Phthalates are manufactured compounds commonly used as plasticizers in polyvinyl chloride products. (1) Dibutyl phthalate (DBP), one of the most abundant phthalates in the environment, is also widely used in personal care products, building materials, cosmetics, and medical supplies, with production exceeding 1 million pounds in 2019. (2) People are ubiquitously exposed to DBP at disproportionately higher levels compared with other phthalates. It was reported that nearly 90% of California children had estimated DBP exposure exceeding the benchmarks of reproductive health. (3) DBP can mimic natural hormone binding and activate the estrogen receptor (ER), which affect the endocrine system and may induce breast cancer. (4−6)

Breast cancer is the most prevalent cancer worldwide, with 2.3 million new cases in 2020. (7) It is also the primary cause of cancer mortality in women (685,000 deaths globally). (7,8) Epidemiological studies have shown that DBP exposure was associated with a nearly 2-fold increased rate of breast cancer. (9,10) Consistent results were also found in several in vitro studies, which showed that DBP can modulate the expression of ERα through DNA hypomethylation or demethylation, even at a very low concentration. (11−14) DBP can also stimulate the aryl hydrocarbon receptor and promote tumorigenesis of ER-negative breast cancer cells. (15) However, most of the studies predominantly focused on gene and protein expression, and the connections between DBP-induced receptor activation and physiological effects in breast cancer remain poorly elucidated, which need further investigations through assessing the downstream metabolic processes. (16−18)

Metabolomics can monitor the holistic changes of metabolites downstream of the genome and proteome, reflecting the physiological activities in cancer. (19,20) Three-dimensional cancer cell spheroid (CCS) is a precise in vitro cancer model for studying the carcinogenic mechanism of environmental pollutants as it closely mimics the complex spatial architecture, microenvironment, and physiological responses of solid tumor tissues. (21) There are proliferative (outer), quiescent (middle), and necrotic (inner) cells in CCS models due to the nutrient and oxygen concentration gradients, and they exert heterogeneity in exogenous stimulus responses, which highlights the importance of considering the various cell statuses in cancer research. Mass spectrometry imaging (MSI) is an innovative label-free technology that can simultaneously obtain chemical information and spatial distribution of diverse molecular species in different regions of CCS which has been used to identify and characterize the biological processes in cancer progression. (22,23) Integrated application of omics and MSI techniques in the CCS model can combine spatial information and in-depth analysis to better understand carcinogenic mechanisms of environmental pollutants... (24)

Figure 1. Growth (A) and areas (B) of breast CCS from day 2 to day 14. Comparison of the areas (C) and cell numbers (D) of breast CCS exposed to various DBP concentrations on day 14. Significantly changed metabolites (E) and lipids (F) in breast CCS were observed after DBP exposure.

Figure 2. DBP-induced disturbance of energy, nucleotide (A), and amino acid (B) metabolism in breast CCS. Comparison of various lipid classes in breast CCS between DBP exposure and control groups (C). Pathway analysis of lipids disturbed by DBP exposure (D).

Figure 3. Optical (A) and segmentation maps (B) of the breast CCS section. Comparison of the distribution and abundance of the significantly changed metabolites (C) and lipids (D) in CCS sections between DBP exposure and control groups. Metabolic and lipidomic networks described the altered pathways in the breast CCS after DBP exposure (E).

This study provided new insights into the metabolic mechanisms of DBP-promoted breast carcinoma development. We inferred that low doses of DBP may activate the PI3K/AKT signaling pathway, (12) which further affected cellular metabolism by enhancing glucose and glutamine uptake and catabolism. (48,52) The high rate of glycolysis and glutaminolysis supported the production of nucleotides, GPs, and amino acids, which provided sufficient energy and building blocks of DNA/RNA and cytomembrane synthesis (Figure 3E). Their concentrated distribution in the peripheral area contributed to the rapid growth and proliferation of breast CCS. Further study will focus on DBP-induced metabolomic responses in human body fluids to further elucidate the underlying mechanisms.