Effect Of Cr(Ⅵ) On Mitochondrial Respiratory Chain Complexes In L-02Hepatpcytes

Objective:To explore the effect of Cr(VI) on the gene expression levels of mitochondria-encoded respiratory chain complex (RCC) subunits, the activities of respiratory chain complexs, the energy metabolism as well as the correlations between them, and to provide the experimental evidence for the molecular mechanism studies of Cr(Ⅵ)-induced mitochondrial energy metabolism dysfunction.Methods:L-02hepatocytes were treated with different concentrations of Cr(VI)(0,2,4,8,16,32,64and128μmol/L) respectively for24h and then were processed for the detection of cell survival rate by MTT assay. Three concentrations (low, moderate and high:2,8and32μmol/L) were chosen for the following studies. Total RNA was extracted from L-02hepatocytes using RNA extraction kit. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was applied to detect the mRNA levels of ND1, ND2, Cyt b, COX Ⅰ-Ⅲ, ATPase6and ATPase8. The content of ATP was measured by bioluminescence technique. The activity of mitochondrial respiratory chain complex V and the level of cellular ROS were determined by ultraviolet spectrophotometry and fluorometric methods respectively.Results:1. Cr(Ⅵ) decreased the survival rate of L-02hepatocytes:In the concentration range of2～128μmol/L, Cr(Ⅵ) significantly decreased the survival rate of L-02hepatocytes (P<0.05) in a dose-response manner. The hepatocytes survival rate and the Cr(Ⅵ) treatment concentrations showed significant negative correlation (r=-0.888, P<0.05).2. Cr(Ⅵ) induced ROS accumulation in L-02hepatocytes:Cr(Ⅵ) increased ROS levels in L-02hepatocytes treated with different concentrations of Cr(Ⅵ).Compared with control, the8and32μmol/L Cr(Ⅵ)-treated groups showed increased ROS levels with the obvious differences (P<0.05). A significant positive correlation has been found between the Cr(Ⅵ)-treated groups and control group (r=0.993, P<0.05).3. The effect of Cr(Ⅵ) on mitochondrial DNA gene expression levels:2μmol/L Cr(Ⅵ) treatment could increase the expression levels of Cyt b、ATPase6and ATPase8, and decrease the expression levels of ND2(P<0.05).8μmol/L Cr(Ⅵ) treatment could increase the expression levels of ND1, ND2, Cyt b, COX Ⅰ and COX Ⅲ, and decrease the expression levels of ATPase6and ATPase8(P<0.05).32μmol/L Cr(Ⅵ) treatment could increase the expression levels of ND1, ND2, COX Ⅰ and COX Ⅲ, and decrease the expression levels of ATPase6and ATPase8(P<0.05).4. The effect of Cr(Ⅵ) on the activities of RCC:Cr(Ⅵ) induced significant inhibition of RCC Ⅰ, Ⅲ, Ⅳand V activities in the2,8and32μmol/L Cr(Ⅵ) treatment groups (P<0.05). The decrease of RCC Ⅱ activities in different Cr(Ⅵ) treatment groups had no significance compared with that of control group (P>0.05).5. The effect of Cr(Ⅵ) on ATP content:Compared with the control group, the different Cr(Ⅵ) treatment groups showed significant decrease of ATP cellular content (P<0.05).Conclusion:Cr(Ⅵ)-induced ROS accumulation is correlated with the gene expression level changes of mitochondria-encoded RCC subunits. A close relationship was found between the gene expression levels of RCC subunits and the activities of RCC Ⅰ-Ⅴ. The translational change of mtDNA expression levels can induce the inhibition of RCC Ⅰ-Ⅴ, which further may account for the Cr(Ⅵ)-induced dysfunction of mitochondrial energy metabolism.