| BackgroundCadmium is an extremely toxic environmental and occupational contaminant, arising primarily from batteries, the food chain, and cigarette smoke. Given its persistence(10-30 years) and its low rate of excretion, this heavy metal is toxic even at low doses for livers where it accumulates. In mammals, it exerts high toxic effects and has been classified as the sixth harm human health of toxic substances by the Agency for Toxic Substances and Diseases Registry. Recently, mitochondrial loss has been revealed to play a prominent role in the observed mitochondrial dysfunction. Growing evidence indicates that Cd exposure leads to mitochondrial loss in liver cells, ref lected by a decrease in both mitochondrial DNA copy number and in the expression of components of the mitochondrial respiratory chain. However, the mechanism by which Cd induces mitochondrial loss during Cd-induced hepatotoxicity is not fully understood. Autophagy, an evolutionarily conserved pathway, plays a crucial role in degrading a wide variety of cellular components, such as peroxisomes, the endoplasmic reticulum, and mitochondria. The specific process that clears the mitochondria from the cell through autophagy has been termed “mitophagy.” In certain circumstances, mitophagy plays a beneficial role in eliminating damaged and unhealthy mitochondria and maintaining the quality of the organelle. In contrast, massive and persistent mitophagy creates the overactivation of intracellular mitochondrial degradation machinery. In particular, increased mitochondrial degradation may contribute to a bioenergetic deficit and cell death. Therefore, the purpose of this study is to investigate how cadmium induces mitochondrial loss in its hepatotoxicity, and to explore whether the activation of Ca2+-DNM1 L dependent mitochondrial fission underlies the Cd caused mitophagy contributes to mitochondrial loss.Methods①The cell viability was applied to evaluate the cytotoxicity of CdCl2 on L02 cell line. Parallel, the effects of Cd on mitochondrial mass levels, the levels of the COX4I1 subunit, mt DNA copy number and ATP concentrations were determined. Mitophagy was evaluated, which may contribute to mitochondrial loss. The expression levels of LC3, SQSTM1 were measured after CdCl2 exposure. The number of autophagic vacuoles was observed by Electron microscopy. Mitochondria-containing autophagosomes and autolysosomes were determined by confocal laser microscopy. Bafilomycin A1(Baf A1), an inhibitor of the lysosomal V-ATPase, used to detect autophagic flux in cadmium-induced hepatotoxicity. To address whether Cd-induced mitophagy was involved in Cd-induced mitochondrial loss and hepatotoxicity, we suppressed mitophagy using siRNA against ATG5. Another possible reason for the observed decrease in the number of mitochondria is impairment of mitochondrial biogenesis. Mitochondrial biogenesis is regulated by PPARGC1 A and TFAM was also determined.② The effects of Cd on mitochondrial dynamics were next examined. The expression levels of DNM1 L, OPA1, MFN1,MFN2,FIS1, responsible for fusion/fission balance, were measured after CdCl2 exposure. Then, the DNM1L-siRNA was transfected into cells. The restoration of mitochondrial dynamics, mitophagy and mitochondrial loss were assayed in transfected cells. Finally, the concentrations of mitochondrial and intracellular calcium were investigated in the Cd-treated L02 cells.③ C57BL6/J mice were injected intraperitoneally(i.p.) with 1 mg/kg Cd for 7 days, amd the mice were injected(i.p.) with 50 mg/kg Mdivi-1 1 h prior to Cd treatment. Mitochondrial morphological alterations were evaluated using Electron microscopy. Mitophagy markers, such as mitochondrial LC3、SQSTM1 and the mitochondrial marker COX4I1 in the livers were analyzed at the same time points at which liver damage were evident.Results① Cd Cl2 treatment significantly increased the cell viability loss in cultured L02 cells. In addition, Cd exposure significantly reduced mitochondrial mass and the levels of the COX4I1 subunit, and decreased mt DNA copy numbers and ATP concentrations in both kind of cells. Cd Cl2 induced a dose-dependent increase in the LC3-II/LC3-I ratio and decreased SQSTM1 levels in L02 cells. The EM studies revealed an increased number of autophagic vacuoles in the CdCl2-treated cells compared with the nontreated control cells. CdCl2 significantly increased the colocalization of GFP-LC3 puncta with mitochondria and colocalization of mitochondria and autolysosomes. Moreover, the presence of 10 nM Baf A1 significantly increased the percentage of cells exhibiting GFP-LC3 positive autophagosomes in L02 cells that were treated with CdCl2. In particular, ATG5 si RNA treatment efficiently prevented the Cd Cl2-treated cells against a loss of mitochondrial mass and inhibited the decrease in both the levels of the mitochondrial protein COX4I1 and mt DNA copy number.These effects were followed by partial improvement in both the ATP level and cell survival. Moreover, the expression of the transcription factor, PPARGC1 A and that of its downstream target TFAM were unaffected by Cd Cl2 treatment.② CdCl2 induced a significant dose-dependent increase in the number of fragmented mitochondria with ring-shaped structures as well as an increase in the number of small-sized mitochondria in the EM images. Time-lapse microscopy revealed that this change in mitochondrial morphology occurred early as 3 h following treatment with Cd. accompanied with the changes of mitochondria morphology, only DNM1 L protein levels were significantly increased in the Cd-treated L02 cells, and no significant changes was detected in the levels of the other mitochondrial fusion/fission proteins. Furthermore, DNM1 L was observed to translocate to the mitochondria following exposure to 12 μM Cd for 12 h. DNM1 L knockdown substantially dereaesed the LC3-II/LC3-I ratio, and increased the mitochondrial mass. Moreover, As early as 2 h after Cd treatment, both [Ca2+]I and [Ca2+]m were significantly increased in a time-dependent manner. In addition, pre-treatment with specific [Ca2+]I chelator, BAPTA-AM, reduced both DNM1 L expression and DNM1 L recruitment into mitochondria. Pre-treatment with an inhibitor of [Ca2+]m uptake, Ru360, also decreased the recruitment of DNM1 L to mitochondria, but had no effect on DNM1 L protein levels. Moreover, pre-treatment with both BAPTA-AM and Ru360 efficiently reduced DNM1L-mediated mitochondrial fragmentation.③ Mdivi-1 partially suppressed the Cd-induced mitochondrial fragmentation in hepatic cells. The expression of mitophagy markers, such as LC3 and the mitochondrial marker COX4I1, were normalized in the Mdivi-1-treated mice compared with the Cd-treated controls. Moreover, the levels of mitochondrial LC3-II rapidly decreased, and the levels of mitochondrial SQSTM1 significantly increased in the Mdivi-1-treated mice. Furthermore, ATP levels were evaluated to determine the effect of Mdivi-1 on Cd-induced liver dysfunction.ConclusionOverall, we provided evidence that Cd induced significant mitochondrial loss through the overactivation of mitophagy during hepatotoxicity. The inhibition of excessive mitophagy may restore mitochondrial mass, compensate for the reduced function of the respiratory chain and maintain overall ATP production. Notably, we demonstrated that Cd induced mitophagy through irreversible mitochondrial fragmentation in a Ca2+-DNM1L-dependent manner. The silencing of DNM1 L expression efficiently prevented mitochondrial fragmentation, thereby attenuating the increased mitophagy that occurs in Cd-induced mitochondrial loss in vitro. Lastly, pharmacological DNM1 L inhibition with Mdivi-1 attenuated the observed abnormal mitophagy and protected against Cd-induced hepatotoxicity in vivo. |
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