Abstract
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 C3H10T½MSC-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 C3H10T½ 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 C3H10T½ cells to adipogenic differentiation through a process involving a crosstalk at GSK3β signaling.
Highlights
IntroductionConstant human exposure to noxious xenobiotics like pesticides is indispensable due to their widespread agricultural and domestic usage often leading to adverse health effects [1, 2]
The cells showed fibroblast-like appearance and remained adhered as flattened structures in control well (CT), following vehicle (DMSO) treatment and CPF treatment at concentrations 0.01, 0.1, 1, 10 and 50 μM
The observations of the present study lend evidence that retinoic acid (RA) directs the undifferentiated C3H10T1⁄2 MSClike cells to undergo adipogenic differentiation which gets accentuated in presence of CPF, an OP pesticide
Summary
Constant human exposure to noxious xenobiotics like pesticides is indispensable due to their widespread agricultural and domestic usage often leading to adverse health effects [1, 2]. Besides being prevalently used in agriculture, these pesticides find their use in household as pet shampoos, and in control of insects in houses and as lawn sprays [5, 6]. This suggests an unprecedented increase in the exposure of OP pesticides with parallel increase in the potential toxic effects on non-target organisms. We propose a cell-based test system for continuous, label-free monitoring of the effect of test substances on stem cell differentiation
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