Assessing developmental toxicity with the boundary of primary cell cultures


In the past 20 years more than 30 diverse platforms has been recommended for assessing developmental toxicity (DT) with the boundary of primary cell cultures, non mammalian embryos, and mammalian embryos or primordial. Invention of embryonic stem cells (ESC) explained the usage of blastocyst derived mouse ESC for in-vitro embryotoxicity testing Hofer, T. et al. (2004)(2). The rat primary embryonic cells based micro mass test was replaced with the differentiating ESC for the prediction of teratogenicity of test compounds Newall, D. R. et al. (1994)(3). In the development of mammalian in-vitro system for teratogenicity testing system, cytotoxicity end point was evaluated with dimethylthiazol- diphenyl tetrazolinm bromide (MTT) assay and it was concluded that ESC were more sensitive than fibroblast or differentiated cells in response to xenobiotics Laschinski, G. et al. (1991)(4). In terms of development of in vitro embryotoxicity system mouse ESC were differentiated in presence of toxicant such as retinoic acid which augmented skeletal muscle differentiation Heuer, J. et al. (1993)(10). However, when embryonic stem cell test (EST) method introduced a combination of cytotoxicity (IC50 for ESC and 3T3), inhibition concentration for differentiation (ID50) and inhibition of the differentiation of ES cells into contracting myocardial with linear biostatistical prediction model revolutionized the embryotoxicity screening Scholz, G. et al. (1999b), Scholz, G. et al. (1999a)(5, 8). In the further improvement of EST molecular end points such as FACS were introduced to reduce the time consumption and high-throughput screening. Seiler, A. et al. (2004)(6)

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To improve the developmental toxicity testing, human embryonic stem cells were introduced for the consideration of safety of patients and consumers in terms of high efficiency in in-vitro toxicological tests and more importantly can avoid the inter species variability. As a first step early developmental markers and cardiac markers were used to determine the toxicity of know toxicant such as retinoic acid and 5 fluro uracil. Adler, S. et al. (2008)(13) But cardiac specific differentiation and analysis of few markers alone not sufficient to make the concrete decision for toxicity determination. Our recent publication regarding multilineage differentiation provided the evidence for the expression of endoderm, ectoderm and mesoderm lineage markers using high sensitive transcriptional profiling and interference of these germ layer markers. These findings will provide substantial evidence to classify the compounds for developmental toxicity Jagtap, S. et al. (2011).

However, in the context of developmental toxicity and species specificity the role of thalidomide is indiscriminate. Thalidomide, a hypnosedative drug causes stunted limb growth (dysmelia) during human embryogenesis Sauer, H. et al. (2000b) and is now used to treat leprosy and multiple myeloma Knobloch, J. et al. (2007b). Although the embropathy of thalidomide has been in focus for over three decades,Stephens, T. D. (1988) its recent use in the inhibition of angiogenesis and immunomodulatory behavior Singhal, S. et al. (1999b) has gained importance Sauer, H. et al. (2000a);D'Amato, R. J. et al. (1994b). It was clinically proven that thalidomide reduces the TNF- and interleukin-12 in patients with chronic Crohn’s disease Bauditz, J. et al. (2002). Anti angiogenic property of thalidomide is another possibility for the teratogenicity and was reported in bFGF induced-rabbit cornea micro pocket assay D'Amato, R. J. et al. (1994a). Clinical evidence also showed the role of thalidomide in advanced and refractory multiple myeloma due to its anti-angiogenic property. Recently it was deciphered that cereblon was identified as molecular target for thalidomide which forms an E3 ubiquitin ligase complex with DDB1 and Cul4A that is important for limb out growth Ito, T. et al. (2010)(20). Although many molecular mechanisms such as oxidative DNA damage, angiogenesis inhibitor, IGF-1, and FGF-2 antagonist proposed for the teratogenecity Stephens, T. D. et al. (2000a)(33), anti-angiogenic property of thalidomide regained the importance of thalidomide in the treatment of refractory multiple myeloma and significant molecule in pharmaceutical industry Zeng, X. et al. (2006), Singhal, S. et al. (1999a)(12, 21). The original application such as sedative effect of thalidomide was found that enantiomer specific and it was proved that enantiomer R or (+) has exhibited positive sedative effect compared to S or (-) (12). In vivo teratogenecity of thalidomide derivative EM12 was conducted with non human primate callithrix jacchus and found 30ug/kg was effective to induce severe skeletal abnormalities and 10ug/kg was considered no observed adverse effect level (NOAEL), this was the lowest dose reported ever for teratogenecity (7). The limb defects were observed in cynomolgus monkeys in response to 15-20 mg of thalidomide treatment during 26-28 days of gestation and transcriptional studies of 6hr treatment of thalidomide down regulated vasculature development gene ontology Ema, M. et al. (2010)(18). The congenital malformations of thalidomide were found prominently in rabbit but where as in rat and hamsters fetal changes were not observed significantly and in mice no fetal malformations were observed. Among the many species studied rabbit is more optimal to study thalidomide effect due to anatomical differences of placentas and its membrane permeability’s Schumacher, H. et al. (1968), Teo, S. K. et al. (2004a), FRATTA, I. D. et al. (1965)(15, 23, and 24). Although many in vivo studies conducted with different concentrations of thalidomide, with best of our knowledge no dose response in vitro studies were performed with either primary cell line or ESCs Ema, M., Ise, R., Kato, H., Oneda, S., Hirose, A., Hirata-Koizumi, M., Singh, A. V., Knudsen, T. B., and Ihara, T. (2010), DELAHUNT, C. S. et al. (1964) (7, 18, and 22).

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Our study was commenced to analyze the dose response assessment of thalidomide mediated DT on multilineage differentiated hESC based on the sensitive transcriptional profiling. Also the effect of thalidomide on multilineage differentiated mESCs and hESC and its cross species relationship of gene expression responses correspond to a powerful approach to study the molecular markers for thalidomide in mouse and human embryonic development. The dose response assessment of thalidomide will reveal the lowest and maximal toxicity responses during hESC embryonic development. A comparative analysis of mouse and human EB development in presence of thalidomide reveals the similarity and species specificity of thalidomide mediated teratogenecity in mouse and human. Finally the sensitive common markers for 14 day old EBs in H9 and CGR8 differentiation and markers for the respective treatment of thalidomide in embryonic development will be non-species specific in embryonic development. The residual species specific markers recapitulate the species specificity of thalidomide observed in in-vivo with human and mouse embryonic development.