Evidence of 2-hydroxylation of estradiol-17 beta 17-glucuronide by male rat liver microsomes

When estradiol-17 beta 17-glucuronide was incubated with male rat liver microsomal preparations with a NADPH-generating system, 2-hydroxyestradiol-17 beta 17-glucuronide was obtained. This 2-hydroxylation was shown to occur without cleavage of the conjugate group. The result clearly indicates that estradiol-17 beta 17-glucuronide could act as substrate for rat liver microsomal 2-hydroxylase.     

Androgen glucuronyl transferase activity in rat liver, evidence for the importance of hepatic tissue in 5 alpha-reduced androgen metabolism

To elucidate the role of the liver in 5 alpha-reduced androgen metabolism, we used a rat liver glucuronyl transferase assay to determine the conversion of 17 beta-hydroxy-5 alpha-androstane-3-one (DHT), 5 alpha-androstane-3 alpha, 17 beta-diol (androstanediol), and androsterone to their glucuronide metabolites. Serum levels of the two isomers of androstanediol glucuronide (androstanediol 3- and 17-glucuronide) were also measured. Using 5 microM unconjugated steroid as substrate, the production rate (pmol/mg/min) for each product from its respective unconjugated steroid was 6.9 +/- 0.4 for DHT glucuronide, 101 +/- 3.3 for androstanediol 3-glucuronide, 71 +/- 2.0 for androstanediol 17-glucuronide, and 181 +/- 11 for androsterone glucuronide. Production rates for androstanediol glucuronide were 800 times greater for rat liver than for rat prostate, when examined under similar conditions. In the presence of either 0 or 5 microM unlabeled androstanediol, about 60% of the androstanediol glucuronide formed by rat liver was androstanediol 3-glucuronide. In normal male rat serum, 69 +/- 8% (mean +/- SEM) of total androstanediol glucuronide was androstanediol 3-glucuronide. We have previously shown that rat prostate forms androstanediol 17-glucuronide, but not androstanediol 3-glucuronide. The results from the present study indicate that rat liver forms both androstanediol glucuronide isomers, and does so in about the same ratio as is found in rat serum. The rate of glucuronidation is also much greater in rat liver than in rat prostate. While other sites of glucuronidation are possible, these results are consistent with the hypothesis that DHT and other unconjugated androgens formed in rat prostate are conjugated to glucuronic acid mainly in the liver.     

Multiplicity of in vitro glucuronidation of 2-hydroxyestriol

In vitro glucuronidation of 2-hydroxyestriol has been investigated by means of HPLC with dual-electrode coulometric detection. When incubated with rat or dog liver microsomal preparation in the presence of UDPGA, 2-hydroxyestriol was transformed into the 2-glucuronide together with a small amount of 16- and/or 17-glucuronides. In contrast, incubation of 2-hydroxyestriol with guinea-pig liver microsomal preparation yielded the 3-glucuronide and a trace amount of the 2-glucuronide, but no ring D glucuronides. Upon pretreatment with 3-methylcholanthrene male rat liver exhibited a marked increase in both 2- and 3-glucuronidation activities, whereas female rat liver showed an elevation only in 2-glucuronidation. On the other hand, in male and female rats pretreatment with phenobarbital caused a relatively small increase in the glucuronidation activity of the liver. In the male guinea-pig, glucuronidation was not affected by pretreatment with either of the two compounds. The present result demonstrates the multiplicity of hepatic 2-hydroxyestriol UDP-glucuronyl-transferase in the rat, guinea-pig and dog.     

Formation of glucuronides of estradiol-17 beta by human mammary cancer cells

The formation of glucuronides of estradiol-17 beta by human mammary cancer cell lines is reported for the first time. When incubated with [3H]estradiol-17 beta (1 nM) for 16 h, ZR-75-1 and T47-D cells formed estradiol-3-glucuronide and estradiol-17 beta-glucuronide in approximately equal proportions, whereas MCF-7 cells formed E2-3-glucuronide only. Yields of monoglucuronides from MCF-7 and ZR-75-1 cells were 0.35 pmol/mg DNA, which represented 20-26% of the yield of estradiol-monosulphates. A HPLC system capable of separating most estradiol monosulphates, monoglucuronides and mixed conjugates, is described.     

Genotoxic effects of estradiol-17beta on human lymphocyte chromosomes

The cytogenetic effect of a hormonal steroid, estradiol-17beta, was assessed in peripheral blood human lymphocyte culture. Sister chromatid exchanges (SCE) and chromosome aberrations (CA) were scored as genetic end points. Significant induction of CA was observed at 25 microg/ml and 50 microg/ml concentrations of estradiol-17beta in the absence of microsomal activation. The drug was effective in all treatments in the presence of rat liver S(9) microsomal fraction (S(9) mix) and exhibited increased frequency of chromosomal aberrations. The drug was effective in increasing the SCE frequency which was found to be maximum at the dose of 50 microg/ml concentration (i.e., 4.34+/-1.22) both with and without metabolic activation. It was found that estradiol-17beta itself and possibly its metabolites are potent mutagens beyond a particular dose in human lymphocytes.     

Determination of cytochrome P450 1A activities in mammalian liver microsomes by high-performance liquid chromatography with fluorescence detection

A sensitive method for the determination of cytochrome P450 (P450 or CYP) 1A activities such as ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD) in liver microsomes from human, monkey, rat and mouse by high-performance liquid chromatography with fluorescence detection is reported. The newly developed method was found to be more sensitive than previous methods using a spectrofluorimeter and fluorescence plate reader. The detection limit for resorufin (signal-to-noise ratio of 3) was 0.80 pmol/assay. Intra-day and inter-day precisions (expressed as relative standard deviation) were less than 6% for both enzyme activities. With this improved sensitivity, the kinetics of EROD and MROD activities in mammalian liver microsomes could be determined more precisely. EROD activities in human and monkey liver microsomes, and MROD activities in liver microsomes from all animal species exhibited a monophasic kinetic pattern, whereas the pattern of EROD activities in rat and mouse liver microsomes was biphasic. In addition, the method could determine the non-inducible and 3-methylcholanthrene-inducible activities of EROD and MROD in rat and mouse liver microsomes under the same assay conditions. Therefore, this method is applicable to in vivo and in vitro studies on the interaction of xenobiotic chemicals with cytochrome CYP1A isoforms in mammals.     

Simultaneous determination of fluoxetine and its metabolite p-trifluoromethylphenol in human liver microsomes using a gas chromatographic-electron-capture detection procedure

An gas chromatography-electron-capture detection method has been developed for simultaneous determination of fluoxetine and p-trifluoromethylphenol (TFMP), an O-dealkylated metabolite of fluoxetine in human liver microsomes. Prior to the analysis, aliquots of alkalinized microsomal mixture were extracted with ethyl acetate solvent containing acetonitrile (10%, v/v) and the derivatizing reagent, pentafluorobenzenesulfonyl chloride (0.1%, v/v). The organ phase was retained and taken to dryness, the residue was reconstituted in methanol, and the aliquot of extracts was injected directly into a gas chromatograph equipped with an electron-capture detector. 2,4-Dichlorophenol was added to the initial incubation mixture and carried through the procedure as the internal standard. The method provided the mean recoveries of up to 103% for fluoxetine and 104% for TFMP. Acceptable relative standard deviations were found for both within-run and day-to-day assays. The practical limit of detection (signal-to-noise ratio=3) was 1.62 ng/ml for TFMP and 6.92 ng/ml for fluoxetine in human liver microsomes, and the limit of quantitation was 8.1 pg for TFMP and 34.6 pg for fluoxetine. The assay is rapid and sensitive and has been applied successfully to simultaneous quantification of fluoxetine and TFMP in human liver microsomes with different CYP2C19 genotypes.     

Properties of a bicarbonate-stimulated ATPase from rat uterus.

Crude subcellular fractions from rat uterus contain a HCO-3 -stimulated Mg2+ -ATPase with properties analogous to those previously reported for the enzyme in gastric mucosa, pancreas, salivary gland and liver lyosome. Estradiol-17 beta treatment of ovariectomized rats resulted in an increase in uterine mitochondrial (HCO-3 +Mg2+)-ATPase and Mg2+ -ATPase activity. In an early response (105 min) to estradiol-17 beta treatment of ovariectomized rats, the lysosomal enzyme, beta-N-acetylglucosaminidase increased in the nuclear and mitochondrial fractions and decreased in the microsomal and supernatant fractions.     

A direct, highly sensitive fluorometric assay for a microsomal cytochrome P450-mediated O-demethylation using a novel coumarin analog as substrate

A highly sensitive fluorometric assay for the determination of monooxygenase activity in liver microsomes is described. The assay is based on the use of 3-chloro-7-methoxy-4-methylcoumarin which is demethylated to 3-chloro-7-hydroxy-4-methylcoumarin. The rate of formation of 3-chloro-7-hydroxy-4-methylcoumarin was recorded as an increase of fluorescence (lambdaA = 380 nm, lambdaF = 480 nm) with time. When 3-chloro-7-methoxy-4-methylcoumarin was incubated in the presence of MgCl2 and NADPH with rat liver microsomes, a continuous increase of the fluorescence could be measured. The reaction proceeded linearly for about 10 min and at least up to a concentration of 0.1 mg/ml of microsomal protein. Besides 3-chloro-7-hydroxy-4-methylcoumarin a hydroxylated derivative of the substrate was formed as a second metabolite during the incubation. Using an excitation wavelength of 380 nm and a fluorescence/emission wavelength of 480 nm, the fluorescence of this substance (lambdaA = 338 nm, lambdaF = 422 nm) amounted only to about 1% of the fluorescence of the main product. The use of 3-chloro-7-methoxy-4-methylcoumarin as substrate enables the fluorometric determination of the O-dealkylation activity of a cytochrome P450-dependent monooxygenase system in rat liver which is inducible by phenobarbital but not by 3-methylcholanthrene.     

An h.p.l.c. assay for protoporphyrinogen oxidase activity in rat liver.

An h.p.l.c. method is described for the assay of protoporphyrinogen oxidase activity in rat liver. A relatively pure protoporphyrinogen IX substrate was obtained by selectively removing any protoporphyrin IX unreduced by sodium amalgam on a small disposable cartridge packed with a strong anion-exchanger. The protoporphyrin IX formed was extracted with dimethyl sulphoxide/methanol (3:7, v/v) containing mesoporphyrin as the internal standard for separation and quantification by reversed-phase chromatography. The Km for protoporphyrinogen was 9.5 +/- 1.6 microM, and the enzyme activities were 0.59 +/- 0.11 nmol of protoporphyrin IX produced/min per mg of mitochondrial protein and 33.5 +/- 2.7 nmol protoporphyrin IX produced/min per g of liver tissue homogenate. The method is applicable to the determination of enzyme activity in small amounts of human liver biopsy.     

The determination of five steroids in avian plasma by radioimmunoassay and competitive protein-binding.

A method has been developed for the simultaneous determination of testosterone, 5alpha-dihydrotestosterone and corticosterone, or of estrone, estradiol-17beta and corticosterone, after separation on a Celite:propylene glycol:ethylene glycol column (6:1.5:1.5 w/v/v). The lower quarter of the column was packed with a Celite: water mixture (3:1 w/v) as a stationary phase (glycol) 'trap'. This effectively prevented leaching of the glycols into the eluate as the concentration of ethyl acetate in the mobile phase was increased to elute the more polar steroids. In addition, a second system utilizing a Celite: ethylene glycol column (2:1 w/v) for the separation of estrone and estradiol-17beta is described. Testosterone, 5alpha-dihydrotestosterone, estrone and estradiol-17beta were measured by radioimmunoassay and corticosterone by a competitive protein-binding technique. Reliability criteria are presented showing that the assay systems used are accurate and reproducible. Plasma-steroid levels of eight avian species are also presented and compared with those found by other investigators.     

Rapid and sensitive high-performance liquid chromatographic assay for 6-hydroxychlorzoxazone and chlorzoxazone in liver microsomes

A high-performance liquid chromatographic assay was developed for the quantitation of chlorzoxazone and its major metabolite 6-hydroxychlorzoxazone. These compounds along with phenacetin, the internal standard, were extracted from incubation mixtures using ether extraction. The extracts were analyzed on a Brownlee Spheri-5 C₈ column with a mobile-phase of acetonitrile–0.5% phosphoric acid (30:70, v/v). The assay utilized UV detection at 287 nm which provided sensitivity and specificity to simultaneously quantify chlorzoxazone and 6-hydroxychlorzoxazone from liver microsomal samples at amounts of 10 ng and greater. The mean correlation coefficient of the standard curves for 6-hydroxychlorzoxazone and chlorzoxazone was 0.998 and 0.993, respectively, over the range of 25–400 ng, and the regression curves were found to be linear at least through 1600 ng. All components eluted within 7 min, resulting in a total analysis time of 8 min. The inter-day and intra-day coefficients of variation were <7 and <3%, respectively. This method provides a rapid, sensitive and cost-effective assay for 6-hydroxychlorzoxazone and chlorzoxazone in liver microsomal incubations.     

Solubilization and characterization of vitamin K epoxide reductase from normal and warfarin-resistant rat liver microsomes

Two procedures have been developed for the solubilization of vitamin K epoxide reductase from rat liver microsomal membranes using the detergent Deriphat 160 at pH 10.8. The methods are applicable to both normal and Warfarin-resistant-strain rat liver microsomes and yield material suitable for further purification. The preparations retain dithiothreitol-dependent vitamin K quinone reductase activity as well as vitamin K epoxide reductase and are free of vitamin K-dependent carboxylase and epoxidase activities. Optimal epoxide reductase activity is obtained at 0.1 M KCl and pH 9 in the presence of sodium cholate. Artifactual formation of vitamin K metabolites was eliminated through the use of mercuric chloride to remove excess dithiothreitol prior to extraction and metabolite assay. Using the solubilized enzyme, valid initial velocities were measured, and reproducible kinetic data was obtained. The substrate initial velocity patterns were determined and are consistent with a ping-pong kinetic mechanism. The kinetic parameters obtained are a function of the cholate concentration, but do not vary drastically from those obtained using intact microsomal membranes. At 0.8% cholate, the enzymes solubilized from normal Warfarin-sensitive- and Warfarin-resistant-strain rat livers exhibit respective values of Vmax = 3 and 0.75 nmol/min/g liver; Km for vitamin K epoxide = 9 and 4 microM; and Km for dithiothreitol of 0.6 and 0.16 mM.     

Rapid high-performance liquid chromatographic method for the separation of hydroxylated testosterone metabolites

A rapid high-performance liquid chromatography (HPLC) method is described for the quantitation of hydroxytestosterone metabolites. The method combines a Hypersil BDS C₁₈ analytical column (10 cm×0.46 cm) and a linear mobile phase (1.25 ml/min) gradient of tetrahydrofuran–acetonitrile–water (10:10:80, v/v) changing to tetrahydrofuran–acetonitrile–water (14:14:72, v/v) over 10 min then remaining isocratic for 3 min. The total run time for the chromatographic separation of eight metabolites of testosterone is 15 min. Detection by UV is linear between 300 ng/ml and 10 μg/ml with a limit of detection on column of 300 ng/ml. A method for the direct HPLC analysis of liver microsomal incubates of [¹⁴C]testosterone is also briefly described and when combined with the HPLC method, offers a distinct advantage over previously reported methods for the rapid screening of testosterone hydroxylase activity in rat and human liver microsomes.     

Estrogen 1,2-epoxides or estrogen quinones/semiquinones

Metabolic activation of estradiol leading to the formation of catechol estrogens is a prerequisite for its genotoxic activity. Both estrogen-o-quinones/semiquinones and estrogen 1,2-epoxides have been proposed to be responsible for this activity. Incubations of [3H]estradiol and [3H]1 alpha,2 alpha-epoxy-4-estrene-3-one-17 beta-ol (ketotautomer of estradiol 1,2-epoxide) with rat liver microsomal and cytosol preparations were carried out in the presence of SKF 525A, ascorbic acid, glutathione and cysteine. Ascorbic acid decreased binding to proteins and aqueous-soluble fraction with both [3H] estradiol and [3H]epoxyestrenolone in incubations with microsomes but no effect with cytosol fraction. Incubations of microsomes with thiols gave water-soluble metabolites which were characterized as 1(4)-thioether derivatives of 2-hydroxyestradiol and incubations of [3H]epoxyestrenolone with cytosol and thiols gave estradiol-2-thioether. Incubations with ascorbic acid and thiols resulted in decreased formation of water-soluble metabolites in microsomal incubations but not in cytosol incubations. These studies indicate that the major pathway for irreversible binding of estrogens to macromolecules involves estrogen-o-quinones/semiquinones and not estrogen 1, 2-epoxide.     

Metabolites of estradiol-17β in guinea pig uterus late in pregnancy

The metabolism of estradiol-17β by the guinea pig uterus late in pregnancy was studied $\text{in vivo}$ and $\text{in vitro}$.Whole uteri were examined for estrogen metabolites one hour following an intravenous injection of [³H]-estradiol-17β or uterine sections were examined after incubation for one hour at 37°C in medium containing [³H]-estradiol-17β.In both instances uterine tissue metabolized estradiol-17g to five products: estrone, estrone-3-sulfate, 17β-estradiol-3-sulfate, estrone-3-glucuronide and 17β-estradiol-3-glucuronide. Of the total radioactive products 11 – 43% were glucuronides, 17 – 26% were sulfates and 4 – 17% was estrone. These results indicate that the guinea pig uterus actively transforms estradiol-17β into glucuronides and sulfates late in pregnancy.     

The effects of adrenal and gonadal steroids and K+-canrenoate on the metabolism of aldosterone by rat liver microsomes

The synthesis of polar aldosterone metabolites by rat liver microsomes at physiological concentrations of aldosterone (21.5 nM), was markedly inhibited by progesterone, testosterone, corticosterone, K+-canrenoate and estradiol-17 beta. In contrast, corticosterone and estradiol-17 beta significantly increased the synthesis of reduced aldosterone metabolites by 8- and 15-fold respectively, the majority of which were 5 alpha-reduced products of aldosterone. In experiments at higher substrate (aldosterone) concentrations (20-200 microM) the synthesis of ring A-reduced aldosterone metabolites by liver microsomes followed Michaelis-Menten kinetics with a Km[app] for aldosterone of 160 microM and Vmax[app] of 12.2 nmoles/mg protein/5 min. In these experiments progesterone, testosterone and K+-canrenoate all competitively inhibited the synthesis of reduced metabolites with inhibition constants (Ki [app]) of 70, 85 and 55 microM respectively; however, corticosterone did not. In contrast, estradiol-17 beta increased the rate of synthesis of reduced products by 40%, lowering the Km[app] to 83 microM.     

25-Hydroxylation of 1 alpha-hydroxyvitamin D-3 in rat and human liver

1 alpha-Hydroxyvitamin D-3 25-hydroxylase activity was measured in subcellular fractions of rat and human liver. The formation of 1,25-dihydroxyvitamin D-3 was determined by high pressure liquid chromatography. In rat liver 1 alpha-hydroxyvitamin D-3 25-hydroxylase activities were found in the purified nuclei, the heavy mitochondrial fraction and the microsomal fraction. The enrichment of 25-hydroxylase activity was highest in the heavy mitochondrial fraction. With this fraction a minimum amount (about 0.5 mg) of protein was required before formation of 1,25-dihydroxyvitamin D-3 could be detected. Above this amount the reaction was linear with amount of protein up to at least 2 mg/ml. The reaction was also linear with time up to 60 min. An apparent Km value of 2 X 10(-5) M was found. The mitochondrial 25-hydroxylase was stimulated by addition of cytosolic protein or bovine serum albumin. The degree of stimulation was dependent on the amount of mitochondrial protein present in the incubation mixture. Maximal stimulation was seen with 0.2 mg/ml of either protein in the presence of 0.5 mg mitochondrial protein. The stimulating effect remained after heating the protein for 5 min at 100 degrees C. The cytosolic protein did not stimulate a reconstituted mitochondrial 1 alpha-hydroxyvitamin D-3 25-hydroxylase. The mitochondrial vitamin D-3 25-hydroxylase was inhibited both by cytosolic protein and by bovine serum albumin. Human liver revealed only one 1 alpha-hydroxyvitamin D-3 25-hydroxylase activity located to the heavy mitochondrial fraction. The results are in agreement with previous studies on the localization of vitamin D-3 25-hydroxylase in rat and human liver. The difference in localization of the 25-hydroxylase between rat and human liver implies that studies on the regulation of the microsomal 25-hydroxylase in rat liver may not be relevant to the situation in human liver.     

A convenient assay for mephenytoin 4-hydroxylase activity of human liver microsomal cytochrome P-450

A simple and rapid method for the determination of (S)-mephenytoin 4-hydroxylase activity by human liver microsomal cytochrome P-450 has been developed. [Methyl-14C] mephenytoin was synthesized by alkylation of S-nirvanol with 14CH3I and used as a substrate. After incubation of [methyl-14C]mephenytoin with human liver microsomes or a reconstituted monooxygenase system containing partially purified human liver cytochrome P-450, the 4-hydroxylated metabolite of mephenytoin was separated by thin-layer chromatography and quantified. The formation of the metabolite depended on the incubation time, substrate concentration, and cytochrome P-450 concentration and was found to be optimal at pH 7.4. The Km and Vmax rates obtained with a human liver microsomal preparation were 0.1 mM and 0.23 nmol 4-hydroxymephenytoin formed/min/nmol cytochrome P-450, respectively. The hydroxylation activity showed absolute requirements for cytochrome P-450, NADPH-cytochrome P-450 reductase, and NADPH in a reconstituted monooxygenase system. Activities varied from 5.6 to 156 pmol 4-hydroxymephenytoin formed/min/nmol cytochrome P-450 in 11 human liver microsomal preparations. The basic system utilized for the analysis of mephenytoin 4-hydroxylation can also be applied to the estimation of other enzyme activities in which phenol formation occurs.     

A sensitive capillary GC assay for the determination of sparteine oxidation products in microsomal fractions of human liver

A sensitive method for the assay of sparteine oxidase activity in vitro by microsomal fractions of human liver is described. The activity of sparteine oxidase was assessed by the formation of 2- and 5-dehydrosparteines, which were estimated by capillary gas chromatography with N2-FID detection. The limit of detection of the two metabolites, 2- and 5-dehydrosparteine, was 10 pmol (2.3 ng) per sample. Sparteine oxidase activity was linear with microsomal protein concentration ranging from 25 to 200 ug and with incubation times between 5 and 60 minutes. Omission of NADPH on incubation under an atmosphere of carbon monoxide inhibited formation of both metabolites, thus indicating that aforementioned metabolites arise in reaction catalyzed by cytochrome P-450. In three liver samples from humans classified as extensive (EM) metabolizers the formation of 2- and 5-dehydrosparteines was observed, 2-dehydrosparteine being the major metabolite. In these samples sparteine oxidase activity was characterised by Vmax = 136 +/- 53 pmol/min/mg and Km = 44 +/- 12 microM for 2-dehydrosparteine formation. For 5-dehydrosparteine formation the following values were obtained: Vmax = 57 +/- 18 pmol/min/mg and Km = 42 +/- 26 microM. In a liver sample from a poor metabolizer (PM) only the formation of 2-dehydrosparteine was detected with the method of analysis used. In this sample a great increase in Km (Km PM = 3033 microM) was noted, while Vmax was very similar to those obtained for 2-dehydrosparteine formation in EM subjects (Vmax PM = 147 pmol min/mg).