A Study Of Nrf2/ARE Signaling Pathways On Oxidative Damage Induced By Nano-TiO₂in HepG₂Cell Line

Objective：Nanoparticle titanium dioxide (Nano-TiO2) is a nanomaterialwidely used in pharmaceuticals, chemical industry, environmental protection,cosmetics, plastics and paints because of its excellent physical and chemicalproperties. However, a number of recent studies show that the toxic effect onorganism produced by oxidative stress in the Nano-TiO2causes DNA damages,cell toxicity, inflammatory reaction, etc. and may produce damage effect onhuman health. Although oxidative stress caused by nanomaterials and theirtoxicity are highly correlated, its targets and regulatory mechanism are notclear. Since Nrf2/ARE signaling pathway is one of the key steps for the cellsto resist oxidative stress, it is of great significance to study the role of Nrf2/ARE signaling pathways in the health damage effect caused by nanomaterialsand its specific mechanism of action. This study aims to take Nrf2/AREsignaling pathway as the breakthrough point to conduct an in-depth study ofthe toxic mechanism of nanomaterials through observation of the effects ofNano-TiO2on the antioxidant function of HepG2cells and DNA damageseffects and identify the future targets and practical and feasible approaches ofprevention and control of cell toxicant function caused by nanomaterials.Methods:1Cell culture and cell modeling: The HepG2cell line (derived fromChina Center for Type Culture Collection (CCTCC) was cultured inDulbecco’s modified Eagle’s medium (DMEM) with10%heat inactivatedfetal calf serum (FCS) at37°C with a5%CO2saturated humidity. Cells in thelogarithmic growth phase were selected and exposed to contaminationtreatment of Nano-TiO2with a particle size of25mm and a concentration0.1μg/mL,1μg/mL and10μg/mL respectively; DMEM was taken as thenegative reference. 2Morphology observation on HepG2cells: the morphology change ofHepG2cells were observed by biological microscope observation.3SOD and GSH-Px activities as well as MDA content were detected bycommercial biochemical kits to evaluate the oxidative damage in HepG2cellsafter Nano-TiO2exposure.4Detect the ROS levels in HepG2cells with DCFH-DA as a fluorescentprobe by using flow cytometry to evaluate the oxidative stress.5Detect the DNA damage by using the SCGE to evaluate the effect ofNano-TiO2on DNA damage in HepG2cells.6The effects of Nano-TiO2on the ARE levels in HepG2cells：The HepG2cells were treated with different concentrations of Nano-TiO2for24h after transfected with pARE-LUC plasmid and pRL-TK plasmid.Protein concentration in the specimen is determined with the gene contained inthe dual-luciferase report and the values of firefly-and RL-luciferaseexpression are expressed respectively with fluorescence intensity/μg proteincontent to determine the effect of Nano-TiO2on the relative fluorescenceintensity of the transfected HepG2cells. The ratio of firefly-luciferase andrenilla-luciferase was calculated to evaluate the ARE levels in HepG2cells.7Nrf2, HO-1, NQO1, γ-GCS gene mRNA expression: the real time PCRdetection technology was used to detect Nrf2, HO-1, NQO1, γ-GCS mRNAexpression.8Nrf2, HO-1, NQO1, γ-GCS protein expression: the Nrf2, HO-1, NQO1and γ-GCS protein expression was detected by Western blot.Results:1The effects of Nano-TiO2on morphology of HepG2cellsThe HepG2cells were spindle, adherent compact growth, high refractiveindex and proliferation. After Nano-TiO2exposure, the number of cellsdecreased, cell shrinkas into circular, the refractive index decreases, theadherent ability drops and most of cells float in the culture fluid.2The effects of Nano-TiO2on oxidative damage in HepG2cellsWith the increase of concentration of Nano-TiO2, MDA content increased, compared with the control group, the differences were statisticallysignificant(P<0.05); the SOD activity gradually reduced, compared with thecontrol group,1and10ug/mL groups were significantly decreased(P<0.05);the GSH-PX activity gradually reduced,compared with the control group, thedifferences were statistically significant (P<0.05); the ROS level of allexposed groups rised, compared with the control group, the differences werestatistically significant (P<0.05).3The effects of Nano-TiO2on DNA damage in HepG2cellsThe olive tail moment of all exposed groups increased. Compared withthe control group, the differences were statistically significant (P<0.05).4The effects of Nano-TiO2on the level expression of ARE in HepG2cellsWith the increase of concentration of Nano-TiO2, relative fluorescenceintensity increased and the differences were statistically significant (P<0.05)and there was a good dose response relationship（r=0.999, P<0.05）. Comparedwith the control group, the expression level of ARE in the HepG2cells risedsignificantly with the increase of the concentration of Nano-TiO2(P<0.05).5The effects of Nano-TiO2on of Nrf2, NQO1, HO-1and γ-GCS mRNAin HepG2cellsCompared with the control group, the expression level of Nrf2andNQO1mRNA in all exposed groups rised significantly along with the increaseof the concentration of Nano-TiO2(P<0.05).The expression level of HO-1mRNA increased with the concentration ofNano-TiO2at a Nano-TiO2concentration of0.1and1μg/mL and a littledecreased at an concentration of Nano-TiO2of10μg/mL. Compared with thecontrol group, the HO-1mRNA were significantly increased after Nano-TiO2exposure (P<0.05).The expression level of γ-GCS mRNA increased at a Nano-TiO2concentration of0.1,1μg/mL Nano-TiO2and decreased at an concentration of10μg/mL Nano-TiO2. Except for the10μg/mL dose group, γ-GCS mRNAwere significantly increased, compared with the control (P<0.05). 6The effects of Nano-TiO2on the expression level of Nrf2, NQO1, HO-1and γ-GCS protein in HepG2cellsThe protein expression level of Nrf2increased at a Nano-TiO2concentration of0.1,1μg/mL and decreased at an concentration of prion of10μg/mL. Except for the10μg/mL dose group, Nrf2protein expression weresignificantly increased, compared with the control (P<0.05).Compared with the control group, the expression level of protein ofNQO1in HepG2cells rised significantly along with the increase of theconcentration of Nano-TiO2(P<0.05).The protein expression level of HO-1and γ-GCS increased with theconcentration of Nano-TiO2at a Nano-TiO2concentration of0.1,1μg/mL anda little decreased at an concentration of Nano-TiO2of10μg/mL.Comparedwith the control, HO-1and γ-GCS protein expression were significantlyincreased after Nano-TiO2exposure(P<0.05).Conclusions:1Nano-TiO2can induce antioxidant function damage and produceoxidative stress in HepG2cells.2Nano-TiO2can induce DNA damage in HepG2cells3After Nano-TiO2expose, antioxidant response elements (ARE) levels inHepG2cells increase to defeat the oxidant damage.4After Nano-TiO2expose, Nrf2/ARE signaling pathways is activated.Nrf2combines with ARE to function as an antioxidant. As a result, itsdownstream target genes NQO1, HO-1and γ-GCS are activated and thetranscriptional expression increases to help protect the cells from undesirablestimulation. When the concentration of Nano-TiO2is too large, someendogenous protective genes may lose the function of protection.