Abstract
The Tox21 program calls for transforming toxicology testing from traditional in vivo tests to less expensive and higher throughput in vitro methods. In developmental toxicology, a spectrum of alternative methods including cell line based tests has been developed. In particular, embryonic stem cells (ESCs) have received widespread attention as a promising alternative model for developmental toxicity assessment. Here, we characterized gene expression changes during mouse ESC differentiation and their modulation by developmental toxicants. C57BL/6 ESCs were allowed to differentiate spontaneously and RNA of vehicle controls was collected at 0, 24, 48, 72, 96, 120 and 168 h after embryoid body (EB) formation; RNA of compound-exposed EBs were collected at 24 h. Samples were hybridized to Affymetrix Mouse Gene 2.0 ST Array; using stringent cut-off criteria of Bonferroni-adjusted p<0.05 and fold change >2.0, a total of 1996 genes were found differentially expressed among the vehicle controls at different time points. Gene ontology (GO) analysis showed these regulated genes were mostly involved in differentiation-related processes such as development, morphogenesis, metabolism, cell differentiation, cell organization and biogenesis, embryonic development, and reproduction. Biomarkers of all three germ layers or of their derivative early cell types were identified in the gene list. Principal component analysis (PCA) based on these genes showed that the unexposed vehicle controls appeared in chronological order in the PCA plot, and formed a differentiation track when connected. Cultures exposed to thalidomide, monobutyl phthalate, or valproic acid deviated significantly from the differentiation track, manifesting the capacity of the differentiation track to identify the modulating effects of diverse developmental toxicants. The differentiation track defined in this study may be further exploited as a baseline for developmental toxicity testing, with compounds causing significant deviation from the differentiation track being predicted as potential developmental toxicants.
Highlights
Toxicity testing has traditionally relied on animal models which are costly, time consuming and low throughput
We further demonstrated that the differentiation track was able to identify the modulating effects of three developmental toxicants: thalidomide (THD), monobutyl phthalate (MBP), and valproic acid (VPA)
We propose the differentiation track defined in this study be further exploited as a baseline for developmental toxicity testing, with compounds causing significant deviation from the differentiation track being predicted as potential developmental toxicants
Summary
Toxicity testing has traditionally relied on animal models which are costly, time consuming and low throughput. It often causes pain and stress to, and frequently involves the sacrifice of, large numbers of laboratory animals This is especially true for reproductive and developmental toxicity testing [1]. With the EU chemicals regulation protocol REACH [2] in force, it has been estimated that over 70–80% of all animals used for safety testing would be used for examining reproductive and developmental toxicity [3,4] Under such circumstances, the Tox program [5] partnered by several US Federal agencies calls for transforming toxicology testing from traditional in vivo tests to less expensive and higher throughput in vitro methods to prioritize compounds for further study, identify mechanisms of action and develop predictive models for adverse health effects in humans. In support of the program, the US FDA is developing alternative models for safety assessment of foods, dietary supplements and cosmetics
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