Mitochondrial P-450 Levels Research Articles

Inhibition of adrenal steroid metabolism by administration of 1-aminobenzotriazole to guinea pigs.

Prior in vitro investigations demonstrated that the P450 suicide substrate, 1-aminobenzotriazole (ABT), was a potent inhibitor of xenobiotic metabolism but had no effect on steroidogenic enzymes in the guinea pig adrenal cortex. Studies were done to determine if ABT administration of guinea pigs in vivo also selectively inhibited adrenal xenobiotic metabolism. At single doses of 25 or 50 mg/kg, ABT effected rapid decreases in spectrally detectable adrenal P450 concentrations. The higher dose caused approx. 75% decreases in microsomal and mitochondrial P450 levels within 2 h. The decreases in P450 were sustained for 24 h but concentrations returned to control levels within 72 h. Accompanying the ABT-induced decreases in adrenal P450 content were proportionately similar decreases in P450-mediated xenobiotic and steroid metabolism. Microsomal benzo(a)pyrene hydroxylase, benzphetamine N-demethylase, 17 alpha-hydroxylase and 21-hydroxylase activities were decreased to 20-25% of control values by the higher dose of ABT. Mitochondrial 11 beta-hydroxylase and cholesterol sidechain cleavage activities were similarly diminished by ABT treatment. Adrenal 3 beta-hydroxysteroid dehydrogenase activity, by contrast, was not affected by ABT, indicating specificity for P450-catalyzed reactions. The results demonstrate that ABT in vivo is a non-selective inhibitor of adrenal steroid- and xenobiotic-metabolizing P450 isozymes. The absence of ABT effects on steroid metabolism in vitro suggests that an extra-adrenal metabolite may mediate the in vivo inhibition of steroidogenesis.

Differential effects of adrenocorticotropic hormone on steroid hydroxylase activities in the inner and outer zones of the guinea pig adrenal cortex

We have studied the effects of ACTH treatment on steroid hydroxylase activities in the inner (zona reticularis) and outer (zona fasciculata plus zona glomerulosa) zones of the guinea pig adrenal cortex. Animals received 5 or 10 U of ACTH daily for 6 days and enzyme activities were then assessed in isolated microsomal or mitochondrial preparations. In control animals, microsomal cytoochrome P-450 concentrations were greater in the inner than outer zone, but mitochondrial P-450 levels were similar in the two zones. Microsomal 17α-hydroxylase and mitochondrial 11β-hydroxylase activities were greater in the outer than inner zone, but microsomal 21-hydroxylase activity was greater in the inner zone. ACTH treatment decreased cytochrome P-450 concentrations in inner but not outer zone microsomes; mitochondrial P-450 levels were unaffected in both zones. ACTH caused a dose-dependent increase in inner zone 17α-hydroxylase activity and decrease in 21-hydroxylase activity without affecting the activity of either enzyme in outer zone microsomes. ACTH also decreased 11β-hydroxylase activity in outer but not inner zone mitochondrial preparations. The net effect of ACTH treatment was to diminish the differences in steroid metabolism between the two zones. The results indicate that the effects of ACTH on steroid hydroxylase activities are both zone- and enzyme-dependent, suggesting the existence of multiple and independent regulatory mechanisms.

Dose-dependent actions of spironolactone on the inner and outer zones of the guinea pig adrenal cortex.

The results of prior in vitro studies indicated that spironolactone (SL) caused far greater degradation of cytochromes P-450 in the outer (zona glomerulosa plus zona fasciculata) than inner (zona reticularis) zone of the guinea pig adrenal cortex and selectively decreased microsomal 17 alpha-hydroxylase activity. Studies were done to determine if the effects of SL in vivo were similarly zone and/or enzyme selective. Administration of high doses of SL (100 mg/kg) to guinea pigs altered the gross appearance of the adrenal glands and caused declines in 17 alpha- and 21-hydroxylase activities in both inner and outer zone microsomal preparations. The losses in enzyme activities were accompanied by decreases in microsomal cytochrome P-450, cytochrome b5 and heme concentrations, and in mitochondrial P-450 levels in both zones. Microsomal P-450(17 alpha) apoprotein levels were also decreased in both zones. A lower dose (25 mg/kg) of SL did not affect adrenal morphology, but decreased microsomal P-450 levels in both zones. Neither mitochondrial P-450 nor microsomal b5 concentrations were affected in either zone. 17 alpha-Hydroxylase activities and P-450(17 alpha) apoprotein concentrations in both zones were decreased by the lower dose of SL, but 21-hydroxylase activity declined in the inner zone only. The results indicate that very high doses of SL have a variety of nonspecific effects on the adrenals which may be the consequence of drug toxicity. Nontoxic doses exert more selective effects on microsomal cytochromes P-450 in both adrenal zones, more closely mimicking the in vitro actions of the drug.

Interaction of bovine renal mitochondrial cytochrome P-450 with antioxidants

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.