogenin (VTG), and estrogen receptor beta (ERbeta) and on the gonadal development in a freshwater rare minnow (Gobiocypris rarus). The mRNA levels of VTG and ERbeta were determined using reverse transcription polymerase chain reaction (RT-PCR) techniques, and VTG protein levels were identified using enzyme-linked immunosorbent assay. A significant increase of VTG expression in the livers of both mature males and females was observed after 14 and 28 d of exposure to 3, 10, and 30 mg/L PFOA, indicating that PFOA could induce VTG synthesis. The expression of ERbeta increased significantly in livers of both mature males and females after a 14-d exposure, although no difference was observed after a 28-d exposure. The development of oocytes in testes exposed to PFOA also provided evidence of estrogenic activity in males. The ovaries of PFOA-exposed females underwent degeneration, as reported in other fish species exposed to environmental estrogens. This preliminary study indicates that PFOA can disturb the activity of estrogen in mature male rare minnows by inducing hepatic estrogen-responsive genes, VTG and ERbeta, and barrier female reproduction.

Morphological and biochemical indices, including hepatosomatic index, gonadosomatic index, and vitellogenin (VTG) induction, were compared between two teleostean species to determine their relative sensitivity of exposure to 17-alpha -ethinylestradiol (EE2). Chinese rare minnow (Gobiocypris rarus) and Japanese medaka (Oryzias latipes) were exposed to aqueous concentrations of EE2 ranging from 0.2 to 100 ng/L for 21 days. A stronger liver response to EE2 in Chinese rare minnow was observed than that in Japanese medaka. In Japanese medaka, significantly increased male GSI could be observed at 50 and 100 ngEE2/L. The semi-quantitative estimation by integrated optic density following electrophoresis showed that exposure of male Chinese rare minnow and Japanese medaka to EE2 resulted in significant (P<0.05) induction of VTG at 0.2 and 2 ngEE2/l, respectively. It indicated that the sensitivity of Chinese rare minnow to the stress of EE2 is about 10-fold greater than that of Japanese medaka.

Adult rare minnow (Gobiocypris rarus) were exposed to 0, 1, 5, and 25 ng/l (nominal concentrations) of 17alpha-ethynylestradiol (EE2) and 3, 10, and 30 microg/l (nominal concentrations) of 4-nonylphenol (NP) under flow-through conditions for a period of 28 d. Low mortality was observed at 5 and 25 ng/l EE2 and the growth of fish reduced significantly at 25 ng/l EE2 compared to controls. However, the gonadosomatic indices (GSI) of male fish were significantly higher in 1 ng/l EE(2) treatments and in 10 and 30 microg/l NP treatments (p<0.05). Renal somatic indices (RSI) of male fish in EE2 treatments were significantly higher than those in controls (p<0.05). In contrast, significantly decreased GSI and RSI of female fish could only be observed in 5 and 25 ng/l EE(2) treatments (p<0.05). Hepatosomatic indices (HSI) of male fish were significantly higher in 25 ng/l EE(2) treatments. However, significantly increased of HSI of female fish could only be observed in 1 ng/l EE(2) treatments. Plasma vitellogenin (VTG) induction could be observed in males after exposed to different concentrations of EE2 and NP, and plasma VTG concentrations in females exposed to 5 and 25 ng/l EE(2) were also significantly higher than in controls (p<0.05). At level higher than 5 ng/l EE2 or 30 microg/l NP, hepatic tissue and renal tissue impairment of males could be observed. The pathological male liver was associated with a hypertrophy of hepatocytes and damages to cellar structure and accumulated eosinophilic material. Renal tissue showed different pathological effects which was reflected by accumulated eosinophilic material, hemorrhages within the kidney tubules and hypertrophy of the tubular epithelia. Also at these levels of exposure, feminization of male fish could be noticed and parts of males manifested the testis-ova phenomenon. Ovaries of female rare minnow in 25 ng/l EE2 treatment group were degenerated. Therefore when exposed to EE2 and NP even at environmental observed concentrations, adverse effects could occur in the reproductive system of adult fishes. The observed hepatic tissue and renal tissue impairment should be due to the induction and accumulation of VTG in organs, especially in males.