Differential Expression of Lonp1 Isoforms in Cancer Cells.

Lonp1 is a mitochondrial protease that degrades oxidized and damaged proteins, assists protein folding, and contributes to the maintenance of mitochondrial DNA. A higher expression of LonP1 has been associated with higher tumour aggressiveness. Besides the full-length isoform (ISO1), we identified two other isoforms of Lonp1 in humans, resulting from alternative splicing: Isoform-2 (ISO2) lacking aa 42-105 and isoform-3 (ISO3) lacking aa 1-196. An inspection of the public database TSVdb showed that ISO1 was upregulated in lung, bladder, prostate, and breast cancer, ISO2 in all the cancers analysed (including rectum, colon, cervical, bladder, prostate, breast, head, and neck), ISO3 did not show significant changes between cancer and normal tissue. We overexpressed ISO1, ISO2, and ISO3 in SW620 cells and found that the ISO1 isoform was exclusively mitochondrial, ISO2 was present in the organelle and in the cytoplasm, and ISO3 was exclusively cytoplasmatic. The overexpression of ISO1 and, at a letter extent, of ISO2 enhanced basal, ATP-linked, and maximal respiration without altering the mitochondria number or network, mtDNA amount. or mitochondrial dynamics. A higher extracellular acidification rate was observed in ISO1 and ISO2, overexpressing cells, suggesting an increase in glycolysis. Cells overexpressing the different isoforms did not show a difference in the proliferation rate but showed a great increase in anchorage-independent growth. ISO1 and ISO2, but not ISO3, determined an upregulation of EMT-related proteins, which appeared unrelated to higher mitochondrial ROS production, nor due to the activation of the MEK ERK pathway, but rather to global metabolic reprogramming of cells.

Fluorescent styrylpyrylium probes for the imaging of mitochondria in live cells.

Eight styrylpyrylium tetrafluoroborate salts have been synthesized and fully optically characterized by UV-vis absorption and fluorescence steady-state/time-resolved spectroscopies. The new dyes exhibit strong emission bands with yellow-orange colours, depending on the substituents present in the structure. Notably, the Stokes shift recorded for some of them exceeds 100 nm, a very valuable feature for biological imaging. Four of them have been assayed as biological imaging agents by confocal laser scanning microscopy (CLSM) in the human hepatoma cell line Hep3B. It has been found that all the compounds efficiently stain intracellular structures which have been identified as mitochondria through colocalization assays with MitoView (a well-known mitochondrial marker) and using carbonyl cyanide m-chlorophenyl hydrazone (CCCP) as a mitochondrial membrane potential uncoupler. Additionally, the potential ability of the studied dyes as cytotoxic drugs has been explored. The inhibitory concentration (IC50) against Hep3B was found to be in the range of 4.2 µM-11.5 µM, similar to other described anticancer drugs for the same hepatoma cell line. The combined features of a good imaging agent and potential anticancer drug make the family of the studied pyrylium salts good candidates for further theranostic studies. Remarkably, despite the extensive use of pyrylium dyes in several scientific areas (from photocatalysis to optics), there is no precedent description of a styrylpyrylium salt with potential theranostic applications.

Hypusinated eIF5A is required for the translation of collagen.

Translation of mRNAs that encode peptide sequences with consecutive prolines (polyproline) requires the conserved and essential elongation factor eIF5A to facilitate the formation of peptide bonds. It has been shown that, upon eIF5A depletion, yeast ribosomes stall in polyproline motifs, but also in tripeptide sequences that combine proline with glycine and charged amino acids. Mammalian collagens are enriched in putative eIF5A-dependent Pro-Gly-containing tripeptides. Here, we show that depletion of active eIF5A in mouse fibroblasts reduced collagen type I α1 chain (Col1a1) content, which concentrated around the nuclei. Moreover, it provoked the upregulation of endoplasmic reticulum (ER) stress markers, suggesting retention of partially synthesized collagen 1 (Col1) in the ER. We confirmed that eIF5A is needed for heterologous collagen synthesis in yeast and, using a double luciferase reporter system, showed that eIF5A depletion interrupts translation at Pro-Gly collagenic motifs. A dramatically lower level of Col1a1 protein was also observed in functional eIF5A-depleted human hepatic stellate cells treated with the profibrotic cytokine TGF-ß1. In sum, our results show that collagen expression requires eIF5A and imply its potential as a target for regulating collagen production in fibrotic diseases.

Mechanisms of action of metformin in type 2 diabetes: Effects on mitochondria and leukocyte-endothelium interactions.

Type 2 diabetes (T2D) is a very prevalent, multisystemic, chronic metabolic disorder closely related to atherosclerosis and cardiovascular diseases. It is characterised by mitochondrial dysfunction and the presence of oxidative stress. Metformin is one of the safest and most effective anti-hyperglycaemic agents currently employed as first-line oral therapy for T2D. It has demonstrated additional beneficial effects, unrelated to its hypoglycaemic action, on weight loss and several diseases, such as cancer, cardiovascular disorders and metabolic diseases, including thyroid diseases. Despite the vast clinical experience gained over several decades of use, the mechanism of action of metformin is still not fully understood. This review provides an overview of the existing literature concerning the beneficial mitochondrial and vascular effects of metformin, which it exerts by diminishing oxidative stress and reducing leukocyte-endothelium interactions. Specifically, we describe the molecular mechanisms involved in metformin's effect on gluconeogenesis, its capacity to interfere with major metabolic pathways (AMPK and mTORC1), its action on mitochondria and its antioxidant effects. We also discuss potential targets for therapeutic intervention based on these molecular actions.

p53 and p53-related mediators PAI-1 and IGFBP-3 are downregulated in peripheral blood mononuclear cells of HIV-patients exposed to non-nucleoside reverse transcriptase inhibitors.

The improved effectiveness and safety of the combined antiretroviral therapy (cART) has largely diminished mortality and AIDS-defining morbidity of HIV-patients. Nevertheless, chronic age-related diseases in these individuals are more common and their underlying pathogenic mechanisms of these actions seem to involve accelerated aging and enhanced inflammation. The present study explores markers of these processes in a heterogenous Spanish HIV cohort using peripheral blood samples of HIV-patients and matched uninfected controls. We isolated periheral blood mononuclear cells (PBMCs) and i) compared the expression of a panel of 14 genes related to inflammation and senescence in PBMCs of HIV-patients vs matched uninfected controls, ii) analyzed the expression in HIV-patients in association with a number of demographic, biochemical and immunological parameters and iii) in relation with the current cART they received. PBMCs of HIV-patients displayed significantly increased expression of general inflammatory genes (IL6, IL18 and CXCL10) and this occurs irrespectively of the antiviral therapy they have been receiving. Conversely, levels of senescence-associated genes TP53, SERPINE1andIGFBP3 were slightly but significantly reduced in patients compared to uninfected matched individuals and this effect is related to NNRTI-containing treatments. The expression of the inflammatory markers IL6, IL18, IL1B, TNFA, RELA, CCL2, CCL20 and CXCL10 displayed correlation with certain demographic, morbidity- and HIV infection-related parameters. The levels of TP53 mRNA were positively associated only with plasma LDL. Correlation analysis between the expressions of pairs of genes revealed a different pattern between HIV-patients and controls. The diminished expression of TP53 and SERPINE1 in HIV-patients was also observed at a protein level, and the correlation between the two proteins (p53 and PAI1) in patients and controls showed the opposite trend. In conclusion, HIV-patients show dysregulation of p53 and p53-related mediators, a phenomenon which may be of pathophysiological relevance and could be related to the shorter health- and/or life-span observed in these individuals.

Self-assembled multivalent (SAMul) ligand systems with enhanced stability in the presence of human serum.

Self-assembled cationic micelles are an attractive platform for binding biologically-relevant polyanions such as heparin. This has potential applications in coagulation control, where a synthetic heparin rescue agent could be a useful replacement for protamine, which is in current clinical use. However, micelles can have low stability in human serum and unacceptable toxicity profiles. This paper reports the optimisation of self-assembled multivalent (SAMul) arrays of amphiphilic ligands to bind heparin in competitive conditions. Specifically, modification of the hydrophobic unit kinetically stabilises the self-assembled nanostructures, preventing loss of binding ability in the presence of human serum - cholesterol hydrophobic units significantly outperform systems with a simple aliphatic chain. It is demonstrated that serum albumin disrupts the binding thermodynamics of the latter system. Molecular simulation shows aliphatic lipids can more easily be removed from the self-assembled nanostructures than the cholesterol analogues. This agrees with the experimental observation that the cholesterol-based systems undergo slower disassembly and subsequent degradation via ester hydrolysis. Furthermore, by stabilising the SAMul nanostructures, toxicity towards human cells is decreased and biocompatibility enhanced, with markedly improved survival of human hepatoblastoma cells in an MTT assay.

Mitophagy in human astrocytes treated with the antiretroviral drug Efavirenz: Lack of evidence or evidence of the lack.

Efavirenz (EFV), a first generation non-nucleoside analogue reverse transcriptase inhibitor widely employed in combination antiretroviral therapy regimens over the last 20 years, has been associated with a wide range of neuropsychiatric effects and has also been linked with HIV-associated neurocognitive disorder (HAND). EFV has been reported to alter mitochondrial dysfunction and bioenergetics in different cell types, including astrocytes. Here, we analyzed whether this mitochondrial effect is associated with alterations in autophagy and, more specifically, mitophagy. U251-MG cells were exposed to EFV (10 and 25 µM; 24 h) and the effect was compared with that of CCCP - an uncoupler of the mitochondrial membrane potential and widely-employed in vitro inducer of mitophagy - and those of the known pharmacological stressors rotenone and thapsigargin, selected due to reported similarities with EFV. EFV induces autophagy with functional autophagic flux despite the accumulated p62/SQSTM1. However, it fails to activate canonical mitophagy (according to mitochondrial mass and expression of mitophagy-related proteins). The fact that EFV-exposed cells display decreased levels of TOM20, an outer mitochondrial membrane protein, together with the association of TOM20 with autophagosomes (LC3), points to an alternative form of mitochondrial degradation. Moreover, the perinuclear mitochondrial cluster in EFV-treated cells differs from that displayed with CCCP. Also, in EFV-treated cells, p62 was associated with mitochondria, which may be related to the mito-protective function of this autophagic protein. In conclusion, these findings add to the existing knowledge of the EFV-triggered mitochondrial interference, a mechanism that may be implicated in the adverse CNS events observed in the clinics.

Synthesis, spectroscopic studies and biological evaluation of acridine derivatives: The role of aggregation on the photodynamic efficiency.

Two new photoactive compounds (1 and 2) derived from the 9-amidoacridine chromophore have been synthesized and fully characterized. Their abilities to produce singlet oxygen upon irradiation have been compared. The synthesized compounds show very different self-aggregating properties since only 1 present a strong tendency to aggregate in water. Biological assays were conducted with two cell types: hepatoma cells (Hep3B) and human umbilical vein endothelial cells (HUVEC). Photodynamic therapy (PDT) studies carried out with Hep3B cells showed that non-aggregating compound 2 showed photoxicity, ascribed to the production of singlet oxygen, being aggregating compound 1 photochemically inactive. On the other hand suspensions of 1, characterized as nano-sized aggregates, have notable antiproliferative activity towards this cell line in the dark.

Role of p62/SQSTM1 beyond autophagy: a lesson learned from drug-induced toxicity in vitro.

BACKGROUND AND PURPOSE: SQSTM1/p62 is a multifunctional, stress-induced, scaffold protein involved in multiple cellular processes including autophagic clearance, regulation of inflammatory responses and redox homeostasis. Its altered function has been associated with different human pathologies, such as neurodegenerative, metabolic and bone diseases (down-regulation), and cancerogenesis (up-regulation). However, its role in the off-target effects of clinically used drugs is still not understood. EXPERIMENTAL APPROACH: We evaluated the expression of p62 in cultured Hep3B cells and their derived ρ° cells (lacking mitochondria), along with markers of autophagy and mitochondrial dysfunction. The effects of efavirenz were compared with those of known pharmacological stressors, rotenone, thapsigargin and CCCP, and we also used transient silencing with siRNA and p62 overexpression. Western blotting, quantRT-PCR and fluorescence microscopy were used to assay these effects and their underlying mechanisms. KEY RESULTS: In Hep3B cells, efavirenz augmented p62 protein content, an effect not observed in the corresponding ρ° cells. p62 up-regulation followed enhanced SQSTM1 expression mediated through the transcription factor CHOP/DDIT3, while other well-known regulators (NF-kB and Nrf2) were not involved. Inhibition of autophagy with 3MA or with transient silencing of Atg5 did not affect SQSTM1 expression in efavirenz-treated cells while p62 overexpression ameliorated the deleterious effect of efavirenz on cell viability. CONCLUSION AND IMPLICATIONS: In our model, p62 exerted a specific, autophagy-independent role and protected against efavirenz-induced mitochondrial ROS generation and activation of the NLRP3 inflammasome. These findings add to the multifunctional nature of p62 and may help to understand the off-target effects of clinically useful drugs.

Lon protease: a novel mitochondrial matrix protein in the interconnection between drug-induced mitochondrial dysfunction and endoplasmic reticulum stress.

BACKGROUND AND PURPOSE: Mitochondria-associated membranes (MAMs) are specific endoplasmic reticulum (ER) domains that enable it to interact directly with mitochondria and mediate metabolic flow and Ca2+ transfer. A growing list of proteins have been identified as MAMs components, but how they are recruited and function during complex cell stress situations is still not understood, while the participation of mitochondrial matrix proteins is largely unrecognized. EXPERIMENTAL APPROACH: This work compares mitochondrial/ER contact during combined ER stress/mitochondrial dysfunction using a model of human hepatoma cells (Hep3B cell line) treated for 24 h with classic pharmacological inducers of ER stress (thapsigargin), mitochondrial dysfunction (carbonyl cyanide m-chlorophenyl hydrazone or rotenone) or both (the antiretroviral drug efavirenz used at clinically relevant concentrations). KEY RESULTS: Markers of mitochondrial dynamics (dynamin-related protein 1, optic atrophy 1 and mitofusin 2) were expressed differently with these stimuli, pointing to a specificity of combined ER/mitochondrial stress. Lon, a matrix protease involved in protein and mtDNA quality control, was up-regulated at mRNA and protein levels under all conditions. However, only efavirenz decreased the mitochondrial content of Lon while increasing its extramitochondrial presence and its localization to MAMs. This latter effect resulted in an enhanced mitochondria/ER interaction, as shown by co-immunoprecipitation experiments of MAMs protein partners and confocal microscopy imaging. CONCLUSION AND IMPLICATIONS: A specific dual drug-induced mitochondria-ER effect enhances the MAMs content of Lon and its extramitochondrial expression. This is the first report of this phenomenon and suggests a novel MAMs-linked function of Lon protease.

Proton Pump Inhibitors Display Antitumor Effects in Barrett's Adenocarcinoma Cells.

Recent evidence has reported that proton pump inhibitors (PPIs) can exert antineoplastic effects through the disruption of pH homeostasis by inhibiting vacuolar ATPase (H+-VATPase), a proton pump overexpressed in several tumor cells, but this aspect has not been deeply investigated in EAC yet. In the present study, the expression of H+-VATPase was assessed through the metaplasia-dysplasia-adenocarcinoma sequence in Barrett's esophagus (BE) and the antineoplastic effects of PPIs and cellular mechanisms involved were evaluated in vitro. H+-VATPase expression was assessed by immunohistochemistry in paraffined-embedded samples or by immunofluorescence in cultured BE and EAC cell lines. Cells were treated with different concentrations of PPIs and parameters of citotoxicity, oxidative stress, and autophagy were evaluated. H+-VATPase expression was found in all biopsies and cell lines evaluated, showing differences in the location of the pump between the cell lines. Esomeprazole inhibited proliferation and cell invasion and induced apoptosis of EAC cells. Production of reactive oxygen species (ROS) seemed to be involved in the cytotoxic effects observed since the addition of N-acetylcysteine significantly reduced esomeprazole-induced apoptosis in EAC cells. Esomeprazole also reduced intracellular pH of tumor cells, whereas only disturbed the mitochondrial membrane potential in OE33 cells. Esomeprazole induced autophagy in both EAC cells, but also triggered a blockade in autophagic flux in the metastatic cell line. These data provide in vitro evidence supporting the potential use of PPIs as novel antineoplastic drugs for EAC and also shed some light on the mechanisms that trigger PPIs cytotoxic effects, which differ upon the cell line evaluated.

Endoplasmic Reticulum and Mitochondria: Independent Roles and Crosstalk in Fatty Liver Diseases and Hepatic Inflammation.

Proper function of the endoplasmic reticulum (ER) and mitochondria is essential for cellular homeostasis and the regulation of metabolic pathways. Perturbation of their function has been linked to pathophysiological states, including metabolic and liver diseases. Fatty liver diseases are a major health problem whose prevalence is dramatically increasing, may be induced by several factors (mainly chronic alcohol consumption, drugs or metabolic alterations), and share common features as lipid deposition, inflammation, oxidative stress and progression to more severe clinical stages, such as fibrosis, cirrhosis or even hepatocellular carcinoma. Besides their independent contributions to metabolic and hepatic pathologies, mitochondria and ER directly interact regulating each other's function, and ER-mitochondria interface is involved in several molecular pathways, as induction of autophagy and triggering of inflammatory cascades. Disturbances in these interactions have already been implicated in different human diseases, and increasing interest is arising in their role on liver illnesses. This review summarizes the current understanding regarding mitochondrial and ER implication in fatty liver diseases, focusing on lipid accumulation and inflammation, and the relevance of both the individual functions of these organelles and of ER-mitochondria interactions in such processes. In addition, it describes the clinical implications and the available therapeutic options targeting directly these organelles or associated molecular mechanisms.

The purine analogues abacavir and didanosine increase acetaminophen-induced hepatotoxicity by enhancing mitochondrial dysfunction.

BACKGROUND: NRTIs are essential components of HIV therapy with well-documented, long-term mitochondrial toxicity in hepatic cells, but whose acute effects on mitochondria are unclear. As acetaminophen-induced hepatotoxicity also involves mitochondrial interference, we hypothesized that it would be exacerbated in the context of ART. METHODS: We evaluated the acute effects of clinically relevant concentrations of the most widely used NRTIs, alone or combined with acetaminophen, on mitochondrial function and cellular viability. RESULTS: The purine analogues abacavir and didanosine produced an immediate and concentration-dependent inhibition of oxygen consumption and complex I and III activity. This inhibition was accompanied by an undermining of mitochondrial function, with increased production of reactive oxygen species and reduction of mitochondrial membrane potential and intracellular ATP levels. However, this interference did not compromise cell survival. Co-administration with concentrations of acetaminophen below those considered hepatotoxic exacerbated the deleterious effects of both compounds on mitochondrial function and compromised cellular viability, showing a clear correlation with diminished glutathione levels. CONCLUSIONS: The simultaneous presence of purine analogues and low concentrations of acetaminophen significantly potentiates mitochondrial dysfunction, increasing the risk of liver injury. This new mechanism is relevant given the liver's susceptibility to mitochondrial dysfunction-related toxicity and the tendency of the HIV infection to increase oxidative stress.

Efavirenz alters mitochondrial respiratory function in cultured neuron and glial cell lines.

BACKGROUND: The NNRTI efavirenz is among the most widely employed antiretroviral drugs. Although it is considered safe, efavirenz has been linked with several adverse effects including neurological manifestations, which appear in the majority of the patients on efavirenz-containing regimens. The molecular mechanisms responsible for these manifestations are not understood, but mounting evidence points to altered brain bioenergetics. METHODS: We evaluated the effect of short-term efavirenz treatment on the mitochondrial respiratory function of cultured glioblastoma and differentiated neuroblastoma cell lines using a Seahorse Extracellular Flux Analyzer. RESULTS: Incubation with efavirenz provoked a significant and concentration-dependent decrease in basal respiration and specifically in ATP production-coupled O2 consumption in both SH-SY5Y and U-251MG cells, with the effect being more pronounced in the latter. In contrast, efavirenz did not alter mitochondrial proton leakage in either of the cell types. Efavirenz led to a decrease in the respiratory control ratio as well as to a reduction in the maximal respiration rate and spare respiratory capacity in both U-251MG and SH-SY5Y cells, the former cells being more susceptible. CONCLUSIONS: These findings reveal that efavirenz specifically alters mitochondrial respiration, which is of relevance for a better understanding of the molecular mechanisms responsible for the efavirenz-associated neurological effects that have been recorded in clinical situations.

Heat Stress Induces Extended Plateau of Hsp70 Accumulation--A Possible Cytoprotection Mechanism in Hepatic Cells.

The relevance of heat preconditioning resides in its ability to protect cells from different kinds of injury by induction of heat shock proteins, a process in which the intensity of heat stress (HS) and duration of subsequent recovery are vital. This study evaluates the effects of moderate HS (45 min/43°C) and the time-dependent changes during recovery period of HSP70, Bcl-2 and p53 gene and protein expression in HepG2 cells. We also evaluated the effects of 0.4 mM aspirin (ASA) as a potential pharmacological co-inducer of HSP, both alone and in a combination with HS (ASA + HS). HS alone and ASA + HS caused a major up-regulation of HSP70 mRNA in the first 2 h, while HSP70 protein increased gradually and was especially abundant from 2 h to 24 h. Regarding Bcl-2, all treatments rendered similar results: gene expression was down-regulated in the first 2 h, after which there was protein elevation (12-48 h after HS). mRNA expression of p53 in HS- and (ASA + HS)-cells was down-regulated in the first 12 h. The immediate decrease of p53 protein after HS was followed by a biphasic increase. In conclusion, 0.4 mM ASA + HS does not act as a co-inducer of HSP70 in HepG2 cells, but promotes Bcl-2 protein expression during prolonged treatment. Our suggestion is that hepatic cells are most vulnerable in the first 2-6 h, but may have a high capacity for combating stress 12-24 h after HS. Finally, short-term exposure HS might be a "physiological conditioner" for liver cells to accumulate HSP and Bcl-2 proteins and thus obtain cytoprotection against an additional stress.

Is glycemic control modulating endoplasmic reticulum stress in leukocytes of type 2 diabetic patients?

Oxidative and endoplasmic reticulum (ER) stress is related to type 2 diabetes (T2D), but the influence of glycemic control on these parameters and its relationship with leukocyte-endothelial interactions is not known. In our study population consisting of 164 diabetic patients, (102 with glycated hemoglobin [HbA1c] <7% and 62 with HbA1c >7%) and 84 nondiabetic controls, we have verified a common anthropometric and metabolic pattern of T2D with dyslipidemia. Inflammatory parameters (high-sensitive C-reactive protein [hs-CRP] and tumor necrosis factor alpha [TNFα]) and E-selectin levels were enhanced in the HbA1c >7% group with regard to controls. O2 consumption and mitochondrial membrane potential were lower in diabetic patients than in controls. Reactive oxygen species (ROS) production was enhanced in diabetic patients than in controls and positively correlated with HbA1c levels. GRP78 levels were higher in both diabetic groups. However, HbA1c <7% patients displayed higher levels of spliced X-box binding protein 1 (sXBP1), whereas HbA1c >7% patients exhibited preferentially enhanced levels of CHOP (CCAAT/enhancer binding protein [C/EBP] homologous protein) and activating transcription factor 6 (ATF6). Reduced leukocyte rolling velocity and increased rolling flux and adhesion were observed in diabetic patients. Our findings lead to the hypothesis of an association between poor glycemic control in T2D and increased leukocyte ROS production and chronic ER stress that could finally promote leukocyte-endothelial interactions, which, in turn, poses a risk of vascular complications for these patients.

Lack of mitochondrial toxicity of darunavir, raltegravir and rilpivirine in neurons and hepatocytes: a comparison with efavirenz.

OBJECTIVES: Growing evidence associates the non-nucleoside reverse transcriptase inhibitor efavirenz with several adverse events. Newer antiretrovirals, such as the integrase inhibitor raltegravir, the non-nucleoside reverse transcriptase inhibitor rilpivirine and the protease inhibitor darunavir, claim to have a better toxicological profile than efavirenz while producing similar levels of efficacy and virological suppression. The objective of this study was to determine the in vitro toxicological profile of these three new antiretrovirals by evaluating their effects on the mitochondrial and cellular parameters altered by efavirenz in hepatocytes and neurons. METHODS: Hep3B cells and primary rat neurons were treated with clinically relevant concentrations of efavirenz, darunavir, rilpivirine or raltegravir. Parameters of mitochondrial function, cytotoxicity and oxidative and endoplasmic reticulum stress were assessed using standard cell biology techniques. RESULTS: None of the new compounds altered the mitochondrial function of hepatic cells or neurons, while efavirenz decreased mitochondrial membrane potential and enhanced superoxide production in both cell types, effects that are known to significantly compromise the functioning of mitochondria, cell viability and, ultimately, cell number. Of the four drugs assayed, efavirenz was the only one to alter the protein expression of LC3-II, an indicator of autophagy, and CHOP, a marker of endoplasmic reticulum stress and the unfolded protein response. CONCLUSIONS: Darunavir, rilpivirine and raltegravir do not induce toxic effects on Hep3B cells and primary rat neurons, which suggests a safer hepatic and neurological profile than that of efavirenz.

Neuronal bioenergetics and acute mitochondrial dysfunction: a clue to understanding the central nervous system side effects of efavirenz.

BACKGROUND: Neurological pathogenesis is associated with mitochondrial dysfunction and differences in neuronal/glial handling of oxygen and glucose. The main side effects attributed to efavirenz involve the CNS, but the underlying mechanisms are unclear. METHODS: Human cell lines and rat primary cultures of neurons and astrocytes were treated with clinically relevant efavirenz concentration. RESULTS: Efavirenz alters mitochondrial respiration, enhances reactive oxygen species generation, undermines mitochondrial membrane potential, and reduces adenosine triphosphate (ATP) levels in a concentration-dependent fashion in both neurons and glial cells. However, it activates adenosine monophosphate-activated protein kinase only in glial cells, upregulating glycolysis and increasing intracellular ATP levels, which do not occur in neurons. To reproduce the conditions that often exist in human immunodeficiency virus-related neuroinflammatory disorders, the effects of efavirenz were evaluated in the presence of exogenous nitric oxide, an inflammatory mediator and mitochondrial inhibitor. The combination potentiated the effects on mitochondrial parameters in both neurons and glial cells, but ATP generation and lactate production were enhanced only in glial cells. CONCLUSIONS: 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.

Profile of stress and toxicity gene expression in human hepatic cells treated with Efavirenz.

Hepatic toxicity and metabolic disorders are major adverse effects elicited during the pharmacological treatment of the human immunodeficiency virus (HIV) infection. Efavirenz (EFV), the most widely used non-nucleoside reverse transcriptase inhibitor (NNRTI), has been associated with these events, with recent studies implicating it in stress responses involving mitochondrial dysfunction and oxidative stress in human hepatic cells. To expand these findings, we analyzed the influence of EFV on the expression profile of selected stress and toxicity genes in these cells. Significant up-regulation was observed with Cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1), which indicated metabolic stress. Several genes directly related to oxidative stress and damage exhibited increased expression, including Methalothionein 2A (MT2A), Heat shock 70kDa protein 6 (HSPA6), Growth differentiation factor 15 (GDF15) and DNA-damage-inducible transcript 3 (DDIT3). In addition, Early growth response protein 1 (EGR1) was enhanced, whereas mRNA levels of the inflammatory genes Chemokine (C-X-C motif) ligand 10 (CXCL10) and Serpin peptidase inhibitor (nexin, plasminogen activator inhibitor type 1), member 1 (SERPINE1) decreased and increased, respectively. This profile of gene expression supports previous data demonstrating altered mitochondrial function and presence of oxidative stress/damage in EFV-treated hepatic cells, and may be of relevance in the search for molecular targets with therapeutic potential to be employed in the prevention, diagnosis and treatment of the hepatic toxicity associated with HIV therapy.