ORIGINAL ABSTRACT

BACKGROUND: The widespread use of pesticides, including glyphosate-based herbicides (e.g., Roundup®, R) and chlorpyrifos-based insecticides (e.g., Clorpi48®, C), has raised concerns about their environmental and human health impacts. Growing evidence links pesticide exposure to cancer development. Given the critical role of immune surveillance in tumor growth control, environmental pollutants such as pesticides should also be evaluated for immunotoxicity. Moreover, while individual pesticides have been extensively studied, the effects of pesticide mixtures on human immune cells remain poorly explored. Here, we assessed the impact of environmentally relevant doses of R, C, or their combination (R+C) on immune cell functions. METHODS: Peripheral blood mononuclear cells (PBMCs), NK cells, and T cells from healthy donors were exposed to environmentally relevant doses of R, C, or R+C. NK cell cytotoxicity, T-bet expression and IFN-g production were analyzed by flow cytometry, and immune synapse formation (LFA-1 localization) and perforin polarization were analyzed by confocal microscopy. T-cell proliferation, Th1 differentiation, and IL-2 signaling were also evaluated by flow cytometry. Oxidative stress was quantified using a fluorometric assay by measuring H2O2 production in PBMCs exposed to R, C, or R+C. Also, the role of oxidative stress in T-cell dysfunction was assessed. RESULTS: The combination of R+C, but not the individual compounds, significantly impaired NK cell cytotoxicity, IFN-g production, and immune synapse formation, as evidenced by disrupted LFA-1 localization and defective perforin polarization. In T cells, R+C exposure inhibited proliferation, Th1 differentiation, IL-2 signaling, and IFN-g secretion by CD8⁺ T cells, all key functions for effective antitumor responses. Mechanistically, oxidative stress contributed to the antiproliferative effect, as scavenging of H2O2 by catalase addition restored T cell proliferation. CONCLUSIONS: Environmentally relevant doses of glyphosate and chlorpyrifos-based pesticide mixtures disrupt innate and adaptive immune effector functions that are critical for the control of neoplastic cells and nascent tumor foci, suggesting that current risk assessments underestimate the immunotoxicity of combined formulations.