Interaction of bovine renal mitochondrial cytochrome P-450 with antioxidants

Abstract

The antioxidants butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), nordihydroguiaretic acid (NDGA), benzyl sulfoxide (BS), ferulic acid (FA), caffeic acid (CA), dimethyl sulfoxide (Me 2SO), protocatechuic acid (PCA), and P-450 inhibitor metyrapone all acted to slow the previously noted loss of vitamin D 3 1α- 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% O 2 vs 19% O 2. These same antioxidants also directly inhibited 1α- and 24-hydroxylase activities. For a single antioxidant, or metyrapone, K i 's for inhibition of both hydroxylases were equal, ED 50's for stabilization of both hydroxylase activities were equal, and K i 's and ED 50's were not significantly different. These antioxidants prevented tert-butylhydroperoxide ( tert-BOOH)-mediated proximal tubule cell death at concentrations i.e., 0.1 m m, which were effective in stabilizing hydroxylase activities. When added together, the antioxidants H 2SeO 3, 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. K i 's for inhibition of hydroxylase activities were very similar in cultured cells and in isolated mitochondria. Antioxidants were shown to inhibit hydroxylase activities, in isolated mitochondria, in a competitive manner by analysis of experiments which varied substrate and antioxidant concentrations. The importance of antioxidants to the maintenance of vitamin D 3 metabolism in vitro is discussed as is relevance of these findings to maintenance of in vivo renal mitochondrial vitamin D 3 metabolism.