Triclosan (TCS), 2,4,6-trichlorophenol (2,4,6-TCP) and 2,4-dichlorophenol (2,4-DCP) frequently co-exist in real-world aquatic environments; the latter two contaminants contributing to TCS photolytic products or chlorinated derivatives. There is a paucity of information regarding their joint toxicity to aquatic organisms leading us to study their effects on the swimming behavior of zebrafish (Danio rerio). Herein, we reported that 0.28 mg/L TDT exposure (mixtures of TCS, 2,4,6-TCP and 2,4-DCP) enhanced 24-hpf embryonic spontaneous movement frequency, 96-hpf larval activity; however, the 0.56 and 1.12 mg/L TDT treatments decreased all of these behavioral endpoints. All adult behavioral tests demonstrated that chronic TDT exposure (0.14 mg/L) led to hyperactivity and restlessness in adult zebrafish. A 0.14 mg/L TD DATE /@ "M/d/yyyy" 11/21/2017T treatment led to anxiety-like behavior in a bottom dwelling test and excessive panic and low hedging capacity in a conditioned place preference test. Social interaction test demonstrated that zebrafish preferred quiet and isolated space in response to TDT stress. Zebrafish memory was significantly decreased in a T-maze experiment. Whole mount in situ hybridization of pax2a and bcl2l11 genes revealed that their differential expression in the brain and skeleton were related to the corresponding phenotypic behavioral abnormality. A series of biomarker and estrogen receptor assays demonstrated that TDT acute exposure caused abnormal energy metabolism and neurological diseases. AO staining revealed that TDT exposure produced vascular ablation in the head, as well as the occurrence of massive apoptosis in the brain. TEM observation showed pyknosis of nucleus following TDT exposure. These results allow assessment of mechanisms for zebrafish abnormal behavior in response to TDT exposure, and are useful for early intervention and gene therapy of contaminant-induced diseases.

Chronic ototoxicity of β-diketone antibiotics (DKAs) to zebrafish (Danio rerio) was explored in detail by following abnormal expressions of two hearing-related miRNAs. Dose-dependent down-regulation of miR-96 and miR-184 was observed in otoliths during embryonic-larval development. Continuous DKA exposure to 120-hpf larva decreased sensitivity to acoustic stimulation. Development of otolith was delayed in treatment groups, showing unclear boundaries and vacuolization at 72-hpf, and utricular enlargement as well as decreased saccular volume in 96-hpf or latter larval otoliths. If one miRNA was knocked-down and another over-expressed, only a slight influence on morphological development of the otic vesicle occurred, but knocked-down or over-expressed miRNA both significantly affected zebrafish normal development. Injection of miR-96, miR-184 or both micRNA mimics to yolk sac resulted in marked improvement of otic vesicle phenotype. However, hair cell staining showed that only the injected miR-96 mimic restored hair cell numbers after DKA exposure, demonstrating that miR-96 played an important role in otic vesicle development and formation of hearing, while miR-184 was only involved in otic vesicle construction during embryonic development. These observations advance our understanding of hearing loss owing to acute antibiotic exposure and provide theoretical guidance for early intervention and gene therapy for drug-induced diseases.

The probiotics, Lactobacillus plantarum ST-III, plays an important role in modulating microbiota and alleviating intestinal metabolic disorders. Herein, we reported that Lactobacillus increases biodiversity of zebrafish gut flora, and attenuates toxic effects from chronic triclosan (TCS) exposure. Lactobacillus-feeding recovered the species and amount of microorganisms in the intestines of zebrafish, and inhibited toxin production by saprophytic bacterial growth. Abnormal physiological indexes and malonaldeyhde content resulting from TCS exposure were effectively alleviated. Additionally, lipid-metabolism disorders, such as increased triglyceride and total cholesterol levels, were attenuated by a probiotics diet. The number of CD4⁺ T cell lymphocytes in the lamina propria of the duodenal mucosa was decreased in zebrafish receiving a Lactobacillus diet compared to the TCS-exposed group, showing a consistent expression trend for six immune genes (NF-κB, IL-1β, TNF-α, lysozyme, TLR4α, IL-10) in the intestinal mucosa. Histopathological observations of intestines, spleen and kidney showed that TCS exposure produced severe damage to the morphology and structure of immune and metabolism-related organs. Lactobacillus was capable of mitigating this damage, but bile salt hydrolase, an active extract of Lactobacillus, was not an effective mitigation strategy. The Lactobacillus-induced decrease in the number of inflammatory cells confirmed its role in preventing inflammatory injury. Three behavioral tests (T-maze, bottom dwelling and social interaction) indicated that a probiotics diet improved zebrafish movement and learning/memory capacity, effectively alleviating anxiety behavior due to TCS exposure. These findings inform development of beneficial strategies to alleviate intestinal metabolic syndromes and neurodegenerative diseases resulting from exposure to environmental contaminants through modifying gut flora with a probiotics diet.

Triclosan (TCS) is a prevalent anthropogenic contaminant in aquatic environments and its chronic exposure can lead to a series of neurotoxic effects in zebrafish. Both qRT-PCR and W-ISH identified that TCS exposure resulted in significant up-regulation of miR-137, but downregulation of its regulatory genes (bcl11aa, MAPK6 and Runx1). These target genes are mainly associated with neurodevelopment and the MAPK signaling pathway, and showed especially high expression in the brain. After overexpression or knockdown treatments by manual intervention of miR-137, a series of abnormalities were induced, such as ventricular abnormality, bent spine, yolk cyst, closure of swim sac and venous sinus hemorrhage. The most sensitive larval toxicological endpoint from intervened miR-137 expression was impairment of the central nervous system (CNS), ventricular abnormalities and notochord curvature. Microinjection of microRNA mimics or inhibitors of miR-137 both caused zebrafish malformations. The posterior lateral line neuromasts became obscured and decreased in number in intervened miR-137 groups and TCS-exposure groups. Up-regulation of miR-137 led to more severe neurotoxic effects than its down-regulation. Behavioral observations demonstrated that both TCS exposure and miR-137 over-expression led to inhibited hearing or vision sensitivity. HE staining indicated that hearing and vision abnormalities induced by long-term TCS exposure originated from CNS injury, such as reduced glial cells and loose and hollow fiber structures. The findings of this study enhance our mechanistic understanding of neurotoxicity in aquatic animals in response to TCS exposure. These observations provide theoretical guidance for development of early intervention treatments for nervous system diseases.