Inhibition of the urea cycle by the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin increases serum ammonia levels in mice.

The aryl hydrocarbon receptor is a ligand-activated transcription factor known for mediating the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. TCDD induces nonalcoholic fatty liver disease (NAFLD)-like pathologies including simple steatosis that can progress to steatohepatitis with fibrosis and bile duct proliferation in male mice. Dose-dependent progression of steatosis to steatohepatitis with fibrosis by TCDD has been associated with metabolic reprogramming, including the disruption of amino acid metabolism. Here, we used targeted metabolomic analysis to reveal dose-dependent changes in the level of ten serum and eleven hepatic amino acids in mice upon treatment with TCDD. Bulk RNA-seq and protein analysis showed TCDD repressed CPS1, OTS, ASS1, ASL, and GLUL, all of which are associated with the urea cycle and glutamine biosynthesis. Urea and glutamine are end products of the detoxification and excretion of ammonia, a toxic byproduct of amino acid catabolism. Furthermore, we found that the catalytic activity of OTC, a rate-limiting step in the urea cycle was also dose dependently repressed. These results are consistent with an increase in circulating ammonia. Collectively, the repression of the urea and glutamate-glutamine cycles increased circulating ammonia levels and the toxicity of TCDD.

Crosstalk of MAP3K1 and EGFR signaling mediates gene-environment interactions that block developmental tissue closure.

Aberrant regulation of signal transduction pathways can adversely derail biological processes for tissue development. One such process is the embryonic eyelid closure that is dependent on the mitogen-activated protein kinase kinase kinase 1 (MAP3K1). Map3k1 KO in mice results in defective eyelid closure and an autosomal recessive eye-open at birth phenotype. We have shown that in utero exposure to dioxin, a persistent environmental toxicant, induces the same eye defect in Map3k1+/- heterozygous but not WT pups. Here, we explore the mechanisms of the Map3k1 (gene) and dioxin (environment) interactions (GxE) underlying defective eyelid closure. We show that, acting through the aryl hydrocarbon receptor, dioxin activates epidermal growth factor receptor signaling, which in turn depresses MAP3K1-dependent Jun N-terminal kinase (JNK) activity. The dioxin-mediated JNK repression is moderate but is exacerbated by Map3k1 heterozygosity. Therefore, dioxin exposed Map3k1+/- embryonic eyelids have a marked reduction of JNK activity, accelerated differentiation and impeded polarization in the epithelial cells. Knocking out Ahr or Egfr in eyelid epithelium attenuates the open-eye defects in dioxin-treated Map3k1+/- pups, whereas knockout of Jnk1 and S1pr that encodes the sphigosin-1-phosphate (S1P) receptors upstream of the MAP3K1-JNK pathway potentiates the dioxin toxicity. Our novel findings show that the crosstalk of aryl hydrocarbon receptor, epidermal growth factor receptor, and S1P-MAP3K1-JNK pathways determines the outcome of dioxin exposure. Thus, gene mutations targeting these pathways are potential risk factors for the toxicity of environmental chemicals.

Cell-specific AHR-driven differential gene expression in the mouse liver cell following acute TCDD exposure.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental contaminant that disrupts hepatic function leading to steatotic liver disease (SLD)-like pathologies, such as steatosis, steatohepatitis, and fibrosis. These effects are mediated by the aryl hydrocarbon receptor following changes in gene expression. Although diverse cell types are involved, initial cell-specific changes in gene expression have not been reported. In this study, differential gene expression in hepatic cell types was examined in male C57BL/6 mice gavaged with 30 µg/kg of TCDD using single-nuclei RNA-sequencing. Ten liver cell types were identified with the proportions of most cell types remaining unchanged, except for neutrophils which increased at 72 h. Gene expression suggests TCDD induced genes related to oxidative stress in hepatocytes as early as 2 h. Lipid homeostasis was disrupted in hepatocytes, macrophages, B cells, and T cells, characterized by the induction of genes associated with lipid transport, steroid hormone biosynthesis, and the suppression of ß-oxidation, while linoleic acid metabolism was altered in hepatic stellate cells (HSCs), B cells, portal fibroblasts, and plasmacytoid dendritic cells. Pro-fibrogenic processes were also enriched, including the induction retinol metabolism genes in HSCs and the early induction of anti-fibrolysis genes in hepatocytes, endothelial cells, HSCs, and macrophages. Hepatocytes also had gene expression changes consistent with hepatocellular carcinoma. Collectively, these findings underscore the effects of TCDD in initiating SLD-like phenotypes and identified cell-specific gene expression changes related to oxidative stress, steatosis, fibrosis, cell proliferation and the development of HCC.

Transcriptome analysis reveal the effect of freshwater sediments containing 2,3,7,8-tetrachlorodibenzo-p-dioxin on the Macrobrachium rosenbergii hepatopancreas, intestine, and muscle.

This research evaluated the hepatopancreas, intestine, and muscle transcriptome alternation of Macrobrachium rosenbergii, and to confirm the relative glycerophospholipid, cytochrome P450 system, and fatty acid metabolism gene expression in sediments containing 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) of 60 ng/sediment (g) and 700 ng/sediment (g) for 90 days of culture. Transcriptome analysis revealed that the TCDD sediment affected the hepatopancreatic metabolism of xenobiotics in M. rosenbergii via the cytochrome P450 system, drug metabolism-other enzymes, drug metabolism-cytochrome P450, chemical carcinogenesis, and lysosome function. Intestinal analysis also showed a similar phenomenon, but this finding was not observed in the muscle tissue. qPCR analysis indicated that the expression levels of APTG4, LPGAT1, ACHE, GPX4, ECHS1, ATP5B, FABP, and ACC in the hepatopancreatic and intestinal tissues decreased, but those in the muscle tissues did not. In summary, TCDD sediment induced tissue metabolism, especially in the hepatopancreas and intestine. TCDD sediment mainly affected the digestive enzyme gene expression with concentration. These results indicated that the presence of TCDD in the sediment played a major role in the hepatopancreatic and intestinal metabolism system of M. rosenbergii.

Polychlorinated Naphthalenes in Foods from the French Market: Occurrence, Dietary Exposure, and Evaluation of Relative Contributions to Dioxin-like Contaminants.

Despite the growing interest in PCNs and the dioxin-like toxicity exhibited by a number of congeners, a comprehensive assessment of their contribution to the cocktail of dioxin-like contaminants is still lacking. To address such a shortcoming, this study investigated the PCN contamination in foodstuffs recently acquired in France, together with that of the regulatory polychlorinated dibenzodioxins/furans (PCDD/Fs) and polychlorinated biphenyls (PCBs). PCNs were ubiquitous at levels (∑70 PCNs = 2.5-150 pg g-1 wet weight) similar to those reported in other countries, with maximum concentrations observed in fish and fishery products from the North-East Atlantic Ocean. Their congener patterns further suggested unintentional releases of PCNs, while those of the other foodstuffs were correlated to the historical PCN profiles. Low risk from dietary exposure was estimated (∑70 PCNs-EDIs of 60-360 pg kg-1 bw d-1, ∑24 PCNs-TEQ-EDIs of 8 × 10-3-2.2 × 10-2 pg TEQ kg-1 bw d-1), with milk and dairy products being the highest contributors, followed by meat and meat products. Finally, the rather high contributions of PCNs to the total PCNs+PCDD/Fs+PCBs concentrations (0.9-50%, average of 9%) and the toxic equivalents (0.2-24%, average of 5%) show that these substances are not minor components of the PCNs+PCDD/Fs+PCBs cocktail.

Gene Target Prediction of Environmental Chemicals Using Coupled Matrix-Matrix Completion.

Human exposure to toxic chemicals presents a huge health burden. Key to understanding chemical toxicity is knowledge of the molecular target(s) of the chemicals. Because a comprehensive safety assessment for all chemicals is infeasible due to limited resources, a robust computational method for discovering targets of environmental exposures is a promising direction for public health research. In this study, we implemented a novel matrix completion algorithm named coupled matrix-matrix completion (CMMC) for predicting direct and indirect exposome-target interactions, which exploits the vast amount of accumulated data regarding chemical exposures and their molecular targets. Our approach achieved an AUC of 0.89 on a benchmark data set generated using data from the Comparative Toxicogenomics Database. Our case studies with bisphenol A and its analogues, PFAS, dioxins, PCBs, and VOCs show that CMMC can be used to accurately predict molecular targets of novel chemicals without any prior bioactivity knowledge. Our results demonstrate the feasibility and promise of computationally predicting environmental chemical-target interactions to efficiently prioritize chemicals in hazard identification and risk assessment.

Dioxin(-like)-Related Biological Effects through Integrated Chemical-wide and Metabolome-wide Analyses.

Dioxin(-like) exposures are linked to adverse health effects, including cancer. However, metabolic alterations induced by these chemicals remain largely unknown. Beyond known dioxin(-like) compounds, we leveraged a chemical-wide approach to assess chlorinated co-exposures and parent compound products [termed dioxin(-like)-related compounds] among 137 occupational workers. Endogenous metabolites were profiled by untargeted metabolomics, namely, reversed-phase chromatography with negative electrospray ionization (C18-negative) and hydrophilic interaction liquid chromatography with positive electrospray ionization (HILIC-positive). We performed a metabolome-wide association study to select dioxin(-like) associated metabolic features using a 20% false discovery rate threshold. Metabolic features were then characterized by pathway enrichment analyses. There are no significant features associated with polychlorinated dibenzo-p-dioxins (PCDDs), a subgroup of known dioxin(-like) compounds. However, 3,110 C18-negative and 2,894 HILIC-positive features were associated with at least one of the PCDD-related compounds. Abundant metabolic changes were also observed for polychlorinated dibenzofuran-related and polychlorinated biphenyl-related compounds. These metabolic features were primarily enriched in pathways of amino acids, lipid and fatty acids, carbohydrates, cofactors, and nucleotides. Our study highlights the potential of chemical-wide analysis for comprehensive exposure assessment beyond targeted chemicals. Coupled with advanced endogenous metabolomics, this approach allows for an in-depth exploration of metabolic alterations induced by environmental chemicals.

Evidence for crosstalk between the aryl hydrocarbon receptor and the translocator protein in mouse lung epithelial cells.

Cellular homeostasis requires the use of multiple environmental sensors that can respond to a variety of endogenous and exogenous compounds. The aryl hydrocarbon receptor (AHR) is classically known as a transcription factor that induces genes that encode drug metabolizing enzymes when bound to toxicants such as 2,3,7,8-tetrachlorodibenzo-ρ-dioxin (TCDD). The receptor has a growing number of putative endogenous ligands, such as tryptophan, cholesterol, and heme metabolites. Many of these compounds are also linked to the translocator protein (TSPO), an outer mitochondrial membrane protein. Given a portion of the cellular pool of the AHR has also been localized to mitochondria and the overlap in putative ligands, we tested the hypothesis that crosstalk exists between the two proteins. CRISPR/Cas9 was used to create knockouts for AHR and TSPO in a mouse lung epithelial cell line (MLE-12). WT, AHR-/-, and TSPO-/- cells were then exposed to AHR ligand (TCDD), TSPO ligand (PK11195), or both and RNA-seq was performed. More mitochondrial-related genes were altered by loss of both AHR and TSPO than would have been expected just by chance. Some of the genes altered included those that encode for components of the electron transport system and the mitochondrial calcium uniporter. Both proteins altered the activity of the other as AHR loss caused the increase of TSPO at both the mRNA and protein level and loss of TSPO significantly increased the expression of classic AHR battery genes after TCDD treatment. This research provides evidence that AHR and TSPO participate in similar pathways that contribute to mitochondrial homeostasis.

Loss of flavin-containing monooxygenase 3 modulates dioxin-like polychlorinated biphenyl 126-induced oxidative stress and hepatotoxicity.

Dioxin-like pollutants (DLPs), such as polychlorinated biphenyl 126 (PCB 126), are synthetic chemicals classified as persistent organic pollutants. They accumulate in adipose tissue and have been linked to cardiometabolic disorders, including fatty liver disease. The toxicity of these compounds is associated with activation of the aryl hydrocarbon receptor (Ahr), leading to the induction of phase I metabolizing enzyme cytochrome P4501a1 (Cyp1a1) and the subsequent production of reactive oxygen species (ROS). Recent research has shown that DLPs can also induce the xenobiotic detoxification enzyme flavin-containing monooxygenase 3 (FMO3), which plays a role in metabolic homeostasis. We hypothesized whether genetic deletion of Fmo3 could protect mice, particularly in the liver, where Fmo3 is most inducible, against PCB 126 toxicity. To test this hypothesis, male C57BL/6 wild-type (WT) mice and Fmo3 knockout (Fmo3 KO) mice were exposed to PCB 126 or vehicle (safflower oil) during a 12-week study, at weeks 2 and 4. Various analyses were performed, including hepatic histology, RNA-sequencing, and quantitation of PCB 126 and F2-isoprostane concentrations. The results showed that PCB 126 exposure caused macro and microvesicular fat deposition in WT mice, but this macrovesicular fatty change was absent in Fmo3 KO mice. Moreover, at the pathway level, the hepatic oxidative stress response was significantly different between the two genotypes, with the induction of specific genes observed only in WT mice. Notably, the most abundant F2-isoprostane, 8-iso-15-keto PGE2, increased in WT mice in response to PCB 126 exposure. The study's findings also demonstrated that hepatic tissue concentrations of PCB 126 were higher in WT mice compared to Fmo3 KO mice. In summary, the absence of FMO3 in mice led to a distinctive response to dioxin-like pollutant exposure in the liver, likely due to alterations in lipid metabolism and storage, underscoring the complex interplay of genetic factors in the response to environmental toxins.

Systematic update to the mammalian relative potency estimate database and development of best estimate toxic equivalency factors for dioxin-like compounds.

The World Health Organization (WHO) assesses potential health risks of dioxin-like compounds using Toxic Equivalency Factors (TEFs). This study systematically updated the relative potency (REP) database underlying the 2005 WHO TEFs and applied advanced methods for quantitative integration of study quality and dose-response. Data obtained from fifty-one publications more than doubled the size of the previous REP database (∼1300 datasets). REP quality and relevance for these data was assessed via application of a consensus-based weighting framework. Using Bayesian dose-response modeling, available data were modeled to produce standardized dose/concentration-response Hill curves. Study quality and REP data were synthesized via Bayesian meta-analysis to integrate dose/concentration-response data, author-calculated REPs and benchmark ratios. The output is a prediction of the most likely relationship between each congener and its reference as model-predicted TEF uncertainty distributions, or the 'best estimate TEF' (BE-TEF). The resulting weighted BE-TEFs were similar to the 2005 TEFs, though provide more information to inform selection of TEF values as well as to provide risk assessors and managers with information needed to quantitatively characterize uncertainty around TEF values. Collectively, these efforts produce an updated REP database and an objective, reproducible approach to support development of TEF values based on all available data.

The aryl hydrocarbon receptor suppresses immunity to oral squamous cell carcinoma through immune checkpoint regulation.

Immune checkpoint inhibitors represent some of the most important cancer treatments developed in the last 20 y. However, existing immunotherapy approaches benefit only a minority of patients. Here, we provide evidence that the aryl hydrocarbon receptor (AhR) is a central player in the regulation of multiple immune checkpoints in oral squamous cell carcinoma (OSCC). Orthotopic transplant of mouse OSCC cells from which the AhR has been deleted (MOC1AhR-KO) results, within 1 wk, in the growth of small tumors that are then completely rejected within 2 wk, concomitant with an increase in activated T cells in tumor-draining lymph nodes (tdLNs) and T cell signaling within the tumor. By 2 wk, AhR+ control cells (MOC1Cas9), but not MOC1AhR-KO cells up-regulate exhaustion pathways in the tumor-infiltrating T cells and expression of checkpoint molecules on CD4+ T cells (PD-1, CTLA4, Lag3, and CD39) and macrophages, dendritic cells, and Ly6G+ myeloid cells (PD-L1 and CD39) in tdLNs. Notably, MOC1AhR-KO cell transplant renders mice 100% immune to later challenge with wild-type tumors. Analysis of altered signaling pathways within MOC1AhR-KO cells shows that the AhR controls baseline and IFNγ-induced Ido and PD-L1 expression, the latter of which occurs through direct transcriptional control. These observations 1) confirm the importance of malignant cell AhR in suppression of tumor immunity, 2) demonstrate the involvement of the AhR in IFNγ control of PD-L1 and IDO expression in the cancer context, and 3) suggest that the AhR is a viable target for modulation of multiple immune checkpoints.

Transgenerational Transmission of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) Effects in Human Granulosa Cells: The Role of MicroRNAs.

Endocrine-disrupting chemicals (EDCs) might contribute to the increase in female-specific cancers in Western countries. 2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD) is considered the "prototypical toxicant" to study EDCs' effects on reproductive health. Epigenetic regulation by small noncoding RNAs (sncRNAs), such as microRNAs (miRNA), is crucial for controlling cancer development. The aim of this study was to analyze transcriptional activity and sncRNA expression changes in the KGN cell line after acute (3 h) and chronic (72 h) exposure to 10 nM TCDD in order to determine whether sncRNAs' deregulation may contribute to transmitting TCDD effects to the subsequent cell generations (day 9 and day 14 after chronic exposure). Using Affymetrix GeneChip miRNA 4.0 arrays, 109 sncRNAs were found to be differentially expressed (fold change < -2 or >2; p-value < 0.05) between cells exposed or not (control) to TCDD for 3 h and 72 h and on day 9 and day 14 after chronic exposure. Ingenuity Pathway Analysis predicted that following the acute and chronic exposure of KGN cells, sncRNAs linked to cellular development, growth and proliferation were downregulated, and those linked to cancer promotion were upregulated on day 9 and day 14. These results indicated that TCDD-induced sncRNA dysregulation may have transgenerational cancer-promoting effects.

A Comprehensive Evaluation of Dioxins and Furans Occurrence in River Sediments from a Secondary Steel Recycling Craft Village in Northern Vietnam.

This first study investigated the presence of dioxins and furans in river sediments around a craft village in Vietnam, focusing on Secondary Steel Recycling. Sediment samples were collected from various locations along the riverbed near the Da Hoi Secondary Steel Recycling village in Bac Ninh province. The analysis was conducted using a HRGC/HRMS-DFS device, detecting a total of 17 dioxin/furan isomers in all samples, with an average total concentration of 288.86 ng/kg d.w. The concentrations of dioxin/furan congeners showed minimal variation among sediment samples, ranging from 253.9 to 344.2 ng/kg d.w. The predominant compounds in the dioxin group were OCDD, while in the furan group, they were 1,2,3,4,6,7,8-HpCDF and OCDF. The chlorine content in the molecule appeared to be closely related to the concentration of dioxins and their percentage distribution. However, the levels of furan isomers did not vary significantly. The distribution of these compounds was not dependent on the flow direction, as they were mainly found in solid waste and are not water-soluble. Although the hepta and octa congeners had high concentrations, when converted to TEQ values, the tetra and penta groups (for dioxins) and the penta and hexa groups (for furans) contributed more to toxicity. Furthermore, the source of dioxins in sediments at Da Hoi does not only originate from steel recycling production activities but also from other combustion sites. The average total toxicity was 10.92 ng TEQ/kg d.w, ranging from 4.99 to 17.88 ng TEQ/kg d.w, which did not exceed the threshold specified in QCVN 43:2017/BTNMT, the National Technical Regulation on Sediment Quality. Nonetheless, these levels are still concerning. The presence of these toxic substances not only impacts aquatic organisms in the sampled water environment but also poses potential health risks to residents living nearby.

Global trends of dioxin and dioxin-like PCBs in animal-origin foods: a systematic review and gap areas.

Dioxins and dioxin-like polychlorinated biphenyls are a group of lipophilic compounds classified under persistent environmental pollutants (POPs). Significant sources of dioxin emissions include industrial effluents, open burning practices, and biomedical and municipal waste incinerators. These emissions will enter the food chain and accumulate in animal-origin foods (AOFs). A systematic review was conducted to analyze the global levels of dioxins and dioxin-like PCBs in AOFs using PRISMA guidelines 2020. The data on the dioxin contamination in AOFs were extracted from 53 publications based on their presence in eggs, meat and meat products, milk and dairy products, marine fish and fish products, and freshwater fish and crabs. A gap analysis was conducted based on the systematic review to understand the grey areas to be focused on the  future. No trend of dioxin contamination in AOFs was observed. A significant gap area was found in the need for nationwide data generation in countries without periodic monitoring of AOFs for dioxin contamination. Source apportionment studies need to be explored for the dioxin contamination of AOFs. Large-scale screening tests of AOFs using DR-CALUX based on market surveys are required for data generation. The outcomes of the study will be helpful for stakeholders and policyholders in framing new policies and guidelines for food safety in AOFs.

Dioxin-elicited decrease in cobalamin redirects propionyl-CoA metabolism to the ß-oxidation-like pathway resulting in acrylyl-CoA conjugate buildup.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental contaminant that induces diverse biological and toxic effects, including reprogramming intermediate metabolism, mediated by the aryl hydrocarbon receptor. However, the specific reprogramming effects of TCDD are unclear. Here, we performed targeted LC-MS analysis of hepatic extracts from mice gavaged with TCDD. We detected an increase in S-(2-carboxyethyl)-L-cysteine, a conjugate from the spontaneous reaction between the cysteine sulfhydryl group and highly reactive acrylyl-CoA, an intermediate in the cobalamin (Cbl)-independent ß-oxidation-like metabolism of propionyl-CoA. TCDD repressed genes in both the canonical Cbl-dependent carboxylase and the alternate Cbl-independent ß-oxidation-like pathways as well as inhibited methylmalonyl-CoA mutase (MUT) at lower doses. Moreover, TCDD decreased serum Cbl levels and hepatic cobalt levels while eliciting negligible effects on gene expression associated with Cbl absorption, transport, trafficking, or derivatization to 5'-deoxy-adenosylcobalamin (AdoCbl), the required MUT cofactor. Additionally, TCDD induced the gene encoding aconitate decarboxylase 1 (Acod1), the enzyme responsible for decarboxylation of cis-aconitate to itaconate, and dose-dependently increased itaconate levels in hepatic extracts. Our results indicate MUT inhibition is consistent with itaconate activation to itaconyl-CoA, a MUT suicide inactivator that forms an adduct with adenosylcobalamin. This adduct in turn inhibits MUT activity and reduces Cbl levels. Collectively, these results suggest the decrease in MUT activity is due to Cbl depletion following TCDD treatment, which redirects propionyl-CoA metabolism to the alternate Cbl-independent ß-oxidation-like pathway. The resulting hepatic accumulation of acrylyl-CoA likely contributes to TCDD-elicited hepatotoxicity and the multihit progression of steatosis to steatohepatitis with fibrosis.

Molecular Evolution of Aryl Hydrocarbon Receptor Signaling Pathway Genes.

The Aryl hydrocarbon receptor is an ancient transcriptional factor originally discovered as a sensor of dioxin. In addition to its function as a receptor of environmental toxicants, it plays an important role in development. Although a significant amount of research has been carried out to understand the AHR signal transduction pathway and its involvement in species' susceptibility to environmental toxicants, none of them to date has comprehensively studied its evolutionary origins. Studying the evolutionary origins of molecules can inform ancestral relationships of genes. The vertebrate genome has been shaped by two rounds of whole-genome duplications (WGD) at the base of vertebrate evolution approximately 600 million years ago, followed by lineage-specific gene losses, which often complicate the assignment of orthology. It is crucial to understand the evolutionary origins of this transcription factor and its partners, to distinguish orthologs from ancient non-orthologous homologs. In this study, we have investigated the evolutionary origins of proteins involved in the AHR pathway. Our results provide evidence of gene loss and duplications, crucial for understanding the functional connectivity of humans and model species. Multiple studies have shown that 2R-ohnologs (genes and proteins that have survived from the 2R-WGD) are enriched in signaling components relevant to developmental disorders and cancer. Our findings provide a link between the AHR pathway's evolutionary trajectory and its potential mechanistic involvement in pathogenesis.

Chorioallantoic membrane assay revealed the role of TIPARP (2,3,7,8-tetrachlorodibenzo-p-dioxin-inducible poly (ADP-ribose) polymerase) in lung adenocarcinoma-induced angiogenesis.

BACKGROUND: The chorioallantoic membrane (CAM) assay is a well-established technique to evaluate tumor invasion and angiogenesis and may overcome the shortcoming of the patient-derived xenograft (PDX) mouse model. Currently, few reports have described lung cancer invasion and angiogenesis in the CAM assay. We therefore used the CAM assay in the evaluation of lung cancer. METHOD: Lung cancer cell line-derived organoids or lung cancer cell lines were transplanted into the CAM on embryonic development day (EDD) 10, and an analysis was performed on EDD 15. Microscopic and macroscopic images and movies of the grafts on the CAM were captured and analyzed. The relationships between the graft and chick vessels were evaluated using immunohistochemistry. RESULTS: We transplanted lung cancer cell lines and cell line-derived organoid into a CAM to investigate angiogenesis and invasion. They engrafted on the CAM at a rate of 50-83%. A549-OKS cells showed enhanced cell invasion and angiogenesis on the CAM in comparison to A549-GFP cells as was reported in vitro. Next, we found that A549-TIPARP cells promoted angiogenesis on the CAM. RNA-seq identified 203 genes that were upregulated more than twofold in comparison to A549-GFP cells. A pathway analysis revealed many upregulated pathways related to degradation and synthesis of the extracellular matrix in A549-TIPARP cells. CONCLUSIONS: The CAM assay can be used to evaluate and research invasion and angiogenesis in lung cancer. The elevated expression of TIPARP in lung cancer may induce angiogenesis by remodeling the extracellular matrix.

Occurrence and Formation Mechanism of PCDD/Fs and SCCPs in Chlorinated Paraffin Products.

Polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) and short-chain chlorinated paraffins (SCCPs) can be formed during the production of chlorinated paraffins (CPs). Detection and accurate quantification of PCDD/Fs in CPs are challenging because of their matrix complexity. Therefore, the occurrence and formation mechanisms of PCDD/Fs from CPs have not been studied extensively in the past. In this study, 15 commercial samples including solid and liquid CPs were collected in 2022 from China. The average ΣSCCP concentrations detected in the solid and liquid CPs were 158 and 137 mg/g, respectively. The average International Toxic Equivalent (I-TEQ) values of 2,3,7,8-PCDD/F in solid and liquid CPs were 15.8 pg I-TEQ/g and 15.0 pg I-TEQ/g, respectively. The solid and liquid CPs had different predominant congener groups for SCCPs and PCDD/Fs. Possible formation routes for the generation of PCDD/Fs were analyzed by screening precursors in paraffin and laboratory-scale thermochemical experiments of CPs. The transformation between 2,3,7,8-PCDD/Fs and non-2,3,7,8-PCDD/Fs was recognized by calculating the successive chlorination preference. The first reported occurrence of PCDD/Fs in CP commercial products indicated that exposure to CPs and downstream products might be an assignable source of PCDD/F emission, which is of great significance to further explore the control factors of PCDD/Fs in the whole life cycle of CPs.

Congener Variation of Genetic Dependent-Developmental Toxicology in Two Emerging Classes of Dioxin-like Compounds.

Emerging classes of dioxin-like compounds (DLCs) like hydroxylated/methoxylated polybrominated diphenyl ethers (HO-/MeO-PBDEs) and polychlorinated diphenyl sulfides (PCDPSs) could lead to diverse adverse outcomes in humans and wildlife, yet knowledge gaps exist in their molecular mechanisms associated with different structures following early life environmental exposure. This study integrated a genetic knockout technique and concentration-dependent reduced zebrafish transcriptome approach (CRZT) to unravel the toxicological pathways underpinning developmental toxicity of four HO-/MeO-PBDEs and five PCDPSs at environmentally relevant doses. Generally, the dependence of aryl hydrocarbon receptor (AhR) on the embryotoxicity and transcriptomic potencies induced by the HO-PBDEs and PCDPSs varied across different congeners. The knockout of the ahr2 gene led to 1.02- to 76.48-fold decreases of DLC-induced embryotoxicities and reduced the transcriptome-based potencies ranging from 1.38 to 2124.74 folds in the CRZT test. The fold changes denoting AhR-mediated potentials significantly increased with the increasing chlorination degrees of MeO-PBDEs and PCDPSs (p < 0.05). Moreover, ahr2 knockout primarily affected the DLC-induced early molecular responses relevant to DNA damage, enzyme activation, and organ development. Our integrated approach revealed the differential role of AhR in mediating the developmental toxicity of emerging DLCs possessing varied structures at environmentally relevant doses.

Mercury and methylmercury differentially modulate hepatic cytochrome P450 1A1 and 1A2 in vivo and in vitro.

The cytochrome P450 1 A (CYP1A) subfamily enzymes are involved in the metabolic activation of several xenobiotics to toxic metabolites and reactive intermediates, resulting ultimately in carcinogenesis. Mercury and halogenated aromatic hydrocarbons (HAHs), typified by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), are persistent environmental pollutants involved in the modulation of aryl hydrocarbon receptor (AHR) gene battery, including cytochrome P450 (CYP) genes. We previously investigated the effect of coexposure to either inorganic or organic mercury (Hg+2 and MeHg) with TCDD on CYP1A1 in vitro. Thus, we examined the impact of coexposure to Hg+2 or MeHg and TCDD on AHR-regulated genes (Cyp1a1/1a2) in vivo and in vitro. Therefore, male C57BL/6 mice were injected intraperitoneally with MeHg or Hg+2 (2.5 mg/kg) in the absence and presence of TCDD (15 µg/kg) for 6 or 24 h. The concentration-dependent effect of MeHg was examined in murine hepatoma Hepa1c1c7 cells. In vivo, both MeHg and Hg2+ inhibited the TCDD-mediated induction of Cyp1a1/1a2 mRNA levels. However, Only Hg2+ was able to inhibit the TCDD-mediated induction at posttranscriptional levels of CYP1A1/1A2 protein and catalytic activity, suggesting differential modulation effects by Hg+2 and MeHg. In addition, the inhibitory role of HO-1 (Heme oxygenase-1) on CYP1A activity induced by TCDD was investigated using a HO-1 competitive inhibitor, tin-mesoporphyrin, that partially restored the MeHg-mediated decrease in CYP1A1 activity. This study demonstrates that MeHg, alongside Hg2+ , can differentially modulate the TCDD-induced AHR-regulated genes (Cyp1a1/1a2) at different expression levels in C57BL/6 mice liver and Hepa1c1c7 cells.