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.

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.

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.

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.

The phylogenomic position of the smooth lanternshark Etmopterus pusillus (Squaliformes: Etmopteridae) inferred from the mitochondrial genome.

In this study, the complete mitogenome of the Smooth lanternshark Etmopterus pusillus (Squaliformes: Etmopteridae) was firstly determined. It was 16,729 bp, consisting of 13 protein-coding genes (PCGs), 22 tRNA genes, 2 rRNA genes and 1 putative control region with typical order to that of most other vertebrates. Its nucleotide base composition is 31.3% A, 23.0% C, 14.3% G and 31.5% T. Two start codons (GTG and ATG) and two stop codons (TAG and TAA/T) were used in the protein-coding genes. The phylogenetic results showed that E. pusillus was clustered to Squaliolus aliae (Dalatiidae) and suggested that Dalatiidae was polyphyletic.