Increased MMAB level in mitochondria as a novel biomarker of hepatotoxicity induced by Efavirenz

Abstract

BackgroundEfavirenz (EFV), a non-nucleoside reverse transcriptase inhibitor (NNRTI), has been widely used in the therapy of human immunodeficiency virus (HIV) infection. Some of its toxic effects on hepatic cells have been reported to display features of mitochondrial dysfunction through bioenergetic stress and autophagy, etc. However, alteration of protein levels, especially mitochondrial protein levels, in hepatic cells during treatment of EFV has not been fully investigated.MethodsWe built a cell model of EFV-induced liver toxicity through treating Huh-7 cells with different concentrations of EFV for different time followed by the analysis of cell viability using cell counting kit -8 (CCK8) and reactive oxygen species (ROS) using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) and MitoSox dye. Proteomic profiles in the mitochondria of Huh-7 cells stimulated by EFV were analyzed. Four differentially expressed proteins were quantified by real time RT-PCR. We also detected the expression of mitochondrial precursor Cob(I)yrinic acid a,c-diamide adenosyltransferase (MMAB) by immunohistochemistry analysis in clinical samples. The expression levels of MMAB and ROS were detected in EFV-treated Huh-7 cells with and without shRNA used to knock down MMAB, and in primary hepatocytes (PHC). The effects of other anti-HIV drugs (nevirapine (NVP) and tenofovirdisoproxil (TDF)), and hydrogen peroxide (H2O2) were also tested. Amino acid analysis and fatty aldehyde dehydrogenase (ALDH3A2) expression after MMAB expression knock-down with shRNA was used to investigate the metabolic effect of MMAB in Huh-7 cells.ResultsEFV treatment inhibited cell viability and increased ROS production with time- and concentration-dependence. Proteomic study was performed at 2 hours after EFV treatment. After treated Huh-7 cells with EFV (2.5mg/L or 10 mg/L) for 2 h, fifteen differentially expressed protein spots from purified mitochondrion that included four mitochondria proteins were detected in EFV-treated Huh-7 cells compared to controls. Consistent with protein expression levels, mRNA expression levels of mitochondrial protein MMAB were also increased by EFV treatment. In addition, the liver of EFV-treated HIV infected patients showed substantially higher levels of MMAB expression compared to the livers of untreated or protease inhibitor (PI)-treated HIV-infected patients. Furthermore, ROS were found to be decreased in Huh-7 cells treated with shMMAB compared with empty plasmid treated with EFV at the concentration of 2.5 or 10 mg/L. MMAB was increased in EFV-treated Huh-7 cells and primary hepatocytes. However, no change in MMAB expression was detected after treatment of Huh-7 cells and primary hepatocytes with anti-HIV drugs nevirapine (NVP) and tenofovirdisoproxil (TDF), or hydrogen peroxide (H2O2), although ROS was increased in these cells. Finally, knockdown of MMAB by shRNA induced increases in the β-Alanine (β-Ala) production levels and decrease in ALDH3A2 expression.ConclusionsA mitochondrial proteomic study was performed to study...

Arsenic trioxide induces apoptosis and the formation of reactive oxygen species in rat glioma cells

“…The generation of ROS was measured as previously described [ 21 ]. Briefly, cell suspensions (2 × 10 6 cells/ml) were exposed to As 2 O 3 at 5.0 μmol/l for 24, 48 and 72 h. To evaluate the major organelles that governed the ROS-mediated stress in glioma cells, C6 and 9 L cells were pretreated with 5 nM antioxidant N-acetylcysteine (NAC) for 2 h, and were exposed to As 2 O 3 at 5.0 μmol/l for 24 h [ 22 ].…”

Protein quality control at the interface of endoplasmic reticulum and mitochondria by Lon protease

“…In this study, Polo et al . () showed that EFV induces both ER stress and mitochondrial dysfunction, but the specific targets of EFV remain unknown (Apostolova et al ., ; Tan et al ., ). A similar class of non‐nucleoside inhibitor rilpivirine does not cause an identical effect suggesting that these drugs may have different molecular targets in cells due to their dissimilar structures.…”

“…It is interesting that the antiretroviral non-nucleoside inhibitor efavirenz (EFV), which is used to treat HIV, induces stress in both mitochondria and ER, causing the depletion of LonP1 in mitochondria and the accumulation of LonP1 outside at MAMs (Polo et al, 2017). In this study, Polo et al (2017) showed that EFV induces both ER stress and mitochondrial dysfunction, but the specific targets of EFV remain unknown (Apostolova et al, 2013;Tan et al, 2017). A similar class of non-nucleoside inhibitor rilpivirine does not cause an identical effect suggesting that these drugs may have different molecular targets in cells due to their dissimilar structures.…”

Effects of ARVs and HIV on mitochondrial function and immune activation in monocyte derived macrophages in vitro

“…In these cases, ROS appears to be increased during HIV infection, although the results are inconsistent 265 . In terms of ART, it has been shown in hepatic cells in vitro that EFV significantly increases cellular ROS and superoxide production 146,257 . TFV has been shown to deplete antioxidant levels leading to increased ROS production and nephrotoxicity 258 .…”

“…Superoxides are produced mainly through ETC complexes I and III, and are used mainly in signaling pathways or to combat invading pathogens, but also have toxic abilities if they accumulate 119,120 . It has been shown in hepatic cells in vitro that EFV significantly increases cellular ROS and superoxide production 146,257 . TFV has been shown to deplete antioxidant levels leading to increased ROS production and nephrotoxicity 258 .…”