| Tetrabromobisphenol A (TBBPA), one of the largest production volume of brominated flame retardants (BFRs), has been detected widely in a variety of environmental media, most organisms and human body. TBBPA has toxicities to mammals and aquatic organisms, including developmental toxicity, cytotoxicity, immunotoxicity, reproduction toxicity, neurotoxicity and endocrine toxicity. Previous research results and literature survey found TBBPA can cause nerve damage and behavior changes, but the results are not consistent each other and lack of mechanism studies. Zebrafish model has already been widely applicated in the environmental chemicals toxicology research field due to the embryo development fast, in vitro fertilization, embryo transparent, anatomical structure and genetic information highly homologous to mammals etc. The developmental nervous system is highly susceptible to environmental pollutants exposure, so the present study chooses zebrafish toxicology research model, which has its unique advantages of mimic the human early stage development, to systematically study the embryos/larvae neurobehavioral toxicity effects, the chemical content in solution and exposed embryos/larvae, and its mechanism under acute TBBPA window exposure. In addition, the developmental and neurobehaviors effects in larvae and adult stages under environmental relevant low doses of TBBPA long-term exposure were evaluated to reveal its environmental safety risk.In the present acute exposure tests, zebrafish embryos exposed to TBBPA during a sensitive window of 8-48 hours post-fertilization (hpf) displayed morphological malformations and mortality. Zebrafish exposed exclusively between 48-96hpf were phenotypically normal. Larval behavior assays indicated that in compared to 48-96hpf exposure group,5μM TBBPA exposed from 8-48hpf can induce:5dpf larval movements speed significantly decreased; 10dpf larval duration in the light area significantly increased in the light/dark exposure test; 11dpf larval shoaling dispersion significantly increased; 12dpf larval mirror attacks significantly increased. So the present study has the conclustion that 8-48hpf is a sensitive exposure for TBBPA inducing developmental and neurobehavioral changes, and 5μM is a sutiable dose for the effects producing and biological or molecular mechanism studies.The mechanism studies included the TBBPA content measurement, biological changes and misexpressed gene screening and pathway analysis. TBBPA concentration detected in the embryo/larvae showed that TBBPA was efficiently quickly absorbed and accumulated in zebrafish embryos, but was eliminated quickly when the exposure solution was removed. TBBPA dose measured in the exposure solution found that TBBPA content in the water decreased 39% after 8-48hpf exposure. Overall, TBBPA can be aborbed and quick eliminated in zebrafish. The biological evaluations found that 5μM TBBPA exposure during the sensitive exposure window induced 24hpf embryos apoptotic cell death significantly increased both at the head and the tail regions using acrdine orange staining; delayed the embryos second motor neurons development at 24hpf in the head region using transgenic Islet line; affected the primary motor neurons development at 48hpf in the head region by significantly decreased the neuron length using transgenic NBT line; And also led to disordered and loosen arrays of fast muscle fibers in 72hpf larvae using F59 immunohistochemistry assay. To further explore TBBPA-induced developmental and neurobehavioral toxicity, RNA-Seq analysis was used to identify early transcriptional changes following TBBPA exposure. In total,1969 transcripts were significantly differentially expressed (P＜0.05, FDR<0.05,1.5-FC) upon TBBPA exposure. Functional and pathway analysis of the TBBPA transcriptional profile identified biological processes involved in nerve development, muscle filament sliding and contraction, and extracellular matrix disassembly and organization changed significantly, which were consistent with the results of apoptotic cell death, motor neuron and muscle fiber development deficits. In addition, TBBPA also led to an elevation in the expression of genes encoding uridine diphosphate glucuronyl transferases (ugt, such as, ugt1a7, ugt2b5, ugt2b3, ugt2a3, ugt5b4, ugt5b6), which could affect thyroxine (T4) metabolism and subsequently lead to neurobehavioral changes.At the same time, environmental relevant low does TBBPA chronic exposure was carried out to evaluate the developmental and neurobehavior effects. In detail, zebrafish were waterborne exposure to 0,5 and 50nM TBBPA during 1-120 days post fertilization (dpf) following by detoxification for four months. The larval behaviors were evaluated in the same way as the sublethal acute exposure, and the male/female adult developments (survival rate, sex ratio, body length/weight and condition factor) and behaviors assessments (free movements speed, movements under light/dark transations, and mirror attacks) were conducted at 9 months old. In contrast to the subacute exposure, low dose TBBPA exposure resulted in:5dpf larval movements speed significantly increased; lOdpf larval duration in the light area significantly decreased in the light/dark exposure test; 11 dpf larval shoaling dispersion significantly decreased; 12dpf larval mirror attacks significantly decreased. At 9 months old, TBBPA treatments did not alter sex ratio, but resulted in reduced zebrafish body weight and length. Adult behavioral assays indicated that TBBPA exposed males had significantly higher average swim speeds and spent significantly more time in high speed darting mode and less time in medium cruising mode compared to control males. In an adult photomotor response assay, TBBPA exposure was associated with hyperactivity in male fish. Female zebrafish responses in these assays followed a similar trend, but the magnitude of TBBPA effects was generally smaller than in males. Social interaction evaluated using a mirror attack test showed that 50nM TBBPA exposed males showed significantly increased mirror attacks, while the effects on females has no significance. Overall, the hyperactivity and social behavior deficits ascribed here to chronic TBBPA exposure was most profound in males.In summary, TBBPA sublethal exposure during a narrow, sensitive developmental window results in neurobehavioral deficits, TBBPA can be easy absorbed and quick eliminated in zebrafish embryos/larvae. TBBPA window exposure induced early stage embryos neurodevelopment related biological changes, various molecules and signal pathways. In addition, environmental relevant low doses TBBPA chronic exposure induced larval behavioral changes, adult developmental and neurobehavioral. deficits. The neurobehavioral effects presented here also inducated that TBBPA exposure at different doses and different developmental stages has variant neurobehavioral outcomes, which verified that TBBPA has a non-monotonic effect. The results here suggest that TBBPA exposure has significantly neurobehavioral toxicity to aquatic organisms, and may pose high environmental safety risk. |
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