Mitochondrial Cytochrome P-450 Content Research Articles

Identification of heterotrimeric G protein α subunits in MC3T3-E1 osteoblastic cells

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.

The influence of NADPH-dependent lipid peroxidation on the progesterone biosynthesis in human placental mitochondria

In an in vitro system consisting of human term placental mitochondria and an NADPH-generating system plus Fe 2+, significant lipid peroxidation was observed along with a concomitant inhibition of progesterone biosynthesis. This inhibition could be markedly blocked by Mn 2+, superoxide dismutase and dimethylfuran, inhibitors of NADPH-dependent lipid peroxidation. In addition, it has been found that malondialdehyde formation is accompanied by a corresponding decrease in placental mitochondrial cytochrome P-450 content. Inhibitors of lipid peroxidation also prevent the loss of cytochrome P-450, further demonstrating a direct relationship between NADPH-dependent lipid peroxidation and degradation of cytochrome P-450 in cell-free systems. These measurements provide the first evidence that the inhibition of progesterone biosynthesis by a NADPH-dependent lipid peroxidation in placental mitochondria is a consequence of cytochrome P-450 degradation due to lipid peroxidation.

Human Chorionic Gonadotropin Treatment Prevents Depressed 17α-Hydroxylase/C17−20 Lyase Activities and Serum Testosterone Concentrations in 23,7,8-Tetrach Iorodibenzo-p-Dioxin-Treated Rats1

It is known that administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes decreased serum testosterone concentrations in the rat. Previous studies in this laboratory have shown that in rats TCDD exposure results in decreased 17 alpha-hydroxylase and C17-20 lyase activities. The decreases in these activities paralleled decreases in testicular microsomal heme and cytochrome P450 contents. As reported herein, neither testicular mitochondrial cytochrome P450 content nor the activity of cholesterol side-chain cleavage was altered in rats exposed to TCDD. Since the production of testosterone in the testis is dependent on LH, it is important to determine the early effects of TCDD on serum LH concentrations in the rat. Male Sprague-Dawley rats were given a single, oral dose of TCDD (50 micrograms/kg). Serum LH concentrations were determined by RIA on Days 1, 2, 3, 5, and 7 following TCDD treatment. Rat serum LH concentrations were decreased to 60% of controls as early as Day 1 and continued to be depressed on Days 2 and 3 at 53% and 59% of control values, respectively. Rat serum LH returned to control values by Day 5 in spite of continued depression of serum testosterone concentrations. The early depression in serum LH levels caused by TCDD may be related to the subsequent androgenic deficiency in the rat. Treatment of rats with hCG was found to be able to prevent the depression of the activities of testicular microsomal 17 alpha-hydroxylase and C17-20 lyase and serum testosterone concentrations caused by TCDD. These data indicate that TCDD decreases serum testosterone by decreasing P450(17 alpha) and C17-20 but not P450sec activities and that hCG treatment prevents the TCDD-induced decrease.

Regulation of progesterone biosynthesis by estrogen during baboon pregnancy: placental mitochondrial cholesterol side-chain cleavage activity in antiestrogen (ethamoxytriphetol, MER-25)-treated baboons.

We determined whether the reduction in placental progesterone (P4) production observed after administration of the antiestrogen ethamoxytriphetol (MER-25) to pregnant baboons was associated with a decline in activity and/or content of the placental mitochondrial cholesterol side-chain cleavage system (P-450scc). Pregnant baboons (Papio anubis) were untreated (n = 9) or administered MER-25 (25 mg/kg BW, orally; n = 6) daily on days 140-170 of gestation (term = 184 days). Placentas were obtained by cesarean section on day 170 of gestation, and P-450scc activity and cytochrome P-450 content were determined on mitochondria-rich fractions. Administration of MER-25 to pregnant baboons resulted in a 40% reduction (P less than 0.01, by Student's t test) in the mean (+/- SE) peripheral serum P4 concentration (6.3 +/- 0.3 ng/ml) compared to that in untreated (10.4 +/- 0.3 ng/ml) baboons. P-450scc activity, as determined by formation of pregnenolone (P5) and P4 during a 30-min incubation (picomoles per mg protein), was 37% lower (P less than 0.01) in placental mitochondria obtained from MER-25-treated baboons (179.8 +/- 25.0) than in that from untreated (285.4 +/- 13.4) baboons. Mitochondrial cytochrome P-450 content, assessed by spectral analysis, was 28% lower (P less than 0.02) in antiestrogen-treated (46.7 +/- 2.1 pmol/mg protein) than in untreated (64.8 +/- 5.2 pmol/mg protein) baboons. The initial (time zero) free cholesterol content (nanomoles per mg protein) of mitochondrial-rich preparations was not significantly different in antiestrogen-treated (189.3 +/- 13.0) and untreated (225.0 +/- 15.1) animals. Collectively, these results suggest that the decline in placental P4 production observed in baboons in response to MER-25 occurs at least in part as a result of a decrease in cytochrome P-450scc activity. The loss in P-450scc activity appears to be an intramitochondrial event and not a result of depletion of the total mitochondrial cholesterol pool. We propose, therefore, that one mechanism by which estrogen may regulate the production of P4 by the placenta during primate pregnancy is via the maintenance of placental mitochondrial cytochrome P-450, the terminal oxidase of cytochrome P-450scc.

Oxidative stress limits vitamin D metabolism by bovine proximal tubule cells in vitro

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 D 3 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 D 3. 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 D 3. 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.

The influence of age on steroidogenic enzyme activities of the rat adrenal gland: enhanced expression of cholesterol side-chain cleavage activity.

The ability of isolated adrenocortical cells to secrete corticosterone in response to ACTH challenge declines as rats age, but the site or mechanism(s) of this impairment is still unknown. To test the functionality of steroidogenic capacity per se, we measured the key enzyme activities involved in corticosterone biosynthesis. We also measured the mitochondrial cytochrome P-450 content and nonsteroidogenic enzymes specific for subcellular fractions. Mitochondria and microsomal fractions were isolated from the adrenals of 2-, 12-, and 18- month-old animals and used for various enzyme measurements.Mitochondrial side-chain cleavage enzyme activity (nanomoles per min mg protein-1) increased from a mean of 0.43 ± 0.06 in 2-month-old rats to 1.26 ± 0.11 and 1.51 ± 0.06 in 12- and 18-month old rats, respectively. After incubation with 5- cholesten-3j8,25-diol (25-hydroxycholesterol; 25 μg/ml) sidechain cleave activity rose to 5.0 ± 0.6, 12.4 ± 1.2, and 16 ± 1.4 nmol min-1 mg protein-1 in adrenal mitochondrial fractions from 2-, 12-, and 18-month-old rats, respectively. In contrast, mitochondrial cytochrome P-450 content did not vary with advancing age. Microsomal δ5-3β-hyroxysteroid dehydrogenase-δ5-δ4- isomerase activities were similar in 2- and 12-month-old rats, but 21-hydroxylase (nanomoles per min mg protein-1) activity was significantly increased in 12-month-old rats (2-month-old, 5.2 ± 0.2; 12-month-old, 7.7 ± 0.5). Finally, mitochondrial 11β- hydroxylase was comparable in both age groups. In addition, activities of mitochondrial nonsteroidogenic enzymes, such as monoamine oxidase, amytal insensitive NADH cytochrome c reductase, cytochrome c oxidase, succinate dehydrogenase, and malate dehydrogenase, did not change with age. It appears from the evidence presented that the activities of the steroidogenic enzymes are not responsible for the diminished capacity in corticosterone production seen with aging in the rat. (Endocrinology120: 2521–2528,1987)

Alterations of heme, cytochrome P-450, and steroid metabolism by mercury in rat adrenal

The treatment of male rats with Hg 2+ resulted in significant alterations in heme and hemoprotein metabolism in the adrenal gland which, in turn, were reflected in abnormal steroidogenic activities and steroid output. Twenty-four hours after the administration of 30 μmol of HgCl 2/kg (sc) the mitochondrial heme and cytochrome P-450 concentrations increased by approximately 50%. Also, Hg 2+ treatment stimulated a porphyrinogenic response which included an 11-fold increase in the activity of δ-aminolevulinate synthetase. The increase in mitochondrial cytochrome P-450 content was reflected in elevated steroid 11β-hydroxylase and cholesterol side-chain cleavage activities. In contrast, Hg 2+ treatment resulted in decreased concentrations of microsomal cytochrome P-450 (−75%) and heme (−45%). Similarly, the reduction in the microsomal cytochrome P-450 content was accompanied by reduced steroid 21α-hydroxylase and benzo[ a]pyrene hydroxylase activities. The mechanisms responsible for the loss of the microsomal cytochrome P-450 content appeared to involve a selective impairment of formation of the holocytochrome as well as an enhanced rate of heme degradation. This suggestion is made on the basis of findings that (a) the decrease in the microsomal cytochrome P-450 content was accompanied by a sevenfold increase in the activity of adrenal heme oxygenase, (b) no decrease in apocytochrome P-450 could be detected in sodium dodecyl sulfate-gel electrophoresis of the solubilized microsomal fractions stained for heme, and (c) the concentration of adrenal microsomal cytochrome b 5 was significantly increased in the Hg 2+-treated animals. It is suggested that Hg 2+ directly caused a defect in adrenal steroid biosynthesis by inhibiting the activity of 21α-hydroxylase. The apparent physiological consequences of this effect included lowered plasma levels of corticosterone and elevated concentrations of progesterone and dehydroepiandrosterone. This abnormal plasma steroid profile is indicative of a 21α-hydroxylase impairment.

Mitochondrial cytochrome P-450 content is not diminished in hCG-desensitized rat testes

Mitochondrial cytochrome P-450 content is not diminished in hCG-desensitized rat testes

Relation of the pituitary gland to gonadal hormone effects on the adrenocortical 11β-hydroxylase system in rats

Studies were conducted to further examine the mechanisms responsible for gonadal hormone effects on the rat adrenocortical 11β-hydroxylase system. Despite higher concentrations of cytochrome P-450 and larger 11-deoxycorticosterone (DOC)-induced difference spectra in adrenal mitochondria from females than males, no sex difference in 11β-hydroxylase activity was observed. The pregnenolone-induced difference spectrum, indicative of cholesterol binding to cytochrome P-450, also was similar in males and females. Testosterone administration to castrated males lowered both 11β-hydroxylase activity and mitochondrial cytochrome P-450 content. Estradiol produced the opposite effects in castrated females. However, when given to ACTH-replaced hypophysectomized rats, neither testosterone nor estradiol affected cytochrome P-450 levels or the rate of 11β-hydroxylation. These observations, taken with the known effects of estradiol and testosterone on ACTH secretion in rats and the effects of ACTH on 11β-hydroxylation, indicate that gonadal hormone effects on the 11β-hydroxylase system are mediated by ACTH.