| Organophosphate Flame Retardants (OPFRs) are a class of widely used flame retardants (FRs), which have been applied to a wide range of fields including building materials, textile, chemical and electronic industries. Restrictions on the use of polybrominated diphenyl ethers (PBDEs) have resulted in the dramatically increased use of alternate flame retardants, notably organophosphate flame retardants. OPFRs used as additives in materials are not chemically bonded, thus they may be easily released from the products and entered the environment. According to the recent literatures, OPFRs have been widely detected in multi-environment media at higher frequency and concentrations, especially in indoor air and surface water as well as drinking water. Futher, they are also found in wildlife, such as fish and birds, even in humans. However, the toxicity data of OPFRs, especially those on developmental toxicity and hepatotoxicity are quite limited. This study focused on the developmental toxicology and hepatotoxicity of 9 frequently used OPFRs (Triethyl phosphate [TEP], tripropyl phosphate [TPrP], Tributyl phosphate [TBP], Tris(butoxyethyl)Phosphate [TBEP], Triphenyl phosphate [TPhP], Cresyl diphenyl phosphate [CDP], Tris(2-chloroethyl)phosphate [TCEP], Tris(clorisopropyl)Phosphate [TCPP], Tris(1,3-dichloropropyl)phosphate [TDCPP]) on zebrafish. Furthermore, we discussed the toxicity effects associated with the structures of OPFRs. The toxicological mechanisms of aryl-OPFRs (TPhP and CDP) on the heart development and TPhP on the hepatic damage were explored.First, acute toxicity of of 9 OPFRs were studied with zebrafish embryos. The results suggested that the values of LC50 ranged from 0.418 mg/L to 1250 mg/L and the logarithmic n-octanol/water partition coefficient (LogKow) had been well documented for its close relationship with toxicity. Moreover, TPhP and CDP showed greater heart developmental toxicity. Then, zebrafish embryos were further exposed to TPhP and CDP with different concentration (0.1,0.5,1.0 mg/L) to explore the effects on cardiac structure and function. The results showed that heart rate was significantly decreased except for 0.1 mg/L TPhP. Also, the SV-BA distance extension and thinner waslls of ventricle and atrium could be observed. Then zebrafish embryos were exposued to 0.5 mg/L TPhP and CDP, and expression levels of key transcriptional factors in heart development including GATA4, NKX2-5, BMP4, TBX5 were measured with RT-PCR. The results revealed that the expressions of NK.X2-5, BMP4, TBX5 were significantly inhibited during 0-72 hpf, which might be the internal cause of the cardiac development defects. Besides, the heart development was found to be easily affected by the xenobiotic in 0-48hpf.To further explore the effects of TPhP on liver, the methods of metabonomics, transcriptome, hepatic histopathological and blood glucose measurement system were developed. When adult zebrafish were exposed to TPhP (0.05 and 0.3 mg/L) for one week, the liver and blood were sampled to the above analysis. The results showed that the metabolomics change of liver had the similar trend in the two exposure group, such as the increase of glucose and the decrease of succinate, fumarate, choline, acetylcarnitine, and several fatty acids, which suggested that the TPhP had a great effect on glucose and lipid metabolism in zebrafish. Transcriptomic analysis revealed that related pathways, such as the glycosphingolipid biosynthesis, PPAR signaling pathway and fatty acid elongation, were significantly affected. What is more, DNA replication, the cell cycle, and non-homologous end-joining and base excision repair were strongly affected, indicating that TPhP hinders the DNA damage repairing system in zebrafish liver cells. Other results such as the increase of blood glucose, the degeneration of fat, and cell apoptosis also suggested that glucose and lipid metabolism as well as the DNA damage repairing system could be affected by the exposure of TPhP.In the end, we developed the method of metabonomics to study the different effects of 9 OPFRs on liver metabolism and analyzed its relationship with the structures of OPFRs. Zebrafish were exposed to the concentration of 1/20 of 96h-LCso for one week. The characteristics of liver metabolism were taken to compare with the control. The result showed that all of the 9 OPFRs could lead to the increase of glucose and the decrease of acetic acid, succinic acid, choline, acetyl carnitine, glutamic acid and glutamine, which indicated that 9 OPFRs could inhibit the metabolism of glucose, fat, and amino acid. Compared to alkyl-OPFRs (TEP, TCrP, TBP, TBEP), aryl-OPFRs (TPhP, CDP) could lead to the decrease of lactate, valine as well as cholesterol ester and the increase of leucine, ARA, and EPA, suggesting that aryl-OPFRs had more significant effect on the metabolism of amino acid and faty acid. While the chloro-OPFRs (TCEP, TCPP) could increase the amount of glycolate, dimethylamine and glucose, and decrease the amout of phosphatidylcholine and trimethylamine oxide. The increasement of glycollic acid esters may be related to the degradation pathway of chlorinated alkanes and chlorinated alkenes and the decrease of trimethylamine oxide may be associated with the activity of intestinal microbial metabolism. Also, the decrease of lecithin would further hinder the metabolism of fat and glucose. The fingerprint characteristics of liver metabolism in zebrfish exposed to TDCPP had obvious difference with other chloro-OPFRs, such as the increase of trimethylamine oxide and lecithin and the decrease of fatty acid, which may be related to the number of chloro group, however, the mechanism should be further explored.This study provided the data support for evaluating the eclogical risk of OPFRs. |
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