A mixture of glyphosate and 2,4-D herbicides enhances the deleterious reproductive outcomes induced by Western diet in obese male mice.

The consumption of Western diet (WD) - enriched in fats and sugars - is associated with overweight, obesity and male reproductive disorders. In addition to WD intake, crops and dairy products display residues of herbicides, including glyphosate and 2,4-D that are widely applied worldwide. The concomitant exposure to WD and herbicides - mimicking contemporary scenarios - is not fully investigated. Thus, we evaluated the effects of glyphosate and 2,4-D, alone or in mixture, on WD-induced alterations in the male genital system. Male C57BL6J mice were submitted to WD (chow containing 20% lard, 0.2% cholesterol, 20% sucrose, and high sugar solution with 23.1 and 18.9 g/L of D-fructose and D-glucose) for 6 months. Concomitantly to WD, the animals received glyphosate (0.05, 5, or 50 mg/kg/day), 2,4-D (0.02, 2 or 20 mg/kg/day) or their mixture (0, 05 + 0.02, 5 + 2, or 50 + 20 mg/kg/day) by intragastrical administration (5×/week). Doses were based on Acceptable Daily Intake (ADIs) or No Observed Adverse Effect Level (NOAEL) values. Herbicide exposure did not alter the WD-induced obesity, hypercholesterolemia and hyperglycemia. WD induced sperm cell abnormalities, reduced the number, volume and area of Leydig cells, enhanced the frequency of epididymal abnormalities, decreased the proliferation in both germinal and epididymal epithelia, and reduced the number of androgen receptor (AR) positive epididymal cells. Remarkably, the herbicide mixtures promoted such WD-induced effects: increased the frequency of sperm cell and epididymal abnormalities (absence of sperm, cytoplasmic vacuoles, and clear cell hypertrophy) (5 + 2 and 50 + 20 doses); decreased Leydig cell nuclei volume and area (5 + 2 and 50 + 20 doses), reduced epididymal cell proliferation (all mixtures), and AR expression (50 + 20 dose). In addition, herbicide mixtures reduced serum testosterone levels (5 + 2 and 50 + 20 doses). Our findings indicate that the mixture of glyphosate and 2,4-D herbicides, mimicking environmentally relevant scenarios, promotes WD-induced changes in the male genital system.