Lead Exposure Disrupts Global DNA Methylation in Human Embryonic Stem Cells and Alters Their Neuronal Differentiation

Abstract

Exposure to lead (Pb) during childhood can result in learning disabilities and behavioral problems. Although described in animal models, whether Pb exposure also alters neuronal differentiation in the developing brains of exposed children is unknown. Here, we investigated the effects of physiologically relevant concentrations of Pb (from 0.4 to 1.9μM) on the capacity of human embryonic stem cells (hESCs) to progress to a neuronal fate. We found that neither acute nor chronic exposure to Pb prevented hESCs from generating neural progenitor cells (NPCs). NPCs derived from hESCs chronically exposed to 1.9μM Pb throughout the neural differentiation process generated 2.5 times more TUJ1-positive neurons than those derived from control hESCs. Pb exposure of hESCs during the stage of neural rosette formation resulted in a significant decrease in the expression levels of the neural marker genes PAX6 and MSI1. Furthermore, the resulting NPCs differentiated into neurons with shorter neurites and less branching than control neurons, as assessed by Sholl analysis. DNA methylation studies of control, acutely treated hESCs and NPCs derived from chronically exposed hESCs using the Illumina HumanMethylation450 BeadChip demonstrated that Pb exposure induced changes in the methylation status of genes involved in neurogenetic signaling pathways. In summary, our study shows that exposure to Pb subtly alters the neuronal differentiation of exposed hESCs and that these changes could be partly mediated by modifications in the DNA methylation status of genes crucial to brain development.

Lead exposure induces changes in 5-hydroxymethylcytosine clusters in CpG islands in human embryonic stem cells and umbilical cord blood

“…Since the WA09 (H9) stem cells have an XX karyotype (i.e., they are from a female embryo), we searched for a potential overlap between female-specific DhMRs identified in the UCB DNA and those detected in hESC DNA after acute exposure to Pb, but found none. Based on this observation, we speculate: 1) prenatal exposure is more similar to a chronic rather than an acute Pb exposure 24 ; and 2) contrary to hESCs, UCB is a mixture of various immune cells and CD34-positive haematopoietic cells, which may have different epigenetic changes and survival capabilities in reponse to Pb exposure.…”

“…hESC culture and Pb exposure The human ESC line WA09 (H9) (Thomson et al, 1998) was obtained from the WiCell Research Institute (Madison, WI, USA) and maintained in a humidified incubator at 37 C with 5% CO 2, as previously described (Senut et al, 2014). Briefly, undifferentiated hESCs (passages 26-39) were cultured in DMEM/F12 supplemented with knockout serum replacement, nonessential amino acids, penicillin/streptomycin, L-Glutamine, 2-mercaptoethanol, and human basic fibroblast growth factor (Life Technologies) on a feeder layer of irradiated mouse embryonic fibroblasts (Globalstem).…”

“…Stock solutions (100-fold concentrated) of Pb acetate (Pb(C 2 H 3 O 2 ) 2 ) (Sigma-Aldrich) were prepared in sterile distilled water. Two physiologically relevant concentrations of Pb acetate chosen on the basis of our previous work 24 were tested in this study: 0.8 mM (16 mg/dL) and 1.5 mM (32 mg/dL). Distilled water was used as a vehicle control.…”

“…22 These Pb-targeted co-regulated regions can influence a variety of biological processes including binding of transcription factors and may serve as putative epigenetic biomarkers for Pb exposure. Although several studies support the role of Pb exposure in altering the DNA methylation profile in humans, 7,23,24 little is known about the effects of Pb exposure on the DNA hydroxymethylation profile.…”

Loss of p15INK4bExpression in Colorectal Cancer is Linked to Ethnic Origin

“…These methylated genes were potentially involved in early stages of adenoma formation and the authors speculated that the observed epigenetic alterations in these markers may be caused in part by the effects of smoking and/or age (Paun et al, 2010). Exposures to environmental toxicants and toxins might cause epigenetic changes (O'Hagan, 2013;Coppede et al, 2014;Senut et al, 2014) and it is clear that many different adverse environmental factors are likely to exist in the East compared to the West as discussed previously (Nieminen et al, 2012). Our data, together with the available literature (Belinsky et al, 2004;Marsit et al, 2006) suggest a link between environmental exposures, epigenetic changes and experimental models and large series of clinical samples.…”