Triclosan induces zebrafish neurotoxicity by abnormal expression of miR-219 targeting oligodendrocyte differentiation of central nervous system.

Triclosan (TCS) is ubiquitous in a wide range of personal care and consumer products, and it is acute/chronic exposure may result in several nervous system disorders. Previous studies demonstrated TCS-induced abnormal expression of miRNAs, but no investigations focused on upstream changes of miRNAs and associated molecular mechanisms. Herein, phenotype observation and behavioral analysis confirmed that TCS exposure (0, 62.5, 125, 250 µg/L) led to developmental neurotoxicity in zebrafish larvae, especially for oligodendrocyte precursor cells (OPCs). High-throughput sequencing demonstrated the critical role of miR-219 in the differentiation of OPCs. Larvae with miR-219 depletion showed the same phenotype caused by TCS. Functional tests with miR-219 knock-down and over-expression showed that miR-219 promoted differentiation of OPCs by acting on myelination inhibitors. The miR-219 also protected against TCS-induced inhibition of cell differentiation. Several epigenetic features were identified to reveal potential upstream regulatory mechanisms of miR-219. In particular, five CpG islands hyper-methylated with increasing TCS concentrations in the promoter region of miR-219. TCS inhibited OPC differentiation by influencing epigenetic effects on miR-219-related pathways, contributing to severe neurotoxicity. These findings enhance our understanding of epigenetic mechanisms affecting demyelination diseases due to TCS exposure, and also provide theoretical guidance for early intervention and gene therapy of environmentally induced diseases.

Identification of receptors for eight endocrine disrupting chemicals and their underlying mechanisms using zebrafish as a model organism.

Herein, eight common endocrine disrupting chemicals (EDCs) were exposed to zebrafish (Danio rerio) to investigate the relationship between different EDCs and their activated estrogen receptors. Under acute exposure, we identified five major malformation types whose incidence and deformity modes differed among EDCs. Luciferase analysis divided the EDC receptors into four categories: (i) triclosan (TCS), 17ß-estradiol (E2) and estriol (E3) mainly activated GPER expression; (ii) bisphenol A (BPA), p-(tert-octyl) phenol (POP), 17α-ethynylestradiol (EE2), E2 and E3 activated ERß expression; (iii) E2 and E3 acted on both GPER and ERß; and (iv) estrone (E1) and 9,9-bis(4-hydroxyphenyl)fluorene (BHPF) had little effect on the two receptors. In vivo immunofluorescence experiments on 96-hpf larvae provided evidence that TCS and POP acted on GPER and ERß, respectively, while E2 acted on the two receptors simultaneously. Luciferase activities in the promoter regions of gper (-986 to -488) and erß (-1998 to -1496) were higher than those in other regions, identifying these key regions as targets for transcription activity. TCS promoted GPER expression by acting on the JUND transcription factor, while POP promoted ERß expression by activating the Foxl1 transcription factor. In contrast, E2 mainly regulated transcription of GPER and ERß by Arid3a. These findings provide compelling evidence that different EDCs possess varying estrogen receptors, leading to differential regulatory pathways and abnormality symptoms. These results offer an experimental strategy and fundamental information to assess the molecular mechanisms of EDC-induced estrogen effects.

Anti-parasitic activities of specific bacterial extracellular products of Streptomyces griseus SDX-4 against Ichthyophthirius multifiliis.

The ciliate Ichthyophthirius multifiliis is one of the most pathogenic parasites of fish maintained in captivity. In this study, effects of bacterial extracellular products of Streptomyces griseus SDX-4 against I. multifiliis were determined. The fermentation liquor of S. griseus was extracted successively in a separating funnel with petroleum ether, ethyl acetate, and n-butanol. In vitro assays revealed that the n-butanol extracts (NBu-E) and ethyl acetate extracts (Eto-E) of S. griseus were observed to be more effective against theronts than the other extracts with median effective concentration (EC50) values of 0.86 and 12.5 mg L(-1), respectively, and significantly reduced the survival of the tomonts and the total number of theronts released by the tomonts (P<0.05). All encysted tomonts were killed when the concentration of NBu-E was 30.0 mg L(-1). Results of in vivo test demonstrated that the number of I. multifiliis trophonts on the grass carp treated with NBu-E was markedly lower compared to the control group at 11 days after exposed to theronts (P<0.05). In the control group, 100% mortality was observed owing to heavy I. multifiliis infection at 11 days after the exposure. On the other hand, only 9.5% mortality owing to parasite infection was recorded in the groups treated with the NBu-E (30 mg L(-1)). The median lethal dose (LD50) of NBu-E for grass carp was 152.4 mg L(-1). Our results indicate that n-butanol extract of S. griseus will be useful in aquaculture for controlling I. multifiliis infections.

Immunotoxicity induced by triclocarban exposure in zebrafish triggering the risk of pancreatic cancer.

Owing to frequent application as a broad-spectrum bactericide, triclocarban (TCC) exposure has raised great concern for aquatic organisms and human health. Herein, based on transcriptome sequencing data analysis of zebrafish, we confirmed that TCC induced oxidative stress and dysimmunity through transcriptional regulation of the related genes. With aid of the Cancer Genome Atlas (TCGA) assembler database, 52 common differentially expressed genes, whose functions were related to immunity, were screened out by virtue of the meta-analysis of pancreatic cancer sample data and differential transcription profiles from TCC-exposed larvae. Acute TCC exposure affected formation of the innate immune cells, delayed mature thymic T-cell development, reduced immunoglobulin M (IgM) levels and promoted excessive release of the pro-inflammatory factors (IL-6, IL-1ß and tnfα). Under TCC exposure, the expressions of the genes associated with immune cell abundance in pancreatic cancer were significantly down-regulated, while the levels of ROS were prominently increased in concomitant with suppressed antioxidant activity. Moreover, a series of marker genes (pi3k, nrf2, keap1, ho-1 and nqo1) in the PI3K/Nrf2 antioxidant-stress pathway were abnormally expressed under TCC exposure. Interestingly, vitamin C decreased the malformation and increased the survival rate of 120-hpf larvae and effectively alleviated TCC-induced oxidative stress and immune responses. Overall, TCC exposure induced immunotoxicity and increased the risk of pancreatic cancer by inhibiting the antioxidant capacity of the PI3K/Nrf2 signal pathway. These observations enrich our in-depth understanding of the effects of TCC on early embryonic-larval development and immune damage in zebrafish.

Identification of three metabolic subtypes in gastric cancer and the construction of a metabolic pathway-based risk model that predicts the overall survival of GC patients.

Gastric cancer (GC) is highly heterogeneous and GC patients have low overall survival rates. It is also challenging to predict the prognosis of GC patients. This is partly because little is known about the prognosis-related metabolic pathways in this disease. Hence, our objective was to identify GC subtypes and genes related to prognosis, based on changes in the activity of core metabolic pathways in GC tumor samples. Differences in the activity of metabolic pathways in GC patients were analyzed using Gene Set Variation Analysis (GSVA), leading to the identification of three clinical subtypes by non-negative matrix factorization (NMF). Based on our analysis, subtype 1 showed the best prognosis while subtype 3 exhibited the worst prognosis. Interestingly, we observed marked differences in gene expression between the three subtypes, through which we identified a new evolutionary driver gene, CNBD1. Furthermore, we used 11 metabolism-associated genes identified by LASSO and random forest algorithms to construct a prognostic model and verified our results using qRT-PCR (five matched clinical tissues of GC patients). This model was found to be both effective and robust in the GSE84437 and GSE26253 cohorts, and the results from multivariate Cox regression analyses confirmed that the 11-gene signature was an independent prognostic predictor (p < 0.0001, HR = 2.8, 95% CI 2.1-3.7). The signature was found to be relevant to the infiltration of tumor-associated immune cells. In conclusion, our work identified significant GC prognosis-related metabolic pathways in different GC subtypes and provided new insights into GC-subtype prognostic assessment.

Toxicity mechanisms regulating bone differentiation and development defects following abnormal expressions of miR-30c targeted by triclosan in zebrafish.

As a ubiquitous environmental estrogen-disrupting chemical, triclosan (TCS) can induce severe osteotoxicity; however, the underlying molecular mechanisms remain uncertain. Herein, we evaluated the toxic effects of TCS on the development of cartilage and osteogenesis in 5-dpf zebrafish. Under TCS exposure from 62.5 to 250 µg/L, several osteodevelopmental malformations were observed, such as defect of craniofacial cartilage, pharyngeal arch cartilage dysplasia, and impairments on skeletal mineralization. Further, the morphology of mature chondrocytes became swollen and deformed, their number decreased, nucleus displacement occurred, and most immature chondrocytes were crowded at both ends of ceratobranchial. SEM observation of larval caudal fin revealed that, the layer of collagen fibers and the mineralized calcium nodules were significantly decreased, with the collagen fibers becoming shorter upon TCS exposure. The activity of bone-derived alkaline phosphatase significantly reduced, and marker functional genes related to cartilage and osteoblast development were abnormally expressed. RNA-seq and bioinformatics analysis indicated, that changes in marker genes intimately related to the negative regulation of miR-30c-5p overexpression targeted by TCS, and the up-regulation of miR-30c induced bone developmental defects by inhibiting the bone morphogenetic protein (BMP) signaling pathway. These findings were confirmed by artificially intervening the expression of miR-30c and using BMP pathway agonists in vivo. In sum, TCS induced osteototoxicity by targeting miR-30c up-regulation and interfering in the BMP signaling pathway. These findings enhance mechanistic understanding of TCS-induced spontaneous bone disorders and bone metastatic diseases. Further research is necessary to monitor chronic TCS-exposure levels in surrounding environments and develop relevant safety precautions based on TCS environmental risk.

Triclosan-induced liver injury in zebrafish (Danio rerio) via regulating MAPK/p53 signaling pathway.

Long-term exposure of triclosan (TCS), an important antimicrobial agent, can lead to deleterious effects on liver growth and development. However, the related mechanisms on TCS-induced hepatocyte injury remain unclear. Herein, we found that after long-time TCS exposure to adult zebrafish (Danio rerio) from 6 hpf (hours post-fertilization) to 90 dpf (days post-fertilization), the body weight and hepatic weight were significantly increased in concomitant with a large amount of lipid droplet accumulation in liver. Also, TCS exposure resulted in occurrence of oxidative stress by increasing the concentrations of malondialdehyde and reducing the activity of superoxide dismutase both in zebrafish larvae (120 hpf) and adult liver. By H&E staining, we observed a series of abnormal phenomena such as severely hepatocellular atrophy and necrosis, as well as prominently increased hepatic plate gap in TCS-exposure treatment groups. Through AO staining, TCS induced obvious apoptosis in larval heart and liver; through TUNEL assay, a concentration-dependent apoptosis was found to mainly occur in adult liver and its surrounding tissues. The mRNA and protein expression of anti-apoptotic protein Bcl-2 decreased, while that of pro-apoptosis protein Bax significantly increased, identifying that liver injury was closely related to hepatocyte apoptosis. The significant up-regulation of MAPK and p53 at both mRNA and protein levels proved that TCS-induced hepatocyte apoptosis was closely related to activating the MAPK/p53 signaling pathway. These results strongly suggest that long-term TCS-exposure may pose a great injury to zebrafish liver development by means of activating MAPK/p53 apoptotic signaling pathway, also lay theoretical foundation for further assessing TCS-induced ecological healthy risk.

Phylogeography and genetic population structure of the spadenose shark (Scoliodon macrorhynchos) from the Chinese coast.

The population structure of Scoliodon macrorhynchos from the Chinese coast was investigated using the mitochondrial control region. All 19 mtDNA haplotypes from 219 sequences were identified. Relatively high average haplotype diversity (0.797) and relatively low average nucleotide diversity (0.0013) were found together with a recent and sudden population expansion. Analysis of the mismatch distributions, neutrality tests and Bayesian skyline plot showed a pattern consistent with a recent population expansion event that may have taken place during the last glacial maximum (LGM). The analysis of molecular variance (AMOVA) showed the low genetic differentiation between the populations, which may be a general feature of sharks living in coastal areas. The phylogenetic and cluster analysis of the mtDNA indicates that two putative groups (K = 2) existed in S. macrorhynchos, showing that the Taiwan Strait acted as a biogeographic barrier during major drops in the sea level in the late Pliocene epoch.

Combined toxicity of triclosan, 2,4-dichlorophenol and 2,4,6-trichlorophenol to zebrafish (Danio rerio).

Triclosan (TCS), 2,4,6-trichlorophenol (2,4,6-TCP) and 2,4-dichlorophenol (2,4-DCP) are the most prevalent chlorinated phenolic pollutants in aquatic environments. Our results showed LC50 and EC50 values of 0.51, 1.11, 2.45mg/L, and 0.36, 0.74, 1.53mg/L for TCS, 2,4,6-TCP and 2,4-DCP, respectively, to 120hpf zebrafish. The highest TCSD (the mixture of TCS, 2,4,6-TCP and 2,4-DCP) toxicity was observed at a TCS:2,4,6-TCP:2,4-DCP concentration ratio of 1:2:4. LC50 and EC50 values of TCSD mixtures for 120-hpf zebrafish were 2.28 and 1.16mg/L, respectively. Two toxicity assessment methods (Toxic Unit and Mixture Toxicity Index) indicated that TCSD interactions produced partly additive toxicity. TCSD exposure decreased zebrafish hatching rate and led to a series of malformations. Following alkaline phosphatase staining, a large area of vascular ablation was observed with almost complete disappearance of vascular branches and a smaller coverage range. Prominent reddening of the yolk sac and visceral mass after oil red O staining implied that TCSD exposure severely affected fat metabolism. Following acridine orange staining, cell death occurred in eyes while high TCSD concentrations (0.84mg/L) induced cardiovascular circulation dysfunction. Alcian blue staining increased the α angle between Meckel's cartilages and ß angle between two ceratobranchial. Basihyal and palatoquadrate became shorter and developmental abnormality or defects occurred in the fifth ceratobranchial. Overall, these results provide a theoretical basis for systematically evaluating the combined toxicity of the prevalent chlorinated phenolic pollutants in real-world aquatic environments.

Mitochondrial genome and phylogenetic position of the tawny nurse shark (Nebrius ferrugineus).

The complete mitochondrial genome of the tawny nurse shark (Nebrius ferrugineus) was first presented in this study. It was 16 693 bp in length with the typical gene order in vertebrates. The overall base composition was 33.6% A, 25.6% C, 12.7% G and 28.1% T. Two start (ATG and GTG) and two stop (TAG and TAA/T--) codons were found in the protein-coding genes. The size of 22 tRNA genes ranged from 67 to 75 bp. The origin of L-strand replication could form a hairpin structure. All nodes strongly supported that N. ferrugineus was placed as sister to Rhincodon typus in the Bayesian tree.

Up-stream mechanisms for up-regulation of miR-125b from triclosan exposure to zebrafish (Danio rerio).

Triclosan (TCS) exposure has widely adverse biological effects such as influencing biological reproduction and endocrine disorders. While some studies have addressed TCS-induced expression changes of miRNAs and their related down-stream target genes, no data are available concerning how TCS impairs miRNA expression leading us to study up-stream regulating mechanisms. Four miRNAs (miR-125b, miR-205, miR-142a and miR-203a) showed differential expression between TCS-exposure treatments and the control group; their functions mainly involved fatty acid synthesis and metabolism. TCS exposure led to the up-regulation of mature miR-125b that was concomitant with consistent changes in pri-mir-125b-1 and pri-mir-125b-3 among its 3 pri-mir-125bs. Up-regulation of miR-125b originated from direct shear processes involving the two up-regulated precursors, but not pri-mir-125b2. Increased expression of pri-mir-125b-1 and pri-mir-125b-3 resulted from nfe2l2- and c/ebpα-integration with positive control elements of promoters for the two precursors. The overexpression of transcriptional factors, nfe2l2 and c/ebpα, initiated the promoter activity for the miR-125b precursor. CpG islands and Nfe2l2 were involved in constitutive expression of mir-125b-1 and mir-125b-3. The activities of two promoter regions, -487 to -1bp for pri-mir-125b1 and -1327 to +14bp for pri-mir-125b-3 having binding sites for NFE2 and Nfe2l2/MAF:NFE2, were higher than other regions, further demonstrating that the transcriptional factor Nfe2l2 was involved in the regulation of pri-mir-125b1 and pri-mir-125b-3. TCS's estrogen activity resulted from its effects on GPER, a novel membrane receptor, rather than the classical ERα and ERß. These results explain, to some extent, the up-stream mechanism for miR-125b up-regulation, and also provide a guidance to future mechanistic study on TCS-exposure.

Joint toxicity of fluoroquinolone and tetracycline antibiotics to zebrafish (Danio rerio) based on biochemical biomarkers and histopathological observation.

Herein, we report on the joint toxicity of four fluoroquinolones and two tetracyclines (ß-diketone antibiotics-DKAs) to zebrafish based on a series of toxicological endpoints and histopathological observations. A positive dose-dependence was observed in DKA-exposure groups with a 72-hpf EC50 of 130.3 mg/L for hatching rate, 120-hpf LC50 of 149.8 mg/L, and 120-hpf EC50 of 135.1 mg/L for malformation rate. When zebrafish at 60 dpf were exposed to a series of DKA concentrations (45, 60 and 90 mg/L) for 7, 14 and 21 days, creatine kinase and AChE activities were significantly induced, and intracellular malondialdehyde increased in all treatments except for the 45 mg/L treatment. The transcription levels of AHRRa from livers were significantly (p < 0.05) up-regulated in all treatments after two months of DKA exposure. CKma expression from skeletal muscle was significantly down-regulated in the 90 mg/L treatment. A remarkable down-regulation of CYP3A65 was observed in the 60 mg/L treatment. DKA exposure resulted in severe tissue damage including mitochondria swelling, reduction of mitochondrial cristae, deepening of mitochondrial cristae bands, and decreasing and even disappearance of the rough endoplasmic reticulum. Total sperm motility was decreased by ca. 30% due to DKA exposure. These results provide important information for toxicity and health risks due to mixed DKA exposure in aquatic environments.

Complete mitogenome of the oriental sole Brachirus orientalis.

The complete mitochondrial genome of the oriental sole Brachirus orientalis was presented in this study. It is 16,600 bp in length, contains 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and 1 control region. A total of 28 bp short overlaps and 23 bp non-coding intergenic spacers were found in the mitogenome. The overall base composition of the L-strand is 30.5% A, 28.7% C, 15.2% G and 25.6% T. Two start codons (ATG and GTG) and three stop codons (AGG, TAG and TAA/T) were found in the protein-coding genes. Twenty-two tRNA genes ranged from 66 bp to 75 bp. Since the tRNA-Ser2 lacks the dihydrouridine arm it can not fold into a typical cloverleaf structure. The control region demonstrates the highest A+T content (66.4%) and the lowest G content (11.8%) in the mitogenome.

Mitochondrial genome of the longtail butterfly ray Gymnura poecilura (Myliobatiformes: Gymnuridae).

The complete mitogenome the longtail butterfly ray (Gymnura poecilura) was first presented in this study. It is 17,874 bp in length, contains 37 genes with the typical gene order and transcriptional direction in vertebrates. The overall base composition is: 28.5% A, 26.5% T, 15.0% G and 30.1% C. There are 26 bp overlaps and 41 bp short intergenic spaces located in 7 and 16 gene junctions, respectively. Two start codons (ATG and GTG) and two stop codons (TAG and TAA/T) were used in protein-coding genes. The origin of L-strand replication (OL) was found between tRNA-Asn and tRNA-Cys genes. The control region has the same A and C contents (28.8%).

Complete mitochondrial genome of the largest living fish: whale shark Rhincodon typus (Orectolobiformes: Rhincodontidae).

The whale shark Rhincodon typus (Pisces: Chondrichthyes, Orectolobiformes, Rhincodontidae) is the largest living fish on Earth. In this study, we presented its complete mitogenome. It is 16,928 bp in length, contains 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and one control region with the typical gene order and transcriptional direction in the vertebrates. Overall base composition of the R. typus mitogenome is 33.5% A, 24.3% C, 12.8% G and 29.5% T. Two start codon (GTG and ATG) and two stop codon patterns (TAG and TAA/T) were found in protein-coding genes. The tRNA-Ser2 could not be folded into the typical cloverleaf secondary structure because of the replacement of its dihydrouridine arm by a simple loop. A termination associated sequences (TAS) and three conserved sequence blocks (CSB1-3) were identified in the control region.

Complete mitochondrial genome and the phylogenetic position of the zebra shark (Stegostoma fasciatum).

The complete mitochondrial genome of the zebra shark (Stegostoma fasciatum) was first determined in this study. The total length of this circle DNA was 16 658 bp, consisted of 37 genes with typical gene order in vertebrate mitogenome. Its nucleotide content was 34.1% A, 25.9% C, 12.5% G and 27.5% T. This mitogenome had 23 bp short intergenic spaces located in 10 gene junctions and 54 bp overlaps located in 11 gene junctions. In the protein-coding genes, two start codons (GTG and ATG) and two stop codons (TAG and TAA/T) were found. The 22 tRNA genes ranged from 66 bp (tRNA-Cys) to 75 bp (tRNA-Leu1). The phylogenetic result showed that S. fasciatum was clustered with the whale shark Rhincodon typus.

Complete mitochondrial genome and the phylogenetic position of the smalleye pygmy shark Squaliolus aliae (Squaliformes: Dalatiidae).

The complete mitogenome of the smalleye pygmy shark Squaliolus aliae (Squaliformes: Dalatiidae) is presented in this study firstly. It is 16,717 bp with a nucleotide base composition of 30.7% A, 26.2% C, 14.7% G and 28.5% T, containing 37 genes and a control region with typical order as vertebrate. There are 23bp short intergenic spaces and 28bp overlaps locating in gene junctions. 2 rRNA genes are located in heavy strand. Twenty-two tRNA genes range from 67bp (tRNA-Cys) to 75 bp (tRNA-Leu1). Two initial codons (ATG and GTG) and two terminal codons (TAA and T) are found in protein-coding genes. The phylogenetic result shows that S. aliae in this study is clustered to the (Somniosus pacificus + Squalidae).

Complete mitochondrial genome and the phylogenetic position of theTaiwan spurdog shark Squalus formosus (Squaliformes: Squalidae).

The complete mitogenome of the Taiwan spurdog shark Squalus formosus (Squaliformes: Squalidae) is determined in this study. It is a circle molecular (16,735 bp), containing 37 genes with typical order to that of most other vertebrates. The nucleotide composition is: A 30.9%; C 24.5%; G 14.2%; T 30.5%. Two start codons (GTG and ATG) and two stop codons (TAG and TAA/T) are used in the protein-coding genes. 22 tRNA genes range from 66 bp (tRNA-Ser2) to 75 bp (tRNA-Leu1). The phylogenetic result shows that S. formosus clusters to the (Squalus acanthias + Cirrhigaleus australis) clade.

Complete mitochondrial genome and the phylogenetic position of the Borneo leg skate Sinobatis borneensis (Rajiformes: Anacanthobatidae).

In this study, the complete mitochondrial genome of the Borneo leg skate Sinobatis borneensis (Rajiformes, Anacanthobatidae) was determined. It had circular molecules (16,701 bp), consisting of 37 genes with a typical gene order in vertebrate mitogenome. In the whole mitogenome, there were 28 bp short intergenic and 31 bp overlaps, respectively, located in 12 and 7 gene junctions. The nucleotide composition was 31.1% A, 26.0% C, 13.9% G and 29.1% T. Two start codons (GTG and ATG) and two stop codons (TAG, TAA/T) were used in the protein-coding genes. The 22 tRNA genes ranged from 66 bp (tRNA-Cys) to 75 bp (tRNA-Leu1 and tRNA-Lys). The phylogenetic result showed that S. borneensis was clustered with the Atlantoraja castelnaui and Pavoraja nitida.

Complete mitochondrial genome and the phylogenetic position of the Leadhued skate Notoraja tobitukai (Rajiformes: Arhynchobatidae).

In this study, the complete mitochondrial genome of the Leadhued skate Notoraja tobitukai (Rajiformes: Arhynchobatidae) was determined. It was a circular molecule (16, 799 bp), consisted of 37 genes with a typical gene order in vertebrate mitogenome. In the whole mitogenome, there were 24 bp short intergenic and 26 bp overlaps. The nucleotide composition was 32.3% A, 22.6% C, 13.1% G and 32.0% T. Two start codons (GTG and ATG) and two stop codons (TAG, TAA/T--) were used in the protein-coding genes. The 22 tRNA genes ranged from 65 bp (tRNA-Cys) to 75 bp (tRNA-Leu1). The phylogenetic result showed that N. tobitukai was clustered with the Pavoraja nitida.