Aspartame carcinogenic potential revealed through network toxicology and molecular docking insights.

The research employed network toxicology and molecular docking techniques to systematically examine the potential carcinogenic effects and mechanisms of aspartame (L-α-aspartyl-L-phenylalanine methyl ester). Aspartame, a commonly used synthetic sweetener, is widely applied in foods and beverages globally. In recent years, its safety issues, particularly the potential carcinogenic risk, have garnered widespread attention. The study first constructed an interaction network map of aspartame with gastric cancer targets using network toxicology methods and identified key targets and pathways. Preliminary validation was conducted through microarray data analysis and survival analysis, and molecular docking techniques were employed to further examine the binding affinity and modes of action of aspartame with key proteins. The findings suggest that aspartame has the potential to impact various cancer-related proteins, potentially raising the likelihood of cellular carcinogenesis by interfering with biomolecular function. Furthermore, the study found that the action patterns and pathways of aspartame-related targets are like the mechanisms of known carcinogenic pathways, further supporting the scientific hypothesis of its potential carcinogenicity. However, given the complexity of the in vivo environment, we also emphasize the necessity of validating these molecular-level findings in actual biological systems. The study introduces a fresh scientific method for evaluating the safety of food enhancers and provides a theoretical foundation for shaping public health regulations.

Revisiting the Sweet Taste Receptor T1R2-T1R3 through Molecular Dynamics Simulations Coupled with a Noncovalent Interactions Analysis.

It is nowadays widely accepted that sweet taste perception is elicited by the activation of the heterodimeric complex T1R2-T1R3, customarily known as sweet taste receptor (STR). However, the interplay between STR and sweeteners has not yet been fully clarified. Here through a methodology coupling molecular dynamics and the independent gradient model (igm) approach we determine the main interacting signatures of the closed (active) conformation of the T1R2 Venus flytrap domain (VFD) toward aspartame. The igm methodology provides a rapid and reliable quantification of noncovalent interactions through a score (Δginter) based on the attenuation of the electronic density gradient when two molecular fragments approach each other. Herein, this approach is coupled to a 100 ns molecular dynamics simulation (MD-igm) to explore the ligand-cavity contacts on a per-residue basis as well as a series of key inter-residue interactions that stabilize the closed form of VFD. We also apply an atomic decomposition scheme of noncovalent interactions to quantify the contribution of the ligand segments to the noncovalent interplay. Finally, a series of structural modification on aspartame are conducted in order to obtain guidelines for the rational design of novel sweeteners. Given that innovative methodologies to reliably quantify the extent of ligand-protein coupling are strongly demanded, this approach combining a noncovalent analysis and MD simulations represents a valuable contribution, that can be easily applied to other relevant biomolecular systems.

Aspartame consumption during pregnancy impairs placenta growth in mice through sweet taste receptor-reactive oxygen species-dependent pathway.

The prevalence of obesity has risen dramatically over recent years, and so has the prevalence of adverse obesity-associated pregnancy outcomes. To combat obesity, the calorie contents of many foods and beverages may be reduced by the use of artificial sweeteners, such as aspartame. However, animal studies suggest that aspartame and its metabolites may exhibit toxicity, and the effects of aspartame on pregnancy are largely unknown. In this study, we treated pregnant mice with aspartame by oral gavage and found that the treatment decreased fasting blood glucose level, whereas systolic blood pressure was elevated. Importantly, the aspartame-treated animals also had low placenta and fetus weights, as well as reduced thickness of the placenta decidua layer. Moreover, aspartame decreased the expression of epithelial-mesenchymal transition proteins and manganese superoxide dismutase (MnSOD) in mouse placentae. In order to clarify the mechanisms though which aspartame affects placenta, we performed experiments on 3A-sub-E trophoblasts. In the cells, aspartame treatments induced cell cycle arrest and reduced the proliferation rate, epithelial-mesenchymal transition, migration activity and invasion activity. We also found that aspartame increased reactive oxygen species (ROS) levels to hyper-activate Akt and downregulate MnSOD expression. Pretreatment with antioxidants or sweet taste receptor inhibitors reversed the effects of aspartame on trophoblast function. We also found that the aspartame metabolite phenylalanine similarly induced ROS production and affected proliferation of trophoblasts. Taken together, our data suggest that aspartame consumption during pregnancy may impact the structure, growth and function of the placenta via sweet taste receptor-mediated stimulation of oxidative stress.

A novel route for aspartame production by combining enzymatic and chemical reactions for industrial use.

Here, we report a novel industrial aspartame production route, involving the enzymatic production of α-l-aspartyl-l-phenylalanine ß-methylester from l-aspartic acid dimethylester and l-phenylalanine by α-amino acid ester acyl transferase. The route also involves the chemical transformation of α-l-aspartyl-l-phenylalanine ß-methylester to α-l-aspartyl-l-phenylalanine methylester hydrochloride (aspartame hydrochloride) in an aqueous solution with methanol and HCl, followed by HCl removal to form aspartame.

Mannosylated Cationic Copolymers for Gene Delivery to Macrophages.

Macrophages are desirable targets for gene therapy of cancer and other diseases. Cationic diblock copolymers of polyethylene glycol (PEG) and poly-L-lysine (PLL) or poly{N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} (pAsp(DET)) are synthesized and used to form polyplexes with a plasmid DNA (pDNA) that are decorated with mannose moieties, serving as the targeting ligands for the C type lectin receptors displayed at the surface of macrophages. The PEG-b-PLL copolymers are known for its cytotoxicity, so PEG-b-PLL-based polyplexes are cross-linked using reducible reagent dithiobis(succinimidyl propionate) (DSP). The cross-linked polyplexes display low toxicity to both mouse embryonic fibroblasts NIH/3T3 cell line and mouse bone marrow-derived macrophages (BMMΦ). In macrophages mannose-decorated polyplexes demonstrate an ≈8 times higher transfection efficiency. The cross-linking of the polyplexes decrease the toxicity, but the transfection enhancement is moderate. The PEG-b-pAsp(DET) copolymers display low toxicity with respect to the IC-21 murine macrophage cell line and are used for the production of non-cross-linked pDNA-contained polyplexes. The obtained mannose modified polyplexes exhibit ca. 500-times greater transfection activity in IC-21 macrophages compared to the mannose-free polyplexes. This result greatly exceeds the targeting gene transfer effects previously described using mannose receptor targeted non-viral gene delivery systems. These results suggest that Man-PEG-b-pAsp(DET)/pDNA polyplex is a potential vector for immune cells-based gene therapy.

Food and Beverage Ingredients Induce the Formation of Silver Nanoparticles in Products Stored within Nanotechnology-Enabled Packaging.

Nanotechnology-based packaging may improve food quality and safety, but packages manufactured with polymer nanocomposites (PNCs) could be a source of human dietary exposure to engineered nanomaterials (ENMs). Previous studies showed that PNCs release ENMs to foods predominantly in a dissolved state, but most of this work used food simulants like dilute acetic acid and water, leaving questions about how substances in real foods may influence exposure. Here, we demonstrate that food and beverage ingredients with reducing properties, like sweeteners, may alter exposure by inducing nanoparticle formation in foods contacting silver nanotechnology-enabled packaging. We incorporated 12.8 ± 1.4 nm silver nanoparticles (AgNPs) into polyethylene and stored media containing reducing ingredients in packages manufactured from this material under accelerated room-temperature and refrigerated conditions. Analysis of the leachates revealed that reducing ingredients increased the total silver transferred to foods contacting PNC packaging (by as much as 7-fold) and also induced the (re)formation of AgNPs from this dissolved silver during storage. AgNP formation was also observed when Ag+ was introduced to solutions of natural and artificial sweeteners (glucose, sucrose, aspartame), commercial beverages (soft drinks, juices, milk), and liquid foods (yogurt, starch slurry), and the amount and morphology of reformed AgNPs depended on the ingredient formulation, silver concentration, storage conditions, and light exposure. These results imply that food and beverage ingredients may influence dietary exposure to nanoparticles when PNCs are used in packaging applications, and the practice of using food simulants may in certain cases underpredict the amount of ENMs likely to be found in foods stored in these materials.

Preparation of an aspartame and N-isopropyl acrylamide copolymer functionalized stationary phase with multi-mode and chiral separation abilities.

A novel multi-mode and chiral separation stationary phase co-modified with copolymer composed of N-isopropyl acrylamide (NIPAM) and aspartame was synthesized by atom transfer radical polymerization (ATRP) reaction. The synthetic material was evaluated using thermogravimetric analysis (TGA), Fourier transform infrared spectrometry (FT-IR) and elemental analysis (EA). Analytes including hydrophobic, hydrophilic, alkaline and acidic compounds were separated well using the prepared stationary phase named Sil-PPAM-NIPAM. Besides, the separation of chiral compounds proved that the developed column also has the potential of chiral separation ability. In summary, the prepared column possesses excellent hydrophilic interaction, ion exchange, reversed-phase and chiral separation modes during the separation of complex and chiral compounds.

Aspartame and Phe-Containing Degradation Products in Soft Drinks across Europe.

Phenylketonuria and tyrosinemia type 1 are treated with dietary phenylalanine (Phe) restriction. Aspartame is a Phe-containing synthetic sweetener used in many products, including many 'regular' soft drinks. Its amount is (often) not declared; therefore, patients are advised not to consume aspartame-containing foods. This study aimed to determine the variation in aspartame concentrations and its Phe-containing degradation products in aspartame-containing soft drinks. For this, an LC-MS/MS method was developed for the analysis of aspartame, Phe, aspartylphenylalanine, and diketopiperazine in soft drinks. In total, 111 regularly used soft drinks from 10 European countries were analyzed. The method proved linear and had an inter-assay precision (CV%) below 5% for aspartame and higher CVs% of 4.4-49.6% for the degradation products, as many concentrations were at the limit of quantification. Aspartame and total Phe concentrations in the aspartame-containing soft drinks varied from 103 to 1790 µmol/L (30-527 mg/L) and from 119 to 2013 µmol/L (20-332 mg/L), respectively, and were highly variable among similar soft drinks bought in different countries. Since Phe concentrations between drinks and countries highly vary, we strongly advocate the declaration of the amount of aspartame on soft drink labels, as some drinks may be suitable for consumption by patients with Phe-restricted diets.

Taste compound - Nanocellulose interaction assessment by fluorescence indicator displacement assay.

Interactions between taste compounds and nanofibrillar cellulose were studied. For this, a new fluorescent indicator displacement method was developed. Two fluorescent indicators, namely, Calcofluor white and Congo red, were chosen because of their specific binding to cellulose and intrinsic fluorescence. Seven taste compounds with different structures were successfully measured together with nanofibrillar cellulose (NFC) and ranked according to their binding constants. The most pronounced interactions were found between quinine and NFC (1.4 × 104 M-1), whereas sucrose, aspartame and glutamic acid did not bind at all. Naringin showed moderate binding while stevioside and caffeine exhibited low binding. The comparison with microcrystalline cellulose indicates that the larger surface area of nanofibrillated cellulose enables stronger binding between the binder and macromolecules. The developed method can be further utilized to study interactions of different compound classes with nanocellulose materials in food, pharmaceutical and dye applications, using a conventional plate reader in a high-throughput manner.

Introducing a New Model of Sweet Taste Receptor, a Class C G-protein Coupled Receptor (C GPCR).

The structure of sweet taste receptor (STR), a heterodimer of class C G-protein coupled receptors comprising T1R2 and T1R3 molecules, is still undetermined. In this study, a new enhanced model of the receptor is introduced based on the most recent templates. The improvement, stability, and reliability of the model are discussed in details. Each domain of the protein, i.e., VFTM, CR, and TMD, were separately constructed by hybrid-model construction methods and then assembled to build whole monomers. Overall, 680 ns molecular dynamics simulation was performed for the individual domains, the whole monomers and the heterodimer form of the VFTM orthosteric binding site. The latter's structure obtained from 200 ns simulation was docked with aspartame; among various binding sites suggested by FTMAP server, the experimentally suggested binding domain in T1R2 was retrieved. Local three-dimensional structures and helices spans were evaluated and showed acceptable accordance with the template structures and secondary structure predictions. Individual domains and whole monomer structures were found stable and reliable to be used. In conclusion, several validations have shown reliability of the new and enhanced models for further molecular modeling studies on structure and function of STR and C GPCRs.

Self-Assembly of Artificial Sweetener Aspartame Yields Amyloid-like Cytotoxic Nanostructures.

Recent reports have revealed the intrinsic propensity of single aromatic metabolites to undergo self-assembly and form nanostructures of amyloid nature. Hence, identifying whether aspartame, a universally consumed artificial sweetener, is inherently aggregation prone becomes an important area of investigation. Although the reports on aspartame-linked side effects describe a multitude of metabolic disorders, the mechanistic understanding of such destructive effects is largely mysterious. Since aromaticity, an aggregation-promoting factor, is intrinsic to aspartame's chemistry, it is important to know whether aspartame can undergo self-association and if such a property can predispose any cytotoxicity to biological systems. Our study finds that aspartame molecules, under mimicked physiological conditions, undergo a spontaneous self-assembly process yielding regular ß-sheet-like cytotoxic nanofibrils of amyloid nature. The resultant aspartame fibrils were found to trigger amyloid cross-seeding and become a toxic aggregation trap for globular proteins, Aß peptides, and aromatic metabolites that convert native structures to ß-sheet-like fibrils. Aspartame fibrils were also found to induce hemolysis, causing DNA damage resulting in both apoptosis and necrosis-mediated cell death. Specific spatial arrangement between aspartame molecules is predicted to form a regular amyloid-like architecture with a sticky exterior that is capable of promoting viable H-bonds, electrostatic interactions, and hydrophobic contacts with biomolecules, leading to the onset of protein aggregation and cell death. Results reveal that the aspartame molecule is inherently amyloidogenic, and the self-assembly of aspartame becomes a toxic trap for proteins and cells, exposing the bitter side of such a ubiquitously used artificial sweetener.

Quantitative comparison of adsorption and desorption of commonly used sweeteners in the oral cavity.

Adsorption-desorption properties of different sweeteners in the oral cavity were evaluated using high performance liquid chromatography-based methodology. Three low calorie artificial sweeteners (aspartame, acesulfame potassium and sucralose), one steviol glycoside (rebaudioside A), and high fructose corn syrup (HFCS) were examined and compared with sucrose at pH 3 and 7 in a model beverage matrix. Results indicated that HFCS had the highest adsorption in the oral cavity, followed by rebaudioside A and the artificial sweeteners. The physicochemical interaction between sweeteners and salivary proteins did not affect the adsorption properties significantly as validated from a series of characterization techniques.

Measuring Artificial Sweeteners Toxicity Using a Bioluminescent Bacterial Panel.

Artificial sweeteners have become increasingly controversial due to their questionable influence on consumers' health. They are introduced in most foods and many consume this added ingredient without their knowledge. Currently, there is still no consensus regarding the health consequences of artificial sweeteners intake as they have not been fully investigated. Consumption of artificial sweeteners has been linked with adverse effects such as cancer, weight gain, metabolic disorders, type-2 diabetes and alteration of gut microbiota activity. Moreover, artificial sweeteners have been identified as emerging environmental pollutants, and can be found in receiving waters, i.e., surface waters, groundwater aquifers and drinking waters. In this study, the relative toxicity of six FDA-approved artificial sweeteners (aspartame, sucralose, saccharine, neotame, advantame and acesulfame potassium-k (ace-k)) and that of ten sport supplements containing these artificial sweeteners, were tested using genetically modified bioluminescent bacteria from E. coli. The bioluminescent bacteria, which luminesce when they detect toxicants, act as a sensing model representative of the complex microbial system. Both induced luminescent signals and bacterial growth were measured. Toxic effects were found when the bacteria were exposed to certain concentrations of the artificial sweeteners. In the bioluminescence activity assay, two toxicity response patterns were observed, namely, the induction and inhibition of the bioluminescent signal. An inhibition response pattern may be observed in the response of sucralose in all the tested strains: TV1061 (MLIC = 1 mg/mL), DPD2544 (MLIC = 50 mg/mL) and DPD2794 (MLIC = 100 mg/mL). It is also observed in neotame in the DPD2544 (MLIC = 2 mg/mL) strain. On the other hand, the induction response pattern may be observed in its response in saccharin in TV1061 (MLIndC = 5 mg/mL) and DPD2794 (MLIndC = 5 mg/mL) strains, aspartame in DPD2794 (MLIndC = 4 mg/mL) strain, and ace-k in DPD2794 (MLIndC = 10 mg/mL) strain. The results of this study may help in understanding the relative toxicity of artificial sweeteners on E. coli, a sensing model representative of the gut bacteria. Furthermore, the tested bioluminescent bacterial panel can potentially be used for detecting artificial sweeteners in the environment, using a specific mode-of-action pattern.

Aspartame as a co-former in co-amorphous systems.

Co-amorphous drug delivery systems are a promising approach to improve the dissolution rate and therefore potentially the oral bioavailability of poorly-water soluble drugs. Several low molecular weight excipients, for instance amino acids, have previously been shown to stabilize the amorphous form and increase the dissolution rate of drugs. In this study, the feasibility of aspartame, a methyl ester of the aspartic acid-phenylalanine dipeptide, as a co-former was investigated and compared with the respective single amino acids, both alone and in combination. The poorly water-soluble compounds mebendazole, tadalafil and piroxicam were chosen as model drugs. In contrast to the single amino acids or the physical mixture of both, all drug-aspartame mixtures became amorphous upon 90 min of ball milling. Only a single glass transition temperature (Tg) was detected by modulated differential scanning calorimetry, which indicates that a homogeneous single-phase co-amorphous system was obtained. Powder dissolution tests showed that the dissolution rates of the drugs from drug-aspartame co-amorphous samples were increased compared to crystalline drugs. Furthermore, supersaturation was observed for the mebendazole-aspartame and tadalafil-aspartame co-amorphous systems. In conclusion, aspartame has been shown to be a promising co-former in co-amorphous systems, superior to the single amino acids or their mixtures.

Enhancement of Z-aspartame synthesis by rational engineering of metalloprotease.

Metalloprotease PT121Y114S, an effective catalyst for Z-aspartame synthesis under the substrate (Z-Asp:l-Phe-OMe) molar ratio of 1:5, was obtained previously. Herein, a computational strategy combining molecular dynamics simulation of the enzyme-substrate complex with binding free energy (ΔG) calculations was established to guide the further engineering of PT121Y114S. One His224 residue proximal to the PT121Y114S active site was selected on the basis of the difference in ΔG decomposition of PT121Y114S toward l-Phe-NH2 and l-Phe-OMe. Site-saturation mutagenesis of His224 resulted in the mutants H224D, H224N, and H224S, which showed great improvement in Z-aspartame synthesis under an economical substrate molar ratio approaching 1:1. Furthermore, the kinetic constants of PT121Y114S and its mutants revealed that the affinity of mutants toward the l-Phe-OMe was significantly higher than that of PT121Y114S. Molecular dynamic simulation revealed that the enhanced synthetic activity may be attributed to the mutation stabilizing the transient state of the enzyme-l-Phe-OMe complex.

A theoretical and experimental study of calcium, iron, zinc, cadmium, and sodium ions absorption by aspartame.

Aspartame (L-Aspartyl-L-phenylalanine methyl ester) is a sweet dipeptide used in some foods and beverages. Experimental studies show that aspartame causes osteoporosis and some illnesses, which are similar to those of copper and calcium deficiency. This raises the issue that aspartame in food may interact with cations and excrete them from the body. This study aimed to study aspartame interaction with calcium, zinc, iron, sodium, and cadmium ions via molecular dynamics simulation (MD) and spectroscopy. Following a 480-ns molecular dynamics simulation, it became clear that the aspartame is able to sequester Fe2+, Ca2+, Cd2+, and Zn2+ ions for a long time. Complexation led to increasing UV-Vis absorption spectra and emission spectra of the complexes. This study suggests a potential risk of cationic absorption of aspartame. This study suggests that purification of cadmium-polluted water by aspartame needs a more general risk assessment.

Influence of Package Visual Cues of Sweeteners on the Sensory-Emotional Profiles of Their Products.

Substantial evidence suggests influence of color, physical state, and other extrinsic features on consumer perception and acceptability of food products. In this study, 560 subjects evaluated liking and emotional responses associated with 5 sweeteners (sucralose, stevia, saccharin, aspartame, and sucrose) under 2 eliciting conditions: control (brand name only) and informed (brand name/packet image), to assess impact of the packet color. For a given condition, 5 identical tea samples each labeled with a sweetener type were rated for sweetness and overall liking (9-point) and emotions (5-point). Nonsignificant interactions between eliciting condition and sweetener type were found for liking attributes and emotions (except peaceful), indicating their independent effects. However, overall differences existed among sweetener types and eliciting conditions based on both hedonic and emotional responses (MANOVA, P < 0.05), suggesting modulating effects of packet color on sweetener type in the sensory-emotion space. The sensory-emotion profile for sucrose was separate from that of nonnutritive sweeteners, with statistically significant Mahalanobis distances among sample centroids. Increases in positive emotion intensities contrasted with a decrease in negative emotion intensities were observed for some sweeteners moving from the control to informed condition. Sweetness liking was strongly correlated with the emotion satisfied (sucralose, saccharin) only in the control condition, whereas it was strongly correlated with the emotions pleased and satisfied (stevia), disgusted (aspartame), and satisfied (sucrose) only in the informed condition. Overall, results suggested that sensory liking and emotions during the consumption experience are related not entirely to the type of sweetener, but also the color of the packet.

Biological fate of low-calorie sweeteners.

With continued efforts to find solutions to rising rates of obesity and diabetes, there is increased interest in the potential health benefits of the use of low- and no-calorie sweeteners (LNCSs). Concerns about safety often deter the use of LNCSs as a tool in helping control caloric intake, even though the safety of LNCS use has been affirmed by regulatory agencies worldwide. In many cases, an understanding of the biological fate of the different LNSCs can help health professionals to address safety concerns. The objectives of this review are to compare the similarities and differences in the chemistry, regulatory status, and biological fate (including absorption, distribution, metabolism, and excretion) of the commonly used LNCSs: acesulfame potassium, aspartame, saccharin, stevia leaf extract (steviol glycoside), and sucralose. Understanding the biological fate of the different LNCSs is helpful in evaluating whether reports of biological effects in animal studies or in humans are indicative of possible safety concerns. Illustrations of the usefulness of this information to address questions about LNCSs include discussion of systemic exposure to LNCSs, the use of sweetener combinations, and the potential for effects of LNCSs on the gut microflora.

Resonant electron capture by aspartame and aspartic acid molecules.

RATIONALE: The processes for dissociative electron capture are the key mechanisms for decomposition of biomolecules, proteins in particular, under interaction with low-energy electrons. Molecules of aspartic acid and aspartame, i.e. modified dipeptides, were studied herein to define the impact of the side functional groups on peptide chain decomposition in resonant electron-molecular reactions. METHODS: The processes of formation and decomposition of negative ions of both aspartame and aspartic acid were studied by mass spectrometry of negative ions under resonant electron capture. The obtained mass spectra were interpreted under thermochemical analysis by quantum chemical calculations. RESULTS: Main channels of negative molecular ions fragmentation were found and characteristic fragment ions were identified. CONCLUSIONS: The СООН fragment of the side chain in aspartic acid is shown to play a key role like the carboxyl group in amino acids and aliphatic oligopeptides. Copyright © 2016 John Wiley & Sons, Ltd.