Interrelationship between RU38486 and the P450 activities in rat liver

Microsomal P450 monooxygenases contribute actively to the biotransformation of the antiglucocorticoid RU38486, an 11 beta-substituted nor-steroid. Pretreatment of adult rats by inducers of specific forms, belonging to different P450 subfamilies, affects the ability of liver microsomes to metabolize RU38486. Phenobarbital and pregnenolone 16 alpha-carbonitrile increase the metabolic activity of liver microsomes whereas methylcholanthrene decreases their capacity to oxidize the steroid. Thus P450 forms IIIA, IIB1,2 and IIC7 are good candidates to be involved in the degradation of this peculiar molecule. Our study has been completed by investigating whether RU38486 would influence the P450 spectrum. Whereas the treatment of rats with either a glucocorticoid (cortisol, dexamethasone) or an antiglucocorticoid (pregnenolone 16 alpha-carbonitrile) has been shown to induce the P450 activity by increasing the hepatic concentration of form IIIA, we observed a slight decrease of the P450 activity by treating the animals with RU38486. Moreover RU38486 was able to antagonize the P450 induction by the other steroids as well as it inhibits the synthesis of various liver enzymes induced by glucocorticoids (for instance tyrosine aminotransferase). These findings may be important for the therapeutic use of RU38486 since its inhibitory effect on P450 activity may be at the origin of drug interactions by modifying the endogenous hormonal status.     

Metabolism and antiproliferative effect of the glucocorticoid antagonist RU38486 in cultured liver and hepatoma cells

In an attempt to elucidate the relationship between the antiglucocorticoid effect and the state of differentiation of the target cells, we studied the metabolism of the potent antagonist in cultured liver and hepatoma cells (HTC, FAZA). After incubation of [3H]RU38486 with the cells for different periods of time, the native steroid and its metabolites were extracted and analyzed by thin layer chromatography. We observed that RU38486 was not metabolized in the transformed cell lines after a 3 h incubation. In contrast RU38486 was extensively metabolized in cultured liver cells. The observed degration could help explain why RU38486 inhibited tyrosine aminotransferase induction in hepatoma cells at a concentration 100 times lower than that needed in liver cells. Moreover this catabolism concerned specifically the antagonist RU38486 since other steroids tested (dexamethasone, promegestone) underwent a much slower degradation. Indirect experiments suggest that the alterations of the RU38486 molecule might be at least partially related to the cytochrome P-450 which is very active in the hepatocytes. This study was paralleled by testing the effect of the antagonist on the growth of hepatoma cells. RU38486 exerted an antiproliferative effect in absence of serum. On the basis of the low metabolism of RU38486 and of its antiproliferative effect in hepatoma cells. one can emphasize that RU38486 might represent a potential drug for use in cancer therapy.     

Dimemorfan N-demethylation by mouse liver microsomal cytochrome P450 enzymes

Dimemorfan (d-3-methyl-N-methylmorphinan), an analogue of dextromethorphan, is commonly used as a non-opioid antitussive. To clarify the contribution of cytochrome P450 (P450) in dimemorfan N-demethylation, effects of selective inducers and inhibitors were studied in ICR mice. Phenobarbital (PB)- and dexamethasone (Dex)-treatments caused 5-fold increases of liver microsomal dimemorfan N-demethylation activity. In untreated mouse liver microsomes, demethylation activity was strongly inhibited by a CYP3A inhibitor, ketoconazole. In PB-and Dex-treated mouse liver microsomes, ketoconazole caused strong inhibition, whereas orphenadrine caused a decrease of less than 20%. Pretreatment of control mouse liver microsomes with anti-CYP3A inhibited demethylation activity, whereas pre-treatment with anti-CYP2B had no effect. In PB-and Dex-treated mouse liver microsomes, the demethylation activity was inhibited by both anti-CYP3A and anti-CYP2B. In control mice, the intrinsic clearance of dimemorfan from N-demethylation was 5.8 microl min(-1)mg protein(-1). In PB- and Dex-treated mice, the correlation coefficient of fitting using one-enzyme and two-enzyme models were similar. The intrinsic clearances of induced mouse liver microsomes were similar. These results revealed that CYP3A played a major role in hepatic demethylation in untreated mice. Both CYP3A and CYP2B were involved in this demethylation in PB- and Dex-treated mice.     

NADPH-cytochrome P-450 reductase is not a component of the liver microsomal steroid 5-alpha reductase

Liver microsomal steroid 5-alpha-reduction is catalyzed by a NADPH-dependent enzyme system. The requirement of NADPH-cytochrome P-450 reductase to shuttle reduction equivalents from NADPH to steroid 5-alpha-reductase was investigated using an inhibitory antibody against NADPH-cytochrome P-450 reductase. This antibody preparation inhibited cytochrome c reduction in microsomes from female rat liver with an I50 of 0.75 mg antibody/mg of microsomal protein. Benzphetamine N-demethylation and testosterone 6-beta-hydroxylation, two cytochrome P-450-mediated oxidative reactions, were inhibited by the antibody. On the other hand, testosterone 5-alpha-reductase was not affected by the antibody. These results suggest that NADPH-cytochrome P-450 reductase is not an obligatory component of the liver microsomal steroid 5-alpha-reduction.     

How the potency of the steroid RU486 is related to P450 activities induced by dexamethasone and phenobarbital in rat hepatoma cells.

In a previous work on rat liver microsomes, we demonstrated that cytochrome P450 isozymes (P450) are engaged in the metabolism of RU486. In order to study the underlying mechanism at the molecular level, our investigations were shifted to a simplified system of cultured hepatoma cells which present a dissociation in the expression of distinct P450 coding genes. Our results show that Fao cells represent a convenient model to study both: (i) the degradation of RU486. Forms IIB1,2 and IIC7, which are present in Fao cells, may contribute to the demethylation of the molecule. Form IIIA, which has not been detected in Fao cells, is probably responsible for its oxidation in the liver; (ii) the effect of RU486 on the expression of P450 enzymes. Unlike other steroids (dexamethasone and pregnenolone 16-carbonitrile), RU486 does not induce P450 activity but inhibits the inducing activity of other agents such as dexamethasone and also phenobarbital. These findings may be important for the therapeutic use of RU486 since its inhibitory effect on P450 activity may be at the origin of drug interactions by modifying the endogenous hormonal status.     

Heparin is not metabolized by the perfused rat liver

The role of the liver in metabolism of heparin was studied using the isolated rat liver perfused in vitro for 10 hr. Porcine intestinal heparin (1000 u) was added to the recirculating liver perfusate, and serial heparin measurements were performed on the liver perfusate every 2 hr, as well as on bile samples secreted by the perfused liver. Heparin concentration remained at a constant level throughout the 10 hr of perfusion, and there was no detectable heparin secreted into bile samples. The findings suggest that hepatic metabolism/clearance plays a minimal role in heparin kinetics in plasma.     

Redox cycling of resorufin catalyzed by rat liver microsomal NADPH-cytochrome P450 reductase

The O-dealkylation of 7-alkoxyresorufins to the highly fluorescent compound, resorufin (7-hydroxyphenoxazone), provides a rapid, sensitive, and convenient assay of certain forms of liver microsomal cytochrome P450. The results of this study indicate that NADPH-cytochrome P450 reductase catalyzes the reduction of resorufin (and the 7-alkoxyresorufins) to a colorless, nonfluorescent compound(s). The reduction of resorufin by NADPH-cytochrome P450 reductase was supported by NADPH but not NADH, and was not inhibited by dicumarol, which established that the reaction was not catalyzed by contaminating DT-diaphorase (NAD[P]H-quinone oxidoreductase). In addition to the rate of reduction, the extent of reduction of resorufin was dependent on the concentration of NADPH-cytochrome P450 reductase. The maintenance of steady-state levels of reduced resorufin required the continuous oxidation of NADPH, during which molecular O2 was consumed. When NADPH was completely consumed, the spectroscopic and fluorescent properties of resorufin were fully restored. These results indicate that the reduction of resorufin by NADPH-cytochrome P450 reductase initiates a redox cycling reaction. Stoichiometric measurements revealed of 1:1:1 relationship between the amount of NADPH and O2 consumed and the amount of H2O2 formed (measured fluorometrically). The amount of O2 consumed during the redox cycling of resorufin decreased approximately 50% in the presence of catalase, whereas the rate of O2 consumption decreased in the presence of superoxide dismutase. These results suggest that, during the reoxidation of reduced resorufin, O2 is converted to H2O2 via superoxide anion. Experiments with acetylated cytochrome c further implicated superoxide anion as an intermediate in the reduction of O2 to H2O2. However, the ability of reduced resorufin to reduce acetylated cytochrome c directly (i.e., without first reducing O2 to superoxide anion) precluded quantitative measurements of superoxide anion formation. Superoxide dismutase, but not catalase, increased the steady-state level of reduced resorufin and considerably delayed its reoxidation. This indicates that superoxide anion is not only capable of reoxidizing reduced resorufin, but is considerably more effective than molecular O2 in this regard. Overall, these results suggest that NADPH-cytochrome P450 reductase catalyzes the one-electron reduction of resorufin (probably to the corresponding semiquinoneimine radical) which can either undergo a second, one-electron reduction (presumably to the corresponding dihydroquinoneimine) or a one-electron oxidation by reducing molecular O2 to superoxide anion.(ABSTRACT TRUNCATED AT 400 WORDS)     

Metabolism of DHEA by cytochromes P450 in rat and human liver microsomal fractions

Administration of dehydroepiandrosterone (DHEA) to rodents produces many unique biological responses, some of which may be due to metabolism of DHEA to more biologically active products. In the current study, DHEA metabolism was studied using human and rat liver microsomal fractions. In both species, DHEA was extensively metabolized to multiple products; formation of these products was potently inhibited in both species by miconazole, demonstrating a principal role for cytochrome P450. In the rat, use of P450 form-selective inhibitors suggested the participation of P4501A and 3A forms in DHEA metabolism. Human liver samples displayed interindividual differences in that one of five subjects metabolized DHEA to a much greater extent than the others. This difference correlated with the level of P4503A activity present in the human liver samples. For one subject, troleandomycin inhibited hepatic microsomal metabolism of DHEA by 78%, compared to 81% inhibition by miconazole, suggesting the importance of P4503A in these reactions. Form-selective inhibitors of P4502D6 and P4502E1 had a modest inhibitory effect, suggesting that these forms may also contribute to metabolism of DHEA in humans. Metabolites identified by LC-MS in both species included 16alpha-hydroxy-DHEA, 7alpha-hydroxy-DHEA, and 7-oxo-DHEA. While 16alpha-hydroxy-DHEA appeared to be the major metabolite produced in rat, the major metabolite produced in humans was a mono-hydroxylated DHEA species, whose position of hydroxylation is unknown.     

Tetrahydrofurane - an inhibitor for ethanol-induced liver microsomal cytochrome P450.

Liver microsomes from ethanol-pretreated rats have been compared with microsomes from male and female controls and phenobarbital- and benzpyrene-pretreated rats. The 0-dealkylation activity for 7-ethoxycoumarin was enhanced after all treatments. Metyrapone selectively inhibited the activity after pretreatment with phenobarbital and naphthoflavone blocked the activity after benzpyrene treatment. Ethanol and even more so tetrahydrofurane inhibited specifically the 0-dealkylation in microsomes from ethanol-pretreated rats. Only in these microsomes tetrahydrofurance produced a pronounced ligand-type optical difference spectrum and concomitantly a new low-spin cytochrome P450 species in the EPR-spectrum. According to inhibition experiments, liver microsomes from male and female rats have a different pattern of cytochrome P450 species.     

Nutritionally triggered alterations in the regiospecificity of arachidonic acid oxygenation by rat liver microsomal cytochrome P450

Cytochrome P450-dependent oxidation of arachidonic acid was studied in liver microsomes from normal fed, protein-energy malnourished, and refed rats. The overall rate of arachidonic acid oxidation was very similar in microsomes from the three groups, but microsomes from malnourished rats showed a higher turnover rate than microsomes from normal fed and refed rats. The regiospecificity of cytochrome P450 oxidation of arachidonic acid was drastically altered by the animal nutritional status. Thus, protein-energy malnutrition results in a clear stimulation of total omega and omega-1 hydroxylation, concomitant with a marked decrease in olefin epoxidation and allyllic oxidations. These changes, as well as the documented biological activity of some of the cytochrome P450 arachidonate metabolites, suggest that protein-energy deficiency might help to select P450 isozymes which are probably involved in key monooxygenation reactions of physiological substrates.     

Inhibition of steroid 5 alpha-reductase and its effects on testosterone hydroxylation by rat liver microsomal cytochrome P-450

It has been shown previously that liver microsomal steroid 5 alpha-reductase activity increases with age in female but not male rats, which coincides with a female-specific, age-dependent decline in the cytochrome P-450-dependent oxidation of testosterone to 1 beta-, 2 alpha-, 2 beta-, 6 alpha-, 6 beta-, 7 alpha-, 15 beta-, 16 alpha-, 16 beta-, and 18-hydroxytestosterone and androstenedione. To determine whether the increase in steroid 5 alpha-reductase activity is responsible for the decrease in testosterone oxidation, we have examined the effects of the steroid 5 alpha-reductase inhibitor, 4-MA (17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one), on the pathways of testosterone oxidation catalyzed by rat liver microsomes. We have also determined which hydroxytestosterone metabolites are substrates for steroid 5 alpha-reductase. At concentrations of 0.1 to 10 microM, 4-MA completely inhibited steroid 5 alpha-reductase activity without inhibiting the pathways of testosterone oxidation catalyzed by liver microsomes from rats of different age and sex, and from rats induced with phenobarbital or pregnenolone-16 alpha-carbonitrile. 4-MA (10 microM) had little or no effect on the oxidation of testosterone catalyzed by liver microsomes from mature male rats (which have low steroid 5 alpha-reductase activity). In contrast, the hydroxylated testosterone metabolites formed by liver microsomes from mature female rats (which have high steroid 5 alpha-reductase activity) accumulated to a much greater extent in the presence of 4-MA. Evidence is presented that 4-MA increases the accumulation of hydroxytestosterones by two mechanisms. First, 4-MA inhibited the 5 alpha-reduction of those metabolites (such as 6 beta-hydroxytestosterone) that were found to be excellent substrates for steroid 5 alpha-reductase. In the absence of 4-MA, these metabolites eventually disappeared from incubations containing liver microsomes from mature female rats. Second, 4-MA inhibited the formation of 5 alpha-dihydrotestosterone, which otherwise competed with testosterone for oxidation by cytochrome P-450. This second mechanism explains why 4-MA increased the accumulation of metabolites (such as 7 alpha-hydroxytestosterone) that were found to be poor substrates for steroid 5 alpha-reductase. Despite its marked effect on the accumulation of hydroxylated testosterone metabolites, 4-MA had no effect on their initial rate of formation by liver microsomes from either male or female rats.(ABSTRACT TRUNCATED AT 400 WORDS)     

Multiple sites of steroid hydroxylation by the liver microsomal cytochrome P-450 system: primary and secondary metabolism of androstenedione

To investigate the potential interaction of the various pathways of androgen hydroxylation, we have conducted studies to identify the profile of products formed during the time course of metabolism of androst-4-ene-3,17-dione (AD). Incubates containing AD, NADPH, and liver microsomes (from rats pretreated with phenobarbital) were sampled at times between 0 and 20 min and the metabolites resolved by reverse-phase (C18) high-performance liquid chromatography. By this method, the pattern of formation and of utilization of eight major primary and secondary metabolites of AD was determined. We report here the formation of two previously unidentified major metabolites of AD: 6 beta,16 alpha-dihydroxyandrost-4-ene-3,17-dione and 6 beta,16 beta-dihydroxyandrost-4-ene-3,17-dione. We propose that liver microsomal cytochromes P-450 can sequentially hydroxylate a single molecule of AD at multiple sites. These hydroxylase activities are presumably a result of multiple cytochrome P-450 isozymes acting on AD resulting in a transient time course for the appearance of some monohydroxylated metabolites. In addition, a unidirectional conversion of the metabolite 16 alpha-hydroxyandrost-4-ene-3,17-dione to 16 beta-hydroxyandrost-4-ene-3,17-dione is described. Evidence is provided to support the role of cytochrome P-450 in catalyzing this reaction.     

EDTA affects cytochrome P450-dependent biotransformation reactions during incubations for the liver microsomal assay

In order to optimize the condition of the liver microsomal assay (LMA), studies were carried out to determine the effects of EDTA on mixed-function oxidase activity and its stability under the exact incubation conditions for the LMA. Aminopyrine N-demethylase (APD) and p-nitroanisole O-demethylase (p-NAD) activities as well as lipid peroxidation development (LP) in S9 liver fractions from beta-naphthoflavone and sodium phenobarbital (beta-NF + PB)- or Aroclor 1254 (AC)-treated mice were examined during a period of preincubation with EDTA ranging from 1 to 40 mM. At 5 mM EDTA, we obtained a strong inhibition of the microsomal LP as well as the greatest value of the mean specific activity (Asp) for both APD and pNAD activities. In agreement with the biochemical data, the presence of 5 mM EDTA in the incubation mixtures for the LMA significantly increased the mitotic gene conversion, mitotic crossing-over and point-reverse mutation of the well-known premutagen cyclophosphamide (30 mM) on the diploid D7 strain of Saccharomyces cerevisiae as the outcome of a greater metabolic activity. We concluded that the systematic use of 5 mM EDTA in LMA mixtures could improve the reliability and sensitivity of such a test.     

Calcitonin gene-related peptide is metabolized by an endopeptidase hydrolyzing substance P

Calcitonin gene-related peptide (CGRP) is cleaved by an endopeptidase, also known to hydrolyze substance P (SP). The enzyme which was isolated from human cerebrospinal fluid, converted rCGRP into two products, clearly separable on HPLC. Amino acid analysis showed cleavage to occur at Leu16-Ser17. The carboxy-terminal fragment, rCGRP-(17-37), was weakly active in inhibiting 125I-rCGRP binding to a rat medulla oblongata membrane preparation, but it showed no binding to spinal cord membranes. The N-terminal fragment, rCGRP-(1-16), had very low or no affinity. Autoradiography with 125I-rCGRP showed distinct labelling of rat dorsal spinal cord, while there was no consistent pattern with 125I-rCGRP-(1-16). In the isolated guinea pig ileum preparation, the two fragments showed no CGRP-like activity. The ability of CGRP to interfere with SP degradation is offered as the explanation why CGRP has been reported to potentiate several biologic actions of SP.     

Adrenodoxin supports reactions catalyzed by microsomal steroidogenic cytochrome P450s

The interaction of adrenodoxin (Adx) and NADPH cytochrome P450 reductase (CPR) with human microsomal steroidogenic cytochrome P450s was studied. It is found that Adx, mitochondrial electron transfer protein, is able to support reactions catalyzed by human microsomal P450s: full length CYP17, truncated CYP17, and truncated CYP21. CPR, but not Adx, supports activity of truncated CYP19. Truncated and the full length CYP17s show distinct preference for electron donor proteins. Truncated CYP17 has higher activity with Adx compared to CPR. The alteration in preference to electron donor does not change product profile for truncated enzymes. The electrostatic contacts play a major role in the interaction of truncated CYP17 with either CPR or Adx. Similarly electrostatic contacts are predominant in the interaction of full length CYP17 with Adx. We speculate that Adx might serve as an alternative electron donor for CYP17 at the conditions of CPR deficiency in human.     

Kinetics of CO and O 2 complexes of rabbit liver microsomal cytochrome P 450

An endonuclease has been isolated and purified from $\text{Escherichia}$$\text{coli}$ which degrades RNA hydrogen bonded to DNA and no other polynucleotide substrates, including double stranded RNA, single stranded RNA, double stranded DNA or single stranded DNA.     

Quaternary structure of the liver microsomal cytochrome P-450

Cytochrome P-450LM2 was isolated from rabbit liver microsomes in a form which was shown to be homogeneous in AcA-22 Ultrogel and ultracentrifugation studies. The molecular mass determined by sedimentation equilibrium roughly corresponded to hexamer composed of 56 kDa monomers. Hexamer structure of the cytochrome was directly demonstrated by electron microscopic study. In the cytochrome P-450LM2 hexamer, monomers seem to be arranged in two layers (three monomers in the layer) in such a way that each monomer occupies a position at the vertices of a triangular antiprism with a 32 point group symmetry.     

Effects of the steroid antagonist RU486 on dimerization of the human progesterone receptor.

We previously reported, using a coimmunoprecipitation assay, that the B form (PR-B) of the human progesterone receptor from T47D human breast cancer cells dimerizes in solution with the A receptor (PR-A) and that the extent of dimerization correlates with receptor binding activity for specific DNA sequences [DeMarzo, A.M., Beck, C.A., O?ate, S.A., & Edwards, D.P. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 72-76]. This suggested that solution dimerization is an intermediate step in the receptor activation process. The present study has tested the effects of the progesterone antagonist RU486 on solution dimerization of progesterone receptors (PR). As determined by the coimmunoprecipitation assay, RU486 binding did not impair dimerization of receptors; rather, the antagonist promoted more efficient solution dimerization than the progestin agonist R5020. This enhanced receptor dimerization correlated with a higher DNA binding activity for transformed receptors bound with RU486. RU486 has been shown previously to produce two other alterations in the human PR when compared with R5020. PR-RU486 complexes in solution exhibit a faster sedimentation rate (6 S) on salt-containing sucrose density gradients than PR-R5020 complexes (4 S), and PR-DNA complexes have a faster electrophoretic mobility on gel-shift assays in the presence of RU486. We presently show that the 6 S PR-RU486 complex is a receptor monomer, not a dimer. The increased sedimentation rate and increased mobility on gel-shift assays promoted by RU486 were also observed with recombinant PR-A and PR-B separately expressed in insect cells from baculovirus vectors. These results suggest that RU486 induces a distinct conformational change both in PR monomers in solution and in dimers bound to DNA. We also examined whether conformational changes in PR induced by RU486 would prevent a PR polypeptide bound to RU486 from heterodimerization with another PR polypeptide bound to R5020. To evaluate this, PR-A and PR-B that were separately bound to R5020 or RU486 in whole cells were mixed in vitro. PR-A-RU486 was capable of dimerization with PR-B-R5020, and this was demonstrated for heterodimers both formed in solution and bound to specific DNA. The capability to form heterodimers in vitro raises the possibility that the antagonist action of RU486 in vivo could in part be imposed in a dominant negative fashion through heterodimerization between one receptor subunit bound to an agonist and another bound to RU486.