Environmentally Relevant Concentrations of Tetrabromobisphenol A Exposure Impends Neurovascular Formation through Perturbing Mitochondrial Metabolism in Zebrafish Embryos and Human Primary Endothelial Cells.

Tetrabromobisphenol A (TBBPA), the most extensively utilized brominated flame retardant, has raised growing concerns regarding its environmental and health risks. Neurovascular formation is essential for metabolically supporting neuronal networks. However, previous studies primarily concerned the neuronal injuries of TBBPA, its impact on the neurovascularture, and molecular mechanism, which are yet to be elucidated. In this study, 5, 30, 100, 300 µg/L of TBBPA were administered to Tg (fli1a: eGFP) zebrafish larvae at 2-72 h postfertilization (hpf). The findings revealed that TBBPA impaired cerebral and ocular angiogenesis in zebrafish. Metabolomics analysis showed that TBBPA-treated neuroendothelial cells exhibited disruption of the TCA cycle and the Warburg effect pathway. TBBPA induced a significant reduction in glycolysis and mitochondrial ATP production rates, accompanied by mitochondrial fragmentation and an increase in mitochondrial reactive oxygen species (mitoROS) production in neuroendothelial cells. The supplementation of alpha-ketoglutaric acid, a key metabolite of the TCA cycle, mitigated TBBPA-induced mitochondrial damage, reduced mitoROS production, and restored angiogenesis in zebrafish larvae. Our results suggested that TBBPA exposure impeded neurovascular injury via mitochondrial metabolic perturbation mediated by mitoROS signaling, providing novel insight into the neurovascular toxicity and mode of action of TBBPA.

Unveiling Distribution of Per- and Polyfluoroalkyl Substances in Matched Placenta-Serum Tetrads: Novel Implications for Birth Outcome Mediated by Placental Vascular Disruption.

The placenta is pivotal for fetal development and maternal-fetal transfer of many substances, including per- and polyfluoroalkyl substances (PFASs). However, the intraplacental distribution of PFASs and their effects on placental vascular function remain unclear. In this study, 302 tetrads of matched subchorionic placenta (fetal-side), parabasal placenta (maternal-side), cord serum, and maternal serum samples were collected from Guangzhou, China. Eighteen emerging and legacy PFASs and five placental vascular biomarkers were measured. Results showed that higher levels of perfluorooctanoic (PFOA), perfluorooctane sulfonic acid (PFOS), and chlorinated polyfluorinated ether sulfonic acids (Cl-PFESAs) were detected in subchorionic placenta compared to parabasal placenta. There were significant associations of PFASs in the subchorionic placenta, but not in the serum, with placental vascular biomarkers (up to 32.5%) and lower birth size. Birth weight was negatively associated with PFOA (ß: -103.8, 95% CI: -186.3 and -21.32) and 6:2 Cl-PFESA (ß: -80.04, 95% CI: -139.5 and -20.61), primarily in subchorionic placenta. Mediation effects of altered placental angiopoietin-2 and vascular endothelial growth factor receptor-2 were evidenced on associations of adverse birth outcomes with intraplacental PFOS and 8:2 Cl-PFESA, explaining 9.5%-32.5% of the total effect. To the best of our knowledge, this study is the first to report on differential intraplacental distribution of PFASs and placental vascular effects mediating adverse birth outcomes and provides novel insights into the placental plate-specific measurement in PFAS-associated health risk assessment.

Parental Exposure to Environmentally Relevant Concentrations of Bisphenol-A Bis(diphenyl phosphate) Impairs Vascular Development in Offspring through DNA/RNA Methylation-Dependent Transmission.

Bisphenol-A bis(diphenyl phosphate) (BDP) has been increasingly detected in indoor environmental and human samples. Little is known about its developmental toxicity, particularly the intergenerational effects of parental exposure. In this study, adult zebrafish were exposed to BDP at 30-30,000 ng/L for 28 days, with results showing that exposure did not cause a transfer of BDP or its metabolites to offspring. Vascular morphometric profiling revealed that parental exposure to BDP at 30 and 300 ng/L exerted significant effects on the vascular development of offspring, encompassing diverse alterations in multiple types of blood vessels. N6-Methyladenosine (m6A) methylated RNA immunoprecipitation sequencing of larvae in the 300 ng/L group revealed 378 hypomethylated and 350 hypermethylated m6A peaks that were identified in mRNA transcripts of genes crucial for vascular development, including the Notch/Vegf signaling pathway. Concomitant changes in 5 methylcytosine (m5C) DNA methylation and gene expression of m6A modulators (alkbh5, kiaa1429, and ythdf1) were observed in both parental gonads and offspring exposed to BDP. These results reveal that parental exposure to low concentrations of BDP caused offspring vascular disorders by interfering with DNA and RNA methylation, uncovering a unique DNA-RNA modification pattern in the intergenerational transmission of BDP's developmental toxicity.

Maternal exposure to nano-titanium dioxide impedes fetal development via endothelial-to-mesenchymal transition in the placental labyrinth in mice.

BACKGROUND: Extensive production and usage of commercially available products containing TiO2 NPs have led to accumulation in the human body. The deposition of TiO2 NPs has even been detected in the human placenta, which raises concerns regarding fetal health. Previous studies regarding developmental toxicity have frequently focused on TiO2 NPs < 50 nm, whereas the potential adverse effects of large-sized TiO2 NPs received less attention. Placental vasculature is essential for maternal-fetal circulatory exchange and ensuring fetal growth. This study explores the impacts of TiO2 NPs (100 nm in size) on the placenta and fetal development and elucidates the underlying mechanism from the perspective of placental vasculature. Pregnant C57BL/6 mice were exposed to TiO2 NPs by gavage at daily dosages of 10, 50, and 250 mg/kg from gestational day 0.5-16.5. RESULTS: TiO2 NPs penetrated the placenta and accumulated in the fetal mice. The fetuses in the TiO2 NP-exposed groups exhibited a dose-dependent decrease in body weight and length, as well as in placental weight and diameter. In vivo imaging showed an impaired placental barrier, and pathological examinations revealed a disrupted vascular network of the labyrinth upon TiO2 NP exposure. We also found an increase in gene expression related to the transforming growth factor-ß (TGF-ß) -SNAIL pathway and the upregulation of mesenchymal markers, accompanied by a reduction in endothelial markers. In addition, TiO2 NPs enhanced the gene expression responsible for the endothelial-to-mesenchymal transition (EndMT) in cultured human umbilical vein endothelial cells, whereas SNAIL knockdown attenuated the induction of EndMT phenotypes. CONCLUSION: Our study revealed that maternal exposure to 100 nm TiO2 NPs disrupts placental vascular development and fetal mice growth through aberrant activation of EndMT in the placental labyrinth. These data provide novel insight into the mechanisms of developmental toxicity posed by NPs.

A single-cell survey unveils cellular heterogeneity and sensitive responses in mouse cortices induced by oral exposure to triphenyl phosphate.

Triphenyl phosphate (TPhP) is a non-halogenated organophosphorus flame retardant, and there is a higher exposure risk in children. TPhP has been found to be neurotoxic upon developmental exposure, yet the specific mechanism remains unclear. To characterize the cellular responses underlying TPhP-induced developmental neurotoxicity, we administered TPhP (0.5, 5 or 50 mg/kg/day) to neonatal mice from postnatal day 10 (P10)-P70. A total of 17,229 cells and 26,338 genes were identified in cortical samples from control and low-dose (the internal doses of metabolite DPhP comparable to human exposure level) groups using single-cell RNA sequencing (scRNA-seq). TPhP exposure led to heterogeneous transcriptional alterations and intercellular crosstalk among neurons, neural stem/progenitor cells (NSPCs), endothelial cells, and immunocytes. Deprivation of NSPCs, loss of mature neurons, and concomitant neuroinflammation mediated by extrinsic and intrinsic immunocytes were found in TPhP-exposed cortices. In addition, we observed blood-brain barrier destruction prior to the anxiety/depression-like neurobehavioral changes. These results reveal the distinctive cellular processes in TPhP's neurodevelopmental toxicity and uncover that the impeded neurogenesis, disrupted vascular barrier, and concomitant neuroinflammation are the sensitive responses to TPhP exposure. Our study paves the way for the application of scRNA-seq in toxicity assessments for emerging neurotoxic pollutants.

Apigenin inhibits cell proliferation, migration, and invasion by targeting Akt in the A549 human lung cancer cell line.

Apigenin (APG), a widely distributed flavonoid in vegetables and fruits, with low toxicity, and a nonmutagenic characteristic, has been reported to have many targets. Evidence indicates that APG can inhibit the proliferation, migration, invasion, and metastasis of some tumor cells, but the mechanism, specifically in lung cancer, is unclear. The phosphoinositide 3-kinase (PI3K)/Akt signaling pathway regulates a diverse set of cellular functions relevant to the growth and progression of lung cancer, including proliferation, survival, migration, and invasion. Our results showed that APG exerted anti-proliferation, anti-migration, and anti-invasion effects in A549 human lung cancer cells by targeting the PI3K/Akt signaling pathway. 3-(4, 5-dimethylthiszol-2-yl)-2, 5-diphenytetrazolium bromide assay and colony formation assay showed that APG suppressed cell proliferation in a dose-dependent and time-dependent manner. Cell motility and invasiveness were assayed using a wound healing and Transwell assay, suggesting that APG inhibited the migration and invasion of A549 cells. Western blot analyses were carried out to examine the Akt signaling pathways. The results confirmed that APG decreased Akt expression and its activation. Then, cells were transfected with Akt-active and Akt-DN plasmids separately. The migration and invasion of A549 cells were significantly changed, constitutively activating Akt or knocking down Akt, indicating that APG can suppress the migration and invasion of lung cancer cells by modulating the PI3K/Akt signaling pathway. Furthermore, the results indicated that APG not only suppressed phosphorylation of Akt, thereby preventing its activation, but also inhibited its downstream gene expression of matrix metalloproteinases-9, glycogen synthase kinase-3ß, and HEF1. Together, APG is a new inhibitor of Akt in lung cancer and a potential natural compound for cancer chemoprevention.

2D-DIGE and MALDI TOF/TOF MS analysis reveal that small GTPase signaling pathways may play an important role in cadmium-induced colon cell malignant transformation.

Cadmium is a toxic heavy metal present in the environment and in industrial materials. Cadmium has demonstrated carcinogenic activity that induces cell transformation, but how this occurs is unclear. We used 2D-DIGE and MALDI TOF/TOF MS combined with bioinformatics and immunoblotting to investigate the molecular mechanism of cadmium transformation. We found that small GTPases were critical for transformation. Additionally, proteins involved in mitochondrial transcription, DNA repair, and translation also had altered expression patterns in cadmium treated cells. Collectively, our results suggest that activation of small GTPases contributes to cadmium-induced transformation of colon cells.

Development of an Automated Morphometric Approach to Assess Vascular Outcomes following Exposure to Environmental Chemicals in Zebrafish.

BACKGROUND: Disruptions in vascular formation attributable to chemical insults is a pivotal risk factor or potential etiology of developmental defects and various disease settings. Among the thousands of chemicals threatening human health, the highly concerning groups prevalent in the environment and detected in biological monitoring in the general population ought to be prioritized because of their high exposure risks. However, the impacts of a large number of environmental chemicals on vasculature are far from understood. The angioarchitecture complexity and technical limitations make it challenging to analyze the entire vasculature efficiently and identify subtle changes through a high-throughput in vivo assay. OBJECTIVES: We aimed to develop an automated morphometric approach for the vascular profile and assess the vascular morphology of health-concerning environmental chemicals. METHODS: High-resolution images of the entire vasculature in Tg(fli1a:eGFP) zebrafish were collected using a high-content imaging platform. We established a deep learning-based quantitative framework, ECA-ResXUnet, combined with MATLAB to segment the vascular networks and extract features. Vessel scores based on the rates of morphological changes were calculated to rank vascular toxicity. Potential biomarkers were identified by vessel-endothelium-gene-disease integrative analysis. RESULTS: Whole-trunk blood vessels and the cerebral vasculature in larvae exposed to 150 representative chemicals were automatically segmented as comparable to human-level accuracy, with sensitivity and specificity of 95.56% and 95.81%, respectively. Chemical treatments led to heterogeneous vascular patterns manifested by 31 architecture indexes, and the common cardinal vein (CCV) was the most affected vessel. The antipsychotic medicine haloperidol, flame retardant 2,2-bis(chloromethyl)trimethylenebis[bis(2-chloroethyl) phosphate], and tert-butylphenyl diphenyl phosphate ranked as the top three in vessel scores. Pesticides accounted for the largest group, with a vessel score of ≥1, characterized by a remarkable inhibition of subintestinal venous plexus and delayed development of CCV. Multiple-concentration evaluation of nine per- and polyfluoroalkyl substances (PFAS) indicated a low-concentration effect on vascular impairment and a positive association between carbon chain length and benchmark concentration. Target vessel-directed single-cell RNA sequencing of fli1a+ cells from larvae treated with λ-cyhalothrin, perfluorohexanesulfonic acid, or benzylbutyl phthalate, along with vessel-endothelium-gene-disease integrative analysis, uncovered potential associations with vascular disorders and identified biomarker candidates. DISCUSSION: This study provides a novel paradigm for phenotype-driven screenings of vascular-disrupting chemicals by converging morphological and transcriptomic profiles at a high-resolution level, serving as a powerful tool for large-scale toxicity tests. Our approach and the high-quality morphometric data facilitate the precise evaluation of vascular effects caused by environmental chemicals. https://doi.org/10.1289/EHP13214.

Environmentally relevant concentrations of fipronil selectively disrupt venous vessel development in zebrafish embryos/larvae.

The pesticide fipronil is widely dispersed in aquatic environments and frequently detected in the general population. Although the adverse effects on embryonic growth by fipronil exposure have been extensively documented, the early responses for its developmental toxicity are largely unknown. In the present study, we explored the sensitive targets of fipronil, focusing on vascular injury using zebrafish embryos/larvae and cultured human endothelial cells. Exposure to 5-500 µg/L fipronil at the early stage impeded the growth of sub-intestinal venous plexus (SIVP), caudal vein plexus (CVP), and common cardinal veins (CCV). The damages on venous vessels occurred at exposure to the environmentally relevant concentration as low as 5 µg/L fipronil, whereas no significant change was observed in general toxicity indexes. In contrast, vascular development of the dorsal aorta (DA) or intersegmental artery (ISA) was not affected. In addition, the mRNA levels of vascular markers and vessel type-specific function genes exhibited significant decreases in venous genes, including nr2f2, ephb4a, and flt4, but no appreciable change in arterial genes. Likewise, the more pronounced changes in cell death and cytoskeleton disruption were shown in human umbilical vein endothelial cells as compared with human aortic endothelial cells. Furthermore, molecular docking supported a stronger affinity of fipronil and its metabolites to the proteins correlated with venous development, such as BMPR2 and SMARCA4. These results reveal the heterogeneity in developing vasculature responsive to fipronil's exposure. The preferential impacts on the veins confer higher sensitivity, allowing them to be appropriate targets for monitoring fipronil's developmental toxicity.

[Artesunate combined with vinorelbine plus cisplatin in treatment of advanced non-small cell lung cancer: a randomized controlled trial].

OBJECTIVE: To our knowledge, there has been no clinical report of artesunate in the treatment of lung cancer. This study was designed to compare the efficacy and toxicity of artesunate combined with NP (a chemotherapy regimen of vinorelbine and cisplatin) and NP alone in the treatment of advanced non-small cell lung cancer (NSCLC). METHODS: One hundred and twenty cases of advanced NSCLC were randomly divided into simple chemotherapy group (control group, n=60) and combined artesunare with chemotherapy group (trial group, n=60). Patients in the control group were treated with NP regimen, including vinorelbine (25 mg/m(2), once-a-day intravenous injection, at the 1st and 8th day) and cisplatin (25 mg/m(2), once-a day intravenous drip, at the 2nd to 4th day). Patients in the trial group were treated with the basal therapy NP (in the same method and doses as control group) and artesunate (120 mg, once-a-day intravenous injection, from the 1st day to 8th day, for 8 days). At least two 21-day-cycles of treatment were performed. The short-term survival rate, disease controlled rate (DCR), time to progression (TTP), mean survival time (MST) and 1-year survival rate were analyzed as the primary end points, and the toxicity and safety were estimated. RESULTS: There were no significant differences in the short-term survival rate, MST and 1-year survival rate between the trial group and the control group, which were 45.1% and 34.5%, 44 weeks and 45 weeks, 45.1% and 32.7%, respectively (P>0.05). The DCR of the trial group (88.2%) was significantly higher than that of the control group (72.7%) (P<0.05), and the trial group's TTP (24 weeks) was significantly longer than that of the control group (20 weeks) (P<0.05). No significant difference was found in toxicity between the two groups, such as myelosuppression and digestion reaction (P>0.05). CONCLUSION: Artesunate can be used in the treatment of NSCLC. Artesunate combined with NP can elevate the short-term survival rate and prolong the TTP of patients with advanced NSCLC without extra side effects.

QM/MM-PB/SA scoring of the interaction strength between Akt kinase and apigenin analogues.

Identification of small-molecule compounds that can bind specifically and stably to protein targets of biological interest is a challenge task in structure-based drug design. Traditionally, several fast approaches such as empirical scoring functions and free energy analysis have been widely used to fulfill for this purpose. In the current study, we raised the rigorous quantum mechanics/molecular mechanics in combination with semi-empirical Poisson-Boltzmann/surface area (QM/MM-PB/SA) as an efficient strategy to characterize the intermolecular interaction between Akt kinase and its small-molecule ligands, although this hybrid approach is computationally expensive as compared to those empirical methods. In a round of experimental activity reproduction test based on a set of known Akt-inhibitor complexes, QM/MM-PB/SA has been shown to perform much better than two widely used scoring functions as well as the sophisticated MM-PB/SA analysis with or without improvement by molecular dynamics (MD) simulations. Next, the QM/MM-PB/SA was employed to screen for strong Akt binders from an apigenin analogue set. Consequently, four compounds, namely apigenin, quercetin, gallocatechin and myricetin, were suggested to have high binding potency to Akt active site. A further kinase assay was conducted to determine the inhibitory activity of the four promising candidates against Akt kinase, resulting in IC50 values of 38.4, 67.5, 157.1 and 25.5 nM, respectively.