Effects Of Cr(â…¥) On Respiratory Function Of Mitochondria In Isolated Rat Hepatocyte

Objective:To explore the effects of Cr(Ⅵ) on respiratory function of mitochondria in isolated rat hepatocyte and investigate the protective effects of NAC on the respiratory dysfunction induced by Cr(Ⅵ).Methods:The mitochondria were randomly divided into eight groups, namely blank control group, NAC control group, ROT control group, ROT+NAC control group, Cr(Ⅵ) treated group, NAC+Cr(Ⅵ) treated group, ROT+Cr(Ⅵ) treated group and ROT+NAC+Cr(Ⅵ) treated group. In vitro, the mitochondria was exposed to Cr(Ⅵ) at 50μmol/L for 30 minutes after 30 mintues'pretreatment of mitochondira respiratiory chain complexⅠinhibitor roteone (ROT) at 10μmol/L and ROS scavenger NAC at 1mmol/L. ROS generatin, PTP open rate, mitochondrial membrane potential (MMP) are determined by a 960MC fluorescence spectrometer. Activities of respiratory chian complexes and ATPase are detected by an UV-7501 spectrophotometer. Oxygen consumption was measured in a Clack-type oxygen electrode. and mutation of mtDNA were investigated through PCR gene amplification technology.Results:1.Effect of Cr(Ⅵ) on generatino of ROS:Compared to control groups, generation of ROS in the Cr(Ⅵ) treated groups increased significantly (P<0.05). Compared to Cr(Ⅵ) alone treated group, generation of ROS in the ROT+Cr(Ⅵ) and NAC+ROT+Cr(Ⅵ) treated groups had a significant increase while in the NAC+Cr(Ⅵ) treated group had a significant decrease.2.Effect of Cr(Ⅵ) on PTP open:PTP open rate in the Cr(Ⅵ) treated groups increased significantly compared with the relative control groups(P<0.05). In NAC+Cr(Ⅵ) treated group, PTP open rate went down compared with Cr(Ⅵ) alone treated group. The pretreatment of ROT increased the PTP open rate.3.Effect of Cr(Ⅵ) on MMP(Δψm):MMP(Δψm) in the groups including Cr(Ⅵ) dropped significantly compared with that in control groups. ROT+Cr(Ⅵ) treated group had the most increase of light density while the light density increased slowly in NAC+Cr(Ⅵ) treated group. 4.Effect of Cr(Ⅵ) on activities of respiratory chain complexesⅠ-Ⅳ: The activities of mitochondria respiratory chain complexesⅠ-Ⅳin each Cr(Ⅵ) treated group decreased significantly(P<0.05):The activities of respiratory chain complexⅠ-Ⅳdeclined by 43.19%,19.2%,71.66% and 59.63%, respectively, in Cr(Ⅵ) treated group; decreased by 61.36%,40%,83.33% and 84.47%, respectively, in ROT+Cr(Ⅵ) treated group compared to that in control group. However, the activities of respiratory chain complexesⅡ-Ⅳincreased by 5%,11.54% and 15.38% respctively in NAC+Cr(Ⅵ) treated group compared with Cr(Ⅵ) alone treated group.5. Effect of Cr(Ⅵ) on activities of ATPase:Compared with the control groups, the activities of Na+K+-ATPase, Ca2+-ATPase and their sum all declined significantly in the groups treated with Cr(Ⅵ)(P<0.05). Compared with the Cr(Ⅵ) alone treated group, all the activities decreased in ROT+Cr(Ⅵ) treated group and increased in NAC+Cr(Ⅵ) treated group. All of them in NAC+ROT+Cr(Ⅵ) treated group were between that in ROT+Cr(Ⅵ) and NAC+Cr(Ⅵ) treated groups.6. Effect of Cr(Ⅵ) on mitochondrial respiratory oxygen consumption:ST3,ST4 and RCR went down significantly in each Cr(Ⅵ) treated group compared to its control group. Compared to Cr(Ⅵ) alone treated group, all of them decreased distinguished in ROT pretreated Cr(Ⅵ) treated group and increased in NAC pretreated Cr(Ⅵ) treated group.7. Effect of Cr(Ⅵ) on mtDNA:ROT+Cr(Ⅵ) treatment lead to gene mutations of the mtDNA ATPase6. NAC inhibited the gene mutations induced by them.Conclusions:Cr(Ⅵ) can induce the genertion of ROS and the open of PTP, decrease the MMP(Δψm) in mitochondria. It also can reduce the activties of the mitochondria electronic transport chain complexes. Preteatment of ROT can aggravate the above changes induced by Cr(Ⅵ) in mitochondria and result in the mutation of ATPase 6. However, NAC can protect mitochondria from the respiratory dysfunction induced by Cr(Ⅵ) in isolated rat hepatocyte mitochondria.