Influence of the antifolate drug Methotrexate on the development of murine neural tube defects and genomic instability.
Impaired folate metabolism is considered a risk factor for neural tube defects (NTDs). However, the relationship between folate deficiency and the risk of NTDs remains unclear, because experimentally induced dietary folate deficiency is insufficient to cause NTDs in non-mutant mice. Methotrexate (MTX) is a specific folate antagonist that competitively inhibits dihydrofolate reductase (DHFR) activity. The objective of this study was to develop a folate dysmetabolism murine model, and study the development of NTDs and its mechanism. Pregnant mice were injected with different doses of MTX [0, 0.5, 1.0, 3.0, 4.5 and 6.0 mg kg(-1) body weight (b/w) intraperitoneally (i.p.)] on gestational day 7.5 and sacrificed on gestational day 11.5. DHFR activity in embryonic tissues was detected, and folate concentrations were analyzed using LC/MS/MS. Copy number variations (CNVs) in neural tube tissues were detected using array comparative genomic hybridization (aCGH). A dose of MTX 4.5 mg kg(-1) b/w, resulted in the highest incidence of NTDs (31.4%) compared with the other groups, and DHFR activities, 5-MeTHF and 5-FoTHF concentrations in embryonic tissues decreased significantly after MTX injection. Furthermore, we found three high-confidence CNVs on chromosome X using aCGH, which was confirmed by RT-PCR and MassARRAY. These results indicate that MTX could cause a folate-associated dysmetabolism, which is similar to that of dietary folate deficiency in mice. The presence of CNVs in neural tube tissues was associated with the development of NTDs.
Department of Biochemistry and Molecular Biology, Shanxi Medical University, Taiyuan 030001, China.
Jie
J
Zhao
Tao
T
Guan
Jianhua
J
Wang
Qian
Q
Xiang
Mingsheng
M
Wang
Xiuwei
X
Wang
Zhen
Z
Guan
Qiu
Q
Xie
Bo
B
Niu
Ting
T
Zhang
D000014:Abnormalities, Drug-Induced / Q000209:etiology / Q000473:pathology; D000818:Animals; D002853:Chromatography, Liquid; D055028:Comparative Genomic Hybridization; D056915:DNA Copy Number Variations / Q000187:drug effects; D004032:Diet; D004305:Dose-Response Relationship, Drug; D047108:Embryonic Development / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000378:metabolism; D005260:Female; D005492:Folic Acid / Q000378:metabolism; D005493:Folic Acid Antagonists / Q000008:administration & dosage / Q000633:toxicity*; D005494:Folic Acid Deficiency / Q000378:metabolism / Q000473:pathology; D042822:Genomic Instability / Q000187:drug effects*; D008297:Male; D008727:Methotrexate / Q000008:administration & dosage / Q000633:toxicity*; D051379:Mice; D008810:Mice, Inbred C57BL; D054259:Neural Tube / Q000187:drug effects / Q000196:embryology / Q000473:pathology; D009436:Neural Tube Defects / Q000139:chemically induced / Q000196:embryology / Q000473:pathology*; D011247:Pregnancy; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism
D004791:Enzyme Inhibitors; D005493:Folic Acid Antagonists; D005492:Folic Acid; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate