Study On The Enrichment Characteristics And Endocrine/metabolic Toxicity Mechanism Of Tetrabromobisphenol A (TBBPA) In Car

Tetrabromobisphenol A（TBBPA）is a widely-used plastic additive.Its primary application is as a reactive flame retardant covalently bound to polymers,which significantly improves the flame retardancy of products.TBBPA production and usage have been increasing annually.However,TBBPA has high bioaccumulation potential and toxicity,and currently,research on the ecological toxicity of TBBPA was limited.Therefore,this study used the common carp（Cyprinus carpio）as a model to investigate the 96h acute toxicity of TBBPA on carp,the accumulation pattern in different tissues,and changes in downstream gene expression of endocrine/metabolism-related thyroid hormone receptor（TR）and aryl hydrocarbon receptor（AhR）signaling pathways.Additionally,the study verified the activation/antagonistic relationship between TBBPA and AhR.The main results of the study are as follows:1.Acute toxicity:The 96h LC₅₀ of TBBPA on carp was 4.2 mg/L.According to the classification standard of acute toxicity in fish,TBBPA is considered a highly toxic substance to carp.2.TBBPA accumulation in tissues:TBBPA accumulation in carp increased with exposure concentration and exposure time.The accumulation pattern in tissues from high to low was liver>gill>brain.The liver had a higher accumulation capacity than metabolic capacity at 14d of exposure to 1.7 mg/L of TBBPA.The accumulation in liver tissue at 14d of exposure to 0.8 mg/L showed a decreasing trend compared to 3d.In gill and brain tissue,TBBPA accumulation increased from 3d to 14d of exposure in each concentration treatment group,but there was no significant difference,indicated that tissue metabolic capacity of the tissue was in a state of equilibrium with the accumulation capacity.Overall,carp tissues have a certain metabolic capacity for TBBPA.3.Effects of TBBPA in regulating the endocrine-related pathway--TR in carp:Exposure to high concentration（2.5 mg/L）of TBBPA resulted in a significant downregulation of the THs responsive genes（dio3b,klf9）in various carp tissues,while the effects of TBBPA on the thrαa,thrαb,and thrβgenes in the TR pathway varied with exposure time,exhibiting a time-dependent relationship.In brain tissue,the expression changes of TR pathway-related genes（thrαa,thrαb,thrβ,dio3b,and klf9）were more significant.Furthermore,under exposure to different concentrations of TBBPA,the TR pathway-related genes in the liver,gills,and brain showed a bidirectional dose-response relationship.These results indicate that dio3b and klf9 may be target genes of TBBPA in the TR pathway.The changes in TR and THs responsive gene expression indicate that exposure to TBBPA in carp can cause endocrine disruption effects mediated by TR in various tissues.4.Effects of TBBPA on the regulation of endocrine-related pathway--AhR in carp:The expression of AhR receptor--ahr2,ahr1a and ahr1b was significantly upregulated in both liver and brain under TBBPA exposure,and the expression of ARNT1 and ARNT2 in gill and brain was significantly upregulated under low dose TBBPA exposure,and the alteration of AhR pathway-related genes was found to be significantly responsive in the brain.The above trends of AhR pathway-related genes suggest that the AhR-mediated pathway may be a potential mediator of TBBPA toxicity,and the significant increase in metabolic-related genes suggests that exposure induces enhanced metabolic capacity and activation of potential defence mechanisms.5.Functional Identification of carp AhRs:Functional characterization of three AhRs and two ARNTs in carp confirmed that all carp AhRs and ARNTs are functional,and ARNTs can assist in the transcriptional activation of carp AhRs.Sensitivity tests of carp AhR subtypes showed that the sensitivity of fish AhRs is determined by species and subtype.Finally,dual luciferase assays in COS-7 cells revealed that TBBPA does not activate AhR transcriptional activity,but can inhibit the induction of reporter gene mediated by AhR after co-exposure with TCDD,indicating that TBBPA is a competitive ligand that inhibits the transcriptional activation of AhR.In summary,this study first explored the toxicity and enrichment patterns of TBBPA in carp,analyzed the in vivo changes of the genes of endocrine/metabolic-related pathway after TBBPA exposure,investigated the mechanisms of interaction between TBBPA and the key metabolism-related receptor--AhR in fish,and finally improved the understanding of TBBPA’s aquatic toxicity and ecological risk.