The role of p53 in death receptor-mediated apoptosis of testicular germ cells in response to mono-(2-ethylhexyl) phthalate treatment

Mono-(2-ethylhexyl) phthalate (MEHP) is the toxic metabolite of the common plasticizer di-(2-ethylhexyl) phthalate (DEHP). Exposure to DEHP or MEHP typically leads to testicular atrophy in laboratory animals. The prevalence of DEHP as an environmental contaminant is therefore cause for concern due to its potential to affect human testicular function and fertility. In the testis, MEHP induces the injury of the supportive cells called Sertoli cells, leading to Fas death receptor-dependent apoptotic elimination of germ cells. This dissertation examines the molecular mechanisms that lead to this loss of germ cells resulting from testicular exposure to MEHP. The p53 tumor suppressor protein is proposed as the cellular stress sensor that sensitizes specific germ cells to Fas-dependent cell death in response to the MEHP-induced Sertoli cell injury. This is based upon recent reports that demonstrate p53’s ability to initiate the localization viii of Fas to the plasma membrane of cells in a transcription-independent manner, in addition to its ability to transcribe various pro-apoptotic proteins including Fas. In experiments involving the exposure of pre-pubertal p53 wild-type and mutant mice (C57 x 129S/v) to a single oral dose of 1g MEHP /kg body weight, we were able to demonstrate that mice lacking the p53 gene were partially protected from the testicular toxicity induced by MEHP. These studies enabled us to establish that a lack of p53 expression affected the activation of the Fas pathway in germ cells after MEHP exposure, partly by affecting Fas membrane localization and also by influencing the cellular retention of an inhibitor of this pathway, the c-FLIP (L) protein. To further clarify these results, we examined the effect of Fas activation in GC-2spd (ts) cells, which are germ cells that demonstrate variable activation of p53 depending on the temperature that they are maintained at. The activation of the p53 protein increased the sensitivity of GC-2 cells to undergo Fas-mediated apoptosis by modulating Fas expression on the germ cell membrane. Additionally, activation of Fas caused an increased tagging of c-FLIP (L) with ubiquitin, indicating its targeting for degradation. Thus the p53 status of GC-2 cells influenced the membrane expression of Fas, subsequently influencing the degradation of the anti-apoptotic protein c-FLIP (L). In summary, the results indicate that p53 promotes Fas activated germ cell apoptosis in response to MEHP-induced Sertoli cell injury, by instigating the degradation of c-FLIP (L) protein via its ubiquitinylation.