Long-Term Exposure to SSRI Citalopram Induces Neurotoxic Effects in Zebrafish.

Residual antidepressants are of increasing concern worldwide, yet critical information on their long-term neurotoxic impacts on nontarget aquatic animals is lacking. Here, we investigated the long-term effects (from 0 to 150 days postfertilization) of the selective serotonin reuptake inhibitor citalopram (0.1-100 µg/L) on motor function, learning, and memory in zebrafish over two generations and explored the reversibility of the effect in F1 larvae. Unlike F0+ larvae, we found that F1+ larvae displayed decreased sensorimotor performance when continuously exposed to citalopram at 100 µg/L. No adverse effects were found in F1- larvae after they were transferred to a clean medium. Whole-mount immunofluorescence assays suggested that the motor impairments were related to axonal projections of the spinal motor neurons (MNs). For F0+ adults, long-term citalopram exposure mainly caused male-specific declines in motor, learning, and memory performance. Analysis of serotonergic and cholinergic MNs revealed no significant changes in the male zebrafish spinal cord. In contrast, the number of glutamatergic spinal MNs decreased, likely associated with the impairment of motor function. Additionally, treatment with 100 µg/L citalopram significantly reduced the number of dopaminergic neurons, but no significant neuronal apoptosis was observed in the adult telencephalon. Overall, this study provides neurobehavioral evidence and novel insights into the neurotoxic mechanisms of long-term citalopram exposure and may facilitate the assessment of the environmental and health risks posed by citalopram-containing antidepressant drugs.

Identification of toxicity factors and causal analysis of toxicity in surface sediments from Liaohe river basin, Northeast China using an effect guidance strategy.

Sediments play a pivotal role in maintaining the aquatic ecological status of rivers. However, the determination of the key toxicants that consider the combined effects of all sediment-related contaminants are still challenging and necessary for an appropriate sediment risk assessment. The effects of sediments on aquatic organisms have been reported in Liaohe River, but their key toxicity factors are not well known. To determine the key toxicity factors, twenty-six surface sediment samples from Liaohe River tributaries in Northeast China were collected. Acute toxicity test of midge larvae results showed that 6 of 26 tributaries had obvious toxic effects, with survival rates of 37%-57% (p < 0.05). The masking test showed that the main pollutants in the surface sediments of T7 and T16 were metals, that of T8 was an organic pollutant, those of T19 and T26 were organic pollutants and ammonia, and those of T17 were heavy metal and ammonia. Chemical analysis showed that the relatively high concentrations of ammonia were only presented in surface sediments of T17, T19, and T26, with PTU of 1.5, 1.2 and 1.1, respectively, whereas heavy metals were markedly high in surface sediments from T7 and T16, with PTU of 0.92 and 0.61, respectively. Interestingly, the observed toxicity in surface sediments agreed with the toxicity predicted by chemical analysis Moreover, the significant correlation between the survival and volume ratio of the sediment and overlying water confirmed ammonia nitrogen was key toxicity factor in T17, T19, and T26, whereas Cu was the key toxicity factor in T7 that cause the biological toxicity. In conclusion, the major toxic factors of ammonia and copper in the sediments were identified. Moreover, our study suggested that effect guidance strategy was an effective method for sediment quality assessment.

Transcriptome profiling reveals toxicity mechanisms following sertraline exposure in the brain of juvenile zebrafish (Danio rerio).

Sertraline (SER) is one of the most commonly detected antidepressants in the aquatic environment that can negatively affect aquatic organisms at low concentrations. Despite some knowledge on its acute toxicity to fish, the effects of chronic SER exposure remain poorly understood along with any underlying mechanisms of SER-induced toxicity. To address this knowledge gap, the effects of chronic exposure to three SER concentrations from low to high were investigated in zebrafish. Juvenile zebrafish were exposed to three concentrations of 1, 10, or 100 µg/L of SER for 28 d, after which indicators of oxidative stress and neurotoxicity in the brain were measured. Superoxide dismutase (SOD) activity was significantly enhanced by SER at 1 up to 100 µg/L, and catalase (CAT) activity was significantly induced by SER at 1 or 10 µg/L. The activity of acetylcholinesterase (AChE) was significantly induced by 10 and 100 µg/L of SER, and the serotonin (5-HT) level was significantly increased by all three concentrations of SER. To ascertain mechanisms of SER-induced toxicity, transcriptomics was conducted in the brain of zebrafish following 100 µg/L SER exposure. The molecular signaling pathways connected with circadian system and the immune system were significantly altered in the zebrafish brain. Based on transcriptomic data, the expression levels of six circadian clock genes were measured, and three genes were significantly altered in relative abundance in fish from all experimental treatments with SER, including cryptochrome circadian regulator 2 (cry2), period circadian clock 2 (per2), and period circadian clock 3 (per3). We hypothesize that the circadian system may be related to SER-induced neurotoxicity and oxidative stress in the central nervous system. This study reveals potential mechanisms and key events (i.e., oxidative stress and neurotoxicity) associated with SER-induced toxicity, and improves understanding of the molecular and biochemical pathways putatively perturbed by SER.

Estimating aquatic plant diversity and distribution in rivers from Jingjinji region, China, using environmental DNA metabarcoding and a traditional survey method.

Traditional survey methods (TSMs) are difficult to use to perform a census of aquatic plant diversity completely in river ecosystems, and improved aquatic plant community monitoring programs are becoming increasingly crucial with a continuous decline in diversity. Although environmental DNA (eDNA) metabarcoding has been applied successfully to assess aquatic biodiversity, limited work has been reported regarding aquatic plant diversity in rivers. In this study, the efficiency of eDNA to estimate the aquatic plant diversity and spatial distribution of rivers from the Jingjinji (JJJ) region was evaluated by comparing results obtained by the TSM. Based on a combination of the two methods, 157 aquatic plant species, including 24 hydrophytes, 61 amphibious plants, and 72 mesophytes, were identified. The spatial patterns in species richness and abundance by eDNA exhibited agreement with the TSM results with a gradual decline from the mountain area (MA) to the agricultural area (AA) and then to the urban area (UA). Compared to the TSM, eDNA identified a significantly greater number of species per site (p < 0.01) and obtained a significantly higher abundance in hydrophytes (p < 0.01), supplementing the unavailable abundance data from the TSM. Furthermore, the aquatic plant assemblages from the different areas were discriminated well using eDNA (p < 0.05), but they were better discriminated by the TSM (p < 0.01). Thus, our study provides more detailed data on aquatic plant diversity in rivers from the JJJ region, which is essential for biodiversity conservation. Our findings also highlight that eDNA can be reliable for evaluating aquatic plant diversity and has the potential to respond to landscape heterogeneity in river ecosystems.

A review on China's constructed wetlands in recent three decades: Application and practice.

Constructed wetlands (CWs) have been introduced to and developed in China for environmental engineering over the most prosperous three decades (1990-2020). To study the origin, development process, and future trend of CWs, this review summarized a wide range of literatures between 1990 and 2020 by Chinese authors. Firstly, the publication number over years, research highlights, and the author contributions with the most published papers in this field were conducted through bibliometric analysis. Secondly, the most principal components of CWs, substrates and macrophytes were summarized and analyzed. Thirdly, the typical application cases from traditional CWs, pond systems to combined pond-wetland systems were presented. In China, CWs were predominately distributed in the east of the so-called 'Hu Huanyong Line'. Therefore CWs were limited by the socio-economic level and climatic conditions. It is unquestionable that the overall level of China's CWs has improved significantly, and one of the most prominent features has started towards the plural pattern development. There has been a trend of large-scale or low-cost CW application in the recent years. However, lifecycle research and management are required for better strategies in the future.

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.

Comparison of the Toxicity Effects of Tris(1,3-dichloro-2-propyl)phosphate (TDCIPP) with Tributyl Phosphate (TNBP) Reveals the Mechanism of the Apoptosis Pathway in Asian Freshwater Clams (Corbicula fluminea).

To compare the toxicities of a chlorinated and a nonchlorinated organophosphorus flame retardant (OPFR) in this study, adult calms (Corbicula fluminea) were exposed to tris(1,3-dichloro-2-propyl)phosphate (TDCIPP) and tributyl phosphate (TNBP) at 20, 200, and 2000 µg/L for 30 days. Toxicity screening using transcriptomics indicated that the apoptosis pathway was significantly affected in the groups exposed to 2000 µg/L TDCIPP and TNBP (p ≤ 0.05), and this finding was further confirmed by the protein interaction network. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay suggested that TDCIPP and TNBP can cause apoptosis. The significant (p ≤ 0.05) increases in the activities of caspases 3 and 8 obtained with all treatments and in that of caspase 9 obtained with 2000 µg/L exposure treatments indicated the presence of mitochondria-dependent and mitochondria-independent apoptosis. Interestingly, a noticeable dose-dependent increase in DNA damage was observed in all treatments, resulting in apoptosis. Therefore, our results demonstrate that TDCIPP and TNBP induce DNA damage and apoptosis in C. fluminea, which indicates that these chemicals pose an ecological risk to benthic organisms. Moreover, through a similar mechanism of action in apoptosis, TDCIPP induced more serious toxicity than TNBP, which indicated that chlorination or differences in structure-specific metabolism could be key factors influencing toxicity.

Chronic thiamethoxam exposure impairs the HPG and HPT axes in adult Chinese rare minnow (Gobiocypris rarus): Docking study, hormone levels, histology, and transcriptional responses.

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.

Transcriptome analysis reveals benzotriazole ultraviolet stabilizers regulate networks related to inflammation in juvenile zebrafish (Danio rerio) brain.

Benzotriazole ultraviolet stabilizers (BUVSs) are widely applied ultraviolet absorbing compounds in industrial materials and personal care products. Due to their ubiquitous use and reports of bio-accumulation in aquatic organisms, these compounds are significant environmental pollutants; however, data are limited for BUVSs toxicity. In this study, juvenile zebrafish (Danio rerio) were exposed to 4 commonly used BUVSs (UV-234, UV-326, UV-329, and UV-P) at one dose of 10 or 100 µg/L for 28 days. To characterize the underlying mechanisms of different BUVSs-induced toxicities, we performed global transcriptome sequencing (RNA-Seq) in the brain (100 µg/L). There were 390, 575, 483, and 470 differentially expressed genes (DEGs) detected following UV-234, UV-326, UV-329, and UV-P exposure at 100 µg/L, respectively. Only 59 genes were identified as DEGs following exposure to each of the BUVSs, suggesting that these chemicals can induce unique responses in fish. Noteworthy was that there were 81 common gene networks (~10%) identified following exposure to BUVSs, many of which were related to inflammation and immune function. Uniquely regulated pathways affected by different BUVSs included those related to mitochondrial respiration, interleukin 1/brain-damaging signaling, dopaminergic signaling, and adrenergic receptor cascades. Furthermore, quantitative PCR (qPCR) results revealed that mgst1 levels were increased in fish from the 100 µg/L UV-329 treatment, while the expression of pck2 was significantly down-regulated in fish from both the 10 and 100 µg/L UV-P treatment. Transcriptomic data suggest that BUVSs can alter mitochondrial bioenergetics, alter expression of a broad range of genes in the oxidative stress response, and can induce pathways related to the immune system in zebrafish brain.

Triphenyl Phosphate (TPHP)-Induced Neurotoxicity in Adult Male Chinese Rare Minnows ( Gobiocypris rarus).

The neurotoxicity of triphenyl phosphate (TPHP) in exposed humans and laboratory animals is under debate. The rapid crossing of the blood-brain barrier (BBB) and high distribution of TPHP in fish brains have raised widespread concerns about potential neurotoxicity. Adult male Chinese rare minnows ( Gobiocypris rarus) were used as a model and exposed to 0, 20, or 100 µg/L TPHP for 28 days. We evaluated the BBB permeability, neuroinflammatory response, cell proliferation and apoptosis, synaptic plasticity and synapse loss in fish brains via the learning/memory performance of fish following 28 days of TPHP exposure. TPHP significantly increased the BBB permeability, activated the neuroinflammatory response, and decreased the tight junction-related mRNA levels of claudin-5α and occludin in the fish brain. In addition, cell proliferation was inhibited by treatment with 100 µg/L TPHP, but no significant apoptosis was observed in the brain. Fish exposed to 100 µg/L TPHP exhibited significantly decreased dendritic arborization in pyramidal neurons in the cerebellum (Ce), and the maze test indicated impaired learning/memory performance. Taken together, these findings provide scientific evidence that TPHP is neurotoxic to fish and further suggest that TPHP may not be a safe alternative for aquatic organisms.

Environmentally Relevant Concentrations of Carbamazepine Caused Endocrine-Disrupting Effects on Nontarget Organisms, Chinese Rare Minnows (Gobiocypris rarus).

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 17ß-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.

The neuropeptides of Asian freshwater clam (Corbicula fluminea) as new molecular biomarker basing on the responses of organophosphate chemicals exposure.

In the present study, to discover new biomarker of Asian freshwater clam (Corbicula fluminea) to assess impact of environmental pollutions, cholecystokinin (CCK), conopressin, and Neuropeptide FF (FFamide) in C. fluminea were selected as potent biomarkers. Therefore, their full-length cDNAs were cloned and characterized to investigate the molecular characteristics and expression patterns of neuropeptides in C. fluminea. According to the sequence analysis, CCK, conopressin, and FFamide encoded proteins of 173, 152, and 90 amino acids, respectively. Moreover, the multiple sequence alignment revealed that the bioactive regions of these neuropeptides were well conserved among different invertebrates. In addition, under basal conditions, CCK, conopressin and FFamide mRNA were mainly expressed in the visceral mass, whereas the FFamide mRNA was rarely detected in the foot and mantle. Exposure to 20 and 200 µg/L Tris (2-butoxyethyl) phosphate (TBOEP) and tri-butyl-phosphate (TBP) exposure significantly up-regulated the expression of the CCK and FFamide mRNAs in the visceral mass (p < 0.05), whereas no significant changes in conopressin mRNA levels were observed in response to any treatment. Therefore, CCK and FFamide of C. fluminea neuropeptides are feasible new biomarkers for screening and assessing responses to organophosphate chemicals.

Phenanthrene-Induced Apoptosis and Its Underlying Mechanism.

Phenanthrene (Phe) is one of the most abundant low-molecular-weight polycyclic aromatic hydrocarbons (PAHs). Widespread human and aquatic organism exposure to Phe has been reported, but the toxic effects of Phe and potential mechanisms are unclear. We focused on the chronic hepatotoxicity of Phe in adult Chinese rare minnows (Gobiocypris rarus) and the underlying mechanisms. The chronic effects of exposing Chinese rare minnows to 8.9, 82.3, or 510.0 µg/L Phe for 30 days were examined by histopathological observation, TUNEL assays, caspase activity assays, and gene expression profiles. The liver lesion frequency and hepatocyte apoptosis were increased in Phe-exposed groups. Caspase 9 and caspase 3 enzyme activity in liver tissues was markedly increased. The expression of miR-17/92 cluster members was significantly increased in the 82.3 and 510.0 µg/L groups. Moreover, the response of primary hepatocytes indicated a significant decrease in the mitochondrial membrane potential (MMP) after a 48 h exposure to Phe. Interestingly, miR-18a was significantly decreased in primary hepatocytes in all treatments. Moreover, molecular docking indicated that Phe might have the same binding domain as pri-miR-18a, forming pi-pi and pi-σ interactions with heterogeneous nuclear ribonucleoprotein (hnRNP) A1. Given the above, Phe caused liver lesions and induced hepatocyte apoptosis through the intrinsic apoptosis pathway, and the interaction of Phe with hnRNP A1 contributes to the suppression of miR-18a expression and hepatocyte apoptosis.

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.

Identification and characterization of novel and conserved microRNAs in several tissues of the Chinese rare minnow (Gobiocypris rarus) based on illumina deep sequencing technology.

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.

Dicamba affects sex steroid hormone level and mRNA expression of related genes in adult rare minnow (Gobiocypris rarus) at environmentally relevant concentrations.

Dicamba is a benzoic acid herbicide that has been detected in surface and ground water. The herbicide has been shown to have cytogeneic and DNA damaging effects and to cause organ damage in mammals; however, little is known about the endocrine disrupting effects of dicamba in fish. In this study, histological changes, plasma vitellogenin (VTG) and sex hormone levels, and mRNA expression of sex steroid hormone-related genes were determined in adult rare minnow exposed to environmentally relevant concentrations of dicamba (0, 0.05, 0.5, 5, and 50 µg/L) for 40 days. The results showed inhibition of spermatogenesis in male testes and ovarian degeneration in females. Plasma 17ß-estradiol (E2) levels were significantly increased in both genders, and plasma VTG levels were significantly increased in males (p<0.05). These results indicate that sex hormone homeostasis and normal reproduction of fish could be affected by dicamba. Moreover, the mRNA levels of vtg were significantly upregulated in the livers and gonads of both male and female rare minnows (p < 0.05). The downregulation of cytochrome P450c19a (cyp19a) and steroidogenic acute regulatory protein (star) mRNA levels, and the upregulation of cytochrome P450c17 (cyp17) mRNA levels were observed in the livers and ovaries (p<0.05). The results of the mRNA analysis suggest that changes in steroid hormone-related gene expression could serve as a regulatory mechanism to maintain sex hormone homeostasis. Overall, dicamba exposure could result in histological lesions, plasma VTG increases, changes in sex hormone levels, and alterations of hormone-related gene expression. Therefore, dicamba should be considered to be a potential endocrine disruptor.

Basal and benzo[a]pyrene-induced expression profile of phase I and II enzymes and ABC transporter mRNA in the early life stage of Chinese rare minnows (Gobiocypris rarus).

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.

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.

Bioaccumulation and debromination of BDE-209 in Japanese medaka (Oryzias Latipes) when continuously exposed to environmental relevant concentrations.

BDE-209 is the most commonly used commercial polybrominated diphenyl ether, and is of particular concern due to its accumulation and debromination to more toxic congeners in aquatic organisms. In this study, Japanese medaka were continuously exposed to BDE-209 with the exposure concentrations ranging from 1 to 1000 ng/L for 15, 30 and 60 days in a flowing-through exposure device. The results showed that BDE-209 could be accumulated in fish muscle at environmental relevant concentrations and its concentration in the muscle increased with the increase of exposure time and reached to a steady state. Toxicokinetic data showed that the dose-dependent half-life of BDE-209 in the muscle of medaka ranged from 16.5 to 19.4 days. Low brominated congeners could be detected, where tri- to hexa-BDEs were predominant congeners with up to 46% to 93% of total PBDEs and lower brominated BDEs may have slower elimination rates. Concentration level of BDE-155 ranged from several ng/g wet weight (ww) to a maximum of 178 ng/g ww. BDE-154 and BDE-153 as intermediates in fish under continuous exposure were negligible. By comparing with previous work, fish may have a different bioaccumulation capacity and metabolic pattern from other species, either because of species difference or the manner of exposures.

Integrative time series of cellular, humoral and molecular response revealed immunotoxicity of bifenthrin to Chinese rare minnow (Gobiocypris rarus) following Pseudomonas fluorescens challenge.

Bifenthrin is a common pesticide that is widespread in aquatic environments. Although it has been shown to be toxic to aquatic organisms, its immunotoxicity and mechanism are unclear. Herein, we reported the immunotoxicity of bifenthrin on adult Chinese rare minnow (Gobiocypris rarus) after 28 days of exposure to different concentrations of bifenthrin (0.1, 0.3, and 1.0 µg/L) and 36-h Pseudomonas fluorescens challenge. Bifenthrin inhibited the fish humoral immune response to bacteria by altering the lymphocyte and neutrophil ratios and decreasing the production of lysozyme, complement component 3, immunoglobulin M, and C-reactive protein, particularly were 1.0 µg/L. Bifenthrin caused intestinal damage and significantly reduced the volume of intestinal mucus at 12 and 36 hours postinjection (hpi) (p < 0.05). Moreover, 1.0 µg/L bifenthrin significantly increased the fish mortality and bacterial loads at 12 and 36 hpi (p < 0.05). RNA-seq analysis revealed several enriched genes involved in pathogen attachment and recognition, inflammatory responses, and complement system at the early-to-mid stage of infection (4-12 hpi). Overall, our results corroborated that bifenthrin induced immunotoxicity in Gobiocypris rarus, resulting in immune dysfunction of fish and increasing their sensitivity to bacterial infection and accelerating mortality. Moreover, 4-12 hpi was better than 36 hpi for analyzing immune responses against pathogen infection in fish exposed to bifenthrin.