Crivello J F
Arch Biochem Biophys. 1986 Aug 1;248(2):551-61. doi: 10.1016/0003-9861(86)90508-4.
The antioxidants butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), nordihydroguiaretic acid (NDGA), benzyl sulfoxide (BS), ferulic acid (FA), caffeic acid (CA), dimethyl sulfoxide (Me2SO), protocatechuic acid (PCA), and P-450 inhibitor metyrapone all acted to slow the previously noted loss of vitamin D3 1 alpha-and 24-hydroxylase activities in cultured bovine proximal tubule cells. The slowing of the loss of hydroxylase activities by antioxidants was increased by culturing cells in 5% O2 vs 19% O2. These same antioxidants also directly inhibited 1 alpha- and 24-hydroxylase activities. For a single antioxidant, or metyrapone, Ki's for inhibition of both hydroxylases were equal, ED50's for stabilization of both hydroxylase activities were equal, and Ki's and ED50's were not significantly different. These antioxidants prevented tert-butylhydroperoxide (tert-BOOH)-mediated proximal tubule cell death at concentrations, i.e., 0.1 mM, which were effective in stabilizing hydroxylase activities. When added together, the antioxidants H2SeO3, uric acid, and trolox c gave slight stabilization of hydroxylase activities without inhibiting hydroxylase activities. Singly, these antioxidants did not stabilize or directly inhibit hydroxylase activities. This antioxidant combination augmented BHA- or BHT-mediated stabilization of both hydroxylase activities independent of any effects on inhibition. But the most potent antioxidants which acted to stabilize hydroxylase activities in culture also directly acted to inhibit hydroxylase activities. Antioxidant effects were additive for both inhibition and stabilization of hydroxylase activities. Stabilization of hydroxylase activities was dissociated from inhibition in the presence of maximal FA, CA, and BHA or FA, CA, and BHT combinations. Bovine renal mitochondrial cytochrome P-450 levels decreased in cultured bovine proximal tubule cells to nondetectable levels by 8 days in culture. When cultures were treated with BHA and BS, mitochondrial P-450 levels were almost twofold greater than in untreated controls. Percentage changes in mitochondrial P-450 levels closely paralleled percentage changes in hydroxylase activities elicited by antioxidant treatment regimes. Antioxidants which were effective inhibitors of hydroxylase activities in cultured bovine proximal tubule cells were also effective in inhibiting hydroxylase activities in isolated proximal tubule mitochondria, supplemented with a NADPH-generating source. Ki's for inhibition of hydroxylase activities were very similar in cultured cells and in isolated mitochondria.(ABSTRACT TRUNCATED AT 400 WORDS)
抗氧化剂丁基羟基茴香醚（BHA）、丁基羟基甲苯（BHT）、去甲二氢愈创木酸（NDGA）、苄基亚砜（BS）、阿魏酸（FA）、咖啡酸（CA）、二甲基亚砜（Me2SO）、原儿茶酸（PCA）以及P - 450抑制剂甲吡酮，均能减缓培养的牛近端肾小管细胞中先前所述的维生素D3 1α - 羟化酶和24 - 羟化酶活性的丧失。与在19%氧气环境中培养细胞相比，在5%氧气环境中培养细胞时，抗氧化剂对羟化酶活性丧失的减缓作用增强。这些相同的抗氧化剂还直接抑制1α - 羟化酶和24 - 羟化酶的活性。对于单一抗氧化剂或甲吡酮而言，抑制两种羟化酶的半数抑制浓度（Ki）相等，稳定两种羟化酶活性的半数有效浓度（ED50）相等，且Ki和ED50无显著差异。这些抗氧化剂在0.1 mM的浓度下可预防叔丁基过氧化氢（tert - BOOH）介导的近端肾小管细胞死亡，该浓度对稳定羟化酶活性有效。抗氧化剂亚硒酸（H2SeO3）、尿酸和生育三烯酚（trolox c）共同添加时，可轻微稳定羟化酶活性而不抑制其活性。单独使用时，这些抗氧化剂既不能稳定也不能直接抑制羟化酶活性。这种抗氧化剂组合可增强BHA或BHT介导的两种羟化酶活性的稳定作用，且与对抑制作用的任何影响无关。但在培养中最有效地稳定羟化酶活性的抗氧化剂也直接抑制羟化酶活性。抗氧化剂对羟化酶活性的抑制和稳定作用具有加和性。在存在最大量的FA、CA与BHA或FA、CA与BHT组合时，羟化酶活性的稳定作用与抑制作用分离。培养的牛近端肾小管细胞中，牛肾线粒体细胞色素P - 450水平在培养8天时降至无法检测的水平。用BHA和BS处理培养物时，线粒体P - 450水平几乎是未处理对照的两倍。线粒体P - 450水平的百分比变化与抗氧化剂处理方案引起的羟化酶活性百分比变化密切平行。在培养的牛近端肾小管细胞中有效抑制羟化酶活性的抗氧化剂，在补充了NADPH生成源的分离近端肾小管线粒体中也能有效抑制羟化酶活性。在培养细胞和分离线粒体中，抑制羟化酶活性的Ki非常相似。（摘要截断于400字）
