We provide evidence that this process promotes cell survival, but exceeding a certain threshold of mitochondrial dysfunction is associated with an autophagic overload or stress. This complex effect could be involved in EFV-related hepatic toxicity and may constitute a new mechanism

implicated in the genesis of drug-generated buy Cabozantinib liver damage. AIDS, acquired immunodeficiency syndrome; ΔΨm, mitochondrial transmembrane potential; EFV, Efavirenz; HAART, highly active antiretroviral therapy; HIV, human immunodeficiency virus; LC3, microtubule-associated protein 1A/1B light chain 3; 3MA, 3-methyladenine; MPT, mitochondrial permeability transition; NAO, 10-N-nonyl acridine orange; NNRTI, nonnucleoside reverse transcriptase inhibitor; NRTI, nucleoside reverse transcriptase inhibitor; Pol-γ, DNA polymerase gamma; PI, propidium iodide; STS, staurosporine; TEM, transmission electron microscopy. Unless stated otherwise, chemical reagents and fluorochromes were purchased from

Sigma-Aldrich (Steinheim, Germany). Efavirenz (Sustiva 600 mg, Bristol-Myers Squibb) was acquired in its clinically available form and dissolved in methanol (3 mg/mL) once insoluble substances had been removed by filtration. The purity (98%-100%) and stability were evaluated by high-performance liquid chromatography (HPLC) and compared with a control solution of EFV (Sequoia Research Products, Pangbourne, UK). The employed range of EFV (10, 25, AZD6738 nmr and 50 μM) MCE is clinically relevant and was chosen considering the important interindividual variability in its pharmacokinetics.16 Although the therapeutic plasma levels of EFV are believed to be 3.17-12.67 μM, as many as

20% of patients exhibit higher levels, with values of 30-50 μM being documented.17-19 0.5% methanol was employed in all EFV treatments and vehicle control experiments, versus which statistical analysis was performed. In most experiments the vehicle-treated were compared to untreated cells and no significant differences in any of the parameters were detected. We used Hep3B cells (American Type Culture Collection [ATCC] HB-8064), which despite constituting a transformed cell line, is considered metabolically competent and, unlike other human hepatoma cell lines, such as HepG2, has an active cytochrome P450 system. Confirmatory experiments were performed in primary human hepatocytes and for gene overexpression we used the human cervical carcinoma cell line HeLa (ATCC CCL-2), as these cells also possess a high mitochondrial content and are frequently employed for transfection (details in Supporting Material). WB was performed using whole-cell protein extracts as described.13 Primary antibodies: anti-Beclin (Abcam), anti-microtubule-associated protein 1A/1B light chain 3 (LC3), and anti-actin (both from Sigma-Aldrich, Steinheim, Germany), all at 1:1,000, and a secondary antibody peroxidase-labeled antirabbit IgG (Vector Laboratories, Burlingame, CA) at 1:5,000.