Detection of the embryotoxic potential of cyclophosphamide by using a combined system of metabolic competent cells and embryonic stem cells.
In order to develop a method for detecting metabolism-mediated embryotoxicity, differentiating embryonic stem (ES) cells were exposed to the well-known proteratogen, cyclophosphamide (CPA). CPA was tested in a scientifically validated embryonic stem-cell test (EST), and in the newly developed reporter-gene assay for developmental cardiotoxicity. Both assays gave false-negative results. Because no metabolic competence (cytochrome P450 [CYP] activity) was found in the ES cells under the selected culture conditions, a simple biotransformation system was combined with the reporter-gene assay. As the metabolic pathway of CPA is well characterised, the genetically engineered mammalian cell line V79, transfected with CYP2B1 cDNA, was selected as a biotransformation system. CYP2B1 is responsible for transforming CPA into teratogenically active metabolites. The supernatants of genetically engineered V79 cells were analysed in the reporter-gene assay for developmental cardiotoxicity. In preliminary experiments, the combined system was able to detect the embryotoxic potential of the proteratogen, CPA.
Bremer
Susanne
S
ECVAM, Institute for Health & Consumer Protection, European Commission Joint Research Centre, 21020 Ispra (VA), Italy.
Pellizzer
Christian
C
Coecke
Sandra
S
Paparella
Martin
M
Catalani
Paolo
P
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D003520:Cyclophosphamide / Q000633:toxicity*; D019362:Cytochrome P-450 CYP2B1 / Q000235:genetics / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004622:Embryo, Mammalian; D005188:False Negative Reactions; D015870:Gene Expression / Q000187:drug effects; D049452:Green Fluorescent Proteins; D007527:Isoenzymes / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D008168:Lung; D008862:Microsomes, Liver; D010078:Oxazines / Q000378:metabolism; D051381:Rats; D013234:Stem Cells / Q000187:drug effects*; D018675:Toxicity Tests; D014162:Transfection
D007527:Isoenzymes; D008164:Luminescent Proteins; D010078:Oxazines; D049452:Green Fluorescent Proteins; C044943:pentoxyresorufin; D003520:Cyclophosphamide; D003577:Cytochrome P-450 Enzyme System; D019362:Cytochrome P-450 CYP2B1