Corexit-EC9527A Disrupts Retinol Signaling and Neuronal Differentiation in P19 Embryonal Pluripotent Cells

Abstract

Corexit-EC9500A and Corexit-EC9527A are two chemical dispersants that have been used to remediate the impact of the 2010 Deepwater Horizon oil spill. Both dispersants are composed primarily of organic solvents and surfactants and act by emulsifying the crude oil to facilitate biodegradation. The potential adverse effect of the Corexit chemicals on mammalian embryonic development remains largely unknown. Retinol (vitamin A) signaling, mediated by all-trans retinoic acid (RA), is essential for neural tube formation and the development of many organs in the embryo. The physiological levels of RA in cells and tissues are maintained by the retinol signaling pathway (RSP), which controls the biosynthesis of RA from dietary retinol and the catabolism of RA to polar metabolites for removal. RA is a potent activating ligand for the RAR/RXR nuclear receptors. Through RA and the receptors, the RSP modulates the expression of many developmental genes; interference with the RSP is potentially teratogenic. In this study the mouse P19 embryonal pluripotent cell, which contains a functional RSP, was used to evaluate the effects of the Corexit dispersants on retinol signaling and associated neuronal differentiation. The results showed that Corexit-EC9500A was more cytotoxic than Corexit-EC9527A to P19 cells. At non-cytotoxic doses, Corexit-EC9527A inhibited retinol-induced expression of the Hoxa1 gene, which encodes a transcription factor for the regulation of body patterning in the embryo. Such inhibition was seen in the retinol- and retinal- induced, but not RA-induced, Hoxa1 up-regulation, indicating that the Corexit chemicals primarily inhibit RA biosynthesis from retinal. In addition, Corexit-EC9527A suppressed retinol-induced P19 cell differentiation into neuronal cells, indicating potential neurotoxic effect of the chemicals under the tested conditions. The surfactant ingredient, dioctyl sodium sulfosuccinate (DOSS), may be a major contributor to the observed effect of Corexit-EC9527A in the cell.

Fig 1. The major regulatory steps in the retinol signaling pathway.

The enzymes that are predominantly expressed in P19 cells are listed in parentheses.

Fig 2. Cytotoxicity of Corexit-9500 and Corexit-9527.

P19 cells exposed to dispersants for 7 or 30 hours were measured for viability using the MTT assay. Relative Cell Viability was calculated by normalizing the MTT readings relative to the control cells (no Corexit exposure), which were set to be 1. LC₅₀ value for Corexit-9500 was derived from the titration curve using the Graphpad Prism software. Values are mean ± s.e.m.; n = 3.

Fig 3. The effect of Corexit-9527 on retinoid-induced Hoxa1 gene expression in P19 cells.

(A) Corexit-9527 inhibited ROH-induced Hoxa1 expression. P19 cells were exposed to the indicated concentrations of Corexit-9527 for 1 or 24 hr followed by 0.3 μM ROH for additional 6 hr. The columns represent the relative Hoxa1 expression levels (left axis) that are normalized to the control (no ROH induction). The bars show cell viability (right axis) at the tested doses of Corexit. Values are mean ± s.e.m.; n = 3. (B) The effect of Corexit-9527 on ROH-, RAL- or RA-induced Hoxa1 expression. P19 cells were exposed to Corexit-9527 for 1 hr and then were induced by indicated retinoids for additional 6 hr. For each retinoid, the Hoxa1 expression levels in the cells that were induced by the cognate retinoid in the absence of Corexit-9527 were set to be 1. Values are mean ± s.e.m.; ***, p < 0.001, n.s., not significant, determined by Student’s t-test using two-tailed distribution and unequal variance; n = 4.

Fig 4. Effect of Corexit-9527 on ROH-induced P19 cell neuronal differentiation.

(A) A simplified workflow for the P19 cell embryoid body formation and neuronal differentiation assays. (B) Phenotypical characteristics of the 3-day EBs and the 4-day EBs-derived cells. Yellow arrows indicate examples of neurons. (C) Expression of several neuronal marker genes in the P19-derived cells post EBs formation and differentiation procedures. The control cells were mock-treated with DMSO vehicles only throughout the experimental procedures. Values are mean ± s.e.m.; n = 4. **, p < 0.01, ***, p < 0.001.

Fig 5. The effect of DOSS on ROH-induced Hoxa1 expression.

P19 cells were exposed to DOSS at 31 or 62 ppm for 24 hr and then induced by 0.33 μM ROH for additional 6 hr. Values are mean ± s.e.m.; ***p < 0.001, n.s., not significant, determined by Student’s t-test using two-tailed distribution and unequal variance, compared to the control (left column); n = 4.