[Folic acid antagonist methotrexate causes the development malformation of heart and down-regulates the BMP2b and HAS2 expressions in zebrafish].
OBJECTIVE
To study the effect of methotrexate (MTX), a folic acid antagonist which can lead to folic acid deficient, on the cardiac development and on the expressions of BMP2b and HAS2 in zebrafish.


METHODS
The zebrafish embryos at 6-48 hrs post fertilization (hpf) were treated with various concentrations of MTX (0.5 x 10(-3), 1.0 x 10(-3) and 2.0 x 10(-3) M). At 48 hpf, the percentage of cardiac malformation and heart rate were recorded. The zebrafish embryos at 6-10 hpf treated with 1.5 x 10(-3) M MTX were used as the MTX treatment group. At 24 and 48 hpf the cardiac morphology was observed under a microscope. The expressions of BMP2b and HAS2 in zebrafish were detected by in situ antisense RNA hybridization and real-time PCR.


RESULTS
6-12 hpf, the early embryonic developmental stage, was a sensitive period that MTX affected cardiac formation of zebrafish. The retardant cardiac development and the evidently abnormal cardiac morphology was found in the MTX treatment group. The results of in situ antisense RNA hybridization showed that the expressions of BMP2b and HAS2 in the zebrafish heart were reduced in the MTX treatment group at 36 and 48 hpf. The real-time PCR results demonstrated that the BMP2b expression decreased at 12, 24, 36 and 48 hpf, and that the HAS2 expression decreased at 24, 36 and 48 hpf in the treatment group compared with the control group without MTX treatment.


CONCLUSIONS
The inhibition of folic acid function may affect cardiac development of early embryos, resulting in a retardant development and a morphological abnormality of the heart in zebrafish, possibly by down-regulating the expressions of BMP2b and HAS2.
Sun
Shu-Na
SN
Children's Hospital of Fudan University, Shanghai, China.
Gui
Yong-Hao
YH
Song
Hou-Yan
HY
Zhong
Tao
T
Wang
Yue-Xiang
YX
Jiang
Qiu
Q
D000014:Abnormalities, Drug-Induced / Q000209:etiology*; D000818:Animals; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000235:genetics*; D015536:Down-Regulation; D005493:Folic Acid Antagonists / Q000633:toxicity*; D005786:Gene Expression Regulation / Q000187:drug effects*; D014453:Glucuronosyltransferase / Q000235:genetics*; D006330:Heart Defects, Congenital / Q000139:chemically induced*; D000076002:Hyaluronan Synthases; D008727:Methotrexate / Q000633:toxicity*; D016133:Polymerase Chain Reaction; D015027:Zebrafish; D029961:Zebrafish Proteins / Q000235:genetics*
D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D005493:Folic Acid Antagonists; D029961:Zebrafish Proteins; C405936:bmp2b protein, zebrafish; D014453:Glucuronosyltransferase; D000076002:Hyaluronan Synthases; C491095:has2 protein, zebrafish; D008727:Methotrexate