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Efavirenz (EFV) is a non-nucleoside reverse transcriptase inhibitor (NNRTI) widely used in human immunodeficiency virus (HIV) infection therapy. It has been associated with hepatotoxic effects and alterations in lipid and body fat composition. Given the importance of the liver in lipid regulation, we have evaluated the effects of clinically used concentrations of EFV on the mitochondria and lipid metabolism of human hepatic cells in vitro. Mitochondrial function was rapidly undermined by EFV to an extent that varied with the concentration employed; in particular, respiration and intracellular adenosine triphosphate (ATP) levels were reduced whereas reactive oxygen species (ROS) production increased. Results in isolated mitochondria suggest that the mechanism responsible for these actions was a specific inhibition of complex I of the respiratory chain. The reduction in energy production triggered a compensatory mechanism mediated by the enzyme adenosine monophosphate-activated protein kinase (AMPK), the master switch of cellular bioenergetics. Fluorescence and nuclear magnetic resonance demonstrated a rapid intracellular increase of neutral lipids, usually in the form of droplets. This was prevented by the AMPK inhibitor compound C and by removal of fatty acids from the culture medium. These effects were not reproduced by Nevirapine, another NNRTI. EFV is clinically coadministered with two nucleoside reverse transcriptase inhibitors. Evaluation of one of the most common combination, EFV/Lamivudine/Abacavir, revealed that the effects of EFV on ROS production were enhanced. Conclusion: Clinical concentrations of EFV induce bioenergetic stress in hepatic cells by acutely inhibiting mitochondrial function. This new mechanism of mitochondrial interference leads to an accumulation of lipids in the cytoplasm that is mediated by activation of AMPK. (HEPATOLOGY 2010;52:115-125) C ontinuous administration of the drugs included under the term highly active antiretroviral therapy has made acquired immune deficiency syndrome a chronic rather than terminal illness. The initial development of these drugs was particularly rapid and focused on clinical efficacy-reduction of mortality-before all other considerations. However, as the disease has come under control, there has been a growing emphasis on the long-term adverse effects induced by this therapy. Efavirenz (EFV) is a non-nucleoside reverse transcriptase inhibitor (NNRTI) widely used in initial therapy for human immunodeficiency virus (HIV) infection. It is administered to adults in a single daily dose of 600 mg, which Abbreviations: 3TC, lamivudine; ABC, abacavir; AMPK, adenosine monophosphate-activated protein kinase; ATP, adenosine triphosphate; DCFH-DA, 2 0 ,7 0 -dichlorodihydrofluorescein diacetate; EFV, Efavirenz; HIV, human immunodeficiency virus; HR-MAS, high resolution magic angle spectroscopy; NNRTIs, nonnucleoside reverse transcriptase inhibitors; NRTI, nucleoside reverse transcriptase inhibitors; NVP, nevirapine; P-AMPK, phosphorylated adenosine monophosphate...

The NNRTI efavirenz has long been one of the most frequently employed antiretroviral drugs in the multidrug regimens used to treat HIV infection, in accordance with its well-demonstrated antiretroviral efficacy and favourable pharmacokinetics. However, growing concern about its adverse effects has sometimes led to efavirenz being replaced by other drugs in the initial treatment selection or to switching of therapy to efavirenz-free regimens in experienced patients. Neurological and neuropsychiatric reactions are the manifestations most frequently experienced by efavirenz-treated patients and range from transitory effects, such as nightmares, dizziness, insomnia, nervousness and lack of concentration, to more severe symptoms including depression, suicidal ideation or even psychosis. In addition, efavirenz has recently been associated with mild/moderate neurocognitive impairment, which is of specific relevance given that half of the patients receiving ART eventually suffer some form of HIV-associated neurocognitive disorder. The mechanisms responsible for efavirenz-induced neurotoxicity are unclear, although growing evidence points to disturbances in brain mitochondrial function and bioenergetics. This review offers a comprehensive overview of the current evidence on the interaction that efavirenz displays with the CNS, including the penetration and concentration of the drug in the brain. We discuss the prevalence, types and specificities of its side effects and recently uncovered cellular mechanisms that may be involved in their development.

BACKGROUND AND PURPOSEEfavirenz (EFV) is widely used in the treatment of HIV-1 infection. Though highly efficient, there is growing concern about EFV-related side effects, the molecular basis of which remains elusive. EXPERIMENTAL APPROACHIn vitro studies were performed to address the effect of clinically relevant concentrations of EFV (10, 25 and 50 mM) on human hepatic cells. KEY RESULTSCellular proliferation and viability were reduced in a concentration-dependent manner. Analyses of the cell cycle and several cell death parameters (chromatin condensation, phosphatidylserine exteriorization, mitochondrial proapoptotic protein translocation and caspase activation) revealed that EFV triggered apoptosis via the intrinsic pathway. In addition, EFV directly affected mitochondrial function in a reversible manner, inducing a decrease in mitochondrial membrane potential and an increase in mitochondrial superoxide production, followed by a reduction in cellular glutathione content. The rapidity of these actions rules out any involvement of mitochondrial DNA replication, which, until now, was thought to be the main mechanism of mitochondrial toxicity of antiretroviral drugs. Importantly, we also observed an increase in mitochondrial mass, manifested as an elevated cardiolipin content and enhanced expression of mitochondrial proteins, which was not paralleled by an increase in the mtDNA/nuclear DNA copy number ratio. The toxic effect of EFV was partially reversed by antioxidant pretreatment, which suggests ROS generation is involved in this effect. CONCLUSION AND IMPLICATIONSClinically relevant concentrations of EFV were shown to be mitotoxic in human hepatic cells in vitro, which may be pertinent to the understanding of the hepatotoxicity associated with this drug.

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Hepatotoxicity is a very common side effect associated with the pharmacological treatment of human immunodeficiency virus (HIV) infection and its pathogenesis is poorly understood. Efavirenz (EFV) is the most widely used nonnucleoside reverse transcriptase inhibitor administered for the control of HIV and some of its toxic effects in hepatic cells have been recently shown to display features of mitochondrial dysfunction. Here we studied the activation of autophagy and, in particular, mitophagy, the main mitochondrial turnover mechanism, in human hepatic cells treated with clinically relevant concentrations of this drug. EFV-treated cells had altered mitochondria, characterized by a relative increase in mitochondrial mass and defective morphology. This was followed by induction of autophagy as shown by the presence of autophagic vacuoles and the presence of the specific autophagic marker proteins microtubule-associated protein 1A/1B light chain 3 and Beclin-1. Importantly, whereas moderate levels of EFV activated autophagy, higher concentrations led to blockage in the autophagic flux, a condition that promotes ''autophagic stress'' and produces severe cellular damage. Finally, pharmacological inhibition of autophagy exacerbated the deleterious effect of EFV on cell survival/proliferation promoting apoptosis, which suggests that autophagy acts as an adaptive mechanism of cell survival. Conclusion: Clinical concentrations of EFV induce autophagy and, in particular, mitophagy in hepatic cells. Activation of this process promotes cell survival, but exceeding a certain threshold of mitochondrial dysfunction is associated with an autophagic overload or stress. This effect could be involved in the EFV-associated hepatotoxicity and may constitute a new mechanism implicated in the genesis of drug-induced liver damage.

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Efavirenz affects the bioenergetics of neurons through a mechanism involving acute mitochondrial inhibition, an action exacerbated in neuroinflammatory conditions. A similar scenario of glial cells survival and degeneration of neurons with signs of mitochondrial dysfunction and oxidative stress has been associated with neurocognitive disorders.