Effects of novel flame retardants tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) and triphenyl phosphate (TPhP) on function and homeostasis in human and rat pancreatic beta-cell lines.

Despite the fact that environmental pollution has been implicated in the global rise of diabetes, the research on the impact of emerging pollutants such as novel flame retardants remains limited. In line with the shift towards the use of non-animal approaches in toxicological testing, this study aimed to investigate the effects of two novel flame retardants tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) and triphenyl phosphate (TPhP) in rat (INS1E) and human (NES2Y) pancreatic beta-cell lines. One-week exposure to 1 µM and 10 µM TDCIPP and TPhP altered intracellular insulin and proinsulin levels, but not the levels of secreted insulin (despite the presence of a statistically insignificant trend). The exposures also altered the protein expression of several factors involved in beta-cell metabolic pathways and signaling, including ATP citrate lyase, isocitrate dehydrogenase 1, perilipins, glucose transporters, ER stress-related factors, and antioxidant enzymes. This study has brought new and valuable insights into the toxicity of TDCIPP and TPhP on beta-cell function and revealed alterations that might impact insulin secretion after more extended exposure. It also adds to the scarce studies using in vitro pancreatic beta-cells models in toxicological testing, thereby promoting the development of non-animal testing strategy for identifying pro-diabetic effects of chemical pollutants.

A quantitative proteomic study reveals oxidative stress and synapse-related proteins contributed to TDCIPP exposure induced neurotoxicity.

Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) has been consistently identified in various environmental media and biological specimens. Current understanding of the in vivo toxicities of TDCIPP is limited, especially for potential for neurotoxic and cognitive impairment effects. To better evaluate the potential adverse effect of the chemical on learning and memory, Sprague Dawley (SD) rats were administered TDCIPP via gavage at doses of 40, 120, and 360 mg/kg/day for a period of 90 days. Quantitative proteomic analysis, immunohistochemistry, and Western blotting were employed to assess alterations in proteins following exposure to TDCIPP. An open field test and the Morris Water Maze were used to assess anxiety and spatial learning memory capacity. Administration of TDCIPP induced anxiety and cognitive impairments in rats. Additionally, a noteworthy decrease in the number of neurons was observed in the hippocampal CA3 and dentate gyrus (DG) regions. Proteomic and bioinformatic analyses revealed dysregulation of numerous hippocampal proteins, particularly those associated with synapses (PKN1) or oxidative stress (GSTM4, NQO1, and BMAL1), which was further confirmed by Western blot analysis. In sum, the cognitive impairment of rats caused by TDCIPP exposure was associated with dysregulation of synaptic and oxidative stress-related proteins.

The regulatory roles of DDIT4 in TDCIPP-induced autophagy and apoptosis in PC12 cells.

Tris (1,3-dichloro-2-propyl) phosphate (TDCIPP) is a commonly used organophosphate-based flame retardant and can bio-accumulate in human tissues and organs. As its structure is similar to that of neurotoxic organophosphate pesticides, the neurotoxicity of TDCIPP has raised widespread concerns. TDCIPP can increase neuronal apoptosis and induce autophagy. However, its regulatory mechanism remains unclear. In this study, we found that the expression upregulation of the DNA Damage-Inducible Transcript 4 (DDIT4) protein, which might play essential roles in TDCIPP-induced neuronal autophagy and apoptosis, was observed in TDCIPP-treated differentiated rat PC12 cells. Furthermore, we determined the protective effect of the DDIT4 suppression on the autophagy and apoptosis induced by TDCIPP using Western blot (WB) and Flow cytometry (FACS) analysis. We observed that TDCIPP treatment increased the DDIT4, the autophagy marker Beclin-1, and the microtubule-associated protein light chain 3-II (LC3II) expressions and decreased the mTOR phosphorylation levels. Conversely, the suppression of DDIT4 expression increased the p-mTOR expression and decreased cell autophagy and apoptosis. Collectively, our results revealed the function of DDIT4 in cell death mechanisms triggered by TDCIPP through the mTOR signaling axis in differentiated PC12 cells. Thus, this study provided vital evidence necessary to explain the mechanism of TDCIPP-induced neurotoxicity in differentiated PC12 cells.

[KEAP1/PGAM5/AIFM1 mediated oxeiptosis pathway in TDCIPP-induced reduction of TM4 cell viability].

OBJECTIVE: To investigate the toxic effects and potential mechanisms of tri(1, 3-dichloro-2-propyl) phosphate(TDCIPP) exposure on the mouse testicular supporting cell line(TM4 cells). METHODS: TM4 cells were treated with different concentrations of TDCIPP(0, 12.5, 25 and 50 µmol/L), or 50 µmol/L TDCIPP combined with antioxidant N-acetylcysteine(NAC) for 24 h. Cell viability was assessed using the CCK8 assay, intracellular ROS levels were detected using the DCFH-DA probe, and the protein levels of oxeiptosis-related proteins, such as KEAP1, PGAM5, AIFM1 and phosphorylated AIFM1(p-AIFM1), were detected using Western blot. RESULTS: TDCIPP dose-dependently reduced TM4 cell viability(P<0.05). ROS levels in TM4 cells treated with 12.5, 25 and 50 µmol/L TDCIPP were 9.44±1.42, 17.25±1.81 and 18.38±2.66, respectively, significantly higher than the control group's 5.08±0.90(P<0.05). ROS levels in the 5 mmol/L NAC+50 µmol/L TDCIPP group were 14.70±0.50, significantly lower than the corresponding TDCIPP group's 26.44±0.73(P<0.05). The activity of TM4 cells in KEAP1siRNA+TDCIPP group and PGAM5siRNA+TDCIPP group were 77.00±1.73 and 76.67±1.53, respectively, significantly higher than TDCIPP group 68.67±1.53(P<0.05). The relative expression of KEAP1 protein in TM4 cells treated with 25 and 50 µmol/L TDCIPP were 0.77±0.04 and 0.82±0.02, respectively, significantly higher than the control group's 0.57±0.01(P<0.05). The relative expression of PGAM5 protein in TDCIPP-treated TM4 cells were 1.17±0.04, 1.38±0.03 and 1.41±0.03, respectively, significantly higher than the control group's 0.81±0.02(P<0.05). The relative expression of AIFM1 protein were 0.42±0.01, 0.63±0.01 and 0.68±0.02, respectively, significantly higher than the control group's 0.34±0.02(P<0.05). The relative expression of p-AIFM1 protein were 1.73±0.02, 1.52±0.02 and 0.73±0.01, respectively, significantly lower than the control group's 2.25±0.02(P<0.05). In the 5 mmol/L NAC+50 µmol/L TDCIPP group, the relative expression of KEAP1, PGAM5 and AIFM1 proteins in TM4 cells were 0.61±0.01, 0.58±0.01 and 0.48±0.03, respectively, significantly lower than the TDCIPP group's 0.86±0.12(P<0.05), 0.74±0.02(P<0.05) and 0.92±0.01(P<0.05). The relative expression of p-AIFM1 protein in the 5 mmol/L NAC+50 µmol/L TDCIPP group was 0.45±0.11, significantly higher than the TDCIPP group's 0.23±0.01(P<0.05). CONCLUSION: The reduction of TM4 cell viability induced by TDCIPP may be related to ROS-mediated regulation of the KEAP1/PGAM5/AIFM1 pathway, leading to oxeiptosis.

[Ferroptosis is involved in testicular injury induced by TDCIPP in adolescent male mice].

Objective: To investigate the role of ferroptosis in testicular injury in adolescent male mice induced by TDCIPP. Methods: In December 2021, 30 healthy 3-week-old male C57BL/6 mice, with a body weight of (13±2) g, were selected and fed adaptive for one week. They were divided into control group, low-dose group, medium-dose group, high-dose group and iron death inhibitor group according to a random number table, with 6 mice in each group. Mice in low, medium and high dose groups were treated with 5, 25 and 125 mg/ (kg·d) TDCIPP for 28 days, respectively, while the control group was treated with the same amount of corn oil for 28 days. The iron death inhibitor group was given 125 mg/ (kg·d) TDCIPP intragastric administration for 28 days, and 30 mg/kg DFO saline solution was intraperitoneally injected three times a week. After the treatment, the mice were killed, the epididymis was separated, and sperm count was performed. HE staining was used to observe the morphological changes of mouse testis, and iron content in testis was detected by tissue iron detection kit. The level of reactive cxygen species, MDA content, and the mitochondrial membrane potential level of mice were detected. Western blot analysis of testicular glutathione peroxidase (GPX4) and internal cyclooxygenase-2 (COX2) protein expression. Results: Compared with the control group, the spermatogenic cells in the testes of mice treated with medium-and high-dose of TDCIPP were disorderly arranged, showing a vacuolar structure. the number of sperm in the epididymis was significantly reduced (P=0.009, 0.004), while the sperm deformity rate was significantly increased (P=0.010, 0.000). Moreover, the content of ROS, iron ion and MDA in the testes increased significantly (P<0.05), and the mitochondrial membrane potential of mouse testicular cells decreased significantly (P<0.05). The expression of GPX4 proteins decreased (P<0.05). while the expression of COX2 increased significantly (P<0.01). Compared with high-dose group group, spermatogenic cells in ferroptosis inhibitor group were closely arranged and normal, and ROS and Fe contents in testicular tissue were significantly decreased (P<0.01) ; GPX4 protein expression was significantly increased while COX2 protein expression was significantly decreased (P<0.05) . Conclusion: Ferroptosis is involved in TDCIPP-induced testicular damage in male pubertal mice.

Tris(1,3-dichloro-2-propyl) phosphate disrupts the trajectory of cytosine methylation within developing zebrafish embryos.

Tris (1,3-dichloro-2-propyl) phosphate (TDCIPP) is an organophosphate ester-based flame retardant widely used within the United States. Within zebrafish, initiation of TDCIPP exposure at 0.75 h post-fertilization (hpf) reliably disrupts cytosine methylation from cleavage (2 hpf) through early-gastrulation (6 hpf). Therefore, the objective of this study was to determine whether TDCIPP-induced effects on cytosine methylation persist beyond 6 hpf. First, we exposed embryos to vehicle or TDCIPP from 0.75 hpf to 6, 24, or 48 hpf, and then conducted bisulfite amplicon sequencing of a target locus (lmo7b) using genomic DNA derived from whole embryos. Within both vehicle- and TDCIPP-treated embryos, CpG methylation was similar at 6 hpf and CHG/CHH methylation were similar at 24 and 48 hpf (relative to 6 hpf). However, relative to 6 hpf within the same treatment, CpG methylation was lower within vehicle-treated embryos at 48 hpf and TDCIPP-treated embryos at 24 and 48 hpf - an effect that was driven by acceleration of CpG hypomethylation. Similar to our previous findings with DNA methyltransferase, we found that, even at high µM concentrations, TDCIPP had no effect on zebrafish and human thymine DNA glycosylase activity (a key enzyme that decreases CpG methylation), suggesting that TDCIPP-induced effects on CpG methylation are not driven by direct interaction with thymine DNA glycosylase. Finally, using 5-methylcytosine (5-mC)-specific whole-mount immunochemistry and automated imaging, we found that exposure to TDCIPP increased 5-mC abundance within the yolk of blastula-stage embryos, suggesting that TDCIPP may impact cytosine methylation of maternally loaded mRNAs during the maternal-to-zygotic transition. Overall, our findings suggest that TDCIPP disrupts the trajectory of cytosine methylation during zebrafish embryogenesis, effects which do not appear to be driven by direct interaction of TDCIPP with key enzymes that regulate cytosine methylation.

Partial dust removal in vehicles does not mitigate human exposure to organophosphate esters.

Organophosphate esters (OPEs) have been detected within car interior dust, suggesting that the indoor microenvironment of vehicles may represent a potential route of human exposure to OPEs. We recently showed that people with longer commutes are exposed to higher concentrations of tris(1,3-dichloro-2-isopropyl)phosphate (TDCIPP) - a widely used OPE - and other studies have suggested that dust removal may lead to lower exposure to chemicals. Therefore, the overall objective of this study was to determine if a decrease in interior car dust results in mitigation of personal OPE exposure. Participants (N = 49) were asked to wear silicone wristbands, and a subset of them wiped interior parts at the front of their vehicles prior to one study week (N = 25) or both study weeks (N = 11). There were no significant differences in total OPE concentrations (77.79-13,660 ng/g) nor individual OPE concentrations (0.04-4852.81 ng/g) across the different wiping groups nor in relation to participant residence ZIP codes and AC/Heater usage. These findings suggest that higher exposure to TDCIPP for participants with longer commutes may be independent of dust located on interior parts at the front of the vehicle. Therefore, our study demonstrates that there is a need for research on the potential contribution of other sources of TDCIPP exposure within car interiors.

Toxicological effects of Tris (1,3-dichloro-2-propyl) phosphate exposure in adult male rats differ depending on the history of exposure in the neonatal period.

There is increasing concern about multiple high concentration exposure to toxins in disaster and emergency situations. However, conventional toxicology testing methods may not adequately address these situations. Thus, we assessed whether the toxic effects of exposure in the adulthood differ depending on the presence or absence of neonatal exposure to Tris (1,3-dichloro-2-propyl) phosphate (TDCIPP) in male rats to investigate the effects of exposure history of chemicals. In the neonatal stage [postnatal days (PNDs) 1-7], animals were treated with either sesame oil (5 ml/kg/day) as a control or TDCIPP (250 mg/kg/day) dissolved in sesame oil. In adulthood (PND 101-107), animals were treated with either sesame oil (5 ml/kg/day) or TDCIPP (650 mg/kg/day). One day after the final administration, dissection was performed, and body and organ weight, hematology, blood biochemistry, and histopathology were examined. The results demonstrated that the toxic effects of TDCIPP exposure in adulthood on adrenal gland size, serum iron content, and unsaturated iron binding capacity were enhanced by TDCIPP exposure in the neonatal stage. From these findings, it was indicated that the toxic effects of TDCIPP exposure in the adult stage are affected by pediatric exposure. These results suggest that the toxic effects of high-dose and long-term unsteady exposure to chemicals in large-scale disasters may change based on the exposure history of chemicals.

Comparison of the mechanisms of estrogen disrupting effects between triphenyl phosphate (TPhP) and tris(1,3-dichloro-2-propyl) phosphate (TDCIPP).

As the typical aryl-organophosphate flame retardants (OPFRs), triphenyl phosphate (TPhP) and tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) were reported to be estrogen disruptors. However, estrogen receptor α (ERα) binding experiments could not explain their biological effects. In this study, their action on ERα, G protein-coupled estrogen receptor (GPER) and the synthesis of 17ß-estradiol (E2) were investigated using in vitro assays and molecular docking. The results showed that TPhP acted as an ERα agonist and recruited steroid receptor co-activator 1 (SRC1) and 3 (SRC3), which was found for the first time. Unlike TPhP, TDCIPP acted as an ERα antagonist. However, both TPhP and TDCIPP activated the estrogen pathway by GPER in SKBR3 cells which were lack of ERα. Although molecular docking results revealed that both TPhP and TDCIPP could dock into ERα and GPER, their substituent groups and combination mode might affect the receptor activation. In addition, by using estrogen biosynthesis assay in H295R cells, both of TPhP and TDCIPP were found to promote E2 synthesis and E2/T ratio involving their different alteration on levels of progesterone, testosterone and estrone, and expression of various key genes. Our data proposed estrogen-disrupting mechanism frameworks of TPhP and TDCIPP. Moreover, our results will contribute to future construction of adverse outcome pathway (AOP) framework of endocrine disruptors.

Tris(1,3-dichloro-2-propyl)phosphate Induces Mass Mortality of Crucian Carp (Carassius carassius) Embryos in Taihu Lake.

Global fishery resources have been declining for decades, leading some fisheries to collapse. Although the decline is partly due to man-made chemical contamination, causal chemicals have been identified in only a few cases. We conducted consecutive 3-year investigations of embryonic mortality in Taihu Lake, China, including heavily contaminated northern areas, including Zhushan (ZS), Meiliang (ML), and Gonghu (GH), and the less polluted southeastern Suzhou (SZ). In 2016, 65.8% of crucian carp (Carassius carassius) embryos collected from ZS died before hatching, a substantially higher mortality rate than those observed in ML (21.7%), GH (15.2%), and SZ (2.2%). In 2017, the embryonic mortality rates were 38.8% in ZS, 1.3% in ML, 6.9% in GH, and 3.5% in SZ, and these rates strongly correlated with the concentrations of tris(1,3-dichloro-2-propyl)phosphate (TDCIPP): 104.2, 1.8, 4.6, and 4.1 ng/g lipid weight (lw) in embryos from ZS, ML, GH, and SZ, respectively. In 2018, embryonic mortality decreased to 4.0% in ZS and 1.2% in GH, consistent with decreases in embryonic TDCIPP concentrations to 17.1 and 1.5 ng/g lw, respectively. Moreover, the TDCIPP concentrations in dead embryos (70.5-216.8 ng/g lw) were much higher than those in live embryos (1.2-10.5 ng/g lw). Embryonic mortality was also observed in well-controlled laboratory experiments in which wild crucian carp were exposed to TDCIPP at concentrations similar to those measured in embryos collected from Taihu Lake, thus confirming TDCIPP as a causal factor in mass crucian carp embryo mortality in Taihu Lake. TDCIPP thus poses a threat to the sustainability of fisheries worldwide, given the high worldwide production volume of this chemical and its embryonic lethal toxicity.

Precision Biotransformation of Emerging Pollutants by Human Cytochrome P450 Using Computational-Experimental Synergy: A Case Study of Tris(1,3-dichloro-2-propyl) Phosphate.

Precision biotransformation is an envisioned strategy offering detailed insights into biotransformation pathways in real environmental settings using experimentally guided high-accuracy quantum chemistry. Emerging pollutants, whose metabolites are easily overlooked but may cause idiosyncratic toxicity, are important targets of such a strategy. We demonstrate here that complex metabolic reactions of tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) catalyzed by human CYP450 enzymes can be mapped via a three-step synergy strategy: (i) screening the possible metabolites via high-throughout (moderate-accuracy) computations; (ii) analyzing the proposed metabolites in vitro by human liver microsomes and recombinant human CYP450 enzymes; and (iii) rationalizing the experimental data via precise mechanisms using high-level targeted computations. Through the bilateral dialogues from qualitative to semi-quantitative to quantitative levels, we show how TDCIPP metabolism especially by CYP3A4 generates bis(1,3-dichloro-2-propyl) phosphate (BDCIPP) as an O-dealkylation metabolite and bis(1,3-dichloro-2-propyl) 3-chloro-1-hydroxy-2-propyl phosphate (alcoholß-dehalogen) as a dehalogenation/reduction metabolite via the initial rate-determining H-abstraction from αC- and ßC-positions. The relative yield ratio [dehalogenation/reduction]/[O-dealkylation] is derived from the relative barriers of H-abstraction at the ßC- and αC-positions by CYP3A4, estimated as 0.002 to 0.23, viz., an in vitro measured ratio of 0.04. Importantly, alcoholß-dehalogen formation points to a new mechanism involving successive oxidation and reduction functions of CYP450, with its precursor aldehydeß-dehalogen being a key intermediate detected by trapping assays and rationalized by computations. We conclude that the proposed three-step synergy strategy may meet the increasing challenge of elucidating biotransformation mechanisms of substantial synthesized organic compounds in the future.

Novel toxicity of tris(1,3-dichloro-2-propyl) phosphate in adult male rats.

The widespread use of tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) as a flame retardant has led to its release to the environment. Thus, the toxicological effects of TDCIPP on humans and animals are of importance. For better understanding of its potential toxicities, TDCIPP (250, 500, or 650 mg/kg/day) or vehicle control was administrated orally to adult male Wistar-Imamichi rats for 7 days. After the final administration of compounds, organ weights, histopathology, blood biochemistry, and hematology were examined. Hepatic toxicity was observed at doses ≥ 500 mg/kg/day of TDCIPP, and renal toxicity was observed at 650 mg/kg/day. The anti-androgenic activity of TDCIPP was previously confirmed in vitro and in vivo, but weights of epididymis, an androgen-dependent organ, were not affected by TDCIPP treatment in adults. Serum alkaline phosphatase activity was significantly decreased in all TDCIPP-treated rats independent of dose. Hemoglobin concentration, hematocrit, red blood cell count, and reticulocyte count were decreased in all TDCIPP-treated rats, but mean corpuscular volume, total iron-binding capacity, and serum iron were normal, suggesting that renal anemia was caused by TDCIPP. Together with previous reports on effects of anti-androgenic substances on red blood cell indices, anemia caused by TDCIPP could be due to its anti-androgenic activity. These considerations will contribute to further assessment of the toxicity of the compound.

Synergistic growth inhibition effect of TiO2 nanoparticles and tris(1,3-dichloro-2-propyl) phosphate on earthworms in soil.

The co-existence of organic pollutants and nanoparticles in the environment may lead to combined biological effects. The joint toxicity of pollutants and nanoparticles has been receiving increasing attention from researchers, but few studies have focused on soil biota due to the complexity of soil matrices. This study investigated the effects of tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) at 0, 5, and 25 mg/kg and nanoparticulate TiO2 (nTiO2) at 0, 500, and 2500 mg/kg in a 3 × 3 factorial arrangement of treatments for 28 days (d) on Eisenia fetida (earthworm). Compared with the control group (the 0 mg/kg TDCIPP + 0 mg/kg nTiO2 treatment), all other single (TDCIPP or nTiO2) and binary (TDCIPP + nTiO2) treatments except for the single 500 mg/kg nTiO2 treatment significantly reduced the weight gain rate of E. fetida. The binary treatments had significantly greater such effect than their corresponding single treatments, exhibiting a synergistic toxicity between TDCIPP and nTiO2 on the growth of E. fetida. Since TDCIPP and nTiO2 had no significant effect on their concentrations in the soil or in E. fetida during binary exposure, the synergistic toxicity could be a result of the superimposition of the toxicity pathways of TDCIPP and nTiO2. Transcriptomic analysis of E. fetida intestinal region revealed that exposure to 25 mg/kg TDCIPP or 2500 mg/kg nTiO2 affected nutrient-related or cell apoptosis and DNA damage related genes, respectively; their co-exposure greatly inhibited genes related to nutrient digestion and absorption, while causing abnormal transcription of genes related to the development and maintenance of E. fetida's muscles, leading to synergistic toxicity. These findings provide new insights into the environmental risks of organophosphorus flame retardants, nanoparticles, and their co-exposure.

Promotion effect of liver tumor progression in male kras transgenic zebrafish induced by tris (1, 3-dichloro-2-propyl) phosphate.

A previous study reported that exposure to tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) could promote the progression of hepatocellular carcinoma (HCC) in female HCC model zebrafish. Due to the existence of gender disparity in the development of HCC between females and males, whether the promotion effect of TDCIPP still exists in male HCC model zebrafish remains unclear. In this study, Tg(fabp10:rtTA2s-M2; TRE2:EGFP-krasG12V), referred as kras transgenic zebrafish which was shown to be an inducible liver tumor model, was applied as experimental model to assess the promotion potential of TDCIPP for HCC in males. In brief, kras males were exposed to 20 mg/L doxycycline (DOX), 0.3 mg/L TDCIPP and a binary mixture of 20 mg/L DOX with 0.3 mg/L TDCIPP, and after exposure liver size, histopathology and transcriptional profiles of liver from these treatments were examined. With the involvement of TDCIPP, the liver size was significantly increased and the lesion of hepatocyte became more aggressive. Furthermore, expressions of genes involved in DNA replication and inflammatory response were simultaneously up-regulated in the treatment of TDCIPP compared with the solvent control and in the treatment of the binary mixture of the two chemicals compared to the single DOX treatment. Overall, our results suggested that TDCIPP had promotion effect on the progression of liver tumor in kras males.

Thyroid disruption properties of three indoor dust chemicals tested in Silurana tropicalis tadpoles.

Indoor dust contains a multitude of industrial chemicals, and ingestion of dust is considered an important exposure route to organic contaminants. Some of these contaminants have been shown to interfere with the thyroid system, which may result in significant consequences on public health. The amphibian metamorphosis is a thyroid hormone-dependent process, which can be used as an in vivo model for studies on thyroid hormone-disrupting potency. Three contaminants of indoor dust were tested on metamorphosing Silurana (Xenopus) tropicalis tadpoles. The tested chemicals were Tris (1,3-dichloroisopropyl) phosphate (TDCiPP), tetrabromobisphenol-A (TBBPA) and propylparaben (PrP). Measurements reflecting general growth, development progress and thyroid epithelial cell height were performed on the exposed tadpoles as well as chemical analyses of the exposure water. It was shown that TDCiPP acts as a thyroid hormone-disrupting chemical in metamorphosing tadpoles by causing increased epithelial cell height in thyroid glands after exposure to a nominal concentration of 0.010 mg/L and in higher concentrations. TBBPA caused reductions in general growth of tadpoles at the nominal concentration 0.125 mg/L, and PrP caused acute toxicity at the nominal concentration 12.5 mg/L. However, no evident indications of specific thyroid-disrupting effects caused by TBBPA or PrP were observed.

Tris(1,3-dichloro-2-propyl)phosphate (TDCIPP) disrupts zebrafish tail fin development.

The flame retardant, tris(1,3-dichloro-2-propyl) phosphate (TDCIPP), is one of the most developmentally toxic organophosphate flame retardants (OPFRs). However, few mechanistic studies on phenotypic malformation caused by TDCIPP have been conducted. This study investigates the molecular mechanism underlying abnormal tail fin development consistently observed in zebrafish embryos exposed to TDCIPP. The results show that the defects in the tail fin (e.g., bent spine, defective caudal fin, and damaged tip) were associated with altered expression of transcription factors. The significant up-regulation of mmp9 and, among insulin-growth factor (IGF) families, igfbp-1a and igfbp1b was observed, whereas alterations in the expression of cdx4, igf1a, ifg1b, igf2b, and vegaa regulating tail development were dependent on time points. In accordance with changes in mRNA gene expression, TDCIPP impaired vessel formation and disorganized muscle in transgenic Tg(fli-GFP) zebrafish larvae. Furthermore, we found that the overexpression of mmp9 caused by TDCIPP was not linked to igfbp-1. Overall, these findings demonstrate that TDCIPP disrupts the progression of tail fin development, accompanied by defects in vessel and muscle formation in developing zebrafish embryos.

Optimization of experimental conditions and measurement of oxygen consumption rate (OCR) in zebrafish embryos exposed to organophosphate flame retardants (OPFRs).

The measurement of oxygen consumption rate (OCR) provides a comprehensive understanding of mitochondrial metabolism. However, no study has been conducted to investigate the mitochondrial dysfunction caused by organophosphate flame retardants (OPFRs). The objectives of this study were to optimize the experimental conditions to measure OCR in zebrafish embryos using the Seahorse XFe 24 Extracellular Flux Analyzer, and to investigate the changes of OCR in zebrafish embryos exposed to OPFRs. We first optimized the experimental conditions such as the number of embryos, concentrations of inhibitors, and time points. We determined the factors, i.e., three embryos, 12.5 µM of oligomycin, 8 µM of carbonyl cyanaide 4-(trifluoromethoxy) phenylhydrazone (FCCP), and 24 hpf (hours post-fertilization) time point, for obtaining the typical pattern of OCR in dechorinated zebrafish embryos. After confirming the determinants upon exposure of triclosan, the inhibition of OCR was measured in zebrafish embryos exposed to two major OPFRs, triphenyl phosphate (TPHP) and tris (1,3-dichloro-2-propyl) phosphate (TDCIPP). We found that significant inhibition of OCR was observed in basal respiration for TPHP, and in basal and maximal respiration for TDCIPP exposure, respectively. We suggest the optimum conditions of the Seahorse XFe 24 analyzer to better evaluate OCR in zebrafish embryos, and demonstrate the potential of TPHP and TDCIPP to cause the disruption of energy metabolism associated with mitochondrial dysfunction.

Early-life exposure to Tris(1,3-dichloroisopropyl) phosphate induces dose-dependent suppression of sexual behavior in male rats.

Exposure to endocrine-disrupting chemicals may adversely affect animals, particularly during development. Tris(1,3-dichloroisopropyl) phosphate (TDCIPP) is an organophosphate with anti-androgen function in vitro that is present in indoor dust at relatively high concentrations. In male rats, androgens are necessary for the development of reproductive organs, as well as the endocrine and central nervous systems. However, we currently do not know the exact effects of TDCIPP exposure through suckling on subsequent reproductive behavior in males. Here, we show that TDCIPP exposure (25-250 mg kg-1 via oral administration over 28 consecutive days post-birth) suppressed male sexual behavior and reduced testes size. These changes were dose-dependent and appeared first in adults rather than in juveniles. These results demonstrate that TDCIPP exposure led to normal body growth and appearance in juveniles, but disrupted the endocrine system and physiology in adults. Therefore, assays should be performed using adult animals to ensure accuracy, and to confirm the influence of chemical substances given during early mammalian life.

Altered Adipogenesis in Zebrafish Larvae Following High Fat Diet and Chemical Exposure Is Visualised by Stimulated Raman Scattering Microscopy.

Early life stage exposure to environmental chemicals may play a role in obesity by altering adipogenesis; however, robust in vivo methods to quantify these effects are lacking. The goal of this study was to analyze the effects of developmental exposure to chemicals on adipogenesis in the zebrafish (Danio rerio). We used label-free Stimulated Raman Scattering (SRS) microscopy for the first time to image zebrafish adipogenesis at 15 days post fertilization (dpf) and compared standard feed conditions (StF) to a high fat diet (HFD) or high glucose diet (HGD). We also exposed zebrafish embryos to a non-toxic concentration of tributyltin (TBT, 1 nM) or Tris(1,3-dichloroisopropyl)phosphate (TDCiPP, 0.5 µM) from 0-6 dpf and reared larvae to 15 dpf under StF. Potential molecular mechanisms of altered adipogenesis were examined by qPCR. Diet-dependent modulation of adipogenesis was observed, with HFD resulting in a threefold increase in larvae with adipocytes, compared to StF and HGD. Developmental exposure to TBT but not TDCiPP significantly increased adipocyte differentiation. The expression of adipogenic genes such as pparda, lxr and lepa was altered in response to HFD or chemicals. This study shows that SRS microscopy can be successfully applied to zebrafish to visualize and quantify adipogenesis, and is a powerful approach for identifying obesogenic chemicals in vivo.

Inhibition of autophagosome-lysosome fusion contributes to TDCIPP-induced Aß1-42 production in N2a-APPswe cells.

Alzheimer's disease is the most common form of dementia and is characterized by cognitive impairment. The disruption of autophagosome-lysosome function has been linked to the pathogenesis of Alzheimer's disease. Tris (1,3-dichloro-2-propyl) phosphate (TDCIPP) is a widely used organophosphorus flame retardant that has the potential to cause neuronal damage. We found that TDCIPP significantly increased the expression of ß-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1), presenilin-1 (PS1) and Aß42. Proteomic studies with TMT labeling revealed changes in the profiles of N2a-APPswe cells after exposure to TDCIPP. Proteomic and bioinformatics analyses revealed that lysosomal proteins were dysregulated in N2a-APPswe cells after treatment with TDCIPP. The LC3, P62, CTSD, and LAMP1 levels were increased after TDCIPP exposure, and dysregulated protein expression was validated by Western blotting. The exposure to TDCIPP led to the accumulation of autophagosomes, and this phenomenon was enhanced in the presence of chloroquine (CQ). Our results revealed for the first time that TDCIPP could be a potential environmental risk factor for AD development. The inhibition of autophagosome-lysosome fusion may have a significant impact on the generation of Aß1-42 in response to TDCIPP.