DNA methylation plays a pivotal role in cancer. The ubiquitous contaminant perfluorooctanesulfonic acid (PFOS) has been epidemiologically associated with breast cancer, and can induce proliferation and malignant transformation of normal human breast epithelial cells (MCF-10A), but the information about its effect on DNA methylation is sparse. The aim of this study was to characterize the whole-genome methylome effects of PFOS in our breast cell model and compare the findings with previously demonstrated DNA methylation alterations in breast tumor tissues. The DNA methylation profile was assessed at single CpG resolution in MCF-10A cells treated with 1 μM PFOS for 72 h by using Enzymatic Methyl sequencing (EM-seq). We found 12,591 differentially methylated CpG-sites and 13,360 differentially methylated 100 bp tiles in the PFOS exposed breast cells. These differentially methylated regions (DMRs) overlapped with 2406 genes of which 494 were long non-coding RNA and 1841 protein coding genes. We identified 339 affected genes that have been shown to display altered DNA methylation in breast cancer tissue and several other genes related to cancer development. This includes hypermethylation of GACAT3, DELEC1, CASC2, LCIIAR, MUC16, SYNE1 and hypomethylation of TTN and KMT2C. DMRs were also found in estrogen receptor genes (ESR1, ESR2, ESRRG, ESRRB, GREB1) and estrogen responsive genes (GPER1, EEIG1, RERG). The gene ontology analysis revealed pathways related to cancer phenotypes such as cell adhesion and growth. These findings improve the understanding of PFOS's potential role in breast cancer and illustrate the value of whole-genome methylome analysis in uncovering mechanisms of chemical effects, identifying biomarker candidates, and strengthening epidemiological associations, potentially impacting risk assessment.

The environmental contaminant dibutyl phthalate (DBP) is reported to be hepatotoxic, but the underlying molecular pathways and pathological processes remain unclear. Here we used RNA-sequencing to characterize persistent hepatic transcriptional effects one week after the conclusion of five weeks oral exposure to 10 mg/kg/day or 100 mg/kg/day DBP in adult male mice. The exploratory transcriptome analysis demonstrated five differentially expressed genes (DEGs) in the 10 mg/kg/day group and 13 in the 100 mg/kg/day group. Gene Set Enrichment Analysis (GSEA), which identifies affected biological pathways rather than focusing solely on individual genes, revealed nine significantly enriched Reactome pathways shared by both DBP treatment groups. Additionally, we found 54 upregulated and one downregulated Reactome pathways in the 10 mg/kg/day DBP group, and 29 upregulated and 13 downregulated pathways in the 100 mg/kg/day DBP group. DBP exposure disrupted several key biological processes, including protein translation, protein folding, apoptosis, Hedgehog signaling, degradation of extracellular matrix and alterations in the energy/lipid metabolism. Subsequent liver tissue analysis confirmed that DBP exposure induced tissue disorganization, oxidative stress, lipid accumulation, increased TNF-α, ATP and glucokinase levels, and affected key metabolic proteins, predominantly in a dose-response manner. Overall, the results show that DBP can cause hepatic stress and damage and suggest a potential role for DBP in the development of non-alcoholic fatty liver disease, the most prevalent liver disease worldwide.

Increased exposure to manmade chemicals may be linked to an increase in immune-related diseases in humans and immune system dysfunction in wildlife. Phthalates are a group of endocrine-disrupting chemicals (EDCs) suspected to influence the immune system. The aim of this study was to characterize the persistent effects on leukocytes in the blood and spleen, as well as plasma cytokine and growth factor levels, one week after the end of five weeks of oral treatment with dibutyl phthalate (DBP; 10 or 100 mg/kg/d) in adult male mice. Flow cytometry analysis of the blood revealed that DBP exposure decreased the total leukocyte count, classical monocyte and T helper (Th) popula-tions, whereas it increased the non-classical monocyte population compared to the vehicle control (corn oil). Immuno-fluorescence analysis of the spleen showed increased CD11b+Ly6G+ (marker of polymorphonuclear myeloid-derived suppressor cells; PMN-MDSCs), and CD43+staining (marker of non-classical monocytes), whereas CD3+ (marker of total T cells) and CD4+ (marker of Th cells) staining decreased. To investigate the mechanisms of action, levels of plasma cytokines and chemokines were measured using multiplexed immunoassays and other key factors were ana-lyzed using western blotting. The observed increase in M-CSF levels and the activation of STAT3 may promote PMN-MDSC expansion and activity. Increased ARG1, NOX2 (gp91phox), and protein nitrotyrosine levels, as well as GCN2 and phosphor-eIRF alpha, suggest that oxidative stress and lymphocyte arrest drive the lymphocyte suppression caused by PMN-MDSCs. The plasma levels of IL-21 (promotes the differentiation of Th cells) and MCP-1 (regulates mi-gration and infiltration of monocytes/macrophages) also decreased. These findings show that adult DBP exposure can cause persistent immunosuppressive effects, which may increase susceptibility to infections, cancers, and immune dis-eases, and decrease vaccine efficacy.