Mengmeng Yao

Metabolomics reveals metabolic changes in male reproductive cells exposed to thirdhand smoke

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.

Metabolomic profiles reveal key metabolic changes in heat stress-treated mouse Sertoli cells

Heat stress (HS) is a potential harmful factor for male reproduction. However, the effect of HS on Sertoli cells is largely unknown. In this study, the metabolic changes in Sertoli cell line were analyzed after HS treatment. Metabolomic analysis revealed that carnitine, 2-hydroxy palmitic acid, nicotinic acid, niacinamide, adenosine monophosphate, glutamine and creatine were the key changed metabolites. We found the expression levels of BTB factors (Connexin43, ZO-1, Vimentin, Claudin1, Claudin5) were disrupted in TM-4 cells after HS treatment, which were recovered by the addition of carnitine. RT-PCR indicated that the mRNA levels of inflammatory cytokines (IL-1α, IL-1β, IL-6) were increased after HS treatment, and their related miRNAs (miR-132, miR-431, miR-543) levels were decreased. Our metabolomic data provided a novel understanding of metabolic changes in male reproductive cells after HS treatment and revealed that HS-induced changes of BTB factors and inflammatory status might be caused by the decreased carnitine after HS treatment.

Effect of perfluorooctane sulfonate on pluripotency and differentiation factors in mouse embryoid bodies

Perfluorooctane sulfonate (PFOS) poses potential risks to early development, but the molecular mechanisms how PFOS affects embryonic development are still unclear. Mouse embryoid bodies (mEBs) provide ideal models for testing safety or toxicity of chemicals in vitro. In this study, mEBs were exposed to PFOS up to 6 days and then their pluripotency and differentiation markers were evaluated. Our data showed that the mRNA and protein levels of pluripotency markers (Oct4, Sox2, Nanog) in mEBs were significantly increased following exposure to PFOS. Meanwhile, the expressions of miR-134, miR-145, miR-490-3p were decreased accordingly. PFOS reduced the mRNA levels of endodermal markers (Sox17, FOXA2), mesodermal markers (SMA, Brachyury) and ectodermal markers (Nestin, Fgf5) in mEBs. Meanwhile, PFOS increased the mRNA and protein levels of polycomb group (PcG) family members (Cbx4, Cbx7, Ezh2). Overall, our results showed that PFOS could increase the expression levels of pluripotency factors and decrease the differentiation markers.

miR-98 and its host gene Huwe1 target Caspase-3 in Silica nanoparticles-treated male germ cells.

Silica nanoparticles (NP) is one of the most commonly used nanomaterials with potential health hazards. However, the effects of Silica NP on germ cells and the underlying mechanisms are still unclear. In this study, GC-2 and TM-4, which are two different types of male germ cells were exposed to Silica NP for 24h, and then general cytotoxicity and multi-parameter cytotoxicity were evaluated. Our results showed that Silica NP could induce apoptosis in GC-2 cells. Transmission electron microscopy (TEM) results showed that Silica NP was localized in the lysosomes of GC-2 cells. High content screening (HCS) showed that Silica NP exposure could increased cell permeabilization and decreased mitochondrial membrane potential in GC-2 cells. The mRNA and protein levels of apoptosis markers (Bax, Caspase-3, Caspase-9) in GC-2 cells were significantly increased, while Bcl-2 was decreased. Accordingly, the expression level of miR-98, which can regulate Caspase-3, was significantly decreased. Huwe1, the host gene of miR-98, was positively associated with miR-98 expression after Silica NP exposure. Dual luciferase reporter assay suggested that miR-98 directly targets Caspase-3. These results suggest that Silica NP induces apoptosis via loss of mitochondrial membrane potential and Caspase-3 activation, while miR-98 plays key role in modulating this effect.

Titanium dioxide nanoparticles induce proteostasis disruption and autophagy in human trophoblast cells

Titanium dioxide nanoparticles (TiO2 NPs) exist in many nano-products and concerns have been raised about their potential toxicity on human beings. One such issue is their potential effects on placental function, and the studies on this topic are limited and the mechanism remains unclear. Here we employed human trophoblast HTR-8/SVneo cells to investigate the effects of TiO2 NPs on trophoblast. Results showed that TiO2 NPs could enter cells and were mostly distributed in lysosomes, with some in the cytoplasm. TiO2 NPs and protein aggregation were found in both fetal bovine serum (FBS) in culture medium and cytoplasm of HTR-8/SVneo cells. In consistence with that, proteostasis of HTR-8/SVneo cells was significantly disrupted and endoplasmic reticulum (ER) stress related markers including PERK, IRE1-α were increased. After high speed centrifugation, the proteins PERK and IRE1-α were dramatically decreased in the highest TiO2 NPs treatment group, which indicated interactions between TiO2 NPs and these two proteins. Meanwhile, the protein expressions of LC3-II/LC3-I and P62, the autophagy biomarkers, were increased and the autophagy flux was not blocked. Cellular ROS stress increased and mitophagy related genes including PINK and Parkin increased along with the increased co-localization of LC3 and mitochondria. Taken together, these results indicated that TiO2 NPs interacted with intracellular proteins and activated ER stress and mitophagy in HTR-8/SVneo cells, which might do damage to placental function.

miR-96-5p and miR-101-3p as potential intervention targets to rescue TiO2 NP-induced autophagy and migration impairment of human trophoblastic cells

Autophagy induced by titanium dioxide nanoparticles (TiO2 NPs) has been realized nowadays, but the underlying mechanisms remain largely unknown. Animal studies have confirmed that autophagy might be an important mechanism to impair placenta development, but the reversal of damage is not clear. Here, we used human HTR-8/SVneo (HTR) cells as a proper model to explore how autophagy is regulated in TiO2 NP-exposed human placenta cells. Our studies showed that TiO2 NPs could enter HTR cells and locate in cytoplasm. Although they did not affect cell viability even under 100 μg ml-1, autophagy was observed and cell migration ability was severely impaired. Further study showed that TiO2 NPs increased the expressions of both miR-96-5p and miR-101-3p and then, they targeted mTOR and decreased the expression of mTOR proteins. In addition, miR-96-5p also targeted Bcl-2 to down-regulate Bcl-2 protein level, which is also a key regulator of autophagy. We proved that when two microRNA inhibitors were added, cell autophagy was, to a greater extent, reversed compared with the result when one inhibitor was added, and the cell migration ability was also reversed to a greater degree. Our studies revealed that TiO2 NPs might impair placenta development via autophagy. Moreover, miR-96-5p as well as miR-101-3p may act as potential targets to reverse TiO2 NP-induced autophagy and placenta dysfunction.

Erratum: Corrigendum: Metabolomics reveals metabolic changes in male reproductive cells exposed to thirdhand smoke

Author(s): Xu, B; Chen, M; Yao, M; Ji, X; Mao, Z; Tang, W; Qiao, S; Schick, SF; Mao, JH; Hang, B; Xia, YScientific Reports 5: Article number: 1551210.1038/srep15512; published online: October222015; updated: April132016 The Acknowledgements section in this Article is incomplete. “This study was supported by National 973 Program (2012CBA01305); National Science Fund for Outstanding Young Scholars (81322039); National Natural Science Foundation (31371524); Distinguished Young Scholars of Jiangsu Province (BK20130041); Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD); New Century Excellent Talents in University (NCET-13-0870) and the California Tobacco-Related Disease Research Program (TRDRP) Grant 19XT-0070 (to B.H.) under U.S. Department of Energy (Contract no. DE-AC02-05CH11231).” should read: “This study was supported by National 973 Program (2012CBA01305); National Science Fund for Outstanding Young Scholars (81322039); National Natural Science Foundation (31371524); Distinguished Young Scholars of Jiangsu Province (BK20130041); Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD); New Century Excellent Talents in University (NCET-13-0870) and the California Tobacco-Related Disease Research Program (TRDRP) Grant 19XT-0070 (to B.H.) under U.S. Department of Energy (Contract no. DE-AC02-05CH11231); The California Consortium on Thirdhand Smoke, California Tobacco-Related Disease Research Program (trdrp.org) grant 20PT-0184 and California Tobacco-Related Disease Research Program grant 21 ST-011).”

Corrigendum: Metabolomics reveals metabolic changes in male reproductive cells exposed to thirdhand smoke.

Author(s): Xu, B; Chen, M; Yao, M; Ji, X; Mao, Z; Tang, W; Qiao, S; Schick, SF; Mao, JH; Hang, B; Xia, YScientific Reports 5: Article number: 1551210.1038/srep15512; published online: October222015; updated: April132016 The Acknowledgements section in this Article is incomplete. “This study was supported by National 973 Program (2012CBA01305); National Science Fund for Outstanding Young Scholars (81322039); National Natural Science Foundation (31371524); Distinguished Young Scholars of Jiangsu Province (BK20130041); Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD); New Century Excellent Talents in University (NCET-13-0870) and the California Tobacco-Related Disease Research Program (TRDRP) Grant 19XT-0070 (to B.H.) under U.S. Department of Energy (Contract no. DE-AC02-05CH11231).” should read: “This study was supported by National 973 Program (2012CBA01305); National Science Fund for Outstanding Young Scholars (81322039); National Natural Science Foundation (31371524); Distinguished Young Scholars of Jiangsu Province (BK20130041); Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD); New Century Excellent Talents in University (NCET-13-0870) and the California Tobacco-Related Disease Research Program (TRDRP) Grant 19XT-0070 (to B.H.) under U.S. Department of Energy (Contract no. DE-AC02-05CH11231); The California Consortium on Thirdhand Smoke, California Tobacco-Related Disease Research Program (trdrp.org) grant 20PT-0184 and California Tobacco-Related Disease Research Program grant 21 ST-011).”