Studies On The Mechanisms Of Mitochondrial Toxicity Induced By Isoniazid In Human Hepatocyte HepG2 Cells

Isoniazid(INH),one of the first-line anti-tuberculosis drugs,has been noted to be associated with hepatotoxicity in clinic.In spite of extensive researches over many decades,the mechanism of INH-induced hepatotoxicity still remains poorly understood.The traditional theory that cytochrome P450 2E1 is involved in INH-induced hepatotoxicity remains controversial,therefore other mechanisms by which INH exerts hepatotoxicity need to be investigated.In the current study,we showed that in vitro treatment of human hepatocarcinoma HepG2 cells with INH induced apoptosis through extrinsic(death receptor)and intrinsic(mitochondrial)pathways.It was characterized by the increased population of apoptotic cells using Annexin V/propidium iodide(PI)double staining by flow cytometry,and by the activations of caspases 8,9,3 and poly(ADP-ribose)-polymerase(PARP)proteins by western blotting.INH treatment also induced autophagy as shown by the up-regulated levels of microtubule-associated protein 1 light chain 3-?(LC3-?),increased GFP-LC3 punctates,and elevated monodansyl cadaverine(MDC)fluorescence intensity.The blockage of autophagy with chloroquine(CQ)exacerbated INH-induced apoptosis significantly.Further study showed that INH treatment down-regulated the protein phosphorylation of mammalian target of rapamycin(mTOR),the key negative regulator of autophagy.In addition,INH induced p38 MAPK(p38 mitogen-activated protein kinase)activation.SB203580,a p38 inhibitor,effectively enhanced INH-induced apoptosis by increasing the cleavages of caspases 9,3 and PARP,but did not affect autophagyRecently,mitochondrial toxicity has been emerging as a new paradigm for INH-induced hepatotoxicity.In this study,we showed that INH impaired mitochondrial biogenesis and dynamics in HepG2 cells.INH reduced mitochondrial membrane potential(MMP),induced mitochondria swelling,and inhibited protein expressions of the three major regulators,silent information regulator two ortholog 1(SIRT1),peroxisome proliferator-activated receptor ?coactivator 1?(PGC1?)and nuclear respiratory factor 1(NRF1),of mitochondrial biogenesis,accompanied by a decrease in the number of mitochondria as demonstrated by reduction of mitochondrial protein cytochrome c oxidase subunit ?(COX ?).INH caused mitochondrial fragmentation involving decreased levels of the fusion protein,MFN2(mitofisin 2)as well as the fission protein,DRP1(dynamin-related protein 1).INH-reduced DRP1 expression was associated with increase in apoptosis,suggesting the existence of pro-survival fission and its involvement in mitochondrial quality control.Inhibition of p38 MAPK exacerbated loss of MMP and mitochondrial swelling,involving decrease in protein expressions of SIRT1,PGC1?,NRF1,and COX ? as well as decrease in DRP1.Mitochondrial homeostasis is achieved by the balance between generation of functional mitochondria by biogenesis and elimination of dysfunctional mitochondria by autophagy.AMP-activated protein kinase(AMPK)can maintain mitochondrial stability through positive control of these two processes.In this study,we showed that AMPK activator acadesine(AICAR)alleviated INH-caused impairment of mitochondrial biogenesis by activation of SIRT1-PGC1? pathway in HepG2 cells.However,mitochondrial instability and apoptosis in HepG2 cells were aggravated by AICAR along with unexpected decrease in INH-induced cytoprotective autophagy.In addition,INH also induced autophagy by activation of AMPK pathway.In summary,we found that INH induced apoptosis in HepG2 cells through death receptor and intrinsic mitochondrial pathways which were characterized by activations of caspases 8,9,3 and PARP.INH induced a protective autophagy against apoptosis in HepG2 cells by inhibition of mTOR pathway and activation of AMPK.INH activated p38 pathway to inhibit apoptosis by down-regulation of mitochondrial apoptotic pathway through inactivation of caspases 9,3 and PARP,but not that of autophagy.Further,INH inhibited mitochondrial biogenesis by inhibition of SIRT1-PGC1? pathway and impaired mitochondrial dynamics by reducing the expressions of MFN2 and DRP1 in HepG2 cells.INH-activated p38 MAPK appeared to alleviate apoptosis of HepG2 cells by inducing mitochondrial biogenesis through partly recovering of SIRT1-PGC1? pathway as well as promoting protective mitochondrial fission by partial recovery of DRP1 protein expression.Therefore,p38 MAPK alleviated INH-caused mitochondrial toxicity and thus apoptosis in HepG2 cells by maintenance of mitochondrial quality control.In addition,AICAR failed to alleviate INH-caused mitochondrial instability in HepG2 cells,due to imbalanced mitochondrial quality control resulting from its inhibitory effect on INH-induced cytoprotective autophagy.