Interaction of prostaglandins with adrenal microsomal cytochrome P-450 in the guinea pig.

Studies were carried out to investigate the effects of prostaglandins (PG) in vitro on adrenal microsomal steroid and drug metabolism in the guinea pig. The addition of PGE1, PGE2, PGA1, PGF1 alpha or PGF2 alpha to isolated adrenal microsomes produced typical type I difference spectra. The sizes of the spectra (delta A385-420) produced by prostaglandins were smaller than those produced by various steroids including progesterone, 17-hydroxyprogesterone and 11 beta-hydroxyprogesterone. However, the affinities of prostaglandins and steroids for adrenal microsomal cytochrome P-450, as estimated by the spectral dissociation constants, were similar. Prior addition of prostaglandins to isolated adrenal microsomes did not affect steroid binding to cytochrome P-450 or the rate of steroid 21-hydroxylation. In contrast, prostaglandins inhibited adrenal metabolism of ethylmorphine and diminished the magnitude of the ethylmorphine-induced spectral change in adrenal microsomes. The results indicate that prostaglandins inhibit adrenal drug metabolism by interfering with substrate binding to cytochrome P-450. Since 21-hydroxylation was unaffected by PG, different cytochrome P-450 moieties are probably involved in adrenal drug and steroid metabolism.     

Cyclopropyl oxiranes: reversible inhibitors of cytosolic and microsomal epoxide hydrolases

A series of aryl- and alkyl-substituted cyclopropyl oxiranes were synthesized as potential suicide inhibitors of mouse liver epoxide hydrolase (EH). The inhibitory potency of each compound and its corresponding alkene precursor was determined with mouse liver EHs using [3H]-cis-stilbene oxide as substrate for microsomal EH (mEH) and for glutathione-S-transferase, and using [3H]-trans-stilbene oxide for cytosolic EH (cEH). The cyclopropyl oxiranes all showed low (26-60% at 5 X 10(-4) M) inhibition of glutathione transferase and moderate inhibition (I50 = 5 X 10(-4) to 6 X 10(-6) M) for cEH and mEH. cis-Phenylcyclopropyl oxirane had an I50 for mEH near that for a commonly used inhibitor, 1,1,1-trichloropropene oxide. Inhibition appeared competitive and reversible, and the cyclopropyl oxiranes appeared to function as alternate substrates. Absence of irreversible inhibition is evidence against a strongly electrophilic epoxide-opening mechanism involving a cyclopropyl carbinyl-homoallyl cation rearrangement. Instead, a concerted mechanism is favored, in which electrophilic opening and hydroxide attack occur in a concerted fashion.     

Interaction of prostaglandins with adrenal microsomal cytochrome P-450 in the guinea pig

Studies were carried out to investigate the effects of prostaglandins (PG) $\text{in vitro}$ on adrenal microsomal steroid and drug metabolism in the guinea pig. The addition of PGE₁, PGE₂, PGA₁, PGF_1α or PGF_2α to isolated adrenal microsomes produced typical type I difference spectra. The sizes of the spectra (ΔA_385–420) produced by prostaglandins were smaller than those produced by various steroids including progesterone, 17-hydroxyprogesterone and 11β-hydroxyprogesterone. However, the affinities of prostaglandins and steroids for adrenal microsomal cytochrome P-450, as estimated by the spectral dissociation constants, were similar. Prior addition of prostaglandins to isolated adrenal microsomes did not affect steroid binding to cytochrome P-450 or the rate of steroid 21-hydroxylation. In contrast, prostaglandins inhibited adrenal metabolism of ethylmorphine and diminished the magnitude of the ethylmorphine-induced spectral change in adrenal microsomes. The results indicate that prostaglandins inhibit adrenal drug metabolism by interfering with substrate binding to cytochrome P-450. Since 21-hydroxylation was unaffected by PG, different cytochrome P-450 moieties are probably involved in adrenal drug and steroid metabolism.     

Binding of thiols to microsomal cytochrome P-450.

Lipophilic thiol compounds interact spectrally with liver microsomes from phenobarbital-pretreated rats by formation of unusual optical difference spectra with peaks at 378, 471, 522 and 593 nm in the oxidized state. The binding kinetics were biphasic. The EPR spectrum of cytochrome P-450 was slightly modified but the magnitude of the low-spin signal was unchanged. n-Octanethiol competitively displaced metyrapone and n-octane from the active site of cytochrome P-450. Other thiols behaved similarly with variations in the magnitude and the affinity of the binding process. Tertiary thiols caused the formation of the high-spin cytochrome P-450 substrate complex, and model studies with myoglobin revealed that steric hindrance prevented the liganding of the tertiary thiol group to the ferric cytochrome P-450. Addition of thiols to dithionite reduced microsomes resulted in relatively small spectral changes with maxima at 449 nm typical for ligand complexes of the ferrous cytochrome. It was concluded that lipophilic thiols can be bound as ligands by at least two species of oxidized cytochrome P-450 which represent, however, not more than about one fifth of the total cytochrome P-450 content in liver microsomes from phenobarbital-pretreated rats.     

The role of cytochrome b5 in adrenal microsomal steroidogenesis.

The role of cytochrome b5 in adrenal microsomal steroidogenesis was studied in guinea pig adrenal microsomes and also in the liposomal system containing purified cytochrome P-450s and NADPH-cytochrome P-450 reductase. Preincubation of the microsomes with anti-cytochrome b5 immunoglobulin decreased both 17 alpha- and 21-hydroxylase activity in the microsomes. In liposomes containing NADPH-cytochrome P-450 reductase and P-450C21 or P-450(17) alpha,lyase, addition of a small amount of cytochrome b5 stimulated the hydroxylase activity while a large amount of cytochrome b5 suppressed the hydroxylase activity. The effect of cytochrome b5 on the rates of the first electron transfer to P-450C21 in liposome membranes was determined from stopped flow measurements and that of the second electron transfer was estimated from the oxygenated difference spectra in the steady state. It was indicated that a small amount of cytochrome b5 activated the hydroxylase activity by supplying additional second electrons to oxygenated P-450C21 in the liposomes while a large amount of cytochrome b5 might suppress the activity through the interferences in the interaction between the reductase and P-450C21.     

Electron paramagnetic resonance spectra of mitochondrial and microsomal cytochrome P-450 from the rat adrenal.

The electron paramagnetic resonance (EPR) spectra of rat adrenal zona fasciculate mitochondria showed peaks corresponding to low spin ferric cytochrome P-450 with apparent g values of 2.424, 2.248 and 1.917, and weak signals due to high spin ferric cytochrome P-450 with gx values of 8.08 and 7.80. The former is attributed to cholesterol side chain cleavage cytochrome P-450, the latter to 11beta-hydroxylase cytochrome P-450. On addition of deoxycorticosterone the g = 7.80 signal was elevated and there was an associated drop in the low spinal signal. As the pH was reduced from 7.4 to 6.1, the g = 8.08 signal increased with again a drop in intensity of the low spin signal. Mitochondria from the zona glomerulosa showed similar spectral properties to those described above. Addition of succinate, isocitrate or pregnenolone caused a loss of the g = 8.08 signal. Addition of calcium increased the magnitude of the g = 8.08 signal, and caused a slight reduction in the magnitude of the low spin signal. Also, addition of deoxycorticosterone, pregnenolone, succinate or isocitrate caused slight shifts of the outer lines of the low spin spectrum. Interaction of mitochondrial cytochrome P-450 with metyrapone and aminoglutethimide modified the low spinal parameters. Adrenal microsomal cytochrome P-450 had low spin ferric g values of 2.417, 2.244 and 1.919 and a high spin ferric gxy values of 7.90 and 3.85, distinct from the values obtained with mitochondria.     

Spectral perturbation of human microsomal cytochrome P-450 by flavonoid binding

Aromatase is the cytochrome P-450 complex responsible for oestrogen biosynthesis in vivo. Inhibitors of this enzyme complex might therefore serve to modulate oestrogen-dependent processes by interfering with the production of oestrogens. Thus, these agents may be useful in reproductive processes and in treating oestrogen-dependent disease states such as breast and endometrial cancer. We have demonstrated that inhibitors such as the naturally occurring flavonoids having 5,7-dihydroxy substituents can bind to human placental cytochrome P-450 with affinity comparable to their ability to inhibit aromatization of androstenedione and testosterone to oestradiol and oestrone, respectively. It appears that the mechanism of this inhibition requires the flavonoid to bind to the active site of the cytochrome P-450 without prior generation of metabolic intermediate products. Our data also suggest that the presently known differences in potency of inhibition of cytochrome P-450-mediated aromatization of steroids by different hydroxylated derivatives of 5,7-dihydroxyflavones may arise from their different binding affinity to the enzyme, particularly those compounds hydroxylated in the C3 position in ring C of the flavonoid nucleus.     

Microsomal and cytosolic epoxide hydrolases in rhesus monkey liver, and in normal and neoplastic human liver

The cytosolic epoxide hydrolase (EH-LC) was observed in rhesus monkey liver cytosol, and in both normal and neoplastic human liver. Microsomal epoxide hydrolase (EH-LM) was detected not only in the microsomes of normal and neoplastic human liver and normal rhesus monkey liver, but also in the cytosol of these tissues. No apparent differences were observed between the EH-LM in liver cytosol and that in microsomes. No major differences were observed between the levels of EH-LM in the cytosol of normal and that in neoplastic human liver.     

Artifacts in isoelectric focusing of the microsomal enzymes cytochrome P-450 and NADPH-cytochrome P-450 reductase.

Highly-purified rat liver microsomal cytochrome P-450 and NADPH-cytochrome P-450 reductase (NADPH-ferricytochrome oxidoreductase, EC 1.6.2.4) preparations gave rise to a large number of bands under a variety of isoelectric focusing conditions, as observed after staining for either zymogen or protein. The binding patterns were not independent of sample concentration and position of application, and eluted bands did not refocus as expected. The artifactual heterogeneity is attributed to strong protein-protein interactions and perhaps to complexation of proteins with carrier ampholytes. These findings suggest caution in using isoelectric focusing to resolve mixtures of membrane proteins.     

Spin state transitions of liver microsomal cytochrome P-450.

Spin state transitions of membrane-bound cytochrome P-450 were investigated by difference spectrophotometry using the 'D'-charge transfer absorbance band at 645 nm as a measure of the amount of hemin iron present in the 5-coordinated state. The magnitude of the 'D'-absorbance band in the absence of exogenous substrates, e.g., the concentration of native high spin cytochrome P-450, was evaluated from the difference in absorbance at 645 nm between ferric cytochrome P-450 and the carbon monoxide derivative of the pigment in its ferrous state. The contribution of the native high spin species to the total cytochrome P-450 content of microsomes was calculated to be between 40% and 65% after induction with phenobarbital and polycyclic hydrocarbons, respectively. Up to 80% of the cytochrome P-450 was found to be present in the high spin state after the addition of exogenous substrates. Further, the steady state concentrations of high spin cytochrome P-450, observed in the presence of reduced pyridine nucleotides, suggest that the rate limiting step for microsomal mixed function oxidation reactions is variable and dependent on the substrate under investigation.     

Differential oxidase activity of hepatic and pulmonary microsomal cytochrome P-450 isozymes after treatment with cytochrome P-450 inducers.

Phenobarbital, 3-methylcholanthrene, acetone and pyrazole were used as inducers of cytochrome P450 and the NADPH-dependent oxidase activity (O-2 production) of pulmonary and hepatic microsomes was determined. Oxidase activity of microsomes from 3-methylcholanthrene-treated rats was significantly decreased as compared to that of controls when expressed on the basis of cytochrome P450 content (30% decrease for liver, 60% decrease for lung). The oxidase activity of liver microsomes from pyrazole-treated rats showed a significant increase, whereas phenobarbital treated microsomes had average superoxide-generating activity. The contribution of cytochromes CYP 1A, CYP 2B and CYP 2E1 to superoxide-generating activity was investigated using monoclonal antibodies. Monoclonal antibody 1-91-3 against CYP 2E1 inhibited superoxide generation by 58% in liver microsomes from pyrazole-treated rats. Monoclonal antibodies 1-7-1 and 2-66-3 against CYP 1A and CYP2B, respectively, had no effect on superoxide generation. These results indicate that different cytochrome P450 isoforms are mainly responsible for differential superoxide generating activities of microsomes and complement the reconstitution study of Morehouse and Aust. Furthermore, our study indicates that CYP 1A1, induced by 3-MC, demonstrates an unusually low oxidase activity.     

Heterogeneity of liver microsomal cytochrome P-450: the spectral characterization of reactants with reduced cytochrome P-450.

The aerobic metabolism of benzphetamine by liver microsomes, during a cytochrome P-450-catalyzed mixed-function oxidation reaction, results in the formation of an easily detected spectral complex with an absorption band maximum at 456 nm. Electron paramagnetic resonance studies, as well as studies with the chemical reductant, sodium dithionite, or the oxidant, potassium ferricyanide, indicate that the spectral complex results from the formation of a product adduct with reduced cytochrome P-450. The spectral properties of this product complex of cytochrome P-450 have been compared to those observed with carbon monoxide, metyrapone, and ethylisocyanide. The reaction of these reagents to specific pools of microsomal cytochrome P-450 permits the identification of at least two major and two minor types of cytochrome P-450 in liver microsomes prepared from phenobarbital-treated rats.     

Electron paramagnetic resonance spectra of mitochondrial and microsomal cytochrome P-450 from the rat adrenal

The electron paramagnetic resonance (EPR) spectra of rat adrenal zona fasciculata mitochondria showed peaks corresponding to low spin ferric cytochrome P-450 with apparent g values of 2.424, 2.248 and 1.917, and weak signals due to high spin ferric cytochrome P-450 with g_x values of 8.08 and 7.80. The former is attributed to cholesterol side chain cleavage cytochrome P-450, the latter to 11β-hydroxylase cytochrome P-450. On addition of deoxycorticosterone the g = 7.80 signal was elevated and there was an associated drop in the low spin signal. As the pH was reduced from 7.4 to 6.1, the g = 8.08 signal increased with again a drop in intensity of the low spin signal. Mitochondria from the zona glomerulosa showed similar spectral properties to those described above. Addition of succinate, isocitrate or pregnenolone caused a loss of the g = 8.08 signal. Addition of calcium increased the magnitude of the g = 8.08 signal, and caused a slight reduction in the magnitude of the low spin signal. Also, addition of deoxycorticosterone, pregnenolone, succinate or isocitrate caused slight shifts of the outer lines of the low spin spectrum. Interaction of mitochondrial cytochrome P-450 with metyrapone and aminoglutethimide modified the low spin parameters. Adrenal microsomal cytochrome P-450 had low spin ferric g values of 2.417, 2.244 and 1.919 and high spin ferric g_xy values of 7.90 and 3.85, distinct from the values obtained with mitochondria.     

Interactions of endogenous and exogenous estrogenic compounds with human placental microsomal cytochrome P-450 (P-450hpm)

Spectral analyses of human placental microsomal cytochrome P-450 (P-450_hpm) revealed that estrone, β-estradiol, and estriol each interacted with the cytochrome to produce Type I difference spectra. β-Estradiol produced the most intense difference spectra and additional studies with this steroid suggested the existence of multiple sites for estrogen binding or of multiple cytochromes in placental microsomes. The magnitude of difference spectra produced by β-estradiol correlated (r = 0.84) with concentrations of P-450_hpm as determined by CO-difference spectra as well as with the intensity of difference spectra produced by androstenedione (r = 0.87). No statistically significant correlations between intensities of β-estradiol-induced binding spectra and placental aryl hydrocarbon hydroxylase activities were observed. The ability of the naturally occurring estrogens to bind to cytochrome P-450 did not appear to be related to their capacity to inhibit the placental aromatization reaction. Diethylstilbestrol, ethinylestradiol, mestranol, progesterone, and pregnenolone all inhibited the placental conversion of androstenedione to estrogens, but these compounds did not produce observable Type I difference spectra. Of the estrogens studied, diethylstilbestrol proved to be the most effective inhibitor of aromatase in vitro causing 99% inhibition at concentrations of 10⁻⁴M. β-Estradiol and ethinylestradiol produced only 60.4 and 81.0% inhibition, respectively, at 10⁻³ M. The results indicated that despite significant binding of endogenous estrogens to P-450_hpm, no evidence for a feedback inhibition of these estrogens on the placental aromatization reaction could be observed.     

Studies on the interaction of steroid substrates with adrenal microsomal cytochrome P-450 (P-450C21) in liposome membranes

Cytochrome P-450 (P-450C21), purified from bovine adrenocortical microsomes, was incorporated into the single bilayer liposomes of egg yolk phosphatidylcholine by gel filtration, using a high pressure liquid chromatography system. Interaction of the steroid substrates, 17 alpha-hydroxyprogesterone and progesterone, with P-450C21 in the liposomes was studied in the equilibrium state by measuring substrate-induced spectral change. The apparent dissociation constant of the P-450C21-substrate complex increased with phosphatidylcholine concentration in the system, showing the substrate to be partitioned between the aqueous and lipid phases. Partition coefficients, determined by equilibrium dialysis and the Hummel-Dreyer method, were 3500 for progesterone and 2000 for 17 alpha-hydroxyprogesterone at 25 degrees C. The binding process of the substrates to P-450C21 in the liposomes and their dissociation were measured by a stopped flow method. The apparent rate of substrate binding to P-450C21 in the liposomes was not effected by substrate partitioning, indicating partitioning to occur much more quickly than substrate binding to P-450C21. Absorption changes observed in the stopped flow experiments were analyzed at a rapid equilibrium of partitioning. Based on these results, the substrate binding site of P-450C21 was concluded to face the lipid phase of the liposome membranes.     

Interactions of various 19-nor steroids with human placental microsomal cytochrome P-450 (P-450hpm).

Each of seven 19-nor steroids exhibited the capacity to facilitate the binding of carbon monoxide (CO) to human placental microsomal cytochrome P-450 although quantitative differences were shown to exist. In every case the facilitation was antagonized by androstenedione. 19-Norandrostenedione produced the most pronounced effect followed by 19-nortestosterone, nandrolone decanoate, norethandrolone, norgestrel, norethynodrel and norethindrone in that order. All steroids investigated produced typical type I binding spectra when added to placental microsomes. Scatched plots also indicated binding of each steroid to two sites--a high-affinity, low-capacity binding site and a low-affinity, high-capacity binding site. Correlations between affinity for either site and capacity to facilitate binding of CO to the cytochrome were not observed nor were there good correlations between maximal absorbance differences (approximately390-420 nm) producible and facilitation capacity. It was therefore concluded that no definitive relationships existed between facilitation capacity and qualitative or quantitative aspects of the steroid-binding spectra. The capacity to facilitate CO binding appeared to reside in the absence of a chemical group substituted at the 10 position on molecules of androgenic steroids since all investigated steroids possessing 10-methyl or other 10-substituted groups either had no effect on the CO-binding spectrum or caused a displacement of CO from ferrous heme. In contrast, all steroids studied that lacked a substitution at C-10 (19-nor steroids) produced a facilitating effect on heme-ligand binding.     

Ligand binding of organic sulfides to microsomal cytochrome P-450.

Octyl methyl-, butyl methyl- and pentamethylene sulfide react with about 50% of oxidized cytochrome P-450 in liver microsomes from phenobarbital-pretreated rats by formation of optical difference spectra with maxima at 435 and 552 nm and concomitant shifts in the electron paramagnetic resonance spectrum. Reduction by NADPH or sodium dithionite yielded a Soret absorption band at 449 nm and alpha and beta bands at 573 and 545 nm, respectively. The ligand metyrapone and the substrate n-octane competitively inhibited the formation of these difference spectra and pentamethylene sulfide was a strong competitive inhibitor of the 0-deakylation of 7-ethoxycoumarin. These results indicate a direct ligand binding of the sulfides to cytochrome P-450 with concomitant blocking of the hydrophobic substrate binding site. Some sulfides did not interact as ligands but as substrates, in variation, however, with the source of microsomes.