RNA transcripts for the quantification of differentiation allow marked improvements in the performance of embryonic stem cell test (EST)
Graphical abstract
Introduction
The lack of in vitro OECD Guidelines for testing toxicity to reproduction and development seems remarkable because the identification of hazards with any of these Guidelines entails the in vivo exposure of animals to the tested chemical with further euthanasia (Estevan et al., 2011). The absence of embryotoxicity tests per se is also remarkable. This means that embryotoxicity has to be assessed within more complete (and expensive) test as toxicity to reproduction. In conclusion, a safe, fast and reliable method that reduces, refines or replaces the use of experimentation animals to test embryotoxicity is strongly desirable for industry and regulatory agencies.
Several years ago, the European Union Reference Laboratory for Alternatives to Animal Testing (EURL-ECVAM) sponsored a blind inter-laboratory validation study of the embryonic stem cell test (EST) (Genschow et al., 2002). The EURL-ECVAM Scientific Advisory Committee considered the EST a scientifically validated procedure ready for consideration for regulatory acceptance and application. However, applicability as a whole in the reproductive toxicity testing context had to wait for the conclusions of another workshop (ESAC, 2002). This second workshop concluded that the EST was not yet ready to replace current animal tests for developmental toxicity and, consequently, it did not recommend its use for regulatory purposes (Spielmann et al., 2006). In addition, a recommendation for improvements in the EST was made in order to allow the higher throughput of test chemicals (Spielmann et al., 2006).
The EST is the only validated method that totally suppresses the use of experimental animals because the biological material employed is cellular established lines, specifically D3 mouse embryonic stem cells, which are used as a model of differentiation cell and 3T3 mouse fibroblasts, employed as a model of non-differentiating cells (Pamies et al., 2011, Sogorb et al., 2014). The EST requires the following three different end-points: IC50D3 and IC503T3, defined as the concentration of the tested compound that brings a 50% reduction in viability (recorded with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay) in D3 and 3T3 cells, respectively, after a 10-day exposure; and ID50, defined as the concentration of the tested compound that brings a 50% reduction in the differentiation of undifferentiated D3 cells into beating cardiomyocytes after a 10-day exposure (Scholz et al., 1999). These three end-points were also introduced into an empirically developed prediction model (PM) with three different mathematical functions to classify the tested chemical according to its in vivo embryotoxic potential into three distinct categories: strong, weak or non-embryotoxicant (Genschow et al., 2000). Under these conditions, the international validation study yielded predictivity percentages of 100%, 69% and 73% for strong, weak and non-embryotoxic chemicals, respectively, with precisions ranging between 68% and 83%, and an overall accuracy of 78% (Genschow et al., 2004).
EURL-ECVAM issued several recommendations for improving the EST. Among them we highlight two: the need for more quantitative end-points (i.e., tissue-specific gene expression markers which help achieve better quantification in differentiation); and the use of end-points for lineages other than mesoderm-derived cardiomyocytes (Spielmann et al., 2006) as the validated protocol assesses only these alterations by morphologically observing contractile cardiomyocytes. In addition, we considered also desirable for throughput purposes to cut test duration (10 days) and to technically simplify procedures because the current protocol establishes D3 cultures in monolayers and in hanging-drops embryoid bodies, which must be individually handpicked up (more than 300 per test).
We have previously demonstrated that by taking advantage of the PM used in the classic EST, cutting exposure to 5 days and using only monolayer cultures, a battery of six different genes biomarkers of differentiation was able to correctly predict the embryotoxic potency of three different compounds (a strong, a weak and a non-embryotoxicant) using IC50D3, IC503T3 and the expression of these biomarker genes as end-points (Romero et al., 2011). We have validated our previous results by extending the battery of genes to 16 and the battery of tested chemicals to seven (2 strong embryotoxicant, 3 weak embryotoxicant and 2 non-embryotoxicants). Our developed protocol shows a 100% concordance between in vivo embryotoxic outcome and in vitro embryotoxicity prediction, and it will allow the design of low density microarrays or other devices, such as prepared PCR plates, for throughput screenings processes.
Section snippets
Chemicals
Seven chemicals used in the prevalidation and validation studies of the EST were used as the model compounds in this study. Table 1 in Romero et al. (2015) displays information about the in vivo embryotoxic properties of these compounds, purity and origin. The other chemicals for cellular cultures were purchased from Sigma-Aldrich (Dulbecco’s modified Eagle Medium (DMEM) supplemented with 4.5 g glucose/l and antibiotics), Invitrogen (β-mercaptoethanol), Gibco (non-essential amino acids and
Cytotoxicity of model compounds
RA displayed the highest cytotoxicity to D3 cells among all seven tested embryotoxic model compounds (Table 2). 5FU and RA (strong embryotoxicants) were also more potent cytotoxicants than the weak and non-embryotoxicants, with IC50 ranging between 5 ng/mL and 20 μg/mL, while the IC50 of all the other model compounds ranged between tens and thousands of μg/mL (Table 2). 5FU and RA were also more cytotoxic (between 2.3 and 4000 times) for D3 embryonic cells than for 3T3 cells (Table 2).
DPH was the
Discussion
We have tested early biomarker genes of differentiation as end-points to be implemented in the classic EST to enhance its performance in order to be considered for regulatory purposes. Our improvements are in terms of: (a) total length of the test (reduced by half); (b) simplicity (use of only monolayer cultures instead of monolayer plus embryoid bodies cultures); (c) predictivity (100% of correct prediction with the battery of model chemicals); and (d) molecular end-points for recording the
Conflict of interest
The authors declare no conflicts of interest.
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2017, ToxicologyCitation Excerpt :A peak in gene expression has been demonstrated in the early cell differentiation stage (Pamies et al., 2010). The relative inhibition of NTE and AChE has been used in in vitro tests for predicting delayed neurotoxicity (Romero et al., 2015); the comparison of the exposure needed for AChE and NTE inhibition with that causing alteration in cell differentiation in vitro, has been proposed and applied for predicting developmental toxicity (Estevan et al., 2013) NTE(+/−) mice survive, but exhibit increased sensitivity to an acute effect of OP compounds, which is distinct from OPIDN. Diminished NTE activity in mice links OP exposure to hyperactivity (Winrow et al., 2003), but not necessarily to attention deficit disorder in humans (Casida and Durkin., 2013).