Tetrahydrobiopterin (BH4) deficiency is caused by genetic abnormalities that impair its biosynthesis and recycling, which trigger neurochemical, metabolic, and redox imbalances. Low BH4 levels are also associated with hypoxia, reperfusion reoxygenation, endothelial dysfunction, and other conditions that are not genetically determined. The exact cause of changes in BH4 in nongenetic disorders is not entirely understood, but a role for oxidant species has been implicated. The oxidation of BH4 generates several products, including 7,8-dihydrobiopterin (BH2), the accumulation of which is predicted in cells with low dihydrofolate reductase activity. The relative efficiency of oxidant species at causing variations in BH4/BH2 levels in cells furnished with several antioxidant enzymes has not yet been systematically analyzed. This study examined the quantitative changes of BH4/BH2 in cells challenged with several oxidants. We showed that BH2 is not a major product of treatments with hydrogen peroxide or RSL3, as indicated by the moderate effect of dihydrofolate reductase-inhibitor methotrexate on the accumulation of BH2. However, we found a net loss in BH4/BH2, suggesting that products other than BH2 were generated. These reactions were further examined in NOX4-expressing HEK cells producing hydrogen peroxide. These cells showed slightly decreased BH4/BH2 ratios compared with HEK wild-type cells, and, again, methotrexate treatment moderately increased BH2 levels. In contrast, peroxynitrite-producing RAW 264.7 cells showed dramatically decreased BH4 levels without BH2 accumulation. Following the activation of peroxynitrite production with PMA in lipopolysaccharide-treated cells, we also found a significant time-dependent decline in GTPCH-I protein levels. We conclude that hydrogen peroxide is the least effective oxidant species at decreasing intracellular BH4 levels, while peroxynitrite is highly effective by targeting GTPCH-I and BH4 directly. Moreover, we conclude that BH4/BH2 levels are not a determinant of RSL3 cytotoxicity.

中文翻译：

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四氢生物蝶呤作为细胞对氧化剂损伤的抵抗力的变阻器


四氢生物蝶呤 （BH4） 缺乏症是由遗传异常引起的，这些异常会损害其生物合成和循环，从而引发神经化学、代谢和氧化还原失衡。低 BH4 水平还与缺氧、再灌注复氧、内皮功能障碍和其他非遗传决定的疾病有关。非遗传性疾病中 BH4 变化的确切原因尚不完全清楚，但已经涉及氧化剂物种的作用。BH4 的氧化产生多种产物，包括 7,8-二氢生物蝶呤 （BH2），在二氢叶酸还原酶活性低的细胞中预测其积累。氧化剂种类在具有几种抗氧化酶的细胞中引起 BH4/BH2 水平变化的相对效率尚未得到系统分析。本研究检查了 BH4/BH2 在受多种氧化剂攻击的细胞中的数量变化。我们表明 BH2 不是过氧化氢或 RSL3 处理的主要产物，正如二氢叶酸还原酶抑制剂甲氨蝶呤对 BH2 积累的中等影响所表明的那样。然而，我们发现 BH4/BH2 存在净损失，这表明产生了 BH2 以外的产品。在产生过氧化氢的 NOX4 表达 HEK 细胞中进一步检查了这些反应。与 HEK 野生型细胞相比，这些细胞显示 BH4/BH2 比率略有降低，并且甲氨蝶呤处理再次适度增加 BH2 水平。相比之下，产生过氧亚硝酸盐的 RAW 264.7 细胞显示 BH4 水平显着降低，而没有 BH2 积累。在脂多糖处理的细胞中用 PMA 激活过氧亚硝酸盐的产生后，我们还发现 GTPCH-I 蛋白水平显着的时间依赖性下降。 我们得出结论，过氧化氢是降低细胞内 BH4 水平最无效的氧化剂种类，而过氧亚硝酸盐通过直接靶向 GTPCH-I 和 BH4 非常有效。此外，我们得出结论，BH4/BH2 水平不是 RSL3 细胞毒性的决定因素。