Research On The Nuclear Receptors Mediated Toxicity Of Typical Organic Pollutants

The ecological and health risk assessment of organic pollutants has been one of the frontier issues and research hotspots in environmental science.Pesticides and polycyclic aromatic hydrocarbons(PAHs)are two typical environmental organic pollutants,and a considerable number of studies have confirmed the presence of their parent compounds and derivatives in various environmental media.Chitin synthetic inhibitors(CSIs),a new class of third generation insecticides,are considered as promising alternatives to conventional insecticides,and have been wildly used in agriculture,forestry and animal husbandry to control diseases,insects and weeds.PAHs are well-recognized persistent toxic substances(PTSs),they are easily converted to various derivatives through photochemical reactions,among which oxygenated PAHs(OPAHs)and nitrated PAHs(NPAHs)with low residue and high toxicity have attracted much attention.At present,toxicological evidence regarding the potential ecological safety and health risks of these organic pollutants is still very limited,especially the systematic research on exposure-effect-mechanism.Therefore,this study has introduced a nuclear receptor mediated high-throughput screening platform to rapidly and efficiently identify the active chemicals and clarify their toxic mechanism,the results will be helpful in establishing the technical support and scientific basis for environmental safety assessment and risk management of organic pollutants.The main achievements are shown as follows:1.China is the leading producer and consumer of pesticides worldwide.CSIs have been widely used in agriculture,forestry and animal husbandry due to their delayed biotoxicity and arthropod specificity.However,accumulating evidence has demonstrated that CSIs may cause detrimental effects on non-target organisms at sublethal concentrations,which highlights the necessity of further ecological and health risk investigations of these chemicals.In this chapter,the agonistic effects of fourteen CSIs on peroxisome proliferator-activated receptor ?(PPAR?)and estrogen receptor ?(ER?)were evaluated using an in vitro dual-luciferase reporter gene assay.Besides,molecular docking was employed to simulate the interactions between active chemicals and receptors,and to explore the possible binding patterns and pockets.In combination with in vivo and in vitro models,the CSIs-induced energy metabolic disorder and the potential molecular mechanism were further elucidated by detecting the expression levels of key signaling molecules and genes.The results showed that five CSIs exhibited apparent PPAR? agonistic effects,the order of relative agonistic potencies according to the 20% relative effective concentration(REC20)was diflubenzuron(DFB)> chlorfluazuron(CFZ)>flucycloxuron(FCX)>noviflumuron(NFM)>flufenoxuron(FF).Surprisingly,none of the fourteen chemicals showed ER? agonistic activities.The results of molecular docking indicated that different binding patterns(hydrogen-or ?-bonding)may stabilize the interactions between five active CSIs and PPAR?.Furthermore,in exposure of DFB,the ATP levels were decreased significantly in both HepG2 cells and zebrafish larvae,and the contents of pyruvate and lactate were increased in a dose-dependent manner.Meanwhile,the m RNA expression of mitochondrial matrix enzyme and four rate-limiting enzymes in the tricarboxylic acid(TCA)cycle were blocked in response to DFB exposure,while two target genes in glycolysis exhibited increased expression.In addition,the pretreatment of PPAR? antagonist(GW9662)effectively alleviated the changes of cellular energy metabolites induced by DFB.In conclusion,these findings suggest that CSIs may act as PPAR? agonists to impede the TCA cycle and accelerate glycolysis progress,and eventually induced energy metabolic disorder.2.Recently,CSIs were reported to exhibit carcinogenic potential.Cyromazine(CMZ)has been classified as a potential carcinogen and its lifetime cancer risk was 0.02 to 1 per million.However,research concerning the carcinogenic risks of CSIs is scarce.Hypoxia signaling pathway has become a hot topic in cancer research,and the activation of hypoxia inducible factor-1?(HIF-1?)is reported to be significantly correlated with tumor metastasis.Therefore,an in vitro reporter gene assay was conducted to screen the HIF-1? agonistic effects of fourteen CSIs.The influences of HIF-1?-agonistic CSIs on directional cell motility and invasion were observed by wound-healing and transwell invasion assays.In addition,the expression of epithelial-to-mesenchymal transition(EMT)and extracellular matrix(ECM)phenotype markers were also examined by quantitative real-time PCR(qRT-PCR).Among the fourteen CSIs,only DFB and triflumuron(TFM)exhibited dose-response HIF-1? agonistic activities at non-cytotoxic concentrations,with REC20 values of 2.11×10-5 and 7.92×10-5 M,respectively.Furthermore,HIF-1?-agonistic CSIs stimulated the invasion and migration of HepG2 cells in a dose-dependent manner.The results of qRT-PCR revealed that both DFB and TFM exposure promoted the activation of EMT and the degradation of ECM,thereby promoting the metastasis of HepG2 cells.Besides,the early embryos have been demonstrated to exhibit similar aggressive phenotypes with cancer cells.Also,DFB exposure caused obvious developmental toxicity in zebrafish embryos/larvae by regulating the expression of EMT and ECM phenotype markers.3.1-Nitropyrene(1-NP)and 3-nitrofluoranthene(3-NFA)exposure were reported to induce an accumulation of lipid droplets in mouse hepatoma cells,which revealing the lipotoxicity of PAHs and their derivatives.In this chapter,nine PAHs and their derivatives with high detection frequencies were selected,and their influences on PPAR? and ER? signaling pathways were investigated by luciferase reporter gene and enzyme-linked immunosorbent assay(ELISA).Thereafter,qRT-PCR and UHPLC-QTOF-MS based lipidomic platform were employed to clarify the cellular lipid metabolic responses to candidate chemicals.Furthermore,oil red O staining was further conducted to examine the abnormal lipid metabolism in the early developmental stage of zebrafish.Interestingly,there were no interference effects of PAHs and OPAHs on the nuclear receptor signaling pathways.As for NPAHs,three of them showed PPAR? agonistic activities and significantly promoted cellular biosynthesis of PPAR?,the order of their agonistic potencies was 9-nitroanthracene(9-NANT)> 2-nitrofluorene(2-NFL)> 3-NFA.Meanwhile,four NPAHs exhibited ER? agonistic properties: 3-NFA>1-NP>2-NFL>9-NANT,and stimulated the secretion of estradiol(E2)in HepG2 cells.The results of qRT-PCR showed that 3-NFA exposure increased the expression of lipid metabolism related genes in HepG2 cells.Moreover,the lipidomic analysis of cell samples revealed that a total of 53 different triacylglycerols(TAGs)and 2 cholesteryl esters(CEs)were evidently increased in response to 3-NFA exposure.Generally,based on responses observed in HepG2 cells,we speculate that 3-NFA might promote lipid accumulation by activating the PPAR? and ER? signaling pathways.Importantly,significant increases in PPAR? and E2 levels,along with transcriptional changes of lipid-sensitive genes,were observed in zebrafish larvae following 3-NFA exposure.Histological observation also showed an excessive lipid droplets deposit in the liver of larvae,thereby further supporting the lipid metabolic perturbation induced by 3-NFA.4.There is broad consensus on the carcinogenicity of PAHs,however,the potential role of HIF-1? is ambiguous.In this chapter,the HIF-1? agonistic effects of PAHs and their derivatives were evaluated using the previously established reporter gene platform.In combination with in vivo and in vitro models,the expression levels of key markers in EMT and ECM were detected to investigate the metastasis-related toxicity induced by active chemicals.Among the nine PAHs and their derivatives,none of the PAHs and OPAHs exhibited disrupting properties against HIF-1?,two NPAHs showed obvious active effects with the REC20 values of 5.91×10-7(1-NP)and 1.49×10-6 M(2-NFL),respectively.Moreover,two candidate NPAHs induced the activation of EMT in both in vivo and in vitro models.Concretely,1-NP and 2-NFL exposure suppressed the m RNA expression of epithelial cell marker(E-cadherin,E-cad),while the levels of mesenchymal phenotypic marker(fibronectin,FN1)were increased evidently.Besides,the balance of matrix metalloproteinase-2(MMP-2)and its tissue inhibitor(TIMP-2)was broken,which leading to an increased ECM degradation.Together,the present study clarifies that NPAHs altered the expression of key genes in EMT and ECM at environmentally relevant levels and that HIF-1? may,at least in part,be responsible for these metastasis-related toxicities.