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Silver Nanoparticles Compromise Female Embryonic Stem Cell Differentiation through Disturbing X Chromosome Inactivation Jie Zhang, Yongjiu Chen, Ming Gao, Zhe Wang, Rui Liu, Tian Xia, and Sijin Liu ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b08604 • Publication Date (Web): 16 Jan 2019 Downloaded from http://pubs.acs.org on January 17, 2019
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Silver Nanoparticles Compromise Female Embryonic Stem Cell Differentiation through Disturbing X Chromosome Inactivation Jie Zhang1,2, Yongjiu Chen1,2, Ming Gao1,2, Zhe Wang3, Rui Liu1,2, Tian Xia1,4, Sijin Liu1,2,*
1 State
Key Laboratory of Environmental Chemistry and Ecotoxicology, Research
Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China. 2
3
University of Chinese Academy of Sciences, Beijing 100049, China.
School of Public Health, Xinxiang Medical University, Xinxiang, Henan Province 453003, China.
4
Division of NanoMedicine, Department of Medicine, University of California Los Angeles, Los Angeles, CA 90095, United States.
*: correspondence to:
[email protected].
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ABSTRACT
The widespread use of silver nanoparticles (AgNPs) has raised substantial health risks to human beings. Despite a wealth of progresses on toxicity studies, the understandings of the adverse effects on fetuses, embryos and early-stage cells are still rather limited, particularly under low-dose exposure settings. Moreover, nearly all previous studies ascribed AgNP-induced toxic effects to oxidative stress. Differently, we here unearthed a mechanism, namely interruption of X chromosome inactivation (XCI) in female mouse embryonic stem cells (mESCs). Albeit no observable cytotoxicity, significant differentiation retardation was found in female mESCs upon low-dose AgNP exposure. Mechanistic investigations uncovered expedited inactivation for the inactive X chromosome (Xi) and attenuated maintenance of active X chromosome (Xa) state during mESC differentiation upon the challenge of low-dose AgNPs, indicative of disordered XCI. Thereby, a few X-linked genes (which are closely involved in orchestrating ESC differentiation) were found to be repressed, partially attributable to reinforced enrichment of histone modification (e.g. histone 3 lysine 27 trimethylation, H3K27me3) on their promoter regions, as the result of disordered XCI. In stark contrast to female mESCs, no impairment of differentiation was observed in male mESCs under low-dose AgNP exposure. All considered, our data unearthed that AgNPs at low concentrations compromised the differentiation program of female mESCs through disturbing XCI. Thus, this work would provide a model for the type of studies necessary to advance the understandings on AgNP-induced developmental toxicity. 2
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KEYWORDS: silver nanoparticles; embryonic stem cells; differentiation; X chromosome inactivation; imbalance.
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Of the nanomaterials currently produced and merchandised, silver nanoparticles (AgNPs) represent the largest family of materials used in personal care, food and biomedical products due to their excellent anti-microbial activity.1 According to a report from the Nanotechnology Consumer Product Inventory, 24% of the nanoenabled commercial products in the market are advertised to contain AgNPs.1 Under this context, widespread use of AgNPs would yield inevitably cumulative exposure to the environment and human beings.2 Till now, internal exposure of AgNPs through various routes has been reported in human blood and urine,3-6 suggesting unavoidable health risks towards humans.7, 8 Based on animal studies, especially mammals, a variety of adverse outcomes have been documented, including hepatoxicity, neurotoxicity, and even reproductive and developmental toxicity.9 Regarding to the latter, a few toxic features have been reported, such as translocation of AgNPs across placenta, impaired testicular structure, compromised sperm/ovarian functionalities, and embryonic development retardation.10, 11 In terms of susceptibility, developing fetal tissues and embryos are more vulnerable to invading foreign particles and chemicals than adults,12, 13
and thus more profound detrimental effects are expected to be incurred.14 However,
the current understandings of the adverse effects induced by AgNPs on fetuses, embryos and early-stage cells are still rather limited, particularly under low-dose exposure, and the underlying mechanisms responsible for their plausible reproductive and developmental toxicity have not been explicitly illustrated so far.
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Relative to mechanistic investigations into AgNP-induced toxicity in adulthood, e.g. hepatoxicity and neurotoxicity, it is fairly difficult to look into the molecular mechanisms underlying the reproductive and developmental toxicity due to the small size of developing embryos from pregnant mice, limited materials from fetal organs and the lack of adequate ex vivo models.13, 15 To circumvent these obstacles, embryonic stem cells (ESCs), derived from the blastocyst stage of early mammalian embryos, represent an ideal in vitro model for the purpose of mechanistic studies at early developmental stages of embryos.13, 16 Different from adult cells, ESCs harbor abilities to propagate for self-renewal and differentiate into other cell types.17 The pluripotency and differentiation processes of ESCs are retained through a complex molecular machinery network; however, this network could be disturbed upon the intrusion of engineered nanoparticles, as described by previous studies.18-20 Although these studies revealed cytotoxicity, DNA damage, and impairment of self-renewal of ESCs upon AgNPs, the molecular mechanisms were mostly attributed to oxidative stress and reactive oxygen species (ROS) production. Additionally, most previous studies used high concentrations that reflect little relevance to the realistic health risks (as summarized in Table S1). In light of the importance of epigenetics in modulating ESC proliferation and differentiation fate, one would expect to see the entanglement of epigenetic mechanisms in AgNP-induced biological effects on ESCs.18 Of the epigenetic machineries, X chromosome inactivation (XCI) is of proven importance in governing 5
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ESC pluripotency and differentiation through finetuning the gene expression dose for those linked to X chromosome.21, 22 In other words, the dosage of genes located in X chromosome contributes to orchestrating the balance between self-renewal and differentiation of ESCs through silencing one copy of X chromosome, and impairment of this regulation would lead to disordered ESC differentiation.23 Nonetheless, whether XCI is affected in ESCs upon treatment of nanomaterials is still a mystery, and whether XCI is attributable to AgNP-induced impairments to ESCs remains to be determined. To this end, the primary objective of the current study is to investigate impacts of AgNPs on ESCs’ abilities in self-renewal and differentiation specifically under lowdose exposure, and to elucidate the entanglement of XCI within the complex molecular network. Our results collectively unearthed the significant alterations to XCI in ESCs upon low-dose AgNP treatment and uncovered the involvement of disordered XCI in repressing ESC differentiation. RESULTS AND DISCUSSION Characterization of AgNPs. As shown in Figure 1A, transmission electronic microscope (TEM) was used to visualize the morphology of polyvinyl pyrrolidone (PVP)-coated AgNPs. Our AgNPs displayed a spherical shape with an average size around 20 nm (Figure 1A and B), which could easily pass the placental barrier, based on previous observations.12, 24, 25 The hydrodynamic diameter of AgNPs was measured to be approximately 59 nm in 6
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water (Figure 1C). To better mimic the condition in culture medium, we measured the hydrodynamic diameter of AgNPs in medium with 15% fetal bovine serum (FBS) and found a reduction of hydrodynamic diameter in medium (~44 nm) compared to that in water (Figure 1C, P
