ORIGINAL ABSTRACT

Western diet (WD) is strongly associated with obesity and metabolic dysfunction and often co-occurs with human exposure to widely used herbicides such as glyphosate (GLP) and 2,4-dichlorophenoxyacetic acid (2,4-D). Both compounds have been suggested as potential endocrine disruptors, including effects on thyroid homeostasis. However, no studies have examined their combined impact in the context of WD-related metabolic disturbances. Here, we investigated the long-term effects of GLP and/or 2,4-D exposure on thyroid-related parameters in male C57BL/6J mice fed a WD for 6 months. Animals received GLP (0.05 or 5 mg/kg/day), 2,4-D (0.02 or 2 mg/kg/day), or their mixtures via intragastric administration, in doses spanning environmentally relevant exposures near the acceptable daily intake (ADI) to levels approaching, but still below, established no-observed-adverse-effect levels (NOAEL). WD feeding successfully induced obesity, glucose intolerance, and hypercholesterolemia; none of these parameters were significantly modified by herbicide exposure. However, GLP at 5 mg/kg increased serum triiodothyronine (T3) levels and promoted thyroid follicular changes, including increased follicle diameter, area, and the area of follicles within the 0-100 μm2 range. TSH levels remained unchanged. Additionally, low-dose GLP (0.05 mg/kg) elevated the percentage of PCNA-positive follicular cells, indicating enhanced cell proliferation. These findings suggest that chronic GLP exposure may exacerbate thyroid morphological and functional alterations even in the absence of changes in systemic metabolic outcomes. Our study provides the first evidence that GLP exposure within toxicologically relevant doses and under a WD setting can modulate thyroid parameters, highlighting the need to consider real-life scenarios in endocrine toxicity assessment.