Research On Endocrine Disruption Effect Induced By Parabens And Its Molecular Mechanism

Parabens(PBs)are alkyl or aryl homologs of the p-hydroxybenzoic acid(p-HBA),which have widely used as preservatives in cosmetics,pharmaceuticals,food,and products for children.PBs have become a global emerging pollutant due to their ubiquity in the environment and humans.Humans are exposed to parabens via the dermal absorption,ingestion,and inhalation,and then bioaccumulation in some tissues,raising concerns about their potential health effects.Epidemiological studies have showed that PBs in human urine has a non-negligible correlation with women’s menstrual cycle,male sperm quality,type 2diabetes mellitus,childhood sensitization and gestational diabetes.Meanwhile,toxicological experimental studies have also demonstrated the association between paraben and human health effects,including reproductive damage,developmental impairment,metabolic disorders,carcinogenic teratogenicity,and endocrine diseases.Although the estrogenic activity of some parabens has been confirmed,the underlying mechanisms on organisms and the structure-estrogenic activity relationship are still largely unclear.In this study,the endocrine disrupting effect of PBs was evaluated by constructing three experimental strategies at in vitro-in vivo-molecular initiating events,and the possible toxic effects and mechanisms of PBs with different side chains were analyzed systematically and comprehensively.It provides help to elucidate the comprehensive toxic mechanism of PBs and provides a theoretical basis for comprehensive safety evaluation and risk management.The following conclusions were obtained:1.To investigate the estrogenic disrupting effect of PBs from the cellular level,PBs with different concentrations and different side chain were exposed to MCF-7 cells,and the results showed that the tested PBs can promoted MCF-7 proliferation by concentrationdependently with the order of Bz P > Bu P > Pr P > Et P > Me P,indicating that the estrogenic effects were mainly determined by the side chain alkyl groups and aromatic groups of PBs compounds,and Bz P with an aromatic ring showed the strongest estrogenic effect among all tested PBs.In order to further study the correlation between the proliferation of MCF-7and estrogen receptor,luciferase activity in MVLN cells were used to investigate the PBs interaction with receptors directly.Consistent with the results of the MCF-7 cell proliferation assay,Bz P showed the strongest estrogenic activity with the significant activation effect observed at 0.01 μM and the EC50 at 0.796 ± 0.307 μM,while Me P with the highest EC50 at 131 ± 20.5 μM.The results showed that the xenoestrogenic effects of parabens at the cellular level are mediated via binding to ERs.2.To explore the endocrine disrupting effect of PBs on transcriptional levels in organisms,zebrafish embryos exposed to different PBs for 120 hpf for transcriptome sequencing analysis.The results showed that 3202 differentially transcripts were identified in zebrafish embryos exposed to Bz P,while just 548 in Me P group.The GO term enrichment analysis showed that the number of GO terms related to endocrine disrupting effect increased as the number of carbon atoms in the alkyl chain increased.Meanwhile,the KEGG enrichment analysis showed that the signaling pathways jointly affected by the tested PBs were endocrine-related signaling pathways,such as steroid hormone synthesis and metabolism pathways,cytochrome P450 metabolic pathways of exogenous substances,and peroxisome proliferator-activated receptor signaling pathways,etc.The expression of sexrelated genes and the changes of sex hormone content were investigated to further verify the results of transcriptomics,showing that PBs significantly increased the expression of ers1,ers2 a,cyp19a1b and vtg in zebrafish embryos.The content analysis of sex hormones in zebrafish juveniles showed that the contents of E2 and VTG increased significantly,and the content of 11-KT decreased significantly in each PBs exposure group,indicating that hormone synthesis and metabolism in zebrafish larvae were significantly affected by PBs.3.Metabolomic analysis was carried out to deeply explore the effect of PBs on the overall metabolic state of zebrafish.The results showed that the PBs could change the expression level of endogenous metabolites in larval zebrafish,and significantly affected differential metabolites,such as tyrosine,phenylalanine,formiminoglutamic acid,arginine,arachidonic acid,phosphocholine,lactic acid,cortisol,and cholesterol,etc.It was suggested that PBs affected tyrosine metabolism,phenylalanine metabolism,arginine and proline metabolism,glycine-alanine-aspartate metabolism,arachidonic acid metabolism,glycerophospholipid metabolism,and TCA cycle.Overall,the endocrine disrupting effect of PBs on zebrafish larvae could be closed related to the disturbances of amino acid,lipid,and energy metabolism,inferring that they have a comprehensive toxic effect on organisms.4.According to above studies,the endocrine disrupting effects of PBs on zebrafish can be preliminary identified.To further investigate their key molecular initiation mechanism,molecular dynamics were employed to analyze the interaction relationship between PBs and classical estrogen receptor ERs,showing a non-negligible interaction between PBs and human and zebrafish estrogen receptors.The stability and binding energy of PBs-ERs complex increased as the side chain length increased from 1 to 4,and Bz P showed the strongest property.The binding free energy of Pr P,Bu P and Bz P to ERs in different species showed significant differences,inclining to bind to zebrafish receptor zf ERβ2.MM-PBSA analysis found that van der Waals force was the main driver of ligand binding to receptors,while polar solvent dissolution was the main inhibitory force.Combining the computational and experimental results,we analyzed the probable structureactivity correlation between the endocrine disrupting activity of parabens.A significant linear correlation between the relative luciferase activity of MVLN by PBs and the calculated binding energies was demonstrated.Similarly,the content of VTG in zebrafish was correlated with the binding free energy of PBs binding to estrogen receptors.In conclusion,the endocrine disrupting effect of PBs was mainly determined by their side chain functional groups,which would be a rational prediction for optimizing the substitution of parabens.