Investigation of the toxic mechanism of aspartame on female infertility using network toxicology and molecular docking approaches.

Nonnutritive sweeteners (NNS) are a class of compounds that affect the intake process and behavior, thus generating new ecological and chemical sensory signals in the brain. [ 1 ] These mainly include aspartame, neotame, and advantame. Among these, aspartame is currently the most widely used nonnutritive sweetener. It is synthesized from phenylalanine and is typically metabolized into 3 components in the gastrointestinal tract: phenylalanine, aspartic acid, and methanol. [ 2 ] Aspartame is approximately 200 times sweeter than sucrose and can activate the body’s taste receptors. [ 3 – 5 ] It is widely consumed by the majority of consumers. [ 6 ] Studies have found that the recommended intake of aspartame is 14% of the acceptable daily intake, approximately 40 mg/kg. [ 7 , 8 ] Since 2000, the low calorie content and high palatability of NNS have led to an explosion in demand for these sweeteners, particularly among children and adolescents, whose consumption has increased by over 200%. [ 9 ] Surveys have shown that between 2009 and 2012, approximately 25% of children and 45% of adults consumed NNS at least once a day. [ 10 ] However, most fast food products and beverages contain aspartame, which means that many children and adults may be consuming more than the recommended intake, thereby increasing the incidence of foodborne illnesses. [ 11 ] The toxic mechanism of aspartame is primarily associated with changes in the structure and function of proteins, as well as mutations in DNA, which subsequently produce cytotoxic methanol and its metabolite formaldehyde, leading to cellular damage or death. [ 12 ] Chronic high-dose intake of aspartame may elevate the risk of various diseases, including obesity, diabetes, metabolic syndrome, as well as cardiovascular and cerebrovascular diseases. [ 11 , 13 , 14 ] In recent years, its safety – especially its potential carcinogenic risk – has garnered widespread attention. [ 15 ] However, less attention has been paid to its impact on other health aspects. Female infertility is a prevalent reproductive system disorder affecting many women of childbearing age. [ 16 ] The incidence of female infertility has been increasing in recent years, significantly threatening women’s physical and mental health. [ 17 ] Currently, the cause of infertility in 15% to 30% of women remains unidentified. [ 18 ] Studies have shown that aspartame and its metabolites may exhibit potential reproductive toxicity, affecting the female reproductive system and resulting in adverse outcomes such as premature endometrial detachment and uterine glandular atrophy. [ 19 ] Excessive consumption of aspartame has been shown to delay the maturation of female oocytes and increase the risk of female infertility by 1.79-fold. [ 20 ] With the advancement of toxicological research methods, particularly the emergence of network toxicology, researchers have started to analyze how chemical substances impact the stability of biological systems from a systems biology perspective. Network toxicology integrates bioinformatics, systems biology, and toxicology to systematically study the interactions between chemicals and biological systems at the molecular level, as well as how these interactions lead to toxic effects. It can be employed to assess the potential mechanisms of action within the “toxicity-drug-ingredient-target” framework and screen for potentially toxic compounds. [ 21 ] Therefore, this study will utilize a combination of network toxicology and molecular docking methods to reveal the toxic mechanism of aspartame in inducing female infertility, characterize its toxicological properties, and predict and identify its potential toxic risks and molecular mechanisms. In addition, this study introduces a novel scientific approach for assessing the safety of food additives and offers new insights for safety research on other sweeteners and food additives. The schematic representation of the comprehensive research methodology is shown in (Fig. 1 ). Schematic illustration of the molecular mechanisms underlying aspartame-induced toxicity and its potential impact on female infertility.

Investigation: Xinye Gao, Xiaobei Pang. Writing – original draft: Tingyuan Yang. Writing – review & editing: Lei Zhang.

The Swiss TargetPrediction ( http://www.swisstargetprediction.ch/ ) and SEA ( http://sea.bkslab.org ) databases were employed in this study. The SMILES sequence of aspartame was retrieved from the PubChem ( https://pubchem.ncbi.nlm.nih.gov/ ) database and then entered into these 2 databases to obtain the prediction results. First, search for “aspartame” in the PubChem database to retrieve its corresponding molecular formula and SMILES structure. Next, download the SDF file of its 2D structure, upload it to Swiss TargetPrediction, set the compound prediction probability threshold to >0, submit the query, and download the results. Use Excel 2021 to select the values with a prediction probability >0 as potential targets of aspartame. Then, upload the SDF file of aspartame to SEA, and apply a Max Tc >0.5 threshold as the screening condition. The resulting targets will be considered as the targets of aspartame. Finally, merge the target results from both databases, eliminate any duplicate targets, and the remaining targets will constitute the target database for aspartame. GeneCards ( https://www.genecards.org/ ) and the TTD database ( https://db.idrblab.net/ttd/ ) were used to search for the keyword “Female Infertility” and obtain targets related to female infertility. Duplicate targets from both databases were eliminated. Furthermore, to ensure that the obtained targets are closely related to female infertility, the threshold for the “score” was set at the median value. Targets with a “score” greater than or equal to the median value were selected to construct a target database for female infertility. Moreover, a Venn diagram was employed to identify potential common targets between the aspartame targets and the female infertility targets. The intersection between the 2 sets was regarded as representing the potential toxic targets of aspartame-induced female infertility. The intersection targets of aspartame-induced female infertility were uploaded to the STRING database ( https://string-db.org/ ), with the species limited to Homo sapiens, and the “Minimum required interaction score” set to >0.6. After performing protein interaction network analysis, the TSV file generated by STRING was downloaded and imported into Cytoscape (3.10.2) software to construct the protein interaction network map. Simultaneously, the CytoNCA plug-in was employed to screen for core targets. The values of betweenness centrality, closeness centrality, degree centrality, eigenvector centrality, local average connectivity, and network centrality were selected if they were greater than or equal to the median value, which was applied to identify the potential core toxic targets of aspartame-induced female infertility. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed on core genes using the RStudio “clusterProfiler” package to investigate the primary functions and associated signaling pathways involved in the progression of female infertility. A threshold of P <.05 was applied, and the top 10 entries for biological process (BP), Cellular Component (CC), and molecular function (MF), as well as the top 20 KEGG pathways, were selected based on the P -value. Subsequently, the RStudio “Enrichplot” package was employed for visualization, and the ggplot2 package was used to enhance the appearance of the results. First, the top 4 core targets with the highest degree values were selected for molecular docking with aspartame. The protein data bank ( https://www.rcsb.org ) database was searched for macromolecular protein structures, and the downloaded protein structures were saved in protein data bank format. Then, the 3D structure of the chemical compound aspartame was searched and downloaded from the PubChem database. ChemBio 3D Ultra software was used to adjust the free energy of the small molecule ligand structure to the minimum energy threshold, and the ligand was saved as a ‘mol2’ format file. Finally, 4 receptor-ligand pairs were obtained for docking: interleukin-1 beta (IL-1β)-aspartame, angiotensin-converting enzyme 2 (ACE2)-aspartame, angiotensin-converting enzyme (ACE)-aspartame, and CTSS-Aspartame. Next, Autodock Tools (1.5.7) software was used to perform operations such as de-watering, hydrogenation, and pocket box settings. Semiflexible docking was performed using Autodock Vina and Perl scripting to calculate the binding energy between small molecule ligands and large molecule protein receptors to determine their stability. Finally, the docking results were visualized using PyMOL software.

Through predictive analysis of databases, such as “Swiss TargetPrediction” and ‘SEA’, it was found that the potential toxicity of aspartame is associated with female infertility. The following section will investigate the potential toxic mechanism of aspartame on female infertility through database searches and literature reviews. The molecular structure and chemical configuration of aspartame are depicted in (Fig. 2 A). (A) Schematic representation of the molecular structure and chemical configuration of aspartame. (B) Venn diagram showing the intersection between predicted molecular targets of aspartame and known biomarkers associated with female infertility. (C) Visualization of the integrated PPI network highlighting target protein associations. (D) Integrated network map illustrating core molecular targets linking aspartame exposure to female infertility. PPI = protein–protein interaction. According to the screening criteria, this study initially identified 81 toxic targets of aspartame in the “Swiss TargetPrediction” database and 55 toxic targets in the “SEA” database. After integrating the results and removing duplicate targets, a total of 115 potential toxic targets for aspartame were identified. Disease targets were filtered using the GeneCards and TTD databases according to the set ‘score’ threshold, yielding a total of 2936 targets highly correlated with female infertility. A Venn diagram was subsequently created using R Studio (venn package) for visualization. The overlapping area of the Venn diagram revealed 46 potential target sites that exhibited a high degree of correlation between the 2 datasets of aspartame and female infertility (Fig. 2 B). The resulting intersection targets were input into the STRING database, with the organism set to “Homo sapiens,” and a protein–protein interaction network analysis was conducted. The threshold for the ‘Minimum required interaction score’ was set to > 0.6, and the results indicated a total of 46 nodes and 94 edges, with an average node degree of 4.09. The Cytoscape (3.10.2) software was then utilized to analyze the topological characteristics of the network nodes, including betweenness centrality, closeness centrality (CC), degree centrality, eigenvector centrality, local average connectivity, and network centrality. After optimization, the protein–protein interaction network map was constructed (Fig. 2 C). The median of the above topological indices was used as the threshold to filter for the core targets (Table 1 ). Key subnetworks were obtained using the ‘CytoNCA’ plug-in, and the intersection with the target set was used to identify the core targets (Fig. 2 D). The results demonstrate that the darker the color and the larger the node area, the higher the degree value. The top 4 core targets, in order of degree value, are IL-1β, ACE2, ACE, and CTSS. Core targets identified in the interaction between aspartame and female infertility. ACE = angiotensin-converting enzyme, ACE2 = angiotensin-converting enzyme 2, CTSS = cathepsin S, IL-1β = interleukin-1 beta. GO and KEGG enrichment analyses were conducted using R Studio on the potential targets related to female infertility induced by aspartame to identify relevant KEGG and GO pathways. A total of 985 BPs, 60 CCs (CC), and 62 MFs (MF) were observed. BP refers to the BPs influenced by genes or gene products, typically resulting from the coordinated activity of one or more MFs. CC represents the specific locations within the cell where gene products are active, while MF refers to the biochemical activities of the gene products themselves. [ 22 ] KEGG pathway enrichment analysis revealed 64 enriched pathways. Among these, the most notable signaling pathways closely related to female infertility were the IL-17 and TNF signaling pathways, which are likely to play significant roles in the toxic effects of aspartame on female infertility (Figs. 3 and 4 ). Top 10 GO functional categories of potential target genes involved in aspartame-induced female infertility. GO = Gene Ontology. KEGG pathway enrichment analysis (top 20) of potential target genes related to aspartame-induced female infertility. Note: (A) KEGG bubble chart; (B) KEGG bar chart. KEGG = Kyoto Encyclopedia of Genes and Genomes. Molecular docking was conducted to dock the 4 core targets with aspartame. The 4 docking groups were IL-1β-Aspartame, ACE2-Aspartame, ACE-Aspartame, and CTSS-Aspartame. Molecular docking was utilized to analyze the interactions between aspartame and these 4 core targets. Autodock Vina and Perl scripting were used to generate the docking results, which were ranked from low to high based on their binding energy. Molecular docking is a valuable tool for predicting the interaction modes between a compound and its target hub nodes. [ 21 ] The core principle behind molecular docking is to evaluate the compound’s toxicity based on its interaction and affinity with the target. A lower ligand-receptor binding energy indicates a stronger interaction and higher affinity between the ligand and the receptor. [ 23 ] The molecular docking results show that the binding energy for all 4 docking results is ≤−5, suggesting that aspartame can spontaneously bind to each core target, [ 24 ] playing a crucial role in the molecular mechanism underlying aspartame-induced female infertility. The docking results were visualized using PyMOL software (Fig. 5 and Table 2 ). Molecular docking results of the interactions between aspartame and key target proteins. ACE = angiotensin-converting enzyme, ACE2 = angiotensin-converting enzyme 2, CTSS = cathepsin S, IL-1β = interleukin-1 beta. (A) Computational molecular docking analysis of the interaction between IL-1β and aspartame. (B) Computational molecular docking analysis of the interaction between ACE2 and aspartame. (C) Computational molecular docking analysis of the interaction between ACE and aspartame. (D) Computational molecular docking analysis of the interaction between CTSS and aspartame. ACE = angiotensin-converting enzyme, ACE2 = angiotensin-converting enzyme 2, CTSS = cathepsin S, IL-1β = interleukin-1 beta.

This study provides compelling evidence linking the artificial sweetener aspartame to the onset of infertility. Network toxicology is a widely used approach for assessing the potential mechanisms of action of toxicants, drugs, components, and their associated targets. [ 15 ] In this study, we employed this technique to investigate the primary targets and potential toxic mechanisms of aspartame in relation to infertility, integrating computational simulation methods. Aspartame may promote the progression of infertility by influencing multiple key signaling proteins and regulators. Key proteins such as IL-1β, ACE2, ACE, CTSS, CTSB, PTGS2, CASP3, ITGβ1, XIAP, and NOS3 are involved in various biological functions, including cell growth, apoptosis, immune response, and others. IL-1β, produced by stromal cells under conditions of high oxidative stress, can induce apoptosis in endometrial epithelial cells. This process accelerates the invasion and implantation of embryonic trophoblast cells, leading to improper decidualization of the uterus and increasing the risk of female infertility. [ 25 ] Elevated levels of IL-1β and IL-6 are observed in patients with PCOS. [ 26 ] Increased IL-1β levels can activate inflammatory factors, inhibit steroid production in ovarian GCs, and cause follicular atresia. [ 27 ] This inhibits oocyte maturation [ 28 ] and results in anovulatory infertility. [ 29 ] In addition, the renin-angiotensin system (RAS) is a paracrine pathway that helps stabilize blood pressure and regulates fertility in the female ovary. ACE, ACE2, and ACE3 are essential components of the RAS. [ 30 ] ACE is expressed in various tissues, including the ovary. Its primary physiological functions include regulating fluid balance, ovarian function, and corpus luteum formation. [ 31 , 32 ] The active enzyme ACE regulates the RAS system, upregulates total serum renin levels, [ 33 ] and enhances RAS activity, which influences the secretion of hormones within the hypothalamic-pituitary-ovarian gonadal axis. [ 34 ] This disruption causes ovulatory abnormalities and increases the risk of PCOS in women. [ 35 ] ACE2 also plays a crucial role in female ovarian steroid production, ovarian development, oocyte maturation, and regulation of ovulation. It can interfere with ovulation and cause follicular atresia in women. [ 36 ] The protein encoded by the CTSS gene, a member of the peptidase C1 family, belongs to the lysosomal cysteine protease family. It plays a crucial role in various physiological activities, including apoptosis, oocyte maturation, and embryo implantation. [ 37 ] Animal studies have shown that the CTSS gene influences estradiol and progesterone secretion in granulosa cells (GCs) of the rabbit ovary. When CTSS levels are reduced, the levels of reproductive genes such as PCNA and the antiapoptotic gene BCL2 decrease in GCs, leading to apoptosis. This process causes follicular atresia and impaired oocyte development, ultimately leading to infertility. [ 38 , 39 ] The results of the KEGG pathway analysis indicated that aspartame primarily induces female infertility through the IL-17 and TNF signaling pathways. IL-17, a well-known target of chronic inflammation, exerts pro-inflammatory, autoimmune, and host defense effects. [ 40 ] Current research on the IL-17 pathway primarily focuses on diseases such as psoriasis, [ 41 ] inflammatory arthritis, [ 42 ] ankylosing spondylitis, [ 43 ] multiple sclerosis, [ 44 ] and inflammatory bowel disease. [ 45 ] In the context of infertility, some studies compared plasma IL-17 levels in 80 infertile women undergoing ovarian stimulation and intracytoplasmic sperm injection (ICSI) with those in 40 women of childbearing age. The results indicated that elevated plasma IL-17 levels may contribute to infertility in female patients undergoing ICSI. [ 46 ] The metabolic regulation of the IL-17 signaling pathway is fundamental to disease development. When IL-17 is improperly transduced, it disrupts cellular and organismal homeostasis, causing damage to tissues and organs with crucial metabolic functions. [ 47 ] Another clinical study found that elevated IL-17 transduction levels trigger an inflammatory response, which is common in many PCOS patients. [ 48 ] Polycystic ovary syndrome (PCOS) has become one of the leading causes of infertility in women of reproductive age. [ 49 ] Additionally, TNF-mediated signaling pathways are closely associated with the proliferation, differentiation, apoptosis, and inflammatory responses of tumor cells. [ 50 , 51 ] Endometrial cancer, ovarian cancer, cervical cancer, and thyroid cancer are major causes of female infertility. [ 50 ] Moreover, endometriosis, a prevalent female reproductive disease, affects the metabolic levels of the liver and adipose tissue and is strongly linked to the inflammatory response mediated by the TNF signaling pathway. [ 52 ] Increased inflammation in the uterine environment can result in the adhesion and ectopic growth of endometrial tissue, as well as inflammatory damage to the uterine stromal tissue, leading to irreversible uterine damage. [ 52 , 53 ] Notably, the TNF-α molecule and TNFR1 together form the TNF-α/NF-κB signaling pathway, producing a trimer, TNFR1. By aggregating downstream signaling proteins, it activates the TNF-κB signaling pathway, promoting inflammatory reactions that can lead to endometriosis and, in turn, increase the risk of female infertility. [ 54 ] In addition, computational simulation techniques were employed to explore the binding affinity and mechanism of aspartame with key infertility-related targets. This computational approach simulates the interaction between the drug molecule and its target protein. A decrease in docking energy indicates a stronger interaction between the molecules, suggesting more stable and safer binding. Our results show that the binding energy of aspartame to IL-1β, ACE2, ACE, and CTSS proteins ranges from −5.5 to −8.4 kcal/mol. The highest binding energy (−8.4 kcal/mol) was observed with ACE, indicating a potentially strong interaction between aspartame and ACE.

This study employed a combination of network toxicology and molecular docking to elucidate the molecular mechanisms through which aspartame induces female infertility. A total of 46 potential target genes related to aspartame-induced female infertility were identified. Enrichment analysis of these potential target genes revealed a possible link between aspartame and female infertility. These findings may offer valuable insights for treating female infertility and developing new drugs. However, despite offering new perspectives on the possible link between aspartame and infertility, there are several limitations that must be acknowledged. First, our study relies on observational biomarker expression patterns, limiting the interpretation of causality. Additionally, we acknowledge the need for supporting epidemiological studies to validate the relationship between aspartame consumption and the risk of infertility.