The effects of solvents on embryonic stem cell differentiation.

Dimethyl sulfoxide (DMSO) and ethanol are common organic solvents used for dissolving lipophilic substances for in vitro testing. However, DMSO is known to induce differentiation in embryonic stem (ES) and embryonic teratocarcinoma (EC) cells. In order to clarify if solvents like DMSO and ethanol have an influence on in vitro developmental toxicity test systems, the presented study has evaluated their effects on differentiation by using different test systems. ES and EC cells were transfected with a construct containing the mTert promoter combined with the green fluorescent protein gene (GFP). A down-regulation of mTert, a marker for undifferentiated cells, results in a lower expression of GFP, which could be measured by flow cytometry. Taking the specific characteristics of ES and EC cells into account this effect could be a hint for the interaction of DMSO with embryonic development. Additionally, the effects of the solvents ethanol and DMSO on Oct-4 expression, another marker for undifferentiated cells, were measured in wild-type ES cells. Both selected molecular markers demonstrated an induction of differentiation after exposure to DMSO; in wild-type ES cells at a concentration of 0.125% and in transgenic EC cells at a concentration of 0.25% DMSO. All other differences from controls, including those which attained a level of statistical significance, were minor or not dosage related in degree, or were not consistent over time and are, therefore, considered to be of little toxicological importance. In addition, a cytotoxicity test demonstrated that the solvents affected the employed molecular markers in non-cytotoxic concentrations. The ES cells were the most sensitive towards the cytotoxic effects of the solvent DMSO while the EC cells were more sensitive when treated with the solvent ethanol.

[1]  H. Spielmann,et al.  The embryonic stem cell test (EST), an in vitro embryotoxicity test using two permanent mouse cell lines : 3T3 fibroblasts and embryonic stem cells , 1997 .

[2]  Elke Genschow,et al.  Embryotoxicity Screening Using Embryonic Stem Cells in vitro: Correlation to in vivo Teratogenicity , 1999, Cells Tissues Organs.

[3]  E. Robertson.  Teratocarcinomas and embryonic stem cells : a practical approach , 1987 .

[4]  J. Fernandez-Solà,et al.  Ethanol inhibits skeletal muscle cell proliferation and delays its differentiation in cell culture. , 2000, Alcohol and alcoholism.

[5]  J. Whitfield,et al.  The differentiation inducer, dimethyl sulfoxide, transiently increases the intracellular calcium ion concentration in various cell types , 1993, Journal of cellular physiology.

[6]  H. Spielmann,et al.  Cytotoxicity test using blastocyst-derived euploid embryonal stem cells: a new approach to in vitro teratogenesis screening. , 1991, Reproductive toxicology.

[7]  L. Hoffman,et al.  Ethanol exposure stimulates cartilage differentiation by embryonic limb mesenchyme cells. , 1996, Experimental cell research.

[8]  S. Jacob,et al.  Pharmacology of DMSO. , 1986, Cryobiology.

[9]  A. Sartorelli,et al.  Differentiation of immortal cells inhibits telomerase activity. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[10]  K. Kitchin,et al.  Further development of rodent whole embryo culture: solvent toxicity and water insoluble compound delivery system. , 1984, Toxicology.

[11]  M. Welsh,et al.  A study of vehicles for dosing rodent whole embryo culture with non aqueous soluble compounds. , 2004, Reproductive toxicology.

[12]  J. Shay,et al.  Regulation of telomerase activity in immortal cell lines , 1996, Molecular and cellular biology.

[13]  C. Grindon.  The fourth EC report on laboratory animal use--is it sufficiently revealing? , 2005, Alternatives to laboratory animals : ATLA.

[14]  T. Hartung,et al.  The Detection of Differentiation-inducing Chemicals by using Green Fluorescent Protein Expression in Genetically Engineered Teratocarcinoma Cells , 2005, Alternatives to laboratory animals : ATLA.

[15]  H. Schöler,et al.  Octamania: the POU factors in murine development. , 1991, Trends in genetics : TIG.

[16]  M. Lako,et al.  mTert expression correlates with telomerase activity during the differentiation of murine embryonic stem cells , 2000, Mechanisms of Development.

[17]  E. Dmitrovsky,et al.  Telomerase activity is repressed during differentiation of maturation-sensitive but not resistant human tumor cell lines. , 1996, Cancer research.

[18]  G. Wallukat,et al.  Pluripotent mouse embryonic stem cells are able to differentiate into cardiomyocytes expressing chronotropic responses to adrenergic and cholinergic agents and Ca2+ channel blockers. , 1991, Differentiation; research in biological diversity.

[19]  J. Shay,et al.  Telomerase activity in human germline and embryonic tissues and cells. , 1996, Developmental genetics.

[20]  J. Kornfehl,et al.  Effects of ethanol treatment of proliferation and differentiation in a head and neck squamous cell carcinoma cell line. , 1999, Alcoholism, clinical and experimental research.

[21]  H. Schöler,et al.  Oct-4 transcription factor is differentially expressed in the mouse embryo during establishment of the first two extraembryonic cell lineages involved in implantation. , 1994, Developmental biology.

[22]  H. Schöler,et al.  Oct‐4: Gatekeeper in the Beginnings of Mammalian Development , 2001, Stem cells.

[23]  B. Fleischmann,et al.  Functional Characteristics of ES Cell–derived Cardiac Precursor Cells Identified by Tissue-specific Expression of the Green Fluorescent Protein , 1998, The Journal of cell biology.