Effects of a Chronic 0.1 μM Arsenic Exposure on P19 Mouse Embryonic Stem Cell Differentiation and Pluripotency

Globally, at least 140 million people are exposed to levels of arsenic greater than the World Health Organization recommended standard of 10 ug/L (10 ppb) through their drinking water and food. Epidemiological studies have correlated arsenic exposure during embryonic and fetal development with reduced birth weight, as well as muscular and neurodevelopmental deficits. The goal of the current study was to determine the mechanisms by which a chronic, low-level arsenic exposure impairs cellular development and differentiation. This was achieved by exposing P19 mouse embryonic stem cells (mESC’s) to 0.1 μM arsenic (7.5 ppb), a concentration lower than the current drinking water standard, continuously for 32 weeks. The P19 stem cell line is commonly used in developmental studies as it can be induced to form cells of all three germ layers. During the current study, a subset of cells were induced to differentiate every 4 weeks of the 32-week arsenic exposure, to assess whether arsenic impaired their ability to form mature cell lineages. Differentiating cells were collected after 5 and 9 days to examine transcript and protein expression. Previous studies of P19 cells exposed to higher arsenic concentrations (0.5 – 1 uM) reported robust differences in cell morphology, as well as reductions of transcription factors involved in neuronal differentiation, and increases in transcription factors crucial for maintaining pluripotency. In this study of 0.1 μM arsenic, only minor and inconsistent changes in morphology were observed to result from exposure. However, arsenic induced consistent overexpression of the pluripotency markers Oct4 and Sox2 by 1.4- to 1.8-fold beginning at week 8, while Sox2 protein expression was increased by 1.4 fold starting at week 16. Elevated expression of pluripotency-associated transcription factors is commonly observed in ESC’s, but also in cancer stem cells (CSC’s). Another feature of CSC’s are protein expression patterns consistent with epithelial-mesenchymal transitions (EMT). A prevailing hallmark of EMT is increased expression of the cell-to-cell adhesion molecule N-cadherin, which is known to be induced by Sox2. Beginning at week 20, N-cadherin levels remained significantly increased by 1.3- to 1.9 fold through the 32-week exposure. Interestingly, as markers of pluripotency and EMT increased, the expression of the doublecortin (DCX) gene, a microtubule-associated protein of immature neurons, was consistently reduced throughout the exposure with significance of up to 2-fold decreases shown at weeks 8 and 20. However, transcript levels of Nrf2 and Hmox1, markers of oxidative stress, and MyoD, a marker of myogenesis, appeared to be unaltered throughout the exposure. In conclusion, this study may have public health ramifications as it was shown arsenic exposure at a level lower than the current drinking water standard maintained pluripotency of ESC’s and reduced neural differentiation. An EMT-like mechanism may be used to impair cellular differentiation as demonstrated by increased N-cadherin expression. Intriguingly, the observed lack of oxidative stress leaves the possibility of another mechanism, such as epigenetics, to be responsible for this reduced differentiation