Presence of a low molecular weight inhibitor of succinate-supported cholesterol side chain cleavage in rat ovaries.

1. Low molecular weight fractions (mol. wt. 3500-10 000) prepared from cytosols of luteinized rat ovaries inhibited succinate-supported cholesterol side chain cleavage by intact ovarian mitochondria utilizing endogenous or exogenous sterol as substrate. 2. The low molecular weight fractions inhibited steroid secretion by collagenase-dispersed ovarian cells stimulated with lutropin or dibutyryl cyclic AMP. 3. Steroidogenesis by intact mitochondria incubated with NADPH was enhanced by the low molecular weight ovarian fraction, but cholesterol side chain cleavage carried out by sonicated mitochondria incubated with NADPH was unaffected. 4. Succinate-supported mitochondrial respiration was stimulated by the low molecular weight factor, apparently by uncoupling of oxidative phosphorylation. The uncoupling seems to be the mechanism by which steroid synthesis is inhibited. 5. The low molecular weight factor was heat-labile and not extracted by activated charcoal. Similar heat-labile material capable of inhibiting succinate-supported mitochondrial steroid synthesis was not found in low molecular weight fractions prepared from rat kidney, liver, spleen, brain, plasma and bovine corpus luteum. 6. Treatment of rats with cycloheximide 1 h before killing resulted in a reduction of inhibitory activity in ovarian low molecular weight cytosolic fractions. 7. We conclude that ovarian cytosols contain a low molecular weight factor, presumably a protein, which inhibits mitochondrial cholesterol side chain cleavage by uncoupling oxidative phosphorylation. The physiological function of this factor remains to be determined.

Metabolism of 25-hydroxycholesterol by rat luteal mitochondria and dispersed cells.

The metabolism of 25-hydroxycholesterol (25-OH-cholesterol) to progestins by mitochondria and dispersed cells prepared from ovaries of PMSG-hCG-primed rats was studied. Mitochondria converted [3H]25-OH-cholesterol into [3H]pregnenolone and [3H]progesterone. Unlabeled 25-OH-cholesterol also stimulated mitochondrial steroidogenesis in a dose-dependent, saturable fashion. A direct relationship between rates of steroid synthesis in the presence of 25-OH-cholesterol and mitochondrial cytochrome P-450 levels was found. Although steroid production and cytochrome P-450 content per milligram protein were higher in mitochrondia prepared from ovaries removed on day 8 post hCG than on either day 1 or day 14, steroid production per nanomole cytochrome P-450 was similar. Treatment of rats with hCG 1 h before killing significantly increased mitrochondrial steroid synthesis from endogenous substrate but had no effect on metabolism of 25-OH-cholesterol. Dispersed cells increased progestin production by 6-fold when incubated with 25-OH-cholesterol. The effects of 25-OH-cholesterol were dose dependent and saturable. While both LH and (Bu)2cAMP stimulated progestin synthesis from endogenous substrate, secretion of progestins with these agents reached levels only 60% of those observed in the presence of 25-OH-cholesterol. Neither LH nor (Bu)2cAMP altered the metabolism of the dydroxysterol by the cells nor did cycloheximide, which substantially inhibited progestin secretion in the absence of the hydroxysterol. However, animoglutethimide did block the stimulation of steroidogenesis by 25-OH-cholesterol. We conclude that 25-OH-cholesterol is an effective steroidogenic substrate for rat luteal tissue. With its use, information regarding the maximal capacity of luteal tissue to produce progestins in vitro can be obtained.

Inhibition of iron-sulfur protein-mediated reduction of cytochrome P-450SCC by specific antibodies.

The mechanism of inhibition of cholesterol side-chain cleavage by specific antibodies was studied systematically. The antibodies had no effect on substrate binding as determined by optical spectroscopy or on the heme environment of the cytochrome P-450 insofar as was detectable by electron paramagnetic resonance spectroscopy. They did not bind to either iron-sulfur protein or its reductase. The antibodies had no effect on chemical reduction of the P-450 or on P-450-CO complex formation. They did inhibit the NADPH-dependent reduction of P-450 and subsequent formation of the P-450-CO complex. This inhibitory effect was concentration dependent and was correlated with the inhibitory effect of the antibodies on enzymatic cholesterol side-chain cleavage. Similar results were obtained using Fab fragments. These results indicate that the antibodies inhibit side-chain cleavage by binding to a region close to the iron-sulfur protein-binding site, thereby preventing transfer of reducing electrons to the cytochrome P-450.

Alteration by perfluorodecalin of hepatic metabolism and excretion of phenobarbital.

The bile duct was cannulated in rats that had been infused intravenously with an emulsion of perfluorodecalin at intervals from 2 to 34 weeks earlier. After injection of [14C]phenobarbital, urine and bile were collected during the next 24 hr and were analyzed for phenobarbital and its metabolites. There was a decrease in the biliary excretion of phenobarbital and its metabolites for several weeks after infusion of perfluorodecalin, but conjugation of the metabolites was not decreased. The reduced excretion returned to normal after about 20 weeks.

In vivo effects of chromium.

The production of reactive oxygen species on addition of hexavalent chromium (potassium dichromate, K2Cr2O7) to lung cells in culture was studied using flow cytometer analysis. A Coulter Epics Profile II flow cytometer was used to detect the formation of reactive oxygen species after K2Cr2O7 was added to A549 cells grown to confluence. The cells were loaded with the dye, 2',7'-dichlorofluorescein diacetate, after which cellular esterases removed the acetate groups and the dye was trapped intracellularly. Reactive oxygen species oxidized the dye, with resultant fluorescence. Increased doses of Cr(VI) caused increasing fluorescence (10-fold higher than background at 200 microM). Addition of Cr(III) compounds, as the picolinate or chloride, caused no increased fluorescence. Electron paramagnetic resonance (EPR) spectroscopic studies indicated that three (as yet unidentified) spectral "signals" of the free radical type were formed on addition of 20, 50, 100, and 200 microM Cr(VI) to the A549 cells in suspension. Two other EPR "signals" with the characteristics of Cr(V) entities were seen at field values lower than the standard free radical value. Liver microsomes from male Sprague-Dawley rats treated intraperitoneally with K2Cr2O7 (130 mumole/kg every 48 hr for six treatments) had decreased activity of cytochromes P4503A1 and/or 3A2, and 2C11. Hepatic microsomes from treated female Sprague-Dawley rats, in contrast, had increased activities of these isozymes. Lung microsomes from male Sprague-Dawley rats had increased activity of P4502C11.

Ecdysone 20-monooxygenase: characterization of an insect cytochrome p-450 dependent steroid hydroxylase.

Ecdysone 20-monooxygenase, the enzyme system that hydroxylates ecdysone at C-20 of the side-chain to form ecdysterone, has been characterized in the fat body of early last instar larvae of the tobacco hornworm, Manduca sexta, using a radioenzymological assay. Ecdysterone was demonstrated to be the product of the enzyme system by high-pressure liquid chromatography, gas-liquid chromatography and mass spectrometry. Differential centrifugation, sucrose-gradient centrifugation, electron microscopy and organelle-marker enzyme analysis revealed that ecdysone 20-monooxygenase activity is associated with the mitochondria. The enzymatic properties of ecdysone 20-monooxygenase are that it is most active in a 0.05 M phosphate buffer, is inhibited by Mg2+ and exhibits pH and temperature optima at 7.5 and 30 degrees C, respectively. The enzyme complex has an apparent Km for ecdysone of 1.60 x 10(-7) M and is competitively inhibited by its product, ecdysterone, with an apparent Ki of 2.72 x 10(-5) M. The cytochrome P-450 nature of this insect steroid hydroxylase was initially suggested by its obligate requirement for NADPH and its inhibition by carbon monoxide, p-chloromercuribenzoate, metyrapone and p-aminoglutethimide but not by cyanide. Difference spectroscopy revealed the presence of cytochrome P-450 in the fat-body mitochondrial fraction. A photochemical action spectrum of ecdysone 20-monooxygenase activity confirmed the involvement of cytochrome P-450 in this monooxygenase system.

Comparison of the induction course, biophysical chemical interactions and photochemical action spectra of phenobarbital and 3-methylcholanthrene induce hepatic microsomal P-450.

A comparison has been made of the physical and chemical properties of hepatic microsomal P-450 and associated enzyme systems from rats treated with phenobarbital or with 3-methylcholanthrene and other polycyclic aryl hydrocarbons. The results of these studies, though preliminary in nature, indicate clearly that the aryl-induced mixed-function oxidase systems differ significantly from the PB-induced ones in time course of induction, spectral properties, hyroxylase and demethylase activities, CO-inhibition of these reactions and light-reversal of the inhibition. The results support and extend the findings of other investigators regarding the differential biophysical and biochemical properties of aryl-induced systems and provide an experimental design for studying these properties in greater depth at the maximum of aryl induction.

Comparative kinetic studies of the dealkylation reaction and steroid hydroxylations in various oxygen and carbon monoxide:oxygen atmospheres.

The time-course kinetics of the cytochrome P-450-catalyzed dealkylations of the exogenous compounds benzphetamine, ethylmorphine, codeine, and 7-ethoxycoumarin were compared to the hydroxylation of the endogenous compound testosterone. Using liver microsomes from phenobarbital-induced rats, the time course of the demethylations of ethylmorphine, codeine, and especially benzphetamine was characterized by a fast initial phase of enzymatic activity and then a steady decline in the rate throughout the remainder of the reaction. In contrast, under the same experimental conditions, both the dealkylation of 7-ethoxycoumarin and the hydroxylation of testosterone showed no initial fast phase of activity and a constant rate of product formation for most of the remainder of the time course. The difference also held for the carbon monoxide inhibition studies in which the degree inhibition of the demethylation reactions by a variety of CO:O2 mixtures was time dependent, in contrast to the constant, time-independent degree of CO inhibition of the other two reactions. The kinetics of the demethylation reactions could not be explained by enzyme destruction, back reaction, or product adduct formation and were further confirmed by measurements of the rate of O2 utilization and NADPH oxidation. The complexity of the demethylation reaction should be taken into consideration in any detailed studies of the monooxygenation reaction system.

A kinetic study comparing the light-reversal properties of carbon monoxide inhibition of ethylmorphine, benzphetamine, and 7-ethoxycoumarin dealkylation with those of hydroxylation of 17-hydroxyprogesterone and testosterone.

The light-reversal properties of carbon monoxide (CO) inhibition of the dealkylation of benzphetamine, ethylmorphine, and 7-ethoxycoumarin by microsomes from phenobarbital (PB)-induced rat livers were compared with those of the 6 beta-, 7 alpha-, and 16 alpha-hydroxylations of testosterone by the same rat hepatic microsomes and C-21 hydroxylation of 17-OH progesterone by steer adrenal microsomes. CO inhibited all reactions studied to essentially the same degree. The significant finding was that the dealkylations were reversed most effectively by light of wavelengths between 440 and 445 nm, rather than around 450 nm, the optimal wavelength for steroid hydroxylations. Moreover, the dealkylations required several-fold higher light intensities for equivalent light reversal. These studies suggest that the heme protein-CO complex responsible for dealkylations has a spectrum corresponding to the shape of the pass band of the 445-nm filter, whereas that of the steroid hydroxylations has its light-reversal maximum at 450 nm and appears to be broader. The measurable differences in the light-reversal properties between the monooxygenations of two groups of substrates, (i) dealkylations and (ii) hydroxylations of lipid substrates, furnish biophysical properties that allow a better characterization of microsomal monooxygenases which should be of value in forwarding progress in the study of these systems.

Biophysical and catalytic properties of the phenobarbital p-hydroxylase--a cytochrome P-450 dependent mixed function oxidase.

1. A sensitive method to detect and quantify the products of phenobarbital (PB) hydroxylation by model chemical systems and by biological systems has been developed. 2. Chemical model systems hydroxylate PB in the p-position of the phenyl ring and form one or two additional oxidation products, while in vitro and in vivo (bile fistula rats) biological systems hydroxylate PB only in the p-position. 3. Phenobarbital hydroxylation rates in vitro are of the order of 0.007 nmol/nmol cytochrome P-450 per min. These values are decreased by pretreatment of the rats with inducing doses of phenobarbital. 4. Enzymes catalysing the p-hydroxylation reaction of phenobarbital are localized in the microsomes and have the biophysical and chemical properties that are usually associated with cytochrome P-450-dependent mixed function oxidases.