Antagonistic Drug Screening On The Combined Toxicity Of Multi-heavy Metals And Underlying Molecular Mechanism

Objective: To investigate the combined toxicity of Nickel(Ni),Zinc(Zn),Lead(Pb),Chromium(Cr),Cadmium(Cd),Copper(Cu),Mercury(Hg)and Manganese(Mn)according to human dietary exposure proportion and to select the effective antagonistic drugs,and then to explore the molecular mechanism.The research results will provide a scientific basis for prevention heavy metals-induced health hazards.Methods: The multi-heavy metal mixture stock solution including Ni,Zn,Pb,Cu,Cd,Hg,Cr and Zn was prepared according to the proportions of daily consumption of each metal through aqua products by an adult in Ningbo area.The effect of multi-heavy metal mixture on cell viability was detected by MTT assay;To screen effective antagonist drugs,HL7702 cells treated with 25.73 mg/L multi-heavy metal mixture alone or supplement with different concentrations of drugs(Epigallocatechin-3-Gallate(EGCG),Luteolin,L(+)-Cysteine,Taurine,Allicin,Curcumin,Dimercaprol,Natrii Dimercaptosuccinas,Sodium Dimercaptosulphonate and Dihydromyricetin);Assays of MDA detection,ROS detection,ATP detection,JC-1 staining,apoptosis detection and western blot analysis were used to explore the molecular mechanism.Results: The MTT results indicated that the heavy-metal mixture could lead to HL7702 cells viability reduction and cell shrinkage.The cytotoxicity of LC25,LC50,LC75 were 20.58,23.16,25.73mg/L,respectively.Luteolin and EGCG showed agonistic effects on the combined toxicity of the multi-heavy metal mixture,and could inhibit the morphological changes of HL7702 cells.Oxidative stress and lipid peroxidation detections showed that the level of cellular oxidation and lipid peroxidation in HL7702 cells were simultaneously increased when treated with multi-heavy metals alone.After addition of luteolin or EGCG,the level of reactive oxygen radicals was decreased.ATP detection assay demonstrated that ATP metabolism disorder occurred in HL7702 cells treated with multi-heavy metals,while this phenomenon could be significantly alleviated by luteolin.The mitochondrial membrane potential detection suggested that mitochondrial function destroyed after heavy metals exposure.Luteolin also showed significant prevention on multi-heavy metal mixture-induced mitochondrial damage.Apoptosis detected by flow cytometry indicated that both EGCG and luteolin could inhibit apoptosis induced by multi-heavy metal mixture.Western blot analysis and immunofluorescence staining showed that multi-heavy metal mixture could increase the ratio of Bax/Bcl-2 and expression level of HO-1 and MT protein,and further resulting in the release of cytochrome C into the cytoplasm,activation of Capase-9,Caspase-3,PARP,p38,JNK,and ERK protein,while luteolin could inhibit these changes,and EGCG could down-regulate the expression of p-p38,p-JNK,p-ERK and HO-1.Conclusions: Heavy metal mixture can induce HL7702 cells oxidative stress,lipid oxidation and mitochondrial function damage,upregulate antioxidant stress related protein HO-1 and MT,then activate MAPK protein signal pathway and Cytochrome c apoptosis signal pathway,induce cleaveage of Capase-9,Caspase-3,PARP protein,thereby induce apoptosis.Both EGCG and luteolin can protect HL7702 cells from heavy metal mixture-induced oxidative stress injury and down-regulation of MAPK signalling pathway;Luteolin can prevent mitochondrial membrane potential damage induced by heavy metal mixture,inhibit Cytochrome c release from mitochondria into cytoplasm,and then reduce the cleavage of Caspase-3 through blocking Cytochrome c/Apaf1/Caspase-9 apoptotic complex formation,thereby inhibit heavy metal mixture-induced HL7702 cell apoptosis.