Investigating the mechanistic link between pesticide DDT and breast cancer through network toxicology, molecular docking, and molecular dynamics simulation.

Introduction
Dichlorodiphenyltrichloroethane (DDT) is an organochlorine pesticide that was widely used due to its effective insecticidal properties. However, as research into its environmental impacts has advanced, the persistence and bioaccumulation of DDT have raised significant concerns1,2. The widespread presence of this compound in soil, water, and the food chain makes human exposure nearly inevitable3,4. Although many countries have restricted or banned the use of DDT, it continues to be employed in certain nations, such as India and South Africa, as part of public health initiatives to combat diseases like malaria5,6. The lipophilic nature of DDT is associated with its potential endocrine-disrupting effects, which may lead to various health issues5,7.
The impact of environmental factors on global cancer incidence has increasingly attracted attention8,9. Studies conducted in the US10 and Taiwan11 indicate a significant association between early-life DDT exposure and breast cancer incidence. Notably, research by Alexandra J. White et al. further reveals a strong association between DDT exposure and estrogen receptor-positive/progesterone receptor-positive (ER+/PR+) breast cancer. Despite multiple studies suggesting that DDT exposure may elevate the risk of breast cancer, the specific mechanisms underlying this association remain unclear11–13.
Recent advancements in bioinformatics technologies have provided new tools for exploring the complex interactions between environmental factors and diseases. By integrating multi-omics data and network analysis, network toxicology has emerged as a key approach for identifying molecular targets and pathways involved in disease etiology. Through the integration of these advanced methodologies, this study aims to elucidate the molecular basis by which exposure to DDT influences the onset and progression of breast cancer. A detailed research workflow is illustrated in Fig. 1. The results of this study not only enhance the understanding of DDT’s role in breast cancer but also provide guidance for pollution reduction efforts to mitigate risk, which is of significant importance for public health.

Methods

Identification of DDT
We retrieved the chemical structure and related molecular information of DDT from the PubChem database (https://pubchem.ncbi.nlm.nih.gov, February 2025). Subsequently, we utilized the ADMETLAB 3.0 platform (https://admetlab3.scbdd.com) and the ProTox 3.0 database (https://tox.charite.de/protox3) to validate the carcinogenicity of this pollutant. We obtained the molecular structures of four breast cancer drugs, including exemestane, carboplatin, fluorouracil, and phosphoramide mustard (the active metabolite of cyclophosphamide), from the PubChem database. Then, we used SWISSADME (http://www.swissadme.ch/) and ProTox 3.0 to compare the physicochemical properties and toxicity profiles of DDT with those of these four anticancer drugs.

Collection of DDT target genes
Potential human target genes for DDT were retrieved from the ChEMBL database (https://www.ebi.ac.uk/chembl, Version ChEMBL_35), the SwissTargetPrediction database (http://www.swisstargetprediction.ch, February 2025), and the STITCH database (http://stitch.embl.de, Version 5.0). The gene data from these three databases were subsequently merged, and duplicate entries were eliminated, resulting in the compilation of a final target gene set for DDT.

Collection of breast cancer-related genes
Genes associated with “breast cancer” were collected from the GeneCard database (https://www.genecards.org, v5.24.0), the OMIM database (https://OMIM.org, February 2025), and the TTD database (https://db.idrblab.net/ttd/, February 2025) using the keyword “breast cancer”. Genes with a Score greater than 10 were selected based on their ranking from GeneCard. After merging the gene lists from the three databases and removing duplicates, a final set of genes related to breast cancer was obtained.

GO/KEGG enrichment analysis
We utilized the “ClusterProfiler,” “Enrichplot,” and “Org.Hs.eg.db” packages in R software (Version 4.4.3, https://www.r-project.org/) to conduct Gene Ontology (GO) functional representation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of intersecting genes between DDT and breast cancer. These analyses uncovered the potential roles of key genes in biological processes, molecular functions, and cellular components, along with their involvement in signaling pathways.

DDT-breast cancer core target screening and PPI network construction
We performed protein-protein interaction (PPI) analysis for the gene segments between DDT and breast cancer using the STRING database (https://cn.string-db.org/, Version 5.0), setting a confidence score threshold of d ≥ 0.4 to select significant interactions. The results were then visualized using Cytoscape 3.10.3 software (https://cytoscape.org/), enabling the construction of a protein interaction network.

Molecular docking
We retrieved the PDB format files for key protein from the RCSB database (https://www.rcsb.org/, February 2025), and the molecular structure files of DDT and four commonly used chemotherapeutic drugs for breast cancer—exemestane, carboplatin, fluorouracil, and phosphoramide mustard—were downloaded from the PubChem database.
The initial structure of the ligand was geometrically optimized using Chem3D software (v20.1.1.125) based on the MM2 force field to ensure the accuracy of its three-dimensional conformation. Subsequently, the optimized ligand structure was subjected to further preprocessing through AutodockTools software (v1.5.7), including the addition of hydrogen atoms and charge assignment. The ligand-binding active site was defined as the docking region. Molecular docking simulations were performed with AutoDock Vina 1.2.5, employing an exhaustive sampling parameter exhaustiveness = 128 to ensure conformational space coverage and docking reliability.
After completing the docking of DDT with the four proteins, we selected the same active sites to perform molecular docking of the four chemotherapeutic drugs with the proteins. The binding energy was utilized to assess the interactions between the ligand and receptor: a binding energy less than 0 kcal/mol indicates spontaneous binding, whereas a binding energy less than − 5 kcal/mol suggests the possibility of binding.

Molecular dynamics simulation
Molecular dynamics simulations were performed using GROMACS 2021 software, a robust computational approach, models the dynamic behavior of molecular systems to predict their physicochemical properties. In this study, MD simulations were employed to investigate the interaction mechanisms between DDT and the breast cancer-related target ESR1. The ligand-protein complex was embedded in a solvation chamber filled with water molecules, and the system’s electrical neutrality was maintained by adding Cl− and Na+ ions. The simulation was conducted at a temperature of 25 °C with a total runtime of 100 nanoseconds (ns). Post-simulation analysis included calculations of root mean square deviation (RMSD), root mean square fluctuation (RMSF), and hydrogen bond properties derived from trajectory data to elucidate the stability and binding characteristics of the DDT-ESR1 interaction.

Result

Toxicity assessment of DDT
We conducted a toxicity assessment of DDT using two toxicity prediction platforms. According to the selection criteria, if carcinogenicity was predicted by either platform, the contaminant was deemed toxic. The results indicated that DDT possessed significant carcinogenicity (Table 1). A comparative analysis was conducted between DDT and four antitumor drugs (exemestane, carboplatin, fluorouracil, and phosphoramide mustard). The results indicated that, compared to the other three antitumor drugs, DDT exhibited the highest similarity to exemestane in physicochemical properties. Both compounds demonstrated relatively high lipophilicity and neurotoxicity. Additionally, they are metabolized by cytochrome CYP2C9 and can activate the estrogen receptor alpha signaling pathway (Fig. 2). Supplementary Fig. 1 displays the physicochemical properties of the five compounds. Supplementary Fig. 2 presents the toxicity radar chart for these compounds.

Collection of DDT target genes
After integrating target prediction data from the ChEMBL, SwissTargetPrediction, and STITCH databases, we identified 26 DDT-related target genes (Supplementary Table 1).

Collection of breast cancer target genes
We retrieved 18,389 relevant genes associated with “breast cancer” from the GeneCard database, 106 related genes from the OMIM database, and 100 relevant genes from the TTD database. To optimize the dataset, we selected genes from GeneCard with a score greater than 10. After removing duplicate sequences, we obtained a total of 3,409 breast cancer-related genes (Supplementary Table 2).

DDT-breast cancer core target screening and PPI network construction
We imported the 12 cross-target genes between DDT and breast cancer into the STRING database for protein-protein interaction (PPI) analysis, setting the confidence threshold to ≥ 0.4 (Fig. 3A). Subsequently, we visualized the PPI network using Cytoscape 3.10.3. In the network, targets were ranked according to their Maximum Clique Centrality (MCC); deeper colors and larger circles indicated stronger interactions with other proteins (Fig. 3B). This visualization provided a clear overview of the interaction relationships among the targets, offering valuable insights for further investigation into the mechanistic links between DDT and breast cancer.

GO/KEGG enrichment analysis
Through cross-analysis of DDT-related genes and breast cancer-related genes, we identified 12 overlapping genes. GO functional enrichment analysis indicated that these genes were primarily involved in processes such as cellular estrogen expression, cell growth, and biosynthesis (Fig. 3C). Furthermore, KEGG pathway analysis revealed significant enrichment in critical pathways, including Chemical carcinogenesis-receptor activation, Breast cancer, Endocrine resistance, Prolactin signaling pathway, and PI3K-Akt signaling pathway (Fig. 3D)14. These findings suggest that DDT may influence the occurrence and progression of breast cancer by regulating intracellular hormone levels, disrupting endocrine functions, and affecting pathways related to carcinogenesis.

Molecular docking
The molecular docking analysis demonstrated that DDT spontaneously binds to all four key proteins binding energies < 0 kcal/mol. Specifically, the binding free energies of DDT with ESR1 (Estrogen Receptor 1), ESR2 (Estrogen Receptor 2), ERBB2 (Erb-B2 Receptor Tyrosine Kinase 2), and AR (Androgen Receptor) were determined as -8.49, -7.34, -7.17, and − 7.78 kcal/mol (Table 2), suggesting that DDT may regulate breast cancer-related biological processes through direct interactions with these critical proteins. The molecular docking and interactions between DDT and four key proteins are shown in Fig. 4.

Further analysis revealed that among four commonly used breast cancer therapeutic agents (Fig. 5), Exemestane exhibited binding energies of -10.29, -10.35, -8.92, and − 10.99 kcal/mol with ESR1, ESR2, ERBB2, and AR, respectively. Notably, Exemestane showed significantly lower binding energies than DDT across all target proteins, suggesting its potential therapeutic advantage for DDT-exposed breast cancer patients. The remaining three agents displayed negative binding energies with key proteins some < − 5 kcal/mol, indicating their possible therapeutic potential in DDT-associated breast cancer cases. Supplementary Fig. 3 shows the interactions of carboplatin with four key proteins, Supplementary Fig. 4 shows the interactions of fluorouracil with four key proteins, and Supplementary Fig. 5 shows the interactions of phosphoramide mustard with four key proteins.

Molecular dynamics simulation
Molecular dynamics simulations revealed structural stabilization after the initial 10 ns, as evidenced by Root Mean Square Deviation (RMSD) values fluctuating within 0.25–0.3 nm (Fig. 6A). Root Mean Square Fluctuation (RMSF) analysis demonstrated higher flexibility (0.5–0.6 nm) in residues at the protein termini compared to the core region, which exhibited smaller fluctuations (0.2–0.3 nm) (Fig. 6B). This pattern aligns with general protein dynamics, characterized by a rigid core and flexible termini. The Radius of Gyration (Rg) decreased from an initial ~ 3.33 nm to a stable value near 3.20 nm (Fig. 6C), signifying that the protein and its DDT complex adopted a more compact and stable conformation. Similarly, the Solvent Accessible Surface Area (SASA) decreased from 420 nm2 to a plateau around 395 nm2 (Fig. 6D). This reduction further supports the formation of a more compact complex structure, leading to decreased solvent exposure. Complete simulation trajectories are provided in Supplementary Table 3.

Discussion
Although the contribution of DDT to global disease control and agricultural productivity cannot be ignored, its use has been banned in multiple countries due to its ecological toxicity and potential risks to human health. Currently, some countries and regions continue to use DDT. Given its long half-life, DDT persists in the environment, leading to lasting pollution and possessing bioaccumulation characteristics15. Therefore, even after the prohibition of DDT, a region may still be affected for an extended period16,17.
Breast cancer is one of the most common malignant tumors worldwide and poses a significant threat to women’s health18. Studies have indicated that genetic factors, lifestyle behaviors, and endocrine factors are recognized as primary high-risk factors for breast cancer19,20. However, recent advancements in understanding environmental factors have revealed that various pollutants present in the environment, such as air pollution, pesticide residues, and harmful substances in building materials, may significantly impact the occurrence and development of breast cancer21–23. These findings underscore the importance of considering a multitude of factors in the prevention and intervention of breast cancer. However, direct research on the relationship between environmental pollutants and the incidence of breast cancer presents challenges. In this study, we adopted a multidisciplinary approach employing network toxicology and molecular docking techniques to elucidate the potential connection between DDT and breast cancer. This research identified key genes and their interaction networks, providing new insights into the role of DDT in breast cancer.
Our study revealed that 12 genes play a critical bridging role between DDT and breast cancer. Through KEGG and GO functional enrichment analyses, we found that DDT may promote the development of breast cancer via multiple mechanisms, including targets were ranked according to their Maximum with endocrine levels, biosynthesis, and chemical carcinogenesis. Notably, AR, ERBB2, ESR1, and ESR2 were identified as key genes within the DDT-breast cancer interaction network. These findings provide new theoretical insights into the molecular mechanisms by which DDT influences breast cancer.
Androgen receptor (AR), classified as a type I nuclear receptor, plays a pivotal role in regulating critical biological processes such as differentiation, proliferation, apoptosis, and angiogenesis24. Studies have demonstrated that AR is expressed in 70–90% of breast cancer cases and plays an essential role in the pathological characteristics and progression of the disease25. Consequently, AR has been proposed as a potential therapeutic target for breast cancer26. Currently, AR antagonists are being evaluated in preclinical and clinical studies, with some research showing promising efficacy27.
ERBB2 (human epidermal growth factor receptor 2, HER2) is a gene that encodes a member of the epidermal growth factor receptor family, playing a crucial role in the regulation of cell proliferation, differentiation, and survival, and is closely associated with the invasiveness of tumor cells28. As a significant driver gene and prognostic indicator in breast cancer,29 ERBB2 not only serves as a key predictive factor for targeted therapies but also has driven the development of related drugs that have substantially altered the diagnostic and therapeutic strategies for breast cancer, thereby improving the prognosis of patients with ERBB2-positive breast cancer30.
Estrogen receptor 1 (ESR1) and estrogen receptor 2 (ESR2) encode estrogen receptor alpha (ER-α) and estrogen receptor beta (ER-β), respectively. Research has indicated that both ESR1 and ESR2 are associated with the risk of breast cancer development; however, the precise mechanisms by which these genes contribute to breast cancer remain incompletely understood31,32. Furthermore, the role of ESR genes may vary across different populations. A study has reported that ESR1 may pose a risk of breast cancer for women of Asian, European, and African descent33. Conversely, ESR2 exhibits contrasting associations in Romanian and Greek populations: in the Romanian cohort, ESR2 is correlated with a higher risk of breast cancer,34 while it is associated with a lower risk in the Greek population35. Based on a study in the U.S. population, 83% of breast cancer patients exposed to DDT were estrogen receptor-positive10. This study found that DDT binds effectively to the ESR1 protein, suggesting that DDT likely promotes breast cancer development by activating the estrogen signaling pathway.
In molecular docking simulations, a lower binding energy between the ligand and the receptor protein typically indicates higher binding stability and stronger affinity. Generally, a binding energy more negative than − 5 kcal/mol is classified as moderate binding affinity, while a value more negative than − 7 kcal/mol is considered strong binding36. Molecular docking demonstrates that the binding energy of DDT to the key protein is more negative than − 7 kcal/mol, suggesting that DDT binds more tightly to this protein, thereby promoting tumorigenesis and progression. Molecular docking with four common antitumor drugs revealed that exemestane exhibits a lower binding energy than DDT to the target protein, indicating stronger binding affinity at the identical site. Molecular dynamics simulations demonstrate that DDT can stably bind to ESR1 through various intermolecular interactions. This finding further supports the hypothesis that DDT may directly interact with these key proteins to modulate their biological functions, which could ultimately contribute to the onset of cancer and increase the risk of breast cancer.
Existing studies have shown that DDT can promote the growth of animal mammary tumors37; multiple retrospective studies and epidemiological investigations have also found a positive correlation between the levels of DDT metabolites in lipids, serum, or plasma and the incidence of breast cancer. 38 Furthermore, two studies have indicated an association between DDT exposure and estrogen receptor-positive breast cancer in women. 39, 40 The above studies strongly confirm the close link between DDT and breast cancer. This study explains the association mechanism between DDT and breast cancer from a bioinformatics perspective, and its results are highly consistent with clinical studies, further verifying the reliability of this research. We hope that this study will provide valuable references for future prevention and treatment strategies of DDT-related breast cancer.
This study presents several advantages: firstly, we employed a comprehensive approach that integrates network toxicology, molecular docking, and molecular dynamics simulations to investigate the relationship between DDT and breast cancer, thereby enhancing the scientific rigor and relevance of the research. Secondly, by focusing on the molecular mechanisms underlying the relationship between DDT and breast cancer, we addressed the existing gap in the molecular understanding of the association between DDT and breast cancer.
This study has several limitations. Firstly, all target information is derived from predictions based on network databases, which may be influenced by algorithmic biases and the quality of the original data. Furthermore, there is currently a lack of direct experimental evidence to validate that DDT mediates its effects on breast cancer through these target genes. We found that exemestane exhibits potential therapeutic effects against breast cancer associated with DDT exposure. However, these findings are based solely on bioinformatics analyses and computational simulations, and should therefore be interpreted with caution. Further experimental studies are warranted to validate these results.

Conclusion
This study systematically reveals the potential association between DDT and breast cancer by integrating approaches of network toxicology, molecular docking, and molecular dynamics simulations. The results indicated that four key genes, namely AR, ERBB2, ESR1, and ESR2, may constitute significant links between DDT exposure and the development of breast cancer. Subsequently, we found that exemestane has potential therapeutic advantages for breast cancer patients exposed to DDT. These findings provide new theoretical insights into the molecular mechanisms by which DDT influences breast cancer progression. Furthermore, these key genes present promising molecular targets for early warning, prognostic assessment, and targeted therapy in breast cancer management.

Future prospects
Future work will employ surface plasmon resonance (SPR) to characterize small molecule-target protein interactions, assess the impact of DDT on mammary cell proliferation and migration using cellular functional assays, and examine relevant protein expression levels via Western blotting (WB).

Supplementary Information
Below is the link to the electronic supplementary material.