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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 (0.1-100 μm) of BaP for 1-48 hours, and found that GJIC exhibited a dose- and time-dependent downregulation. Treatment with 10 μm BaP increased apoptosis, intracellular Ca 2+ level ([Ca 2+ ] i ) and calmodulin (CaM) protein expression, and decreased the protein level of connexin 43 (Cx43) (also known as gap junction α-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 [Ca 2+ ] 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 Ca 2+ /CaM signaling, and that BaP-induced GJIC suppression increases apoptosis in TM4 cells., (Copyright © 2018 John Wiley & Sons, Ltd.)

Cadmium (Cd), a toxic heavy metal, is known to induce renal toxicity by primarily targeting at renal proximal tubule. Endoplasmic reticulum (ER) stress and gap junctional intercellular communication (GJIC) regulate many pathophysiological processes. Yet, how ER stress and GJIC regulate Cd-induced nephrotoxicity remain elusive. In this study, we treated human proximal tubule (HK-2) cells with 1 μM CdCl 2 every other day for 12 days and found that Cd significantly increased cell apoptosis at 10 and 12 days. This cytotoxicity correlated with activation of ER stress and apoptotic signaling evidenced by upregulation of inositol-requiring enzyme 1 (IRE1α), splice X-box binding protein-1 (XBP-1s), and apoptosis signal-regulating kinase 1 (ASK1) proteins. Interestingly, the AKT signaling was activated at 2- and 4-day and then inhibited at 10- and 12-day of Cd treatment; by contrast, Cd decreased GJIC levels at 2- and 4-day followed by a significant increase at 10- and 12-day treatment. Activation of AKT by SC79 or inhibition of GJIC by 18α-glycyrrhetinic acid (18α-GA) completely abolished Cd-induced AKT inhibition and IRE1α-ASK1 activation. Importantly, pretreatment with ER stress inhibitor or 18α-GA significantly mitigated Cd-induced apoptosis. These results suggest that GJIC collaborates with AKT signaling and ER stress in regulating prolonged Cd-treatment-induced apoptosis in HK-2 cells., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Titanium dioxide nanoparticles (TiO2 NPs) have been widely used for many years. Their toxic effects on the male reproductive system had been reported, but the underlying mechanisms were still not clear. Here we utilized two germ cell lines (GC-2 and TM4) to explore the possible toxic effects of TiO2 NPs on male reproductive system. Our results showed that TiO2 NPs did not affect cell viability but induced cell apoptosis of both GC-2 and TM4 cells up to 100 μg/ml. Microtubule networks and microtubule dynamics of GC-2 but not TM4 cells were changed. The microfilaments arrangement of TM4 cells altered after treated with TiO2 NPs, and the phagocytosis activity of TM4 cells decreased significantly. Although the microfilament network of GC-2 cells seemed normal, the migration ability of GC-2 cells was significantly impaired. Totally TiO2 NPs is toxic to GC-2 cells by inducing cell apoptosis, disturbing microtubule arrangement and microtubule dynamic, and impairing cell migration ability. In addition, they altered the microfilament network and reduced the phagocytic activity of TM4 cells. We firstly reported that cytoskeletons (microtubules and microfilaments) in different cells showed different responses to TiO2 NPs, which might mediate different toxic mechanisms.

Thirdhand smoke (THS) is a new term for the toxins in cigarette smoke that linger in the environment long after the cigarettes are extinguished. The effects of THS exposure on male reproduction have not yet been studied. In this study, metabolic changes in male germ cell lines (GC-2 and TM-4) were analyzed after THS treatment for 24 h. THS-loaded chromatography paper samples were generated in a laboratory chamber system and extracted in DMEM. At a paper: DMEM ratio of 50 μg/ml, cell viability in both cell lines was normal, as measured by the MTT assay and markers of cytotoxicity, cell cycle, apoptosis and ROS production were normal as measured by quantitative immunofluorescence. Metabolomic analysis was performed on methanol extracts of GC-2 and TM-4 cells. Glutathione metabolism in GC-2 cells, and nucleic acid and ammonia metabolism in TM-4 cells, was changed significantly by THS treatment. RT-PCR analyses of mRNA for enzyme genes Gss and Ggt in GC-2 cells, and TK, SMS and Glna in TM-4 cells reinforced these findings, showing changes in the levels of enzymes involved in the relevant pathways. In conclusion, exposure to THS at very low concentrations caused distinct metabolic changes in two different types of male reproductive cell lines.

Perfluorooctane sulfonate (PFOS) poses potential risks to reproduction and development. Mouse embryonic stem cells (mESCs) are ideal models for developmental toxicity testing of environmental contaminants in vitro. However, the mechanism by which PFOS affects early embryonic development is still unclear. In this study, mESCs were exposed to PFOS for 24 h, and then general cytotoxicity and pluripotency were evaluated. MTT assay showed that neither PFOS (0.2 µM, 2 µM, 20 µM, and 200 µM) nor control medium (0.1% DMSO) treatments affected cell viability. Furthermore, there were no significant differences in cell cycle and apoptosis between the PFOS treatment and control groups. However, we found that the mRNA and protein levels of pluripotency markers (Sox2, Nanog) in mESCs were significantly decreased following exposure to PFOS for 24 h, while there were no significant changes in the mRNA and protein levels of Oct4. Accordingly, the expression levels of miR-145 and miR-490-3p, which can regulate Sox2 and Nanog expressions were significantly increased. Chrm2, the host gene of miR-490-3p, was positively associated with miR-490-3p expression after PFOS exposure. Dual luciferase reporter assay suggests that miR-490-3p directly targets Nanog. These results suggest that PFOS can disturb the expression of pluripotency factors in mESCs, while miR-145 and miR-490-3p play key roles in modulating this effect.

The present study aimed to investigate the molecular mechanism and the effect of Saponin from Tupistra chinensis Baker (STCB) on the proliferation and apoptosis of ovarian cancer cells. To investigate the inhibitory effect of STCB on the proliferation of ovarian cancer cells, SKOV3 cells were cultured and the methyl thiazolyl tetrazolium assay was used. Flow cytometry was also used to analyze the cell cycle distribution and apoptotic rate. Ki‐67, cyclin D1, cleaved caspase‐3, cleaved caspase‐9, β‐catenin, and c‐Myc protein expressions were detected by western blot. Ovarian cancer cells were treated with STCB and Wnt pathway activator lithium chloride (LiCl). These methods were also used to determine the proliferation, cell cycle distribution, and apoptosis of ovarian cancer cells. In STCB‐treated group, the proliferation inhibition and apoptosis rate, the proportion of G0‐G1 phase, and the expression level of cleaved caspase‐3 and 9 of ovarian cancer cells were significantly increased. Similarly, the expression of Ki‐67, cyclin D1, β‐catenin, and c‐Myc were significantly decreased (p <.05). The results also showed that in STCB‐LiCl‐treated group, while the proliferation inhibition rate of ovarian cancer cells, the proportion of G0‐G1 cells, the expression level of cleaved caspase‐3 and 9, and the apoptosis rate (p <.05) were significantly decreased, the expression level of Ki‐67, cyclin D1, β‐catenin, and c‐Myc was significantly increased. STCB induced G0‐G1 phase arrest, inhibited cell proliferation, and promoted apoptosis of ovarian cancer cells by inhibiting Wnt/β‐catenin pathway. [ABSTRACT FROM AUTHOR]

Connexin 43 (Cx43) is believed to play a role in the mechanisms of toxicity of many chemical species, include cadmium (Cd). In this study, human renal proximal tubule (HK-2) cells were exposed to Cd (1 μM, 10 days). Of the 584 protein residues detected using a Phospho Explorer antibody microarray (PEX100), more than half changed their levels of phosphorylation after chronic Cd exposure. Cx43 siRNA attenuated Cd-induced apoptosis and inhibited proliferation, while also attenuating changes in the levels of phosphorylation of many protein residues. According to DAVID Bioinformatics Resources analysis and KEGG PATHWAY database, AKT signal pathway may be the important one. Focusing on the AKT pathway confirmed that Cx43 mediated increased levels of p-PTEN Ser380/Ser382/Thr383 and decreased levels of p-AKT Thr308 , p-AKT Tyr326 , p-ASK1 Ser83 , and p-p27 Thr187 , thereby possibly contributing to the Cd-induced apoptosis and inhibited proliferation. These results suggested that AKT pathway was the dominant pathway involved in Cx43-mediated chronic Cd toxicity. [ABSTRACT FROM AUTHOR]