Use of two validated in vitro tests to assess the embryotoxic potential of mycophenolic acid.
Mycophenolate mofetil is a widely used immunosuppressive drug that recently has been categorized as a human teratogen. Animal experiments indicate a teratogenic potential of the drug, but so far, it has not been studied in embryotoxicity in vitro assays. The aim of this study was to evaluate the in vitro embryotoxic potential of mycophenolic acid and investigate the ability of such tests to detect its embryotoxic potential. We used two validated assays: the rat whole embryo culture and the murine embryonic stem cell test. Rat embryos cultured from gestational day 9.5 for 48 h with the drug showed dysmorphogenic development already at a concentration of 250 microg mycophenolic acid/l medium. At concentrations of 750 microg/l and more, all rat embryos exhibited malformations. The main effects were defective yolk sac blood circulation, neural tube defects (open cranial neural pore), malformations of the head with missing eye anlagen and heart anomalies. Moreover, the exposed embryos showed a concentration-dependent decrease in protein content, crown-rump length, number of somites and morphological score. The murine embryonic stem cell test was slightly more sensitive. Proliferation and differentiation of the ES-D3-cells were significantly impaired at concentrations of 31 and 125 microg mycophenolic acid/l medium, respectively. In the differentiation assay, at a concentration of 125 microg mycophenolic acid/l medium and more, the number of wells with differentiated cardiomyocytes significantly decreased. Additionally, a cytotoxicity assay with balb/c 3T3 mouse fibroblasts was used to compare the proliferation and vitality of embryonic cells with adult cells. In the balb/c 3T3 cytotoxicity assay, the number of vital mouse fibroblasts significantly decreased at a mycophenolic acid concentration of 62 microg/l and more. In conclusion, by using the two validated in vitro tests, we showed that mycophenolic acid exhibits a pronounced embryotoxic potential at cytotoxic concentrations. This result from validated in vitro tests is of special interest, because it supports the use of the tests to detect human teratogens.
Institute of Clinical Pharmacology and Toxicology, Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, Garystr. 5, 14195, Berlin, Germany.
Kathrin
K
Eckardt
Ralf
R
Stahlmann
D000818:Animals; D000853:Anophthalmos / Q000139:chemically induced; D041702:BALB 3T3 Cells; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004622:Embryo, Mammalian / Q000033:anatomy & histology / Q000187:drug effects*; D047108:Embryonic Development / Q000187:drug effects*; D053595:Embryonic Stem Cells / Q000187:drug effects*; D006330:Heart Defects, Congenital / Q000139:chemically induced; D007166:Immunosuppressive Agents / Q000633:toxicity*; D066298:In Vitro Techniques; D051379:Mice; D009173:Mycophenolic Acid / Q000633:toxicity*; D009436:Neural Tube Defects / Q000139:chemically induced; D051381:Rats; D017208:Rats, Wistar; D013723:Teratogens / Q000633:toxicity*; D018675:Toxicity Tests / Q000379:methods*
D007166:Immunosuppressive Agents; D013723:Teratogens; D009173:Mycophenolic Acid