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As an extensively employed plasticizer in industrial applications, di-2-ethylhexyl phthalate (DEHP) can induce apoptosis of mouse Leydig cells, yet the precise mechanism remains elusive. In the current study, we identified that DEHP could specially induced apoptosis in the Leydig cells of the testis tissue, accompanied with the upregulation of apoptosis-related protein in the TGF-β signaling pathway (ARTS) in the cells. Overexpression of ARTS significantly induced apoptosis of TM3 cells, while knockdown of ARTS inhibited apoptosis. Furthermore, DEHP-induced apoptosis of TM3 cells could be alleviated by knockdown of ARTS, which indicated that ARTS was involved in DEHP-induced apoptosis of mouse Leydig cells. Bioinformation assay predicts that there are four potential p53-responsive elements (p53-REs) located at − 6060, − 5726, − 5631 and − 5554 before the transcription start site of ARTS gene, implying that gene transcription of ARTS could be regulated by p53. Interestingly, DEHP was shown to specifically upregulate the expression of p53 in the Leydig cells of the testis tissue and TM3 cells. Consistently, p53 was proved to bind to the RE4 site of the ARTS gene promoter and transcriptionally activated the promoter-driven expression of the luciferase reporter gene. Overexpression of p53 could induce apoptosis of TM3 cells; while knockdown of p53 could not only rescue DEHP-induced apoptosis of the cells, but also inhibit DEHP-caused upregulation of ARTS. Meanwhile, we showed that oxidative stress could induce apoptosis of TM3 cells, accompanied with the increased protein levels of p53 and ARTS; while inhibition of oxidative stress dramatically alleviated DEHP-induced apoptosis and the up-regulation of p53 and ARTS. Taken together, these results indicated that DEHP-induced oxidative stress activates the p53-ARTS cascade to promote apoptosis of mouse Leydig cells.

As one of the most important phthalates, di-isononyl phthalate (DINP) has been widely used as a common plasticizer in the food and personal care products sectors. In our previous study, we found that DINP can induce autophagy of ovarian granulosa cells; while the underlying mechanism is unclear. In the study, we showed that DINP exposure could induce autophagy of ovarian granulosa cells and KGN cells, accompanied with the increase in the mRNA and protein level of DDIT4. Furthermore, overexpression of DDIT4 were shown to induce autophagy of KGN cells; while knockdown of DDIT4 inhibited DINP-induced autophagy, implying that DDIT4 played an important role in DINP-induced autophagy of ovarian granulosa cells. There were three putative binding sites of transcription factor ATF4 in the promoter region of DDIT4 gene, suggesting that DDIT4 might be regulated by ATF4. Herein, we found that overexpression of ATF4 could upregulate the expression of DDIT4 in KGN cells, while knockdown of ATF4 inhibited its expression. Subsequently, ATF4 was identified to bind to the promoter region of DDIT4 gene and promote its transcription. The expression of ATF4 was also increased in the DINP-exposed granulosa cells, and ATF4 overexpression promoted autophagy of KGN cells; whereas knockdown of ATF4 alleviated DINP-induced upregulation of DDIT4 and autophagy of the cells. Taken together, DINP triggered autophagy of ovarian granulosa cells through activating ATF4/DDIT4 signals.