Oxidative stress limits vitamin D metabolism by bovine proximal tubule cellsin vitro

Abstract

When bovine proximal tubule cells are placed in primary culture, they are subject to elevated oxidative stress which acts to limit the expression of mitochondrial vitamin D₃ 1α- and 24-hydroxylase activities. This increased oxidative stress was demonstrated by increased production of cell and mitochondrial membrane lipid hyperperoxides (LOOH). This increased production was prevented by the addition of the antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT). Cell and mitochondrial membrane LOOH increased from 1 to 2 pmol/mg protein on the day of plating to 70–90 pmol/mg protein after 6 days in culture. Pretreatment of cultures with BHA and BHT resulted in membrane LOOH of 15–20 pmol/mg protein after 6 days. Mitochondrial LOOH production was greater than total cell LOOH after 6 days. The increase in cellular oxidative stress was paralleled by decreases in both 1α- and 24-hydroxylase activities toward 25-OH D₃. Mitochondrial hydroxylase activities were inversely proportional to the increase in mitochondrial membrane LOOH production. Mitochondrial cytochrome P-450 content, determined spectrophotometrically, was decreased over time in culture. Mitochondrial cytochrome P-450 content determined by a specific polyclonal antibody in an enzyme-linked immunosorbant assay also decreased over time in culture. Specificity of polyclonal antibodies, raised against rat liver microsomal cytochrome P-450 RLM5, was demonstrated by the immunosequestration of both 1α- and 24-hydroxylase activities from a partially purified preparation of renal mitochondrial cytochrome P-450. BHA showed the loss of 1α- and 24-hydroxylase activities and mitochondrial P-450 content measured by all criteria. These experiments indicate that oxidative stress-mediated changes in hydroxylase activities are mediated directly by changes in hydroxylase content and not at distal sites. A partially purified preparation of bovine proximal tubule mitochondrial cytochrome P-450, with purified renal ferre-doxin, ferredoxin reductase, and NADPH, expressed both 1α- and 24-hydroxylase activities toward 25-OH D₃. LOOH, derived from mitochondrial membranes of 5-day-old cultures, when added to this mixture, caused a dose-dependent decrease in both activities. These experiments suggested that an increase in mitochondrial LOOH production resulted in a loss of 1α- and 24-hydroxylase activities. 1α-Hydroxylase was more sensitive to the effects of LOOH treatment than 24-hydroxylase. At a ratio of LOOH: P-450 of 5:1 (molar), all 1α-hydroxylase activity was lost but 50% of the 24-hydroxylase activity remained. At 10:1 LOOH: P-450 all hydroxylase activities were lost. Co-addition of BHA or BHT protected both hydroxylase activities against added LOOH. Previously, it had been demonstrated that antioxidants acted to stabilize, in primary culture, bovine proximal tubule 1α- and 24-hydroxylase activities (J. F. Crivello (1985) Arch. Biochem. Biophys.248, 551–561). This report suggests that antioxidant effects are twofold. Antioxidants can directly inhibit both hydroxylase activities and induce a Type II spectral response in partially purified preparations of renal mitochondrial cytochrome P-450. In addition, antioxidants can directly inhibit production of mitochondrial LOOH, and in the presence of LOOH, protect both hydroxylases against LOOH-mediated inactivation. The importance of LOOH to the maintenance of vitamin D metabolism in vivo and in vitro is discussed in light of these results.