Abstract for TestSmart--A Humane and Efficient Approach to Screening Information Data Sets (SIDS) Data

Prevalidation of the Embryonic Stem Cell Test (EST) and Status of the ECVAM Validation on Three In Vitro Embryotoxicity Tests

Manfred Liebsch
ZEBET

Note: The following manuscript on the "EST" by Scholz et al. has been recently accepted by Acta Anatomica. Since it most closely matches the presentation given by Manfred Liebsch at the CAAT TestSmart meeting on 27 April 1999 in Parallel Session II "In Vitro Developmental Toxicity" it is used instead of drafting a new manuscript. Due to confidentiality restrictions the currently running ECVAM Validation Study on three embryotoxicity tests was only mentioned in the presentation, but no data were shown.

EMBRYOTOXICITY SCREENING USING ES CELLS IN VITRO: CORRELATION TO IN VIVO TERATOGENICITY

Gabriele Scholz¹, Ingeborg Pohl¹, Elke Genschow², Martina Klemm¹ and Horst Spielmann¹

¹Center for Documentation and Evaluation of Alternative Methods to Animal Experiments (ZEBET) at the ²Federal Institute for Health Protection of Consumers and Veterinary Medicine (BgVV), Berlin, Germany.

short title: embryotoxicity screening using ES cells in vitro

key words: in vitro differentiation, embryonic stem cell test (EST), cardiomyocytes, cytotoxicity, embryotoxicity, prediction model

Abstract

Blastocyst-derived totipotent embryonic stem (ES) cells of the mouse can be induced to differentiate in culture into a variety of cell types, including cardiac muscle cells. The embryonic stem cell test (EST) that makes use of the differentiation of ES cells into cardiomyocytes in a standardised in vitro model, was developed to offer an alternative method to comprehensive in vivo studies in reproductive toxicology about toxic effects of chemicals. ES cells of the mouse cell line D3 are investigated for their preserved capability to differentiate following drug exposure, and both ES cells and differentiated fibroblast cells of the mouse cell line 3T3 are comparatively analysed for effects on viability. The following endpoints are used to classify the embryotoxic potential of chemicals into three classes of in vitro embryotoxicity (non, weak or strong embryotoxic). These endpoints are: (i) the inhibition of differentiation of ES cells into cardiomyocytes after 10 days of treatment and the decrease of viability (cytotoxicity) of (ii) 3T3 cells and (iii) ES cells after 10 days of treatment, determined by an MTT test. 50% inhibition concentrations for differentiation (ID₅₀) and cytotoxicity (IC₅₀D3 and IC₅₀3T3) are calculated from concentration-response curves. Applying linear analysis of discriminance, a biostatistical prediction model (PM) was developed. This procedure identified three variables, the lg (IC₅₀D3), the lg(IC₅₀3T3) and the relative distance between IC₅₀3T3 and ID₅₀, that improved the separation of the three classes of embryotoxicity compared to the prediction model that was originally proposed after test development (Spielmann et al, 1997). Unlike the original PM, the improved PM incorporates as one variable the relative distance between IC₅₀3T3 and ID₅₀, instead of the ratio ID₅₀/ICM₅₀D3, that was used previously.

Introduction

Developing organisms undergo rapid and complex changes within a relatively short period of time. Therefore, in mammals any agent administered during pregnancy to the mother under appropriate conditions of time and dosage may interfere with embryonic development and induce embryolethality, growth ratardation or teratogenic effects, with structural and functional abnormalities in the offspring. Persistent lesions, e.g. general growth retardation or delayed organ growth are defined as embryotoxic effects. Currently, according to the great number of existing industrial chemicals that are commercially available, screening tests or multigeneration studies must be conducted to provide information on the toxic effects on specific elements of the highly complicated reproductive cycle according to guidelines of the OECD (OECD Guidelines No. 414 (1981), 415 (1983), 416 (1983) 421 (1995), 422 (1996)). For chemicals used as drugs, segment studies have to be conducted covering preconceptional exposure as well as pre- and postnatal development including the lactation period (guidelines of the International Conference on Harmonisation, ICH, 1993).

These in vivo protocols are time consuming, expensive and they are carried out on high numbers of laboratory animals. One important feature of the in vivo test protocols is the assessment of maternal toxicity in comparison to adverse effects in the offspring. Today, these two important aspects can to some extent be covered by in vitro approaches. In developmental toxicology, many in vitro alternatives to testing in animals have been developed using a wide spectrum of cell and tissue cultures, e.g. permanent cell lines, cultures of primary embryonic cells and cultures of non mammalian tissue and mammalian embryos (for a review, see Brown et al, 1995, Spielmann 1998). For example, in the rat limb bud micromass (MM) assay, effects on viability of primary limb bud cell cultures (cytotoxicity) are compared to effects on the differentiation of these cells into chondrocytes (Flint and Orton, 1984). In the postimplantation rat whole embryo culture (WEC) assay, both general growth retardation and specific malformations of the cultivated embryos are assessed (for a review, see Spielmann, 1998). One of the more recently developed in vitro approaches is based on blastocyst-derived pluripotent embryonic stem (ES) cells of the mouse. In contrast to the WEC and MM tests, the embryonic stem cell test (EST, Spielmann et al, 1997) has the advantage of of using established cell lines without the need to sacrifice pregnant animals.

Since cultivation and maintanance of ES cells in the undifferentiated state was first reported by Evans and Kaufman (1981), cultivation and differentiation of ES cells is today a widely applied method in mammalian developmental biology. ES cells can be genetically manipulated to generate transgenic or "knock out" mice (Thomas and Capecci, 1987) and in vitro cell culture models were established to study myogenesis, angiogenesis, hematopoiesis, neurogenesis and cardiogenesis in the mouse (Rohwedel et al, 1994; Kolossov et al, 1998; Wiles and Keller, 1991; Heuer et al, 1994; Strübing et al, 1995; Wobus et al, 1991; Maltsev et al, 1994).

The differentiation of ES cells into cardiac cells (Doetschman et al, 1985) has been used in investigations on prenatal pharmacology, electrophysiology and molecular genetics (Wobus et al, 1991; Maltsev et al, 1993; Metzger et al, 1996; Kolossov et al, 1998). The EST developed at ZEBET was designed to adopt the ES cell cultures to embryotoxicity studies by using an endpoint related to prenatal differentiation in vivo, the differentiation of ES cells into contracting cardiac muscle cells. The inhibition of this specific differentiation by embryotoxic agents was compared to cytotoxic effects in the ES cells (Laschinski et al, 1991). To improve the predictive potential of this in vitro embryotoxicity test, in the EST, the influence of potentially embryotoxic chemicals on the viability of ES cells and on their capability to differentiate in vitro (Wobus et al, 1991; Rohwedel et al, 1994; Spielmann et al, 1997) is examined in the permanent mouse ES cell line D3 and compared to the effect on viability of differentiated, fibroblast cells of the line 3T3.

Despite the use of some established alternative embryotoxicity tests like the MM test and the WEC test in the drug and chemical industry for screening of new chemicals that are structurally related to established embryotoxic chemicals, none of these in vitro tests has to date sufficiently been validated for regulatory purposes according to the recommendations of the European Centre for the Validation of Alternative Methods, ECVAM (Ispra, Italy; Balls et al, 1995). Therefore, in developmental toxicity testing there is a strong demand for validated in vitro tests using mammalian embryos as well as primary cultures of embryonic cells and permanent cell lines. The limiting factors for in vitro screening are obvious when taking into account the complexity of normal differentiation and development of mammalian embryos.

Nevertheless, three of the most promising in vitro tests are currently undergoing formal validation in a ring trial funded by ECVAM: the WEC Test, the MM test and the embryonic stem cell test (EST). This validation study is aimed at predicting the embryotoxic potential of a set of test chemicals characterised by high quality in vivo embryotoxicity data in laboratory animals and humans (Scholz et al, 1998, Genschow et al, 1999).

In this study the performance of the EST to predict the embryotoxic potential of test chemicals is described and we report about the development of an improved prediction model for the EST, derived from results obtained in our laboratory in a prevalidation trial that preceded the currently running validation study. The importance of sound biometrical prediction models as a means for an objective evaluation and classification of the performance of in vitro tests in validation studies is discussed.

Test Procedure

BALB/c 3T3 cells (clone 31, ICN Flow, Eschwege, Germany) are routinely maintained in DMEM supplemented with 10% FCS, 4 mM Glutamine, 50 U/ml Penicillin and 50 Mg/ml Streptomycin. ES cells (line D3, a donation from Prof. R. Kemler, MPI, Freiburg, Germany) are maintained in DMEM supplemented with 20% FCS, 2 mM glutamine, 50 U/ml Penicillin, 50 Mg/ml Streptomycin 1% non essential amino acids, 0.1% β-mercaptoethanol and 1000 U/ml leukeamia inhibitory factor (LIF).

To determine cytotoxic effects on ES and 3T3 cells, MTT-tests were performed in the absence of LIF as described previously (Spielmann et al, 1997). Briefly, 500 cells are seeded into each well of a 96 well microtiter plate and grown in the presence of a concentration range of the test chemical. A negative control containing solvent diluted in medium is also included. Each test concentration and negative control is tested in 6 replicate wells. After 10 days of culture with 2 changes of medium (containing the appropriate concentration of test chemical) on day 3 and 5, the viability of the cells is determined using an MTT test, which is based on the capacity of mitochondrial dehydrogenase enzymes in living cells to convert the yellow substrate 3-(4,5-di-methylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) into a dark blue formazan product, which is detected quantitatively using a microplate ELISA reader. Cytotoxicity is expressed as the concentration decreasing viability to 50% of the control level (IC₅₀3T3 and IC₅₀D3; determined from a concentration response curve).

To detect effects on differentiation of ES cells into cardiomyocytes, differentiation assays were performed as described previously (Spielmann et al, 1997). Briefly, 750 cells in drops of 20 µl are seeded (in the absence of LIF to allow differentiation) into the lid of a culture dish and grown for 3 days in a "hanging drop" culture in the presence of a concentration range of test chemical. During this period the cells will form aggregates called embryoid bodies (EBs). After 3 days of "hanging drop" culture the EBs are transferred to bacterial petri dishes containing the appropriate concentration of test chemical for another 2 days. Bacterial petri dishes are used to avoid adherence and outgrowth of the EBs during this stage of the culture. On day 5 EBs are seeded singly into one well of a 24 well tissue culture plate (containing the appropriate concentration of test chemical) to allow adherence and outgrowth of the EBs and development of contracting cardiomyocytes. One 24 well plate is used for each concentration of test chemical and one for the solvent control. Differentiation is assessed by microscopic inspection of the EBs on day 10. The percentage of wells of each plate in which contracting cardiomyocytes have developed is determined and compared to the percentage of wells in which cardiomyocytes developed on the solvent control plate. The inhibition of differentiation (ID₅₀) is expressed as the concentration of test chemical inhibiting the development into contracting cardiomyocytes by 50% (calculated from the concentration response curve).

Results

In a recent prevalidation study that was conducted according to the recommendations of the European Centre for the Validation of Alternative Methods ECVAM (Curren et al, 1995), chemicals assigned to the three classes of embryotoxicity were tested under blind conditions in order to prove that the assay is reproducible and transferable to other laboratories and that it is able to predict embryotoxic potential of a given set of chemicals. In embryotoxicity testing the selection of test chemicals is particularly difficult, since the number of chemicals which are backed by high quality in vivo data is rather limited. For the prevalidation trial, only test chemicals were used for which sufficient high quality in vivo data were available from both testing in animals and human pregnancy. These were selected from a list of chemicals recommended by the US Teratology Society for in vitro teratogenesis test validation (Smith et al, 1983). Nine test chemicals and a positive control of known embryotoxic potential in vivo were selected for testing during the prevalidation study (Table 1), mainly consisting of drugs which have been used in human pregnancy, e.g. in case of bacterial infections or the therapy of cancer. The prevalidation trial was conducted in two European and one US laboratory, yet the exact results obtained during this interlaboratory study are reported elsewhere (Scholz et al, 1999; in press). This report emphasises exclusively on the results of the lead laboratory of the study, since only the results obtained in our laboratory were used as the basis for the development of the improved PM.

Table 1: Training Set of Test Chemicals

Test Chemical	Abbr.	CAS no.	Embryo- toxicity	In vivo activity species	Use
Penicillin G	Pen	69-57-8	non	-	antibiotic
Isoniacide	Iso	54-85-3	non	-	tubercolostatic
Ascorbic acid	Asc	134-03-2	non	-	vitamin
Diphenylhydantoin	DPH	630-93-3	weak	human, animal	anticonvulsant
Caffeine	Caf	58-08-2	weak	human, animal	central stimulant, drug
Dexamethasone	Dex	50-02-2	weak	human, animal	antiasmathic, glucocorticoid
Cytosine-arabinoside	AraC	69-74-9	strong	human, animal	cytostatic
all-trans-Retinoic acid	RA	302-79-4	strong	human, animal	acne treatment
Hydroxyurea	HU	127-07-1	strong	animal	cytostatic
5-Fluorouracil	5-FU	51-21-8	strong	animal	cytostatic

Training set of chemicals. The table summarises the characteristics of the training set of chemicals used in the study. For the 9 test chemicals and the positive control 5-fluorouracil (5-FU). The embryotoxicity in vivo and their use in humans are given. Test chemicals with known embryotoxic potential in vivo were selected from a published list recommended by the US Teratology Society for the validation of in vitro embryotoxicity tests (Smith et al, 1983). All chemicals were purchased from Sigma-Aldrich Chemie GmbH, Steinheim, Germany. Chemicals were dissolved according to a strategy developed for dissolving of coded chemicals, which has been included in the standard protocol. Briefly, a hierarchical approach was used to find an appropriate concentration in a suitable solvent , starting with PBS or medium as solvent, then 50% (vol/vol) Ethanol in PBS, then DMSO, and finally pure Ethanol.

Most of the chemicals tested (Table 1) can be categorized in vitro roughly according to typical concentration response curves obtained for the differentiation of ES cells and for the viability of ES and 3T3 cells in the presence of a concentration range of test chemical.

Strong embryotoxic chemicals are usually inhibiting differentiation of ES cells at very low concentrations and they also show a high cytotoxic potential at low concentrations both to 3T3 mouse fibroblast cells and ES cells, e.g. cytostatic drugs (e.g. 5-fluorouracil, see also Figure 2A). Chemicals characterised by a high teratogenic potential, e.g. retinoic acid, are inhibiting differentiation at very low concentrations, while cytotoxic effects are apparent at concentrations that are several orders of magnitude higher (Figure 1C). On the other hand, weak embryotoxic chemicals are effective in the EST at intermediate concentrations, where the differentiation of ES cells into cardiomyocytes is more sensitive than growth inhibition, e.g. in case of the anticonvulsant drug diphenylhydantoin (Figure 1B). Chemicals that do not exhibit any embryotoxic potential, e.g. the sweetener saccharine or the antibiotic penicillin G are not inhibiting growth nor differentiation of ES cells at concentrations of more than 500 µg/ml. Other non embryotoxic chemicals, however, as for instance the vitamin and food additive ascorbic acid, exert cytotoxic effects at relatively low concentrations on differentiatred 3T3 cells, whereas cell differentiation and/or growth of ES cells are less sensitive (Figure 1A). The biostatistical evaluation was conducted on the limited set of 9 chemicals tested in our laboratory during prevalidation of the EST (Scholz et al, in press). However, the evaluation of the improved PM must be performed with the results of a new set of test chemicals.

Figure 1

Figure 1a

Figure 1b

Figure 1c

Typical concentration response curves. Diagram (A) shows concentration response curves for the three endpoints of the EST: cytotoxicity of D3 and 3T3 cells and inhibition of differentiation of D3 cells. The curves were obtained from two individual experiments using the non embryotoxic ascorbic acid (Asc). (B) shows concentration response curves of two individual experiments using the weak embryotoxic diphenylhydantoin (DPH). The lower section (C) shows concentration response curves of two individual experiments using the strong embryotoxic chemical retinoic acid (RA).

In the procedure of validation, in vitro tests have to prove their reliability and relevance. For the first requirement, the reliability, individual laboratories verify a sufficiently high level of exactness by repeating results independently (Balls et al, 1990 & 1995). The second requirement, the relevance of the in vitro test for a specific application, is estimated by applying a biostatistically based prediction model (PM). For developing a scientifically acceptable in vitro embryotoxicity test the PM should allow to discriminate between three classes of embryotoxicity: non, weak and strong embryotoxic, according to in vivo data.

The PM originally developed for the EST (Spielmann et al, 1997) is based on a mathematical calculation, the linear discriminant analysis, taking into account three endpoints used to discriminate between the three embryotoxicity classes: cytotoxicity determined with 3T3 fibroblasts and ES cells of the mouse and differentiation of ES cells into contracting cardiomyocytes after a 10 day culture period.

The concentration response curves for several chemicals were analysed in order to determine appropriate endpoints that allow to discriminate between the three classes of embryotoxic chemicals. First, 50% inhibition concentrations (IC₅₀) were calculated from the concentration response curves for cytotoxicity for D3 cells and 3T3 fibroblasts and for differentiation of D3 cells, in order to evaluate if individual endpoints alone sufficiently allow to discriminate between the three classes of embryotoxicity.

Figures 1 and 2 show that the variability at the 50% inhibition concentration is higher than at the 25% viability/differentiation concentration (IC₂₅). Therefore, the discriminating potential of the two endpoints (IC₅₀, IC₂₅) must be evaluated. Although the IC₂₅ showed a better performance in the analysis of discriminance -- and therefore provided a better discriminative power -- the IC₅₀ was chosen as the appropriate endpoint for the following reasons: When testing chemicals under blind conditions, it is sometimes difficult to completely cover the entire range of the concentration response curve between 100% and 0% both for viability and differentiation. Furthermore, given the intrinsic variability of the assay and the limited number of concentrations that can be tested within one experiment, the dilution factor should be sufficiently small to achieve the most exact measurement of the endpoint.

Endpoints and variable used in the improved predictionmodel (iPM). Diagram shows concentration response curves for the three endpoints cytotoxicity of D3 and 3T3 cells and inhibition of differentiation of D3 cells. The curves were obtained from two individual experiments using the positive control chemical 5-FU. Endpoints (IC50D3, IC503T3, ID50) and the variable ( ) are marked by arrows.

Therefore, the concentration response curves may not reach 25% viability or differentiation in each case, whereas the 50% inhibition concentrations are more easily met. Thus, the 50% viability or differentiation (IC₅₀, ID₅₀) was applied as endpoint in the linear discriminant analysis in the improved prediction model.

Yet the endpoint value (IC₅₀, ID₅₀) cannot be used directly as a variable in the linear analysis of discriminance. In order to convert the numerical value of the endpoint into a linear model the logarithm of the 50% inhibition concentrations is calculated. The term "endpoint" is used only for the 50% cytotoxic concentrations (IC₅₀3T3, IC₅₀D3) and the 50% differentiation concentration (ID₅₀), that are directly calculated from the concentration response curve. The term "variable" is applied on all mathematical calculations (logarithm, relative distance) of the original endpoints. The improvement of the PM described here is based on the same endpoints as the original PM, but uses a slightly different variable.

In order to include occasionally divergent test results on viability and differentiation into the model, the relative distance between two of the endpoints (IC₅₀3T3, IC₅₀D3) was used as a variable (Figure 2A). This variable is independent from the absolute concentration values, since this information is already given by the two other variables, the lg(IC₅₀3T3) and the lg(IC₅₀D3) values.

The in vitro test provided a total of 9 different experimental variables (lg(IC₅₀3T3); lg(IC₅₀D3); lg(ID₅₀); lg(IC₂₅3T3); lg(IC₂₅D3); lg(ID₂₅), relative distance between IC₅₀3T3 and IC₅₀D3; relative distance between IC₅₀3T3 and ID₅₀, and relative distance between IC₅₀D3 and ID₅₀) each of which may contribute to distinguish between the three groups of embryotoxic chemicals. Consequently, a stepwise selection of variables was performed (procedure: "Stepwise" of SPSS, Noruis 1994). This procedure identifies the best variable to discriminate between the selected classes. Subsequently, in a stepwise fashion, each of the remaining variables is separately added to the model and rejected again, if it does not improve the separation of three classes of embryotoxicity significantly.

Three variables were accepted in the analysis of discriminance which improved the distinction between the three embryotoxic classes compared to the original model. The linear discriminant functions of the PM for the EST incorporating the variables selected are shown in Table 3.

Table 2: Classification of the Training Set of Chemicals

Embryotoxicity in vivo	No. of chemicals	No. of cases	Predication in vitro
			1	2	3
Class 1: not embryotoxic	3	9	9 100.0%	0 0%	0 0%
Class 2: weak embryotoxic	3	9	1 11.1%	8 88.9%	0 0%
Class 3: strong embryotoxic	4	12	0 0%	1 8.3%	11 91.7%

Classification of the training set of chemicals. The 10 chemicals of the training set were repeatedly tested, resulting in 30 individual experiments (number of cases). Correct classifications are marked by a grey box. 100% of the non embryotoxic chemicals, 89% of the weak embryotoxic chemicals and 92% of the strong embryotoxic chemicals were classified correctly. In total 94% of all cases were classified correctly. To identify the endpoints, linear discriminant analysis was used (SPSS, stepwise discriminant analysis, Noru_is 1994; Backhaus et al, 1996; Bortz 1993).

Table 3: Improved Prediction Model (iPM) for the EST

(A) Linear Discriminant Functions I, II and III

Function I	5.92 Ig(IC₅₀ 3T3) + 3.50 Ig(IC₅₀D3 - 5.31	C₅₀3T3 - ID₅₀	- 15.7
		___________
		IC₅₀ 3T3
Function I	3.65 Ig(IC₅₀ 3T3) + 2.39 Ig(IC₅₀D3 - 2.03	C₅₀3T3 - ID₅₀	- 6.85
		___________
		IC₅₀ 3T3
Function III	-0.125 Ig(IC₅₀ 3T3) + 1.92 Ig(IC₅₀D3 - 1.50	C₅₀3T3 - ID₅₀	- 2.67
		___________
		IC₅₀ 3T3

(B) Classification Criteria

class 1	not embryotoxic	if I>II and I>III
class 2	weak embryotoxic	if II>I and II>III
class 3	strong embryotoxic	if III>I and III>II

Improved prediction model (iPM) for the EST. (A) shows the linear discriminant functions I, II and III; the classification criteria are shown in (B).

The following procedure is ensued to classify the chemicals according to the improved PM: 50% inhibition concentrations (IC₅₀3T3, IC₅₀D3, ID₅₀) and the relative distance between IC₅₀3T3 and ID₅₀ are determined and employed in the three linear discriminant functions. A chemical is classified as not embryotoxic, if the result of equation I exceeds the results of equation II and III. A chemical is classified as weak embryotoxic; if the result of equation II exceeds the results of equations I and III, the chemical is weak embryotoxic. Finally, if the result of equation III exceeds the results of equations I and II, the chemical is classified strong embryotoxic.

The classification of the so called "learning sample" (Table 2) according to the improved prediction model provided 94% correct classifications. However, a model usually fits the learning sample better than it will fit new chemicals. Thus the percentage of correctly classified cases is an overly optimistic estimation and a lower rate for correct classifications must be expected when new chemicals are evaluated with the model. Currently, a set of 20 chemicals is tested in a validation study of three in vitro embryotoxicity tests, including the EST. The results of this study will be used to evaluate the improved PM.

Discussion

Teratogenic chemicals are distributed to numerous types of chemicals and function via diverse pathways, either highly specific receptor signalling pathways, or more general mechanisms. Although the molecular functions of several teratogens are the subject of intensive research, such as the retinoic acid receptor pathway or the arylhydrocarbon receptor pathway, the mechanisms of how adverse effects are affecting normal mammalian development are highly complex and have not sufficiently been identified. Therefore, establishing a highly specific, mechanistic test is impossible to date. Taking into account known mechanisms of embryotoxicity we have used the differentiation of ES cells into contracting cardiomyocytes as a means to measure differentiation and early mammalian development. Our results show that a variety of strong and weak teratogens can correctly be identified in the EST, even chemicals which do not specifically act on early heart development in vivo. For instance, in vivo exposure of early postimplantation rodent embryos to all-trans retinoic acid (RA) frequently results in craniofacial and defects of the central nervous system. Exposure at later developmental stages is often associated with limb and genitourinary defects. Adverse effects on the development of the heart, however, are observed only very rarely (Kochhar 1997). Nevertheless, RA is a highly potent inhibitor of cardiomyocyte differentiation in our in vitro model, and we did not obtain false negative results in the EST so far during test development and prevalidation. However, it must be kept in mind that teratogens which specifically interfere with the development of certain tissues, e.g. the palate, may not be detected in the EST.

The EST and the improved PM for the EST are based on the different responsiveness of pluripotent embryonic stem cells and differentiated fibroblast cells to teratogenic/embryotoxic agents. This difference, especially with respect to the cytotoxicity tests with the two cell lines, may to some extent be influenced by the different media requirements and cell growth characteristics of D3 and 3T3 cells. On the one hand, media for 3T3 cells contain less serum (10% FCS) than media for the D3 cells (20% FCS). Since it is known that chemicals can have different affinities for serum proteins, the concentration of free, and active, compounds may be different in the presence of different serum concentrations, and therefore a higher sensitivity of 3T3 cells in the cytotoxicity test would be expected. On the other hand, 3T3 cells have a longer doubling time than D3 cells and display contact inhibition, which could also explain a higher sensitivity of D3 cells in cytotoxicity tests in some cases. To elucidate the different responsiveness of D3 compared to 3T3 cells, additional analytical investigations on the serum binding affinities of test chemicals would be necessary, which was beyond the scope of this study Since the EST is the only mammalian in vitro alternative to animal testing in developmental toxicology to date which is based on established cell lines and does not require pregnant animals, this test offers the possibility to be optimised for automated high throughput screening systems (HTPS) in toxicity testing of drugs and other chemicals for regulatory purposes. An earlier and slightly different approach to use ES cells in an in vitro embryotoxicity test, which used colony formation as a morphological endpoint of differentiation, showed a very poor prediction and had not been developed further (Newall & Beedles, 1994).

Attempts to further optimise the EST test protocol are currently under way in order to identify endpoints of differentiation other than microscopic evaluation of contracting areas. This aim may be achieved by using promoter/reporter gene expression techniques and stably transfected ES cells which are expressing e.g. green fluorescent protein (GFP) under the control of a developmentally regulated promoter, such as the cardiac a-actin promoter which is activated during cardiac muscle cell development (Kolossov et al, 1998). Another approach may focus on endogenous gene expression by RT-PCR methods, for which protocols and primers exist to detect early markers of gene expression in early development as well as gene expression patterns in developing embryoid bodies with a very limited amount of ES cells (Wiles 1993). Such molecular techniques would offer the advantage to replace the subjective evaluation through an experienced person by an objective measurement that can be automatised, which is an important prerequisite for HTPS. Furthermore, the assay duration could be reduced, from 10 to e.g. 7 or even 5 days.

The inclusion of other endpoints in addition to cardiac muscle cell development is another important consideration in order to reduce the risk to obtain false negative results. For instance, early heamatopoiesis and blood cell development can be induced in ES cell cultures under defined culture conditions and by the addition of specific growth factors.

Thus, there are several promising strategies to adapt or extend in vitro ES cell differentiation techniques in order to reduce animal testing in reproductive toxicology and at the same time to gain further insight into mechanisms of action of teratogens on the cellular and molecular level.

Acknowledges

This study was funded by the European Centre for the Validation of Alternative Methods (ECVAM) at the EU Joint Research Centre (JRC) in Ispra, Italy, through a subcontract of JRC Grant No. 11279-95010FIED ISP GB awarded to Microbiological Associates Ltd., Stirling (UK).

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Abbreviations

ES cells, embryonic stem cells; EST, Embryonic Stem Cell Test; LIF, Leukaemia Inhibitory Factor; SOP, Standard Operating Procedure; MTT, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide; EB, embryoid body; PM, prediction model; MM, micromass; WEC, whole embryo culture; HTS, high throughput screening

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