Developmental Toxicity Effects Of Di(2-ethylhexyl) Phthalate (DEHP) On MicroRNA Expression Of Mouse Testes

In recent years, a rise in the amounts of environmental pollutants has resulted inan increased incidence of reproductive tract disorders. Developing organisms aremore prone to be affected by exposure to toxic compounds, and this is a cause forconcern since proper development of the testes during male fetal growth is essentialfor the proper functioning of the reproductive system in adulthood. The incidence oftesticular cancer and male infertility are increasing, and seem to be a result fromdisturbance during early development; additionally, occurrence of reproductive tractmalformations in infants is also ascending.Di-(2ethylhexyl) phthalate (DEHP) has been associated with the manifestation ofdetrimental effects of the reproductive system. Numerous studies have demonstratedharmful effects of prenatal exposure to DEHP through animal models; however themechanism in which toxicity develops remains unclear.In this study, pregnant micewere randomly assigned to one of three treatment groups or a control group and doseddaily with different DEHP doses(60,175or500mg/kg) or a vehicle, through oralgavage from GD4.5to GD17.5. On GD18.5females were sacrificed and fetusesremoved for further analysis. There were no significant changes in the weight offetuses, the male-female ratio, or the incidence of resorptions and fetal deaths amongthe groups compared to control. There was, however, an incidence of gross externalmalformations only present in the highest dose group. Histological analysis of thetestes revealed a significant and dose-dependent increase in the number ofmultinucleated gonocytes, as well as an increased number of nuclei permultinucleated cell with increasing treatment dose. Analysis of serum testosteroneshowed an increase in concentration in the two highest dose groups compared tocontrol. As a novel endpoint, miRNA expression profiles of the fetal testes wereassessed through microarray and25mouse miRNAs showed statistically significantdysregulation compared to control after treatment with DEHP. Eight of thedysregulated miRNAs were corroborated by RT-PCR, which confirmed three of themiRNAs, namely miR-195, miR-185and miR-451showed overexpression comparedto control. The putative targets of these miRNAs were searched in three webprediction sites and the validated targets for each miRNA were also found. Functionaland pathway analysis of the predicted and validated targets revealed that these genesare involved in the regulation of the cell cycle, cell proliferation, and apoptosis. Theseresults are consistent with the biological processes that take place in the testes during embryonic development where the cell cycle is strictly controlled. Thus, increasedexpression of miR-195, miR-185and miR-451may be involved in the developmentof toxicity from DEHP exposure by affecting the proliferation or differentiation of thedifferent cell types within thedeveloping testes; which renders these miRNAs aspotential biomarkers for DEHP induced toxicity in testis.