Developmental Immunotoxicity of Low-dose Inorganic Arsenic Reprograms Macrophages Inducing Tumor-promoting Phenotypes

Illingworth, E. J.; Sanchez, S. S.; Rychlik, K. A.; Kleensang, A.; Lopez-Cecetaite, G. A.; Rodriguez Steube, M. C.; Mathur, A.; Sadhukhan, P.; Medina-Cleghorn, D. A.; Nomura, D.; Smith, M. T.; Hoque, M. O.; Sille, F. C. M.

doi:10.1101/2025.07.14.663593

bioRxiv · 10.1101/2025.07.14.663593

Developmental Immunotoxicity of Low-dose Inorganic Arsenic Reprograms Macrophages Inducing Tumor-promoting Phenotypes

Illingworth, E. J.·Sanchez, S. S.·Rychlik, K. A.·Kleensang, A.·Lopez-Cecetaite, G. A.·Rodriguez Steube, M. C.·Mathur, A.·Sadhukhan, P.·Medina-Cleghorn, D. A.·Nomura, D.·Smith, M. T.·Hoque, M. O.·Sille, F. C. M.

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Abstract

In many regions around the world, including the United States, inorganic arsenic (iAs) contaminates groundwater used for drinking, food production, and irrigation. Although the World Health Organization has set a safety limit of 10 {micro}g/L for arsenic in drinking water, an estimated 200 million people worldwide are still exposed to arsenic concentrations above this threshold. Eliciting a broad range of adverse health effects, arsenic is a known carcinogen classified by the International Agency for Research on Cancer (IARC) and causes increased susceptibility to infectious diseases, highlighting its role as an immunotoxicant. The purpose of this study is to elucidate the effects of arsenic on the innate immune system, namely macrophages, using in vitro exposure models. Bone marrow-derived macrophages (BMDMs) were cultured from adult male and female C57/BL6 mice. These naive macrophages ("M0" BMDMs) were exposed in vitro to a non-cytotoxic dose of iAs (0.1 {micro}M sodium (meta)arsenite) during the 7 day period of macrophage differentiation and stimulated for 24 hrs with LPS and IFN{gamma} (to induce "M1" pro-inflammatory activation) or IL-4 and IL-13 (to induce "M2" anti-inflammatory activation). In a parallel chronic exposure model, RAW 264.7 (RAW) macrophages were cultured in vitro with iAs for 70 days. Culture supernatant analysis for nitric oxide and cytokine secretion revealed sex-dependent differences in immune response between exposure models, as well as between iAs-exposed and nonexposed macrophages, with and without stimulation. Additionally, iAs-exposed macrophages exhibited increased lipid droplet formation and altered lipidomic and metabolomic profiles, as determined by LC/MS. Flow cytometric analysis further revealed changes in macrophage polarization markers in a sex- and stimulation-dependent manner, with M2-related markers being upregulated in iAs-exposed conditions. Finally, to assess the effects of iAs on macrophages in the context of cancer, we demonstrated that iAs-exposed macrophages displayed increased migration toward cancer cell-conditioned media, and promoted cancer cell proliferation. These results suggest that dysregulated macrophage polarization due to iAs exposure could impact susceptibility to diseases. This research contributes to our understanding of the full spectrum of adverse health effects of iAs exposure and may aid in the development of therapeutics for iAs-induced diseases, including cancer.

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Illingworth, E. J., Sanchez, S. S., Rychlik, K. A., Kleensang, A., Lopez-Cecetaite, G. A., Rodriguez Steube, M. C., Mathur, A., Sadhukhan, P., Medina-Cleghorn, D. A., Nomura, D., Smith, M. T., Hoque, M. O., Sille, F. C. M.. 2025-07-18. Developmental Immunotoxicity of Low-dose Inorganic Arsenic Reprograms Macrophages Inducing Tumor-promoting Phenotypes. https://doi.org/10.1101/2025.07.14.663593

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