Detection of the embryotoxic potential of cyclophosphamide by using a combined system of metabolic competent cells and embryonic stem cells

Altern Lab Anim. 2002 Jan-Feb;30(1):77-85. doi: 10.1177/026119290203000108.

Abstract

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.

MeSH terms

  • Animals
  • Cell Line
  • Cricetinae
  • Cricetulus
  • Cyclophosphamide / toxicity*
  • Cytochrome P-450 CYP2B1 / genetics
  • Cytochrome P-450 CYP2B1 / metabolism
  • Cytochrome P-450 Enzyme System / metabolism
  • Embryo, Mammalian*
  • False Negative Reactions
  • Gene Expression / drug effects
  • Green Fluorescent Proteins
  • Isoenzymes / metabolism
  • Luminescent Proteins / genetics
  • Lung
  • Microsomes, Liver
  • Oxazines / metabolism
  • Rats
  • Stem Cells / drug effects*
  • Toxicity Tests*
  • Transfection

Substances

  • Isoenzymes
  • Luminescent Proteins
  • Oxazines
  • Green Fluorescent Proteins
  • pentoxyresorufin
  • Cyclophosphamide
  • Cytochrome P-450 Enzyme System
  • Cytochrome P-450 CYP2B1