An in Vitro Model of Aspartame Cytotoxicity via Heterologous Expression of Nmda Receptors

Abstract

Within the last 40 years, aspartame (L-aspartyl-L-phenylalanyl-methylester) became a very popular artificial sweetener, being introduced in over 6000 food products worldwide, including solid foods, beverages, and drugs for oral administration. However, its safety and toxicity have been subject of concern since its discovery, due to potentially harmful effects of its intestinal decomposition products: aspartate, phenylalanine, and methanol. Neuropsychological effects have been reported occasionally, such as headache, blurred vision, insomnia, torpor, memory loss, nausea, speech impairment, personality changes, loss of energy, hyperactivity, hearing problems. Our aim in the present study was to determine the effects of aspartame at various concentrations on the viability of cells transiently transfected with the combination of N-methyl-Daspartate (NMDAR) ionotropic glutamate receptor subunits NR1+NR2b, as well as to prove direct activation of NMDAR currents by aspartame via whole-cell patch-clamp experiments. CHO-K1 cells transfected with NR1+NR2b via plasmid constructs containing the two genes fused with enhanced green fluorescent protein (eGFP) gene were exposed for 2 -4 h to aspartame in progressive decimal dilutions (0.1 µM to 10 mM) and assessed by flow cytometry after propidium iodide (PI) staining, showing increased percentages of dead cells (PI+) at aspartame concentrations of 1 µM or higher, while nontransfected cells featured increased PI+ percentages at aspartame concentrations above 1 mM. We also showed in HEK293T cells transfected by electroporation with the same plasmid combination (NR1+NR2b) transient outward NMDAR currents at +40 mV elicited by brief pulses of glutamate 100 µM or aspartame 100 µM. RezumatÎn ultimii 40 ani, aspartamul a devenit un îndulcitor artificial popular, fiind introdus la nivel mondial în peste 6000 alimente, băuturi și medicamente pentru administrare orală. Totuși, siguranța și toxicitatea sa au fost subiecte de interes încă de la descoperire, datorită potențialelor efecte nocive ale produșilor săi de descompunere intestinală: aspartat, fenilalanină, metanol. Efecte neuropsihice au fost raportate uneori, precum cefalee, vedere neclară, insomnie, torpoare, pierderi de memorie, greață, tulburări de vorbire, schimbarea personalității, pierderea energiei, hiperactivitate, tulburări auditive. Scopul nostru a fost de a determina efectele aspartamului la diverse concentrații asupra viabilității celulelor transfectate tranzient cu combinația de subunități de receptor de N-metil-D-aspartat (NMDAR) NR1+NR2b, şi de a demonstra direct activarea de către aspartam a NMDAR în experimente de whole-cell patch-clamp. Celule CHO-K1 transfectate cu NR1+NR2b prin plasmide conținând cele 2 gene cuplate cu eGFP au fost expuse 2 -4 h la diluții decimale progresive de aspartam (0,1 µM-10 mM) și măsurate prin citometrie în flux după marcare cu iodură de propidiu (PI), arătând procentaje crescute de celule moarte (PI+) la concentrații de aspartam peste 1 µM, în timp ce celulele netransfectate au avut p...