Establishment of an Integrated Nontarget and Suspect Screening Workflow for Organophosphate Diesters (Di-OPEs) and Identification of Seven Previously Unknown Di-OPEs in Food Contact Plastics.

In this study, an innovative, integrated nontarget and suspect screening workflow was developed for identifying organophosphate diesters (di-OPEs) using high-resolution mass spectrometry. The workflow featured the utilization of 0.02% acetic acid as a mobile-phase additive, differentiated screening methods for alkyl and aryl di-OPEs, and a combination of electrospray negative ionization and positive ionization. Using this workflow, 18 di-OPEs were identified in the extracts of 75 food contact plastic (FCP) samples sourced from South China. Among these, six alkyl and one aryl di-OPEs were previously unknown (one unequivocal identification and six probable structures based on diagnostic evidence). (Semi)quantification revealed that bis(2,4-di-tert-butylphenyl) phosphate was the major di-OPE in FCPs, with a median concentration of 1079 ng/g (range: 23.4-158,414 ng/g). The migration efficiencies of di-OPEs from an FCP sample to four kinds of food simulants were between 2.58 and 54.3%. This study offered a useful workflow for the comprehensive profiling of di-OPEs in FCPs.

Amniogenesis in Human Amniotic Sac Embryoids after Exposures to Organophosphate Flame Retardants.

BACKGROUND: Amniogenesis is a key event in biochemical pregnancy, and its failure may result in human embryonic death. However, whether and how environmental chemicals affect amniogenesis remain largely unknown. OBJECTIVES: The objective of the present study was to screen chemicals that may disrupt amniogenesis in an amniotic sac embryoid model and to investigate the potential mechanism of amniogenesis failure, with a focus on organophosphate flame retardants (OPFRs). METHODS: This study developed a high-throughput toxicity screening assay based on transcriptional activity of octamer-binding transcription factor 4 (Oct4). For the two positive OPFR hits with the strongest inhibitory activity, we used time-lapse and phase-contrast imaging to assess their effects on amniogenesis. Associated pathways were explored by RNA-sequencing and western blotting, and potential binding target protein was identified through a competitive binding experiment. RESULTS: Eight positive hits exhibiting Oct4 expression were identified, with 2-ethylhexyl-diphenyl phosphate (EHDPP) and isodecyl diphenyl phosphate (IDDPP) showing the strongest inhibitory activity. EHDPP and IDDPP were found to disrupt the rosette-like structure of the amniotic sac or inhibit its development. Functional markers of squamous amniotic ectoderm and inner cell mass were also found disrupted in the EHDPP- and IDDPP-exposed embryoids. Mechanistically, embryoids exposed to each chemical exhibited abnormal accumulation of phosphorylated nonmuscle myosin (p-MLC-II) and were able to bind to integrin ß1 (ITGß1). CONCLUSION: The amniotic sac embryoid models suggested that OPFRs disrupted amniogenesis likely by inhibiting the ITGß1 pathway, thus providing direct in vitro evidence associating OPFRs with biochemical miscarriage. https://doi.org/10.1289/EHP11958.

Screening of Organophosphate Flame Retardants with Placentation-Disrupting Effects in Human Trophoblast Organoid Model and Characterization of Adverse Pregnancy Outcomes in Mice.

BACKGROUND: Abnormal placental development may result in adverse pregnancy outcomes and metabolic diseases in adulthood; however, it remains unknown whether and how xenobiotics affect human placentation. OBJECTIVES: This study aimed to screen and identify placentation-disrupting chemicals in commonly used organophosphate flame retardants (OPFRs) and, if identified, to investigate potential adverse effects on placentation in relation to adverse pregnancy outcomes and metabolic disorder in offspring in mice. METHODS: We devised a high-throughput immunofluorescence screening assay based on human trophoblast organoids and used it to screen OPFRs that inhibit the proliferation of organoids. One identified chemical was assessed for its effects on placentation by evaluating villous cytotrophoblasts, syncytiotrophoblasts, and extravillous trophoblasts using immunofluorescence and a mitochondrial stress test after 2 d of exposure. A 10-d exposure study was further performed to observe the dynamic effect of the OPFR on the structure of the organoids. RNA-sequencing and western blotting experiments were performed to explore the associated pathways, and a potential binding protein was identified by immunoprecipitation and in vitro kinase activity assays. Animal studies were performed to determine whether the findings in organoids could be replicated in mice and to observe adverse pregnancy outcomes. RESULTS: The proliferation of organoids exposed to three aryl-OPFRs was significantly lower than the proliferation of control organoids. Further analysis demonstrated that one such chemical, 2-ethylhexyl-diphenyl phosphate (EHDPP), disrupted placentation in organoids. Mechanistically, EHDPP interfered with insulin-like growth factor 1 receptor (IGF1R) to inhibit aerobic respiration. Mice exposed to EHDPP at a physiological human concentrations exhibited immature and mature placental disorders, which correlated with fetal growth restriction, implantation failure, stillbirth, and impaired glucose tolerance. CONCLUSIONS: The human trophoblast organoid model showed that the commonly used OPFRs disrupted placentation via IGF1R, indicating that its use may contribute to adverse pregnancy outcomes and metabolic disorders in offspring. https://doi.org/10.1289/EHP10273.

Triphenyl phosphate delayed pubertal timing and induced decline of ovarian reserve in mice as an estrogen receptor antagonist.

Although concerns have been raised about the adverse effects of triphenyl phosphate (TPhP) on female fertility, its risk to ovarian functioning remains unknown. In this study, female C57BL/6 mice at postnatal day 21 were exposed on a daily basis to TPhP dose of 2, 10, and 50 mg/kg for 40 days. A significant delay in pubertal timing was observed in the mice exposed to 50 mg/kg of TPhP. An estrogen-responsive reporter transgenic mice assay demonstrated that TPhP significantly downregulated the estrogen receptor (ER) signaling by 45.1% in the whole body in the 50 mg/kg group, and by 14.7-43.7% in the uterus for all exposure groups compared with the control. This strong antagonistic activity of TPhP toward ER explained the delay in pubertal timing. A significant reduction in the number of follicles in all stages was observed in mice after being exposed to TPhP for 40 days at concentrations of 10 and 50 mg/kg, resulting in a decline of the ovarian reserve. The elevation of the follicle-stimulating hormone concentration may have contributed to this phenomenon, as controlled by the antagonistic activity of TPhP toward ER in the brain. The toxic effects of TPhP on ovarian functioning highlight this chemical as a potential risk factor for female fertility.

Maternal Transfer of 2-Ethylhexyl Diphenyl Phosphate Leads to Developmental Toxicity Possibly by Blocking the Retinoic Acid Receptor and Retinoic X Receptor in Japanese Medaka (Oryzias latipes).

2-Ethylhexyl diphenyl phosphate (EHDPP) has been detected in wild fish with high concentrations, which may pose a risk in the embryo development considering its potential maternal transfer. In this study, EHDPP was demonstrated to elicit antagonistic activity to medaka retinoic acid receptor (mRAR) and retinoic X receptor (mRXR) with 50% inhibitory concentration of 18 and 36 µM, respectively. After adult female medaka were exposed to EHDPP at 156, 405, and 1161 ng/L for 35 days, the embryonic EHDPP concentrations (364-4824 ng/g lipid weight (lw)) were higher than those in the maternal tissues (15.0-4166 ng/g lw), showing notable maternal transfer. The embryonic concentration of EHDPP decreased limitedly during 1-2 day post-fertilization (dpf, the main developmental window of eye) but then decreased sharply after 2 dpf. The transcript abundance of cyp26a1 was inhibited and subsequent increasing embryonic all-trans RA level was observed in embryos, showing RAR/RXR antagonistic activity. These results may specifically contribute to the increased eye deformity incidences in all exposure groups (up to 8.0%; 51/637) relative to the control (1.0%, 7/733). The response behavior of the larvae to light stimulation was impaired in a dose-dependent manner, demonstrating a vision disorder. Because such developmental toxicities were observed at the environmental level, EHDPP may pose a threat to the survival of wild larvae and therefore a population risk for wild fish.

Triphenyl phosphate modulated saturation of phospholipids: Induction of endoplasmic reticulum stress and inflammation.

Although triphenyl phosphate (TPHP) has been reported to disrupt lipid metabolism, the effect of TPHP on lipid saturation remains unexplored. In this study, a lipidomic analysis demonstrated decreases in the levels of poly-unsaturated phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS) in RAW264.7 murine macrophage cells exposed to 10 µM TPHP. The expression of the gene encoding lysophosphatidylcholine acyltransferase 3 (Lpcat3) was significantly downregulated by 0.76 ± 0.03 and 0.70 ± 0.08-fold in 10 and 20 µM TPHP exposure groups, relative to the control group. This finding explains the observed decrease in lipid saturation. Correspondingly, exposure to 10 and 20 µM TPHP induced endoplasmic reticulum (ER) stress and inflammatory responses, which have been linked to metabolic dysfunction such as insulin resistance and hypertriglyceridemia. Therefore, TPHP may pose a risk to human health by promoting metabolic diseases.

Cytoplasm lipids can be modulated through hormone-sensitive lipase and are related to mitochondrial function in porcine IVM oocytes.

Intracellular lipids provide energy for oocyte maturation and development. Triglycerides are the main components of cytoplasm lipid droplets, and hydrolysis of triglycerides requires several lipase-mediated steps. The aim of this study was to determine the effects of the ß-adrenoceptor agonist isoproterenol (ISO) and the hormone-sensitive lipase (HSL) inhibitor CAY10499 on the IVM of porcine oocytes. ISO (5mg L-1) and CAY10499 (20mg L-1) had positive and negative effects respectively on invitro oocyte maturation and subsequent embryo development. The rates of polar body extrusion, cleavage and blastocyst formation were significantly higher in the ISO-treated group than the control and CAY10499-treated groups. ISO treatment also upregulated intracellular cAMP levels in comparison with the control group, while CAY10499 significantly increased the triglyceride content of matured oocytes when compared with other groups, consistent with the observed decrease in LIPE (HSL) mRNA levels. Furthermore, the inhibitory effects of CAY10499 included decreases in mitochondrial membrane potential and mitochondrial temperature. These results indicate that ISO has a positive effect on the IVM of porcine oocytes, and that intracellular lipid metabolism can be modulated by CAY10499 through inhibition of HSL and is closely related to mitochondrial function.

Triphenyl Phosphate at Environmental Levels Retarded Ovary Development and Reduced Egg Production in Japanese Medaka (Oryzias latipes).

Since triphenyl phosphate (TPhP) elicits both antiestrogenic activities via blocking the estrogen receptor (ER) and estrogenic activity by elevating 17ß-estradiol (17ß-E2) synthesis, its adverse effect on female reproduction is uncertain. In this study, we exposed Japanese medaka to TPhP at 131, 363, and 1773 ng/L for 100 days following hatching. TPhP significantly induced ovary retardation in all exposure groups (incidence: from 11.9 to 37.8%) and reduced egg production by 38.9 and 50.9% in the 363 and 1773 ng/L exposure groups, respectively. Vitellogenin (vtg) transcription was significantly downregulated by 35.4-57.4% after TPhP exposure, explaining the ovary retardation. Considering that 17ß-E2 was only significantly decreased in the 1773 ng/L exposure group, ER antagonism could be the dominant contributor to the inhibition of vtg transcription and female reproductive toxicity of TPhP. As 4-hydroxyphenyl diphenyl phosphate, a metabolite of TPhP, was detected in livers with similar concentration [68.4-1237 ng/g lipid weight (lw)] to that of TPhP (485-1594 ng/g lw) and elicited medaka ER antagonistic activity (50% inhibitory concentration = 78.1 µM), TPhP and its metabolite should both contribute to the reproductive inhibition. We demonstrate that TPhP at environmentally relevant concentrations is toxic to female reproduction, which poses an ecological risk to wild fish at the population level.

Screening of House Dust from Chinese Homes for Chemicals with Liver X Receptors Binding Activities and Characterization of Atherosclerotic Activity Using an in Vitro Macrophage Cell Line and ApoE-/- Mice.

BACKGROUND: Atherosclerotic cardiovascular disease has become the leading cause of death worldwide, and environmental pollutants are increasingly recognized as risk factors for atherosclerosis. Liver X receptors (LXRs) play a central role in atherosclerosis; however, LXR activity of organic pollutants and associated potential risk of atherosclerosis have not yet been characterized. OBJECTIVES: This study aimed to explore whether LXR-antagonistic chemicals are present in indoor house dust and, if so, to characterize this activity in relation to changes in macrophages in vitro and cardiovascular disease indicators in vivo in an atherosclerosis ApoE-/- mouse model. METHODS: We used a His-LXRα-pull-down assay and a nontarget high-resolution mass spectrometry method to screen house dust collected from Chinese homes for LXRα- and LXRß-antagonist activity. A chemical identified in this manner was assessed for its ability to induce cholesterol efflux and foam cell formation in RAW264.7 macrophages, to down-regulate the expression of two LXR-dependent genes, ABCA1 and ABCG1, and finally to induce atherosclerotic lesions in vivo using an ApoE-/- mouse model. RESULTS: We identified the flame retardants triphenyl phosphate (TPHP) and 2-ethylhexyl diphenyl phosphate (EHDPP) in house dust samples and demonstrated their ability to antagonize LXRs. The potency of TPHP was similar to that of the LXR-antagonist SR9238. TPHP could also inhibit cholesterol efflux and promote foam cell formation in RAW264.7 macrophages and mouse peritoneal macrophages and significantly promoted atherosclerotic lesion formation in the ApoE-/- mouse model. CONCLUSIONS: We found LXR-antagonist chemicals in environmental samples of indoor dust from Chinese homes. One of the chemicals, TPHP, was able to promote the development of atherosclerotic lesions in the ApoE-/- mouse model. These results highlight the need to assess the LXR-antagonist activities of pollutants in future environmental management programs. https://doi.org/10.1289/EHP5039.

RNA-Seq transcriptome profiling of mouse oocytes after in vitro maturation and/or vitrification.

In vitro maturation (IVM) and vitrification have been widely used to prepare oocytes before fertilization; however, potential effects of these procedures, such as expression profile changes, are poorly understood. In this study, mouse oocytes were divided into four groups and subjected to combinations of in vitro maturation and/or vitrification treatments. RNA-seq and in silico pathway analysis were used to identify differentially expressed genes (DEGs) that may be involved in oocyte viability after in vitro maturation and/or vitrification. Our results showed that 1) 69 genes were differentially expressed after IVM, 66 of which were up-regulated. Atp5e and Atp5o were enriched in the most significant gene ontology term "mitochondrial membrane part"; thus, these genes may be promising candidate biomarkers for oocyte viability after IVM. 2) The influence of vitrification on the transcriptome of oocytes was negligible, as no DEGs were found between vitrified and fresh oocytes. 3) The MII stage is more suitable for oocyte vitrification with respect to the transcriptome. This study provides a valuable new theoretical basis to further improve the efficiency of in vitro maturation and/or oocyte vitrification.