Effects of organophosphorus flame retardant EHDPP on mouse retinal photoreceptor cells: Oxidative stress, apoptosis, and proinflammatory response.

2-Ethylhexyl diphenyl phosphate (EHDPP) is a frequently utilized organophosphorus flame retardant (OPFR) and has been extensively detected in environmental media. Prolonged daily exposure to EHDPP has been linked to potential retinal damage, yet the adverse impacts on the retina are still generally underexplored. In this research, we explored oxidative stress, inflammation, and the activating mechanisms initiated by EHDPP in mouse retinal photoreceptor (661 W) cells following a 24 h exposure period. Our research demonstrated that EHDPP led to a decline in cell viability that was directly proportional to its concentration, with the median lethal concentration (LC50) being 88 µM. Furthermore, EHDPP was found to elevate intracellular and mitochondrial levels of reactive oxygen species (ROS), trigger apoptosis, induce cell cycle arrest at the G1 phase, and modulate the expression of both antioxidant enzymes (Nrf2, HO-1, and CAT) and pro-inflammatory mediators (TNF-α, IL-1ß, and IL-6) within 661 W cells. These findings indicate that retinal damage triggered by EHDPP exposure could be mediated via the Nrf2/HO-1 signaling pathway in these cells. Collectively, our investigation revealed that oxidative stress induced by EHDPP is likely a critical factor in the cytotoxic response of 661 W cells, potentially leading to damage in retinal photoreceptor cells.

Mechanistic insights into cadmium exacerbating 2-Ethylhexyl diphenyl phosphate-induced human keratinocyte toxicity: Oxidative damage, cell apoptosis, and tight junction disruption.

Organophosphate flame retardants 2-ethylhexyldiphenyl phosphate (EHDPP) and cadmium (Cd) are ubiquitous in environmental matrices, and dermal absorption is a major human exposure pathway. However, their detrimental effects on the human epidermis remain largely unknown. In this study, human keratinocytes (HaCaT cells) were employed to examine the toxicity and underlying mechanisms of co-exposure to EHDPP and Cd. Their influence on cell morphology and viability, oxidative damage, apoptosis, and tight junction were determined. The results showed that co-exposure decreased cell viability by >40 %, induced a higher level of oxidative damage by increasing the generation of reactive oxygen species (1.3 folds) and inhibited CAT (79 %) and GPX (90 %) activities. Moreover, Cd exacerbated EHDPP-induced mitochondrial disorder and cellular apoptosis, which was evidenced by a reduction in mitochondrial membrane potential and an elevation of cyt-c and Caspase-3 mRNA expression. In addition, greater loss of ZO-1 immunoreactivity at cellular boundaries was observed after co-exposure, indicating skin epithelial barrier function disruption, which may increase the human bioavailability of contaminants via the dermal absorption pathway. Taken together, oxidative damage, cell apoptosis, and tight junction disruption played a crucial role in EHDPP + Cd triggered cytotoxicity in HaCaT cells. The detrimental effects of EHDPP + Cd co-exposure were greater than individual exposure, suggesting the current health risk assessment or adverse effects evaluation of individual exposure may underestimate their perniciousness. Our data imply the importance of considering the combined exposure to accurately assess their health implication.

Stormwater Bioretention Cells Are Not an Effective Treatment for Persistent and Mobile Organic Compounds (PMOCs).

Bioretention cells are a stormwater management technology intended to reduce the quantity of water entering receiving bodies. They are also used to reduce contaminant releases, but their performance is unclear for hydrophilic persistent and mobile organic compounds (PMOCs). We developed a novel eight-compartment one-dimensional (1D) multimedia model of a bioretention cell ("Bioretention Blues") and applied it to a spike and recovery experiment conducted on a system near Toronto, Canada, involving PMOC benzotriazole and four organophosphate esters (OPEs). Compounds with (log DOC) (organic carbon-water distribution coefficients) < ∼2.7 advected through the system, resulting in infiltration or underdrain flow. Compounds with log DOC > 3.8 were mostly sorbed to the soil, where subsequent fate depended on transformation. For compounds with 2.7 ≤ log DOC ≤ 3.8, sorption was sensitive to event size and compound-specific diffusion parameters, with more sorption expected for smaller rain events and for compounds with larger diffusion coefficients. Volatilization losses were minimal for all compounds tested. Direct uptake by vegetation also played a negligible role regardless of the compounds' physicochemical properties. Nonetheless, model simulations showed that vegetation could play a role by increasing transpiration, thereby increasing sorption to the bioretention soil and reducing PMOC release. Model results suggest design modifications to bioretention cells.

Effects of 2-ethylhexyl diphenyl phosphate (EHDPP) on glycolipid metabolism in male adult zebrafish revealed by targeted lipidomic analyses.

The present study aims to evaluate the effects of 2-ethylhexyldiphenyl phosphate (EHDPP) on glycolipid metabolism in vivo. Adult male zebrafish were exposed to various concentrations (0, 1, 10, 100 and 250 µg/L) of EHDPP for 28 days, and changes in lipid and glucose levels were measured. Results indicated significant liver damages in the 100 and 250 µg/L EHDPP groups, which both exhibited significant decreases in hepatic somatic index (HSI), elevated activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in serum and liver, as well as hepatocyte vacuolation and nuclear pyknosis. Exposure to 100 and 250 µg/L EHDPP led to significant reductions in serum and liver cholesterol (TC), triglycerides (TGs), and lipid droplet deposition, indicating a significant inhibition of EHDPP on hepatic lipid accumulation. Lipidomic analyses manifested that 250 µg/L EHDPP reduced the levels of 103 lipid metabolites which belong to glycerides (TGs, diglycerides, and monoglycerides), fatty acyles (fatty acids), sterol lipids (cholesterol, bile acids), sphingolipids, and glycerophospholipids, and downregulated genes involved in de novo synthesis of fatty acids (fas, acc, srebp1, and dagt2), while upregulated genes involved in fatty acid ß-oxidation (pparα and cpt1). KEGG analyses revealed that EHDPP significantly disrupted glycerolipid metabolism, steroid biosynthesis and fatty acid biosynthesis pathways. Collectively, the results showed that EHDPP induced lipid reduction in zebrafish liver, possibly through inhibiting lipid synthesis and disrupting glycerolipid metabolism. Our findings provide a theoretical basis for evaluating the ecological hazards and health effects of EHDPP on glycolipid metabolism.

Bioaccumulation and thyroid endcrione disruption of 2-ethylhexyl diphenyl phosphate at environmental concentration in zebrafish larvae.

2-ethylhexyl diphenyl phosphate (EHDPP) strongly binds to transthyretin (TTR) and affects the expression of genes involved in the thyroid hormone (TH) pathway in vitro. However, it is still unknown whether EHDPP induces endocrine disruption of THs in vivo. In this study, zebrafish (Danio rerio) embryos (< 2 h post-fertilization (hpf)) were exposed to environmentally relevant concentrations of EHDPP (0, 0.1, 1, 10, and 100 µg·L-1) for 120 h. EHDPP was detected in 120 hpf larvae at concentrations of 0.06, 0.15, 3.71, and 59.77 µg·g-1 dry weight in the 0.1, 1, 10, and 100 µg·L-1 exposure groups, respectively. Zebrafish development and growth were inhibited by EHDPP, as indicated by the increased malformation rate, decreased survival rate, and shortened body length. Exposure to lower concentrations of EHDPP (0.1 and 1 µg·L-1) significantly decreased the whole-body thyroxine (T4) and triiodothyronine (T3) levels and altered the expressions of genes and proteins involved in the hypothalamic-pituitary-thyroid axis. Downregulation of genes related to TH synthesis (nis and tg) and TH metabolism (dio1 and dio2) may be partially responsible for the decreased T4 and T3 levels, respectively. EHDPP exposure also significantly increased the transcription of genes involved in thyroid development (nkx2.1 and pax8), which may stimulate the growth of thyroid primordium to compensate for hypothyroidism. Moreover, EHDPP exposure significantly decreased the gene and protein expression of the transport protein transthyretin (TTR) in a concentration-dependent manner, suggesting a significant inhibitory effect of EHDPP on TTR. Molecular docking results showed that EHDPP and T4 partly share the same mode of action of binding to the TTR protein, which might result in decreased T4 transport due to the binding of EHDPP to the TTR protein. Taken together, our findings indicate that EHDPP can cause TH disruption in zebrafish and help elucidate the mechanisms underlying EHDPP toxicity.

Effects of 2-ethylhexyl diphenyl phosphate exposure on the glucolipid metabolism and cardiac developmental toxicity in larval zebrafish based on transcriptomic analysis.

2-Ethylhexyl diphenyl phosphate (EHDPP) is an organophosphorus type of flame retardant. It is mainly used as a flame-retardant plasticizer in the production of flexible polyvinyl chloride. EHDPP is widely present in environment, particularly in aquatic environment. In this study, we reported that EHDPP exposure significantly affected glucose and lipid metabolism in zebrafish larvae, which was reflected by changes in the transcription of relevant genes and decreased levels of glucose, pyruvate, and triglycerides. In addition, the transcriptomic analysis revealed that the differentially expressed genes could enrich various endpoints in zebrafish larvae. Interestingly, EHDPP exposure could not only change the transcription of genes related to glucolipid metabolism but also cause cardiotoxicity by affecting the transcription of genes related to calcium signaling pathways in zebrafish larvae. To support these findings, we confirmed that these genes involved in cardiac morphology and development were significantly upregulated in zebrafish larvae after EHDPP exposure. More importantly, the distance and overlapping area of the atrium and ventricle were also changed in the EHDPP-exposed zebrafish larvae of transgenic Tg (myl7: EGFP). Overall, our study revealed that EHDPP exposure could affect various endpoints related to glucolipid metabolism and cardiac development in the early developmental stages of zebrafish.

Human exposure to organophosphate esters in water and packed beverages.

Drinks are an essential part of human diet, which makes them a source of human exposure to plasticizers such as organophosphate esters (OPEs). The current study provides new information about sixteen OPE levels in 75 different samples (tap water, packed water, cola drinks, juice, wine and hot drinks). Tap water mean levels (40.9 ng/L) were statistically higher than packed water mean levels (4.82 ng/L), mainly due to the contribution of tris(2-chloroisopropyl) phosphate (TCIPP) and tris(2-butoxyethyl) phosphate (TBOEP) that may come from PVC water pipes. Over 90% of samples presented at least one OPE, where regular cola drinks had the highest mean concentrations (2876 ng/L). There was a significantly higher presence of OPEs in added sugar beverages than sugar free drinks, especially for 2-ethylhexyl diphenyl phosphate (EHDPP), which might be related not only to packaging materials but to the added sugar content. Estimated daily intakes (EDIs) in normal and high-exposure scenarios were 2.52 ng/kg bw/day and 7.43 ng/kg bw/day, respectively. Human risk associated with beverages ingestion showed regular cola drinks, juice and tap water as the groups with the highest hazard quotients (HQs). Although OPE exposure was below to safety limits, it should be noted that EHDPP values for regular cola group must be cause of concern, and other routes of exposure such as food ingestion or air inhalation should be also considered.

Downregulation of miRNA-155-5p contributes to the adipogenic activity of 2-ethylhexyl diphenyl phosphate in 3T3-L1 preadipocytes.

2-Ethylhexyl diphenyl phosphate (EHDPP) is a commonly used organophosphorus flame retardant and food packaging material. Because of its high lipophilic and bioaccumulative properties, adipocytes are the primary target of EHDPP. However, the toxicity of EHDPP on preadipocytes and the potential mechanism have not been fully elucidated. MicroRNAs (miRNAs) are thought to be an important mediator that contribute to the toxicity of environmental contaminants. To identify the miRNAs specifically responsible for EHDPP exposure and their role in EGDPP's toxicity in preadipocytes, the adipogenic effects and miRNA expression profiling were performed on 3T3-L1 preadipocytes exposed to EHDPP. EHDPP at concentrations of 1-10 µM promoted adipocyte differentiation, as evidenced by lipid staining, triglyceride content, and expression of adipogenesis markers. MiRNA-seq analysis revealed that 7 differentially expressed miRNAs were recognized under EHDPP exposure, with miR-155-5p being the top down-regulated miRNA. Quantitative reverse transcription PCR (RT-qPCR) analysis showed that miR-155-5p level fell sharply during the first 2 days and continued to fall dose-dependently throughout the EHDPP exposure period. MiR-155-5p inhibition promotes adipocyte differentiation, whereas its overexpression counteracted EHDPP-induced adipogenesis. Luciferase reporter assay identified CCAAT/enhancer-binding protein beta (C/EBPß) as a target of miR-155-5p in 3T3-L1 preadipocytes in response to EHDPP. Taken together, EHDPP exposure down-regulated miR-155-5p, which then increased C/EBPß and peroxisome proliferator-activated receptor γ (PPARγ) expression and promoted adipogenesis in preadipocytes.

Human CYP enzyme-activated clastogenicity of 2-ethylhexyl diphenyl phosphate (a flame retardant) in mammalian cells.

2-Ethylhexyl diphenyl phosphate (EHDPP) is a common flame retardant and environmental pollutant, exposing humans with endocrinal disrupting potentials. Its mutagenicity, especially following metabolism, remains unclear. In this study, molecular docking analysis indicated that EHDPP was a potential substrate for several human CYP enzymes except for CYP1A1. Among V79-derived cell lines genetically engineered for the expression of each CYP, EHDPP (6 h exposure/18 h recovery) did not induce micronuclei in the V79 or V79-derived cells expressing human CYP1A1, however, it was positive in V79-derived cell lines expressing human CYP2E1, 3A4, and 2B6. In a human hepatoma (HepG2) cell line, EHDPP (48 h exposure) moderately induced micronuclei, which was blocked by 1-aminobenzotriazole (ABT, 60 µM, inhibitor of CYPs); pretreating HepG2 cells with bisphenol AF, another organic pollutant as inducer of CYPs (0.1 µM for 16 h), significantly potentiated micronuclei formation by EHDPP, threshold being decreased from 10 to 1.25 µM. This effect was blocked by ABT, drastically reduced by ketoconazole (inhibiting CYP3A expression/activity), and moderately inhibited by trans-1,2-dichloroethylene (selective CYP2E1 inhibitor). Immunofluorescent centromere protein B staining indicated that EHDPP-induced micronuclei in V79-derived cell lines expressing human CYP2E1 and 3A4 were predominantly centromere-negative, and that in HepG2 cells pretreated with bisphenol AF (for inducing multiple CYPs) were purely centromere-negative. In bisphenol AF-pretreated HepG2 cells EHDPP potently induced DNA breaks, as indicated by the comet assay and Western blot analysis of γ-H2AX. In conclusion, our study suggests that EHDPP is potently clastogenic, following activation by several human CYP enzymes, CYP3A4 being a major one.

Evaluation of hepatotoxicity induced by 2-ethylhexyldiphenyl phosphate based on transcriptomics and its potential metabolism pathway in human hepatocytes.

Increasing use of organophosphorus flame retardants (OPFRs) has aroused great concern to their uncertain environment risk, especially to human health risk. In our study, hepatotoxicity screening of six aryl-OPFRs, potential hepatotoxicity mechanism of 2-ethylhexyldiphenyl phosphate (EHDPP) using RNA-sequencing and its metabolites were investigated in human hepatocytes (L02). The toxicity results demonstrated that EHDPP should be prioritized for further research with the highest toxicity. Further RNA-seq results through GO and KEGG enrichment analysis indicated that exposure to 10 mg/L of EHDPP significantly affected energy homeostasis, endoplasmic reticulum (ER) stress, apoptosis, cell cycle, and inflammation response in cells. The top 12 hub genes were validated by RT-qPCR and conformed to be mainly related to glycolysis and ER stress, followed by cell cycle and inflammation response. Western blot, apoptosis detection, glycolysis stress test, and cell cycle analysis were further performed to verify the above main pathways. Additionally, it was found in the metabolism experiment that detoxification of EHDPP by phase I and phase II metabolism in cells wasn't significant until 48 h with a metabolic rate of 6.12%. EHDPP was stable and still dominated the induction of toxicity. Overall, this study provided valuable information regarding the toxicity and potential metabolism pathway of EHDPP.

Adipogenic activity of 2-ethylhexyl diphenyl phosphate via peroxisome proliferator-activated receptor γ pathway.

Recent studies have shown that exposure to some organophosphates, such as triphenyl phosphate (TPHP) and diphenyl phosphate (DPHP), can affect adipogenesis in preadipocytes. 2-Ethylhexyl diphenyl phosphate (EHDPP), an organophosphate, is frequently detected in various environmental media. However, there is less information about the toxicity effects and the mechanism by which EHDPP affects preadipocytes. In the present study, we investigated whether EHDPP could induce differentiation in 3T3-L1 preadipocytes through the peroxisome proliferator-activated receptor γ (PPARγ) signaling pathway. The fluorescence competitive binding assay and the dual-luciferase reporter gene assay were used to assess the binding affinity and activation of PPARγ, and the results showed that EHDPP can bind to the ligand binding domain of PPARγ (PPARγ-LBD) and activate PPARγ in vitro. Exposure to EHDPP for 10 days extensively induced adipogenesis in 3T3-L1 preadipocytes as assessed by lipid accumulation and gene expression of adipogenic markers of fatty acid binding protein 4 (FABP4), lipoprotein lipase (Lpl), adiponectin (Adip), and fatty acid synthase (Fasn). Furthermore, the preadipocytes differentiation was blocked by the PPARγ-specific antagonist GW9662, indicating that the PPARγ signaling pathway plays an important part in 3T3-L1 cell differentiation induced by EHDPP. Taken together, EHDPP can bind to PPARγ-LBD, activate PPARγ receptor, and induce cell differentiation via the PPARγ signaling pathway in 3T3-L1 preadipocytes.

Biomonitoring of organophosphate triesters and diesters in human blood in Jiangsu Province, eastern China: Occurrences, associations, and suspect screening of novel metabolites.

Since organophosphate (OP) triesters are ubiquitous in environmental matrices, there is an increasing concern regarding human exposure to OP triesters or their metabolites. In this study, we measured levels of 16 OP triesters and 4 OP diesters in n = 99 human blood samples of non-occupationally exposed adults (aged 18-87) from Jiangsu Province, eastern China. Based on the measured concentrations, statistical difference and correlativity were calculated to characterize the population diversity and potential sources of OP triester and diester. Di (2-ethylhexyl) phosphate (DEHP) and 2-ethylhexyl diphenyl phosphate (EHDPP) were found in many participants' blood, with median concentrations of 1.2 (range: n.d. - 44.7, detection frequency: 99%) and 0.85 (n.d. - 28.8, 68%) ng mL-1, respectively. Blood samples of older participants contained significantly lower concentrations of OP diesters or triesters than their younger counterparts (p < 0.01). Regional- and age-specific differences in the blood concentrations of OP triesters and diesters were attributed to disparities in environmental exposure intensity. EHDPP and tris (phenyl) phosphate (TPHP), the predominant OP triesters, exhibited significant positive correlation (p < 0.01, r = 0.84) suggestive of analogous transport behavior from similar exposure sources to humans. The increased correlations between diphenyl phosphate (DPHP) and TPHP as well as with EHDPP as observed from the multivariate regression suggests that DPHP could be derived from the metabolism of both TPHP (the crucial precursor) and EHDPP. When the blood samples were subsequently screened using high-resolution spectrometry, we detected five novel OP metabolites: glucuronide conjugates of hydroxylated DEHP (OH-DEHP glucuronide conjugate), 2-ethylhexyl monophenyl phosphate (EHMPP), hydroxylated EHMPP (OH-EHMPP), dihydroxylated bis(2-butoxyethyl) phosphate (di-OH-BBOEP), and dihydroxylated tris(butyl) phosphate (di-OH-TNBP). Overall, this study provides novel information regarding the occurrence of OP triesters and diesters, and further suggested several novel OP metabolites in human blood.