Retinoic acid exacerbates chlorpyrifos action in ensuing adipogenic differentiation of C3H10T½ cells in a GSK3β dependent pathway

The cell differentiation can be exploited as a paradigm to evaluate the effects of noxious chemicals, on human health, either alone or in combinations. In this regard, the effect of a known cell differentiation agent, retinoic acid (RA) was analyzed in the presence of a noxious chemical chlorpyrifos (CPF), an organophosphate (OP), the receptors of which have recently been localized to mesenchymal stem cells (MSCs). The observed imbalance of adipogenic to skeletal differentiation by CPF together with conundrum about adipogenic potential of RA prompted us to delineate their combinatorial effects on C(3)H(10)T½MSC-like undifferentiated cells. Based on MTT assay, the cellular viability was retained by CPF at concentrations ranging from 0.01–50μM, beyond which it caused cytotoxicity. These non-toxic concentrations also mildly interfered with adipogenesis of C(3)H(10)T½ cells following exposure to adipogenic cocktail. However, upon exposure to RA alone, these MSCs adopted elongated morphology and accumulated lipid vesicles, by day 20, as discerned by phase-contrast and transmission electron microscopy (TEM), in concert with enhanced Oil Red O stained cells. This effect got strongly augmented upon exposure to combination of CPF and RA in a dose-dependent manner. Simultaneous up-regulation in perilipin-1 (PLIN1) and adipsin (ADN) genes, additionally reiterated the adipogenic differentiation. Mechanistically, GSK3β pathway was found to be a major player, whereby inhibiting it with lithium chloride (LiCl) resulted in complete blockage of lipid accumulation, accompanied by complete down regulation of PLIN1 and ADN gene expression. In conclusion, these observations for the first time, lend evidence that exposure of CPF accompanied by RA directs commitment of C(3)H(10)T½ cells to adipogenic differentiation through a process involving a crosstalk at GSK3β signaling.