Damage To MtDNA In Liver Injury Of Patients With Extrahepatic Cholestasis: The Protective Effects Of Mitochondrial Transcription Factor A

Background and ObjectsExtrahepatic cholestasis could be caused by conditions that block the normal flow ofbile, such as tumors and gallstones. These extrahepatic biliary obstructions result in anintrahepatic accumulation of toxic hydrophobic bile acids that leads to hepatocyte death interms of apoptosis and necrosis, and eventual liver fibrosis and cirrhosis. Evidence from invivo and in vitro experiments demonstrates that oxidative stress and mitochondrialdysfunction are involved in the pathogenesis of chronic liver cholestasis.Mitochondrial DNA (mtDNA) is highly susceptible to oxidative stress and mtDNAdamage leads to mitochondrial dysfunction. In addition to regulating expression of genesencoded by mtDNA, mitochondrial transcription factor A (TFAM) plays multiplepathophysiologic roles in mtDNA maintenance, including nucleoid formation, mtDNAstabilization, mtDNA transcription and preferentially recognition of damaged mtDNA.These findings suggest that TFAM may have a close relationship to the mtDNA alterationsobserved in extrahepatic cholestatic patients. This study aimed to investigate the mtDNAalterations that occurred during liver injury in patients with extrahepatic cholestasis. Wethen investigated the possible relationship of the TFAM with mtDNA damage, as mediatedby GCDCA, and the possible protective effects of TFAM over-expression.Materials and Methods1. Twelve patients admitted for surgery because of obstructive jaundice wereincluded in the study. Of the patients, six had pancreatic cancer, four had periampullarytumor, and two had cholangiocarcinoma. Liver tissue of the obstructive jaundice patientswas obtained during nonhepatic major abdominal surgery. Control liver tissue (controlgroup, n=10) was obtained from nonjaundiced patients with pancreatic tumors (n=4) andfrom patients undergoing cholecystectomy for gallstones (n=6). All subjects included inthe study were negative for viral hepatitis infection, liver autoimmune or metabolic disorders and were not under treatment with hepatotoxic drugs. Patients reporting recentabuse of alcohol were also excluded. After collection, the liver samples were immediatelyimmersed in liquid nitrogen and were stored at-80℃until being processed forbiochemical investigations. Cells from the human normal liver cell line L02werepurchased from the Cell Bank of Institute of Biochemistry and Cell Biology (Shanghai,China).2. Mitochondrial8-hydroxydeoxyguanosine (8-OHdG) levels, reactive oxygenspecies (ROS) production, adenosine triphosphate (ATP) content, mitochondrialmembrane potential (△Ψm) andcell viability were detected by assay kits both in thepatients with extrahepatic cholestasis and L02cells treated with various concentrationsof glycochenodeoxycholic acid (GCDCA).3. Real-time PCR was utilized to detect mtDNA copy number, levels of mtDNAtranscripts and TFAM mRNA. The real-time PCR was conducted with an iQ5Real-Time PCR Detection System using the SYBR Green I detection method.4. Western blot was analysed levels of TFAM protein both in the patients withextrahepatic cholestasis and L02cells treated with GCDCA.5. Cells were transfected with pEGFP-N1-TFAM, pEGFP-N1-ΔC-TFAM or emptypEGFP-N1vector employing Optimum-Minimum Essential Medium withLipofectamine2000. The over-expression of pEGFP-N1-TFAM and pEGFP-N1-ΔC-TFAM were detected by western blot analysis and immunofluorescence as describedabove. For immunofluorescence, transfected cells with EGFP were incubated withMitoTracker Red CMXRos probe to label mitochondria.Results1. Oxidative stress and mitochondrial dysfunction in the liver injury of patients withextrahepatic cholestasis. Light microscopy showed that hepatic cord disturbance andintracellular cholestasis in cholestatic patients. Scattered hepatocytes were observed withswelling, denaturationand necrosis. In cholestatic patients, the malondialdehyde (MDA)levels exhibited a significant four times higher than those in the control group. In contrast,the ATP concentration significantly decreased by37%in patients versus controls.Extrahepatic cholestatic patients presented a significant increase in mitochondrial8-OHdGlevels and the decreases in mtDNA copy number, mtDNA transcripts levels and mtDNA nucleoid structure.2. The deleterious effects of GCDCA on cell viability were presented in adose-dependent manner in L02cells. ROS production increased by1.5-,1.9-,2.0-, and2.1-fold compared with controls after cells were treated for6h with25,50,75and100μM GCDCA, respectively. In contrast, ATP levels and△Ψm were reduced in adose-dependent manner in L02cells treated with GCDCA. Following oxidative stress andmitochondrial dysfunction, confocal laser microscopy detection and ELISA showed thatthe levels of8-OHdG in mtDNA were significantly increased at6h after L02cells treatedwith various concentrations of GCDCA. In addition, real-time PCR analysis showed thatGCDCA(50,75and100μM) significantly decreased the levels of mtDNA copy numberand mtDNA transcripts, a similar change to what was observed in cholestatic patients.Moreover, the spotted staining indicative of nucleoid structure was also significantlydisturbed in GCDCA-treated cells.3. The levels of TFAM mRNA and protein were significantly reduced in cholestaticpatients compared with those in controls. In parallel, at6h after L02cells were treatedwith GCDCA (50,75,100μM), the TFAM mRNA levels decreased by27,32, and60%of that of controls and the TFAM protein levels were significantly reduced than thosein controls, respectively.4. TFAM over-expression efficiently reversed the increased in8-OHdG levels in L02cells treated with75μM GCDCA for6h. In parallel, the decreased levels of mtDNA copynumber and mtDNA transcripts were obviously recovered after TFAM was over-expressed.In addition, confocal analysis showed that TFAM over-expression also prevented thedisruptions of the nucleoid structure of the mtDNA. The protective effects of TFAM onmtDNA are useful for mitochondrial turnover. Indeed, mitochondrial dysfunction, asindicated by the ROS production, ATP levels and△Ψm was significantly attenuated inTFAM over-expressed cells treated by GCDCA.5. we deleted the C-terminal tail of TFAM to generate ΔC-TFAM, which has nofunctional regions that stimulate mtDNA transcription but can still maintain the nucleoidstructure and mtDNA copy number. As a result, ΔC-TFAM over-expression could notrescue the decrease in mtDNA transcripts at all. Compared with wild-type TFAM,ΔC-TFAM was less effective in reversing mtDNA damage and mitochondrial dysfunction in GCDCA-treated L02cells.ConclusionsOur study clearly demonstrated that mtDNA damage is involved in liver damage inextrahepatic cholestasis patients and GCDCA hepatotoxicity. The mtDNA damage isattributable to the loss of TFAM. TFAM has mtDNA-protective effects against thehepatotoxicity of bile acid during cholestasis. The protective effects of TFAM againstGCDCA-hepatotoxiciy were attributable to its multiple roles in the maintenance ofmtDNA. Without its C tail, ΔC-TFAM is less effective against the hepatotoxicity ofglycochenodeoxycholic acid.