Effects Of Manganese Chloride And Divalent Metal Coexposure On Metal Homeostasis And Oxidative Damage In A549 Cells

Objective:To explore the regulatory functions of metal transporters on metal homeostasis and oxidative damage,we investigated metal concentrations,oxidative damage and expression of transporters in in vitro A549cells in presence of MnCl₂（Manganese Chloride）and/or other divalent metals.We aimed to evaluate the effects of other divalent metals on Mn（Manganese）-induced cyto-toxicity and its underlying toxicological mechanism,and to provide a scientific basis for the prevention and treatment of toxic effects caused by occupational manganese exposure.Methods:（1）After A549 cells were treated with 0,200 and 400μM MnCl₂ for 24 h,survival rates were determined by MTT assay.（2）After cells were treated with different concentrations of ZnSO₄（Zinc Sulfate）,FeSO₄（Ferrous Sulfate）,CuCl₂（Copper Chloride）,Mg Cl₂（Magnesium Chloride）,Ni Cl₂（Nickel Chloride）,Co Cl₂（Cobalt Chloride）and Cd Cl₂（Cadmium Chloride）for 24 h,respectively,survival rates were determined by MTT assay to choose the maximum inactive dose.Cells were exposed to manganese chloride and/or other divalent metals for 24 hr,and then were collected for subsequent experiments.（3）After cells were lysed by repeated freezing and thawing,intracellular concentrations of the above metals were detected by ICP-MS（Inductive Coupled Plasma Mass Spectrometer）.Protein contents were determined through BCA protein concentration assay kit supplied by Thermo Fisher.（4）ROS（Reactive oxygen species）levels were determined by DCFH-DA fluorescent probe,and SOD（Superoxide dismutase）activity was determined via WST-1 assay kit.（5）Total RNA was extracted by Trizol and then reversed to c DNA.The m RNA levels of the transporters SLC11A2（Solute carrier family 11 member 2,encoding DMT1）,SLC39A8（Solute carrier family 39 member 8,encoding ZIP8）,SLC39A14（Solute carrier family 39 member 14,encoding ZIP14）,SLC40A1（Solute carrier family 40 member 1,encoding FPN1）,ATP2C1(ATPase secretory pathway Ca²⁺transporting 1,encoding SPCA1)and ATP13A2（ATPase cation transporting 13A2）were detected via real-time fluorescence quantitative PCR（RT-q PCR）.Results:（1）Cell survival rates were decreased in a concentration-dependent and time-dependent manner upon Mn exposure.And it was decreased significantly（P<0.01）at the concentrations≥200μM compared to control group.Cell survival rates were significantly different among all groups（P<0.01）from 24 hr-incubation time.（2）Cell survival rates were decreased by increasing concentrations of other divalent metals.And survival rates were decreased significantly by treatment with Zn≥50μM,Fe≥30μM,Cu≥30μM,Mg≥200μM,Ni≥50μM,Co≥30μM,Cd≥2.0μM,respectively.（3）Intracellular Mn concentration was significantly increased（P<0.05）with the increase of Mn treatment,but it was more significantly decreased（P<0.05）with Zn-added treatment,while it was more significantly increased with the addition of other divalent metals（P<0.05）.（4）Intracellular divalent metals concentrations were increased with the increase of other divalent metals.Intracellular Zn and Mg concentrations were not affected by the increase of Mn concentration（P>0.05）,however intracellular concentrations of the other divalent metals were significantly decreased（P<0.05）.（5）Compared with the control,ROS level was significantly increased and SOD activity was decreased upon Mn exposure,while Zn addition treatment caused ROS level decreased（P<0.01）and SOD activity increased,respectively.In contrast,the other divalent metals-added treatment induced ROS level significantly increased and SOD activity significantly decreased（P<0.05）.（6）Compared with control group,DMT1,ZIP8,ZIP14,FPN1,SPCA1 and ATP13A2 m RNA levels were significantly increased（P<0.05）under Mn treatment.DMT1 m RNA was significantly increased by Cd-added treatment,while significantly decreased with the addition of the other divalent metals.ZIP8m RNA was significantly increased with the addition of Cu and Mg（P<0.05）,while ZIP14 m RNA had no significant difference with the addition of Co,besides that,ZIP8 and ZIP14 m RNA with the addition of the other divalent metals showed similar trend to DMT1 m RNA.Moreover,FPN1 m RNA was significantly decreased by adding Cu,Ni and Co treatment,while was significantly increased with the addition of the other divalent metals（P<0.05）;except that SPCA1 m RNA was significantly decreased with the addition of Fe and Mg while significantly increased by Cu-added treatment,ATP13A2 m RNA was significantly increased by Co-added treatment,while there was no significant difference with the addition of Zn and Cu,SPCA1 and ATP13A2 m RNA showed similar trend to FPN1 m RNA with the addition of the other divalent metals.Conclusions:（1）Intracellular Mn concentration was increased as Mn treatment increased,however,this increase was inhibited by Zn-added treatment,while was promoted by the other divalent metals-added treatment respectively.Other divalent metals treatment increased their intracellular individual metal concentrations respectively,which were inhibited in presence of Mn treatment,except for Zn or Mg.（2）Zn treatment decreased oxidative stress,while Mn and the other divalent metals treatment respectively increased oxidative stress.Zn-added treatment inhibited Mn-induced oxidative stress,however the other divalent metals-added treatment promoted Mn-induced oxidative stress.（3）m RNA expressions of transporters DMT1,ZIP8,ZIP14,FPN1,SPCA1and ATP13A2 were affected by Mn exposure together with other divalent metals.These transporters may play a regulatory role in the effect of other divalent metal on manganese homeostasis and Mn-caused mitochondrial oxidative damage.