Role Of Oxidative Stress Pathway In Flutamide-induced Hepatic Mitochondrial Toxicity

ObjectiveFlutamide is a widely used nonsteroidal anti-androgen drug for prostate cancer therapy,but its clinical application is restricted by the obvious liver injury.Increasing evidence suggests that flutamide-induced liver injury is associated with mitochondrial oxidative stress,though the precise mechanism is poorly understood.Nrf2/HO-1 pathway is a key antioxidative pathway and play an important role in drug-induced hepatotoxicity and mitochondrial oxidative stress.ERK1/2/PGC-1? pathway is important in regulation of mitochondrial biogenesis.Activation of Nrf2 and PGC-1? pathways can protect liver from oxidative stress injury.Flutamide can inhibit Nrf2/HO-1 pathway and activate ERK1/2/PGC-1? pathway,but the mechanism underlying flutamide induced liver injury is unclear.This study was designed to observe hepatotoxicity and mitochondrial oxidative stress of flutamide in human HepG2 cells,estabilish a connection between Nrf2/HO-1 and ERK1/2/PGC-1? pathways and flutamide-induced hepatic injury,and elucidate the mechanism of flutamide-induced liver toxicity.The implementation of this subject will provide a new method for the prediction of drug-induced hepatotoxicity.Methods1.HepG2 Cells were treated with flutamide.Cytotoxicity,mitochondrial damage,perturbation of Nrf2/HO-1 and ERK1/2/PGC-1? pathways were observed to estabilish a connection between oxidative stress pathway and mitochondrial injury.Cytotoxicity was evaluated by determination of cell viability using CCK-8 and lactate dehydrogenase(LDH)leakage using LDH assay kit.Mitochondrial toxicity was evaluated by hydrogen peroxide using a hydrogen peroxide assay kit,mitochondrial membrane potential using flow cytometer,ATP production using an ATP colorimetric assay,and Mitochondrial DNA copy number using Real-Time PCR.Perturbations of Nrf2/HO-1 and ERK1/2/PGC-1? pathways were analyzed with the protein expression of Nrf2,HO-1,SOD2,ERK1,PGC-1?,NRF1,TFAM and COXIV using western blot analysis.2.Role of the Nrf2/HO-1 pathway in flutamide-induced mitochondrial toxicity was further determined.Nrf2 or HO-1 knockdown cell model,HO-1 inhibitor Snpp and HO-1 activator Copp were used to observe the effect of flutamide on mitochondrial function and Nrf2/HO-1 pathway.3.Evaluated the role of the ERK1/2 and Nrf2/HO-1 pathway in flutamide-induced mitochondrial biogenesis.ERK1/2 inhibitor PD98059,ERK1/2 activator Curcumin,Nrf2 knockdown cell model,HO-1 inhibitor Snpp and HO-1 activator Copp were used to observe the effect of flutamide on mtDNA copy number and Nrf2/HO-1 and ERK1/2/PGC-1? pathways.Results1.Characterization of the hepatotoxicity of flutamide.24 h treatment of flutamide in Hep G2 cells concentration-dependently reduced cell viability and increased LDH leakage(tipping point was 50 ?M),increased ROS accumulation(tipping point was 25 ?M,less than concentration in cytotoxicity),decreased mitochondrial membrane potential and ATP level(tipping point was 25 ?M).Flutamide also increased first and then decreased mtDNA copy number(increase in 25 ?M and decrease in 75 ?M)indicated that mtDNA copy number increased within a given concentration range with the increase of ROS accumulation.Forthermore,flutamide concentration-dependently perturbed Nrf2/HO-1 and ERK1/2/PGC-1? pathways.Protein expression of Nrf2 and HO-1 increased first and then decreased(tipping point was 12.5 ?M),PGC-1? and ERK1/2 also increased first and then decreased(tipping point was 50 ?M),COXIV increased from 25 ?M.The results demonstrated that there was a certain correlation between oxidative stress pathway and mitochondrial toxicity,and provided experiment reference to certify the connection between toxicity pathways perturbation and cytotoxicity outcome.2.Connection between toxicity pathways perturbation and flutamideinduced hepatic mitochondrial toxicity.Nrf2 or HO-1 knockdown cell model was established using Lipfectamine 3000 to introduced Nrf2 or HO-1 siRNA into HepG2 cells.24 h treatment of 50 ?M flutamide-induced hydrogen peroxide accumulation,mitochondrial membrane potential loss,ATP depletion and protein expression of Nrf2 and HO-1 descending were exacerbated in Nrf2 knockdown cells.In addition,hydrogen peroxide accumulation,mitochondrial membrane potential loss,ATP depletion and protein expression of HO-1 descending were deteriorated in HO-1 knockdown cells.The results suggested that Nrf2 and HO-1 were important in regulating antioxidative injury.Forthmore,flutamide induced increase of hydrogen peroxide accumulation,mitochondrial membrane potential loss,ATP depletion and protein expression of Nrf2 and HO-1 descending after pre-treatment of HO-1 inhibitor Snpp,but induced decrease of hydrogen peroxide accumulation,mitochondrial membrane potential loss,ATP depletion and protein expression of Nrf2 and HO-1 descending after pre-treatment of HO-1 activator Copp.The results suggested that Nrf2/HO-1 pathway played important roles in up-regulating oxidative stress.3.Connection between toxicity pathway perturbation and flutamide-induced mitochondrial biogenesis.24 h treatment of 50 ?M of flutamide-induced mtDNA copy number and protein expression of ERK1/2 and PGC-1? increase were declined after pre-treatment of ERK1/2 inhibitor PD98059,but were ascended after pre-treatment of ERK1/2 activator Curcumin.Forthmore,flutamide-induced mtDNA copy number and protein expression of ERK1/2 and PGC-1? increase were decreased in Nrf2 knockdown cells.In addition,flutamide induced rise of mtDNA copy number and protein expression of ERK1/2 and PGC-1? increase after pre-treatment of Snpp,but induced reduction of mtDNA copy number and protein expression of ERK1/2 and PGC-1? increase after pre-treatment of Copp.The results indicated that activation of ERK1/2/PGC-1? pathway increased mitochondrial biogenesis,and vice versa.Additionally,Nrf2 inhibition decreased mitochondrial biogenesis.Taken together,these findings certified the causal relationship between oxidative stress pathway and mitochondrial toxicity.Conclusions1.Flutamide concentration-dependently affected hepatotoxicity and oxidative stress pathway(Nrf2/HO-1 pathway or ERK1/2/PGC-1? pathway)alteration,induced mitochondrial biogenesis changing and mitochondrial dysfunction.Mitochondrial oxidative stress may be the key event in flutamide-induced hepatotoxicity.2.Activation/Inhibition of Nrf2/HO-1 pathway induced hepatic mitochondrial oxidative stress alleviation/aggravation.Activation/Inhibition of ERK1/2/PGC-1? pathway induced hepatic mitochondrial biogenesis increase/decrease.Nrf2/HO-1 and ERK1/2/PGC-1? pathways involved in regulating of flutamide-induced mitochondrial toxicity.Besides,causal relationship between oxidative stress pathway and mitochondrial toxicity was proved.3.Oxidative stress pathway(Nrf2/HO-1 pathway or ERK1/2/PGC-1? pathway)alteration is implicated as a MIE(Molecular Initiating Event)in flutamide-induced mitochondrial toxicity,and could be recognized as part of mitochondrial toxicity AOP(Adverse Outcome Pathways),thus providing a new method for prediction of drug-induced mitochondrial toxicity.