Bruce-Gregorios J H, Agarwal R P, Oracion A, Ramirez A, Lin L
Department of Pathology, University of Miami School of Medicine, FL 33101.
J Neuropathol Exp Neurol. 1991 Nov;50(6):770-8. doi: 10.1097/00005072-199111000-00008.
There is increasing evidence to indicate that astrocytes are primary targets for methotrexate (MTX) neurotoxicity. However, the mechanism by which MTX exerts its deleterious effect on astroglial cells is not known. Methotrexate acts by inhibiting dihydrofolate reductase and in other cell systems has been reported to inhibit thymidylate synthesis, purine synthesis or both. To determine the mechanism involved in MTX-induced toxicity to the nervous system, RNA synthesis was studied in two week-old primary astrocyte cultures by measuring [3H]Uridine (Urd) incorporation 24 hours after exposure to varying concentrations of MTX. De novo purine synthesis was also studied by measuring incorporation of [14C]glycine and [14C]formate in cultured astrocytes. The radioactivity level of incorporated Urd in culture decreased to 48%, 53% and 43% after exposure to 1, 10 and 100 microM MTX. Total [14C]glycine incorporation was not affected while incorporation of [14C]formate was almost completely inhibited by MTX. The MTX-induced inhibition of [3H]Urd incorporation was not reversed by concomitant addition of exogenous purine bases (1 and 10 microM adenine, guanine and hypoxanthine) or nucleosides (1 and 10 microM adenosine, guanosine and inosine) to the MTX-treated cultures. On the other hand, addition of formyl-tetrahydrofolate reversed the MTX-induced reduction in [3H]Urd incorporation, indicating that the RNA inhibition was due to depletion of folate-dependent substrates for purine synthesis. Our results provide evidence that inhibition of purine and RNA synthesis may be the underlying mechanism involved in MTX-induced injury to the astrocytes, and may be important in the pathogenesis of MTX encephalopathy.
越来越多的证据表明，星形胶质细胞是甲氨蝶呤（MTX）神经毒性的主要靶点。然而，MTX对星形胶质细胞产生有害作用的机制尚不清楚。甲氨蝶呤通过抑制二氢叶酸还原酶发挥作用，并且在其他细胞系统中据报道可抑制胸苷酸合成、嘌呤合成或两者。为了确定MTX诱导的神经系统毒性所涉及的机制，通过在暴露于不同浓度的MTX 24小时后测量[3H]尿苷（Urd）掺入，对两周大的原代星形胶质细胞培养物中的RNA合成进行了研究。还通过测量培养的星形胶质细胞中[14C]甘氨酸和[14C]甲酸的掺入来研究嘌呤的从头合成。暴露于1、10和100微摩尔/升MTX后，培养物中掺入的Urd放射性水平分别降至48%、53%和43%。总[14C]甘氨酸掺入不受影响，而[14C]甲酸的掺入几乎被MTX完全抑制。MTX诱导的[3H]Urd掺入抑制在MTX处理的培养物中同时添加外源性嘌呤碱（1和10微摩尔/升腺嘌呤、鸟嘌呤和次黄嘌呤）或核苷（1和10微摩尔/升腺苷、鸟苷和肌苷）后并未逆转。另一方面，添加甲酰四氢叶酸可逆转MTX诱导的[3H]Urd掺入减少，表明RNA抑制是由于嘌呤合成中叶酸依赖性底物的耗竭。我们的结果提供了证据，即嘌呤和RNA合成的抑制可能是MTX诱导星形胶质细胞损伤的潜在机制，并且可能在MTX脑病的发病机制中起重要作用。
