RNA transcripts for the quantification of differentiation allow marked improvements in the performance of embryonic stem cell test (EST)

doi:10.1016/j.toxlet.2015.08.008

Toxicology Letters

Volume 238, Issue 3, 4 November 2015, Pages 60-69

https://doi.org/10.1016/j.toxlet.2015.08.008 Get rights and content

Highlights

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We used RNA transcripts as biomarkers for monitoring D3 cell differentiation in EST.
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We reduced total length of EST by half simplifying technical procedures.
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We obtained 100% concordance between in vitro prediction and in vivo performance.
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Our proposal would enhance the EST capability for high throughput testing.

Abstract

Embryonic stem cell test (EST) is an in vitro validated assay for testing embryotoxicity. The EST needs improvements before being used for regulatory purposes, but also needs technical simplification for use in high throughput screenings. We propose the quantification in alterations of the differentiation of D3 monolayer cells cultures through the expression of biomarker genes in a shorter (5-day) and technically simpler (we use only monolayer cultures) test. We have defined a set of sixteen different genes biomarkers of ectoderm (Nrcam, Nes, Shh and Pnpla6), endoderm formation (Flk1 and Afp), mesoderm formation (Mesp1, Vegfa, Myo1e and Hdac7) and general cellular processes (Cdk1, Myc, Jun, Mixl, Cer and Wnt3). These, together with alterations in the viability of D3 and 3T3 cells and the prediction model of a classic EST, enhance the features of EST determinations to 100% concordance between in vivo-in vitro predictions with a set of seven different chemicals used in the validation of a classic EST. In conclusion, the proposed changes implemented in the classic EST confer it more reliability, speed and technical simplicity, which brings the EST closer to high throughput processes and regulatory purposes.

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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Furthermore, for such toxicological studies, exploring mouse embryonic stem cells (mESCs) are suggested to be useful as it provides data consistent to in vivo ones and better sensitivity to cytotoxic chemicals including teratogens compared with mouse fibroblasts (Laschinski et al., 1991) Testing toxic chemicals on mESCs also believed to be helpful not only in maintaining several generations without the loss of differentiation and genetic manipulation but also allow us to developmental toxicity assays. For these reasons, mESC-based toxicity assays are now routinely used for high-throughput toxicity screening of organic chemicals as embryonic stem cell test (EST) (Chandler et al., 2011; Romero et al., 2015; Yao et al., 2016; Worley and Parker, 2019). Nevertheless, more studies are required to ascertain the relationship between environmental chemical exposure and expression of miRNAs for identifying specific miRNAs that can be used as diagnostic markers for defining the exposure of toxic chemicals.
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We employed mouse embryonic stem cells (mESCs) because their pluripotency makes them an ideal model for assessing developmental toxicity (Faiola et al., 2015; Liu et al., 2017; Yao et al., 2016). Since developmental toxicity can be predicted by measuring the alterations in expression of differentiation biomarkers (Estevan et al., 2014; Pamies et al., 2014; Romero et al., 2015; Yin et al., 2015, 2018), five molecular markers were used to validate the neural differentiation propensity of mESCs, including: Sox1 and Sox3, the earliest known neural ectoderm markers which are critical for neural ectoderm commitment in the mouse embryo (Wood and Episkopou, 1999); Pax6, which encodes a homeobox and paired domain containing transcription regulator that acts as a key transcription factor in the development of the central nervous system; Nestin, which expresses in both neural ectoderm and further developed cell types (Suter et al., 2009); and a typical dendritic marker, microtubule associated protein 2 (Map2) which encodes a cytoskeleton related protein involved in microtubule assembly (Xu et al., 2012). Moreover, the phenotype of the differentiated cells was characterized by immunofluorescence staining with a MAP2 antibody, to show more visually the effects of F-53B and PFOS treatment on neural development in vitro.
F-53B, as an alternative to the persistent organic pollutant perfluorooctane sulfonate (PFOS), is amply used in the electric plating industry. F-53B and PFOS have similar physicochemical, biochemical and physiological properties, due to the similarity in their chemical structure. Thus, they may also possess similar toxicities. Although epidemiological studies and in vivo assays have shown that prenatal exposure to PFOS may impair the development of the nervous system, toxicity data for F-53B are still scarce. In this study, we employed an embryonic stem cell (ESC) in vitro differentiation system, to detect the potential developmental neural toxicity of F-53B and PFOS, at human exposure relevant doses. We demonstrated that during early mouse ESC (mESC) neural differentiation, F-53B and PFOS disrupted the expression of neural marker genes and affected the morphology of the differentiated cells. However, the very same treatments did not cause any cytotoxic effects. In conclusion, our ESC in vitro differentiation system was able to prove for the first time that F-53B and PFOS at human exposure relevant concentrations, could alter the expression of differentiation biomarkers, indicating a potential developmental neural toxicity. Based on our findings, it is reasonable to deduce that excessive exposure to F-53B and PFOS may cause severe dysfunctions during early stages of embryo development.
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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).
Organophosphorus compounds (OPs) are a large and diverse class of chemicals mainly used as pesticides and chemical weapons. People may be exposed to OPs in several occasions, which can produce several distinct neurotoxic effects depending on the dose, frequency of exposure, type of OP, and the host factors that influence susceptibility and sensitivity. These neurotoxic effects are mainly due to the interaction with enzyme targets involved in toxicological or detoxication pathways. In this work, the toxicological relevance of known OPs targets is reviewed. The main enzyme targets of OPs have been identified among the serine hydrolase protein family, some of them decades ago (e.g. AChE, BuChE, NTE and carboxylesterases), others more recently (e.g. lysophospholipase, arylformidase and KIA1363) and others which are not molecularly identified yet (e.g. phenylvalerate esterases). Members of this family are characterized by displaying serine hydrolase activity, containing a conserved serine hydrolase motif and having an alpha-beta hydrolase fold. Improvement in Xray-crystallography and in silico methods have generated new data of the interactions between OPs and esterases and have established new methods to study new inhibitors and reactivators of cholinesterases. Mass spectrometry for AChE, BChE and APH have characterized the active site serine adducts with OPs being useful to detect biomarkers of OPs exposure and inhibitory and postinhibitory reactions of esterases and OPs. The purpose of this review is focus specifically on the interaction of OP with esterases, mainly with type B-esterases, which are able to hydrolyze carboxylesters but inhibited by OPs by covalent phosphorylation on the serine or tyrosine residue in the active sites. Other related esterases in some cases with no-irreversible effect are also discussed. The understanding of the multiple molecular interactions is the basis we are proposing for a multi-target approach for understanding the organophosphorus toxicity.

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RNA transcripts for the quantification of differentiation allow marked improvements in the performance of embryonic stem cell test (EST)

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Chemicals

Cytotoxicity of model compounds

Discussion

Conflict of interest

Toxicol. Lett.

Toxicol. In Vitro

Methods

Reprod. Toxicol.

Toxicol. In Vitro

Toxicol. Lett.

Reprod. Toxicol.

Toxicol. Lett.

Reprod. Toxicol.

Toxicol. Appl. Pharmacol.

Toxicol. Lett.

Organophosphorus pesticide chlorpyrifos and its metabolites alter the expression of biomarker genes of differentiation in D3 mouse embryonic stem cells in a comparable way to other model neurodevelopmental toxicants

Chem. Res. Toxicol.