Transcriptomics in developmental toxicity testing
H. M. Bolt11Leibniz Institut für Arbeitsforschung an der TU Dortmund, Leibniz Research Centre for Working Environment and Human Factors (IfADo), Ardeystrasse 67, 44139 Dortmund, Germany
EXCLI J 2013;12:Doc1027
Reproductive toxicity testing is one of the most complex, expensive and labor intensive fields of toxicology (Leist et al., 2013; Wobus and Löser, 2011; Hengstler, 2011; Krause et al., 2013). The catastrophic consequences of thalidomide-induced teratogenesis (Schmahl et al., 1996; Sterz et al., 1987) drastically demonstrate the fundamental importance of reliable developmental toxicity tests for human safety (van Thriel and Stewart, 2012; van Thriel et al., 2012; Frimat et al., 2010; Kadereit et al., 2012; Marques et al., 2012; Duydu et al., 2011). Currently, large efforts are undertaken to establish in vitro test systems of developmental toxicity (Krug et al., 2013; Strikwold et al., 2013; Seiler et al., 2011; Bolt, 2013). Recently, human embryonic stem cell based in vitro test systems have been established that recapitulate critical periods of human early development (Krug et al., 2013; Zimmer et al., 2011; 2012). During this differentiation period the differentiating stem cells are exposed to test compounds to study their influence on genome-wide expression patterns. Evaluation of the deregulated genes is usually based on methods of pattern analysis and identification of overrepresented motifs which initially has been introduced for characterization of tumor tissue (Kammers et al., 2011; Lohr et al., 2012; Botling et al., 2013; Schmidt et al., 2008, 2012; Cadenas et al., 2010). These studies have clearly shown that compounds known to induce developmental toxicity cause different alterations in gene expression than negative control compounds (Krug et al., 2013; Krause et al., 2013). Despite of this success stem cell based in vitro studies are still not broadly applied in routine toxicity testing. The majority of currently published studies are still performed in vivo (e.g. Gao et al., 2012; Saegusa et al., 2012; Ogawa et al., 2012; Romano et al., 2012; Lim et al. 2007; Burns and Korack, 2012; Shiraki et al., 2012; Balansky et al., 2012). Of course in vitro systems still have the limitation that it is difficult to derive NOAELS (Godoy et al., 2013). Although currently large efforts are undertaken to define in vivo relevant concentrations for in vitro testing (Mielke et al., 2011) and to correlate in vitro and in vivo data (Heise et al., 2012; Schug et al., 2013) the use of in vitro systems in the risk evaluation process is still controversial. Their application for harzard identification and to filter problematic compounds is more generally accepted. Although the recently published transcriptomic studies in developing stem cells represent a critical progress they still leave some important questions open:
- How are the compound induced gene expression alterations linked to adverse effects? Which expression responses represent reversible 'harmless' efforts of the cells to reestablish their equilibrium? Which genes, in contrast, indicate mechanisms leading to reversible consequences?
- What is the optimal concentration range for transcriptomics studies? Is it acceptable to use the EC10 as practiced in most studies? Or do already slightly cytotoxic concentrations induce cell death associated expression signatures which dilute the specific sigals?
- Do differentiating embryonic stem cells in vitro show waves of development with susceptible periods similar to the in vivo situation?
Answers to these critical questions would certainly improve the general acceptance of the recently established FP7 ESNATS in vitro test systems (Bolt, 2013; Leist et al., 2013) in developmental toxicity.
1. Balansky R, D'Agostini F, Micale RT, La Maestra S, Steele VE, De Flora S. Dose-related cytogenetic damage in pulmonary alveolar macrophages from mice exposed to cigarette smoke early in life. Arch Toxicol. 2012;86:509-16.
2. Bolt HM. Developmental neurotoxicity testing with human embryonic stem cell-derived in vitro systems: the novel FP7 ESNATS tests are available. Arch Toxicol. 2013;87:5-6.
3. Botling J, Edlund K, Lohr M, Hellwig B, Holmberg L, Lambe M, et al. Biomarker discovery in non-small cell lung cancer: integrating gene expression profiling, meta-analysis, and tissue microarray validation. Clin Cancer Res. 2013;19:194-204.
4. Burns KA, Korach KS. Estrogen receptors and human disease: an update. Arch Toxicol. 2012;86:1491-504.
5. Cadenas C, Franckenstein D, Schmidt M, Gehrmann M, Hermes M, Geppert B, et al. Role of thioredoxin reductase 1 and thioredoxin interacting protein in prognosis of breast cancer. Breast Cancer Res. 2010;12:R44.
6. Duydu Y, Başaran N, Üstündağ A, Aydin S, Ündeğer Ü, Ataman OY, et al. Reproductive toxicity parameters and biological monitoring in occupationally and environmentally boron-exposed persons in Bandirma, Turkey. Arch Toxicol. 2011;85:589-600.
7. Frimat JP, Sisnaiske J, Subbiah S, Menne H, Godoy P, Lampen P, et al. The network formation assay: a spatially standardized neurite outgrowth analytical display for neurotoxicity screening. Lab Chip. 2010;10:701-9.
8. Gao X, Wang Q, Wang J, Wang C, Lu L, Gao R, et al. Expression of calmodulin in germ cells is associated with fenvalerate-induced male reproductive toxicity. Arch Toxicol. 2012;86:1443-51.
9. Godoy P, Hewitt NJ, Albrecht U, Andersen ME, Ansari N, Bhattacharya S, et al. Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME. Arch Toxicol. 2013;87:1315-530.
10. Heise T, Schug M, Storm D, Ellinger-Ziegelbauer H, Ahr HJ, Hellwig B, et al. In vitro - in vivo correlation of gene expression alterations induced by liver carcinogens. Curr Med Chem. 2012;19:1721-30.
11. Hengstler JG. Cutting-edge topics in toxicology. EXCLI J. 2011;10:117-9.
12. Kadereit S, Zimmer B, van Thriel C, Hengstler JG, Leist M. Compound selection for in vitro modeling of developmental neurotoxicity. Front Biosci (Landmark Ed). 2012;17:2442-60.
13. Kammers K, Lang M, Hengstler JG, Schmidt M, Rahnenführer J. Survival models with preclustered gene groups as covariates. BMC Bioinformatics. 2011;12:478.
14. Krause KH, van Thriel C, De Sousa PA, Leist M, Hengstler JG. Monocrotophos in Gandaman village: India school lunch deaths and need for improved toxicity testing. Arch Toxicol. 2013;87:1877-81.
15. Krug AK, Balmer NV, Matt F, Schönenberger F, Merhof D, Leist M. Evaluation of a human neurite growth assay as specific screen for developmental neurotoxicants. Arch Toxicol. 2013;87:2215-31.
16. Leist M, Ringwald A, Kolde R, Bremer S, van Thriel C, Krause KH, et al. Test systems of developmental toxicity: state-of-the art and future perspectives. Arch Toxicol. 2013;87:2037-42.
17. Lim J, DeWitt JC, Sanders RA, Watkins JB 3rd, Henshel DS. Suppression of endogenous antioxidant enzymes by 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced oxidative stress in chicken liver during development. Arch Environ Contam Toxicol. 2007;52:590-5.
18. Lohr M, Köllmann C, Freis E, Hellwig B, Hengstler JG, Ickstadt K, et al. Optimal strategies for sequential validation of significant features from high-dimensional genomic data. J Toxicol Environ Health A. 2012;75:447-60.
19. Marques RC, Dórea JG, Leão RS, Dos Santos VG, Bueno L, Marques RC, et al. Role of methylmercury exposure (from fish consumption) on growth and neurodevelopment of children under 5 years of age living in a transitioning (tin-mining) area of the western Amazon, Brazil. Arch Environ Contam Toxicol. 2012;62:341-50.
20. Mielke H, Anger LT, Schug M, Hengstler JG, Stahlmann R, Gundert-Remy U. A physiologically based toxicokinetic modelling approach to predict relevant concentrations for in vitro testing. Arch Toxicol. 2011;85:555-63.
21. Ogawa B, Wang L, Ohishi T, Taniai E, Akane H, Suzuki K, et al. Reversible aberration of neurogenesis targeting late-stage progenitor cells in the hippocampal dentate gyrus of rat offspring after maternal exposure to acrylamide. Arch Toxicol. 2012;86:779-90.
22. Romano MA, Romano RM, Santos LD, Wisniewski P, Campos DA, de Souza PB, et al. Glyphosate impairs male offspring reproductive development by disrupting gonadotropin expression. Arch Toxicol. 2012;86:663-73.
23. Saegusa Y, Fujimoto H, Woo GH, Ohishi T, Wang L, Mitsumori K, et al. Transient aberration of neuronal development in the hippocampal dentate gyrus after developmental exposure to brominated flame retardants in rats. Arch Toxicol. 2012;86:1431-42.
24. Schmahl HJ, Dencker L, Plum C, Chahoud I, Nau H. Stereoselective distribution of the teratogenic thalidomide analogue EM12 in the early embryo of marmoset monkey, Wistar rat and NMRI mouse. Arch Toxicol. 1996;70:749-56.
25. Schmidt M, Böhm D, von Törne C, Steiner E, Puhl A, Pilch H, et al. The humoral immune system has a key prognostic impact in node-negative breast cancer. Cancer Res. 2008;68:5405-13.
26. Schmidt M, Hellwig B, Hammad S, Othman A, Lohr M, Chen Z, et al. A comprehensive analysis of human gene expression profiles identifies stromal immunoglobulin κ C as a compatible prognostic marker in human solid tumors. Clin Cancer Res. 2012;18:2695-703.
27. Schug M, Stöber R, Heise T, Mielke H, Gundert-Remy U, Godoy P, et al. Pharmacokinetics explain in vivo/in vitro discrepancies of carcinogen-induced gene expression alterations in rat liver and cultivated hepatocytes. Arch Toxicol. 2013;87:337-45.
28. Seiler A, Oelgeschläger M, Liebsch M, Pirow R, Riebeling C, Tralau T, et al. Developmental toxicity testing in the 21st century: the sword of Damocles shattered by embryonic stem cell assays? Arch Toxicol. 2011;85:1361-72.
29. Shiraki A, Akane H, Ohishi T, Wang L, Morita R, Suzuki K, et al. Similar distribution changes of GABAergic interneuron subpopulations in contrast to the different impact on neurogenesis between developmental and adult-stage hypothyroidism in the hippocampal dentate gyrus in rats. Arch Toxicol. 2012;86:1559-69.
30. Sterz H, Nothdurft H, Lexa P, Ockenfels H. Teratologic studies on the Himalayan rabbit: new aspects of thalidomide-induced teratogenesis. Arch Toxicol. 1987;60:376-81.
31. Strikwold M, Spenkelink B, Woutersen RA, Rietjens IM, Punt A. Combining in vitro embryotoxicity data with physiologically based kinetic (PBK) modelling to define in vivo dose-response curves for developmental toxicity of phenol in rat and human. Arch Toxicol. 2013;87:1709-23.
32. Van Thriel C, Stewart JD. Developmental neurotoxicity: the case of perfluoroalkylated compounds. Arch Toxicol. 2012;86:1333-4.
33. Van Thriel C, Stewart JD. Developmental neurotoxicity: the case of perfluoroalkylated compounds. Arch Toxicol. 2012;86:1333-4.
34. Van Thriel C, Westerink RH, Beste C, Bale AS, Lein PJ, Leist M. Translating neuro-behavioural endpoints of developmental neurotoxicity tests into in vitro assays and readouts. Neurotoxicology. 2012;33:911-24.
35. Wobus AM, Löser P. Present state and future perspectives of using pluripotent stem cells in toxicology research. Arch Toxicol. 2011;85:79-117.
36. Zimmer B, Kuegler PB, Baudis B, Genewsky A, Tanavde V, Koh W, et al. Coordinated waves of gene expression during neuronal differentiation of embryonic stem cells as basis for novel approaches to developmental neurotoxicity testing. Cell Death Differ. 2011;18:383-95.
37. Zimmer B, Lee G, Balmer NV, Meganathan K, Sachinidis A, Studer L, et al. Evaluation of developmental toxicants and signaling pathways in a functional test based on the migration of human neural crest cells. Environ Health Perspect. 2012;120:1116-22.