Tris(1,3-dichloro-2-propyl) Phosphate Exposure Research Articles

Assessments of the effects of tris(1,3-dichloro-2-propyl) phosphate (TDCPP) on human intestinal health from the aspects of intestinal flora changes and cytotoxicity to human cells

Organophosphorus flame retardants (OPFRs) are now drawing the public's attention due to their potential toxicity. Given that contaminated food may result in the ingestion of OPFRs to the human intestine, further investigation is required to determine the potential adverse effects of these compounds on human intestinal health. The present study aimed to comprehensively assess the effect of tris(1,3-dichloro-2-propyl) phosphate (TDCPP), a typical OPFR, on human intestinal health by evaluating both intestinal flora and human cell Caco-2. Based on the results, TDCPP exposure altered the composition of intestinal flora and increased the proportion of pathogenic bacteria. PICRUSt2 analysis revealed that certain pathways were affected by TDCPP, and the resulting metabolic disorders might cause health problems. Orthologous genes of glutathione S-transferase and multidrug efflux system were up-regulated, demonstrating that the bacteria resisted TDCPP to maintain their vitality. Compared to the other two OPFRs, TDCPP induced greater cytotoxicity, and the results were consistent with the dose-effect relationship. Three OPFRs, especially TDCPP, caused the release of lactate dehydrogenase, accumulation of ROS, decline in mitochondrial membrane potential and increase in intracellular Ca2+, which could consequently induce cell death. The simultaneous effects of TDCPP on both intestinal cells and intestinal flora are likely to engender more severe intestinal health issues.

Pyrroloquinoline quinone promotes growth and intestinal health and alleviates tris(1,3-dichloro-2-propyl) phosphate-induced hepatic oxidative stress in zebrafish

Pyrroloquinoline quinone (PQQ) is a newly discovered redox coenzyme possessing multiple biological activities, whose role in nutritional and physiological regulation in fish remains elusive. In this study, we explored how the dietary PQQ influenced the growth, intestinal health and oxidative stress of liver triggered by tris(1,3-dichloro-2-propyl) phosphate (TDCPP) in zebrafish, which was accomplished through a long-term feeding trial (experiment 1) and a short-term stress trial (experiment 2). In experiment 1, two-month-old zebrafish were fed experimental diets either involving 0.4 mg/kg (low PQQ, LP) or 1.6 mg/kg PQQ·Na2 (high PQQ, HP), or a basal diet (CNT) for four weeks. It showed that compared to the zebrafish in the CNT group, those in the LP and HP groups exhibited remarkably improved growth performance, whose feed conversion ratio was lower, while specific growth rate and weight gain rate higher. No significant intestinal pathological symptoms were found among groups, albeit the prominently increased villus height of zebrafish in LP and HP groups than the CNT zebrafish. Dietary PQQ led to great phylum- and genus-level modulation of intestinal microflora and made it more balanced. In experiment 2, four-month-old adult zebrafish were fed a basal diet (control group), a basal diet under TDCPP exposure (TDCPP group), a HP diet under TDCPP exposure (TDCPP+PQQ group), or a HP diet (PQQ group) for one week. After the stress trial, significant intrahepatic pathological symptoms were found in the TDCPP group, while TDCPP+PQQ and PQQ groups exhibited no significant stress injury. Further analysis implied that dietary PQQ significantly alleviated the TDCPP-triggered intrahepatic oxidative stress of zebrafish by elevating the antioxidation-related gene levels, potentiating the antioxidant enzyme activities and maintaining appropriate level of apoptosis. Taken together, this study suggested that PQQ could improve growth performance and intestinal health and alleviate the TDCPP-induced liver oxidative stress in zebrafish.

Tris(1,3-dichloro-2-propyl) phosphate is a metabolism-disrupting chemical in male mice

Tris(1,3-dichloro-2-propyl) phosphate (TDCPP) is an organophosphate flame retardant. The primary TDCPP metabolite, bis(1,3-dichloro-2-propyl) phosphate (BDCPP), is detectable in the urine of over 90 % of Americans. Epidemiological studies show sex-specific associations between urinary BDCPP levels and metabolic syndrome, which is an established risk factor for type 2 diabetes, heart disease, and stroke. We used a mouse model to determine whether TDCPP exposure disrupts glucose homeostasis. Six-week old male and female C57BL/6J mice were given ad libitum access to diets containing vehicle (0.1 % DMSO) and TDCPP resulting in the following treatment groups: 0 mg/kg/day, 0.02 mg/kg/day, 1 mg/kg/day, or 100 mg/kg/day. After being on the experimental diet for five weeks without interruption, body composition was analyzed, glucose and insulin tolerance tests were performed, and fasting glucose and insulin levels were quantified. TDCPP at 100 mg/kg/day caused male sex-specific adiposity, fasting hyperglycemia, and insulin resistance. TDCPP-induced modulation of nuclear receptor activation was investigated using an in vitro screen to identify potential mechanisms of metabolic disruption. TDCPP activated farnesoid X receptor (FXR) and pregnane X receptor (PXR), and inhibited the androgen receptor (AR). PXR target genes, but not FXR target genes, were upregulated in livers from mice exposed to 100 mg TDCPP/kg/day. Interestingly, PXR target genes were differentially expressed in livers from both males and females. It remains to be determined whether TDCPP-induced metabolic disruption occurs via modulation of nuclear receptor activity. Taken together, these studies build upon the association of TDCPP exposure and metabolic syndrome in humans by identifying sex-specific effects of TDCPP on glucose homeostasis in mice.

Organophosphorus flame retardant TDCPP induces neurotoxicity via mitophagy-related ferroptosis in vivo and in vitro

Tris (1,3-dichloro-2-propyl) phosphate (TDCPP) has neurotoxicity, but its mechanism remains unclear. Evidence recently showed that ferroptosis might be associated with TDCPP-induced neurotoxicity. To explore the role and underlying mechanism of ferroptosis in TDCPP-induced neurotoxicity, the occurrence of ferroptosis was examined in mice and PC12 cells upon TDCPP exposure. The mechanism of TDCPP-induced ferroptosis was clarified in vitro combined with the RNA sequencing assay. The in vivo results showed that orally TDCPP exposure (100 mg/kg, 30 d) inhibited the learning and memory ability of mice, reduced hippocampus neurons, induced malondialdehyde (MDA) accumulation, and decreased glutathione (GSH) and superoxide dismutase (SOD) levels in the hippocampus. Moreover, TDCPP exposure (100 mg/kg, 30 d) altered the ferroptosis and autophagy-related protein abundances in the hippocampus. The in vitro results showed that TDCPP exposure (0, 5, 20, 50, 100, and 200 μM) for 24 h induced dose-dependent cell death in PC12 cells, and the cell death was ameliorated by the co-treatment with ferrostatin-1 (1 μM, 24 h). Similarly, TDCPP exposure (0, 50, 100, and 200 μM) for 24 h increased the levels of MDA and LPO, but decreased the reduced GSH in PC12 cells. Furthermore, TDCPP exposure (0, 50, 100, and 200 μM) for 24 h altered the ferroptosis and autophagy-related protein abundances in PC12 cells. The RNA-sequencing revealed that TDCPP exposure (100 μM, 24 h) induced mitophagy activation in SH-SY5Y cells. Meanwhile, the in vitro experiments confirmed that TDCPP exposure (0, 50, 100, and 200 μM) for 24 h increased abundances of mitophagy-related protein phosphatase and tensin homolog induced kinase 1(PINK1), Parkinson protein 2 E3 ubiquitin-protein ligase (PARKIN), inositol 1,4,5-trisphosphate receptor type 1 (IP3R1), and voltage-dependent anion channel 1 (VDAC1) in PC12 cells. Moreover, TDCPP treatment (100 μM, 24 h) increased the mitochondrial recruitment of PARKIN, decreased the mitochondrial membrane potential (MMP) level, and increased the Fe2+ level in mitochondria. In addition, decreased ATP levels and increased reactive oxygen species (ROS) levels were observed in PC12 cells upon TDCPP exposure (0, 50, 100, and 200 μM) for 24 h. In summary, ferroptosis was associated with TDCPP-induced neurotoxicity, and the mechanism might be related to PINK1/PARKIN-mediated mitophagy initiated by mitochondrial damage.

Organelle specific fluorescent phenomics and transcriptomic profiling to evaluate cellular response to tris(1,3 dichloro 2 propyl)phosphate

Tris(1,3-dichloro-2-propyl)phosphate (TDCPP) has been suspected to cause toxicity invertebrates, but its phenotypic effects and the underlying regulatory mechanism have not been fully revealed. Generally, cellular responses tightly control and affect various phenotypes. The scope of the whole organism or cellular toxicological phenotyping, however, has been limited, and quantitative analysis methods using phenotype data have not been fully established. Here, we demonstrated that fluorescence imaging of sub-organelle-based phenomic analysis together with transcriptomic profiling can enable a comprehensive understanding of correlations between molecular and phenomic events. To reveal the cellular response to TDCPP exposure, we obtained three sub-organelle images as fluorescent phenotypes. Transcriptomic perturbation data were measured from the RNA-seq experiment, and both profiling results were analyzed together. Interestingly, organelle phenomic data showed a unique fluorescent intensity increase in the endoplasmic reticulum (ER), and pathway analysis using transcriptomic data also revealed that ER was significantly enriched in gene ontology terms. Following the series of analyses, RNA-seq data also revealed potential carcinogenic effects of TDCPP. Our multi-dimensional profiling approach for organophosphate chemicals can uniquely correlate phenotypic changes with transcriptomic perturbations.

Organophosphorus Flame Retardant TDCPP Displays Genotoxic and Carcinogenic Risks in Human Liver Cells.

Tris(1,3-Dichloro-2-propyl)phosphate (TDCPP) is an organophosphorus flame retardant (OPFR) widely used in a variety of consumer products (plastics, furniture, paints, foams, and electronics). Scientific evidence has affirmed the toxicological effects of TDCPP in in vitro and in vivo test models; however, its genotoxicity and carcinogenic effects in human cells are still obscure. Herein, we present genotoxic and carcinogenic properties of TDCPP in human liver cells (HepG2). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and neutral red uptake (NRU) assays demonstrated survival reduction in HepG2 cells after 3 days of exposure at higher concentrations (100–400 μM) of TDCPP. Comet assay and flow cytometric cell cycle experiments showed DNA damage and apoptosis in HepG2 cells after 3 days of TDCPP exposure. TDCPP treatment incremented the intracellular reactive oxygen species (ROS), nitric oxide (NO), Ca2+ influx, and esterase level in exposed cells. HepG2 mitochondrial membrane potential (ΔΨm) significantly declined and cytoplasmic localization of P53, caspase 3, and caspase 9 increased after TDCPP exposure. qPCR array quantification of the human cancer pathway revealed the upregulation of 11 genes and downregulation of two genes in TDCPP-exposed HepG2 cells. Overall, this is the first study to explicitly validate the fact that TDCPP bears the genotoxic, hepatotoxic, and carcinogenic potential, which may jeopardize human health.

Tris(1,3-dichloro-2-propyl) phosphate reduces longevity through a specific microRNA-mediated DAF-16/FoxO in an unconventional insulin/insulin-like growth factor‑1 signaling pathway

Tris(1,3-dichloro-2-propyl) phosphate (TDCPP) has received concerns due to its frequent detection in environmental media and biological samples. Our previous study has indicated TDCPP reduced the lifespan of Caenorhabditis elegans (C. elegans) by triggering an unconventional insulin/insulin-like growth factor signaling (IIS) pathway. This study continued to investigate the possible deleterious effects of TDCPP relating to longevity regulation signal pathways and biological processes. Specifically, this study uniquely performed small RNA transcriptome sequencing (RNA-seq), focusing on the underlying mechanisms of TDCPP-reduced the longevity of C. elegans in-depth in microRNAs (miRNAs). Based on Small RNA-seq results and transcript levels of mRNA involved in the unconventional IIS pathway, a small interaction network of miRNAs-mRNAs following TDCPP exposure in C. elegans was preliminarily established. Among them, up-regulated miR-48 and miR-84 (let-7 family members) silence the mRNA of daf-16 (the crucial member of the FoxO family and pivotal regulator in longevity) via post-transcription and translation dampening abilities, further inhibit its downstream target metallothionein-1 (mtl-1), and ultimately contributed to the reduction of nematode longevity and locomotion behaviors. Meanwhile, the high binding affinities of TDCPP with miRNAs cel-miR-48–5p and cel-miR-84–5p strongly support their participation in the regulation of nematode mobility and longevity. These findings provide a comprehensive analysis of TDCPP-reduced longevity from the perspective of miRNAs.

Tris(1,3‐Dichloro‐2‐Propyl)Phosphate Is an Endocrine Disrupting Compound Causing Sex‐Specific Changes in Body Composition and Insulin Sensitivity

Organophosphate flame retardants are used on polyurethane foams and off-gassing leads to ubiquitous environmental exposure. Tris(1,3-dichloro-2-propyl)phosphate (TDCPP) is one of several organophosphate flame retardants, and its use and accumulation in indoor spaces has been steadily increasing over the past decade. However, the impact of TDCPP exposure on human health is unknown, especially as it relates to metabolic disorders. The objective of the present study, therefore, was to test whether TDCPP exposure causes metabolic disruption. Beginning at 6 weeks of age, male and female C57BL/6 mice (N = 4-10 per group) were given ad libitum access to purified diets with either vehicle (0.1% w/w DMSO) or one of three doses of TDCPP (Low, 0.02 mg/kg/day; Medium, 1 mg/kg/day; High, 50 mg/kg/day). After 5 weeks of dietary exposure, body weights were comparable between groups, yet, body composition as determined by quantitative magnetic resonance revealed dose-dependent increase in percent body fat and decrease in percent lean mass in male mice. Intraperitoneal glucose tolerance testing showed no effect of TDCPP exposure. However, insulin tolerance tests showed severe insulin resistance in the High dose males while females were unaffected. In vitro reporter assays were used to determine TDCPP (1 nM, 0.1 µM, and 100 µM) agonist activity on 26 nuclear receptors. Reporter cells expressed either native receptor or receptor hybrids in which Gal4 DNA binding domain replaced native DNA binding domains. Firefly luciferase was functionally linked downstream of receptor-specific response elements or Gal4 upstream activating sequence. Of note, TDCPP did not affect cell viability at any of the concentrations tested. Compared to DMSO vehicle control, the 100 µM TDCPP caused activation of both human and mouse pregnane X receptors (16-fold increase) and farnesoid X receptor (2.5-fold increase). Interestingly, the 100 µM TDCPP also caused a 60% reduction in estrogen receptor alpha and vitamin D receptor luminescence. Taken together, these studies show TDCPP is an endocrine disrupting compound that causes sex-specific body composition maladaptations and insulin resistance. Future studies will determine whether these nuclear receptors are mediating metabolic disruption in vivo.

Proteomic analysis and biochemical alterations in marine mussel gills after exposure to the organophosphate flame retardant TDCPP

Organophosphate flame retardants (OPFRs) are (re-)emergent environmental pollutants increasingly being used because of the restriction of other flame retardants. The chlorinated OPFR, tris(1,3-dichloro-2-propyl) phosphate (TDCPP) is among those of highest environmental concern, but its potential effects in the marine environment have rarely been investigated. We exposed a widely used sentinel marine mussel species, Mytilus galloprovincialis, to 10 μg L−1 of TDCPP during 28 days and studied: (i) the kinetics of bioaccumulation and elimination of the compound, (ii) the effect on two molecular biomarkers, glutathione S-transferase (GST) and acetylcholinesterase (AChE) activities, and (iii) proteomic alterations in the gills, following an isobaric labeling quantitative shotgun proteomic approach, at two exposure times (7 and 28 days). Uptake and elimination of TDCPP by mussels were very fast, and the bioconcentration factor of this compound in mussels was 147 L kgww-1, confirming that this compound is not very bioaccumulative, as predicted by its chemical properties. GST activity was not affected by TDCPP exposure, but AChE activity was inhibited by TDCPP at both 7 and 28 days of exposure. Proteomic analysis revealed subtle effects of TDCPP in mussel gills, since few proteins (less than 2 % of the analysed proteome) were significantly affected by TDCPP, and effect sizes were low. The most relevant effects detected were the up-regulation of epimerase family protein SDR39U1, an enzyme that could be involved in detoxification processes, at both exposure times, and the down-regulation of receptor-type tyrosine-protein phosphatase N2-like (PTPRN2) after 7 days of exposure, which is involved in neurotransmitter secretion and might be related to the neurotoxicity described for this compound. Exposure time rather than TDCPP exposure was the most important driver of protein abundance changes, with 33 % of the proteome being affected by this factor, suggesting that stress caused by laboratory conditions could be an important confounding factor that needs to be controlled in similar ecotoxicology studies. Proteomic data are available via ProteomeXchange with identifier PXD019720.

Tris(1,3-dichloro-2-propyl)phosphate Reduces the Lifespan via Activation of an Unconventional Insulin/Insulin-Like Growth Factor-1 Signaling Pathway.

Tris(1,3-dichloro-2-propyl)phosphate (TDCPP) is an environmental contaminant that has attracted increasing concern due to its presence in environmental media and biological samples. Our previous study demonstrated that exposure to TDCPP reduced the lifespan of Caenorhabditis elegans, but the mechanisms, including the relevant signaling pathways, are unclear. The current study found that TDCPP exposure triggers an unconventional insulin/insulin-like growth factor signaling (IIS) pathway, not by disrupting the insulin-like growth factor-1 receptor DAF-2/IGF1R but by inhibiting the downstream tumor-suppressor factor DAF-18/PTEN. This inhibition reduces PI(3,4,5)P3 (PIP3) dephosphorylation, causing buildup that increases the activation of the Akt/Protein Kinase B (PKB) family of serine/threonine kinases. This activation induces DAF-16/FoxO phosphorylation and promotes the sequestration of DAF-16/FoxO in the cytoplasm, reducing the lifespan of nematodes. Our results have important diagnostic and therapeutic implications for controlling TDCPP-related diseases, especially those originating with IIS pathway components.

Neonatal exposure to organophosphorus flame retardant TDCPP elicits neurotoxicity in mouse hippocampus via microglia-mediated inflammation in vivo and in vitro.

Tris(1,3-dichloro-2-propyl) phosphate (TDCPP) is a phosphorus-based flame retardant common in consumer goods and baby products. Concerns have been raised about TDCPP exposure and neurodevelopmental toxicity. However, the mechanism and early response for TDCPP-induced neurotoxicity are poorly understood. This study investigates the role of microglia-mediated neuroinflammation in TDCPP-induced neurotoxicity in mice and primary cells. TDCPP was administered to C57BL/6 pups (0, 5, or 50mg/kg/day) via an oral gavage from postnatal days 10-38 (28days). The results showed that TDCPP exposure for 28days altered the gene expression of neuronal markers Tubb3, Nefh, and Nes, and led to apoptosis in the hippocampus. The mRNA levels of pro-inflammatory factors Il-1β, Tnfα and Ccl2 dose dependently increased in the hippocampus at both 24h and 28days following exposure, accompanied by microglia activation characterized by an amoeboid-like phenotype. In in vitro studies using the primary microglia isolated from neonatal mice, exposure to TDCPP (0-100μM) for 24h resulted in cellular activation. It also increased the expression of genes responsible for inflammatory responses including surface markers and pro-inflammatory cytokines. These changes occurred in a dose-dependent fashion. Neurite outgrowth of primary mouse hippocampal neurons was inhibited by treatment with the conditioned medium harvested from microglia exposed to TDCPP. These results reveal that neonatal exposure to TDCPP induces neuronal damage through microglia-mediated inflammation. This provides insight into the mechanism of TDCPP's neurodevelopmental toxicity, and suggests that microglial cell is a sensitive responder for OPFRs exposure.

Metabolomics reveals that tris(1,3-dichloro-2-propyl)phosphate (TDCPP) causes disruption of membrane lipids in microalga Scenedesmus obliquus

Tris(1,3-dichloro-2-propyl)phosphate (TDCPP) is one of the most widely used organophosphate ester flame retardants. The presence of TDCPP in surface waters and aquatic organisms have been reported worldwide, yet the ecological risk of TDCPP on microalgae is rarely studied. We investigated the biotransformation of TDCPP and its toxicity on the microalga Scenedesmus obliquus using an untargeted metabolomics approach. Exposure to TDCPP resulted in a dose-response decrease of micoalgal biomass. In the presence of microalgae, TDCPP concentration in the media decreased by 25.3–40.6% after 5 days. TDCPP metabolites were identified in the media including hydrolysis and hydroxyl-substituted dechlorination products. A dose-response separation of metabolic profiles of microalgae was observed, with effect seen at the lowest concentration of 10 µg/L tested, which is slightly higher than environmentally relevant concentrations. Differentiated metabolites identified include 52 lipids and 6 polar metabolites. Analysis of altered lipid pathways suggests that microalgal cells reinforce thylakoid membranes (function to protect photosynthesis) by compromising the integrity of plasma membrane (function to protect cellular substances) and extraplastidial cellular membranes. Changes in the polar metabolites might indicate osmotic stress and improved NO signaling after TDCPP exposure. Consistent with perturbation of membrane lipids, further experiment confirmed that exposure to 10 mg/L TDCPP resulted in significant (p < 0.01) plasma membrane damage. This study indicates biotransformation and the membrane damage toxicity mechanism of TDCPP on S. obliquus, demonstrating the usefulness of metabolomics for the toxicity mechanism elucidation of emerging pollutants.

Tris(1,3-dichloro-2-propyl) phosphate accelerated the aging process induced by the 4-hydroxynon-2-enal response to reactive oxidative species in Caenorhabditis elegans

Tris(1,3-dichloro-2-propyl) phosphate (TDCPP) has been frequently detected in environmental media and biological samples. However, knowledge of its adverse health consequences is limited. In the current study, Caenorhabditis elegans (C. elegans, L1 larvae) were exposed to TDCPP at environmentally relevant concentrations (control, 0.1, 1, 100 and 1000 μg L−1) for 72 h to explore any association between TDCPP and the aging process. Some of the degenerative age-related indicators were observed, including locomotion behaviors and lifespan. As crucial biomarkers of aging, the accumulation of lipofuscin, and lipid peroxidation (LPO) products exemplified by 4-hydroxynon-2-enal (4-HNE) were detected. This product forms as a result of oxidative stress, as confirmed by an N-acetyl-L-cysteine (NAC) pharmacological assay. Moreover, a significant increase in reactive oxide species (ROS) production in a dose-dependent manner using a fluorescent probe was observed. For the underlying molecular mechanism of the above aging phenotypes, significantly upregulated transcription of genes related to antioxidant systems, especially a subset of glutathione S-transferase (gst-5, gst-6, gst-9, gst-10, gst-19, gst-24, gst-26, gst-29, gst-33, and gst-38), was found by RNA-Seq and further confirmed by RT-qPCR. The elevated glutathione S-transferase (GST) was attributed to the significant increase in 4-HNE because mutations in gst-5 and gst-24 inhibited the conjugation of GSTs with 4-HNE. Therefore, GST play an indispensable role in the detoxification process of TDCPP exposure and further confirmed LPO accumulation at the molecular mechanism level. In conclusion, TDCPP accelerated the aging process induced by the LPO products, 4-HNE, response to reactive oxidative species in C. elegans.

Effects of tris(1,3-dichloro-2-propyl) phosphate (TDCPP) and triphenyl phosphate (TPP) on sex-dependent alterations of thyroid hormones in adult zebrafish

Organophosphate flame retardants (OPFRs) have been widely used as alternatives to polybrominated diphenyl ethers for fire prevention. OPFRs are suspected of causing potential thyroid disruption in humans. In fish, their thyroid hormone modulation is reported but the mechanisms of this modulation are less understood. Thyroid-disturbing effects of OPFRs were evaluated using adult zebrafish (Danio rerio) following 14d exposure to tris(1,3-dichloro-2-propyl) phosphate (TDCPP) or triphenyl phosphate (TPP). Plasma concentrations of thyroid hormones were measured and transcriptions of several genes involved in thyroid function were quantified in brain, thyroid, and liver. Exposure to TDCPP or TPP led to significant decreases in plasma triiodothyronine (T3) and thyroxine (T4) concentrations in the male fish, while the increases were observed in the female fish. Exposure to the OPFRs also altered the transcription of regulatory genes and receptors in hypothalamus, pituitary, and thyroid of the fish in sex-dependent manner. In the male fish, transcriptions of corticotropin-releasing hormone (crh) and thyroid-stimulating hormone (tsh) in the brain were significantly up-regulated, probably as a compensation for hypothyroidism, but thyroglobulin (tg) and deiodinase 2 (dio2) were down-regulated in thyroid or liver. In contrast, in the females, transcriptions of crh and tsh genes were significantly down-regulated. These observations show that TDCPP and TPP exposure can lead to sex-dependent disruptions of thyroid hormone balances in the adult zebrafish through alterations of the hypothalamus-pituitary-thyroid (HPT) axis.

Oxidative stress, cell cycle arrest, DNA damage and apoptosis in adult zebrafish (Danio rerio) induced by tris(1,3-dichloro-2-propyl) phosphate

Tris(1,3-dichloro-2-propyl)phosphate (TDCPP) is an additive flame retardant of high production volume, and frequently detected in biota and environment. However, knowledge on its potential risk and toxicological mechanism still remains limited. In this study, DNA damage, transcriptomic responses and biochemical changes in the liver of zebrafish (Danio rerio) induced by TDCPP were investigated. Zebrafish was exposed to 45.81μg/L (1/100 (96h-LC50)) and 229.05μg/L (1/20 (96h-LC50)) TDCPP for 7 d. The reactive oxygen species (ROS) and GSH contents, in addition to antioxidant enzyme activities in the liver changed significantly, and the mRNA levels of genes related to oxidative stress were alerted in a dose-dependent and/or sex-dependent manner after exposure to TDCPP. Significant DNA damage in zebrafish liver was found, and olive tail moment increased in a concentration-dependent manner. Moreover, exposure of TDCPP at 45.81μg/L level activated the cell cycle arrest, DNA repair system and apoptosis pathway in male zebrafish, and 229.05μg/L TDCPP exposure inhibited those pathways in both male and female zebrafish. The cell apoptosis was confirmed in TUNEL assay as higher incidence of TUNEL-positive cells were observed in zebrafish exposed to 229.05μg/L TDCPP. Our results also indicated that males were more sensitive to TDCPP exposure compared with females. Taken together, our results showed that TDCPP could induce oxidative stress, cell cycle arrest, DNA damage and apoptosis in adult zebrafish liver in sex- and concentration-dependent manners.

Effects of organophosphorus flame retardant TDCPP on normal human corneal epithelial cells: Implications for human health

Tris(1,3-dichloro-2-propyl) phosphate (TDCPP) is one of the most detected organophosphorus flame retardants (OPFRs) in the environment, especially in indoor dust. Continuous daily exposure to TDCPP-containing dust may adversely impact human cornea. However, its detrimental effects on human corneal epithelium are largely unknown. In this study, we investigated the cell apoptosis in normal human corneal epithelial cells (HCECs) after TDCPP exposure and elucidated the underlying molecular mechanisms. Our data indicated a dose-dependent decrease of cell viability after TDCPP exposure with LC50 at 202 μg/mL. A concentration-dependent apoptotic sign was observed in HCECs after exposing to ≥2 μg/mL TDCPP. Endoplasmic reticulum stress induction was evidenced by up-regulation of its biomarker genes (ATF-4, CHOP, BiP, and XBP1). Furthermore, alternation of Bcl-2/Bax expression, mitochondrial membrane potential loss, cellular ATP content decrease, and caspase-3 and -9 activity increase were observed after exposing to 2 or 20 μg/mL TDCPP. Taken together, the data implicated the involvement of endoplasmic reticulum stress in TDCPP-induced HCEC apoptosis, probably mediated by mitochondrial apoptotic pathway. Our findings showed TDCPP exposure induced toxicity to human cornea. Due to TDCPP's presence at high levels in indoor dust, further study is warranted to evaluate its health risk on human corneas.

Developmental exposure to the organophosphorus flame retardant tris(1,3-dichloro-2-propyl) phosphate: Estrogenic activity, endocrine disruption and reproductive effects on zebrafish

Tris(1,3-dichloro-2-propyl) phosphate (TDCPP) is an organophosphate flame retardant that is detectable in the environment and biota, prompting concern over its risk to wildlife and human health. Our objective was to investigate whether long-term exposure to low concentrations of TDCPP can affect fish reproduction. Zebrafish embryos were exposed to low concentrations (0, 4, 20 and 100μg/L) of TDCPP from 2h post-fertilization until sexual maturation. Exposure to TDCPP significantly increased plasma estradiol and testosterone levels in females, but had no effect in males. TDCPP exposure also caused a significant reduction in fecundity as indicated by decreased egg production. Real-time PCR was performed to examine selected genes in the hypothalamic–pituitary–gonadal (HPG) axis and liver. Principle component analysis (PCA) showed that sex hormone levels and fecundity were related to the mRNA level of several genes in the HPG axis. Furthermore, hepatic vitellogenin (vtg1 and vtg3) expression was upregulated in both females and males, suggesting TDCPP has estrogenic activity. Histological examination revealed promotion of oocyte maturation in the females, but retardation of spermiation in males. Reduced egg quality (e.g., egg diameter) and increased malformation rates were observed in the F1 generation. Chemical analysis showed significant levels of TDCPP and its metabolite bis(1,3-dichloro-2-propyl) phosphate in the gonads of males and females. In conclusion, long-term exposure to low concentrations of TDCPP impairs fish reproduction.

In Ovo Effects of Two Organophosphate Flame Retardants—TCPP and TDCPP—on Pipping Success, Development, mRNA Expression, and Thyroid Hormone Levels in Chicken Embryos

Tris(1-chloro-2-propyl) phosphate (TCPP) and tris(1,3-dichloro-2-propyl) phosphate (TDCPP) are organic flame retardants detected in the environment and biota for which avian toxicological data are limited. In this study, domestic chicken eggs were injected with TCPP or TDCPP (maximum dose = 51,600 and 45,000ng/g egg, respectively) to determine dose-dependent effects on pipping success, development, hepatic messenger RNA (mRNA) expression levels of genes associated with xenobiotic metabolism and the thyroid hormone (TH) pathway, and TH levels following 20-22 days of incubation. Neither compound reduced pipping success; however, TCPP significantly delayed pipping at 9240 and 51,600ng/g and reduced tarsus length at 51,600ng/g. TDCPP exposure resulted in significant decreases in head plus bill length, embryo mass, and gallbladder size at 45,000ng/g and reduced plasma free T4 levels at 7640ng/g. Type I deiodinase, liver fatty acid-binding protein, and cytochrome P450 (CYP) 3A37 mRNA levels were significantly induced by TCPP, whereas TDCPP induced CYP3A37 and CYP2H1. Chemical analysis of egg contents at incubation days 0, 5, 11, 18, and 19 revealed that > 92% of the injected TCPP or TDCPP concentration was detectable up to day 5; however, < 1% was detected by day 19. The observed phenotypic responses to TCPP and TDCPP exposure may be associated with disruption of the TH axis, which is critical for normal growth and development in birds. The effects of TDCPP on the gallbladder indicate that the disturbance of lipid metabolism is a likely mechanism of toxicity.

Exposure of zebrafish embryos/larvae to TDCPP alters concentrations of thyroid hormones and transcriptions of genes involved in the hypothalamic–pituitary–thyroid axis

Tris(1,3-dichloro-2-propyl) phosphate (TDCPP) has been frequently detected in the environment and in various biota, including fish, and has been implicated in disruption of the thyroid endocrine system. In the present study, zebrafish (Danio rerio) embryos were exposed to different concentrations of TDCPP (10, 50, 100, 300 and 600μg/L) from 2h post-fertilization (hpf) to 144hpf. Developmental endpoints, and whole-body concentrations of thyroid hormones and transcriptional profiles of genes involved in the hypothalamic–pituitary–thyroid (HPT) axis were examined. Exposure to TDCPP caused a dose-dependent developmental toxicity, including decreased body weight, reduced hatching, survival and heartbeat rates, and increased malformation (spinal curvature). Treatment with the positive control chemical 3,3′,5-triiodo-l-thyronine (T3) significantly decreased whole-body thyroxin (T4) concentrations, increased whole-body T3 concentrations, and upregulated mRNA expression involved in the HPT axis as a compensatory mechanism. These results suggested that the HPT axis in 144-hpf zebrafish larvae was responsive to chemical exposure and could be used to evaluate the effects of chemicals on the thyroid endocrine system. TDCPP exposure significantly decreased whole-body T4 concentrations and increased whole-body T3 concentrations, indicating thyroid endocrine disruption. The upregulation of genes related to thyroid hormone metabolism (dio1 and ugt1ab) might be responsible for decreased T4 concentrations. Treatment with TDCPP also significantly increased transcription of genes involved in thyroid hormone synthesis (tshβ, slc5a5 and tg) and thyroid development (hhex, nkx2.1 and pax8) as a compensatory mechanism for decreased T4 concentrations. Taken together, these results suggest that TDCPP alters the transcription of genes involved in the HPT axis and changes whole-body concentrations of thyroid hormones in zebrafish embryos/larvae, thus causing an endocrine disruption of the thyroid system.

Early Zebrafish Embryogenesis Is Susceptible to Developmental TDCPP Exposure

Background: Chlorinated phosphate esters (CPEs) are widely used as additive flame retardants for low-density polyurethane foams and have frequently been detected at elevated concentrations within indoor environmental media.Objectives: To begin characterizing the potential toxicity of CPEs on early vertebrate development, we examined the developmental toxicity of four CPEs used in polyurethane foam: tris(1,3-dichloro-2-propyl) phosphate (TDCPP), tris(2-chloroethyl) phosphate (TCEP), tris(1-chloro-2-propyl) phosphate (TCPP), and 2,2-bis(chloromethyl)propane-1,3-diyl tetrakis(2-chlorethyl) bis(phosphate) (V6).Methods: Using zebrafish as a model for vertebrate embryogenesis, we first screened the potential teratogenic effects of TDCPP, TCEP, TCPP, and V6 using a developmental toxicity assay. Based on these results, we focused on identification of susceptible windows of developmental TDCPP exposure as well as evaluation of uptake and elimination of TDCPP and bis(1,3-dichloro-2-propyl)phosphate (BDCPP, the primary metabolite) within whole embryos. Finally, because TDCPP-specific genotoxicity assays have, for the most part, been negative in vivo and because zygotic genome remethylation is a key biological event during cleavage, we investigated whether TDCPP altered the status of zygotic genome methylation during early zebrafish embryogenesis.Results: Overall, our findings suggest that the cleavage period during zebrafish embryogenesis is susceptible to TDCPP-induced delays in remethylation of the zygotic genome, a mechanism that may be associated with enhanced developmental toxicity following initiation of TDCPP exposure at the start of cleavage.Conclusions: Our results suggest that further research is needed to better understand the effects of a widely used and detected CPE within susceptible windows of early vertebrate development.