Miriam Polo

Involvement of Nitric Oxide in the Mitochondrial Action of Efavirenz: A Differential Effect on Neurons and Glial Cells

The anti-human immunodeficiency virus (HIV) drug efavirenz (EFV) alters mitochondrial function in cultured neurons and glial cells. Nitric oxide (NO) is a mediator of mitochondrial dysfunction associated with HIV central nervous system symptoms. We show that EFV promotes inducible nitric oxide synthase (iNOS) expression in cultured glial cells and generated NO undermines their mitochondrial function, as inhibition of NOS partially reverses this effect. EFV inhibits mitochondrial Complex I in both neurons and glia; however, when the latter cells are treated for longer periods, other mitochondrial complexes are also affected in accordance with the increased NO production. These findings shed light on the mechanisms responsible for the frequent EFV-associated neurotoxicity.

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.

Mitochondrial (dys)function – a factor underlying the variability of efavirenz-induced hepatotoxicity?

The non‐nucleoside analogue reverse transcriptase inhibitor efavirenz is associated with hepatic toxicity and metabolic disturbances. Although the mechanisms involved are not clear, recent evidence has pinpointed a specific mitochondrial action of efavirenz accompanied by the induction of an endoplasmic reticulum (ER) stress/unfolded protein response in human hepatic cells. The aim of this study was to further investigate the involvement of this organelle by evaluating efavirenzs effects in cells lacking functional mitochondria (rho°) and comparing them with those of the typical mitotoxic agent rotenone, a standard complex I inhibitor, and the ER stress inducer thapsigargin.

The Pivotal Role of Nitric Oxide: Effects on the Nervous and Immune Systems

Nitric oxide (NO) has an important role in physiological and pathological processes in general, and in particular plays a homeostatic role in the nervous and immune systems. The many different physiological functions of NO include those of a mediator of blood vessel dilation, neurotransmitter, neuromodulator and inductor of mitochondrial biogenesis. In addition, NO can transform into highly reactive and harmful molecules producing an impairment of the DNA, lipids or proteins, and thus altering their function. This dual action of NO, by which it plays an important role in homeostasis and aids the development of pathological processes, makes this molecule an interesting target for medical therapies, especially with respect to the nervous and immune systems. This review describes the multiple roles of NO played out in the nervous and immune systems during different physiological and pathophysiological processes.

Different origin of adipogenic stem cells influences the response to antiretroviral drugs

Lipodystrophy (LD) is a main side effect of antiretroviral therapy for HIV infection, and can be provoked by nucleoside reverse transcriptase inhibitors (NRTIs) and protease inhibitors (PIs). LD exists in different forms, characterized by fat loss, accumulation, or both, but its pathogenesis is still unclear. In particular, few data exist concerning the effects of antiretroviral drugs on adipocyte differentiation. Adipose tissue can arise either from mesenchymal stem cells (MSCs), that include bone marrow-derived MSCs (hBM-MSCs), or from ectodermal stem cells, that include dental pulp stem cells (hDPSCs). To analyze whether the embryonal origin of adipocytes might impact the occurrence of different phenotypes in LD, we quantified the effects of several antiretroviral drugs on the adipogenic differentiation of hBM-MSCs and hDPSCs. hBM-MSCs and hDPSCs were isolated from healthy donors. Cells were treated with 10 and 50 μM stavudine (d4T), efavirenz (EFV), atazanavir (ATV), ritonavir (RTV), and ATV-boosted RTV. Viability and adipogenesis were evaluated by staining with propidium iodide, oil red, and adipoRed; mRNA levels of genes involved in adipocyte differentiation, i.e. CCAAT/enhancer-binding protein alpha (CEBPα) and peroxisome proliferator-activated receptor gamma (PPARγ), and in adipocyte functions, i.e. fatty acid synthase (FASN), fatty acid binding protein-4 (FABP4), perilipin-1 (PLIN1) and 1-acylglycerol-3-phosphate O-acyltransferase-2 (AGPAT2), were quantified by real time PCR. We found that ATV, RTV, EFV, and ATV-boosted RTV, but not d4T, caused massive cell death in both cell types. EFV and d4T affected the accumulation of lipid droplets and induced changes in mRNA levels of genes involved in adipocyte functions in hBM-MSCs, while RTV and ATV had little effects. All drugs stimulated the accumulation of lipid droplets in hDPSCs. Thus, the adipogenic differentiation of human stem cells can be influenced by antiretroviral drugs, and depends, at least in part, on their embryonal origin.

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 livers susceptibility to mitochondrial dysfunction-related toxicity and the tendency of the HIV infection to increase oxidative stress.

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

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.

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

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.

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

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.

Toxicological properties of two fluorescent carbon quantum dots with onion ring morphology and their usefulness as bioimaging agents

In the present work, two carbon quantum dots with onion ring morphology, C-NOR and C-NOR(Eu) with an average size of 40 nm differing in the absence or presence of Eu3+ as Lewis acids during their preparation were synthesized and fully characterized by several techniques. These nanoparticles can be internalized into human HeLa and Hep3B carcinoma cells where they exhibit interesting photoluminescent properties, in the same manner as in solution, confirming their utility as bioimaging agents. To address this possibility, a complete in vitro toxicological study has been performed here. Viability, proliferation, apoptosis and oxidative stress assessments upon limited or continuous exposure were done. It was observed that both nanoparticles did not show toxicity in both situations at low concentration, although some toxicity has been determined at higher concentrations under continuous exposure. These results support the possible use of C-NOR and C-NOR(Eu) nanoparticles as bioimaging agents.