Benzo[a]pyrene-decreased gap junctional intercellular communication via calcium/calmodulin signaling increases apoptosis in TM4 cells

The mechanism of male reproductive toxicity induced by benzo[a]pyrene (BaP) is poorly understood. Gap junctional intercellular communication (GJIC) is known to play a critical role in maintaining spermatogenesis. The aim of the present study was to determine the toxic effects of BaP in Sertoli cells, and to explore the possibility and potential mechanisms of BaP-induced changes in the level of GJIC, and the relationship between GJIC and BaP-induced apoptosis. We treated mouse Sertoli cell lines (TM4) with different concentrations (0.1-100m) of BaP for 1-48hours, and found that GJIC exhibited a dose- and time-dependent downregulation. Treatment with 10m BaP increased apoptosis, intracellular Ca2+ level ([Ca2+](i)) and calmodulin (CaM) protein expression, and decreased the protein level of connexin 43 (Cx43) (also known as gap junction -1 protein [GJA1]) in TM4 cells. However, BaP had no effect on the phosphorylation of Cx43 at Ser279/282, Ser255, Ser368 or Ser262. Downregulation of [Ca2+](i) by BAPTA-AM significantly attenuated the BaP-induced GJIC suppression, Cx43 protein decrease and CaM protein increase. Interestingly, inhibition of CaM expression by W7 partially recovered BaP-induced GJIC inhibition, but had no effect on BaP-induced Cx43 protein decrease. Pretreatment with the GJIC activator retinoic acid significantly mitigated BaP-induced apoptosis. In conclusion, these results suggest that BaP can decrease GJIC via Ca2+/CaM signaling, and that BaP-induced GJIC suppression increases apoptosis in TM4 cells. The mechanism of male reproductive toxicity induced by benzo[a]pyrene (BaP) is unclear. In this study, BaP increased apoptosis, [Ca2+](i) level and calmodulin (CaM) expression, and decreased gap junctional intercellular communication (GJIC) level and Cx43 expression, but did not affect the Cx43 phosphorylation at certain sites in TM4 cells. [Ca2+](i) chelator attenuated BaP-induced changes of GJIC, Cx43 and CaM. CaM inhibition partially recovered GJIC, but did not affect Cx43. Moreover, GJIC activator mitigated BaP-induced apoptosis. Thus, BaP-decreased GJIC via Ca2+/CaM signaling increases apoptosis in TM4 cells.