Simazine-induced decreased Rxfp1 and Nos2 expression and reduced NO production in testicular cells in vitro.

Rat Leydig cells (LC540) were exposed to simazine (0, 0.01, 0.1, or 1 µM) for 36 h, and the mRNA levels of Rxfp1 (A) and Nos2 (B) were analyzed by qRT-PCR. The relative expression levels of Rxfp1 and Nos2 normalized to β-actin are shown. (C) LC540 cells were exposed to various concentrations of simazine for 36 h, and the levels of NO produced were determined by measuring nitrite concentration. (D) LC540 knockdown cells were prepared by transfection with a scrambled sequence or siRNAs for Rxfp1, and reduced Rxfp1 expression was demonstrated by western blotting. Subsequently, knocked-down cells were treated with simazine for 36 h, and the levels of NO produced were determined. For all experiments (A-D), three independent experiments were performed in triplicate, and different letters denote significant values (p<0.05).