Zhilei Mao

Metabolomic Analysis Reveals Metabolic Changes Caused By Bisphenol A in Rats

Bisphenol A (BPA) is a widely used material known to cause adverse effects in humans and other mammals. To date, little is known about the global metabolomic alterations caused by BPA using urinalysis. Sprague-Dawley rats were orally administrated BPA at the levels of 0, 0.5 μg/kg/day and 50 mg/kg/day covering a low dose and a reference dose for 8 weeks. We conducted a capillary electrophoresis in tandem with electrospray ionization time-of-flight mass spectrometry based nontargeted metabolomic analysis using rat urine. To verify the metabolic alteration at both low and high doses, reverse transcription-polymerase chain reaction (RT-PCR) and western blotting were further conducted to analyze hepatic expression of methionine adenosyltransferase Iα (Mat1a) and methionine adenosyltransferase IIα (Mat2a). Hepatic S-adenosylmethionine (SAMe) was also analyzed. A total of 199 metabolites were profiled. Statistical analysis and pathway mapping indicated that the most significant metabolic perturbations induced by BPA were the increased biotin and riboflavin excretion, increased synthesis of methylated products, elevated purine nucleotide catabolism, and increased flux through the choline metabolism pathway. We found significantly higher mRNA and protein levels of Mat1a and Mat2a, and significantly higher SAMe levels in rat liver at both low and high doses. These two genes encode critical isoenzymes that catalyze the formation of SAMe, the principal biological methyl donor involved in the choline metabolism. In conclusion, an elevated choline metabolism is underlying the mechanism of highly methylated environment and related metabolic alterations caused by BPA. The data of BPA-elevated accepted biomarkers of injury indicate that BPA induces DNA methylation damage and broad protein degradation, and the increased deleterious metabolites in choline pathway may also be involved in the toxicity of BPA.

Effect of bisphenol A on pluripotency of mouse embryonic stem cells and differentiation capacity in mouse embryoid bodies.

Bisphenol A (BPA) poses potential risks to reproduction and development. However, the mechanism of BPAs effects on early embryonic development is still unknown. Embryonic stem cells (ESC) and embryoid bodies (EB) provide valuable in vitro models for testing the toxic effects of environmental chemicals in early embryogenesis. In this study, mouse embryonic stem cells (mESC) were acutely exposed to BPA for 24h, and general cytotoxicity and the effect of BPA on pluripotency were then evaluated. Meanwhile, mouse embryoid bodies (mEB) were exposed to BPA up to 6 days and their differentiation capacity was evaluated. In mESC and mEB, we found that BPA up-regulated pluripotency markers (Oct4, Sox2 and Nanog) at mRNA and/or protein levels. Moreover, BPA increased the mRNA levels of endodermal markers (Gata4,Sox17) and mesodermal markers (Sma,Desmin), and reduced the mRNA levels of ectodermal markers (Nestin,Fgf5) in mEB. Furthermore, microRNA(miR)-134, an expression inhibitor of pluripotency markers including Oct4, Sox2 and Nanog, was decreased both in BPA-treated mESC and mEB. These results firstly indicate that BPA may disturb pluripotency in mESC and differentiation of mEB, and may inhibit ectodermal lineage differentiation of mEB while miR-134 may play a key role underlying this effect.

Parental phenols exposure and spontaneous abortion in Chinese population residing in the middle and lower reaches of the Yangtze River

Widespread use of phenols has led to ubiquitous exposure to phenols. In experimental animals, phenols increased resorptions, reduced live litter size and fetal body weights. However, there are limited epidemiological evidences of the relationships between exposure to phenols and pregnancy outcomes. We evaluated the associations between parental urinary levels of various phenols and spontaneous abortion in a Chinese population residing in the middle and lower reaches of the Yangtze River. A case-control study was conducted that included 70 case couples with medically unexplained spontaneous abortion and 180 control couples who did not have a history of spontaneous abortion and had at least one living child. Both parental urinary phenols were measured by ultra-high performance liquid chromatography-tandem mass spectrometry including bisphenol A (BPA), benzophenone-3 (BP-3), 2,3,4-trichlorophenol (2,3,4-TCP), pentachlorophenol (PCP), 4-n-octylphenol (4-n-OP) and 4-n-nonylphenol (4-n-NP). Compared with the low exposure group, there was an increased risk of spontaneous abortion with high paternal urinary PCP concentration [odds ratio (OR)=2.09, 95% Confidence Interval (CI), 1.05-4.14], and maternal exposure to 4-n-OP and alkylphenol(s) also significantly increased the risk of spontaneous abortion (OR=2.21, 95% CI, 1.02-4.80; OR=2.81, 95% CI, 1.39-5.65, respectively). Our study firstly provides the evidence that paternal PCP exposure, maternal 4-n-OP and alkylphenol(s) exposure are associated with spontaneous abortion in humans.

Perfluorooctane Sulfonate Disturbs Nanog Expression through miR-490-3p in Mouse Embryonic Stem Cells

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.

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.

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).”