Immunotoxic Effects Of 2,4-Ditert-Butyl Phenol And Its Mechanism On Danio Rerio

2,4-ditert-butyl phenol(2,4-DTBP),a typical synthetic phenolic antioxidant,has been widely used in food packaging plastics,petrochemicals,and personal care products.It has become an important emerging pollutant due to its wide distribution,environmental persistence and biological enrichment.However,the related toxicity studies are very limited.In this paper,zebrafish was used to preliminary explore the toxic effects of 2,4-DTBP.The 96 h-LC₅₀ and the related teratogenic effects of zebrafish embryos was firstly tested,then embryos was exposed to 6 days at sublethal levels(0.01?M,0.1?M and 1?M),observations and statistics the development/behavior target,the results showed that 2,4-DTBP has apparent developmental toxicity,affecting the process of zebrafish incubation most apparent indexes and significant change in its motion and anxiety behavior;Transcriptome sequencing was performed on larvae exposed to0.01?M and 0.1?M.Results showed that 2,4-DTBP mainly affected immune-related pathways,leading to inhibition of innate immune pathways such as NF-?B,and significantly enriched genes related to intestinal barrier.In order to further elucidate the immunotoxicity and mechanism,larvae and adult zebrafish were exposed to 2,4-DTBP at 6 d and 28 d,respectively.The intestinal immunotoxicity of zebrafish exposed to 2,4-DTBP at different concentrations was investigated at the gene,protein,tissue and individual levels.Finally,based on the existing data,the toxicity effect framework of 2,4-DTBP was preliminarily established based on AOPs,which provided data support for further study on the ecological risk of this compound.The main conclusions are as follows:(1)The 96h-LC₅₀ of 2,4-DTBP on zebrafish embryos(wild AB)was 3.91?M,which was higher than that of the same type of synthetic phenolic antioxidants.(2)Sublethal levels of 2,4-DTBP have apparent developmental toxicity on zebrafish embryos/larvae.It inhibits early spontaneous movement(28-29 hpf),hatching rate(56 hpf)and heart rate(72 hpf).0.01 and 0.1?M 2,4-DTBP induced abnormal activity of motor behavior of zebrafish larvae(144 hpf),and 1?M 2,4-DTBP induced a significant increase in dark avoidance behavior during 0-15 min,showing anxiety-like behavior.Quantitative PCR results of key genes in the dopamine and serotonin pathways showed that 5ht1ab and drd1 expression levels were significantly up-regulated after exposure.(3)The results of transcriptome showed that 2,4-DTBP mainly affected the inflammatory and immune-related pathways,and the NF-?B pathway was significantly inhibited by 0.01?M and 0.1?M exposure groups.Subnetwork enrichment analysis showed that genes were significantly enriched in four cellular processes:intestinal barrier,tissue repair,tissue remodeling,and immune regulation.These results suggest that 2,4-DTBP may disrupt the intestinal barrier of larva and induce immunotoxicity by affecting immune-related pathways.(4)2,4-DTBP significantly inhibited IL1B-MYD88-NF-?B pathway related genes in larvae and adult fish,and exposure to 0.01?M significantly decrseaed the expression of nfkb gene in larvae and female adult fish.The content of NF-?B protein in 0.1?M and 1?M was significantly decreased.Pathological sections showed that the intestinal barrier of larvae and adult was destroyed,mainly manifested by the abnormal shape of intestinal villi.At the individual level,0.1?M and 1?M exposure significantly reduced the feeding capacity of larvae and adults.Acute infection with high concentration of Ecoli could cause pericardial edema and abdominal dilation of larvae.Adult zebrafish exposed for more than 20 days will cause gradual death of 0.1?M and 1?M 2,4-DTBP.In conclusion,2,4-DTBP may induce immunosuppressive toxicity on zebrafish by inhibiting NF-?B pathway,disrupt intestinal homeostasis of larval and adult zebrafish,and lead to decreased feeding ability of zebrafish,and cause death of zebrafish after long-term exposure.