Similarly to other investigations the present study

Similarly to other investigations, the present study found that ZEA in high concentrations decreases viability of both normal (Liu et al., 2018, Zheng et al., 2018a, Zheng et al., 2018b) and cancer Prednisolone (Khosrokhavar et al., 2009, Kowalska et al., 2017). The toxic effect of ZEA observed by Liu et al. is associated with visible changes in the morphology of cells i.e. fragmentation of nuclei, indicating DNA damage caused by ZEA, which is in accordance with the results of our study (Liu et al., 2018). The addition of selective ERβ inhibitor PHTPP increased the decrease in cell viability what suggests that lack of active ERβ might increase toxic effects of ZEA, what then confirms the protective role of ERβ in prostate cells (Silva et al., 2018). The decrease in cell viability was associated with oxidative stress caused by the presence of ZEA and similarly to other studies we also observed a dose-dependent increase in ROS positive cells caused by ZEA (Fan et al., 2017). Que and al. reported that ERβ activation decreases the oxidative stress in cells, our results are also similar to that observation (Que et al., 2017). Moreover, the present study showed that the addition of PHTPP caused a significant increase in the number of ROS positive cells, indicating that the lack of active ERβ increases oxidative stress in cells caused by ZEA. In our experiments, the induction of oxidative stress was also associated with a decrease in CAT and SOD activity, and although it was not significant, the higher decrease in SOD activity was visible for ZEA+PHTPP treatment. The results are similar to the previous ones, where ZEA induced the decrease in CAT and SOD activity (Muthulakshmi et al., 2018). It was shown before in the study on prostate cancer cells (Kowalska et al., 2017) and by Fan et al. as well (Fan et al., 2017) as well that ZEA-induced oxidative stress is associated with mitochondrial function and damage. In this study, we observed that the expression of mitochondrial SOD (SOD2) was seven-fold decreased after ZEA treatment and nine-fold decreased after ZEA+PHTPP treatment, which seems to confirm the observed by us increase in ROS positive cells after ZEA+PHTPP treatment as compared to ZEA alone. The observed increase in oxidative stress was also associated with decreased migration of cells as well as cell cycle disturbances. The migration of PNT1A cells was significantly decreased after both ZEA and ZEA+PHTPP treatments in two highest concentrations and correlated with the decreased ZEB2 expression. As we showed that ZEA affected cell cycle regulation, similarly, a cell cycle arrest effect of ZEA was observed in Sertoli cells, where ZEA caused a statistically significant increase in the number of cells in the G2/M cell cycle phase (Zheng et al., 2018a, Zheng et al., 2018b). The G2/M cell cycle arrest is also associated with the lowered expression of CDK2 and CDK4. The addition of PHTPP caused significant decrease in the number of G2/M cells as compared to ZEA treatment, visible also in the expression of CDC2, the increase in the number of cells in the G0/G1 cell cycle phase was also apparent, but it was still lower as compared to control cells. Interestingly, we observed that simultaneous treatment of cells with ZEA and PHTPP caused an increase in the number of cells in the sub-G0 cell cycle phase visible on the representative results, indicating that lack of ERβ might trigger a different effect of ZEA-induced changes in cell cycle progression. Due to the fact that chronic inflammation in prostate is associated with a higher cancer risk as well as the fact that ROS generation might be also associated with reactive nitric species (RNS) generation (Ying and Hofseth, 2007), we evaluated the effect of ZEA and involvement of ERβ on the expression of iNOS (NOS2) and eNOS (NOS3). We observed that ZEA in a dose-dependent manner increases the expression of iNOS, a similar effect was observed for addition of PHTPP, however, it was presented to a lower extent. The induction of oxidative stress by ZEA is also visible in the NOS3 expression, which was increased after ZEA treatment and interestingly this effect was not visible in cells treated with ZEA and PHTPP. NOS3 was previously reported to be potentially modulated by estrogens via ERβ in prostate cancer (Re et al., 2018). In this study, we observed that the lack of active ERβ increases the expression of NOS3 for control and E2 treated cells. Our results suggest that the observed ZEA-induced ROS generation might be associated with RNS species too.