Di Renzo Francesca, Cappelletti Graziella, Broccia Maria L, Giavini Erminio, Menegola Elena
Department of Biology, University of Milan, Via Celoria, 26. 20133 Milan, Italy.
Toxicol Appl Pharmacol. 2007 Apr 15;220(2):178-85. doi: 10.1016/j.taap.2007.01.001. Epub 2007 Jan 12.
Histone deacetylases (HDAC) control gene expression by changing histonic as well as non histonic protein conformation. HDAC inhibitors (HDACi) are considered to be among the most promising drugs for epigenetic treatment for cancer. Recently a strict relationship between histone hyperacetylation in specific tissues of mouse embryos exposed to two HDACi (valproic acid and trichostatin A) and specific axial skeleton malformations has been demonstrated. The aim of this study is to verify if boric acid (BA), that induces in rodents malformations similar to those valproic acid and trichostatin A-related, acts through similar mechanisms: HDAC inhibition and histone hyperacetylation. Pregnant mice were treated intraperitoneally with a teratogenic dose of BA (1000 mg/kg, day 8 of gestation). Western blot analysis and immunostaining were performed with anti hyperacetylated histone 4 (H4) antibody on embryos explanted 1, 3 or 4 h after treatment and revealed H4 hyperacetylation at the level of somites. HDAC enzyme assay was performed on embryonic nuclear extracts. A significant HDAC inhibition activity (compatible with a mixed type partial inhibition mechanism) was evident with BA. Kinetic analyses indicate that BA modifies substrate affinity by a factor alpha=0.51 and maximum velocity by a factor beta=0.70. This work provides the first evidence for HDAC inhibition by BA and suggests such a molecular mechanism for the induction of BA-related malformations.
组蛋白去乙酰化酶（HDAC）通过改变组蛋白以及非组蛋白的蛋白质构象来控制基因表达。HDAC抑制剂（HDACi）被认为是癌症表观遗传治疗中最有前景的药物之一。最近已证明，暴露于两种HDACi（丙戊酸和曲古抑菌素A）的小鼠胚胎特定组织中的组蛋白高度乙酰化与特定的轴向骨骼畸形之间存在密切关系。本研究的目的是验证硼酸（BA）是否通过类似机制发挥作用：抑制HDAC和组蛋白高度乙酰化，因为BA在啮齿动物中诱导的畸形与丙戊酸和曲古抑菌素A相关的畸形相似。在妊娠第8天，给怀孕小鼠腹腔注射致畸剂量的BA（1000 mg/kg）。在处理后1、3或4小时取出的胚胎上，用抗高度乙酰化组蛋白4（H4）抗体进行蛋白质印迹分析和免疫染色，结果显示在体节水平存在H4高度乙酰化。对胚胎核提取物进行HDAC酶活性测定。BA具有明显的HDAC抑制活性（与混合型部分抑制机制相符）。动力学分析表明，BA使底物亲和力改变了α = 0.51倍，最大速度改变了β = 0.70倍。这项工作首次证明了BA对HDAC的抑制作用，并提出了BA相关畸形诱导的这种分子机制。
