Maurício Angelo Q, Lopes George K B, Gomes Cezar S, Oliveira Ricardo G, Alonso Antonio, Hermes-Lima Marcelo
Oxyradical Research Group, Departamento de Biologia Celular, Universidade de Brasília, Brazil.
Biochim Biophys Acta. 2003 Mar 17;1620(1-3):15-24. doi: 10.1016/s0304-4165(02)00502-0.
Previous work from our laboratory demonstrated that pyridoxal isonicotinoyl hydrazone (PIH) has in vitro antioxidant activity against iron plus ascorbate-induced 2-deoxyribose degradation due to its ability to chelate iron; the resulting Fe(III)-PIH(2) complex is supposedly unable to catalyze oxyradical formation. A putative step in the antioxidant action of PIH is the inhibition of Fe(III)-mediated ascorbate oxidation, which yields the Fenton reagent Fe(II) [Biochim. Biophys. Acta 1523 (2000) 154]. In this work, we demonstrate that PIH inhibits Fe(III)-EDTA-mediated ascorbate oxidation (measured at 265 nm) and the formation of ascorbyl radical (in electron paramagnetic resonance (EPR) studies). The efficiency of PIH against ascorbate oxidation, ascorbyl radical formation and 2-deoxyribose degradation was dose dependent and directly proportional to the period of preincubation of PIH with Fe(III)-EDTA. The efficiency of PIH in inhibiting ascorbate oxidation and ascorbyl radical formation was also inversely proportional to the Fe(III)-EDTA concentration in the media. When EDTA was replaced by the weaker iron ligand nitrilotriacetic acid (NTA), PIH was much more effective in preventing ascorbate oxidation, ascorbyl radical formation and 2-deoxyribose degradation. Moreover, the replacement of EDTA with citrate, a physiological chelator with a low affinity for iron, also resulted in PIH having a higher efficiency in inhibiting iron-mediated ascorbate oxidation and 2-deoxyribose degradation. These results demonstrate that PIH removes iron from EDTA (or from either NTA or citrate), forming an iron-PIH complex that cannot induce ascorbate oxidation effectively, thus inhibiting iron-mediated oxyradical formation. These results are of pharmacological relevance because PIH has been considered for experimental chelating therapy in iron-overload diseases.
我们实验室之前的研究表明，吡哆醛异烟酰腙（PIH）具有体外抗氧化活性，可抵抗铁加抗坏血酸诱导的2-脱氧核糖降解，这是因为它具有螯合铁的能力；由此产生的Fe(III)-PIH(2)复合物据推测无法催化氧自由基的形成。PIH抗氧化作用的一个假定步骤是抑制Fe(III)介导的抗坏血酸氧化，该氧化反应会产生芬顿试剂Fe(II)[《生物化学与生物物理学报》1523 (2000) 154]。在本研究中，我们证明PIH可抑制Fe(III)-EDTA介导的抗坏血酸氧化（在265 nm处测量）以及抗坏血酸自由基的形成（在电子顺磁共振（EPR）研究中）。PIH对抗坏血酸氧化、抗坏血酸自由基形成和2-脱氧核糖降解的效率呈剂量依赖性，且与PIH与Fe(III)-EDTA的预孵育时间成正比。PIH抑制抗坏血酸氧化和抗坏血酸自由基形成的效率也与培养基中Fe(III)-EDTA的浓度成反比。当用较弱的铁配体次氮基三乙酸（NTA）取代EDTA时，PIH在防止抗坏血酸氧化、抗坏血酸自由基形成和2-脱氧核糖降解方面更有效。此外，用对铁亲和力低的生理性螯合剂柠檬酸盐取代EDTA，也导致PIH在抑制铁介导的抗坏血酸氧化和2-脱氧核糖降解方面具有更高的效率。这些结果表明，PIH从EDTA（或从NTA或柠檬酸盐）中去除铁，形成一种不能有效诱导抗坏血酸氧化的铁-PIH复合物，从而抑制铁介导的氧自由基形成。这些结果具有药理学相关性，因为PIH已被考虑用于铁过载疾病的实验性螯合治疗。
