The chick cardiomyocyte micromass system and stem cell differentiation along specific pathways : prediction of embryotoxic effects and their mechanism

Malformations in humans at birth have been recorded since ancient times. These malformations are anatomical or physiological anomalies present at the time of birth that may be caused by genetic or environmental factors or a combination of both. The pathogenesis is only known in 10%, of which 1% or less are caused by drugs and medications. Certain disease states, like maternal epilepsy and depression during gestation itself, contribute to abnormal development. Further, this dilemma is augmented by the use of medications during pregnancy. The antiepileptic (AEDs) and antidepressant drugs (ADPs) with a history of producing malformed neonates are mostly classified as moderate teratogens. This study was designed to evaluate teratogenic potential at the cellular and molecular levels of AEDs and ADPs on cardiomyocytes at different stages of development and the neural stem cell derived neurons using in vitro systems. In the micromass system (MM), five day old embryonic chick cardiomyocytes were cultured to form beating foci, while embryonic stem cell were differentiated into contracting cardiomyocytes (ESDC) using the hanging drop method. In a third in vitro system early chick Neural Stem Cells (NSC) were diverted to a neuronal lineage. Drug toxic effects were estimated on cultured cell viability and protein content. The effects on gap junctions (Cx43) in cardiomyocytes and neurofilament (NF) in NSC were also evaluated because of their important role in cell differentiation and regulation. Oxidative stress, being the potential source of xenobiotic toxicity induction, was also analysed and toxic effects were counteracted using antioxidants and other molecules. In AEDs, valproic acid (VPA) mainly targeted the cardiomyocyte differentiation and contractile activity with reduced Cx43 turnover. In NSC the VPA effects were different and it did not inhibit the neuronal differentiation. With carbamazepine (CBZ) the low doses showed no effect on NSC compared to high doses. In ESDC, the contractile activity stops at a 200µM dose with reduced cell viability and proliferation. Cx43 phosphorylation was reduced after CBZ treatment which might have affected the contractile activity. An increase ROS production with CBZ treatment was recorded, which was protected either by the addition of Ascorbic acid (AA) or superoxide dismutase (SOD). The other AEDs, Phenytoin (PHT) and Primidone (PRM), mainly affected the cardiomyocyte contractile activity with some chronic exposure effects. In ADP, bupropion (BPN) severely affects cell proliferation in all systems. The NF-L was not statistically reduced in neurons but Cx43 expression in cardiomyocytes declined which might result in reduced contraction. The other ADP, lithium carbonate showed developmental stage dependent effect on cardiogenesis, where contractile activity ceased completely at higher dose in the ESDC with increased cell proliferation. Lithium mimics the Wnt/β-catenin pathway and also inhibits the PI cycle, effects which were reversed by the addition of myo-inositol in the ESDC system. In NSC the lithium showed no significant inhibitory effects on neural differentiation at and above drug serum therapeutic concentrations. The active constituents of the herbal antidepressant drug St. John’s wort, hypericin and hyperforin, showed synergistic inhibition of contractile activity with reduced proliferation at higher doses in the MM system. Drug interference at the molecular level during development may induce modification at the gene and protein levels with altered signalling. The tissue specific effects depend on the drug mechanism, while increased oxidative stress in part has a contribution in initiating the embryopathies. By identifying the exact mechanism of toxicity induction, the molecular mechanism can be protected against and thus abnormal development be avoided