Responses Of Inflammatory Signaling Pathways To MCLR And TiO₂NPs Exposure In Hepatocytes

Increasing evidence linking micropollutant exposure with biotoxicity necessitates an urgent need for better understanding the responses of early regulating signaling pathways, which will help specify and predict the health risks of micropollution. The purpose of this study was to understand the hepatitis risk of microcontaminants by activation of inflammatory signaling pathways under in vitro exposure condition. In consideration of the chemical damage from microcystins ubiquitously existing in southern aquatic environment and physical damage from widely applicated nanomaterials, the differential responses of inflammatory signaling pathways to these two kind micropollution in primary and inmortalized hepatocyte models were investigated. Temporal and dose-dependent activation of mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways were determined in response to single and combined exposure of microcystins and titanium dioxide nanoparticles (TiO2NPs), where typical MCLR and anatase, rutile were selected. In addition, ultrastructure observation by AFM and TEM disclosed mophological damage of hepatocytes exposure to MCLR and TiO2NPs, deciphering their possible contribution to dysfunction of signal transduction. Our study provides new insight on adverse outcome pathways for monitoring toxicity of exogenous stimulators at sub-cytotoxic levels. The main findings are as follows:(1)Activation of inflammatory MAPK and NF-κB signaling pathways by MCLR mediates the hepatitis risk of MCLR.Both primary mouse hepatocytes (PMHs) and human hepatoma cell line HepG2 were exposed to environmental relevant concentrations of 1-1000μg/L MCLR for 24 h. PMHs were more sensitive than HepG2, which elicited significant cytotoxicity at 25μg/L MCLR exposure indicated by protein degradation with EC50 values of 11.86±1.01-14.49±3.74 μg/L. The results of western blotting and luciferase reporter assay showed that MAPK and NF-κB pathways were significantly activated by sub-cytotoxic levels of MCLR in both cell types (p<0.05). Activation of MAPK and NF-κB pathways regulated downstream IL-6 cytokine expression, confirming the hepatitis risk of MCLR. Furthermore, the responses of MAPK and NF-κB pathways to combined exposure of MCLR and proinflammatory TNFa suggested that pre-exposure to MCLR sensitized hepatocytes and promoted TNFa-induced activation of MAPK/NF-κB pathways and IL-6 expression, uncovering the secondary exposure risk of MCLR under proinflammatory condition. Signal transduction dysregulation induced by MCLR was accompanied with mitochondria damage observed by TEM, which showed cristae and matrix lost in a progressive and dose-dependent manner.(2) Differential dynamic activation of MAPK and NF-κB signaling pathways are regulated by agglomeration of TiO2NPs.In HepG2 cells,5-160μg/mL TiO2NPs exposure significantly activated MAPK and NF-κB signaling pathways in a dose-dependent manner (p<0.05). However, the dynamic activation profiles of inflammatory signaling pathways were unique to each TiO2 NPs. p38 was phosphorylated by both TiO2 NPs in a dual-peak pattern at the early and middle exposure stages, while activation of ERK1/2 exhibited unimodal pattern, the maximum activation of which occurred earlier by rutile than by anatase. For NF-κB activation, rutile phosphorylated IκBα in a bell curve-like pattern and to a higher degree than anatase with a sigmoidal activation profile. This discrepancy was attributed to the second size effects of particle agglomerations, which showed that smaller agglomerated rutile had higher activation potential, and was consistent with its more pronounced cytotoxicity and immunogenicity. The results indicated that differential activation of MAPK and NF-κB pathways could be early predictors for distinct hepatitis risk of two agglomerated TiO2NPs. Furthermore, cell elasticity indicated by Young’s modulus and adhesion force increased accompanied with mitochondria damage, which might contribute to indirect signal transduction through deformation of cytoskeleton during nanoparticles endocytosis at biomembrane interface.(3) MAPK and NF-κB signaling pathways respond synergistically or antagonistically to combined exposure of MCLR and TiO2NPs.Phosphorylation of ERK1/2 and p38 pathways in HepG2 cells were significantly elevated under composite exposure of MCLR (10-1000 μg/L) and TiO2NPs(10-40 μg/mL) (p<0.05). For NF-κB pathway, MCLR weakened TiO2NPs induced IκBα phosphorylation. The dynamic responses of signaling pathways also confirmed that combined exposure of MCLR and TiO2NPs advanced maximum activation of ERK1/2 from 12 h to 8 h, and p38 from 4 h to 1 h, while the dynamic phosphorylation of IκBα was not affacted. Response surface analysis of signaling pathway activation as well as coefficient of drug interaction (CDI) demonstrated that MCLR and TiO2NPs synergistically activated MAPK pathways, while antagonistically activated NF-κB pathway. DLS results showed a significant reduction of TiO2NPs Zeta potential and anatase agglomeration size with MCLR addition, which might influence the cellular transportation of MCLR adsorbed on TiO2NPs and the specific combined responses of inflammatory signaling pathways compared to single exposure.