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Teratogenic Substances
Toxicology in Vitro 13 (1999) 619±623 www.elsevier.com/locate/toxinvit
The Relative Semi-quanti®cation of mRNA Expression as a Useful Toxicological Endpoint for the Identi®cation of Embryotoxic/Teratogenic Substances 1
K. BIGOT1,2,*, J. DE LANGE1, G. ARCHER1, R. CLOTHIER2 and S. BREMER1
ECVAM, JRC Institute of Health and Consumer Protection, Ispra, Italy and 2FRAME Alternatives Laboratory, School of Biomedical Sciences, Nottingham University, UK
AbstractÐEmbryonic stem cells, which resemble the undierentiated cells of the epiblast in a blastocyst, are able to dierentiate into derivatives of the primary germ layers, including cardiomyocytes. The eects of embryotoxic/teratogenic compounds on the dierentiation of cells was examined by semiquanti®cation of the mRNA expression using the RT±PCR protocol. Alpha and beta myosin heavy chain (a-MHC and b-MHC, respectively) mRNA expression were chosen as tissue-speci®c markers, characteristic of early cardiac muscle development. Nine chemicals were investigated, chosen according to their in vivo embryotoxic/teratogenic potential in mice. The teratogens all-trans retinoic acid (RA), 5¯uorouracil (5-FU), hydroxyurea (HU), diphenylhydantoin (DPH) and caeine (Ca) caused a signi®cant reduction in MHC mRNA expression at a dose lower than that required for cytotoxicity. Saccharin (Sacc) had a similar eect, while penicillin G (PenG), isoniazid (Iso) and cytarabine (Ara-C) only showed an eect on MHC mRNA expression when the cells had a signi®cant loss in viability. The inability to identify the strong teratogen Ara-C is related to the inappropriate target tissue. # 1999 Elsevier Science Ltd. All rights reserved Keywords: embryonic stem cells; teratogenicity; embryotoxicity; PCR analysis. Abbreviations: 5-FU = 5-¯uorouracil; Ara-C = cytarabine; Ca = caeine; DPH = diphenylhydantoin; ESC = embryonic stem cells; HU = hydroxyurea; Iso = isoniazid; MHC = myosin heavy chain; PenG = penicillin G; PBS = phosphate buered saline; RA = all-trans retinoic acid; RT±PCR = reverse transcriptase±polymerase chain reaction; Sacc = saccharin.
INTRODUCTION
The mouse D3 embryonic stem cells (ESC) are pluripotent, non-transformed cells derived from the inner cell mass of a mouse blastocyst. They have been shown to dierentiate in vitro into a wide variety of cell types such as cardiomyocytes, neurocytes and haematopoietic cells (Doetschman et al., 1985; Keller et al., 1993; Wobus et al., 1988). To obtain precise information of the eect of chemicals on developing tissues during embryonic stem cell dierentiation, quanti®cation of mRNA expression using the reverse transcriptase±polymerase chain reaction (RT±PCR) assay has been employed. *Corresponding author at: ECVAM TP 580, Institute for Health and Consumer Protection, Joint Research Centre, 21020 Ispra (VA) Italy.
Robbins et al. (1990) showed that the ES cells are capable of simulating aspects of cardiogenesis. They are able to undergo in vitro dierentiation and then show spontaneous contractile activity after day 7. Hence, the ES cells dierentiating into cardiomyocytes provide a model to study early cardiac muscle development and to perform embryotoxicological investigations, employing the analysis of a- and bmyosin heavy chain (MHC) gene expression, characteristic of atrial and ventricular cells during early embryonic heart development. To evaluate this approach, nine compounds were selected according to their in vivo embryotoxic/teratogenic potential in mice (non, weak/moderate or strong teratogenic). The cells were exposed to the test compounds, and the following endpoints were examined for their ability to distinguish potential
0887-2333/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. Printed in Great Britain PII: S0887-2333(99)00027-2
620
K. Bigot et al.
teratogens: (a) mRNA expression modulation; (b) general cell viability; and (c) presence of contracting myocardial cells.
MATERIALS AND METHODS
Analysis System (Fotodyn Inc., USA). The image analyser package NIH.Image 1.61 was then used to quantify and estimate the relative ratio between the tissue-speci®c and the non-speci®c gene signals to test the gene expression modulation. Cytotoxicity assay
Cell culture and induction of dierentiation The mouse D3 embryonic stem (ES) cells (ATCC Cat. No. CRL-1934) were maintained in the undifferentiated state by including myeloid leukaemia inhibitory factor (LIF; GIBCO Brl UK) at 1000 U/ ml in the culture media (Williams et al., 1988). To induce cardiomyocyte dierentiation by cell aggregation and produce so-called `embryoid bodies' (EBs), the hanging drop culture was used (Rudnicki and McBurney, 1987). Brie¯y, 20-ml samples containing 750 cells were placed in a hanging drop on the lid of a humidi®ed petri dish containing phosphate buered saline (PBS), this being considered as day 0 of the dierentiating state. After 3 days, the embryoid bodies (EBs) were resuspended in 5 ml medium in bacteriological petri dishes (Greiner GmbH, Germany) and cultured in suspension for a further 2 days. 5-day-old EBs were plated separately into individual wells of a 24-microwell plate for further dierentiation. Quanti®cation of mRNA 12 EBs were collected at each time interval for each test. The cells were lysed in a buer based on guanidinium thiocyanate salt and the cell extract was puri®ed for poly(A)+ mRNA using oligo(dT)cellulose anity. Poly(A)+ mRNA was reversetranscripted and ampli®ed by using oligonucleotide primers speci®c for a-MHC and b-MHC transcripts (Robbins et al., 1990). The housekeeping gene hypoxanthine phosphoribosyltransferase (HPRT) was used as an internal standard. The reverse transcription and the ampli®cation were performed according to the method of Robbins et al. (1991). The ampli®ed products were stained with ethidium bromide on an agarose gel. The running gels were TM photographed using FOTO/Analyst Image
Cells were incubated in the presence of the test substance for a period of 10 days, after which cytotoxicity was assessed using the MTT assay. Brie¯y, 20 ml MTT solution (5 mg/ml in PBS) was added to each well and the cultures were incubated for 2 hr. At the end of this incubation, the culture medium was removed and replaced with 130 ml MTT desorb solution (0.7% SDS in isopropanol). The plate was shaken on a plate mixer for up to 2 hr. The absorbance was then measured using a SPECTRAmax 250 spectrophotometer at 570 nm (Molecular Devices Corp., USA). Chemicals The well-known teratogen all-trans retinoic acid (Sigma, USA), which causes speci®c developmental abnormalities in musculoskeletal and cardiovascular systems in vivo (Lammer et al., 1985), was used to assess the incubation time required for modulation of dierentiation, viability and inhibition of contracting myocardial cells. The chemicals (Table 1; Sigma, USA) were added to the culture medium for the full culture period. Five to six concentrations per chemical, with a 1.47±2-fold dilution factor, were chosen. These concentrations ranged from one which did not have a signi®cant eect on the dierentiation to one which completely decreased cell viability. A range®nder was performed prior to two runs with a narrowerrange trials. The medium containing the investigated compounds was refreshed on days 3 and 5. Working solutions were prepared freshly from the stocks and were used within 1 hr of dilution. If a solvent was used, the solvent concentration was kept constant. A non-solvent control group was included to con®rm non-toxicity of the solvent used. Compounds sensitive to photodegradation
Table 1. Results for the tested compounds using embryonic stem cells
Teratogenic status in vivo Strong
Weak/moderate Non
Chemicals 5-Fluorouracil Retinoic Acid Arabinoside Furanoside Hydroxyurea Diphenylhydantoin Caeine Isoniazid Pencillin G Saccharin
Cytotoxicity
Morphological inspection
IC50 D3
ID50
0.39 2 0.04 3.8 2 0.3 0.212 0.07 992 15 490 2 80 2400 2 100 5700 2 300 4500 2 800 26,8002 500
0.25 20.01* 0.30 20.01* 0.38 20.02 38 29* 74 22* 1600 2150 8300 23400 2700 2800 8300 2600*
Eect on MHC mRNMA expression IC50 a-MHC
IC50 b-MHC
0.25 20.03* 0.37 20.07* 0.41 20.02 42 210* 92 212* 1500 2290 14800 22000 6000 21300 10,700 22500*
0.22 20.01* 0.25 20.08* 0.43 20.02 44 211* 120 215* 1600 2380 15600 21600 6080 21400 13,200 23600*
Values (nm) are means 2 SE (n = 3); *Signi®cantly lower than the IC50 D3 concentration (PE 0.05, Student's t-test).
Embryotoxicological investigation with PCR analysis
621
(Iso, RA, PenG) were manipulated to avoid prolonged exposure to the light. RESULTS
Incubation time From kinetic analysis of MHC mRNA expression over a time period of 12 days in the presence of RA, MHC mRNA expression analysis and the morphological observation of contracting myocardial cells, made in parallel, gave the same conclusion, an incubation time of 10 days is required to reveal the full toxic potential of compounds (Plate 1, Fig. 1). This time period is used to screen the selected compounds. Screening test The chemicals show a cytotoxicity as determined by the MTT assay, in the range from 0.39 nM to 26.8 mM. The ranking in terms of the chemical toxicity was approximately the same as that noted for the down regulation of MHC mRNA expression and loss of contracting myocardial cells from the culture (Table 1). Six of the nine chemicals, 5-FU, RA, HU, DPH, Ca and Sacc, have eects on the MHC mRNA expression at concentrations lower than those which cause cytotoxicity (Table 1; Fig. 2). Iso, PenG and Ara-C appear to have no eects on MHC mRNA expression, or contracting myocardial cells, until the chemicals are applied at concentrations greater than the cytotoxic IC50 concentration (Fig. 2). DISCUSSION
The approach for identifying potential teratogens depends on the inhibition of tissue-speci®c mRNA expression. Nine test chemicals were selected from those recommended for evaluation in a teratogenicity validation trial (Smith et al., 1983). Two chemicals, RA and DPH, caused a range of defects in vivo, including eects on cardiac tissues (Geelen, 1979; Watkinson and Millicovski, 1983). Three of
Fig. 1. Cardiomyocyte development in dierentiating ES cells treated with all-trans retinoic acid (RA) dissolved in ethanol (EtOH), illustrated either by the morphological inspection (A), or by the ratio of a-MHC to HPRT (housekeeping gene) (B) or by the ratio of b-MHC to HPRT (C).
the teratogens have been shown to give multiple eects, either by alterations in dierentiation and growth (5-FU and HU; Dagg, 1960; Seller and Perkins, 1983), or by a generalized inhibition of growth (caeine; Piersma et al., 1993). Ara-C has a more restrictive eect (Ortega et al., 1991), while Sacc, Iso and PenG are not known to cause terato-
Plate 1. Representation of an electrophoresis gel of PCR products obtained from cells after 7 days of exposure to alltrans retinoic acid.
622
K. Bigot et al.
Fig. 2. Relationship between IC50 values determined via MTT assay on D3 cells and MHC mRNA expression analysis. Mean2SE, n = 3. According to the Student's t-test, IC50 a-MHC and IC50 b-MHC are signi®cantly lower (*P E0.05) than IC50 D3 for the following chemicals: 5-FU, RA, HU, DPH. Chemicals below the line of equality have a greater embryotoxic/teratogenic potential than cytotoxic potential.
genic/embryotoxic eects in mice (Boucher and Delost, 1964; Kalter, 1972; Tanaka, 1964). Sacc, however, is known to be carcinogenic in mice (Arnold, 1984). The six chemicals which were below the line of equality (Fig. 2) were predicted to have teratogenic/ embryotoxic potential, since the inhibition of mRNA occurred at doses signi®cantly lower than those causing cytotoxicity. RA and DPH appear well below the line, indicating their potentially strong teratogenic/embryotoxic eects on cardiac tissues. 5-FU, HU and Ca were also located below but closer to the line, indicating their general in vivo alteration in dierentiation and/or growth. Sacc, classi®ed in vivo as a non-teratogen, was located below the line, indicating a teratogenic/embryotoxic potential, which may re¯ect its carcinogenic eects. The strong teratogen Ara-C was not detected, which may re¯ect a dierent target tissue and indicates that analysis of other cell lineages is required. As downregulation of mRNA could also occur due to reduced viability of the cells when exposed to cytotoxic levels of the test agents, it is important to distinguish the embryotoxic/teratogenic concentration from the cytotoxic concentration. The loss of observable contracting myocardial cells also con®rmed the embryotoxic/teratogenic action on the cardiomyocytes. This assay system is potentially more speci®c and quanti®able than previous screening systems, for example the micromass assay (Flint, 1993) or the
generalized eects on stem cells (Newall and Beedles, 1994), The studies need to be extended so that teratogens that target other cell lineages can be identi®ed. Methods for dierentiating ES cells along other lineages exist (Doetschman et al., 1985). Such a testing approach should identify not only potential teratogens, but also their probable mode of action and target tissues. AcknowledgementsÐThis study was supported by the European Centre for the Validation of Alternative Methods (ECVAM) at the Joint Research Centre (JRC) of the EU in Ispra/Italy.
REFERENCES
Arnold D. L. (1984) Toxicology of saccharin. Fundamental and Applied Toxicology 4, 674±685. Boucher D. and Delost P. (1964) Developpement postnatal des descendants issus de meres traitees par la penicilline au cours de la gestation chez la souris. C.R. Societe de Biologie (Paris) 158, 528±532. Dagg C. P. (1960) Sensitive stages for the production of developmental abnormalities in mice with 5-¯uorouracil. American Journal of Anatomy 106, 89±96. Doetschman T. C., Eistetter H., Katz M., Schmidt W. and Kemler R. (1985) The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. Journal of Embryology and Experimental Morphology 87, 27±45. Flint O. P. (1993) In vitro tests for teratogens: desirable endpoints, test batteries and current status of the micromass teratogen test. Reproductive Toxicology 7, 103±11.
Embryotoxicological investigation with PCR analysis Geelen J. A. G. (1979) Hypervitaminosis A induced teratogenesis. CRC Critical Reviews in Toxicology 6, 351± 375. Kalter H. (1972) Nonteratogenicity of isoniazid in mice. Teratology 5, 259. Keller G., Kennedy M., Papayannopoulou T. and Wiles M. V. (1993) Hematopoietic commitment during embryonic stem cell dierentiation in culture. Molecular Cellular Biology 13, 473±486. Lammer E. J., Chen D. T., Hoar R. M., Agnish N. D., Benke P. J., Braun J. T., Femho P. M., Grix A. W., Jr, Lott I. T., Richard J. M. and Sun S. C. (1985) Retinoic acid embryopathy. New England Journal of Medicine 313, 837±841. Mosmann T. (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of Immunological Methods 65, 55±63. Newall D. R. and Beedles K. E. (1994) The stem cell testA novel in vitro assay for teratogenic potential. Toxicology in Vitro 8, 697±701. Ortega A., Puig M. and Domingo J. L. (1991) Maternal and developmental toxicity of low doses of cytosine arabinoside in mice. Teratology 44, 379±384. Piersma A. H., Haakmat A. S. and Hagenaars A. M. (1993) In vitro assays for the developmental toxicity of xenobiotic compounds using dierentiating embryonic carcinoma cells in culture. Toxicology in Vitro 7, 615±621. Robbins J., Gulick J., Sanchez A., Howles P. and Doetschman T. (1990) Mouse embryonic stem cells express the cardiac myosin heavy chain genes during
623
development in vitro. Journal of Biological Chemistry 265, 11905±11909. Rudnicki M. A. and McBurney M. W. (1987) Cell culture methods and induction of dierentiation of embryonal carcinoma cell lines. In Teratocarcinoma and Embryonic Stem Cells: A Practical Approach. Edited by E. J. Robertson, pp. 19±49. IRL Press, Washington, DC. Seller M. J. and Perkins K. J. (1983) Eect of Hydroxyurea on neural tube defects in the curly-tail mouse. Journal of Craniofacial Genetics and Developmental Biology 3, 11±17. Smith M. K., Kimmel G. L., Kochhar D. M., Shepard T. H., Spielberg S. P. and Wilson J. G. (1983) A selection of candidate compounds for in vitro teratogenesis test validation. Teratogenesis, Carcinogenesis, and Mutagenesis 3, 461±480. Tanaka R. (1964) LD50 of saccharin or cyclamate for mice embryos in the 7th day of pregnancy (fetal median lethal dose: FLD50). Journal of the Iwate Medicine Association 16, 330±337. Watkinson W. P. and Millicovsky G. (1983) Eect of phenytoin on maternal heart rate in A/J mice: possible role in teratogenesis. Teratology 28, 1±8. Williams R. C., Hilton D. J., Peace S., Willson T. A., Stewart C. C., Gearing D. P., Wagner E. F., Metcalf D., Nicola N. A. and Gough N. M. (1988) Myeloid leukaemia inhibitory factor maintains the development potential of embryonic stem cells. Nature 336, 684±687. Wobus A. M., Grosse R. and Schoeneich J. (1988) Speci®c eects of nerve growth factor on the dierentiation pattern of mouse embryonic stem cells in vitro. Biomedica et Biochimica Acta 47, 965±973.
The Relative Semi-quanti®cation of mRNA Expression as a Useful Toxicological Endpoint for the Identi®cation of Embryotoxic/Teratogenic Substances 1
K. BIGOT1,2,*, J. DE LANGE1, G. ARCHER1, R. CLOTHIER2 and S. BREMER1
ECVAM, JRC Institute of Health and Consumer Protection, Ispra, Italy and 2FRAME Alternatives Laboratory, School of Biomedical Sciences, Nottingham University, UK
AbstractÐEmbryonic stem cells, which resemble the undierentiated cells of the epiblast in a blastocyst, are able to dierentiate into derivatives of the primary germ layers, including cardiomyocytes. The eects of embryotoxic/teratogenic compounds on the dierentiation of cells was examined by semiquanti®cation of the mRNA expression using the RT±PCR protocol. Alpha and beta myosin heavy chain (a-MHC and b-MHC, respectively) mRNA expression were chosen as tissue-speci®c markers, characteristic of early cardiac muscle development. Nine chemicals were investigated, chosen according to their in vivo embryotoxic/teratogenic potential in mice. The teratogens all-trans retinoic acid (RA), 5¯uorouracil (5-FU), hydroxyurea (HU), diphenylhydantoin (DPH) and caeine (Ca) caused a signi®cant reduction in MHC mRNA expression at a dose lower than that required for cytotoxicity. Saccharin (Sacc) had a similar eect, while penicillin G (PenG), isoniazid (Iso) and cytarabine (Ara-C) only showed an eect on MHC mRNA expression when the cells had a signi®cant loss in viability. The inability to identify the strong teratogen Ara-C is related to the inappropriate target tissue. # 1999 Elsevier Science Ltd. All rights reserved Keywords: embryonic stem cells; teratogenicity; embryotoxicity; PCR analysis. Abbreviations: 5-FU = 5-¯uorouracil; Ara-C = cytarabine; Ca = caeine; DPH = diphenylhydantoin; ESC = embryonic stem cells; HU = hydroxyurea; Iso = isoniazid; MHC = myosin heavy chain; PenG = penicillin G; PBS = phosphate buered saline; RA = all-trans retinoic acid; RT±PCR = reverse transcriptase±polymerase chain reaction; Sacc = saccharin.
INTRODUCTION
The mouse D3 embryonic stem cells (ESC) are pluripotent, non-transformed cells derived from the inner cell mass of a mouse blastocyst. They have been shown to dierentiate in vitro into a wide variety of cell types such as cardiomyocytes, neurocytes and haematopoietic cells (Doetschman et al., 1985; Keller et al., 1993; Wobus et al., 1988). To obtain precise information of the eect of chemicals on developing tissues during embryonic stem cell dierentiation, quanti®cation of mRNA expression using the reverse transcriptase±polymerase chain reaction (RT±PCR) assay has been employed. *Corresponding author at: ECVAM TP 580, Institute for Health and Consumer Protection, Joint Research Centre, 21020 Ispra (VA) Italy.
Robbins et al. (1990) showed that the ES cells are capable of simulating aspects of cardiogenesis. They are able to undergo in vitro dierentiation and then show spontaneous contractile activity after day 7. Hence, the ES cells dierentiating into cardiomyocytes provide a model to study early cardiac muscle development and to perform embryotoxicological investigations, employing the analysis of a- and bmyosin heavy chain (MHC) gene expression, characteristic of atrial and ventricular cells during early embryonic heart development. To evaluate this approach, nine compounds were selected according to their in vivo embryotoxic/teratogenic potential in mice (non, weak/moderate or strong teratogenic). The cells were exposed to the test compounds, and the following endpoints were examined for their ability to distinguish potential
0887-2333/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. Printed in Great Britain PII: S0887-2333(99)00027-2
620
K. Bigot et al.
teratogens: (a) mRNA expression modulation; (b) general cell viability; and (c) presence of contracting myocardial cells.
MATERIALS AND METHODS
Analysis System (Fotodyn Inc., USA). The image analyser package NIH.Image 1.61 was then used to quantify and estimate the relative ratio between the tissue-speci®c and the non-speci®c gene signals to test the gene expression modulation. Cytotoxicity assay
Cell culture and induction of dierentiation The mouse D3 embryonic stem (ES) cells (ATCC Cat. No. CRL-1934) were maintained in the undifferentiated state by including myeloid leukaemia inhibitory factor (LIF; GIBCO Brl UK) at 1000 U/ ml in the culture media (Williams et al., 1988). To induce cardiomyocyte dierentiation by cell aggregation and produce so-called `embryoid bodies' (EBs), the hanging drop culture was used (Rudnicki and McBurney, 1987). Brie¯y, 20-ml samples containing 750 cells were placed in a hanging drop on the lid of a humidi®ed petri dish containing phosphate buered saline (PBS), this being considered as day 0 of the dierentiating state. After 3 days, the embryoid bodies (EBs) were resuspended in 5 ml medium in bacteriological petri dishes (Greiner GmbH, Germany) and cultured in suspension for a further 2 days. 5-day-old EBs were plated separately into individual wells of a 24-microwell plate for further dierentiation. Quanti®cation of mRNA 12 EBs were collected at each time interval for each test. The cells were lysed in a buer based on guanidinium thiocyanate salt and the cell extract was puri®ed for poly(A)+ mRNA using oligo(dT)cellulose anity. Poly(A)+ mRNA was reversetranscripted and ampli®ed by using oligonucleotide primers speci®c for a-MHC and b-MHC transcripts (Robbins et al., 1990). The housekeeping gene hypoxanthine phosphoribosyltransferase (HPRT) was used as an internal standard. The reverse transcription and the ampli®cation were performed according to the method of Robbins et al. (1991). The ampli®ed products were stained with ethidium bromide on an agarose gel. The running gels were TM photographed using FOTO/Analyst Image
Cells were incubated in the presence of the test substance for a period of 10 days, after which cytotoxicity was assessed using the MTT assay. Brie¯y, 20 ml MTT solution (5 mg/ml in PBS) was added to each well and the cultures were incubated for 2 hr. At the end of this incubation, the culture medium was removed and replaced with 130 ml MTT desorb solution (0.7% SDS in isopropanol). The plate was shaken on a plate mixer for up to 2 hr. The absorbance was then measured using a SPECTRAmax 250 spectrophotometer at 570 nm (Molecular Devices Corp., USA). Chemicals The well-known teratogen all-trans retinoic acid (Sigma, USA), which causes speci®c developmental abnormalities in musculoskeletal and cardiovascular systems in vivo (Lammer et al., 1985), was used to assess the incubation time required for modulation of dierentiation, viability and inhibition of contracting myocardial cells. The chemicals (Table 1; Sigma, USA) were added to the culture medium for the full culture period. Five to six concentrations per chemical, with a 1.47±2-fold dilution factor, were chosen. These concentrations ranged from one which did not have a signi®cant eect on the dierentiation to one which completely decreased cell viability. A range®nder was performed prior to two runs with a narrowerrange trials. The medium containing the investigated compounds was refreshed on days 3 and 5. Working solutions were prepared freshly from the stocks and were used within 1 hr of dilution. If a solvent was used, the solvent concentration was kept constant. A non-solvent control group was included to con®rm non-toxicity of the solvent used. Compounds sensitive to photodegradation
Table 1. Results for the tested compounds using embryonic stem cells
Teratogenic status in vivo Strong
Weak/moderate Non
Chemicals 5-Fluorouracil Retinoic Acid Arabinoside Furanoside Hydroxyurea Diphenylhydantoin Caeine Isoniazid Pencillin G Saccharin
Cytotoxicity
Morphological inspection
IC50 D3
ID50
0.39 2 0.04 3.8 2 0.3 0.212 0.07 992 15 490 2 80 2400 2 100 5700 2 300 4500 2 800 26,8002 500
0.25 20.01* 0.30 20.01* 0.38 20.02 38 29* 74 22* 1600 2150 8300 23400 2700 2800 8300 2600*
Eect on MHC mRNMA expression IC50 a-MHC
IC50 b-MHC
0.25 20.03* 0.37 20.07* 0.41 20.02 42 210* 92 212* 1500 2290 14800 22000 6000 21300 10,700 22500*
0.22 20.01* 0.25 20.08* 0.43 20.02 44 211* 120 215* 1600 2380 15600 21600 6080 21400 13,200 23600*
Values (nm) are means 2 SE (n = 3); *Signi®cantly lower than the IC50 D3 concentration (PE 0.05, Student's t-test).
Embryotoxicological investigation with PCR analysis
621
(Iso, RA, PenG) were manipulated to avoid prolonged exposure to the light. RESULTS
Incubation time From kinetic analysis of MHC mRNA expression over a time period of 12 days in the presence of RA, MHC mRNA expression analysis and the morphological observation of contracting myocardial cells, made in parallel, gave the same conclusion, an incubation time of 10 days is required to reveal the full toxic potential of compounds (Plate 1, Fig. 1). This time period is used to screen the selected compounds. Screening test The chemicals show a cytotoxicity as determined by the MTT assay, in the range from 0.39 nM to 26.8 mM. The ranking in terms of the chemical toxicity was approximately the same as that noted for the down regulation of MHC mRNA expression and loss of contracting myocardial cells from the culture (Table 1). Six of the nine chemicals, 5-FU, RA, HU, DPH, Ca and Sacc, have eects on the MHC mRNA expression at concentrations lower than those which cause cytotoxicity (Table 1; Fig. 2). Iso, PenG and Ara-C appear to have no eects on MHC mRNA expression, or contracting myocardial cells, until the chemicals are applied at concentrations greater than the cytotoxic IC50 concentration (Fig. 2). DISCUSSION
The approach for identifying potential teratogens depends on the inhibition of tissue-speci®c mRNA expression. Nine test chemicals were selected from those recommended for evaluation in a teratogenicity validation trial (Smith et al., 1983). Two chemicals, RA and DPH, caused a range of defects in vivo, including eects on cardiac tissues (Geelen, 1979; Watkinson and Millicovski, 1983). Three of
Fig. 1. Cardiomyocyte development in dierentiating ES cells treated with all-trans retinoic acid (RA) dissolved in ethanol (EtOH), illustrated either by the morphological inspection (A), or by the ratio of a-MHC to HPRT (housekeeping gene) (B) or by the ratio of b-MHC to HPRT (C).
the teratogens have been shown to give multiple eects, either by alterations in dierentiation and growth (5-FU and HU; Dagg, 1960; Seller and Perkins, 1983), or by a generalized inhibition of growth (caeine; Piersma et al., 1993). Ara-C has a more restrictive eect (Ortega et al., 1991), while Sacc, Iso and PenG are not known to cause terato-
Plate 1. Representation of an electrophoresis gel of PCR products obtained from cells after 7 days of exposure to alltrans retinoic acid.
622
K. Bigot et al.
Fig. 2. Relationship between IC50 values determined via MTT assay on D3 cells and MHC mRNA expression analysis. Mean2SE, n = 3. According to the Student's t-test, IC50 a-MHC and IC50 b-MHC are signi®cantly lower (*P E0.05) than IC50 D3 for the following chemicals: 5-FU, RA, HU, DPH. Chemicals below the line of equality have a greater embryotoxic/teratogenic potential than cytotoxic potential.
genic/embryotoxic eects in mice (Boucher and Delost, 1964; Kalter, 1972; Tanaka, 1964). Sacc, however, is known to be carcinogenic in mice (Arnold, 1984). The six chemicals which were below the line of equality (Fig. 2) were predicted to have teratogenic/ embryotoxic potential, since the inhibition of mRNA occurred at doses signi®cantly lower than those causing cytotoxicity. RA and DPH appear well below the line, indicating their potentially strong teratogenic/embryotoxic eects on cardiac tissues. 5-FU, HU and Ca were also located below but closer to the line, indicating their general in vivo alteration in dierentiation and/or growth. Sacc, classi®ed in vivo as a non-teratogen, was located below the line, indicating a teratogenic/embryotoxic potential, which may re¯ect its carcinogenic eects. The strong teratogen Ara-C was not detected, which may re¯ect a dierent target tissue and indicates that analysis of other cell lineages is required. As downregulation of mRNA could also occur due to reduced viability of the cells when exposed to cytotoxic levels of the test agents, it is important to distinguish the embryotoxic/teratogenic concentration from the cytotoxic concentration. The loss of observable contracting myocardial cells also con®rmed the embryotoxic/teratogenic action on the cardiomyocytes. This assay system is potentially more speci®c and quanti®able than previous screening systems, for example the micromass assay (Flint, 1993) or the
generalized eects on stem cells (Newall and Beedles, 1994), The studies need to be extended so that teratogens that target other cell lineages can be identi®ed. Methods for dierentiating ES cells along other lineages exist (Doetschman et al., 1985). Such a testing approach should identify not only potential teratogens, but also their probable mode of action and target tissues. AcknowledgementsÐThis study was supported by the European Centre for the Validation of Alternative Methods (ECVAM) at the Joint Research Centre (JRC) of the EU in Ispra/Italy.
REFERENCES
Arnold D. L. (1984) Toxicology of saccharin. Fundamental and Applied Toxicology 4, 674±685. Boucher D. and Delost P. (1964) Developpement postnatal des descendants issus de meres traitees par la penicilline au cours de la gestation chez la souris. C.R. Societe de Biologie (Paris) 158, 528±532. Dagg C. P. (1960) Sensitive stages for the production of developmental abnormalities in mice with 5-¯uorouracil. American Journal of Anatomy 106, 89±96. Doetschman T. C., Eistetter H., Katz M., Schmidt W. and Kemler R. (1985) The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. Journal of Embryology and Experimental Morphology 87, 27±45. Flint O. P. (1993) In vitro tests for teratogens: desirable endpoints, test batteries and current status of the micromass teratogen test. Reproductive Toxicology 7, 103±11.
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