Differential response of neurons and glia to the mitochondrial effects produced by the antiretroviral drug Efavirenz.

HA Funes1,2, A Blas-Garcia1, N Apostolova1, JV Esplugues1,2. 1University of Valencia. Facultaty of Medicine., Pharmacology 46010, Spain, 2Fundación Hospital Universitario Dr. Peset, 46017, Spain

Background: Antiretroviral therapy has made treatment of human immunodeficiency virus (HIV) a chronic rather than terminal illness. As the clinical outcome of patients has improved there has been growing emphasis on the long term adverse effects of these drugs. Efavirenz (EFV), the most widely employed non-nucleoside reverse transcriptase inhibitor (NNRTI), has been associated with the development of adverse neuronal events. The underlying molecular mechanisms of these effects are unknown, though reports of mitotoxicity could be relevant in this respect.

Aim: To assess the effect of plasmatic concentrations of EFV (10 and 25 µM) on the mitochondrial and bioenergetic functions of human glioblastoma cells (U-251MG) and human neuroblastoma cells (SH-SY5Y) in which neuronal differentiation has previously been induced.

Results: Cellular proliferation and viability were reduced in a concentration-dependent manner. In addition, EFV directly affected mitochondrial function, inducing a reduction in O2 consumption, a decrease in mitochondrial membrane potential (∆Ψm), and an increase in mitochondrial reactive oxygen species (ROS) production in both cell types. Interestingly, a drop in the level of intracellular adenosine triphosphate (ATP) was only observed in neuroblastoma cells. Human glioblastoma cells displayed an increased concentration of ATP due to active glycolysis, evident in an enhanced lactate production. We also analysed the impact of nitric oxide (NO), a mediator present in many inflammatory conditions in neuronal cells, on the mitochondrial effect induced by EFV. To do this, both cell types were exposed to exogenous nitric oxide (NO) following treatment with DETA-NO, a common NO-donor. Fluorescence microscopy demonstrated that co-treatment of both cell types with EFV and DETA-NO led to a lower ∆Ψm and higher ROS with respect to cells treated with NO or EFV individually. Additionally, it was observed that co-treatment with NO and EFV produced a more marked decrease in the number of neuroblastoma cells than individual treatment which pointed to an increased toxicity. All data were expressed as mean±SEM. One-way ANOVA followed by Newman-keuls post hoc test analysis were done and statistical significance was set up at *p<0.05 or **<0.01 (vs. vehicle). All experiments were performed in groups of n≥4.

Conclusions: Clinically relevant plasma concentrations of EFV are mitotoxic in human glioma and neuroblastoma cell lines, an effect that is enhanced by the presence of NO in both cell types. In addition, inhibition of mitochondrial function by EFV up-regulates the glycolytic pathway in human glioblastoma cells. These findings could help to improve understanding of the neuronal side effects associated with EFV in HIV patients with dementia and/or those suffering disruption of the blood-brain-barrier (BBB) and presence of inflammation as a result of encephalopathy.