Long-term arsenic exposure impairs differentiation in mouse embryonal stem cells

Abstract

Arsenic is a contaminant found in many foods and drinking water. Exposure to arsenic during development can cause improper neuronal progenitor cell development, differentiation, and function, while in vitro studies have determined that acute arsenic exposure to stem and progenitor cells reduced their ability to differentiate. In the current study, P19 mouse embryonal stem cells were exposed continuously to 0.1-μM (7.5 ppb) arsenic for 32 weeks. A cell lineage array examining messenger RNA (mRNA) changes after 8 and 32 weeks of exposure showed that genes involved in pluripotency were increased, whereas those involved in differentiation were reduced. Therefore, temporal changes of select pluripotency and neuronal differentiation markers throughout the 32-week chronic arsenic exposure were investigated. Sox2 and Oct4 mRNA expression were increased by 1.9- to 2.5-fold in the arsenic-exposed cells, beginning at Week 12. Sox2 protein expression was similarly increased starting at Week 16 and remained elevated by 1.5-fold to sixfold. One target of Sox2 is N-cadherin, whose expression is a hallmark of epithelial-mesenchymal transitions (EMTs). Exposure to arsenic significantly increased N-cadherin protein levels beginning at Week 20, concurrent with increased grouping of N-cadherin positive cells at the perimeter of the embryoid body. Expression of Zeb1, which helps increase the expression of Sox2, was also increased started at Week 16. In contrast, Gdf3 mRNA expression was reduced by 3.4- to 7.2-fold beginning at Week 16, and expression of its target protein, phospho-Smad2/3, was also reduced. These results suggest that chronic, low-level arsenic exposure may delay neuronal differentiation and maintain pluripotency.

Keywords: Gdf3; N-cadherin; P19 cells; Sox2; Zeb1; arsenic; pluripotency; stem cell.

FIGURE 1

Morphology of Day 9 cells exposed to 0 or 0.1-μM arsenic. Representative images of Day 9 cells at Weeks 0, 16, 24, and 32 of exposure are shown. Arrows show elongating and differentiating cells

FIGURE 2

Acute arsenic exposure alters the expression of neural and oxidative stress-related transcripts. Levels of mRNA following a 9-day acute exposure to 0.1- and 0.5-μM arsenic exposure were quantified by qPCR for Oct4 (A), Sox2 (B), Ascl1 (C), Gdf3 (D), Dcx (E), and Gad2 (F). Data were normalized to the geometric mean of Gapdh and β2-microglobulin and is expressed as the average 2-ddCt for each group (n = 6 for controls and n = 3 for each exposure group). Statistical differences (*) were determined using analysis of variance followed by Bonferroni’s test (p ≤ 0.05)

FIGURE 3

Chronic exposure to arsenic alters the expression of pluripotency markers. Transcript levels from Day 9 differentiated cells after 0, 4, 12, 16, 24, and 28 weeks of continuous exposure to 0.1-μM arsenic were quantified by qPCR for Sox2 (A), Oct4 (B), and Gdf3 (C). At each time point, data were normalized to the geometric mean of Gapdh and β2-microglobulin using the ddCt method. Expression fold changes were compared with transcript levels at Week 0 (n = 3 flasks per exposure group). Statistical differences (*) were determined using Student’s t-test (p ≤ 0.05)

FIGURE 4

Sox2 expression is increased due to arsenic exposure. Representative images of Sox2 expression in Day 5 control and 0.1 μM arsenic-exposed embryoid bodies at Weeks 0, 16, 24, and 32 of exposure (A). Relative fluorescence was determined in ImageJ and is expressed as the integrated density per cell (n = 3 per exposure group). Statistical differences (*) were determined using Student’s t-test at p ≤ 0.05 (B). Immunoblotting of Sox2 and Gapdh protein expression in the Day 9 control and 0.1 μM arsenic-exposed cells (C). Protein levels were assessed by densitometry, normalized to Gapdh, and relative fold expression (n = 3 per exposure group) was assessed using the integrated density function in Bio-Rad Image Lab (D). Statistical differences (*) were determined using Student’s t-test (p ≤ 0.05)

FIGURE 5

Arsenic exposure results in increased expression and altered patterning of N-cadherin. (A) Representative images of N-cadherin expression in Day 5 control and 0.1 μM arsenic-exposed embryoid bodies at Weeks 0, 16, 20, and 28 of exposure. (B) Relative fluorescence was determined in ImageJ and is expressed as an integrated density value (IDV) (n = 3 per exposure group). Statistical differences (*) were determined using Student’s t-test at p ≤ 0.05

FIGURE 6

Chronic arsenic exposure does not alter E-cadherin expression. (A) Representative images of E-cadherin expression in Day 5 control and 0.1 μM arsenic-exposed embryoid bodies at Weeks 0, 16, 20, and 28 of exposure. (B) Relative fluorescence was determined in ImageJ and is expressed as the integrated density per cell (n = 3 per exposure group). Statistical differences (*) were determined using Student’s t-test at p ≤ 0.05

FIGURE 7

Arsenic impairs transcription of Zeb1. Transcript levels in Day 5 embryoid bodies after 0, 4, 8, 12, 16, 20, 24, and 28 weeks of continuous exposure to 0.1-μM arsenic were quantified by qPCR. At each time point, Zeb1 levels were normalized to the geometric mean of Gapdh and β2-microglobulin using the ddCt method. Expression fold changes were compared with transcript levels at Week 0 (n = 3 flasks per exposure group). Statistical differences (*) were determined using Student’s t-test (p ≤ 0.05)