Effects of TPPU on ASD-like behavioral abnormalities in juvenile offspring after maternal glyphosate exposure. (A) Schedule of treatment and behavioral tests. Water or glyphosate (0.098%) was given to pregnant mice. Vehicle (5 mL/kg/d) or TPPU (3 mg/kg/d) was administered orally to pregnant mice from E5 to P21. Subsequently, all mice received normal water. Grooming test and three-chamber social interaction test were performed from P28 to P35. (B) Change of body weight of mothers (n = 5 or 6). Two-way ANOVA (glyphosate, F1,17 = 7.66, P = 0.013; TPPU, F1,17 = 9.14, P = 0.008; interaction, F1,17 = 1.59, P = 0.225). (C) Grooming test. Treatment with TPPU significantly attenuated the increased grooming time in juvenile offspring after maternal glyphosate exposure. Two-way ANOVA (glyphosate, F1,36 = 14.19, P = 0.001; TPPU, F1,36 = 25.34, P < 0.001; interaction, F1,36 = 11.31, P = 0.002). Data are shown as mean ± SEM (n = 10; ***P < 0.01 compared to glyphosate + vehicle group). (D) Three-chamber social interaction test. (Left) Three-way ANOVA (glyphosate, F1,56 = 9.948, P = 0.003; TPPU, F1,56 = 0.301, P = 0.585; stranger, F1,56 = 135.27, P < 0.001; interaction [glyphosate × TPPU], F1,56 = 0.845, P = 0.362; interaction [glyphosate × stranger], F1,56 = 38.61, P < 0.001; interaction [TPPU × stranger], F1,56 = 3.593, P = 0.063; interaction [glyphosate × TPPU × stranger], F1,56 = 11.06, P = 0.002). (Right) Three-way ANOVA (glyphosate, F1,56 = 4.168, P = 0.046; TPPU, F1,56 = 21.96, P < 0.001; stranger, F1,56 = 104.28, P < 0.001; interaction [glyphosate × TPPU], F1,56 = 1.902, P = 0.173; interaction [TPPU × stranger], F1,56 = 3.870, P = 0.054; interaction [glyphosate × stranger], F1,56 = 13.10, P = 0.001; interaction [glyphosate × TPPU × stranger], F1,56 = 19.69, P < 0.001). Data are shown as mean ± SEM (n = 8; ***P < 0.01). N.S., not significant.